Vol. 108, No. 2, 1982 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS September 30, 1982 Pages 479-405 AMINO GROUP MODIFICATION INHIBITS RISTOCETIN COFACTOR ACTIVITY OF HUMAN VON WILLEBRAND FACTOR Samuel A. Santoro Division of Laboratory Medicine Washington University School of Medicine St. Louis, Missouri 63110 Received July 26, 1982 SUMMARY: Purified von Willebrand factor rapidly loses activity when treated under mild conditions with the highly specific amino group reagent trinitrobenzenesulfonic acid. Greater than 90 percent inhibition of activity is achieved by modification of only 7 percent of the amino groups. Other modifications such as acetylation and succinylation also abolish activity. It is unlikely that the essential rapidly reacting amino groups function simply in an electrostatic manner since modifi- cations such as amidination and methylation which produce derivatives which retain positive charge are also inactive or nearly so. von Willebrand factor circulates in plasma as a heterogeneous series of polymers, composed of peptide chains of 230,000 daltons which are covalently linked by disulfide bonds (l-3). As part of the factor VIII complex von Willebrand factor is associated in plasma with another lower molecular weight peptide which carries factor VIII procoagulant activity. A number of observations have implicated von Willebrand factor in the adhesion of platelets to components of the vascular subendothelium which become exposed by endothelial injury (4-g). Relatively little, however, is known regarding detailed structural and functional correlations within this important molecule. MATERIALS AND METHODS Materials - Trinitrobenzenesulfonic acid was obtained from Eastman, methyl acetimidate hydrocloride from Pierce Chemical Company and sodium borohydride, formaldehyde, acetic anhydride and succinic anhydride from Fisher Scientific. Ristocetin was purchased from Helena Laboratories. Sepharose 48 and cyanogen bromide activated Sepharose 48 were obtained from Pharmacia. Gelatin was prepared by heat denaturing purified lathy- ritic rat skin acid soluble collagen (10) in a boiling water bath. Other chemicals employed were of reagent grade. 479 0006-291X/82/180479-07$01.00/0 Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.