We have expressed in Escherichia coli the domain of von Willebrand factor (vWF) containing the binding site for platelet glycoprotein (GP) Ib and used it to study the regulation of vWF-platelet interaction. The recombinant fragment, comprising residues 445-733 of the mature vWF subunit and designated rvWF445-733, did not have the native conformation of the corresponding domain in the intact molecule because, in order to prevent formation of random aggregates, the seven cysteine residues in the sequence were reduced and alkylated. Unlike native vWF, rvWF445-733 bound to GP Ib in the absence of any modulator, suggesting that the lack of disulfide bonds and/or carbohydrate side chains within this domain may expose platelet interaction sites. In the presence of two modulators, the glycopeptide ristocetin and the snake protein botrocetin, rvWF445-733 inhibited native vWF binding to GP Ib as well as platelet aggregation mediated by vWF, suggesting that both the fragment and the native molecule interact with the same site on platelets. This conclusion was also supported by the observation that the recombinant fragment competed with the binding to platelets of an anti-GP Ib monoclonal antibody known to inhibit vWF binding. Botrocetin formed a complex with rvWF445-733, but the affinity of this interaction was approximately 25-fold lower than with native vWF. However, the complexes of botrocetin with either rvWF445-733 or multimeric native vWF bound to GP Ib with similar dissociation constant. Therefore, conformational attributes of vWF regulate its affinity for botrocetin, but once the complex is formed, interaction with GP Ib is independent of native vWF conformation. These findings provide insights into the regulation of vWF-platelet interaction.

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Lyophilized plasma derivatives are more stable to heat than when they are in the liquid state. Commercial Factor VIII (antihemophilic factor) was seeded with a measured quantity of hepatitis B virus. The contaminated material was then lyophilized and subjected to heat of 60 degrees C for 30 hr. Chimpanzees were inoculated with the heat-treated antihemophilic factor or sham-treated antihemophilic factor that had been held at 4 degrees C. Surprisingly, hepatitis B virus survived the heating procedure with no apparent loss in titer: the incubation period to appearance of HBsAg was that expected for the challenge dose of virus. Even more surprising, one chimpanzee (the recipient of the unheated antihemophilic factor) also developed non-A, non-B hepatitis and two chimpanzees (recipients of the heated antihemophilic factor) also developed delta hepatitis. Neither of these agents was a contaminant of the hepatitis B virus challenge pool, since the purity of this hepatitis B virus pool was established previously in chimpanzees. Thus, both a non-A, non-B agent and the delta agent apparently contaminated the commercial antihemophilic factor. This is the first direct evidence for contamination of antihemophilic factor with the delta agent and confirms previous seroepidemiologic evidence for its presence in pooled plasma derivatives. Subsequent inactivation studies were performed with antihemophilic factor experimentally contaminated with the Hutchinson strain of non-A, non-B hepatitis virus. In these studies, heating at 60 degrees C for 30 hr in the dry state rendered antihemophilic factor free of detectable non-A, non-B hepatitis virus.

The binding of von Willebrand factor (vWF) to platelet glycoprotein (GP) Ib receptor is one of the initial events in thrombus formation. Previous studies have shown that RG12986, a reduced and alkylated recombinant fragment of vWF (Ser445-Val733), can inhibit binding of native vWF to GP Ib and offers potential as an anti-thrombotic agent. We have now evaluated a series of deletion mutants of RG12986 and found that reduced and alkylated rvWF508-704 is close to the minimal sequence with optimal RG12986-like activity (IC50 for inhibition of GP Ib-dependent platelet aggregation in the absence of modulators: 0.022 microM +/- 0.01, n = 3) and that it too binds directly to GP Ib. Under in vitro conditions, with no exogenous modulators present and in the absence of shear stress, oxidized rvWF508-704 (containing a disulfide bond between Cys508 and Cys659) is approximately 5-fold less active than reduced and alkylated rvWF508-704; the two fragments, however, display comparable activity in the presence of the modulator botrocetin. The smaller rvWF508-704 fragment offers distinct advantages over RG 12986. In particular, removal of non-active NH2 and COOH terminal sequences may reduce the risk of antigenicity and may contribute to rendering the molecule mostly monomeric in solution, as opposed to the monomer-dimer equilibrium previously described for RG12986.

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MonoclateR is a highly purified human factor VIII:C preparation obtained by immunoaffinity chromatography of plasma cryoprecipitat followed by chromatography on aminohexyl sepharose. The resulting product is devoid of alloantigens, can be stabilized in the absence of extraneous human proteins and has a specific activity higher than 3000 U/mg.To ascertain product integrity, we set out to compare MonoclateR preparations with either unfractionated human plasma, cryoprecipitateor commercially available lyophilized antihemophilia factor (AHF) preparations by Western blots. As probes, we utilized a series of monoclonal antibodies to defined factor VIII:C epitopes. The MonoclateR VIII:C chain composition appeared indistinguishable from that in the native state of in commercial, clinically useful preparations.Patients with hemophilia are often afflicted with an impairment of cell-mediated immunity, when treated repeatedly with plasma cryoprecipitate preparations. Those preparations, containing at best 0.2% factor VIII:C in protein content, were found in our hands to dramatically inhibit both human mixed lymphocyte reactions and the phytohemagglutinin induced human lymphocyte mitogenesis. We obtained 50% inhibition at about 1 mg/ml protein concentration. These effects were not due to cytotoxicity nor related to procoagulant potency.MonoclateR, in contrast, was totally devoid of immunosuppressive activity at all concentrations tested. This in vitro finding may be related to clinical observation of corrections in immune impairment in adult hemophiliacs treated with MonoclateR. With this immunological profile, MonoclateR represents in our opinion, a promising novel treatment modality for hemophilia A.

We expressed a recombinant peptide fragment (Ser445-Val733) of human von Willebrand factor (vWF), containing the binding domain for the platelet receptor of GP Ib, in E. coli. This 33 kD peptide blocks binding of the intact vWF molecule to GP Ib in the presence of modulators. Thus, it offers potential as an antithrombotic agent. High level expression was achieved in a plasmid construct driven by the bacteriophage T7 promoter. The peptide was solubilized from inclusion bodies in strong chaotrope, then reduced and alkylated. Following purification, formulation at pH 3.5, and lyophilization, the reconstituted experimental product (RG 12986) exists as an equilibrium of monomer and dimer species. When formulated above pH 5.0, soluble aggregates are formed; these solutions have less bioactivity than RG 12986. Interestingly, the non-aggregated state of RG 12986 remains conserved following dilution and incubation with platelet-poor plasma. The overall purification/low pH formulation strategies may be applicable to other E. coli recombinant proteins having a tendency to aggregate following removal of chaotrope near physiologic pH when in a concentrated format.