HYDROLYSIS OF PLATELET VITRONECTIN BY CALPAIN

Vitronectin (Vn) is not only a major adhesive glycoprotein present in platelets but also regulates proteolytic enzyme cascades, including th e blood coagulation, fibrinolytic, and complement systems. In human pl atelet lysates prepared by freeze-thawing or by the addition of nonion ic detergent, the Vn antigen content was drastically reduced in compar ison with lysates prepared in tire presence of SDS, suggesting that Vn is hydrolyzed by platelet-associated enzymes. Exogenously added purif ied human Vn and Vn present in plasma were also cleaved by these enzym e systems. Degradation was mediated by a nonsecreted or membrane-assoc iated protease system that was inhibited by E-64, EDTA, and leu-peptin but not inhibitors of serine and aspartic proteases, suggesting an in volvement of calcium-dependent cysteine proteases. Consistently, calpa statin inhibited the hydrolysis of Vn, suggesting that Vn is a substra te for calpain. This was confirmed in a purified system. Vn was cleave d by calpains I and II in a dose- and time-dependent manner, resulting in defined Vn fragments with similar electrophoretic mobility in comp arison with those detected in platelet lysates. Functional characteriz ation of the calpain-hydrolyzed Vn revealed that while the type 1 plas minogen activator inhibitor binding activity was unchanged, the hepari n and cell binding functions were destroyed. These results suggest tha t calpains released upon platelet membrane damage or upon tissue injur y and necrosis differentially regulate functional domains of the Vn mo lecule.