Nicholas J. Greco

Proteolysis of platelet cortactin by calpain.

Cortactin, a substrate of pp60c- src and a potent filamentous actin binding and cross-linking protein, is abundant in circulating platelets. After stimulation of platelet aggregation with collagen, cortactin undergoes a dramatic increase in tyrosine phosphorylation followed by a rapid degradation. The cleavage of platelet cortactin was detected in lysates prepared using either Triton-containing buffer or SDS-sample buffer. However, the degradation of cortactin was not observed in platelets derived from a Glanzmann’s patient, who lacked functional integrin αIIbβ3 (GPIIb-IIIa). In addition, the proteolysis of cortactin was abolished by treating platelets before but not after collagen stimulation with EGTA or calpeptin. Furthermore, recombinant cortactin was digested by μ-calpain in vitro in a dose-dependent manner, indicating that cortactin is a substrate for calpain. We also observed that the calpain-mediated digestion in vitro is dependent on the presence of a sequence containing a proline-rich region and multiple tyrosine residues that are phosphorylated by pp60c- src . Tyrosine phosphorylation by pp60c- src up-regulates the activity of calpain toward cortactin. Our data suggest that the calpain-mediated proteolysis of tyrosine-phosphorylated cortactin may provide a mechanism to remodel irreversibly the cytoskeleton in response to platelet agonists.

Human Platelet Thrombin Receptors and the Two Receptor Model for Platelet Activation

The interaction of proteolytically-active α-thrombin with platelets and other cells of the vasculature, such as endothelial cells and smooth muscle cells, plays a major role in both normal hemostasis and atherosclerosis.1,2 Despite extensive studies in numerous laboratories extending back over thirty years, major questions regarding the mechanism of these interactions remain unresolved. Furthermore, since thrombin can also induce Chemotaxis and adhesion of inflammatory cells, and fibroblast mitogenesis, the importance of elucidating the nature of its receptor, or receptors, extends far beyond its role in platelet activation. However, this review will be restricted mainly to considerations of thrombin receptors in human platelets.