MICROVESICLE RELEASE IS ASSOCIATED WITH EXTENSIVE PROTEIN-TYROSINE DEPHOSPHORYLATION IN PLATELETS STIMULATED BY A23187 OR A MIXTURE OF THROMBIN AND COLLAGEN

Phosphatidylserine exposure and microvesicle release give rise to proc oagulant activity during platelet activation. We have previously shown that whereas the Ca2+ ionophore A23187 and 2,5-di-(t-butyl)-1,4-benzo hydroquinone, a Ca2+-ATPase inhibitor, induce phosphatidylserine expos ure, only the former triggers microvesicle release. We now report that microvesicle formation with ionophore A23187 is specifically associat ed with mu-calpain activation, increased protein tyrosine phosphatase (PTP) activity and decreased tyrosine phosphorylation. The degree to w hich calpain and individual PTPs were activated in response to A23187 depended on the extent of bivalent cation chelation in the external me dium. EGTA (2 mM) blocked or severely retarded their activation, and a ddition of extracellular Ca2+ in excess (2 mM) resulted in virtually i mmediate tyrosine dephosphorylation. Dephosphorylation was correlated with an increase in total PTP activity in platelet lysates. In platele ts stimulated by a combination of thrombin and collagen, only the subp opulation undergoing microvesicle release and isolated by their bindin g to annexin-V-coated magnetic beads exhibited protein tyrosine dephos phorylation. Detection of PTP activity in an 'in-gel' assay showed the Ca2+-dependent appearance of active low-molecular-mass bands at 38, 3 6 and 27 kDa. Individual PTPs varied in their protease sensitivity to changes in intracellular Ca2+ levels. For example, PTP1B was a more se nsitive substrate than SH2-domain-containing tyrosine phosphatase-1 fo r mu-calpain cleavage. Incubation of platelets with the PTP inhibitors , phenylarsine oxide and benzylphosphonic acid acetoxymethyl ester, le d to increased tyrosine phosphorylation and the surface expression of aminophospholipids but little microvesicle formation. Furthermore, mic rovesicle release in response to ionophore A23187 was inhibited. We co nclude that platelet microvesicle formation is associated with extensi ve protein tyrosine dephosphorylation.