OF WORK: The licensing of products in the United States for the treatment of von Willebrand Disease (vWD) has been problematic. Among the reasons for this is because of lack in Vitro test which correlates with clinical efficacy. To explore new test methodology, we investigated the PFA-100 system as a device for detection of vWF activity in factor VIII concentrates. Results: Seven factor VIII concentrates from 7 different manufacturers, and plasma cryoprecipitate were used in this study. VWF activities were tested by ristocetin cofactor (RCof) assay, for collagen binding, and for vWF antigen by ELISA. VWF multimers were detected by the electrophoresis of vWF on high resolution agarose gels (2%), followed by Western-blotting. To determine the relationship of closure time and vWF activity of factor VIII concentrates, a mixture of type III vWD blood (no detectable level of vWF) and factor VIII concentrate was incubated at room temperature for 10 minutes prior to testing on the PFA-100. Each dilution was tested on the PFA machine in duplicate with the collagen/epinephrine and collagen/ADP cartridges. We found that vWF with high molecular weight multimers plays an essential role for platelet anchorage to collagen and platelet aggregation in the PFA-100 system. Factor VIII concentrates with only low molecular weight multimers of vWF did not support the occlusion. There was no good correlation between the closure time and vWF:RCof activity of factor VIII concentrates. One unit of vWF:RCof activity did not always give identical closure time for the different factor VIII concentrates, suggesting that the shear stress assay measures the property of vWF which was not assessed by the RCof test. The relationship of the closure time and clinical outcome is under investigation. B. Standardization of Recombinant Factor VIII SUMMARY OF WORK: Several studies have shown that recombinant FVIII products often have discrepant potencies when different assay methods are applied. In the case of one recombinant product, use of the chromogenic assay, mandated in Europe, would mean that 30-50% less FVIII would be delivered to patients on a molar basis than if one applied the standard one-stage clotting assay used in the United States. It is important for us to discern the mechanism responsible for such discrepancies, to achieve standardization of potency measurements for recombinant FVIII. Results: Thrombin catalyzes the activation of FVIII by cleaving several peptide bonds located on the heavy and light chains of the FVIII molecule. To evaluate the effect of the acidic regions of FVIII on thrombin-catalyzed activation, we first asked whether there is a complementary binding site(s) on thrombin in addition to the catalytic site for FVIII. (-Thrombin, blocked at its active site by D-Phe-Pro-Arg Chloromethyl Ketone (PPACK), inhibited thrombin activation of FVIII. This suggests that a site(s) on thrombin, other than the active site, is needed for FVIII activation. Thrombin-catalyzed cleavages of both the heavy and light chains of FVIII were inhibited by a sulfated polypeptide segment of the FVIII heavy chain, residues 714-740 (F8II). F8II did not inhibit either the amidolytic or the clotting activity of thrombin. This further supports the involvement of sites on thrombin, other than the active site, in FVIII activation. To investigate the potential importance of the fibrinogen-recognition exosite of thrombin on FVIII cleavage, the C-terminal dodecapeptide of hirudin named Hirugen, which binds to the exosite, was tested as an inhibitor of thrombin proteolysis of FVIII. The peptide only partially inhibited the digestion of FVIII, mainly on the light chain, by (-thrombin. Furthermore, (-thrombin, which lacks the fibrinogen-recognition exosite, cleaved both the heavy and light chains of FVIII, at positions similar to those cleaved by (-thrombin. We conclude that a novel recognition site on thrombin, in addition of the fibrinogen-recognition exosite and the active site of thrombin, is involved in activation of FVIII. An acidic region (residues 714-740) of FVIII binds to one of the sites on thrombin involved in the rate-limiting step of the reaction.
