Differential regulation of platelet aggregation and aminophospholipid exposure by calpain.

Aggregation, exposure of procoagulant phospholipids and shedding of microparticles are platelet responses that depend on activating conditions. To determine how these different responses are interconnected, we simultaneously measured fibrinogen (Fg) binding and aminophospholipid exposure on activated platelets by means of flow cytometry. Low calcium ionophore (A23187) concentrations induced Fg binding but not annexin V binding. In contrast, high A23187 concentrations induced annexin V binding but not Fg binding. Collagen, both alone and in the presence of thrombin, induced both Fg and annexin V binding. Dual labelling was found on 38 +/- 9% of platelets stimulated by thrombin plus collagen. The regulatory role of calpain in these platelet functions was investigated. When calpain was partially inhibited by 2 microg/ml calpeptin, Fg binding still occurred but aminophospholipid exposure was limited. By contrast, complete inhibition of calpain by 100 microg/ml calpeptin or E64d decreased Fg binding but enhanced aminophospholipid exposure. In these latter conditions, cytosolic calcium-extruding systems were inhibited. The results suggest that (i) conditions that favour aminophospholipid exposure tend to decrease the aggregation process and (ii) calpain determines the switch to either aggregation or aminophospholipid exposure by controlling intracellular calcium.

Integrin alpha(IIb)beta3 signals lead cofilin to accelerate platelet actin dynamics.

Cofilin, in its Ser3 dephosphorylated form, accelerates actin filament turnover in cells. We report here the role of cofilin in platelet actin assembly. Cofilin is primarily phosphorylated in the resting platelet as evidenced by a specific antibody directed against its Ser3 phosphorylated form. After stimulation with thrombin under nonstirring conditions, cofilin is reversibly dephosphorylated and transiently incorporates into the actin cytoskeleton. Its dephosphorylation is maximal 1-2 min after platelet stimulation, shortly after the peak of actin assembly occurs. Cofilin rephosphorylation begins 2 min after activation and exceeds resting levels by 5-10 min. Cofilin is dephosphorylated with identical kinetics but fails to become rephosphorylated when platelets are stimulated under stirring conditions. Cofilin is normally rephosphorylated when platelets are stimulated in the presence of Arg-Gly-Asp-Ser (RGDS) peptide or wortmannin to block alpha(IIb)beta3 cross-linking and signaling or in platelets isolated from a patient with Glanzmann thrombasthenia, which express only 2-3% of normal alpha(IIb)beta3 levels. Furthermore, actin assembly and Arp2/3 complex incorporation in the platelet actin cytoskeleton are decreased when alpha(IIb)beta3 is engaged. Our results suggest that cofilin is essential for actin dynamics mediated by outside-in signals in activated platelets.