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 the blood coagulation, fibrinolytic, and complement systems. In human platelet lysates prepared by freeze-thawing or by the addition of nonionic detergent, the Vn antigen content was drastically reduced in comparison with lysates prepared in the presence of SDS, suggesting that Vn is hydrolyzed by platelet-associated enzymes. Exogenously added purified human Vn and Vn present in plasma were also cleaved by these enzyme systems. Degradation was mediated by a nonsecreted or membrane-associated protease system that was inhibited by E-64, EDTA, and leupeptin but not inhibitors of serine and aspartic proteases, suggesting an involvement of calcium-dependent cysteine proteases. Consistently, calpastatin inhibited the hydrolysis of Vn, suggesting that Vn is a substrate for calpain. This was confirmed in a purified system. Vn was cleaved by calpains I and II in a dose- and time-dependent manner, resulting in defined Vn fragments with similar electrophoretic mobility in comparison with those detected in platelet lysates. Functional characterization of the calpain-hydrolyzed Vn revealed that while the type 1 plasminogen activator inhibitor binding activity was unchanged, the heparin and cell binding functions were destroyed. These results suggest that calpains released upon platelet membrane damage or upon tissue injury and necrosis differentially regulate functional domains of the Vn molecule.
Seiffert
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Department of Vascular Biology, Scripps Research Institute, La Jolla, California 92037, USA. seiffert@scripps.edu
D000911:Antibodies, Monoclonal; D001792:Blood Platelets / Q000378:metabolism*; D002154:Calpain / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006868:Hydrolysis; D015151:Immunoblotting; D007700:Kinetics; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D019096:Vitronectin / Q000097:blood*
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D019096:Vitronectin; D002154:Calpain