This proposal is aimed toward an understanding of a transglutaminase (TGase) recently isolated and characterized in our laboratory from human platelets and smooth muscle tissue, that is different from factor XIIIa. The TGases also express proteolytic activity toward the cytoskeletal protein components that serve as suitable substrates for cross-linking. Calmodulin consistently was found to stimulate TGase activity, even when proteolysis was prevented by specific inhibitors. In view of the substrate preference of TGase and protease for cytoskeletal proteins, the proposed studies are based on the working hypothesis that cross-linking (with or without proteolysis) of cytoskeletal proteins may occur with transmembrane protein components as part of a mechanism for the clustering or capping of receptors that mediate transmittal of their stimuli into the platelet cytoplasm. For this purpose, the calmodulin-TGase will be characterized under physiological conditions whereby platelets will be stimulated and aggregated by thrombin in addition to have membrane receptors (glycoproteins IIb-IIIa) capped by ConA. The formation of hybrid complexes formed between the membrane receptors with the cytoskeletal protein components, alpha-actinin, vinculin, actin, actin-binding protein and spectrin, will be established. Biochemically, experiments are proposed to dissect the CaM-TGase from the protease activity and establish if these two entities reside in the same molecule or are independently active subunits or constitute two entirely separate molecules sharing binding affinity for each other and/or for calcium and calmodulin as the modulator. The active sites of the TGase will be analyzed for the presence of free thiol cystein group and the mode of cross-linking of the specific substrates will be examined for the covalent formation of epsilon(gamma-glutamyl)-lysine linkages. Antibodies will be sought for the TGase and protease entities and immunolocalization studies will ensue. These studies may clarify the complex interactions that occur between the membrane of platelets with the internal cytoskeleton that are active in supporting platelet function during aggregation and/or thrombus formation.
