We investigated the binding characteristics of angiotensin receptors and used this assay to determine the predominant enzyme capable of converting angiotensin I in the human left ventricle. In homogenates of human left ventricle, 125 I-[Sar 1 ,Ile 8 ]angiotensin II bound with sub-nanomolar affinity, with a corresponding K D of 0.42±0.09nM, a B max of 11.2±2.3fmolċmg -1 protein and a Hill slope of 1.04±0.04. The rank order of inhibitory potency of competing ligands for the 125 I-[Sar 1 ,Ile 8 ]angiotensin II binding site was CGP42112 > angiotensin II⩾ angiotensin III = angiotensin I > losartan. The angiotensin type II (AT 2 ) receptor predominated in the human left ventricle over the angiotensin type I (AT 1 ) receptor, with an approximate AT 1 /AT 2 receptor ratio of 35:65. No specific 125 I-angiotensin IV binding sites could be detected in the human left ventricle. Using competitive radioligand binding assays, we were able to demonstrate that the chymase/cathepsin G enzyme inhibitor chymostatin was more potent than the angiotensin-converting enzyme (ACE) inhibitor captopril at inhibiting the conversion of angiotensin I in the human left ventricle. Aprotonin (an inhibitor of cathepsin G but of not chymase) had no effect on angiotensin I conversion, suggesting that the majority of the conversion was mediated by chymase. Thus, although the current therapies used for the renin-angiotensin system have focused on ACE inhibitors and AT 1 receptor antagonists, the left ventricle of the human heart expresses mainly AT 2 receptors and the tissue-specific conversion of angiotensin I occurs predominantly via chymase rather than ACE.

1. Close contact between platelets and neutrophils modulates their cellular interactions in thrombotic and inflammatory states, with stimulation of P-selectin expression on platelets by agonists such as thrombin and neutrophil-derived cathepsin G being critical in mediating platelet—neutrophil adhesion. This study compared the effects of thrombin and cathepsin G on platelet P-selectin expression and on P-selectin-mediated platelet—neutrophil adhesion. 2. Washed platelets and platelet—neutrophil mixed cell suspensions (platelet/neutrophil ratio, 10:1) were incubated with either the supernatant of activated neutrophils, purified cathepsin G or thrombin. Platelet P-selectin expression and platelet adhesion to neutrophils was quantified by flow fluorocytometric analysis. 3. The supernatant from activated neutrophils stimulated platelet P-selectin expression comparable to that produced by purified cathepsin G or thrombin. P-selectin expression induced by both activated neutrophil supernatant and purified cathepsin G was completely inhibited by α 1 -antichymotrypsin, a specific inhibitor of cathepsin G. Unlike thrombin, which induced maximum platelet P-selectin expression by 10 min, sustained to 120 min, cathepsin G induced an initial large increase in platelet P-selectin expression, followed by a progressive reduction over 30–60 min to baseline levels. 4. Co-incubation of neutrophils with thrombin-stimulated platelets resulted in a significant increase in P-selectin-mediated platelet—neutrophil adhesion, which was completely inhibited by preincubation of neutrophils with anti-sialyl Lewis x monoclonal antibody. Thrombin produced maximum platelet—neutrophil adhesion by 10 min which remained stable over 120 min. In contrast, cathepsin G-stimulated platelets did not adhere to neutrophils over 120 min of co-incubation. Addition of cathepsin G to thrombin-stimulated platelets caused a progressive reduction over 30–60 min to baseline levels of platelet—neutrophil adhesion. 5. Neutrophil-derived cathepsin G is a potent platelet activator, but unlike thrombin it causes a time-dependent loss of platelet P-selectin expression and inhibits P-selectin-mediated platelet—neutrophil adhesion. Therefore, cathepsin G may modulate thrombin-mediated platelet—neutrophil adhesive interactions in inflammation and thrombosis.