I. Assessment of von Willebrand Factor (vWF) Activity in Therapeutic Concentrates. A. Development of an international von Willebrand factor (vWF) concentrate standard. SUMMARY OF WORK: This work is a joint effort among CBER/FDA, National Institute for Biological Standards and Control (NIBSC), and the Science and Standard Committee (SSC) on the development of an international standard for vWF concentrates. The project has been separated into three phases. Phase (I) is initial characterization of vWF concentrates. The vWF concentrates with the best combination of relevant characteristics will be selected as candidates for the standard. Two or three candidates after phase (I) will be selected for next phase study. Phase (II) is a pilot testing of candidates. Twelve to 20 laboratories, randomly numbered from 1 to 20, will participate in the study. Coded vials will be distributed to each laboratory as needed. Each laboratory with their own assay procedures will test the properties of vWF. Phase (III) is a production phase of the standard. The manufacturer of the proposed standard after the Phase (II) pilot study will produce one lot of the vWF concentrate with sufficient amount of material for an international vWF concentrate standard. This proposed international standard will be calibrated in a collaborative study. Result: Materials offered from 5 manufacturers were used for trial fills. The initial characterazation of the 5 vWF concentrates were performed in three collaborative sites (CBER/FDA, NIBSC and LFB). The results of this phase (I) study were reported in the SSC annual meeting on August 15, 1999 in Washington DC. B. Standardization of Testing Method for vWF:RcoF Activity. SUMMARY OF WORK: The ristocetin cofactor activity assay is a quantitative, functional assay used to determine vWF levels in plasma or factor VIII concentrates. In the presence of the antibiotic ristocetin, fresh or fixed platelets will agglutinate via the vWF and GPIb interaction on the platelet surfaces. The agglutination slope, as measured on the aggregometer, is proportional to the vWF "activity". The ristocetin cofactor activity assay is widely used and it is probably the most accepted assay for vWF activity at current time. However, the assay variations are big. In order to reduce the variations, we need to use ristocetin cofactor assay in a " standardized manner". As a part of the effort to standardize the vWF:RcoF activity assay, and because of the necessity to prepare a CBER Standard Operating Procedure (SOP) for testing vWF:RcoF activities of conformance lots, we have compiled information on what assays have been performed in local and national laboratories. Copies of procedures and comments from laboratory supervisors were received. In addition, experts were invited to present their positions, rationales, and experiences regarding the benefits and liabilities of using ristocetin cofactor activity as a means of defining vWF activity.The information was used to help developing a CBER SOP for testing vWF:RcoF activity from factor VIII concentrates. The procedure has been validated. The inter-assay variation for the slope of the dose-response relationship is less than 20%, which is generally acceptable for biological assays of this type. The average r2 for Curve Fit Coefficient is 0.9478 (range: 0.8478 - 0.9873, n=8). C. The Correlation of Closure Time and vWF Activities of Factor VIII/vWF Concentrates. SUMMARY OF WORK: 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. II. 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. &#61537;-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 &#61537;-thrombin. Furthermore, &#61543;-thrombin, which lacks the fibrinogen-recognition exosite, cleaved both the heavy and light chains of FVIII, at positions similar to those cleaved by &#61537;-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.