Characterization of 25 monoclonal antibodies to factor VIII-von Willebrand factor: relationship between ristocetin-induced platelet aggregation and platelet adherence to subendothelium

Abstract

We have studied the role of factor VIII-von Willebrand factor (FVIII- vWF) in both platelet adherence to subendothelium and ristocetin- induced platelet aggregation using monoclonal antibodies to human FVIII- vWF. Twenty-five monoclonal antibodies were obtained, two of which were directed to the factor VIII moiety of FVIII-vWF; one of these two completely inhibited the procoagulant activity (FVIII:C). The remaining 23 monoclonal antibodies were directed to the von Willebrand factor moiety of FVIII-vWF. The ability of the latter monoclonal antibodies to inhibit platelet adherence to arterial subendothelium was investigated with a perfusion model. According to the number of platelets adhering to the subendothelium, three groups of monoclonal antibodies could be discerned: (A) antibodies not affecting platelet adherence; (B) antibodies that inhibited platelet adherence to the level as observed when von Willebrand's disease plasma was tested; and (C) antibodies that completely inhibited both platelet adherence to subendothelium and ristocetin-induced platelet aggregation. The two antibodies present in group C competed for the same or closely related epitope(s) present on FVIII-vWF. These results demonstrate that a domain is present on the FVIII-vWF molecule that is associated both with ristocetin-induced aggregation and with the ability of FVIII-vWF to support platelet adherence to the subendothelium. Based on these observations, it is concluded that ristocetin-induced binding of FVIII-vWF to platelets reflects, at least in part, a physiologic mechanism regulating the function of FVIII-vWF in primary hemostasis.

Immortalisation of human bone marrow endothelial cells: characterisation of new cell lines

“…HBMEC were immortalised with the use of the amphotrophic helper-free retrovirus pLXSN16E6/E7 [13], as described previously [24]. The packaging cell line PA317, which produces the amphotrophic helper-free retrovirus pLXSN16E6/E7 [17], was kindly provided by Dr D. A. Galloway (Fred Hutchinson Cancer Research Center, Seattle, WA, USA). After the immortalisation procedure, the obtained cell colonies were further expanded.…”

Indications for a protective function of beta2‐glycoprotein I in thrombotic thrombocytopenic purpura

“…Murine polyclonal antibody b 2 GPI 3B7 was produced by immunizing mice with human b 2 GPI. Mouse-anti-human VWF RAG201, directed against VWF A3 domain, and RAG 35, directed against VWF A1 domain, were produced as previously described (Stel et al, 1984). Mouse anti-human CD235a, as marker for erythrocytes, was purchased from BD Bioscience (Erembodegem-Aalst, Belgium).…”

“…Expressed proteins were purified from serum-free medium using an affinity-column coupled with anti-human VWF RAG-201. Recombinant VWF A1-domain containing the R1306Q mutation was produced and purified as described before (Stel et al, 1984;Huizinga et al, 2002).…”

Perturbation of cultured human vascular endothelial cells by phorbol ester or thrombin alters the cellular von Willebrand factor distribution

“…The von Willebrand factor ELISA had a similar configuration as the immunoradiometric assay; rabbit polyclonal antibodies, monospecific for von Willebrand factor, served as solid phase, and the murine monoclonal antibody CLB-RAg-35 (Stel et al, 1984) against von Willebrand factor was used as indicator, in conjunction with peroxidase-labeled sheep antimouse IgG (Institut Pasteur, Marnes-la-Garenne, France). The response in this assay of serial dilutions of endothelial cell-conditioned media and extracellular matrix extracts paralleled those of the standard, normal plasma.…”

“…The matrix extracts were precleared three times by incubation with aspecific mouse IgG coupled to Sepharose, followed by passage over a gelatin Sepharose column to remove fibronectin. The precleared extracts were incubated with Sepharose coupled CLB-RAG-35, 38 and 201, all three monoclonal antibodies directed against von Willebrand factor (Stel et al, 1984), that were purified from ascites fluid by chromatography over a protein A Sepharose column (Pharmacia, Uppsala, Sweden). The immunoprecipitates were analyzed by electrophoresis on 5% polyacrylamide gels in the presence of sodium dodecylsulfate and 0mercaptoethanol (Laemmli, 1970).…”