We have used a general quenched-flow approach to study platelet function as early as 0.3 s after stimulation with three types of human thrombin: alpha-thrombin, gamma-thrombin, which is proteolytically active but does not bind to the high-affinity sites, and di-isopropyl fluorophosphate-derivatized (DIP)-alpha-thrombin, an active site-inhibited analogue that does bind to the high-affinity site. Large doses of gamma-thrombin evoked moderate aggregation and serotonin release, but minimal phosphorylation of the 20 and 47 kDa proteins. The initial (1.5-3.0 s) increase in cystolic free calcium concentration ([Ca2+]i) indicated by Indo-1 was also diminished, but by 5 s was nearly as high (1.0 microM) as with alpha-thrombin. A large dose of DIP-alpha-thrombin, on the other hand, induced minimal aggregation, serotonin secretion and [Ca2+]i response within 6 s. There was, however, a transient dephosphorylation of the 20 kDa protein. When combined, gamma- and DIP-alpha-thrombin were approximately additive in their ability to induce aggregation and serotonin secretion, but strongly synergistic in phosphorylating the 20 and 47 kDa proteins. The [Ca2+]i increase was not, however, enhanced over that induced by gamma-thrombin alone. These results demonstrate that phosphorylation of either the 20 or 47 kDa proteins is not correlated with [Ca2+]i dynamics and is neither required nor directly involved in platelet aggregation and secretion induced by thrombin. The high-affinity binding activity of thrombin is not necessary for rapid platelet Ca2+ influx, aggregation and serotonin release within the first critical seconds of activation.
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September 1989
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Research Article|
September 01 1989
Effects of separate proteolytic and high-affinity binding activities of human thrombin on rapid platelet activation. A quenched-flow study
G D Jones
;
G D Jones
1Department of Biochemistry, University of Virginia, Charlottesville, VA 22908, U.S.A.
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D J Carty
;
D J Carty
1Department of Biochemistry, University of Virginia, Charlottesville, VA 22908, U.S.A.
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D L Freas
;
D L Freas
1Department of Biochemistry, University of Virginia, Charlottesville, VA 22908, U.S.A.
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J T Spears
;
J T Spears
1Department of Biochemistry, University of Virginia, Charlottesville, VA 22908, U.S.A.
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A R L Gear
A R L Gear
1Department of Biochemistry, University of Virginia, Charlottesville, VA 22908, U.S.A.
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Biochem J (1989) 262 (2): 611–616.
Citation
G D Jones, D J Carty, D L Freas, J T Spears, A R L Gear; Effects of separate proteolytic and high-affinity binding activities of human thrombin on rapid platelet activation. A quenched-flow study. Biochem J 1 September 1989; 262 (2): 611–616. doi: https://doi.org/10.1042/bj2620611
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