EFFECT OF MULTIMERIZATION OF HUMAN AND RECOMBINANT VON-WILLEBRAND-FACTOR ON PLATELET-AGGREGATION, BINDING TO COLLAGEN AND BINDING OF COAGULATION-FACTOR-VIII

The smallest circulating von Willebrand factor (VWF) molecule is a dim er composed of two identical subunits containing binding sites for hep arin, collagen, platelet glycoproteins and coagulation factor VIII (FV III). Interdimeric disulfide linking leads to multimers composed of up to 40 dimers. vWF serves as a carrier of FVIII and is required for no rmal interactions of platelets with the subendothelium of the injured vessel wall. Von Willebrand factor was purified from human plasma cryo precipitate and fermentation supernatant of recombinant CHO cells by a nion exchange chromatography. Heparin affinity chromatography was used to isolate vWF polymers of different degree of multimerization. Analy sis of collagen binding and platelet aggregation revealed that these a ctivities increase with increasing degree of multimerization of vWF. B inding of FVIII to vWF was studied by real-time biospecific interactio n analysis and surface plasmon technology. The binding data showed tha t the binding of FVIII is independent of vWF multimerization. Using re combinant FVIII and recombinant VWF, real-time biospecific interaction analysis resulted in a potential stoichiometry of 2 to 2.5 vWF-subuni ts per bound FVIII molecule. The kinetic analysis of the VWF-FVIII int eraction resulted in a binding rate constant of about 3 x 10(6) M(-1) s(-1) and an equilibrium dissociation constant of about 0.4 x 10(-9) M .