THROMBOSIS RESEARCH 22; 267-279, 1981 0049-3848/81/090267-13$02.00/O Printed in the USA. Copyright (c) 1981 Pergsmon Press Ltd. All rights reserved. THE RELEASE OF HEPARIN BINDING PEPTIDES FROM PLATELET THROMBOSPONDIN BY PROTEOLYTIC ACTION OF THROMBIN, PLASMIN AND TRYPSIN John W. Lawler* and Henry S. Slayter Sidney Farber Cancer Institute and Department of Physiology, Harvard zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCB Medical School, Boston, Massachusetts 02115 (Received 17.11.1980; in revised form 21.4.1981. Accepted by Editor J.B. Smith) zyxwvutsrqponmlkjihgfedcbaZYX ABSTRACT When purified human platelet thrombospondin is exposed to thrombin (4 Units/ml) or plasmin (2 Units/ml) for prolonged periods of time proteolytic cleavages occur which result in a decrease in the molec- ular weight of the 145,000 dalton thrombospondin chains to 115,000 daltons. Low molecular weight fragments produced by thrombin were observed in reduced samples at 30,000, 25,000, 18,500 and 12,500 daltons. Heparin-Sepharose affinity chromatography of thrombin, plasmin or trypsin-treated thrombospondin indicates that the 30,000 dalton polypeptide contains the heparin binding site. The heparin- binding polypeptides separate into multiple bands on 10% discontin- uous sodium dodecyl sulfate polyacrylamide gel electrophoresis and during isoelectric focusing. The results of thrombin treatment of thrombospondin in the supernatant from thrombin-treated platelets suggests that some of the proteolytic cleavages are contingent upon the presence of calcium and a nondialyzable component of the native supernatant. INTRODUCTION Exposure of human blood platelets to thrombin rapidly initiates the mobi- lization of platelet granules and the subsequent secretion of platelet-specific proteins (1). zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Among the released proteins there is a 420,000 dalton glyco- protein, designated thrombospondin , which is comprised of three polypeptides of equivalent molecular weight (2,3). Subcellular fractionation studies and analysis of platelets from patients with gray platelet syndrome have demon- *To whom all inquiries should be addressed at St. Elizabeth's Hospital, Department of Research, Boston, MA 02135 Key words: Platelet Thrombospondin; Thrombin; Plasmin; Heparin-binding 267