Studies with a murine monoclonal antibody that abolishes ristocetin- induced binding of von Willebrand factor to platelets: additional evidence in support of GPIb as a platelet receptor for von Willebrand factor

Abstract

A murine monoclonal antibody directed at or near a platelet membrane receptor for the von Willebrand factor was produced by the hybridoma technique. Purified F(ab')2 fragments and/or intact antibody completely blocked the agglutination of platelets induced by both ristocetin and bovine von Willebrand factor and the binding of von Willebrand factor antigen to platelets. The antibody also decreased platelet retention, prevented the reduction in platelet electrophoretic mobility caused by bovine von Willebrand factor, and decreased the serum prothrombin time. Radiolabeled F(ab')2 fragments bound to or approximately 2.5 X 10(4) sites on normal platelets with high affinity (KD or approximately 1.5 X 10(-8) M); there was no binding to platelets from 2 patients with the Bernard-Soulier syndrome. Immunoprecipitation and affinity chromatography studies indicated that the antibody binds to glycoprotein lb at a site contained on the externally oriented portion of the GPIb alpha chain (glycocalicin). An unidentified mol wt or approximately 20,000 molecule labeled by periodate/NaB3H4 coprecipitated and copurified with GPIb.

Tumor‐cell‐induced platelet aggregation is a glycoprotein‐dependent and lipoxygenase‐associated process

“…MAb 6D1 against platelet GPIb, the characteristics of which have been previously described (Coller et al, 1983), was a gift from Dr. B. Coller, Stony Brook, NY. The concentration used (10 pg/ml of 6D 1 MAb) was the minimum amount required to completely inhibit ristocetin-induced platelet aggregation, while not altering ADP-, collagen-or sodium-arachidonate-induced platelet aggregation.…”

Evidence for a factor promoting the conversion of VWF from low and intermediate to high molecular mass polymers on the platelet membrane

“…In the supernatant ob- ristocetin on vWF molecules. We propose that on the membrane surface of resting platelets there is a factor capable of promoting the organization of IMW-and LMW-vWF polymers toward structures having a higher molecular mass; ristocetin, present in samples 3 and 4 because of the large excess employed in the agglutination process, on complexing with the newly formed HMW-vWF polymers allows the activitation and the consequent aggregation of WP [7][8][9][10][11]. This idea is in accordance with the parallel decrease in the vWF activity in the supernatants (from 25% of sample 2 to 12% and 7°7o after one and two hours of incubation, respectively).…”

“…Under these conditions, with increasing incubation time, a progressive decrease in the concentration of LMWand IMF-vWF polymers was observed, accom- case, allows the accumulation of the species that, in the experiment of fig.2, were removed because of the aggregation process. The possibility that, in the experiment shown in fig.3, the HMW-vWF polymers present in the supernatants after incubations could be due to the dismission of the content of the platelets ozgranules by an incidental activation is to be excluded either because of our routine aggregometric control during incubation, and because, in this hypothesis, the platelet activation should have been followed by aggregation [7][8][9][10][11], by which, after centrifugation, the electrophoretic pattern and the activity assay of the supernatants should have been similar to those of the experiment represented in fig.2.…”

“…In plasma, vWF circulates as a heterogeneous mixture of disulfide-bridged polymers ranging from dimers (Mr 25X104) to multimers (Mr>2× 107) and its role is to promote the initial step in hemostatic plug formation [4] by interacting with the subendothelial structures (collagen) of injured vessel and with the glycoprotein IIb-IIIa of activated platelets [5,6]. In the presence of the antibiotic ristocetin, vWF is capable of interacting with resting platelets too: in this case the platelet membrane glycoprotein Ib is responsible for the interaction with the ristocetin-vWF complex [7][8][9][10][11] by which platelets are activated and aggregate. Glycoprotein Ib retains the capability to interact with the ristocetin-vWF complex also when platelets are metabolically Correspondence address: A. Ferri, Istituto di Chimica Biologica, UniversitA degli Studi, Via L. Borsari 46, 1-44100 Ferrara, Italy inactive such as after treatment with formaldehyde [12,13]; in this case agglutination of FFP occurs.…”

A variant form of Bernard-Soulier syndrome: mild haemostatic defect associated with partial platelet GPIb deficiency

“…Subsequently, a molecular defect involving the platelet membrane glycoprotein (GP) I complex was established (Nurden & Caen 1975), and the weight of evidence suggests that the major defect is an absence of GPlb, although deficiencies of GPIX and GPV coexist (N urden & Caen 1978;N urden et al 1981;Clemetson ct al. 1-982;Berndt et al 1983;Collier et al 1983). GPlb and GPIX exist together as a physical complex but no physical association of GPV with either GPlb or GPIX has yet been proved.…”