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 (PIP(2)), apparently because of a shift in the equilibria between PIP(2) 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 microM) to inhibit protein phosphorylation, okadaic acid (1 microM) to inhibit protein phosphatases, genistein (100 microM) to inhibit protein tyrosine phosphorylation, PGE (10 microM) 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 microM) inhibited the decrease in PIP(2) 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)beta(3) 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 PIP(2) caused by binding of polymerizing fibrin to alpha(IIb)beta(3) and an associated increase in cytoskeletal actin, which may be involved in reorganization of the cytoskeleton for platelet-mediated clot retraction.