Platelets co-stimulated with collagen and thrombin display a number of unusual features. As initially described, these dual activated platelets, referred to a COAT-platelets (collagen and thrombin stimulated-platelets), express high levels of surface-bound factor V. This sub-population of platelets represents approximately 30 percent of the total and is most prominent among young platelets. COAT-platelets are also observed upon activation with thrombin plus convulxin, an agonist for glycoprotein VI; however, no single agonist examined was able to produce COAT-platelets. The functional significance of FV on COAT-platelets was shown by demonstrating high factor V activity, preferential binding of factor Xa and significant prothrombinase activity. In addition, COAT-platelets were found to have several other alpha-granule proteins including von Willebrand factor, fibrinogen, fibronectin, thrombospondin and alpha2-antiplasmin, bound at high levels. Unexpectedly, COAT-platelet formation is prevented by transglutaminase inhibitors including dansyl cadaverine, putrescine, and acetyl-casein, and a synthetic peptide substrate for transglutaminases is incorporated in COAT- platelets. The platelet component serving as the acyl acceptor for the transglutaminase reaction was found to be serotonin, and multi-valent serotonin-adducts of albumin were effective inhibitors of COAT-platelet formation. Fibrinogen isolated from COAT-platelets was also found to have conjugated serotonin. This proposal will further characterize COAT-platelets by identifying serotonin-adducts of other alpha-granule proteins found on COAT- platelets, by characterizing the serotonin binding sites present on COAT-platelets, and by evaluating the physiological manipulation of COAT-platelets in experimental animals.