Phosphatidylserine exposure and microvesicle release give rise to procoagulant activity during platelet activation. We have previously shown that whereas the Ca2+ ionophore A23187 and 2,5-di-(t-butyl)-1,4-benzohydroquinone, a Ca2+-ATPase inhibitor, induce phosphatidylserine exposure, only the former triggers microvesicle release. We now report that microvesicle formation with ionophore A23187 is specifically associated with µ-calpain activation, increased protein tyrosine phosphatase (PTP) activity and decreased tyrosine phosphorylation. The degree to which calpain and individual PTPs were activated in response to A23187 depended on the extent of bivalent cation chelation in the external medium. EGTA (2 mM) blocked or severely retarded their activation, and addition of extracellular Ca2+ in excess (2 mM) resulted in virtually immediate tyrosine dephosphorylation. Dephosphorylation was correlated with an increase in total PTP activity in platelet lysates. In platelets stimulated by a combination of thrombin and collagen, only the subpopulation undergoing microvesicle release and isolated by their binding to annexin-V-coated magnetic beads exhibited protein tyrosine dephosphorylation. Detection of PTP activity in an ‘in-gel ’ assay showed the Ca2+-dependent appearance of active low-molecular-mass bands at 38, 36 and 27 kDa. Individual PTPs varied in their protease sensitivity to changes in intracellular Ca2+ levels. For example, PTP1B was a more sensitive substrate than SH2-domain-containing tyrosine phosphatase-1 for µ-calpain cleavage. Incubation of platelets with the PTP inhibitors, phenylarsine oxide and benzylphosphonic acid acetoxymethyl ester, led to increased tyrosine phosphorylation and the surface expression of aminophospholipids but little microvesicle formation. Furthermore, microvesicle release in response to ionophore A23187 was inhibited. We conclude that platelet microvesicle formation is associated with extensive protein tyrosine dephosphorylation.
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Research Article|
August 01 1998
Microvesicle release is associated with extensive protein tyrosine dephosphorylation in platelets stimulated by A23187 or a mixture of thrombin and collagen
Jean-Max PASQUET;
Jean-Max PASQUET
1UMR 5533 CNRS, Hôpital Cardiologique, 33604 Pessac, France
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Jeanne DACHARY-PRIGENT;
Jeanne DACHARY-PRIGENT
1UMR 5533 CNRS, Hôpital Cardiologique, 33604 Pessac, France
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Alan T. NURDEN
Alan T. NURDEN
1
1UMR 5533 CNRS, Hôpital Cardiologique, 33604 Pessac, France
1To whom correspondence should be addressed (e-mail: Alan.Nurden@cnrshl.u-bordeaux.fr).
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Publisher: Portland Press Ltd
Received:
January 05 1998
Revision Received:
April 03 1998
Accepted:
April 27 1998
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1998
1998
Biochem J (1998) 333 (3): 591–599.
Article history
Received:
January 05 1998
Revision Received:
April 03 1998
Accepted:
April 27 1998
Citation
Jean-Max PASQUET, Jeanne DACHARY-PRIGENT, Alan T. NURDEN; Microvesicle release is associated with extensive protein tyrosine dephosphorylation in platelets stimulated by A23187 or a mixture of thrombin and collagen. Biochem J 1 August 1998; 333 (3): 591–599. doi: https://doi.org/10.1042/bj3330591
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