SummaryThe platelet cytoskeleton contains two actin filament-based components. One is the cytoplasmic actin filaments which fill the cytoplasm and mediate contractile events. The other is the membrane skeleton, which coats the plasma membrane and regulates properties of the membrane such as its contours and stability. In the unstimulated platelet, only 30-40% of the actin is polymerized into filaments; the rest is thought to be prevented from polymerizing by the association of thymosin β4 with monomeric actin and by the association of gelsolin with the barbed ends of pre-existing actin filaments. When platelets are activated, there is a rapid increase in actin polymerization; new filaments fill the extending filopodia and form a network at the periphery of the platelet. As a result of activation, myosin binds to cytoplasmic actin filaments, causing them to move towards the center of the platelet. As platelets aggregate, additional cytoskeletal reorganizations occur: GP Ilb-IIIa associates with adhesive ligand in a platelet aggregate; this results in the association of GP Ilb-IIIa, membrane skeleton proteins, and signaling molecules with cytoplasmic actin. Future studies should help to elucidate the significance of the cytoskeleton in regulating signal transduction events in platelets.

The zeta-form 14-3-3 protein (14-3-3zeta) regulates protein kinases and interacts with several signaling molecules. We reported previously that a platelet adhesion receptor, glycoprotein (GP) Ib-IX, was associated with a 29-kDa protein with partial sequences identical to 14-3-3zeta. In this study, the interaction between GPIb-IX and recombinant 14-3-3zeta is reconstituted. Further, we show that the 14-3-3zeta binding site in GPIb is within a 15 residue sequence at the C terminus of GPIb-alpha, as indicated by antibody inhibition and direct binding of 14-3-3zeta to synthetic GPIb-alpha cytoplasmic domain peptides. The 14-3-3zeta binds to recombinant wild type GPIb-IX but not to the GPIb-alpha mutants lacking C-terminal 5 or more residues, suggesting that the C-terminal 5 residues of GPIb-alpha are critical. Similarity between the GPIb-alpha C-terminal sequence and the serine-rich regions of Raf and Bcr kinases suggests a possible serine-rich recognition motif for the 14-3-3 protein.

Abstract. Calpain (a Ca2+-dependent protease) is present in many cell types. Because it is present in the cytosol, the potential exists that it may regulate critical intracellular events by inducing crucial proteolytic cleavages. However, the concentrations of Ca 2 § required to activate calpain are higher than those attained in the cytoplasm of most cells. Thus, the physiological importance of calpain and the mechanisms involved in its activation have remained elusive. In this study, we show that calpain rapidly moved to a peripheral location upon the addition of an agonist to suspensions of platelets, but it remained unactivated. We provide three lines of evidence that calpain was subsequently activated by a mechanism that required the binding of an adhesive ligand to the major platelet integrin, glycoprotein (GP) IIb-IIIa: calpain activation was prevented by RGDS, a tetrapeptide that inhibits the binding of adhesive ligand to GP Ub-l/Ia; it was also prevented by monoclonal antibodies that inhibit adhesive ligand binding to GP llb-IIIa; and its activation was markedly reduced in platelets from patients whose platelets have greatly reduced levels of functional GP llb-IIIa. Thus, in platelets, binding of the extracellular domain of GP l/b-IIIa to its adhesive ligand can initiate a transmembrane signal that activates intraceUular calpain. Because calpain is present in focal contacts of adherent cells, the interaction of integrins with adhesive ligands in the extracellular matrix may regulate activation of calpain in other cell types as well.

Experiments were performed to determine whether platelets contain a membrane skeleton. Platelets were labeled by a sodium periodate/sodium [3Hjborohydride method and 1ysed with Triton X-100. Much of the filamentous actin could be sedimented at low g forces (15,600 g, 4 min), but some of the actin filaments required high-speed centrifugation for their sedimentation (100,000 g, 3 h). The latter filaments differed from those in the low-speed pellet in that they could not be depolymerized by Ca2+ and could not be sedimented at low g forces even from Triton X-100 lysates of platelets that had been activated with thrombin. Actin-binding protein sedimented with both types of filaments, but 3H-labeled membrane glycoproteins were recovered mainly with the high-speed filaments. The primary 3H-labeled glycoprotein recovered with this "membrane skeleton" was glycoprotein (GP) lb. Approximately 70% of the platelet GP lb was present in this skeleton. Several other minor glycoproteins, including >50% of the GP Ia and small amounts of three unidentified glycoproteins of M, > 200,000, were also recovered with the membrane skeleton. The Triton X-100 insolubility of GP Ib, GP Ia, a minor membrane glycoprotein of 250,000 Mr, and actinbinding protein resulted from their association with actin filaments as they were rendered Triton X-100-soluble when actin filaments were depolymerized with deoxyribonuclease I and coisolated with actin filaments on sucrose gradients. When isolated platelet plasma membranes were extracted with Triton X-100, actin, actin-binding protein, and GP lb were recovered as the Triton X-100 residue. These studies show that unstimulated platelets contain a membrane skeleton composed of actin filaments and actin-binding protein that is distinct from the rest of the cytoskeleton and is attached to GP Ib, GP Ia, and a minor glycoprotein of 250,000 M, on the plasma membrane.