Evaluation of Ristocetin–Willebrand Factor Assay and Ristocetin-induced Platelet Aggregation

Abstract

Normal subjects, patients with various bleeding disorders, and patients with von Willebrand's disease were studied. All patients with von Willebrand's disease had decreased levels of ristocetin-Willebrand factor (range, 0 to 41%) as compared with all other subjects (range, 79 to 202%). Ristocetin-induced platelet aggregation of platelet-rich plasma was abnormal in all patients with von Willebrand's disease tested, and it was possible to correct this abnormal response by addition of normal platelet-poor plasma. Abnormal ristocetin-induced platelet aggregation was seen in patients with intrinsic platelet disorders or, on some occasions, in normal patients who had ingested aspirin. Ristocetin-induced platelet aggregation is not diagnostic, but it may be useful as a simple screening test for patients with possible von Willebrand's disease. In conjunction with other tests, the assay for ristocetin-Willebrand factor will be useful in diagnosis and evaluation of these patients.

Binding and covalent cross-linking of purified von Willebrand factor to native monomeric collagen.

“…The ability ofnative vWF polymers to agglutinate formalin-fixed platelets was determined in the presence of 1 mg/ml ristocetin (30,31), and agglutination was monitored on a dual-channel aggregometer (Payton Assoc., Inc., Buffalo, NY).…”

Structural basis of von Willebrand factor binding to platelet glycoprotein Ib and collagen. Effects of disulfide reduction and limited proteolysis of polymeric von Willebrand factor.

“…These were prepared as previously described (20) and resuspended in 50 mM Tris, 0.15 M NaCl, 0.02% sodium azide, pH 7.4 at a final concentration of 6 X 105 platelets per microliter. The ability of native vWF polymers to agglutinate formalin-fixed platelets was determined in the presence of 1 mg/ml ristocetin (21,22) and agglutination was monitored on a Payton dual-channel aggregometer (Payton Scientific, Inc., Buffalo, NY). Sodium dodecyl sulfate polyacryl-vWF purification.…”

“…4-ml fractions, collected at a 70 ml/h flow rate, were analyzed for ristocetin-dependent platelet agglutinating activity (21), protein concentration by the method of Lowry (24), and for purity by 5% SDS-PAGE after disulfide bond reduction, as described above (23). Those fractions demonstrating a single 220,000-D band on 5% SDS-PAGE were pooled and concentrated by precipitation with 35% (wt/vol) ammonium sulfate followed by centrifugation at 34,800 g for 30 min at 4VC.…”

von Willebrand protein facilitates platelet incorporation in polymerizing fibrin.

“…vWFwas prepared from a cryoprecipitate ofhuman plasma as described previously (33). vWF activity was determined by the ristocetin assay (34,35) using FP. Only the earliest eluting fractions from the Sephacryl S-1000 column ofvWF were used in the experiments presented here to minimize fibrinogen and fibronectin contamination.…”