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The hyaluronic acid binding serine protease (PHBSP), an enzyme with the ability to activate the coagulation factor FVII and the plasminogen activator precursors and to inactivate factor VIII and factor V, could be isolated from human plasma in the presence of 6M urea as a single-chain zymogen, whereas under native conditions only its activated two-chain form was obtained. The total yield of proenzyme (proPHBSP) was 5-6 mg/l, corresponding to a concentration of at least 80-100nM in plasma. Upon removal of urea, even in the absence of charged surfaces a rapid development of amidolytic activity was observed that correlated with the appearance of the two-chain enzyme. The highest activation rate was observed at pH 6. ProPHBSP processing was concentration-dependent following a second order kinetic and was accelerated by catalytic amounts of active PHBSP, indicating an intermolecular autocatalytic activation. Charged macromolecules like poly-L-lysine, heparin, and dextran sulfate strongly accelerated the autoactivation, suggesting that in vivo proPHBSP activation might be a surface-bound process. The intrinsic activity of the proenzyme was determined to be 0.25-0.3%, most likely due to traces of PHBSP. The presence of physiological concentrations of known plasma inhibitors of PHBSP, like alpha2 antiplasmin and C1 esterase inhibitor, but not antithrombin III/heparin, slowed down zymogen processing. Our in vitro data suggest that the autoactivation of proPHBSP during plasma fractionation is induced by the removal of inhibitors of PHBSP and is accelerated by charged surfaces of the chromatographic resins.

To cite this article: Hubbard AR, Dodt J, Lee T, Mertens K, Seitz R, Srivastava A, Weinstein M, On Behalf of the Factor VIII and Factor IX Subcommittee of the Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis. Recommendations on the potency labelling of factor VIII and factor IX concentrates. J Thromb Haemost 2013; 11: 988-9.This document addresses two main objectives of product labelling -the first being to define the quantity of the active substance in the vial for manufacturing and marketing purposes, and the second to guide physicians on the dose to be used for treatment that would correlate with recovery data measured in clinical laboratories. For the latter to be effective, the labelling of potency needs to correlate with methods that are predominantly used in hospital laboratories. These correlations may be difficult to establish for individual products and should be resolved at the level of the manufacturers and regulators. This document relates to the potency labelling of all new factor VIII (FVIII) and factor IX (FIX) concentrate products, including plasma-derived, recombinant and modified products. The following recommendations are made in recognition that the potency measurement of some new products might be highly dependent on the choice of assay methods and reagents. Manufacturer's characterization of new product potencyDecisions on potency labelling should be supported by a thorough characterization of the in vitro properties of the new product. This will include testing against the current WHO International Standards (WHO IS) for FVIII or FIX concentrate to establish if valid estimates are possible using both one-stage clotting and chromogenic methods. Methodologies should follow the SSC recommendations and relevant monograph methods where applicable (e.g. pre-dilution in appropriate factor-deficient plasma; albumin in dilution buffers; specified activation times). Best practice for testing includes multiple dilutions (at least three) of standard and product samples to enable evaluation of statistical validity. Potency estimates based on single dilutions of test samples should be avoided and are not acceptable for product labelling. (Chromogenic assays for FIX should be included for information even though the method has only recently become available.)Evaluation by the one-stage clotting method should be performed using different APTT reagents (e.g. silica-based and ellagic acid). It is anticipated that the potency of modified products (e.g. pegylated molecules) by the onestage clotting method may be highly dependent on the choice of APTT reagent in some cases.In addition to potency assessment relative to the WHO IS Concentrate, assays should also be conducted using a plasma reference. This information may not be used in connection with product potency assignment but could be useful when considering the use of a plasma reference to monitor the recovery of new products in clinical laboratories. Data from other analytical methods (e.g. global tests...

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A questionnaire was sent out in 1993 to more than 350 European institutions caring for patients with hemorrhagic disorders with the request to provide data of patients with congenital factor XIII deficiency, to pursue the following aims: (1) establish a registry of congenital factor XIII deficiency patients, (2) promote exchange between clinicians and basic researchers, (3) improve diagnostic and therapeutic approaches, and (4) stimulate research on gene defects and their impact on factor XIII function. So far, 72 patient questionnaires from 60 families have been collected. Their bleeding pattern is typical, with frequent involvement of the umbilical cord and the central nervous system. Forty-nine patients receive regular factor XIII replacement, but obviously some patients with mild symptoms do not require prophylactic substitution, despite low factor XIII levels. On the other hand, 18 patients had factor XIII activities of > or = 5% of normal, but only 3 of those patients were reported to have no bleeding symptoms. Furthermore, 17 symptomatic, apparently heterozygous relatives in eight families were observed. Seven out of 30 females aged over 18 years had experienced spontaneous abortions; wound healing problems were seen in 26 patients. Currently, a second questionnaire is being distributed to obtain more detailed information on bleeding and other symptoms, diagnostic approaches, and exclusion of concurrent other bleeding diatheses. Future activities will be validation and standardization of assays, and study of gene defects and their impact on the structure of factor XIII and symptoms of patients. We intend to expand the survey to countries outside Europe.