In this study, we characterised the mechanisms of Rac GTPase activation in human platelets stimulated by two physiological agonists, either thrombin, acting through membrane receptors coupled to heterotrimeric G-proteins, or collagen which is known to mobilise a tyrosine kinase-dependent pathway. Both agonists induced a rapid activation of Rac that was not significantly affected by the inhibition of integrin K K IIb L L 3 engagement. Using pharmacological inhibitors, we found that phospholipase C activation and calcium mobilisation were essential for platelet Rac activation by either thrombin or collagen whereas protein kinase C inhibition was without effect. In contrast to Rac, Cdc42 activation was independent of phospholipase C activation, indicating that the two GTPases are differently regulated. We also found that phosphoinositide 3-kinase was not required for Rac activation in response to thrombin but was involved in its activation by collagen. ß

IntroductionThe interactions between the plasma membrane and the cytoskeleton are essential for a wide variety of cellular processes such as endocytosis, cell morphology, cell adhesion, formation of cell-cell contacts and cell motility. These interactions are regulated by signaling complexes formed by the assembly of transmembrane and cytosolic proteins and lipids (Engqvist-Goldstein and Drubin, 2003;Fais and Malorni, 2003;Sechi and Wehland, 2000). These complexes are thought to participate in the control of the elongation of actin filaments leading to the formation of membrane protrusions, or in the contraction of the actomyosin network leading to membrane retraction. Blood platelets represent an attractive model to investigate these mechanisms as the actin cytoskeleton and its interactions with the plasma membrane are critical for their physiological functions (Hartwig et al., 1999). Indeed, platelets must respond rapidly to a vessel injury by a series of coordinated events including adhesion, shape change, spreading, aggregation and clot retraction. αIIbβ3 is the most prominent platelet integrin, and, upon activation by 'insideout' signaling, is capable of binding to several adhesive proteins, including fibrinogen, to support platelet aggregation. During aggregation, αIIbβ3 is also involved in transmitting an 'outside-in' signal leading to the formation of a network of signaling and structural proteins strongly interacting with the newly remodeled actin cytoskeleton (Hartwig et al., 1999). These protein complexes were previously co-isolated with the cytoskeleton as a Triton X-100 insoluble pellet obtained at low speed centrifugation (15,000 g) from aggregated platelets (Fox, 1993). The characterization of this cellular fraction led to the identification of several key signaling molecules recruited to αIIbβ3-based cytoskeletal structures, where they are thought to play an essential role in stabilization of platelet aggregation (Hartwig et al., 1999).At the sites of interaction with the cytoskeleton, the plasma membrane is subjected to important mechanical forces, especially during platelet fibrin clot retraction, a postaggregation event involved in the maintenance of hemostasis. During retraction, integrins take part in the formation of a transmembrane linkage between proteins from the extracellular matrix and from the actomyosin filament network, allowing the transmission of contractile forces. It is currently hypothesized that the areas of membrane interacting with the cytoskeleton have a specific lipid composition adapted to these forces. In this context, cholesterol and sphingolipid enriched membrane microdomains, known as detergent resistant membranes (DRMs) or lipid rafts, are of major interest. Their lipid composition confers a restricted fluidity state called a liquid ordered phase to these membrane microdomains (Brown and London, 2000). Although the existence of rafts has been 759 Dynamic connections between actin filaments and the plasma membrane are crucial for the regulation of blood platelet funct...