Abstract. Talin is a high molecular weight protein localized at adhesion plaques in fibroblasts. It binds vinculin and integrin and appears to participate in generating a transmembrane connection between the extracellular matrix and the cytoskeleton. We have recently shown that talin is an abundant protein in platelets, cells highly specialized for regulated adhesion. Although talin constitutes >3 % of the total protein in intact human platelets, its location within the cells had not been defined. In the work reported here, we have investigated the distribution of talin in resting and activated human platelets by immunofluorescence and immunoelectron microscopy. We have found that talin undergoes an activation-dependent change in its subcellular location. In resting platelets, which are nonadhesive, talin is uniformly distributed throughout the cytoplasm. In contrast, in thrombin-and glassactivated, substratum-adherent platelets, talin is concentrated at the cytoplasmic face of the plasma membrane. This dramatic, regulated redistribution of talin raises the possibility that talin plays a role in the controlled development of platelet adhesion.

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Abstract. Activation of blood platelets triggers a series of responses leading to the formation and retraction of blood clots. Among these responses is the establishment of integrin-mediated transmembrane connections between extracellular matrix components and the actin cytoskeleton of the platelet. Here we report that a specific subpopulation of the major platelet integrin, glycoprotein IIb-IHa (GPIlb-HIa) (also referred to as ottr~3 integrin), becomes incorporated into the detergent-insoluble actin cytoskeleton of platelets during the platelet activation response. The cytoskeletal association of GPIIb-lIla is independent of platelet aggregation and fibrin sedimentation and is sensitive to cytochalasin D treatment. As determined by Western immunoblot analysis, ~,,22 % of the total cellular GPIIb-IIIa becomes associated with the actin cytoskeleton upon thrombin activation in a manner that is independent of the detection of talin, ot-actinin, or vinculin in the complex. We found that the cytoskeleton-associated GPllb-llla is derived from an intracellular source since it is not available for lactoperoxidase-catalyzed radioiodination before platelet activation. Two intracellular sources of GPIIb-IIIa are present in resting platelets: GPl/b-IIIa associated with the a-granule secretory compartment as well as surface-inaccessible domains of the surface-connected canalicular system. Interestingly, a-granule secretion, which occurs in thrombin-activated platelets and results in the translocation of intracellular GPHb-INa to the plasma membrane, appears to be required for the cytoskeleton incorporation of GPllb-IIIa that we observe. Collectively, our data provide evidence that a subpopulation of GPIIb-INa derived from an intracellular source is selectively linked to the actin cytoskeleton of platelets upon thrombin activation in the absence of platelet aggregation.

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