Von Willebrand factor-dependent agglutination of washed fixed human platelets by insoluble collagen isolated from bovine aorta.
Adult bovine aortic tissue was homogenized in a neutral phosphate buffer containing proteinase inhibitors. The insoluble residue was rehomogenized in Tris-buffered 6 mol/L guanidinium chloride (pH 7.4). An insoluble fibrillar protein, floating above the main pellet after recentrifugation, was harvested. This material agglutinated washed fixed human platelets in the presence of either normal human plasma or purified von Willebrand factor (vWF). No such reaction was seen when either buffer or plasma from patients with severe von Willebrand's disease was added instead. The extent of platelet agglutination was measured photometrically, similarly to the ristocetin cofactor assay. The agglutination reaction was strongest at neutral pH and was impaired after either addition of EDTA or previous digestion of the fibrillar material by collagenase or pepsin. By light microscopy platelets were seen to adhere onto isolated fibers. Amino acid composition, subunit polypeptides, substrate properties, and interaction with fibronectin of this fibrillar protein were comparable to those of collagen. Therefore, we tentatively denote the induction of platelet agglutination by vWF protein in the described test system as "vWF-collagen cofactor" activity. Comparison of this activity in 65 plasma samples, containing various concentrations of vWF, with ristocetin cofactor activity showed good correlation between results obtained in both tests (r = 0.91).
D000372:Agglutination Tests; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001011:Aorta / Q000032:analysis; D002417:Cattle; D003094:Collagen / Q000302:isolation & purification / Q000502:physiology*; D004492:Edetic Acid / Q000494:pharmacology; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D003012:Microbial Collagenase; D009391:Nephelometry and Turbidimetry; D009994:Osmolar Concentration; D010434:Pepsin A; D010973:Platelet Adhesiveness; D011506:Proteins / Q000032:analysis; D012310:Ristocetin / Q000494:pharmacology; D012995:Solubility; D014841:von Willebrand Factor / Q000502:physiology*
D000596:Amino Acids; D011506:Proteins; D014841:von Willebrand Factor; D012310:Ristocetin; D003094:Collagen; D004492:Edetic Acid; D010434:Pepsin A; D003012:Microbial Collagenase