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Abstract
Author(s) -
A. Bouziane,
F. Picard,
S. Weber,
M. Fontenay
Publication year - 2003
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/j.1538-7836.2003.tb00066_3.x
Subject(s) - medicine
Platelet microparticles (PMP) are present in the circulation of patients who have an increased risk of thromboembolic diseases. Simultaneously to phospholipid flip-flop that supports the procoagulant activity, blebs are formed at the plasma membrane. We have previously demonstrated that early platelet shape change is coordinated by a Cdc42/Rac1/p21-activated kinase (PAK)-dependent actin reorganization (Blood, 2002; 100: 4462-9). Whether this pathway is required for PMP shedding is not known and was investigated in the present work. Platelet suspensions were stimulated either with a mixture of 10 μM TRAP and 10 μg mL-1 collagen (TC) or with 1 μM A23187 in the presence of 70 μM calpain inhibitor, calpeptin, and 1 mM extracellular calcium and the percentages of PMP represented 18 ± 5 and 54 ± 16% of CD41a-positive elements, respectively. Cdc42-GTP and Rac1-GTP trapped on PAK binding domain-GST beads were fully activated by TC although this activation was transient in response to A23187. TC, but not A23187, stimulated the in vitro kinase activity of PAK and the phosphorylation of p38 MAPK. Toxin B (1 μg mL-1) from C. difficile that prevents TC-induced activation of Cdc42 and Rac1, inhibited p38 phosphorylation and decreased the amount of polymerized actin. However, toxin B did not decrease the shedding of PMP. Western blot analysis of PMP, isolated by centrifugation at 100 000 x g, showed that they expressed GpIIbIIIa which confirmed that PMP were generated after blebbing of the plasma membrane. However, PMP did not contain actin, although they expressed two actinbinding proteins, WASP and cortactin. Our data show that massive PMP shedding after A23187 stimulation does not correlate with the activation of the Cdc42/Rac1/PAK pathway and that inhibition of Cdc42 and Rac1 does not influence PMP generation. Furthermore, actin does not colocalize with WASP and cortactin that could represent a cytosolic pool of inactive proteins. These results suggest that PMP shedding is independent of actin reorganization downstream of Cdc42 and Rac1.