Joan E. B. Fox

Regulation of Platelet Function by the Cytoskeleton

The platelet cytoskeleton contains two actin filament-based components. One is the cytoplasmic actin filaments that fill the cytoplasm and mediate contractile events. The other is the membrane skeleton that coats the plasma membrane and regulates properties of the membrane such as its contours and stability and the lateral distribution of membrane glycoproteins. Recent work reviewed in this article indicates that the GP IIb-IIIa complex can associate with the membrane skeleton. Upon platelet activation, GP IIb-IIIa becomes competent to bind its adhesive ligand, fibrinogen. This induces a reorganization of the cytoskeleton such that the membrane skeletal proteins with which GP IIb-IIIa is associated become associated with underlying cytoplasmic filaments. As in focal contacts of cultured cells, this ligand-induced association of GP IIb-IIIa with cytoplasmic actin filaments regulates the ability of GP IIb-IIIa to bind adhesive ligand. Intracellular enzymes that are activated as a consequence of ligand binding to the GP IIb-IIIa complex include tyrosine kinase(s) and calpain, making these potential candidates for enzymes inducing the two-way signaling across the membrane. Additional candidates include phosphoinositide 3-kinase and protein kinase C, other enzymes that have been detected in focal contacts of aggregating platelets. Future studies identifying interactions between the GP IIb-IIIa complex and membrane skeletal proteins should help to further elucidate the significance of the GP IIb-IIIa in cytoskeleton interaction in regulating integrin-mediated transmembrane signaling in platelets.

Dystrophin-related Protein in the Platelet Membrane Skeleton INTEGRIN-INDUCED CHANGE IN DETERGENT-INSOLUBILITY AND CLEAVAGE BY CALPAIN IN AGGREGATING PLATELETS

The platelet membrane is lined with a membrane skeleton that associates with transmembrane adhesion receptors and is thought to play a role in regulating the stability of the membrane, distribution and function of adhesive receptors, and adhesive receptor-induced transmembrane signaling. When platelets are lysed with Triton X-100, cytoplasmic actin filaments can be sedimented by centrifugation at low g-forces (15,600 × g) but the membrane skeleton requires 100,000 × g. The present study shows that DRP (dystrophin-related protein) sediments from lysed platelets along with membrane skeleton proteins. Sedimentation results from association with the membrane skeleton because DRP was released into the detergent-soluble fraction when actin filaments were depolymerized. Interaction of fibrinogen with the integrin αβ induces platelet aggregation, transmembrane signaling, and the formation of integrin-rich cytoskeletal complexes that can be sedimented from detergent lysates at low g-forces. Like other membrane skeleton proteins, DRP redistributed from the high-speed pellet to the integrin-rich low-speed pellet of aggregating platelets. One of the signaling enzymes that is activated following αβ-ligand interactions in a platelet aggregate is calpain; DRP was cleaved by calpain to generate a 140-kDa fragment that remained associated with the low-speed detergent-insoluble fraction. These studies show that DRP is part of the platelet membrane skeleton and indicate that DRP participates in the cytoskeletal reorganizations resulting from signal transmission between extracellular adhesive ligand and the interior of the cell.

Hydrolysis of Cytoskeletal Proteins by the Ca2+-Dependent Protease During Platelet Activation

During platelet stimulation, the cytosolic Ca2+ concentration increases to micromolar levels. One consequence of this increase is that the Ca2+-dependent protease within platelets is activated. Activation of the Ca2+-dependent protease results in hydrolysis of actin-binding protein and P235. Actin-binding protein and P235 can both affect the organization of actin, thus activation of the Ca2+-dependent protease may provide a regulatory mechanism by which stimulus-induced changes in the organization of actin filaments could be directed. Although both actin-binding protein and P235 affect actin polymerization, stimulus-induced actin polymerization occurs before hydrolysis of actin-binding protein or P235 can be detected, thus it seems unlikely that hydrolysis of these proteins affects actin polymerization. Actin-binding protein also cross-links actin filaments into networks, a function that is lost when it is hydrolyzed by the Ca2+-dependent protease. Thus, hydrolysis of actin-binding protein may result in disruption of the actin filament networks that form early during platelet activation and permit the reorganization of filaments into the bundles present at later stages of platelet activation.