IDENTIFICATION AND CHARACTERIZATION OF THE CYTOPLASMIC ANTIPROTEINASE(CAP) IN HUMAN PLATELETS - EVIDENCE FOR THE INTERACTION OF CAP WITH ENDOGENOUS PLATELET PROTEINS

To define the presence and potential role of platelet-associated prote ase inhibitors, we initiated a study designed to characterize the plat elet components that are responsible for the formation of two SDS-stab le complexes of approximately 58 and 70 kDa initially observed followi ng the incubation of I-125-thrombin and human platelets. We demonstrat e that thermal-mediated unfolding of the 58-kDa complex between I-125- thrombin and a nonsecreted platelet protein leads to an apparent molec ular mass of 70 kDa. This platelet component is functionally and immun ologically indistinguishable from the cytoplasmic antiproteinase (CAP) , also known as placental thrombin inhibitor, a recently cloned member of the ovalbumin family of intracellular serpins (serine proteinase i nhibitors), CAP-specific mRNA and antigen were detected in human plate lets, suggesting that CAP synthesis occurs concurrent with platelet de velopment. Utilizing quantitative immunoblotting, CAP antigen was esti mated at 1.014 +/- 0.181 mu g/10(9) nonstimulated platelets. After pla telet activation with the calcium ionophore A23187, CAP antigen was de tected in released microparticles at approximately 0.195 +/- 0.031 mu g/10(9) platelets and a fraction of platelet CAP was proteolytically m odified. We provide evidence that these lower molecular mass species a rise by cleavage of CAP at or near the reactive site loop. Most import antly, molecular sieving chromatography indicates the presence of an a pproximately 68-kDa SDS-labile complex between cleaved CAP and a cellu lar component in A23187-stimulated platelets, suggesting a physiologic al target of this intracellular serpin and a potential role for this i nhibitor in regulating proteolytic activity that may be formed during platelet activation.