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 dimer composed of two identical subunits containing binding sites for heparin, collagen, platelet glycoproteins and coagulation factor VIII (FVIII). Interdimeric disulfide linking leads to multimers composed of up to 40 dimers. vWF serves as a carrier of FVIII and is required for normal interactions of platelets with the subendothelium of the injured vessel wall. Von Willebrand factor was purified from human plasma cryoprecipitate and fermentation supernatant of recombinant CHO cells by anion exchange chromatography. Heparin affinity chromatography was used to isolate vWF polymers of different degree of multimerization. Analysis of collagen binding and platelet aggregation revealed that these activities increase with increasing degree of multimerization of vWF. Binding of FVIII to vWF was studied by real-time biospecific interaction analysis and surface plasmon technology. The binding data showed that the binding of FVIII is independent of vWF multimerization. Using recombinant FVIII and recombinant vWF, real-time biospecific interaction analysis resulted in a potential stoichiometry of 2 to 2.5 vWF-subunits per bound FVIII molecule. The kinetic analysis of the vWF-FVIII interaction 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.
Fischer
B E
BE
IMMUNO AG, Biomedical Research Center, Orth/Donau, Austria.
Kramer
G
G
Mitterer
A
A
Grillberger
L
L
Reiter
M
M
Mundt
W
W
Dorner
F
F
Eibl
J
J
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001704:Biopolymers; D015374:Biosensing Techniques; D016466:CHO Cells; D002846:Chromatography, Affinity; D003094:Collagen / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D016895:Culture Media, Serum-Free; D019281:Dimerization; D005169:Factor VIII / Q000378:metabolism*; D005285:Fermentation; D006493:Heparin / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D010974:Platelet Aggregation / Q000187:drug effects*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D014841:von Willebrand Factor / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D001704:Biopolymers; D016895:Culture Media, Serum-Free; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D014841:von Willebrand Factor; D005169:Factor VIII; D006493:Heparin; D003094:Collagen