Abstract
Platelets are small anucleate blood cells supporting vascular function. They circulate in a quiescent state monitoring the vasculature for injuries. Platelets adhere to injury sites and can be rapidly activated to secrete granules and to form platelet/platelet aggregates. These responses are controlled by signalling networks that include G proteins and their regulatory guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Recent proteomics studies have revealed the complete spectrum of G proteins, GEFs, and GAPs present in platelets. Some of these proteins are specific for platelets and very few have been characterised in detail. GEFs and GAPs play a major role in setting local levels of active GTP-bound G proteins in response to activating and inhibitory signals encountered by platelets. Thus, GEFs and GAPs are highly regulated themselves and appear to integrate G protein regulation with other cellular processes. This review focuses on GAPs of small G proteins of the Arf, Rab, Ras, and Rho families, as well as of heterotrimeric G proteins found in platelets.
Introduction to platelet signalling
Platelets circulate in the bloodstream supporting vascular integrity, haemostasis, immune defence, and tissue repair [1]. Platelets are anucleate and have a lifespan of around 10 days. Core structural components include an open canalicular system which is continuous with the plasma membrane and a dense tubular system involved in Ca2+ storage. Platelets contain 50–80 alpha granules, 3–8 dense granules, and 1–3 lysosomal granules, as well as 5–8 mitochondria [2,3]. Each granule type carries specific regulatory molecules which upon exocytosis stimulate a positive feedback loop recruiting more platelets to lesion sites and supporting their activation. Platelet alpha granules contain more than 300 different proteins while dense granules contain ATP, ADP, Ca2+ ions, and pyrophosphates which are essential for normal haemostasis [9]. Activated platelets change their shape, they adhere to sites of vascular injury and bind to each other to form aggregates [4]. These processes require rapid and extensive remodelling of the cytoskeleton. Furthermore, activated platelets alter the composition of the outer layer of their plasma membrane by exposing phosphatidylserine thus supporting binding and activation of clotting factors leading to extracellular fibrin fibre formation [5]. Platelet functions are tightly controlled by activating and inhibitory signalling networks (Figure 1) [6].
Overview of platelet signalling
Platelet can be activated by von Willebrand factor (vWF) binding to the glycoprotein (GP) complex GPIb/V/IX, collagen binding to GPVI, and podoplanin binding to the C-type lectin-like receptor (CLEC-2) which activate tyrosine kinase signalling leading to activation of phospholipase C (PLC), formation of inositol 1,4,5-triphosphate (IP3) and 1,2-diacylglycerol (DAG) from phosphatidylinositol 4,5-bisphosphate (PIP2), activation of protein kinase C (PKC), and release of Ca2+ ions from intracellular stores. Thromboxane A2 (TXA2), thrombin, and adenosine-diphosphate (ADP) activate TP, protease-activated receptors (PAR1, PAR4 in humans), and purinergic P2Y1 and P2Y12 G-protein coupled receptors leading to activation of α and βγ subunits of heterotrimeric G proteins by exchange of Gα-bound guanosine-5'-diphosphate, GDP, by guanosine-5'-triphoshate, GTP followed by PLC activation and Ca2+ signalling, activation of guanine-nucleotide exchange factors (GEF) of the small G protein RhoA, activation of phosphatidylinositol 3-kinase (PI3K), or inhibition of adenylate cyclase (AC), as indicated, and platelet activation. Gα-GTP is turned into inactive Gα-GDP by regulator of G protein signalling proteins (RGS). RhoA-GTP triggers a range of cytoskeletal rearrangements and RhoA-GTP is turned into inactive RhoA-GDP by GTPase-activation proteins (RhoGAP). Endothelium-derived prostacyclin (PGI2) inhibits platelets through activation of the IP receptor coupled to the stimulatory Gαs protein leading to formation of 3'5'-cyclic adenosine monophosphate (cAMP) from adenosine-5'-triphosphate (ATP), activation of protein kinase A (PKA) and phosphorylation of a wide range of substrate proteins. In parallel, endothelial nitric oxide (NO) diffuses through the plasma membrane and activates NO-sensitive guanylate cyclase (GC) to generate 3'5'-cyclic guanosine monophosphate (cGMP) which activates protein kinase G (PKG) to phosphorylate substrates leading to platelet inhibition. Platelet activation and inhbition pathways interact at multiple levels. Platelet activation leads to activation of the Rap1 G protein which enables the transition of inactive to active integrin αIIbβ3 (αIIbβ3) leading to fibrinogen binding, platelet aggregation, and further platelet activation. Rap1 is regulated by specific GEF and GAP proteins. Arf family G proteins are involved in endocytosis of integrin αIIbβ3 and in the regulation of the cytoskeleton, and in other membrane and vesicle transport processes. Rab family G proteins are required for the generation, transport and release of platelet granules, and are probably involved in many other membrane and vesicle transport processes as well as in endocytosis. Arf and Rab proteins are controlled by dedicated GEFs and GAPs. Created with Biorender.com.
Platelet can be activated by von Willebrand factor (vWF) binding to the glycoprotein (GP) complex GPIb/V/IX, collagen binding to GPVI, and podoplanin binding to the C-type lectin-like receptor (CLEC-2) which activate tyrosine kinase signalling leading to activation of phospholipase C (PLC), formation of inositol 1,4,5-triphosphate (IP3) and 1,2-diacylglycerol (DAG) from phosphatidylinositol 4,5-bisphosphate (PIP2), activation of protein kinase C (PKC), and release of Ca2+ ions from intracellular stores. Thromboxane A2 (TXA2), thrombin, and adenosine-diphosphate (ADP) activate TP, protease-activated receptors (PAR1, PAR4 in humans), and purinergic P2Y1 and P2Y12 G-protein coupled receptors leading to activation of α and βγ subunits of heterotrimeric G proteins by exchange of Gα-bound guanosine-5'-diphosphate, GDP, by guanosine-5'-triphoshate, GTP followed by PLC activation and Ca2+ signalling, activation of guanine-nucleotide exchange factors (GEF) of the small G protein RhoA, activation of phosphatidylinositol 3-kinase (PI3K), or inhibition of adenylate cyclase (AC), as indicated, and platelet activation. Gα-GTP is turned into inactive Gα-GDP by regulator of G protein signalling proteins (RGS). RhoA-GTP triggers a range of cytoskeletal rearrangements and RhoA-GTP is turned into inactive RhoA-GDP by GTPase-activation proteins (RhoGAP). Endothelium-derived prostacyclin (PGI2) inhibits platelets through activation of the IP receptor coupled to the stimulatory Gαs protein leading to formation of 3'5'-cyclic adenosine monophosphate (cAMP) from adenosine-5'-triphosphate (ATP), activation of protein kinase A (PKA) and phosphorylation of a wide range of substrate proteins. In parallel, endothelial nitric oxide (NO) diffuses through the plasma membrane and activates NO-sensitive guanylate cyclase (GC) to generate 3'5'-cyclic guanosine monophosphate (cGMP) which activates protein kinase G (PKG) to phosphorylate substrates leading to platelet inhibition. Platelet activation and inhbition pathways interact at multiple levels. Platelet activation leads to activation of the Rap1 G protein which enables the transition of inactive to active integrin αIIbβ3 (αIIbβ3) leading to fibrinogen binding, platelet aggregation, and further platelet activation. Rap1 is regulated by specific GEF and GAP proteins. Arf family G proteins are involved in endocytosis of integrin αIIbβ3 and in the regulation of the cytoskeleton, and in other membrane and vesicle transport processes. Rab family G proteins are required for the generation, transport and release of platelet granules, and are probably involved in many other membrane and vesicle transport processes as well as in endocytosis. Arf and Rab proteins are controlled by dedicated GEFs and GAPs. Created with Biorender.com.
Platelet activation can be initiated by subendothelial collagen exposed at injury sites providing a binding site for von Willebrand factor (vWF). vWF binding to the glycoprotein (GP) receptor complex GPIb-V-IX establishes platelet tethering but cannot support stable adhesion. Instead, it facilitates platelet binding to collagen via the tyrosine kinase linked GPVI receptor [7]. GPVI is covalently bound to the Fc gamma receptor (FcRγ) and binding of collagen to GPVI induces phosphorylation of an immunoreceptor tyrosine-based activation motif (ITAM) in FcRγ by Src family kinases (Figure 1). This phosphorylation activates the tyrosine kinase Syk leading to hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by phospholipase C γ2 (PLCγ2) resulting in the second messengers 1,2-diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3). DAG activates protein kinase C (PKC) whereas IP3 increases intracellular Ca2+ both leading to platelet activation and granule release [8]. ADP, a dense granule component, binds to and activates the G-protein coupled receptors (GPCR) P2Y12 and P2Y1 leading to amplified platelet activation through reduced cyclic adenosine monophosphate (cAMP) synthesis, phosphatidylinositol 3-kinase (PI3K) activation and through increased Ca2+ signalling (Figure 1, see chapter on heterotrimeric G proteins below for further details). Other amplifiers of platelet activation include thromboxane A2 (TXA2) which activates the thromboxane-prostanoid (TP) GPCR and thrombin, produced in the clotting cascade, which activates platelets through PAR1 and PAR4 GPCRs [9]. Platelet activation leads to a conformational change in the abundant fibrinogen receptor at the plasma membrane, integrin αIIbβ3, which mediates platelet aggregation.
Platelet inhibition is maintained predominantly by the activation of two cyclic nucleotide signalling pathways, one involving cAMP which is activated by endothelium-derived prostacyclin (PGI2), and the other cyclic guanosine monophosphate (cGMP) activated by endothelial nitric oxide (NO) (Figure 1). These pathways are likely to be constitutively active due to the continuous production and release of PGI2 and NO from the endothelium. PGI2 binds to the IP receptor, a GPCR coupled to Gαs which activates adenylate cyclase (AC). AC uses ATP to synthesise cAMP which binds to the regulatory subunit of protein kinase A (PKA) allowing the catalytic subunits to phosphorylate numerous substrate proteins and inhibit platelet activation. NO activates the cGMP pathway following its diffusion through the membrane and subsequent binding to NO-dependent guanylate cyclase which activates protein kinase G (PKG). There is significant overlap between the substrates phosphorylated by PKG and PKA. The inhibitory effects of these pathway are so potent that activation of either pathway can reverse platelet aggregation resulting in dissolution of pre-existing platelet aggregates [10].
Activating and inhibitory pathways converge on guanine-nucleotide binding proteins (G proteins or GTPases) of the Ras superfamily which exert their functions through interactions of the GTP-bound G proteins with downstream effector proteins (Figure 1) [11]. G proteins require guanine nucleotide exchange factors (GEFs) to allow GTP to replace GDP in the active centre. GTPase-activating proteins (GAPs) play a critical role in activating the low endogenous GTPase activities of many G proteins by turning the active GTP-bound forms into their inactive GDP-bound versions. Thus, GAPs effectively serve as off-switches of G protein signalling [12]. This review will provide a broad overview of GAPs of Ras superfamily small G proteins and of alpha subunits of heterotrimeric (αβγ) G proteins found in human platelets. A few GAPs will be described in more detail focusing on available platelet data. Emerging roles of GAPs beyond G protein regulation will be highlighted. Information on platelet GEFs of the Rho family, a member of the Ras superfamily, is included in other reviews [13–16].
Expression of G proteins and GAPs in platelets
The Ras superfamily of small G proteins encompasses Arf, Rab, Ras, and Rho family proteins and each family has its dedicated GAPs. Similarly, the activity of heterotrimeric (αβγ) G proteins is controlled by regulator of G protein signalling (RGS) proteins. Comprehensive proteomics mapping [17,18] has revealed the G proteins and GAPs expressed by human platelets including protein copy numbers per platelet (Tables 1 and 2). G protein specificities have only been established for a few GAPs and even fewer studies have investigated platelet specific roles of GAPs. GAPs have been identified that regulate Rap1, RhoA, Rac1, and Gαi and Gαq in platelets; however, many orphan G proteins are still present, especially in the Arf and Rab families. Almost all ArfGAPs encoded by the human genome, except ADAPs [19], and most Ras/RapGAPs are expressed in platelets. In contrast, platelets express only about half of the encoded RabGAPs, RhoGAPs, and RGSs. Similar numbers of Arf, Ras and Gα proteins and their GAPs can be found, whereas more Rabs and fewer Rhos compared to their GAPs are expressed. A low Rho/RhoGAP ratio might indicate that individual Rho proteins are targeted by more than one RhoGAP possibly contributing to the creation of diverse zones of local Rho protein activity [20,21]. G protein expression levels vary more than 10-fold with certain G proteins ranging between 10,000 up to above 100,000 copies per platelet. In contrast, GAP copy numbers are generally below 10,000 copies per platelet [18]. A comparison of tissue expression patterns suggests that the G proteins Rab27B, Rab32, Rab37, RhoF, Gα13, Gαq (Table 1), and the GAP family members RASA3, Rap1GAP2, RhoGAP6, RhoGAP18, and RGS18 are particularly highly expressed in platelets possibly indicating platelet specific functions (Table 2).
UniProt . | Gene Name . | Protein name . | Protein copy number per platelet . | Platelet specificity . | Platelet studies . |
---|---|---|---|---|---|
Arf family | |||||
P84077 | ARF1 | ADP-ribosylation factor 1 | 49771 | ||
P61204 | ARF3 | ADP-ribosylation factor 3 | 44255 | ||
P18085 | ARF4 | ADP-ribosylation factor 4 | 33268 | ||
P84085 | ARF5 | ADP-ribosylation factor 5 | 36244 | + | |
P62330 | ARF6 | ADP-ribosylation factor 6 | 6370 | 22916275 26738539 | |
Q13795 | ARFRP1 | ADP-ribosylation factor-related protein 1 | 1495 | ||
P40616 | ARL1 | ADP-ribosylation factor-like protein 1 | 2274 | ||
P36404 | ARL2 | ADP-ribosylation factor-like protein 2 | 922 | ||
P36405 | ARL3 | ADP-ribosylation factor-like protein 3 | 3335 | ||
P40617 | ARL4A | ADP-ribosylation factor-like protein 4A | - | ||
P56559 | ARL4C | ADP-ribosylation factor-like protein 4C | - | ||
P49703 | ARL4D | ADP-ribosylation factor-like protein 4D | - | ||
Q9Y689 | ARL5A | ADP-ribosylation factor-like protein 5A | Detected | ||
Q96KC2 | ARL5B | ADP-ribosylation factor-like protein 5B | Detected | ||
A6NH57 | ARL5C | Putative ADP-ribosylation factor-like protein 5C | - | ||
Q9H0F7 | ARL6 | ADP-ribosylation factor-like protein 6 | Detected | ||
Q96BM9 | ARL8A | ADP-ribosylation factor-like protein 8A | 4977 | ||
Q9NVJ2 | ARL8B | ADP-ribosylation factor-like protein 8B | 5894 | ||
Q6T311 | ARL9 | ADP-ribosylation factor-like protein 9 | - | ||
Q8N8L6 | ARL10 | ADP-ribosylation factor-like protein 10 | - | ||
Q969Q4 | ARL11 | ADP-ribosylation factor-like protein 11 | - | ||
Q5H913 | ARL13A | ADP-ribosylation factor-like protein 13A | - | ||
Q3SXY8 | ARL13B | ADP-ribosylation factor-like protein 13B | Detected | ||
Q8N4G2 | ARL14 | ADP-ribosylation factor-like protein 14 | - | ||
Q9NXU5 | ARL15 | ADP-ribosylation factor-like protein 15 | 1838 | ||
Q0P5N6 | ARL16 | ADP-ribosylation factor-like protein 16 | - | ||
Q8IVW1 | ARL17A | ADP-ribosylation factor-like protein 17 | - | ||
Rab family | |||||
P62820 | RAB1A | Ras-related protein Rab-1A | 24879 | ||
Q9H0U4 | RAB1B | Ras-related protein Rab-1B | Detected | + | 29632235 |
P61019 | RAB2A | Ras-related protein Rab-2A | 11553 | ||
Q8WUD1 | RAB2B | Ras-related protein Rab-2B | 7403 | ||
P20336 | RAB3A | Ras-related protein Rab-3A | 3735 | ||
P20337 | RAB3B | Ras-related protein Rab-3B | - | ||
Q96E17 | RAB3C | Ras-related protein Rab-3C | 4173 | ||
O95716 | RAB3D | Ras-related protein Rab-3D | Detected | ||
P20338 | RAB4A | Ras-related protein Rab-4A | 6013 | 10938270 | |
P61018 | RAB4B | Ras-related protein Rab-4B | 6242 | ||
P20339 | RAB5A | Ras-related protein Rab-5A | 6189 | 34732055 | |
P61020 | RAB5B | Ras-related protein Rab-5B | 7661 | ||
P51148 | RAB5C | Ras-related protein Rab-5C | 12138 | ||
P20340 | RAB6A | Ras-related protein Rab-6A | 20898 | ||
Q9NRW1 | RAB6B | Ras-related protein Rab-6B | 27455 | ||
Q9H0N0 | RAB6C | Ras-related protein Rab-6C | - | ||
Q53S08 | RAB6D | Ras-related protein Rab-6D | - | ||
P51149 | RAB7A | Ras-related protein Rab-7a | 18761 | ||
Q96AH8 | RAB7B | Ras-related protein Rab-7b | - | ||
O14966 | RAB29 | Ras-related protein Rab-7L1 | Detected | ||
P61006 | RAB8A | Ras-related protein Rab-8A | 17938 | + | 23140275 |
Q92930 | RAB8B | Ras-related protein Rab-8B | 16055 | ||
P51151 | RAB9A | Ras-related protein Rab-9A | 1737 | ||
Q9NP90 | RAB9B | Ras-related protein Rab-9B | 2464 | ||
P61026 | RAB10 | Ras-related protein Rab-10 | 23418 | ||
P62491 | RAB11A | Ras-related protein Rab-11A | 27698 | ||
Q15907 | RAB11B | Ras-related protein Rab-11B | 25996 | ||
Q6IQ22 | RAB12 | Ras-related protein Rab-12 | 2122 | ||
P51153 | RAB13 | Ras-related protein Rab-13 | 11203 | ||
P61106 | RAB14 | Ras-related protein Rab-14 | 23180 | ||
P59190 | RAB15 | Ras-related protein Rab-15 | 8452 | ||
Q9H0T7 | RAB17 | Ras-related protein Rab-17 | - | ||
Q9NP72 | RAB18 | Ras-related protein Rab-18 | 4663 | ||
A4D1S5 | RAB19 | Ras-related protein Rab-19 | - | ||
Q9NX57 | RAB20 | Ras-related protein Rab-20 | 1462 | ||
Q9UL25 | RAB21 | Ras-related protein Rab-21 | 5612 | ||
Q9UL26 | RAB22A | Ras-related protein Rab-22A | 1906 | ||
Q9ULC3 | RAB23 | Ras-related protein Rab-23 | Detected | ||
Q969Q5 | RAB24 | Ras-related protein Rab-24 | 885 | ||
P57735 | RAB25 | Ras-related protein Rab-25 | - | ||
Q9ULW5 | RAB26 | Ras-related protein Rab-26 | - | ||
P51159 | RAB27A | Ras-related protein Rab-27A | 7688 | 12070017 | |
O00194 | RAB27B | Ras-related protein Rab-27B | 35939 | ++ | 17384153 |
P51157 | RAB28 | Ras-related protein Rab-28 | Detected | ||
Q15771 | RAB30 | Ras-related protein Rab-30 | 4724 | + | |
Q13636 | RAB31 | Ras-related protein Rab-31 | 2157 | 35839075 | |
Q13637 | RAB32 | Ras-related protein Rab-32 | 8860 | ++ | 31399401 |
Q14088 | RAB33A | Ras-related protein Rab-33A | 1964 | ||
Q9H082 | RAB33B | Ras-related protein Rab-33B | 1589 | ||
Q9BZG1 | RAB34 | Ras-related protein Rab-34 | Detected | ||
Q15286 | RAB35 | Ras-related protein Rab-35 | 5614 | ||
O95755 | RAB36 | Ras-related protein Rab-36 | - | ||
Q96AX2 | RAB37 | Ras-related protein Rab-37 | 9153 | ++ | |
P57729 | RAB38 | Ras-related protein Rab-38 | 4450 | + | 31399401 |
Q14964 | RAB39A | Ras-related protein Rab-39A | Detected | ||
Q96DA2 | RAB39B | Ras-related protein Rab-39B | Detected | ||
Q8WXH6 | RAB40A | Ras-related protein Rab-40A | - | ||
Q12829 | RAB40B | Ras-related protein Rab-40B | - | ||
Q96S21 | RAB40C | Ras-related protein Rab-40C | - | ||
P0C0E4 | RAB40AL | Ras-related protein Rab-40A-like | - | ||
Q5JT25 | RAB41 | Ras-related protein Rab-41 | - | ||
Q8N4Z0 | RAB42 | Ras-related protein Rab-42 | - | ||
Q86YS6 | RAB43 | Ras-related protein Rab-43 | Detected | ||
Q7Z6P3 | RAB44 | Ras-related protein Rab-44 | - | ||
Q5HYI8 | RABL3 | Rab-like protein 3 | 1041 | ||
Q3YEC7 | RABL6 | Rab-like protein 6 | 1360 | ||
Q8IZ41 | RASEF | Ras and EF-hand domain-containing protein | - | ||
Q9UBK7 | RABL2A | Rab-like protein 2A | - | ||
Q9UNT1 | RABL2B | Rab-like protein 2B | Detected | ||
Ras family | |||||
Q9NYS0 | NKIRAS1 | NF-kappa-B inhibitor-interacting Ras-like protein 1 | - | ||
Q9NYR9 | NKIRAS2 | NF-kappa-B inhibitor-interacting Ras-like protein 2 | Detected | ||
P11233 | RALA | Ras-related protein Ral-A | 3363 | 29437579 | |
P11234 | RALB | Ras-related protein Ral-B | 6791 | + | 29437579 |
P62834 | RAP1A | Ras-related protein Rap-1A | 124810 | 30131434 | |
P61224 | RAP1B | Ras-related protein Rap-1b | 154326 | + | 30131434 |
P10114 | RAP2A | Ras-related protein Rap-2a | 2921 | ||
P61225 | RAP2B | Ras-related protein Rap-2b | 6248 | + | 18582561 |
Q9Y3L5 | RAP2C | Ras-related protein Rap-2c | 3054 | ||
Q7Z444 | ERAS | GTPase ERas | - | ||
P01112 | HRAS | GTPase HRas | Detected | ||
P01116 | KRAS | GTPase KRas | 6581 | ||
Q9NYN1 | RASL12 | Ras-like protein family member 12 | - | ||
O14807 | MRAS | Ras-related protein M-Ras | - | ||
P01111 | NRAS | GTPase NRas | 6581 | ||
Q9H628 | RERGL | Ras-related and estrogen-regulated growth inhibitor-like protein | - | ||
Q96A58 | RERG | Ras-related and estrogen-regulated growth inhibitor | - | ||
Q92963 | RIT1 | GTP-binding protein Rit1 | 1415 | ||
Q99578 | RIT2 | GTP-binding protein Rit2 | - | ||
P62070 | RRAS2 | Ras-related protein R-Ras2 | Detected | 29883056 | |
P10301 | RRAS | Ras-related protein R-Ras | 2648 | ||
Q92737 | RASL10A | Ras-like protein family member 10A | - | ||
Q96S79 | RASL10B | Ras-like protein family member 10B | - | ||
Q6T310 | RASL11A | Ras-like protein family member 11A | - | ||
Q9BPW5 | RASL11B | Ras-like protein family member 11B | - | ||
Rho family | |||||
P60953 | CDC42 | Cell division control protein 42 homolog | 27939 | 20139097 | |
P63000 | RAC1 | Ras-related C3 botulinum toxin substrate 1 | 32870 | 16195235 18704480 | |
P15153 | RAC2 | Ras-related C3 botulinum toxin substrate 2 | 27923 | 16195235 | |
P60763 | RAC3 | Ras-related C3 botulinum toxin substrate 3 | Detected | ||
O94844 | RHOBTB1 | Rho-related BTB domain-containing protein 1 | - | ||
Q9BYZ6 | RHOBTB2 | Rho-related BTB domain-containing protein 2 | - | ||
P61586 | RHOA | Transforming protein RhoA | 31315 | 22045984 | |
P62745 | RHOB | Rho-related GTP-binding protein RhoB | Detected | ||
P08134 | RHOC | Rho-related GTP-binding protein RhoC | 25384 | ||
O00212 | RHOD | Rho-related GTP-binding protein RhoD | - | ||
Q9HBH0 | RHOF | Rho-related GTP-binding protein RhoF | 5257 | ++ | 23359340 |
P84095 | RHOG | Rho-related GTP-binding protein RhoG | 5498 | 24106270 | |
Q15669 | RHOH | Rho-related GTP-binding protein RhoH | - | ||
Q9H4E5 | RHOJ | Rho-related GTP-binding protein RhoJ | - | ||
P17081 | RHOQ | Rho-related GTP-binding protein RhoQ | Detected | ||
Q7L0Q8 | RHOU | Rho-related GTP-binding protein RhoU | - | ||
Q96L33 | RHOV | Rho-related GTP-binding protein RhoV | - | ||
Q92730 | RND1 | Rho-related GTP-binding protein Rho6 | - | ||
P52198 | RND2 | Rho-related GTP-binding protein RhoN | - | ||
P61587 | RND3 | Rho-related GTP-binding protein RhoE | - | ||
Galpha family | |||||
P29992 | GNA11 | Guanine nucleotide-binding protein subunit alpha-11 | Detected | ||
Q03113 | GNA12 | Guanine nucleotide-binding protein subunit alpha-12 | Detected | ||
Q14344 | GNA13 | Guanine nucleotide-binding protein subunit alpha-13 | 6145 | ++ | 14528298 15326177 |
O95837 | GNA14 | Guanine nucleotide-binding protein subunit alpha-14 | - | ||
P30679 | GNA15 | Guanine nucleotide-binding protein subunit alpha-15 | Detected | ||
P63096 | GNAI1 | Guanine nucleotide-binding protein G(i) subunit alpha-1 | 10226 | ||
P04899 | GNAI2 | Guanine nucleotide-binding protein G(i) subunit alpha-2 | 14795 | 20852125 | |
P08754 | GNAI3 | Guanine nucleotide-binding protein G(i) subunit alpha-3 | 8712 | ||
P38405 | GNAL | Guanine nucleotide-binding protein G(olf) subunit alpha | - | ||
P09471 | GNAO1 | Guanine nucleotide-binding protein G(o) subunit alpha | Detected | ||
P50148 | GNAQ | Guanine nucleotide-binding protein G(q) subunit alpha | 14839 | ++ | 9296496 15326177 |
Q5JWF2 | GNAS1 | Guanine nucleotide-binding protein G(s) subunit alpha isoforms XLas | - | ||
P63092 | GNAS2 | Guanine nucleotide-binding protein G(s) subunit alpha isoforms short | 2874 | 18812479 32647264 | |
P11488 | GNAT1 | Guanine nucleotide-binding protein G(t) subunit alpha-1 | Detected | ||
P19087 | GNAT2 | Guanine nucleotide-binding protein G(t) subunit alpha-2 | - | ||
A8MTJ3 | GNAT3 | Guanine nucleotide-binding protein G(t) subunit alpha-3 | - | ||
P19086 | GNAZ | Guanine nucleotide-binding protein G(z) subunit alpha | 4436 | + |
UniProt . | Gene Name . | Protein name . | Protein copy number per platelet . | Platelet specificity . | Platelet studies . |
---|---|---|---|---|---|
Arf family | |||||
P84077 | ARF1 | ADP-ribosylation factor 1 | 49771 | ||
P61204 | ARF3 | ADP-ribosylation factor 3 | 44255 | ||
P18085 | ARF4 | ADP-ribosylation factor 4 | 33268 | ||
P84085 | ARF5 | ADP-ribosylation factor 5 | 36244 | + | |
P62330 | ARF6 | ADP-ribosylation factor 6 | 6370 | 22916275 26738539 | |
Q13795 | ARFRP1 | ADP-ribosylation factor-related protein 1 | 1495 | ||
P40616 | ARL1 | ADP-ribosylation factor-like protein 1 | 2274 | ||
P36404 | ARL2 | ADP-ribosylation factor-like protein 2 | 922 | ||
P36405 | ARL3 | ADP-ribosylation factor-like protein 3 | 3335 | ||
P40617 | ARL4A | ADP-ribosylation factor-like protein 4A | - | ||
P56559 | ARL4C | ADP-ribosylation factor-like protein 4C | - | ||
P49703 | ARL4D | ADP-ribosylation factor-like protein 4D | - | ||
Q9Y689 | ARL5A | ADP-ribosylation factor-like protein 5A | Detected | ||
Q96KC2 | ARL5B | ADP-ribosylation factor-like protein 5B | Detected | ||
A6NH57 | ARL5C | Putative ADP-ribosylation factor-like protein 5C | - | ||
Q9H0F7 | ARL6 | ADP-ribosylation factor-like protein 6 | Detected | ||
Q96BM9 | ARL8A | ADP-ribosylation factor-like protein 8A | 4977 | ||
Q9NVJ2 | ARL8B | ADP-ribosylation factor-like protein 8B | 5894 | ||
Q6T311 | ARL9 | ADP-ribosylation factor-like protein 9 | - | ||
Q8N8L6 | ARL10 | ADP-ribosylation factor-like protein 10 | - | ||
Q969Q4 | ARL11 | ADP-ribosylation factor-like protein 11 | - | ||
Q5H913 | ARL13A | ADP-ribosylation factor-like protein 13A | - | ||
Q3SXY8 | ARL13B | ADP-ribosylation factor-like protein 13B | Detected | ||
Q8N4G2 | ARL14 | ADP-ribosylation factor-like protein 14 | - | ||
Q9NXU5 | ARL15 | ADP-ribosylation factor-like protein 15 | 1838 | ||
Q0P5N6 | ARL16 | ADP-ribosylation factor-like protein 16 | - | ||
Q8IVW1 | ARL17A | ADP-ribosylation factor-like protein 17 | - | ||
Rab family | |||||
P62820 | RAB1A | Ras-related protein Rab-1A | 24879 | ||
Q9H0U4 | RAB1B | Ras-related protein Rab-1B | Detected | + | 29632235 |
P61019 | RAB2A | Ras-related protein Rab-2A | 11553 | ||
Q8WUD1 | RAB2B | Ras-related protein Rab-2B | 7403 | ||
P20336 | RAB3A | Ras-related protein Rab-3A | 3735 | ||
P20337 | RAB3B | Ras-related protein Rab-3B | - | ||
Q96E17 | RAB3C | Ras-related protein Rab-3C | 4173 | ||
O95716 | RAB3D | Ras-related protein Rab-3D | Detected | ||
P20338 | RAB4A | Ras-related protein Rab-4A | 6013 | 10938270 | |
P61018 | RAB4B | Ras-related protein Rab-4B | 6242 | ||
P20339 | RAB5A | Ras-related protein Rab-5A | 6189 | 34732055 | |
P61020 | RAB5B | Ras-related protein Rab-5B | 7661 | ||
P51148 | RAB5C | Ras-related protein Rab-5C | 12138 | ||
P20340 | RAB6A | Ras-related protein Rab-6A | 20898 | ||
Q9NRW1 | RAB6B | Ras-related protein Rab-6B | 27455 | ||
Q9H0N0 | RAB6C | Ras-related protein Rab-6C | - | ||
Q53S08 | RAB6D | Ras-related protein Rab-6D | - | ||
P51149 | RAB7A | Ras-related protein Rab-7a | 18761 | ||
Q96AH8 | RAB7B | Ras-related protein Rab-7b | - | ||
O14966 | RAB29 | Ras-related protein Rab-7L1 | Detected | ||
P61006 | RAB8A | Ras-related protein Rab-8A | 17938 | + | 23140275 |
Q92930 | RAB8B | Ras-related protein Rab-8B | 16055 | ||
P51151 | RAB9A | Ras-related protein Rab-9A | 1737 | ||
Q9NP90 | RAB9B | Ras-related protein Rab-9B | 2464 | ||
P61026 | RAB10 | Ras-related protein Rab-10 | 23418 | ||
P62491 | RAB11A | Ras-related protein Rab-11A | 27698 | ||
Q15907 | RAB11B | Ras-related protein Rab-11B | 25996 | ||
Q6IQ22 | RAB12 | Ras-related protein Rab-12 | 2122 | ||
P51153 | RAB13 | Ras-related protein Rab-13 | 11203 | ||
P61106 | RAB14 | Ras-related protein Rab-14 | 23180 | ||
P59190 | RAB15 | Ras-related protein Rab-15 | 8452 | ||
Q9H0T7 | RAB17 | Ras-related protein Rab-17 | - | ||
Q9NP72 | RAB18 | Ras-related protein Rab-18 | 4663 | ||
A4D1S5 | RAB19 | Ras-related protein Rab-19 | - | ||
Q9NX57 | RAB20 | Ras-related protein Rab-20 | 1462 | ||
Q9UL25 | RAB21 | Ras-related protein Rab-21 | 5612 | ||
Q9UL26 | RAB22A | Ras-related protein Rab-22A | 1906 | ||
Q9ULC3 | RAB23 | Ras-related protein Rab-23 | Detected | ||
Q969Q5 | RAB24 | Ras-related protein Rab-24 | 885 | ||
P57735 | RAB25 | Ras-related protein Rab-25 | - | ||
Q9ULW5 | RAB26 | Ras-related protein Rab-26 | - | ||
P51159 | RAB27A | Ras-related protein Rab-27A | 7688 | 12070017 | |
O00194 | RAB27B | Ras-related protein Rab-27B | 35939 | ++ | 17384153 |
P51157 | RAB28 | Ras-related protein Rab-28 | Detected | ||
Q15771 | RAB30 | Ras-related protein Rab-30 | 4724 | + | |
Q13636 | RAB31 | Ras-related protein Rab-31 | 2157 | 35839075 | |
Q13637 | RAB32 | Ras-related protein Rab-32 | 8860 | ++ | 31399401 |
Q14088 | RAB33A | Ras-related protein Rab-33A | 1964 | ||
Q9H082 | RAB33B | Ras-related protein Rab-33B | 1589 | ||
Q9BZG1 | RAB34 | Ras-related protein Rab-34 | Detected | ||
Q15286 | RAB35 | Ras-related protein Rab-35 | 5614 | ||
O95755 | RAB36 | Ras-related protein Rab-36 | - | ||
Q96AX2 | RAB37 | Ras-related protein Rab-37 | 9153 | ++ | |
P57729 | RAB38 | Ras-related protein Rab-38 | 4450 | + | 31399401 |
Q14964 | RAB39A | Ras-related protein Rab-39A | Detected | ||
Q96DA2 | RAB39B | Ras-related protein Rab-39B | Detected | ||
Q8WXH6 | RAB40A | Ras-related protein Rab-40A | - | ||
Q12829 | RAB40B | Ras-related protein Rab-40B | - | ||
Q96S21 | RAB40C | Ras-related protein Rab-40C | - | ||
P0C0E4 | RAB40AL | Ras-related protein Rab-40A-like | - | ||
Q5JT25 | RAB41 | Ras-related protein Rab-41 | - | ||
Q8N4Z0 | RAB42 | Ras-related protein Rab-42 | - | ||
Q86YS6 | RAB43 | Ras-related protein Rab-43 | Detected | ||
Q7Z6P3 | RAB44 | Ras-related protein Rab-44 | - | ||
Q5HYI8 | RABL3 | Rab-like protein 3 | 1041 | ||
Q3YEC7 | RABL6 | Rab-like protein 6 | 1360 | ||
Q8IZ41 | RASEF | Ras and EF-hand domain-containing protein | - | ||
Q9UBK7 | RABL2A | Rab-like protein 2A | - | ||
Q9UNT1 | RABL2B | Rab-like protein 2B | Detected | ||
Ras family | |||||
Q9NYS0 | NKIRAS1 | NF-kappa-B inhibitor-interacting Ras-like protein 1 | - | ||
Q9NYR9 | NKIRAS2 | NF-kappa-B inhibitor-interacting Ras-like protein 2 | Detected | ||
P11233 | RALA | Ras-related protein Ral-A | 3363 | 29437579 | |
P11234 | RALB | Ras-related protein Ral-B | 6791 | + | 29437579 |
P62834 | RAP1A | Ras-related protein Rap-1A | 124810 | 30131434 | |
P61224 | RAP1B | Ras-related protein Rap-1b | 154326 | + | 30131434 |
P10114 | RAP2A | Ras-related protein Rap-2a | 2921 | ||
P61225 | RAP2B | Ras-related protein Rap-2b | 6248 | + | 18582561 |
Q9Y3L5 | RAP2C | Ras-related protein Rap-2c | 3054 | ||
Q7Z444 | ERAS | GTPase ERas | - | ||
P01112 | HRAS | GTPase HRas | Detected | ||
P01116 | KRAS | GTPase KRas | 6581 | ||
Q9NYN1 | RASL12 | Ras-like protein family member 12 | - | ||
O14807 | MRAS | Ras-related protein M-Ras | - | ||
P01111 | NRAS | GTPase NRas | 6581 | ||
Q9H628 | RERGL | Ras-related and estrogen-regulated growth inhibitor-like protein | - | ||
Q96A58 | RERG | Ras-related and estrogen-regulated growth inhibitor | - | ||
Q92963 | RIT1 | GTP-binding protein Rit1 | 1415 | ||
Q99578 | RIT2 | GTP-binding protein Rit2 | - | ||
P62070 | RRAS2 | Ras-related protein R-Ras2 | Detected | 29883056 | |
P10301 | RRAS | Ras-related protein R-Ras | 2648 | ||
Q92737 | RASL10A | Ras-like protein family member 10A | - | ||
Q96S79 | RASL10B | Ras-like protein family member 10B | - | ||
Q6T310 | RASL11A | Ras-like protein family member 11A | - | ||
Q9BPW5 | RASL11B | Ras-like protein family member 11B | - | ||
Rho family | |||||
P60953 | CDC42 | Cell division control protein 42 homolog | 27939 | 20139097 | |
P63000 | RAC1 | Ras-related C3 botulinum toxin substrate 1 | 32870 | 16195235 18704480 | |
P15153 | RAC2 | Ras-related C3 botulinum toxin substrate 2 | 27923 | 16195235 | |
P60763 | RAC3 | Ras-related C3 botulinum toxin substrate 3 | Detected | ||
O94844 | RHOBTB1 | Rho-related BTB domain-containing protein 1 | - | ||
Q9BYZ6 | RHOBTB2 | Rho-related BTB domain-containing protein 2 | - | ||
P61586 | RHOA | Transforming protein RhoA | 31315 | 22045984 | |
P62745 | RHOB | Rho-related GTP-binding protein RhoB | Detected | ||
P08134 | RHOC | Rho-related GTP-binding protein RhoC | 25384 | ||
O00212 | RHOD | Rho-related GTP-binding protein RhoD | - | ||
Q9HBH0 | RHOF | Rho-related GTP-binding protein RhoF | 5257 | ++ | 23359340 |
P84095 | RHOG | Rho-related GTP-binding protein RhoG | 5498 | 24106270 | |
Q15669 | RHOH | Rho-related GTP-binding protein RhoH | - | ||
Q9H4E5 | RHOJ | Rho-related GTP-binding protein RhoJ | - | ||
P17081 | RHOQ | Rho-related GTP-binding protein RhoQ | Detected | ||
Q7L0Q8 | RHOU | Rho-related GTP-binding protein RhoU | - | ||
Q96L33 | RHOV | Rho-related GTP-binding protein RhoV | - | ||
Q92730 | RND1 | Rho-related GTP-binding protein Rho6 | - | ||
P52198 | RND2 | Rho-related GTP-binding protein RhoN | - | ||
P61587 | RND3 | Rho-related GTP-binding protein RhoE | - | ||
Galpha family | |||||
P29992 | GNA11 | Guanine nucleotide-binding protein subunit alpha-11 | Detected | ||
Q03113 | GNA12 | Guanine nucleotide-binding protein subunit alpha-12 | Detected | ||
Q14344 | GNA13 | Guanine nucleotide-binding protein subunit alpha-13 | 6145 | ++ | 14528298 15326177 |
O95837 | GNA14 | Guanine nucleotide-binding protein subunit alpha-14 | - | ||
P30679 | GNA15 | Guanine nucleotide-binding protein subunit alpha-15 | Detected | ||
P63096 | GNAI1 | Guanine nucleotide-binding protein G(i) subunit alpha-1 | 10226 | ||
P04899 | GNAI2 | Guanine nucleotide-binding protein G(i) subunit alpha-2 | 14795 | 20852125 | |
P08754 | GNAI3 | Guanine nucleotide-binding protein G(i) subunit alpha-3 | 8712 | ||
P38405 | GNAL | Guanine nucleotide-binding protein G(olf) subunit alpha | - | ||
P09471 | GNAO1 | Guanine nucleotide-binding protein G(o) subunit alpha | Detected | ||
P50148 | GNAQ | Guanine nucleotide-binding protein G(q) subunit alpha | 14839 | ++ | 9296496 15326177 |
Q5JWF2 | GNAS1 | Guanine nucleotide-binding protein G(s) subunit alpha isoforms XLas | - | ||
P63092 | GNAS2 | Guanine nucleotide-binding protein G(s) subunit alpha isoforms short | 2874 | 18812479 32647264 | |
P11488 | GNAT1 | Guanine nucleotide-binding protein G(t) subunit alpha-1 | Detected | ||
P19087 | GNAT2 | Guanine nucleotide-binding protein G(t) subunit alpha-2 | - | ||
A8MTJ3 | GNAT3 | Guanine nucleotide-binding protein G(t) subunit alpha-3 | - | ||
P19086 | GNAZ | Guanine nucleotide-binding protein G(z) subunit alpha | 4436 | + |
G protein family members were obtained from UniProt and compared with platelet proteome data. Shown are all proteins encoded by the human genome. Protein copy numbers per platelet are given as far as available. ‘Detected’ indicates expressed proteins where a copy number has not yet been determined. All found proteins have also been confirmed at the transcriptome level of megakaryocytes or platelets. ‘-’ indicates that proteins could not be detected in platelets. Platelet specificity was determined as high expression in platelets compared to other human tissues according to https://www.proteomicsdb.org and http://www.humanproteomemap.org (‘+’ indicates within the top 5 highly expressing tissues, ‘++’ indicates platelets as highest expressing tissue in both databases). References for platelet studies on G proteins are given as PubMed ID numbers (PMID).
UniProt . | Gene . | Protein name . | Protein copy number per platelet . | Platelet specificity . | Phospho sites . | G-protein specificity (cell-based assays) . | Functions and regulation (platelet studies) . |
---|---|---|---|---|---|---|---|
ArfGAP, PS50115 | |||||||
Q15027 | ACAP1 | Arf-GAP with coiled-coil, ANK repeat and PH domain-containing protein 1 | 1150 | 6 | Arf6 (11062263) | ||
Q15057 | ACAP2 | Arf-GAP with coiled-coil, ANK repeat and PH domain-containing protein 2 | 1057 | 13 | Arf6 (11062263) | ||
Q96P50 | ACAP3 | Arf-GAP with coiled-coil, ANK repeat and PH domain-containing protein 3 | Detected | 3 | |||
O75689 | ADAP1 | Arf-GAP with dual PH domain-containing protein 1 | - | ||||
Q9NPF8 | ADAP2 | Arf-GAP with dual PH domain-containing protein 2 | - | ||||
Q9UPQ3 | AGAP1 | Arf-GAP with GTPase, ANK repeat and PH domain-containing protein 1 | Detected | 7 | |||
Q99490 | AGAP2 | Arf-GAP with GTPase, ANK repeat and PH domain-containing protein 2 | 1199 | 15 | |||
Q96P47 | AGAP3 | Arf-GAP with GTPase, ANK repeat and PH domain-containing protein 3 | Detected | 8 | |||
Q96P64 | AGAP4 | Arf-GAP with GTPase, ANK repeat and PH domain-containing protein 4 | - | ||||
A6NIR3 | AGAP5 | Arf-GAP with GTPase, ANK repeat and PH domain-containing protein 5 | - | ||||
Q5VW22 | AGAP6 | Arf-GAP with GTPase, ANK repeat and PH domain-containing protein 6 | - | ||||
Q5VUJ5 | AGAP7 | Putative Arf-GAP with GTPase, ANK repeat and PH domain-containing protein 7 | - | ||||
Q5VTM2 | AGAP9 | Arf-GAP with GTPase, ANK repeat and PH domain-containing protein 9 | - | ||||
P52594 | AGFG1 | Arf-GAP domain and FG repeat-containing protein 1 | 1825 | 10 | |||
O95081 | AGFG2 | Arf-GAP domain and FG repeat-containing protein 2 | Detected | 2 | |||
Q96P48 | ARAP1 | Arf-GAP with Rho-GAP domain, ANK repeat and PH domain-containing protein 1 | 3061 | 17 | |||
Q8WZ64 | ARAP2 | Arf-GAP with Rho-GAP domain, ANK repeat and PH domain-containing protein 2 | - | ||||
Q8WWN8 | ARAP3 | Arf-GAP with Rho-GAP domain, ANK repeat and PH domain-containing protein 3 | - | ||||
Q8N6T3 | ARFGAP1 | ADP-ribosylation factor GTPase-activating protein 1 | Detected | 17 | Arf1 (19015319, 36513395) | ||
Q8N6H7 | ARFGAP2 | ADP-ribosylation factor GTPase-activating protein 2 | 899 | 13 | Arf1 (19015319, 36513395) | ||
Q9NP61 | ARFGAP3 | ADP-ribosylation factor GTPase-activating protein 3 | Detected | 21 | Arf1, Arl1 (19015319, 33715220) | ||
Q9ULH1 | ASAP1 | Arf-GAP with SH3 domain, ANK repeat and PH domain-containing protein 1 | 3588 | + | 18 | Binds Crkl and to focal adhesions (12522101) | |
O43150 | ASAP2 | Arf-GAP with SH3 domain, ANK repeat and PH domain-containing protein 2 | 4132 | + | 10 | ||
Q8TDY4 | ASAP3 | Arf-GAP with SH3 domain, ANK repeat and PH domain-containing protein 3 | Detected | 5 | |||
Q9Y2X7 | GIT1 | ARF GTPase-activating protein GIT1 | 3149 | 30 | Arf1, Arf6 (28981399) | Binds RhoGEF6 and RhoGEF7, interacts with integrin αIIbβ3 (26507661, 18211801) | |
Q14161 | GIT2 | ARF GTPase-activating protein GIT2 | 1071 | 28 | |||
Q8IYB5 | SMAP1 | Stromal membrane-associated protein 1 | 890 | 0 | |||
Q8WU79 | SMAP2 | Stromal membrane-associated protein 2 | 1717 | 5 | |||
TBC_RabGAP, PS50086 | |||||||
Q96CN4 | EVI5L | EVI5-like protein | 648 | 2 | |||
O60447 | EVI5 | Ecotropic viral integration site 5 protein homolog | - | ||||
Q5TC63 | GRTP1 | Growth hormone-regulated TBC protein 1 | Detected | 1 | |||
Q5R372 | RABGAP1L | Rab GTPase-activating protein 1-like | 615 | 5 | |||
Q9Y3P9 | RABGAP1 | Rab GTPase-activating protein 1 | 1619 | 9 | |||
Q2NKQ1 | SGSM1 | Small G protein signalling modulator 1 | - | ||||
O43147 | SGSM2 | Small G protein signalling modulator 2 | - | ||||
Q96HU1 | SGSM3 | Small G protein signalling modulator 3 | - | ||||
Q86TI0 | TBC1D1 | TBC1 domain family member 1 | Detected | 24 | |||
Q9BYX2 | TBC1D2 | TBC1 domain family member 2A | - | ||||
Q9UPU7 | TBC1D2B | TBC1 domain family member 2B | Detected | 10 | |||
Q8IZP1 | TBC1D3 | TBC1 domain family member 3 | - | ||||
A6NDS4 | TBC1D3B | TBC1 domain family member 3B | - | ||||
Q6IPX1 | TBC1D3C | TBC1 domain family member 3C | - | ||||
A0A087WVF3 | TBC1D3D | TBC1 domain family member 3D | - | ||||
A0A087X179 | TBC1D3E | TBC1 domain family member 3E | - | ||||
A6NER0 | TBC1D3F | TBC1 domain family member 3F | - | ||||
Q6DHY5 | TBC1D3G | TBC1 domain family member 3G | - | ||||
P0C7X1 | TBC1D3H | TBC1 domain family member 3H | - | ||||
A0A087WXS9 | TBC1D3I | TBC1 domain family member 3I | - | ||||
A0A087X1G2 | TBC1D3K | TBC1 domain family member 3K | - | ||||
B9A6J9 | TBC1D3L | TBC1 domain family member 3L | - | ||||
O60343 | TBC1D4 | TBC1 domain family member 4 | Detected | 42 | Rab10 (33175605) | ||
Q92609 | TBC1D5 | TBC1 domain family member 5 | 1046 | 27 | Rab7B (30111580) | ||
Q9P0N9 | TBC1D7 | TBC1 domain family member 7 | Detected | 2 | |||
O95759 | TBC1D8 | TBC1 domain family member 8 | - | ||||
Q0IIM8 | TBC1D8B | TBC1 domain family member 8B | Detected | 2 | |||
Q6ZT07 | TBC1D9 | TBC1 domain family member 9 | Detected | 0 | |||
Q66K14 | TBC1D9B | TBC1 domain family member 9B | 1012 | 18 | |||
Q9BXI6 | TBC1D10A | TBC1 domain family member 10A | Detected | 14 | Rab35 (28566286) | ||
Q4KMP7 | TBC1D10B | TBC1 domain family member 10B | 827 | 22 | Rab27b (23671284) | ||
Q8IV04 | TBC1D10C | Carabin | Detected | 2 | |||
O60347 | TBC1D12 | TBC1 domain family member 12 | - | ||||
Q9NVG8 | TBC1D13 | TBC1 domain family member 13 | 2656 | + | 1 | Rab35 (22762500) | |
Q9P2M4 | TBC1D14 | TBC1 domain family member 14 | Detected | 5 | |||
Q8TC07 | TBC1D15 | TBC1 domain family member 15 | 2534 | 14 | |||
Q8TBP0 | TBC1D16 | TBC1 domain family member 16 | - | ||||
Q9HA65 | TBC1D17 | TBC1 domain family member 17 | Detected | 5 | Rab8 (22854040) | ||
Q8N5T2 | TBC1D19 | TBC1 domain family member 19 | - | ||||
Q96BZ9 | TBC1D20 | TBC1 domain family member 20 | 1069 | 0 | Rab1A (22854043) | ||
Q8IYX1 | TBC1D21 | TBC1 domain family member 21 | - | ||||
Q8WUA7 | TBC1D22A | TBC1 domain family member 22A | 1138 | 8 | |||
Q9NU19 | TBC1D22B | TBC1 domain family member 22B | Detected | 8 | |||
Q9NUY8 | TBC1D23 | TBC1 domain family member 23 | 1187 | 7 | |||
Q3MII6 | TBC1D25 | TBC1 domain family member 25 | Detected | 1 | |||
Q86UD7 | TBC1D26 | TBC1 domain family member 26 | - | ||||
Q9UFV1 | TBC1D29P | Putative TBC1 domain family member 29 | - | ||||
Q9Y2I9 | TBC1D30 | TBC1 domain family member 30 | - | ||||
Q96DN5 | TBC1D31 | TBC1 domain family member 31 | - | ||||
Q8TEA7 | TBCK | TBC domain-containing protein kinase-like protein | Detected | 1 | |||
P35125 | USP6 | Ubiquitin carboxyl-terminal hydrolase 6 | - | ||||
Q92738 | USP6NL | USP6 N-terminal-like protein | Detected | 26 | |||
RasGAP, PS50018 | |||||||
Q5VWQ8 | DAB2IP | Disabled homolog 2-interacting protein | - | ||||
Q14C86 | GAPVD1 | GTPase-activating protein and VPS9 domain-containing protein 1 | 1283 | 34 | |||
P46940 | IQGAP1 | Ras GTPase-activating-like protein IQGAP1 | 1040 | 17 | No GAP activity, Cdc42/Rac1 effector (34830479) | Inhibits alpha granule release (15026422) | |
Q13576 | IQGAP2 | Ras GTPase-activating-like protein IQGAP2 | 8102 | + | 19 | No GAP activity, Cdc42/Rac1 effector (34830479) | Interacts with arp3 and actin (12515716) |
Q86VI3 | IQGAP3 | Ras GTPase-activating-like protein IQGAP3 | - | ||||
P21359 | NF1 | Neurofibromin | 628 | 20 | HRas (9668168) | ||
O95294 | RASAL1 | RasGAP-activating-like protein 1 | - | ||||
Q9UJF2 | RASAL2 | Ras GTPase-activating protein nGAP | Detected | 26 | |||
Q86YV0 | RASAL3 | RAS protein activator like-3 | 573 | 25 | |||
C9J798 | RASA4B | Ras GTPase-activating protein 4B | - | ||||
P20936 | RASA1 | Ras GTPase-activating protein 1 | 2921 | + | 5 | ||
Q15283 | RASA2 | Ras GTPase-activating protein 2 | Detected | 5 | |||
Q14644 | RASA3 | Ras GTPase-activating protein 3 | 8293 | ++ | 12 | Rap1, H-Ras (16431904) | Reduces Rap1-GTP, inhibits platelet adhesion and aggregation, binds PIP3, linked to P2Y12 receptor via PI3K (24967784, 25705885, 27903653) |
O43374 | RASA4 | Ras GTPase-activating protein 4 | - | ||||
Q96PV0 | SYNGAP1 | Ras/Rap GTPase-activating protein SynGA | - | ||||
RapGAP, PS50085 | |||||||
Q5VVW2 | GARNL3 | GTPase-activating Rap/Ran-GAP domain-like protein 3 | - | ||||
Q6GYQ0 | RALGAPA1 | Ral GTPase-activating protein subunit alpha-1 | 669 | 28 | |||
Q2PPJ7 | RALGAPA2 | Ral GTPase-activating protein subunit alpha-2 | Detected | 12 | |||
Q86X10 | RALGAPB | Ral GTPase-activating protein subunit beta | 884 | 7 | RalA (29915037) | ||
P47736 | RAP1GAP | Rap1 GTPase-activating protein 1 | - | ||||
Q684P5 | RAP1GAP2 | Rap1 GTPase-activating protein 2 | 2327 | ++ | 22 | Rap1 (15632203) | Inhibited by S9 phosphorylation, activated by PKA/PKG mediated S7 phosphorylation, binds 14-3-3, binds Slp1 and stimulates dense granule release (15632203, 18039662, 19528539) |
O43166 | SIPA1L1 | Signal-induced proliferation-associated 1-like protein 1 | Detected | 65 | |||
Q9P2F8 | SIPA1L2 | Signal-induced proliferation-associated 1-like protein 2 | - | ||||
O60292 | SIPA1L3 | Signal-induced proliferation-associated 1-like protein 3 | - | ||||
Q96FS4 | SIPA1 | Signal-induced proliferation-associated protein 1 | Detected | 12 | |||
P49815 | TSC2 | Tuberin | 593 | 35 | |||
RhoGAP, PS50238 | |||||||
Q9Y3L3 | SH3BP1 | SH3 domain-binding protein 1 | 595 | 14 | |||
Q12979 | ABR | Active breakpoint cluster region-related protein | Detected | 5 | Rac1 (32203420) | ||
Q96P48 | ARAP1 | Arf-GAP with Rho-GAP domain, ANK repeat and PH domain-containing protein 1 | 3061 | 17 | Rac1, Cdc42 (32203420) | ||
Q8WZ64 | ARAP2 | Arf-GAP with Rho-GAP domain, ANK repeat and PH domain-containing protein 2 | - | ||||
Q8WWN8 | ARAP3 | Arf-GAP with Rho-GAP domain, ANK repeat and PH domain-containing protein 3 | - | ||||
P11274 | BCR | Breakpoint cluster region protein | 793 | 47 | Rac1 (32203420) | ||
Q6ZT62 | BARGIN | Bargin | - | ||||
P15882 | CHN1 | N-chimaerin | - | ||||
P52757 | CHN2 | Beta-chimaerin | - | ||||
Q8WUY9 | DEPDC1B | DEP domain-containing protein 1B | - | ||||
O94988 | FAM13A | Protein FAM13A | - | ||||
Q9NYF5 | FAM13B | Protein FAM13B | - | ||||
Q9P107 | GMIP | GEM-interacting protein | 1228 | 17 | RhoA, Rac1, Cdc42 (32203420) | ||
P32019 | INPP5B | Type II inositol 1,4,5-trisphosphate 5-phosphatase | 1629 | 1 | |||
B2RTY4 | MYO9A | Unconventional myosin-IXa | Detected | 14 | |||
Q13459 | MYO9B | Unconventional myosin-IXb | 1428 | 40 | RhoA (33268376) | Activated by PKA/PKG mediated S1354 phosphorylation (32692911) | |
Q01968 | OCRL | Inositol polyphosphate 5-phosphatase OCRL | 850 | 3 | RhoA (33528045) | Reduces RhoA-GTP, augments Rac1-GTP and myosin light chain phosphorylation, inhibits filopodia, stimulates spreading, clot retraction and thrombus formation (33528045, 36176266) | |
O60890 | OPHN1 | Oligophrenin-1 | 1527 | 3 | RhoA, Rac1, Cdc42 (25556321) | Reduces RhoA-GTP, Rac1-GTP, and Cdc42-GTP, inhibits adhesion and granule release (25556321) | |
P27986 | PIK3R1 | Phosphatidylinositol 3-kinase regulatory subunit alpha | 1904 | 17 | |||
O00459 | PIK3R2 | Phosphatidylinositol 3-kinase regulatory subunit beta | Detected | 12 | |||
Q15311 | RALBP1 | RalA-binding protein 1 | 785 | 14 | Rac1 (32203420) | ||
Q9H0H5 | RACGAP1 | Rac GTPase-activating protein 1 | - | ||||
Q07960 | ARHGAP1 | Rho GTPase-activating protein 1 | 9290 | + | 8 | RhoA, Cdc42 (32203420) | |
P98171 | ARHGAP4 | Rho GTPase-activating protein 4 | 1400 | 6 | Rac1 (32203420) | ||
Q13017 | ARHGAP5 | Rho GTPase-activating protein 5 | Detected | 16 | RhoA (32203420) | ||
O43182 | ARHGAP6 | Rho GTPase-activating protein 6 | 4151 | ++ | 9 | RhoA (32203420) | Binds COPD and COPI vesicles, might regulate protein transport (37409526) |
Q96QB1 | DLC1 | Rho GTPase-activating protein 7 | - | ||||
P85298 | ARHGAP8 | Rho GTPase-activating protein 8 | - | ||||
Q9BRR9 | ARHGAP9 | Rho GTPase-activating protein 9 | Detected | 3 | Rac1 (32203420) | ||
A1A4S6 | ARHGAP10 | Rho GTPase-activating protein 10 | 1114 | 1 | RhoA (32203420) | ||
Q6P4F7 | ARHGAP11A | Rho GTPase-activating protein 11A | - | ||||
Q3KRB8 | ARHGAP11B | Inactive Rho GTPase-activating protein 11B | - | ||||
Q8IWW6 | ARHGAP12 | Rho GTPase-activating protein 12 | Detected | 23 | Rac1 (32203420) | ||
Q53QZ3 | ARHGAP15 | Rho GTPase-activating protein 15 | 833 | 11 | Rac1 (32203420, 32839212) | ||
Q68EM7 | ARHGAP17 | Rho GTPase-activating protein 17 | 1603 | 21 | Rac1, RhoA (22975681) | Regulated by tyrosine phosphorylation (22975681), activated by PKA/PKG mediated S702 phosphorylation (26507661) | |
Q8N392 | ARHGAP18 | Rho GTPase-activating protein 18 | 7100 | ++ | 8 | RhoC (25425145), RhoA (32016689) | |
Q14CB8 | ARHGAP19 | Rho GTPase-activating protein 19 | - | ||||
Q9P2F6 | ARHGAP20 | Rho GTPase-activating protein 20 | - | ||||
Q5T5U3 | ARHGAP21 | Rho GTPase-activating protein 21 | 886 | 46 | RhoA (32203420, 33727037), Cdc42 (33727037) | Inhibits alpha granule release and aggregation (33727037) | |
Q7Z5H3 | ARHGAP22 | Rho GTPase-activating protein 22 | - | ||||
Q9P227 | ARHGAP23 | Rho GTPase-activating protein 23 | - | ||||
Q8N264 | ARHGAP24 | Rho GTPase-activating protein 24 | - | ||||
P42331 | ARHGAP25 | Rho GTPase-activating protein 25 | 1054 | 11 | Rac1 (36190314) | ||
Q9UNA1 | ARHGAP26 | Rho GTPase-activating protein 26 | Detected | 1 | RhoA (32203420) | ||
Q6ZUM4 | ARHGAP27 | Rho GTPase-activating protein 27 | Detected | 11 | Rac1 (32203420) | ||
Q9P2N2 | ARHGAP28 | Rho GTPase-activating protein 28 | - | ||||
Q52LW3 | ARHGAP29 | Rho GTPase-activating protein 29 | - | ||||
Q7Z6I6 | ARHGAP30 | Rho GTPase-activating protein 30 | Detected | 15 | RhoA, Rac1, Cdc42 (32203420) | ||
Q2M1Z3 | ARHGAP31 | Rho GTPase-activating protein 31 | - | ||||
A7KAX9 | ARHGAP32 | Rho GTPase-activating protein 32 | Detected | 40 | |||
O14559 | ARHGAP33 | Rho GTPase-activating protein 33 | - | ||||
Q9NRY4 | ARHGAP35 | Rho GTPase-activating protein 35 | 783 | 23 | RhoA, Rac1 (32203420) | ||
Q6ZRI8 | ARHGAP36 | Rho GTPase-activating protein 36 | - | ||||
Q9C0H5 | ARHGAP39 | Rho GTPase-activating protein 39 | - | ||||
Q5TG30 | ARHGAP40 | Rho GTPase-activating protein 40 | - | ||||
A6NI28 | ARHGAP42 | Rho GTPase-activating protein 42 | - | ||||
Q17R89 | ARHGAP44 | Rho GTPase-activating protein 44 | - | ||||
Q92619 | ARHGAP45 | Rho GTPase-activating protein 45 (HMHA1) | 3891 | 25 | |||
Q7Z6B7 | SRGAP1 | SLIT-ROBO Rho GTPase-activating protein 1 | Detected | 9 | Rac1 (32203420) | ||
O75044 | SRGAP2 | SLIT-ROBO Rho GTPase-activating protein 2 | Detected | 23 | Rac1, Cdc42 (32203420, 31880824) | ||
O43295 | SRGAP3 | SLIT-ROBO Rho GTPase-activating protein 3 | - | ||||
Q9Y3M8 | STARD13 | StAR-related lipid transfer protein 13 | Detected | 8 | RhoA (32203420) | ||
Q92502 | STARD8 | StAR-related lipid transfer protein 8 | Detected | 3 | RhoA, Cdc42 (32203420, 25673874) | ||
Q6ZW31 | SYDE1 | Rho GTPase-activating protein SYDE1 | - | ||||
Q5VT97 | SYDE2 | Rho GTPase-activating protein SYDE2 | - | ||||
Q8N103 | TAGAP | T-cell activation Rho GTPase-activating protein | - | ||||
RGS, PS50132 | |||||||
Q08116 | RGS1 | Regulator of G-protein signalling 1 | - | ||||
P41220 | RGS2 | Regulator of G-protein signalling 2 | - | ||||
P49796 | RGS3 | Regulator of G-protein signalling 3 | Detected | 8 | Gαi, Gαo (33007266) | ||
P49798 | RGS4 | Regulator of G-protein signalling 4 | - | ||||
O15539 | RGS5 | Regulator of G-protein signalling 5 | - | ||||
P49758 | RGS6 | Regulator of G-protein signalling 6 | 2434 | + | 0 | Gαo (32513692, 33007266) | |
P49802 | RGS7 | Regulator of G-protein signalling 7 | - | ||||
P57771 | RGS8 | Regulator of G-protein signalling 8 | - | ||||
O75916 | RGS9 | Regulator of G-protein signalling 9 | - | ||||
O43665 | RGS10 | Regulator of G-protein signalling 10 | 4608 | + | 5 | Gαq, Gαi, Gαo (30150297, 33007266) | Inhibits Gαq and Gαi signalling, aggregation, granule release, regulated by spinophilin and 14-3-3 (27829061, 30150297) |
O94810 | RGS11 | Regulator of G-protein signalling 11 | - | ||||
O14924 | RGS12 | Regulator of G-protein signalling 12 | - | ||||
O14921 | RGS13 | Regulator of G-protein signalling 13 | - | ||||
O43566 | RGS14 | Regulator of G-protein signalling 14 | Detected | 12 | Gαi1 (30093406, 33007266) | ||
O15492 | RGS16 | Regulator of G-protein signalling 16 | - | ||||
Q9UGC6 | RGS17 | Regulator of G-protein signalling 17 | - | ||||
Q9NS28 | RGS18 | Regulator of G-protein signalling 18 | 4463 | ++ | 3 | Gαi, Gαq (33007266) | Inhibits Gαi and Gαq signalling, regulated by spinophilin and 14-3-3, inhibited by S49/S218 phosphorylation, activated by PKA/PKG mediated S216 phosphorylation (22210881, 26407691, 22234696, 24244663) |
P49795 | RGS19 | Regulator of G-protein signalling 19 | 1086 | 5 | Gαz (33007266) | ||
O76081 | RGS20 | Regulator of G-protein signalling 20 | - | ||||
Q2M5E4 | RGS21 | Regulator of G-protein signalling 21 | - | ||||
Q8NE09 | RGS22 | Regulator of G-protein signalling 22 | - | ||||
Q15835 | GRK1 | Rhodopsin kinase GRK1 | - | ||||
P25098 | GRK2 | Beta-adrenergic receptor kinase 1 | 1409 | 7 | |||
P35626 | GRK3 | Beta-adrenergic receptor kinase 2 | Detected | 3 | |||
P32298 | GRK4 | G protein-coupled receptor kinase 4 | - | ||||
P34947 | GRK5 | G protein-coupled receptor kinase 5 | 1557 | 3 | Inhibits PAR1 receptor signalling (34581777) | ||
P43250 | GRK6 | G protein-coupled receptor kinase 6 | 2005 | 2 | Inhibits PAR1 and P2Y12 receptor signalling (31899801) | ||
Q8WTQ7 | GRK7 | Rhodopsin kinase GRK7 | - | ||||
O15085 | ARHGEF11 | Rho guanine nucleotide exchange factor 11 | - | ||||
O15169 | AXIN1 | Axin-1 | Detected | 8 | |||
Q9Y2T1 | AXIN2 | Axin-2 | - | ||||
Q9Y5W8 | SNX13 | Sorting nexin-13 | Detected | 0 | |||
Q9Y5W7 | SNX14 | Sorting nexin-14 | Detected | 1 | |||
Q9H3E2 | SNX25 | Sorting nexin-25 | - | ||||
O43572 | AKAP10 | A-kinase anchor protein 10, mitochondrial | 819 | 6 | Rab4, Rab11 (19797056) |
UniProt . | Gene . | Protein name . | Protein copy number per platelet . | Platelet specificity . | Phospho sites . | G-protein specificity (cell-based assays) . | Functions and regulation (platelet studies) . |
---|---|---|---|---|---|---|---|
ArfGAP, PS50115 | |||||||
Q15027 | ACAP1 | Arf-GAP with coiled-coil, ANK repeat and PH domain-containing protein 1 | 1150 | 6 | Arf6 (11062263) | ||
Q15057 | ACAP2 | Arf-GAP with coiled-coil, ANK repeat and PH domain-containing protein 2 | 1057 | 13 | Arf6 (11062263) | ||
Q96P50 | ACAP3 | Arf-GAP with coiled-coil, ANK repeat and PH domain-containing protein 3 | Detected | 3 | |||
O75689 | ADAP1 | Arf-GAP with dual PH domain-containing protein 1 | - | ||||
Q9NPF8 | ADAP2 | Arf-GAP with dual PH domain-containing protein 2 | - | ||||
Q9UPQ3 | AGAP1 | Arf-GAP with GTPase, ANK repeat and PH domain-containing protein 1 | Detected | 7 | |||
Q99490 | AGAP2 | Arf-GAP with GTPase, ANK repeat and PH domain-containing protein 2 | 1199 | 15 | |||
Q96P47 | AGAP3 | Arf-GAP with GTPase, ANK repeat and PH domain-containing protein 3 | Detected | 8 | |||
Q96P64 | AGAP4 | Arf-GAP with GTPase, ANK repeat and PH domain-containing protein 4 | - | ||||
A6NIR3 | AGAP5 | Arf-GAP with GTPase, ANK repeat and PH domain-containing protein 5 | - | ||||
Q5VW22 | AGAP6 | Arf-GAP with GTPase, ANK repeat and PH domain-containing protein 6 | - | ||||
Q5VUJ5 | AGAP7 | Putative Arf-GAP with GTPase, ANK repeat and PH domain-containing protein 7 | - | ||||
Q5VTM2 | AGAP9 | Arf-GAP with GTPase, ANK repeat and PH domain-containing protein 9 | - | ||||
P52594 | AGFG1 | Arf-GAP domain and FG repeat-containing protein 1 | 1825 | 10 | |||
O95081 | AGFG2 | Arf-GAP domain and FG repeat-containing protein 2 | Detected | 2 | |||
Q96P48 | ARAP1 | Arf-GAP with Rho-GAP domain, ANK repeat and PH domain-containing protein 1 | 3061 | 17 | |||
Q8WZ64 | ARAP2 | Arf-GAP with Rho-GAP domain, ANK repeat and PH domain-containing protein 2 | - | ||||
Q8WWN8 | ARAP3 | Arf-GAP with Rho-GAP domain, ANK repeat and PH domain-containing protein 3 | - | ||||
Q8N6T3 | ARFGAP1 | ADP-ribosylation factor GTPase-activating protein 1 | Detected | 17 | Arf1 (19015319, 36513395) | ||
Q8N6H7 | ARFGAP2 | ADP-ribosylation factor GTPase-activating protein 2 | 899 | 13 | Arf1 (19015319, 36513395) | ||
Q9NP61 | ARFGAP3 | ADP-ribosylation factor GTPase-activating protein 3 | Detected | 21 | Arf1, Arl1 (19015319, 33715220) | ||
Q9ULH1 | ASAP1 | Arf-GAP with SH3 domain, ANK repeat and PH domain-containing protein 1 | 3588 | + | 18 | Binds Crkl and to focal adhesions (12522101) | |
O43150 | ASAP2 | Arf-GAP with SH3 domain, ANK repeat and PH domain-containing protein 2 | 4132 | + | 10 | ||
Q8TDY4 | ASAP3 | Arf-GAP with SH3 domain, ANK repeat and PH domain-containing protein 3 | Detected | 5 | |||
Q9Y2X7 | GIT1 | ARF GTPase-activating protein GIT1 | 3149 | 30 | Arf1, Arf6 (28981399) | Binds RhoGEF6 and RhoGEF7, interacts with integrin αIIbβ3 (26507661, 18211801) | |
Q14161 | GIT2 | ARF GTPase-activating protein GIT2 | 1071 | 28 | |||
Q8IYB5 | SMAP1 | Stromal membrane-associated protein 1 | 890 | 0 | |||
Q8WU79 | SMAP2 | Stromal membrane-associated protein 2 | 1717 | 5 | |||
TBC_RabGAP, PS50086 | |||||||
Q96CN4 | EVI5L | EVI5-like protein | 648 | 2 | |||
O60447 | EVI5 | Ecotropic viral integration site 5 protein homolog | - | ||||
Q5TC63 | GRTP1 | Growth hormone-regulated TBC protein 1 | Detected | 1 | |||
Q5R372 | RABGAP1L | Rab GTPase-activating protein 1-like | 615 | 5 | |||
Q9Y3P9 | RABGAP1 | Rab GTPase-activating protein 1 | 1619 | 9 | |||
Q2NKQ1 | SGSM1 | Small G protein signalling modulator 1 | - | ||||
O43147 | SGSM2 | Small G protein signalling modulator 2 | - | ||||
Q96HU1 | SGSM3 | Small G protein signalling modulator 3 | - | ||||
Q86TI0 | TBC1D1 | TBC1 domain family member 1 | Detected | 24 | |||
Q9BYX2 | TBC1D2 | TBC1 domain family member 2A | - | ||||
Q9UPU7 | TBC1D2B | TBC1 domain family member 2B | Detected | 10 | |||
Q8IZP1 | TBC1D3 | TBC1 domain family member 3 | - | ||||
A6NDS4 | TBC1D3B | TBC1 domain family member 3B | - | ||||
Q6IPX1 | TBC1D3C | TBC1 domain family member 3C | - | ||||
A0A087WVF3 | TBC1D3D | TBC1 domain family member 3D | - | ||||
A0A087X179 | TBC1D3E | TBC1 domain family member 3E | - | ||||
A6NER0 | TBC1D3F | TBC1 domain family member 3F | - | ||||
Q6DHY5 | TBC1D3G | TBC1 domain family member 3G | - | ||||
P0C7X1 | TBC1D3H | TBC1 domain family member 3H | - | ||||
A0A087WXS9 | TBC1D3I | TBC1 domain family member 3I | - | ||||
A0A087X1G2 | TBC1D3K | TBC1 domain family member 3K | - | ||||
B9A6J9 | TBC1D3L | TBC1 domain family member 3L | - | ||||
O60343 | TBC1D4 | TBC1 domain family member 4 | Detected | 42 | Rab10 (33175605) | ||
Q92609 | TBC1D5 | TBC1 domain family member 5 | 1046 | 27 | Rab7B (30111580) | ||
Q9P0N9 | TBC1D7 | TBC1 domain family member 7 | Detected | 2 | |||
O95759 | TBC1D8 | TBC1 domain family member 8 | - | ||||
Q0IIM8 | TBC1D8B | TBC1 domain family member 8B | Detected | 2 | |||
Q6ZT07 | TBC1D9 | TBC1 domain family member 9 | Detected | 0 | |||
Q66K14 | TBC1D9B | TBC1 domain family member 9B | 1012 | 18 | |||
Q9BXI6 | TBC1D10A | TBC1 domain family member 10A | Detected | 14 | Rab35 (28566286) | ||
Q4KMP7 | TBC1D10B | TBC1 domain family member 10B | 827 | 22 | Rab27b (23671284) | ||
Q8IV04 | TBC1D10C | Carabin | Detected | 2 | |||
O60347 | TBC1D12 | TBC1 domain family member 12 | - | ||||
Q9NVG8 | TBC1D13 | TBC1 domain family member 13 | 2656 | + | 1 | Rab35 (22762500) | |
Q9P2M4 | TBC1D14 | TBC1 domain family member 14 | Detected | 5 | |||
Q8TC07 | TBC1D15 | TBC1 domain family member 15 | 2534 | 14 | |||
Q8TBP0 | TBC1D16 | TBC1 domain family member 16 | - | ||||
Q9HA65 | TBC1D17 | TBC1 domain family member 17 | Detected | 5 | Rab8 (22854040) | ||
Q8N5T2 | TBC1D19 | TBC1 domain family member 19 | - | ||||
Q96BZ9 | TBC1D20 | TBC1 domain family member 20 | 1069 | 0 | Rab1A (22854043) | ||
Q8IYX1 | TBC1D21 | TBC1 domain family member 21 | - | ||||
Q8WUA7 | TBC1D22A | TBC1 domain family member 22A | 1138 | 8 | |||
Q9NU19 | TBC1D22B | TBC1 domain family member 22B | Detected | 8 | |||
Q9NUY8 | TBC1D23 | TBC1 domain family member 23 | 1187 | 7 | |||
Q3MII6 | TBC1D25 | TBC1 domain family member 25 | Detected | 1 | |||
Q86UD7 | TBC1D26 | TBC1 domain family member 26 | - | ||||
Q9UFV1 | TBC1D29P | Putative TBC1 domain family member 29 | - | ||||
Q9Y2I9 | TBC1D30 | TBC1 domain family member 30 | - | ||||
Q96DN5 | TBC1D31 | TBC1 domain family member 31 | - | ||||
Q8TEA7 | TBCK | TBC domain-containing protein kinase-like protein | Detected | 1 | |||
P35125 | USP6 | Ubiquitin carboxyl-terminal hydrolase 6 | - | ||||
Q92738 | USP6NL | USP6 N-terminal-like protein | Detected | 26 | |||
RasGAP, PS50018 | |||||||
Q5VWQ8 | DAB2IP | Disabled homolog 2-interacting protein | - | ||||
Q14C86 | GAPVD1 | GTPase-activating protein and VPS9 domain-containing protein 1 | 1283 | 34 | |||
P46940 | IQGAP1 | Ras GTPase-activating-like protein IQGAP1 | 1040 | 17 | No GAP activity, Cdc42/Rac1 effector (34830479) | Inhibits alpha granule release (15026422) | |
Q13576 | IQGAP2 | Ras GTPase-activating-like protein IQGAP2 | 8102 | + | 19 | No GAP activity, Cdc42/Rac1 effector (34830479) | Interacts with arp3 and actin (12515716) |
Q86VI3 | IQGAP3 | Ras GTPase-activating-like protein IQGAP3 | - | ||||
P21359 | NF1 | Neurofibromin | 628 | 20 | HRas (9668168) | ||
O95294 | RASAL1 | RasGAP-activating-like protein 1 | - | ||||
Q9UJF2 | RASAL2 | Ras GTPase-activating protein nGAP | Detected | 26 | |||
Q86YV0 | RASAL3 | RAS protein activator like-3 | 573 | 25 | |||
C9J798 | RASA4B | Ras GTPase-activating protein 4B | - | ||||
P20936 | RASA1 | Ras GTPase-activating protein 1 | 2921 | + | 5 | ||
Q15283 | RASA2 | Ras GTPase-activating protein 2 | Detected | 5 | |||
Q14644 | RASA3 | Ras GTPase-activating protein 3 | 8293 | ++ | 12 | Rap1, H-Ras (16431904) | Reduces Rap1-GTP, inhibits platelet adhesion and aggregation, binds PIP3, linked to P2Y12 receptor via PI3K (24967784, 25705885, 27903653) |
O43374 | RASA4 | Ras GTPase-activating protein 4 | - | ||||
Q96PV0 | SYNGAP1 | Ras/Rap GTPase-activating protein SynGA | - | ||||
RapGAP, PS50085 | |||||||
Q5VVW2 | GARNL3 | GTPase-activating Rap/Ran-GAP domain-like protein 3 | - | ||||
Q6GYQ0 | RALGAPA1 | Ral GTPase-activating protein subunit alpha-1 | 669 | 28 | |||
Q2PPJ7 | RALGAPA2 | Ral GTPase-activating protein subunit alpha-2 | Detected | 12 | |||
Q86X10 | RALGAPB | Ral GTPase-activating protein subunit beta | 884 | 7 | RalA (29915037) | ||
P47736 | RAP1GAP | Rap1 GTPase-activating protein 1 | - | ||||
Q684P5 | RAP1GAP2 | Rap1 GTPase-activating protein 2 | 2327 | ++ | 22 | Rap1 (15632203) | Inhibited by S9 phosphorylation, activated by PKA/PKG mediated S7 phosphorylation, binds 14-3-3, binds Slp1 and stimulates dense granule release (15632203, 18039662, 19528539) |
O43166 | SIPA1L1 | Signal-induced proliferation-associated 1-like protein 1 | Detected | 65 | |||
Q9P2F8 | SIPA1L2 | Signal-induced proliferation-associated 1-like protein 2 | - | ||||
O60292 | SIPA1L3 | Signal-induced proliferation-associated 1-like protein 3 | - | ||||
Q96FS4 | SIPA1 | Signal-induced proliferation-associated protein 1 | Detected | 12 | |||
P49815 | TSC2 | Tuberin | 593 | 35 | |||
RhoGAP, PS50238 | |||||||
Q9Y3L3 | SH3BP1 | SH3 domain-binding protein 1 | 595 | 14 | |||
Q12979 | ABR | Active breakpoint cluster region-related protein | Detected | 5 | Rac1 (32203420) | ||
Q96P48 | ARAP1 | Arf-GAP with Rho-GAP domain, ANK repeat and PH domain-containing protein 1 | 3061 | 17 | Rac1, Cdc42 (32203420) | ||
Q8WZ64 | ARAP2 | Arf-GAP with Rho-GAP domain, ANK repeat and PH domain-containing protein 2 | - | ||||
Q8WWN8 | ARAP3 | Arf-GAP with Rho-GAP domain, ANK repeat and PH domain-containing protein 3 | - | ||||
P11274 | BCR | Breakpoint cluster region protein | 793 | 47 | Rac1 (32203420) | ||
Q6ZT62 | BARGIN | Bargin | - | ||||
P15882 | CHN1 | N-chimaerin | - | ||||
P52757 | CHN2 | Beta-chimaerin | - | ||||
Q8WUY9 | DEPDC1B | DEP domain-containing protein 1B | - | ||||
O94988 | FAM13A | Protein FAM13A | - | ||||
Q9NYF5 | FAM13B | Protein FAM13B | - | ||||
Q9P107 | GMIP | GEM-interacting protein | 1228 | 17 | RhoA, Rac1, Cdc42 (32203420) | ||
P32019 | INPP5B | Type II inositol 1,4,5-trisphosphate 5-phosphatase | 1629 | 1 | |||
B2RTY4 | MYO9A | Unconventional myosin-IXa | Detected | 14 | |||
Q13459 | MYO9B | Unconventional myosin-IXb | 1428 | 40 | RhoA (33268376) | Activated by PKA/PKG mediated S1354 phosphorylation (32692911) | |
Q01968 | OCRL | Inositol polyphosphate 5-phosphatase OCRL | 850 | 3 | RhoA (33528045) | Reduces RhoA-GTP, augments Rac1-GTP and myosin light chain phosphorylation, inhibits filopodia, stimulates spreading, clot retraction and thrombus formation (33528045, 36176266) | |
O60890 | OPHN1 | Oligophrenin-1 | 1527 | 3 | RhoA, Rac1, Cdc42 (25556321) | Reduces RhoA-GTP, Rac1-GTP, and Cdc42-GTP, inhibits adhesion and granule release (25556321) | |
P27986 | PIK3R1 | Phosphatidylinositol 3-kinase regulatory subunit alpha | 1904 | 17 | |||
O00459 | PIK3R2 | Phosphatidylinositol 3-kinase regulatory subunit beta | Detected | 12 | |||
Q15311 | RALBP1 | RalA-binding protein 1 | 785 | 14 | Rac1 (32203420) | ||
Q9H0H5 | RACGAP1 | Rac GTPase-activating protein 1 | - | ||||
Q07960 | ARHGAP1 | Rho GTPase-activating protein 1 | 9290 | + | 8 | RhoA, Cdc42 (32203420) | |
P98171 | ARHGAP4 | Rho GTPase-activating protein 4 | 1400 | 6 | Rac1 (32203420) | ||
Q13017 | ARHGAP5 | Rho GTPase-activating protein 5 | Detected | 16 | RhoA (32203420) | ||
O43182 | ARHGAP6 | Rho GTPase-activating protein 6 | 4151 | ++ | 9 | RhoA (32203420) | Binds COPD and COPI vesicles, might regulate protein transport (37409526) |
Q96QB1 | DLC1 | Rho GTPase-activating protein 7 | - | ||||
P85298 | ARHGAP8 | Rho GTPase-activating protein 8 | - | ||||
Q9BRR9 | ARHGAP9 | Rho GTPase-activating protein 9 | Detected | 3 | Rac1 (32203420) | ||
A1A4S6 | ARHGAP10 | Rho GTPase-activating protein 10 | 1114 | 1 | RhoA (32203420) | ||
Q6P4F7 | ARHGAP11A | Rho GTPase-activating protein 11A | - | ||||
Q3KRB8 | ARHGAP11B | Inactive Rho GTPase-activating protein 11B | - | ||||
Q8IWW6 | ARHGAP12 | Rho GTPase-activating protein 12 | Detected | 23 | Rac1 (32203420) | ||
Q53QZ3 | ARHGAP15 | Rho GTPase-activating protein 15 | 833 | 11 | Rac1 (32203420, 32839212) | ||
Q68EM7 | ARHGAP17 | Rho GTPase-activating protein 17 | 1603 | 21 | Rac1, RhoA (22975681) | Regulated by tyrosine phosphorylation (22975681), activated by PKA/PKG mediated S702 phosphorylation (26507661) | |
Q8N392 | ARHGAP18 | Rho GTPase-activating protein 18 | 7100 | ++ | 8 | RhoC (25425145), RhoA (32016689) | |
Q14CB8 | ARHGAP19 | Rho GTPase-activating protein 19 | - | ||||
Q9P2F6 | ARHGAP20 | Rho GTPase-activating protein 20 | - | ||||
Q5T5U3 | ARHGAP21 | Rho GTPase-activating protein 21 | 886 | 46 | RhoA (32203420, 33727037), Cdc42 (33727037) | Inhibits alpha granule release and aggregation (33727037) | |
Q7Z5H3 | ARHGAP22 | Rho GTPase-activating protein 22 | - | ||||
Q9P227 | ARHGAP23 | Rho GTPase-activating protein 23 | - | ||||
Q8N264 | ARHGAP24 | Rho GTPase-activating protein 24 | - | ||||
P42331 | ARHGAP25 | Rho GTPase-activating protein 25 | 1054 | 11 | Rac1 (36190314) | ||
Q9UNA1 | ARHGAP26 | Rho GTPase-activating protein 26 | Detected | 1 | RhoA (32203420) | ||
Q6ZUM4 | ARHGAP27 | Rho GTPase-activating protein 27 | Detected | 11 | Rac1 (32203420) | ||
Q9P2N2 | ARHGAP28 | Rho GTPase-activating protein 28 | - | ||||
Q52LW3 | ARHGAP29 | Rho GTPase-activating protein 29 | - | ||||
Q7Z6I6 | ARHGAP30 | Rho GTPase-activating protein 30 | Detected | 15 | RhoA, Rac1, Cdc42 (32203420) | ||
Q2M1Z3 | ARHGAP31 | Rho GTPase-activating protein 31 | - | ||||
A7KAX9 | ARHGAP32 | Rho GTPase-activating protein 32 | Detected | 40 | |||
O14559 | ARHGAP33 | Rho GTPase-activating protein 33 | - | ||||
Q9NRY4 | ARHGAP35 | Rho GTPase-activating protein 35 | 783 | 23 | RhoA, Rac1 (32203420) | ||
Q6ZRI8 | ARHGAP36 | Rho GTPase-activating protein 36 | - | ||||
Q9C0H5 | ARHGAP39 | Rho GTPase-activating protein 39 | - | ||||
Q5TG30 | ARHGAP40 | Rho GTPase-activating protein 40 | - | ||||
A6NI28 | ARHGAP42 | Rho GTPase-activating protein 42 | - | ||||
Q17R89 | ARHGAP44 | Rho GTPase-activating protein 44 | - | ||||
Q92619 | ARHGAP45 | Rho GTPase-activating protein 45 (HMHA1) | 3891 | 25 | |||
Q7Z6B7 | SRGAP1 | SLIT-ROBO Rho GTPase-activating protein 1 | Detected | 9 | Rac1 (32203420) | ||
O75044 | SRGAP2 | SLIT-ROBO Rho GTPase-activating protein 2 | Detected | 23 | Rac1, Cdc42 (32203420, 31880824) | ||
O43295 | SRGAP3 | SLIT-ROBO Rho GTPase-activating protein 3 | - | ||||
Q9Y3M8 | STARD13 | StAR-related lipid transfer protein 13 | Detected | 8 | RhoA (32203420) | ||
Q92502 | STARD8 | StAR-related lipid transfer protein 8 | Detected | 3 | RhoA, Cdc42 (32203420, 25673874) | ||
Q6ZW31 | SYDE1 | Rho GTPase-activating protein SYDE1 | - | ||||
Q5VT97 | SYDE2 | Rho GTPase-activating protein SYDE2 | - | ||||
Q8N103 | TAGAP | T-cell activation Rho GTPase-activating protein | - | ||||
RGS, PS50132 | |||||||
Q08116 | RGS1 | Regulator of G-protein signalling 1 | - | ||||
P41220 | RGS2 | Regulator of G-protein signalling 2 | - | ||||
P49796 | RGS3 | Regulator of G-protein signalling 3 | Detected | 8 | Gαi, Gαo (33007266) | ||
P49798 | RGS4 | Regulator of G-protein signalling 4 | - | ||||
O15539 | RGS5 | Regulator of G-protein signalling 5 | - | ||||
P49758 | RGS6 | Regulator of G-protein signalling 6 | 2434 | + | 0 | Gαo (32513692, 33007266) | |
P49802 | RGS7 | Regulator of G-protein signalling 7 | - | ||||
P57771 | RGS8 | Regulator of G-protein signalling 8 | - | ||||
O75916 | RGS9 | Regulator of G-protein signalling 9 | - | ||||
O43665 | RGS10 | Regulator of G-protein signalling 10 | 4608 | + | 5 | Gαq, Gαi, Gαo (30150297, 33007266) | Inhibits Gαq and Gαi signalling, aggregation, granule release, regulated by spinophilin and 14-3-3 (27829061, 30150297) |
O94810 | RGS11 | Regulator of G-protein signalling 11 | - | ||||
O14924 | RGS12 | Regulator of G-protein signalling 12 | - | ||||
O14921 | RGS13 | Regulator of G-protein signalling 13 | - | ||||
O43566 | RGS14 | Regulator of G-protein signalling 14 | Detected | 12 | Gαi1 (30093406, 33007266) | ||
O15492 | RGS16 | Regulator of G-protein signalling 16 | - | ||||
Q9UGC6 | RGS17 | Regulator of G-protein signalling 17 | - | ||||
Q9NS28 | RGS18 | Regulator of G-protein signalling 18 | 4463 | ++ | 3 | Gαi, Gαq (33007266) | Inhibits Gαi and Gαq signalling, regulated by spinophilin and 14-3-3, inhibited by S49/S218 phosphorylation, activated by PKA/PKG mediated S216 phosphorylation (22210881, 26407691, 22234696, 24244663) |
P49795 | RGS19 | Regulator of G-protein signalling 19 | 1086 | 5 | Gαz (33007266) | ||
O76081 | RGS20 | Regulator of G-protein signalling 20 | - | ||||
Q2M5E4 | RGS21 | Regulator of G-protein signalling 21 | - | ||||
Q8NE09 | RGS22 | Regulator of G-protein signalling 22 | - | ||||
Q15835 | GRK1 | Rhodopsin kinase GRK1 | - | ||||
P25098 | GRK2 | Beta-adrenergic receptor kinase 1 | 1409 | 7 | |||
P35626 | GRK3 | Beta-adrenergic receptor kinase 2 | Detected | 3 | |||
P32298 | GRK4 | G protein-coupled receptor kinase 4 | - | ||||
P34947 | GRK5 | G protein-coupled receptor kinase 5 | 1557 | 3 | Inhibits PAR1 receptor signalling (34581777) | ||
P43250 | GRK6 | G protein-coupled receptor kinase 6 | 2005 | 2 | Inhibits PAR1 and P2Y12 receptor signalling (31899801) | ||
Q8WTQ7 | GRK7 | Rhodopsin kinase GRK7 | - | ||||
O15085 | ARHGEF11 | Rho guanine nucleotide exchange factor 11 | - | ||||
O15169 | AXIN1 | Axin-1 | Detected | 8 | |||
Q9Y2T1 | AXIN2 | Axin-2 | - | ||||
Q9Y5W8 | SNX13 | Sorting nexin-13 | Detected | 0 | |||
Q9Y5W7 | SNX14 | Sorting nexin-14 | Detected | 1 | |||
Q9H3E2 | SNX25 | Sorting nexin-25 | - | ||||
O43572 | AKAP10 | A-kinase anchor protein 10, mitochondrial | 819 | 6 | Rab4, Rab11 (19797056) |
Proteins containing GAP domains were obtained from UniProt and compared with platelet proteome data. Shown are all proteins encoded by the human genome. Protein copy numbers per platelet are given as far as available. ‘Detected’ indicates expressed proteins where a copy number has not yet been determined. All found proteins have also been confirmed at the transcriptome level of megakaryocytes or platelets. ‘-’ indicates that proteins could not be detected in platelets. Platelet specificity was determined as high expression in platelets compared to other human tissues according to https://www.proteomicsdb.org and http://www.humanproteomemap.org (‘+’ indicates within the top 5 highly expressing tissues, ‘++’ indicates platelets as highest expressing tissue in both databases). Phosphorylation (Phospho) sites refers to sites identified by proteomics and by low throughput studies. Shown are numbers of phosphorylation sites found in any cell type with a minimum of at least 5 references according to PhosphoSite (https://www.phosphosite.org/). References for G protein specificities of GAPs are given as PubMed ID numbers (PMID). Detailed studies refers to platelet data, listed are PMIDs.
GAP domain structure and function
GAPs are defined as proteins that contain catalytic GAP domains and GAP domain profiles have been defined and are accessible through the Prosite database (ARFGAP, PS50115; TBC_RABGAP, PS50086; RAS_GTPASE_ACTIV_2, PS50018; RAPGAP, PS50085; RHOGAP, PS50238; RGS, PS50132). In general, GAPs bound to their small G proteins contribute catalytic site residues to stimulate GTP hydrolysis. For example, GAPs for Ras and Rho GTPases contribute an essential arginine, called arginine-finger, whereas RabGAPs use an arginine/glutamine dual-finger, and GAPs for Rap and Ral use an asparagine, called asparagine-thumb [12,22,23]. RGS domains do not appear to contribute catalytic site residues but stabilize a pre-transition state of Gα [24,25]. Although minimal sequences exhibiting GAP activity have been defined, in some cases other regions or domains outside of the core GAP domain contribute supporting or inhibitory functions. For example, autoinhibition is a common feature of RhoGAPs [26] which has been confirmed for the platelet GAPs RhoGAP6 [27] and RASA3 [28]. The pleckstrin homology (PH) domain of the ArfGAP ASAP1 supports Arf binding whereas its predicted N-terminal Bin/amphiphysin/Rvs (BAR) domain inhibits GAP activity (Figure 2A) [29]. In contrast, nonmuscle myosin 2A (NM2A), a BAR domain binding partner, stimulates ASAP1 activity, possibly by relieving the autoinhibition [30]. Other examples for regulatory components within GAPs include the RhoA regulator RhoGAP35 which exhibits a protrusion localization sequence, including two pseudo GTPase regions (Figure 2D), involved in localization to lamellipodia in cancer cells that inhibits GAP activity [31].
Domain structures of platelet GAPs
Shown are GAPs of Arf (A), Rab (B), Ras/Rap (C), Rho (D), and alpha subunits of heterotrimeric (E) G proteins found in human platelets. Domains, domain boundaries, and protein lenghts are given according to UniProt, and proteins are sorted according to their expression levels (Table 2). Domain names and abbreviations are given.
Shown are GAPs of Arf (A), Rab (B), Ras/Rap (C), Rho (D), and alpha subunits of heterotrimeric (E) G proteins found in human platelets. Domains, domain boundaries, and protein lenghts are given according to UniProt, and proteins are sorted according to their expression levels (Table 2). Domain names and abbreviations are given.
Other domains contained in GAPs
GAPs are typically large proteins including multiple domains (Figure 2). Only few GAPs appear to consist of a catalytic GAP domain alone. These catalytic domain only GAPs include the most highly expressed platelet RabGAP TBC1D13 as well as RGSs 10, 18, and 19 (Figure 2B, 2E). All other RabGAPs and RGSs as well as ArfGAPs, Ras/RapGAPs, and RhoGAPs (Figure 2A, 2C, 2D) contain a diverse set of additional domains. A prominent feature found in most GAPs are domains that can mediate lipid and membrane binding, as for example, PH (most ArfGAPs), C2 (Ras/RapGAPs) and BAR domains (RhoGAP). Membrane binding might be required for the interaction with G proteins as most G proteins are attached to membranes via lipid anchors [11]. Furthermore, a variety of protein/protein interaction domains are present in these GAPs including ankyrin repeats (ArfGAPs), Src homology 2 (SH2), Src homology 3 (SH3), WW, and post synaptic density protein, Drosophila disc large tumor suppressor, and zonula occludens-1 protein (PDZ) domains (Figure 2). Protein interaction domains might localise GAPs to specific G proteins, to other GAPs and GEFs, or to adapter proteins and thereby contribute to the formation of G protein networks required for the coordination of multiple platelet functions [21,32]. Another characteristic seen in platelet GAPs is their ability to regulate other cellular processes independent of their GAP activity. For example sequences outside the catalytic GAP domain enable interactions of Rap1GAP2 with Slp1 to modulate dense granule release (see below for details, Figure 3B) [33]. RhoGAP6 enhances protein transport through the secretory pathway and its RhoGAP activity actually inhibits this effect (Figure 3C) [27].
Most GAPs contain sites for possible post-translational modifications. For example, all platelets GAPs are predicted to contain 10 phosphorylation sites per protein on average (Table 2), estimate based on a conservative approach with a minimum of 5 references according to PhosphoSite) pointing towards complex regulation by multi-site phosphorylation [34]. GAP phosphorylation has been linked to changes in GAP activity during platelet activation and inhibition. Many G proteins are turned into their active GTP-bound versions during platelet activation. Maintenance of GTP-bound G proteins requires reduced GAP activities whereas classical platelet inhibitors tend to stimulate GAPs. This has been observed for RASA3, Rap1GAP2, RhoGAP17, Myo9B, RGS10 and RGS18 (see below for details). These activity changes have been linked to phosphorylation and dephosphorylation of regulatory sites involving binding of 14-3-3 adapter proteins. The adapter protein 14-3-3 is common phosphorylation dependent interaction partner of many GAPs including Rap1GAP2, RhoGAP6, RGS10 and RGS18 (Figure 3) and 14-3-3s play an established role in regulating platelet functions [35].
Arf proteins and ArfGAPs in platelets
Arf proteins
G proteins of the Arf family are generally known has signalling molecules controlling diverse cellular functions including vesicle formation and membrane trafficking as well as remodelling of the actin cytoskeleton and cell adhesion [36]. Arf1, 3, 4, and 5 are expressed in high amounts in platelets [18] (Table 1) and are thought to have overlapping roles in vesicle formation and transport to and from the Golgi and in recycling endosomes [37–39]. For example, Arf1 initiates vesicle formation by recruiting coat protein complex I (COPI) and clathrin adapter proteins (AP-1, AP-3, and AP-4) to membranes. Coat proteins then complete vesicle formation and select protein and lipid cargo for vesicular transport [37]. Of all 27 Arf proteins found in platelets only Arf6 has been studied is some detail. Arf6 has a role in controlling signalling in the cell periphery. In platelets, Arf6 is involved in endocytosis of membrane receptors including the fibrinogen receptor integrin αIIbβ3, the P2Y12 ADP receptor [40] and Toll-like receptors 7 and 9 [41]. Reduced Arf6 expression in mouse platelets resulted in impaired fibrinogen endocytosis and uptake into alpha granules which correlated with increased platelet spreading and clot retraction [42]. In contrast with many other small G proteins Arf6 is typically found in its GTP-bound form in resting platelets. Arf6 transitions to the inactive GDP-bound form during platelet activation by collagen and thrombin [43], although one study has also described the opposite effect [44]. The ArfGAP regulating Arf6 in platelets is unknown. In vitro studies using purified proteins have provided evidence for specificity of GIT1, ACAP1, and ACAP2 towards Arf6 [45,46], and these three GAPs are present in platelets (Figure 2A). High Arf6-GTP levels might be maintained by cytohesin-2 which has been identified as GEF for Arf6 in platelets [47].
ArfGAPs
Interestingly, the most highly expressed ArfGAPs in platelets, ASAP1, ASAP2, and GIT1 (Figure 2A) have been described as integrin and receptor regulators [36,48]. Integrin regulation is thought to be mediated by effects on membrane trafficking and endocytosis possibly via Arf6 or other Arfs, or through Rac1 signalling. ASAP1 localizes to platelet focal adhesions through binding of the adapter protein CrkL [49]. GIT1 currently is the most extensively studied ArfGAP in platelets. Platelet activation has been shown to induce tyrosine phosphorylation of GIT1 probably through integrin αIIbβ3 induced outside-in signalling mediated by Src kinase [50]. Tyrosine phosphorylation correlates with translocation of GIT from a membrane to a cytoskeletal fraction together with integrin β3. As GIT1 is known to bind constitutively to the Rac1 regulators RhoGEF6 [51] and RhoGEF7 [50] in platelets cytoskeletal recruitment could contribute to Rac1 and possibly Arf6 mediated actin rearrangements during platelet activation. ARAP1, another highly expressed platelet ArfGAP (Figure 2A), contains a RhoGAP domain as well as a Ras-association region and might support the coordination of the function of multiple G proteins including Rap1, Rac1, RhoA, Cdc42, and Arf proteins [52]. Similar to other ArfGAPs ARAP1 contains PH domains (Figure 2A) which might facilitate close association to membrane surfaces required for effective Arf interaction [53].
In summary, platelet Arfs are only marginally described apart from Arf6 which has a role in receptor endocytosis and in the regulation of the actin cytoskeleton. As ArfGAPs appear to be recruiting additional G protein regulators future studies should consider analysing Arfs in combination with their GAPs (and GEFs) to improve understanding of the specific roles of Arf proteins in platelet signalling [36].
Rab proteins and RabGAPs in platelets
Rab proteins
Similar to Arfs Rab proteins are involved in controlling intracellular membrane and vesicle traffic. Rabs tend to associate with specific vesicles thus conferring membrane identity and enabling directed transport between donor and acceptor compartments, both in secretion as well as in endocytosis and recycling pathways [54,55]. For example, active Rab27-GTP localised at the cytosolic surface of platelet dense granules can bind to the effector protein Munc13-4 supporting tethering of the granule to the plasma membrane [56]. Tethering is followed by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) mediated membrane fusion and secretion of granule content [57]. Prominent vesicle/granule associated SNAREs in platelets are VAMP-8 and VAMP-7 which interact with target membrane SNAREs like syntaxin-11 and SNAP-23 as well as Munc18 proteins. The Ras family G protein Ral might be another component of the secretory complex through interactions with SNARE binding exocyst factors. However, mouse knockout studies suggest only minor roles for RalA and RalB in granule release, but a possible role for Ral in P-selectin translocation to the plasma membrane has been proposed [58]. Rab27B is the most highly expressed Rab in platelets [18] (Table 1) and its role in dense granule release has been confirmed [59]. Similar to Arf6 Rab27 is present predominantly in the GTP-bound form in unstimulated platelets, whereas granule release leads to a decrease of Rab27-GTP levels [60]. A possible GAP that could mediate Rab27 inactivation is TBC1D10B which has been shown to have activity towards Rab27B in other cells [61]. Rab8 might also support tethering of dense granules to the plasma membrane by interacting with synaptotagmin-like protein 4 [62]. Regarding the role of Rab proteins in the biogenesis of dense granules data have been obtained from Hermansky–Pudlak syndrome (HPS) patients and mouse models. Combined deficiency of Rab32 and Rab38 in mice leads to defective dense granule formation and impaired platelet function similar to defects seen in HPS patients [63]. Alpha granules were not affected by Rab32/38 deficiency and platelet counts were not changed indicating that dense granule deficiency does not necessarily impact on platelet biogenesis. These data support earlier findings obtained using the megakaryocytic cell line MEG-01 suggesting that Rab32 and Rab38 enable the transport of dense granule components from early endosomes via AP-3 and Rab7 labelled vesicles towards dense granule precursors [64]. Little data are available regarding biogenesis, tethering and release of alpha granules. Rab1B is down-regulated in patients with alpha granule defects due to deficiency of the transcription factor RUNX1 and Rab1B was shown to be involved in ER-Golgi transport of alpha granule content in megakaryocytic cells; however, changes in alpha granule release were not reported [65]. Endocytosis of extracellular proteins contributes to alpha granule content [40]. Rab31 appears to play an important role in this process as endosomal trafficking is impaired and alpha granule proteins like VWF accumulate in early endosomes of megakaryocytes lacking Rab31 [66]. Furthermore, endocytosed fibrinogen was detected in Rab5 positive granules equivalent to early endosomes, followed by Rab7 positive late endosomes, and finally P-selectin positive alpha granules of megakaryocytes [67]. Rab5 has also been shown to regulate endocytosis and trafficking of the GPIb receptor in megakaryocytes which was linked to proplatelet formation [68]. Alpha granule release might involve Rab4 based on studies using permeabilized platelets [69]. Further characterization of the localization and function of Rab proteins and their GEFs and GAPs might help in clarifying open questions regarding alpha granule subtypes with different release kinetics [70].
Rab regulated endocytotic pathways contribute to the recycling of plasma membrane proteins. Fibrinogen labelled integrin αIIbβ3, passes through a Rab4-positive early endosome compartment followed by colocalization with Rab11-positive structures which are considered recycling endosomes [42]. Similarly, the P2Y12 ADP receptor was shown to be recycled via a Rab5/Rab11 pathway in transfected cells [71]. Recycling pathways have also been shown to play a role in virus entry into platelets. HIV virions sequentially pass through Rab4 and Rab7 compartments in platelets finally accumulating in microtubule-associated protein 1A/1B-light chain 3 (LC3) positive compartments similar to those found in phagocytes [41]. Related pathways may be used for the internalization of influenza and/or SARS-COV-2 viruses leading to platelet activation [72,73]. Rab11A and B are actually among the most highly expressed Rabs in platelets (Table 1) pointing towards a prominent role for recycling pathways in platelet function. Furthermore, Rab11 might be cooperating closely with Arf6 in controlling integrin traffic as has been shown for neurons [74].
RabGAPs
A PubMed search using the terms ‘RabGAP’ and ‘platelets’ did not detect any platelet or megakaryocyte studies at this time. However, one might speculate about potential functions of the top three RabGAPs expressed in platelets based on data from other cells (Figure 2B). TBC1D13, the most highly expressed RabGAP, has been shown to interact with Rab10, to have GAP activity towards Rab35, and to regulate trafficking of membrane proteins towards the plasma membrane [75]. Both, Rab10 and Rab35, are also expressed in platelets (Table 1). TBC1D15, the second highest RabGAP, serves as GAP for Rab7 and regulates mitochondrial fission and regeneration of lysosomes [76,77]. And RabGAP1, the third most highly expressed RabGAP, is involved in recycling of integrins to the plasma membrane [78] pointing to potential cross-talk between RabGAP1 and some of the ArfGAPs like ASAP1 and GIT1 mentioned above.
Taken together, Rabs have been assigned specific roles in the biogenesis and release of dense granules as well as in endocytosis. Further analysis of the localization and function of Rab proteins and their GAPs is required to better understand alpha granules and their possible heterogeneity, as well as membrane recycling pathways, and might help in deciphering the exact roles of the multiple membrane bound compartments found in platelets including the canalicular systems.
Ras/Rap proteins and Ras/RapGAPs in platelets
Ras/Rap proteins
The Ras family proteins Rap1A and Rap1B are the most highly expressed small G proteins in platelets. With estimated copy numbers of 120,000 and 150,000 each per platelet [18], they outnumber other G-proteins by about 10-fold (Table 1). The key role of Rap1 proteins appears to be regulation of integrin αIIbβ3 activation and platelet aggregation [79]. Rap1-GTP interacts with talin at the plasma membrane triggering a major conformational change in integrin αIIbβ3 enabling fibrinogen binding and cross-linking of platelets. Platelet activation correlates with increased levels of Rap1-GTP whereas cyclic nucleotide mediated platelet inhibition reduces Rap1-GTP [80,81]. Reduced Rap1 activation for example due to mutation of the gene for CalDAG-GEFI, a Rap1 GEF, causes bleeding in humans and mice [82–86] which has been confirmed in Rap1A and Rap1B knockout mouse models [87–89]. Additional roles for the Rap1A isoform beyond integrin regulation include cross-activation of the Rho family G protein Rac1 as well as a regulation of alpha granule release in response to GPVI activation through unknown mechanisms [88]. Other Ras/Rap1 proteins expressed at higher levels in platelets include Rap2A, B, and C, RalA and B (mentioned above), and the prototypic Ras proteins KRas and NRas. The functions of these G proteins in platelets are not clear. However, RRas2 (TC21), a Ras family member detected in platelets, regulates Rap1B and integrin αIIbβ3 downstream of the GPVI receptor [90].
Ras/RapGAPs
RASA3 (GAP1-IP4BP) has been confirmed as GAP of Rap1 in megakaryocytes and platelets (Figures 2C and 3A). Complete deletion of RASA3 in megakaryocytes is embryonically lethal due to bleeding and defects in the development of the lymphatic system [91]. Megakaryocytes expressing catalytically inactive or reduced levels of RASA3 exhibit altered adhesive properties, reduced motility and structural alterations [92–94]. As expected, integrin αIIbβ3 was found to be constitutively activated in these cells which correlated with increased Rap1-GTP levels [94]. Basal and ADP stimulated levels of Rap1GTP and active αIIbβ3 were also found to be elevated in mature mouse platelets expressing reduced levels of H794L mutant RASA3 [91]. In particular, RASA3 inhibition was suggested to play a specific role in platelet activation downstream of the P2Y12 Gi coupled ADP receptor as P2Y12 and PI3K inhibitors lost their in efficiency in RASA3 mutant platelets. Interestingly, RASA3 was also detected in a proteomics approach aimed at identifying phosphatidylinositol 3,4,5-tris-phosphate (PI(3,4,5)P3)-binding proteins in platelets [95]. ADP binding to the P2Y12 receptor increased the association of RASA3 with the plasma membrane resulting in reduced GAP activity. These studies led to the proposal that the P2Y12 receptor might regulate RASA3 through Gi mediated activation of PI3K leading to the production of PI(3,4,5)P3 followed by recruitment and inactivation of RASA3 at the plasma membrane (Figure 3A). In this way, RASA3 mediated turnover of Rap1-GTP could be prevented during ADP-induced platelet activation [95]. RASA3 has been described as dual specificity GAP that is able to regulate both, Rap1 and Ras [96] and PI(3,4,5)P3 binding could potentially affect the specificity of RASA3 towards Rap1 or Ras. The N-terminal C2 and C-terminal PH domains of RASA3 involved in membrane binding are required for GAP activity towards Rap1, whereas the isolated GAP domain might be sufficient for activity towards Ras [28,97]. Thus, one might speculate that membrane recruitment could shift the activity of RASA3 away from Rap1 towards Ras. However, RASA3 has also been shown to bind to both, PI(4,5)P2 as well as PI(3,4,5)P3 via its PH domain [98]. In this study, constitutive association of transfected RASA3 with the plasma membrane was observed and PI3K inhibitors had no effect on plasma membrane binding.
Examples of regulation and function of platelet GAPs
(A) RASA3 is a Rap1GAP which is inhibited by binding to phosphatidylinositol 3,4,5-tris-phosphate (PIP3) at the plasma membrane. PIP3 is generated by phosphatidylinositol 3-kinase (PI3K) downstream of the P2Y12 receptor for ADP. In this way ADP could lead to inhibition of RASA3 leading to elevated Rap1-GTP levels stimulating integrin activation and aggregation. (B) Rap1GAP2 is inhibited by binding of the adapter protein 14-3-3 to phosphorylated serine 9 of Rap1GAP2. cAMP- and cGMP-dependent protein kinase (PKA, PKG) mediated phosphorylation of serine 7 of Rap1GAP2 inhibits 14-3-3 binding resulting in increased GAP activity, formation of inactive Rap1-GDP and inhibition of aggregation. Rap1GAP2 also interacts with synaptotagmin-like protein 1 (Slp1) through a TKxT motif in the C-terminal part of Rap1GAP2 (T524-K525-X-T527). Slp1 inhibits dense granule release whereas binding of Rap1GAP2 to Slp1 reverses this inhibition. (C) The Rho-specific GAP RhoGAP6 interacts with δ-COP, a core component of the COPI vesicle coat, through a triple tryptophane motif ((W-W)3). Through this interaction GAP6 is able to regulate intracellular protein transport along the secretory pathway. The exact role of COPI in platelets is not known. At the same time RhoGAP6 is regulated by 14-3-3 binding to serine 37 which has a negative impact on the δ-COP interaction. (D) The Rac1-specific GAP RhoGAP17 is regulated by PKA and PKG mediated phosphorylation of serine 702 which interferes with binding to the actin-regulating Cdc42-interacting protein 4 (CIP4). CIP4 is also an effector of Rac1-GTP. Serine 702 phosphorylation stimulates the GAP function of RhoGAP17 presumably by interfering with an inhibitory function of CIP4. In addition, Src family kinases (SFK) can phosphorylate RhoGAP17 on tyrosines 124 and 314 leading to GAP activation. (E) RGS18 has GAP activity towards Gαq and Gαi. RGS18 is negatively regulated by 14-3-3 and spinophilin. During platelet inhibition PKA and PKG mediated phosphorylation of serine 216 on RGS18 triggers the dephosphorylation of the 14-3-3 binding phospho-serine 218 on RGS18 resulting in detachment of 14-3-3 and activation of RGS18 function. PKA mediated phosphorylation of serine 94 on spinophilin contributes to dissolution of the RGS18/14-3-3/spinophilin complex. Spinophilin also interacts with the serine/threonine phosphatase PP1 and the tyrosine phosphatase SHP-1. PP1 binding to spinophilin is negatively regulated by the SHP-1. During platelet activation SHP-1 detaches from spinophilin enabling PP1 recruitment, de-phosphorylation of PKA/PKG-dependent regulatory sites, enhanced 14-3-3 and spinophilin binding and reduced RGS activity. P refers to protein phosphorylations detected in platelets. Double arrowhead lines refer to protein/protein interactions.
(A) RASA3 is a Rap1GAP which is inhibited by binding to phosphatidylinositol 3,4,5-tris-phosphate (PIP3) at the plasma membrane. PIP3 is generated by phosphatidylinositol 3-kinase (PI3K) downstream of the P2Y12 receptor for ADP. In this way ADP could lead to inhibition of RASA3 leading to elevated Rap1-GTP levels stimulating integrin activation and aggregation. (B) Rap1GAP2 is inhibited by binding of the adapter protein 14-3-3 to phosphorylated serine 9 of Rap1GAP2. cAMP- and cGMP-dependent protein kinase (PKA, PKG) mediated phosphorylation of serine 7 of Rap1GAP2 inhibits 14-3-3 binding resulting in increased GAP activity, formation of inactive Rap1-GDP and inhibition of aggregation. Rap1GAP2 also interacts with synaptotagmin-like protein 1 (Slp1) through a TKxT motif in the C-terminal part of Rap1GAP2 (T524-K525-X-T527). Slp1 inhibits dense granule release whereas binding of Rap1GAP2 to Slp1 reverses this inhibition. (C) The Rho-specific GAP RhoGAP6 interacts with δ-COP, a core component of the COPI vesicle coat, through a triple tryptophane motif ((W-W)3). Through this interaction GAP6 is able to regulate intracellular protein transport along the secretory pathway. The exact role of COPI in platelets is not known. At the same time RhoGAP6 is regulated by 14-3-3 binding to serine 37 which has a negative impact on the δ-COP interaction. (D) The Rac1-specific GAP RhoGAP17 is regulated by PKA and PKG mediated phosphorylation of serine 702 which interferes with binding to the actin-regulating Cdc42-interacting protein 4 (CIP4). CIP4 is also an effector of Rac1-GTP. Serine 702 phosphorylation stimulates the GAP function of RhoGAP17 presumably by interfering with an inhibitory function of CIP4. In addition, Src family kinases (SFK) can phosphorylate RhoGAP17 on tyrosines 124 and 314 leading to GAP activation. (E) RGS18 has GAP activity towards Gαq and Gαi. RGS18 is negatively regulated by 14-3-3 and spinophilin. During platelet inhibition PKA and PKG mediated phosphorylation of serine 216 on RGS18 triggers the dephosphorylation of the 14-3-3 binding phospho-serine 218 on RGS18 resulting in detachment of 14-3-3 and activation of RGS18 function. PKA mediated phosphorylation of serine 94 on spinophilin contributes to dissolution of the RGS18/14-3-3/spinophilin complex. Spinophilin also interacts with the serine/threonine phosphatase PP1 and the tyrosine phosphatase SHP-1. PP1 binding to spinophilin is negatively regulated by the SHP-1. During platelet activation SHP-1 detaches from spinophilin enabling PP1 recruitment, de-phosphorylation of PKA/PKG-dependent regulatory sites, enhanced 14-3-3 and spinophilin binding and reduced RGS activity. P refers to protein phosphorylations detected in platelets. Double arrowhead lines refer to protein/protein interactions.
Recently, a PI3K independent pathway of Rap1 regulation downstream of P2Y12 has been proposed [99], which could potentially involve Rap1GAP2, another highly expressed GAP of Rap1 in human platelets (Figure 2C) [100]. Mouse platelets contain only low levels of Rap1GAP2, whereas humans express Rap1GAP2 at the highest level in platelets compared with other tissues [101] (Table 2). Rap1GAP2 is regulated by activating and inhibitory signalling pathways (Figure 3B). During platelet activation Rap1GAP2 is phosphorylated on serine 9 leading to binding of the adapter protein 14-3-3. 14-3-3 binding correlates with reduced Rap1GAP2 function [102], however, the kinase mediating serine 9 phosphorylation has not yet been identified. In contrast, inhibitory cyclic nucleotide pathways phosphorylate Rap1GAP2 on serine 7 leading to detachment of 14-3-3 (Figure 3B). Thus platelet activators appear to reduce Rap1GAP2 activity to retain high Rap1-GTP levels required for aggregation, whereas platelet inhibitors like the endothelium-dependent cyclic nucleotide pathways reverse this effect. In addition, a TKxT motif in the C-terminal part of Rap1GAP2 (amino acids 524-527) was shown to mediate binding of Rap1GAP2 to synaptotagmin-like protein 1 (Slp1), a protein regulating dense granule release (Figure 3B) [33]. Thus, Rap1GAP2 might link two important platelet responses, aggregation via Rap1 regulation as well as dense granule release via Slp1 binding.
The RasGAP domain of IQGAPs is lacking GAP activity (Figure 2C). Instead, IQGAPs are acting primarily as scaffolds linking receptors to intracellular signalling networks [103]. IQGAPs bind to Rho family G proteins like Racs, RhoG and Cdc42 stabilizing their active GTP-bound forms as well as to Arf6, Rap1 and GDP-bound Rab27a [104,105]. Studies of IQGAP1 deficient platelets indicate that IQGAP1 inhibits intracellular calcium elevation in response to thrombin exposure. IQGAP1 also inhibits alpha granule release and attenuates the development of a procoagulant plasma membrane [106]. IQGAP2, a particularly highly expressed protein in platelets, interacts with the actin-binding protein Arp3 in thrombin treated platelets [107].
In summary, Rap1 has attracted a lot of attention as highly expressed G protein and integrin αIIbβ3 regulator leading to the discovery of RASA3 and Rap1GAP2 as major RapGAPs. However, many open questions remain regarding the regulation of these GAPs and further studies are required to identify the specific roles of the other Ras proteins like Ral, Rap2 or NRas.
Rho proteins and RhoGAPs in platelets
Rho proteins
Rac1, Rac2, Cdc42, RhoA, RhoC, and RhoG are the most highly expressed Rho family proteins in platelets, and RhoF is strikingly platelet specific [18] (Table 1). These G proteins appear to have multiple roles in controlling platelet signalling, cytoskeletal reorganization, granule release, aggregation, and clot retraction [13]. Rac1 is essential for signalling downstream of GPVI and CLEC2 receptors activation [108], and both, Rac1 and Rac2, control lamellipodia formation and platelet spreading and are required for the stabilization of platelet aggregates [109]. Cytoplasmic FMR1 Interacting Protein 1 (CYFIP1) has been identified as Rac1 effector mediating lamellipodia formation in platelets [110]. Cdc42 has inhibitory roles in alpha and dense granule release correlating with increased aggregation of knockout platelets [111] although opposing data have been obtained in a different Cdc42 knockout model [112]. RhoA plays a role in shape change, alpha and dense granule release, and clot retraction downstream of Gα13-coupled thrombin and TXA2 receptors of mature platelets [113]. Target proteins mediating the effects of RhoA-GTP include the Rho-associated protein kinases 1 and 2 (ROCK1/2); however, deletion of these proteins only partly copied the RhoA knockout phenotype [114,115]. For example, loss of ROCK2 lead to reduced Thr853 phosphorylation of myosin phosphatase target 1 and to reduced platelet adhesion [115]. Deletion of other RhoA effectors like Protein diaphanous homolog 1 (mDia1) and FH1/FH2 domain-containing protein 1 (Fhod1) did not result in platelet defects suggesting multiple compensatory Rho effector pathways [116]. A normal phenotype was also described for RhoF mouse knockout platelets again indicating compensatory mechanisms [117]; however, RhoF is expressed only at very low levels in mouse compared to human platelets [101]. On the other hand, RhoG is present at high levels in mouse platelets and RhoG knockout leads to defect in alpha and dense granule release specifically in response to GPVI activation [118,119]. Platelet activation is often associated with the formation of the GTP-bound versions of Rho family proteins, whereas endothelial platelet inhibitors tend to keep these proteins inactive [10].
RhoGAPs
Thirty-five RhoGAPs have been detected in platelets [18] (Table 2 and Figure 2D), and the G protein/GAP ratio appears to be particularly low for this group of proteins suggesting that Rho proteins might be regulated by more than one GAP per G protein. The specificities of all RhoGAPs towards RhoA, Rac1, and Cdc42 have recently been studied by Müller et al. using a cell-based assay and these data are included in Table 2 [26]. However, as only RhoA, Rac1, and Cdc42 were tested, activities towards other Rho family G proteins remain unknown. Using affinity purification of overexpressed GAPs and GEFs coupled to mass spectrometry Müller et al. also provided evidence for interactions among GAP family members as well as between GAPs and GEFs. In addition, information on spatial distribution was obtained, indicating that many RhoGAPs associate with the actin cytoskeleton, at least in transfected cells.
RhoGAP1 (ARHGAP1) and 18 (ARHGAP18) are the most highly expressed RhoGAPs in platelets (Table 2 and Figure 2D). RhoGAP1 contains a CRAL-TRIO domain (BNIP-2 and Cdc42GAP Homology domain (BCH) subclass) that can bind RhoA with its prenylation moiety resulting in GAP activation [120]. Interestingly, an amino acid change at the start of the catalytic GAP domain of RhoGAP1 (L263F) was identified as putative causal variant in a patient with a primary platelet secretion defect [121]. RhoGAP18 is known to contribute to localized RhoA regulation in distinct cellular compartments [122–124]; however, platelet functions of RhoGAP18 have not yet been described. RhoGAP6 (ARHGAP6), another highly expressed RhoGAP, has been shown to bind to δ-COP, a component of the COPI coat, in platelets (Figure 3C). COPI is usually thought to be involved in vesicle transport between the Golgi and the ER and COPI function requires Arf1 and ArfGAP1-3 [37,125]. But only ArfGAP2 appears to be expressed in platelets at detectable levels (Figure 2A). δ-COP binding involves three tryptophan motifs (LIG_deltaCOP1_diTrp_1 according to the ELM resource [126]) in the C-terminal part of RhoGAP6 (amino acids 947-958). This tryptophan motif has previously been characterized only in ArfGAP1 [27]. The δ-COP interaction is inhibited by 14-3-3 binding to phosphorylated serine 37, and RhoGAP6 stimulates vesicle transport and protein secretion in transfected cells which depends on COPI binding but not on GAP activity (Figure 3C). Taken together RhoGAP6 might link RhoA regulation with protein transport processes in platelets.
Other RhoGAPs that have been studied in platelets include oligophrenin 1 (OPHN1), RhoGAP21 (ARHGAP21), Oculocerebrorenal syndrome of Lowe 1 and Inositol 5-phosphatase (OCRL), RhoGAP17 (ARHGAP17), and Myo9B (Table 2). Oligophrenin 1 inhibits lamellipodia formation and adhesion, alpha and dense granule release and thrombus formation under low shear rates, whereas platelet aggregation does not appear to be regulated according to a mouse study [127]. In this study evidence was provided for GAP activity of oligophrenin towards RhoA, Rac1, as well as Cdc42; however, other Rho family proteins have not been tested. Similarly, investigation of RhoGAP21 in mouse platelets revealed a role for this GAP in attenuating alpha granule release, platelet aggregation, and thrombus formation which was linked to inactivation of RhoA and Cdc42 [128]. In the GAP domain of OCRL, the catalytic arginine is replaced by a glutamine and no GAP activity can be detected, nevertheless OCLR has been shown to bind to Rac1-GTP [129]. OCRL also contains a central inositol 5-phosphatase phosphatase domain processing PIP2 into phosphatidylinositol 4-phosphate (PI4P). Defects in OCRL cause a rare inherited disorder which is associated with a bleeding phenotype. A recent study of platelets from these patients showed reduced adhesion to collagen surfaces, enhanced filopodia and reduced lamellipodia formation on fibrinogen, which correlated with reduced Rac1-GTP and enhanced RhoA-GTP levels but reduced myosin light chain phosphorylation, clot retraction, and thrombus formation [130]. Similar findings were obtained by pharmacological inhibition of OCRL’s phosphatase activity including enhanced filopodia and actin nodule formation, reduced spreading and reduced myosin light chain phosphorylation [131]. Taken together, OCRL might regulate actin dynamics by Rac1 binding as well as by lowering PIP2 levels in the plasma membrane with follow-on effects on PIP2-binding proteins. RhoGAP17 is another GAP with activities towards Rac1 and RhoA found in platelets. RhoGAP17 has been identified as a target of various signalling pathways. PKA and PKG phosphorylate RhoGAP17 on serine 702 leading to detachment of the Cdc42-interacting protein 4 (CIP4) from RhoGAP17 [51]. CIP4 is an effector of Rac1-GTP and Cdc42-GTP involved in proplatelet formation [132,133]. Loss of CIP4 binding to RhoGAP17 correlates with enhanced inhibition of cell migration possibly through increased GAP activity leading to reduced Rac1-GTP levels [51]. Thus, PKA/G-mediated detachment of CIP4 might activate RhoGAP17 and terminate Rac1 signalling towards CIP4 (Figure 3D). Interestingly, RhoGAP17 can also be activated by tyrosine phosphorylation (Y124 and Y314) through Src family kinases during platelet activation [134] suggesting that both, platelet activation and inhibition pathways, might lead to RhoGAP17 activation. These phosphorylations could potentially have additive effects on GAP activity levels; however, cross-regulation of RhoGAP17 phosphorylation sites has not been investigated. PKA/G also phosphorylate the RhoGAP and myosin motor protein Myo9B which moves along actin filaments and has been implied in lamellipodia formation in various cells [135,136]. Phosphorylation of Myo9B on serine 1354 enhanced the GAP activity of Myo9B towards RhoA [137] possibly contributing to a modulation of local RhoA activity around actin filaments [138].
In summary, Rho family G proteins are appearing to be regulated by a complex set of RhoGAPs integrating numerous additional domains and functions. Specific combinations of Rhos and GAPs/GEFs might be involved in controlling distinct cellular processes beyond the classical roles of RhoA, Cdc42, and Rac1 in stress fibre, filopodia and lamellipodia formation.
Heterotrimeric G proteins and RGSs in platelets
Heterotrimeric G proteins
Heterotrimeric (αβγ) G proteins transduce signals from ligand binding GPCRs to downstream targets at the plasma membrane [139]. Platelets express all 3 Gαi isoforms (Gαi1-3) and Gαq at highest levels [18] (Table 1). Gα13, Gαz (another Gi family member), and Gαs are less prominent. The most highly expressed corresponding beta and gamma subunits are β1, β4, β2, β5, and γ11, γ5, γ10 (in descending order of expression level). Activated GPCRs act as GEFs and GTP-bound α subunits dissociate from βγ subunits to interact with downstream effector targets. Activation of the P2Y12 receptor by ADP induces Gαi2- and Gαz-GTP formation which inhibits AC leading to reduced cAMP synthesis and facilitating platelet activation and aggregation [140–142]. Overexpression of a GAP resistant Gαi2 mutant led to reduced cAMP levels, and increased aggregation in response to GPCR agonists confirming the inhibitory role of Gαi in platelets [143]. The P2Y1 ADP receptor as well as thrombin and TXA2 receptors activate Gαq (Figure 1) [144,145] leading to stimulation of PLCβ followed by the release of Ca2+ ions from intracellular stores and platelet activation [146,147]. The PAR1 thrombin receptor and the TP receptor are also linked to Gα13 which activates RhoA [148] probably through direct interaction with a RhoGEF like RhoGEF1 (p115RhoGEF) [149]. Information on the role of Gs, linked to the prostacyclin receptor (IP receptor), has been obtained using Gs-deficient platelets from patients with pseudohypoparathyroidism types Ia [150,151]. In these platelets reduced Gs expression results in attenuated responses to IP receptor activation including reduced AC stimulation, followed by reduced cAMP/PKA activation and reduced inhibition of platelet aggregation. Gs overexpression has been associated with an increased bleeding risk [152]. Only few studies have addressed roles of Gβγ subunits of heterotrimeric G proteins in platelets. Gβ1 was shown to interact with the catalytic subunit of protein phosphatase 1 and with PLCβ3 [153], and a small molecule inhibitor of Gβγ inhibits platelet aggregation and granule release [154]. Gβγ subunits contribute to PI3K activation downstream of Gi-coupled receptors [155,156] and to AC activation downstream of Gs-coupled receptors [157]. Multiple combinations of α, β and γ subunits are expected to exist leading to many possible functions [158].
RGSs
GAP roles are provided by RGS proteins some of which have been studied in platelets (Table 2 and Figure 2E). Only 20 (RGS1-20) of the 35 proteins containing RGS or RGS-homology (RH) domains are considered canonical RGS proteins based on structural and functional (GAP activity) similarities [25,159]. RGS18 is the most highly expressed and platelet-specific RGS (Table 2) and exhibits selectivity towards Gαq and Gαi (Figure 3E) [159]. Knockout mouse studies revealed a role for RGS18 in megakaryocyte development and platelet production [160,161]. Mature platelets lacking RGS18 are hyperactive exhibiting increased basal as well as thrombin and TXA2 stimulated alpha and dense granule release, integrin activation and aggregation. RGS18 interacts with Gαq and Gαi in human platelets, as well as with the adapter proteins spinophilin (neurabin-2, PPP1R9B), and 14-3-3 [162–165]. A regulatory cycle was proposed for RGS18 leading to RGS inhibition during platelet activation (associated with 14-3-3 binding to phosphorylated serines 49 and 218 and loss of spinophilin binding) and activation during platelet inhibition (associated with cyclic nucleotide mediated loss of 14-3-3 due to S216 phosphorylation) (Figure 3E) [10] PKA-mediated phosphorylation of serine 94 on spinophilin contributes to dissolution of the RGS18/14-3-3/spinophilin complex [165]. Recruitment of the serine/threonine phosphatase PP1 to spinophilin is regulated by the tyrosine phosphatase SHP-1 [166] and might play a role in dephosphorylating the regulatory sites on RGS18 [163]. A recent study has revealed further complexity of heterotrimeric G protein regulation as Gαq was shown to interact with either RGS10 or RGS18 or with PLCβ [167]. RGS10 is another via Gαq/Gαi-specific abundantly expressed RGS in platelets which has been shown to negatively regulate release of calcium ions from intracellular stores, alpha granule release, aggregation, and thrombus formation downstream of thrombin, TXA2, and ADP receptors in mice and to be regulated by spinophilin and 14-3-3 binding similar to RGS18 [168–170]. RGS16 has been described as a platelet regulator [171]; however, proteomics have provided little evidence for RGS16 expression in platelets (Table 2).
In addition to the classical RGS proteins a few related proteins containing RH domains are expressed in platelets. The G protein–coupled receptor kinases (GRK) appear to have some GAP activity towards Gα, but many of these have numerous ways of regulating receptor functions [25]. GRK2 has been shown to specifically terminate P2Y12 and P2Y1 coupled Gq and possibly Gi signalling in platelets [172]. Of note, in this study, GRK2 was seen to bind to Gβq but not the Gαq or Gαi subunits. In contrast, GRK6 regulates PAR1 receptors. GRK6 has been shown to bind to the PAR1 receptor in thrombin treated platelets which was linked to increased receptor phosphorylation but GRK6 might also regulate PAR4, at least in mice [173]. GRK5 also regulates PAR1 and PAR4 mediated platelet activation in a mouse knockout model where lack of GRK5 led to increased thrombin-induced aggregation. The common intronic GRK5 variant rs1088643 linked to reduced transcript numbers is associated with increased thrombin-induced platelet reactivity in humans [174,175]. As GRK5 was shown to bind to the PAR1 receptor GRK5 might de-activate PAR signalling through phosphorylation and β-arrestin mediated receptor internalization.
Taken together, RGS proteins are emerging as essential regulators of GPCR function in platelets. RGS18 has been shown to regulate transmission of Gq and Gi mediated thrombin and TXA2 receptor signalling in many studies. Further analyses are required to clarify GPCR specificities and regulatory complexes forming around RGSs.
Platelet G proteins and GAPs as therapeutic targets
Platelets play a major role in arterial and venous thrombotic disease. However, current antiplatelet therapies are targeting only a restricted set of molecules like the P2Y12 ADP receptor, cyclooxygenase-1 required for TXA2 synthesis, or the cAMP degrading phosphodiesterase type 3 [176,177]. Furthermore, the major drawback of any anti-haemostatic approach, an increased bleeding risk, has not been overcome. G proteins and their regulators might represent a new class of targets for antiplatelet therapy.
The approval of sotorasib in 2021 as first G protein inhibitor targeting K-Ras in cancer patients indicates the feasibility of targeting these types of proteins [178]. Both, GTP- and GDP-bound states of K-Ras can be targeted by small molecules, and drugs are being developed that interfere with effector and/or GEF and GAP binding sites. Highly expressed and platelet specific G proteins like Rap1B or Rab27B could be promising targets for the development of similar drugs for antiplatelet therapy. In principal, it should also be possible to identify small molecules that stimulate GAP functions, for example, by interfering with negative regulatory regions. The platelet specific Rap1GAP RASA3 could potentially be activated by interference with the regulatory PH domain of RASA3 whereas blocking 14-3-3 interaction would activate Rap1GAP2 (Figure 3A,B). Similarly, impeding interactions between RGS18 and spinophilin or 14-3-3 would be expected to lead to platelet inhibition (Figure 3E) and the development of 14-3-3 interaction inhibitors is an area of active research [179]. As GAPs are regulated by complex phosphorylation patters targeting protein kinases might be another way of influencing platelet function. Kinase inhibitors have become a major area of drug development since the discovery and clinical implementation of imatinib as highly effective inhibitor of the tyrosine kinase BCR-ABL in cancer therapy. Around 20 kinase inhibitors are already in clinical use and 20% of the entire kinome of 560 kinases is being targeted in clinical trials [180]. Studies are also underway addressing the potential of PI3K and tyrosine kinase inhibitors for platelet inhibition [176]. Kinases appear to have a major role in regulating GAPs (Table 2), however, further studies are required to identify specific kinase/GAP interactions with therapeutic potential in platelets.
Conclusion
Platelets contain a unique set of G proteins and G protein regulators which are likely to be adapted to the specialised functions of this cell type. So far only few platelet GAPs have been studied in detail. The multi-domain structure of GAPs might contribute to local control of G proteins and facilitate complex connections between G proteins of different families required for the regulation of receptor function, membrane and vesicle traffic and the cytoskeleton. Platelets represent a unique cell model for signalling research as gene expression changes do not need to be considered. In addition to platelet focused omics studies careful validation of the specificities, control mechanisms, and localization of individual proteins will be essential to build a solid foundation for improved understanding of platelet function, meaningful modelling of signalling networks, and for the identification of new therapeutic targets in vascular disease.
Competing Interests
The authors declare that there are no competing interests associated with the manuscript.
Funding
The authors would like to thank UCD School of Medicine and UCD Conway Institute for funding and continued support, and three anonymous reviewers for their helpful comments and suggestions.
CRediT Author Contribution
Lorna O'Donoghue: Conceptualization, Formal analysis, Visualization, Writing—original draft, Writing—review & editing. Albert Smolenski: Conceptualization, Formal analysis, Visualization, Writing—original draft, Writing—review & editing.
Abbreviations
- AP
adapter protein
- cGMP
cyclic guanosine monophosphate
- CIP4
Cdc42-interacting protein 4
- COPI
coat protein complex I
- CYFIP1
cytoplasmic FMR1 interacting protein 1
- DAG
1,2-diacylglycerol
- FcRγ
Fc gamma receptor
- Fhod1
FH1/FH2 domain-containing protein 1
- GAP
GTPase-activating protein
- GEF
guanine nucleotide exchange factor
- HPS
Hermansky–Pudlak syndrome
- IP3
inositol 1,4,5-triphosphate
- OCRL
Oculocerebrorenal syndrome of Lowe 1 and Inositol 5-phosphatase
- PKG
protein kinase G
- ROCK1/2
Rho-associated protein kinases 1 and 2
- Slp1
synaptotagmin-like protein 1
- SNARE
soluble N-ethylmaleimide-sensitive factor attachment protein receptor