Binding of polymerizing fibrin to integrin alpha IIb beta3 on chymotrypsin-treated rabbit platelets decreases phosphatidylinositol 4,5-bisphosphate and increases cytoskeletal actin

Abstract

Incubation of polymerizing fibrin with washed, chymotrypsin-treated (CT) rabbit platelets resulted in the formation of platelet-fibrin clots, a decrease of 27.4% (P < 0.001, n = 20) in the amount of phosphatidylinositol 4,5-bisphosphate (PIP) and an increase of 37.2% (P < 0.001, n = 20) in phosphatidylinositol 4-phosphate (PIP2), apparently because of a shift in the equilibria between PIP2 and PIP toward PIP. In contrast, incubation of fibrinogen with CT-platelets resulted in agglutination of platelets, but no changes in the phosphoinositides. Preincubation of CT-platelets with staurosporine (1 mu M) to inhibit protein phosphorylation, okadaic acid (1 mu M) to inhibit protein phosphatases, genistein (100 mu M) to inhibit protein tyrosine phosphorylation, PGE (10 mu M) to increase cAMP and cause Ca2+ sequestration or with wortmannin (50 nM) to inhibit phosphoinositide 3-kinase, did not inhibit the polymerizing fibrin-induced decrease in PIP2 and increase in PIP. Preincubation with cytochalasin E (CE, 5 mu M) inhibited the decrease in PIP2 by 57% (P < 0.01, n = 8), but not completely. CE did not affect the resting levels of PIP2. Thus, the state of the actin cytoskeleton appears to affect signalling from the integrin receptor alpha IIb beta3 to the enzymes of phosphoinositide interconversion. Platelet cytoskeleton actin content increased by 16.4 4.1% (P < 0.01, n = 5) because of polymerizing fibrin binding to CT-platelets. This is the first demonstration of a pathway involving a decrease in PIP2 caused by binding of polymerizing fibrin to alpha IIb beta3 and an associated increase in cytoskeletal actin, which may be involved in reorganization of the cytoskeleton for platelet-mediated clot retraction.