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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:618.)
© 2001 American Heart Association, Inc.

Thrombosis

Platelet Adhesion Enhances the Glycoprotein VI–Dependent Procoagulant Response

Involvement of p38 MAP Kinase and Calpain

Pia Siljander; Richard W. Farndale; Marion A. H. Feijge; Paul Comfurius; Snjezana Kos; Edouard M. Bevers; Johan W. M. Heemskerk

From the Wihuri Research Institute (P.S.), and the Electron Microscopy Unit (P.S.), Institute of Biotechnology, University of Helsinki, Helsinki, Finland; the Departments of Biochemistry and Human Biology (P.S., M.A.H.F., P.C., S.K., E.M.B., J.W.M.H.), University of Maastricht, Maastricht, The Netherlands; and the Department of Biochemistry (P.S., R.W.F.), University of Cambridge, United Kingdom.

Correspondence to Pia Siljander, Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, UK. E-mail prms2{at}mole.bio.cam.ac.uk

Abstract—In the final stages of activation, platelets express coagulation-promoting activity by 2 simultaneous processes: exposure of aminophospholipids, eg, phosphatidylserine (PS), at the platelet surface, and formation of membrane blebs, which may be shed as microvesicles. Contact with collagen triggers both processes via platelet glycoprotein VI (GPVI). Here, we studied the capacity of 2 GPVI ligands, collagen-related peptide (CRP) and the snake venom protein convulxin (CVX), to elicit the procoagulant platelet response. In platelets in suspension, either ligand induced full aggregation and high Ca²⁺ signals but little microvesiculation or PS exposure. However, most of the platelets adhering to immobilized CRP or CVX had exposed PS and formed membrane blebs after a prolonged increase in cytosolic [Ca²⁺]_i. Platelets adhering to fibrinogen responded similarly but only when exposed to soluble CRP or CVX. By scanning electron microscopic analysis, the bleb-forming platelets were detected as either round, spongelike structures with associated microparticles or as arrays of vesicular cell fragments. The phosphorylation of p38 mitogen-activated protein kinase (MAPK) elicited by CRP and CVX was enhanced in fibrinogen-adherent platelets compared with that in platelets in suspension. The p38 inhibitor SB203580 and the calpain protease inhibitor calpeptin reduced only the procoagulant bleb formation, having no effect on PS exposure. Inhibition of p38 also downregulated calpain activity. We conclude that the procoagulant response evoked by GPVI stimulation is potentiated by platelet adhesion. The sequential activation of p38 MAPK and calpain appears to regulate procoagulant membrane blebbing but not PS exposure.

Key Words: platelets • procoagulant activity • adhesion • glycoprotein VI • signal transduction

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