David L. Aronson

Pharmacokinetics of von Willebrand factor and factor VIIIC in patients with severe von Willebrand disease (type 3 VWD): estimation of the rate of factor VIIIC synthesis

Nine patients (10 infusions) with a confirmed diagnosis of type 3 VWD were infused with von Willebrand factor (human), a preparation of von Willebrand factor (VWF) with a very low factor VIII content. Each patient was infused with one dose of approximately 50 or 100 iu ristocetin cofactor activity (VWF:RiCoF) per kg body weight. Bleeding times were performed during the 24 h period after infusion. Plasma samples were obtained over the 96 h period after infusion and were analysed for factor VIII coagulant activity (FVIIIC), VWF:RiCoF, von Willebrand factor antigen (VWF:Ag), and multimers. The FVIIIC data were analysed by non‐linear least‐squares analysis assuming constant FVIIIC ‘synthesis’ and exponential decay. The VWF data were fitted for exponential decay. The average decay rates for FVIIIC, VWF:RiCoF and VWF:Ag were 0.041, 0.061 and 0.056 respectively. The average calculated ‘synthesis’ rate for FVIIIC was 6.4 u/dl/h. The synthesis of FVIIIC was slightly faster and the decay slightly slower following the infusion of 100 iu VWF:RiCoF/kg than of 50 iu VWF:RiCoF/kg. Correction of the bleeding time was strongly dose dependent. At 4 h post infusion the median bleeding time was 9 min following a dose of 50 iu VWF:RiCoF/kg versus 3 min with a dose of 100 iu VWF:RiCoF/kg. There was no decrease in the bleeding time until the level of VWF:Ag or VWF:RiCoF reached >100 u/dl.

Molecular weights of human prothrombin and thrombin by electron irradiation.

The enzyme thrombin and its precursor, prothrombin, play central roles in the process of blood coagulation. The availability of purified thrombin and prothrombin has made possible studies of the sizes of these two molecules by ultracentrifugation and other methods (1-5). There is some disagreement among the published figures, and there are little data on human materials. An attempt has been made to estimate the molecular weights of human thrombin and human prothrombin by applying target theory (6). The advantages of this technique are that only small amounts of material are necessary and that, as a rule, the presence of impurities does not affect the molecular weight estimates.

Ultracentrifugal Analysis of Factor VIII and von Willebrand Factor in Therapeutic Preparations

Plasma and therapeutic preparations of factor VIII (1 recombinant factor VIII and two monoclonally purified plasma‐derived factor VIII preparations, Kogenate, and AHF‐M and Monoclate, respectively) were centrifuged in a sucrose density gradient, and the fractions were analyzed for factor VIII and von Willebrand factor (vWF). The residual vWF in the monoclonally purified factor VIII preparations sediments more slowly than the vWF of plasma. In the absence of added vWF, the factor VIII in all preparations sediments more slowly than plasma factor VIII. These same preparations of factor VIII added to hemophilic plasma as a source of vWF sediment differently. The addition of either recombinant factor VIII or AHF‐M results in sedimentation of the factor VIII with the plasma vFW and in a position indistinguishable from factor VIII in plasma. In contrast, when Monoclate is added to hemophilic plasma in vitro, the factor VIII sediments more slowly than the vWF of the hemophilic plasma. However, 5 min after the infusion of Monoclate into a patient with hemophilia A, the factor VIII sediments with the plasma vWF. These results indicate that the addition of recombinant factor VIII and AHF‐M results in random binding to all vWF multimers of plasma, while there is little exchange between the added factor VIII in Monoclate and the plasma vWF in vitro. In contrast, when the Monoclate is infused, there is rapid binding of factor VIII to the plasma vWF. This is presumably due to tight binding of the factor VIII to the vWF in Monoclate, whereas the factor VIII in Kogenate and AHF‐M, containing no or very low amounts of vWF, are free to bind to plasma vWF. In vivo there are other conditions, as yet uninvestigated, which allow for the binding of factor VIII to vWF.

von Willebrand factor‐cleaving protease content of Cohn plasma fractions

The clinical syndrome thrombotic thrombocytopenic purpura (TTP) has been associated with ultra-large multimers of von Willebrand factor (VWF) in the circulation and a deficiency of the protease that cleaves VWF at a tyrosine-methionine bond [1]. This deficiency has two origins, either as an autoimmune disease that produces an inhibitory autoantibody, or as a genetic mutation that leads to decreased function. Congenital TTP can be treated with plasma infusions at biweekly intervals [2], while the acquired form is treated with plasma exchange. We have assayed Cohn fractions produced from citrated plasma for their VWF-cleaving protease (VWFCP) content using minor modifications of a collagen-binding assay for VWFCP [3]. Cohn fractions (frozen paste, supplied by Dr Tom Zimmerman, Bayer, NC) were dissolved in a small volume of Tris-buffered saline (total protein: ≈ 100 mg/ml), centrifuged, and the protein concentration estimated by measuring the absorbance at 280 nm ( A 280 ). A 50μ l sample was added to 450 μ l of purified VWF (1 U/ml) previously dialysed against 1·5M urea (LFB, Lille, France). Mixtures were then incubated for 2 h at 37 ° and the collagen binding of the remaining VWF and a baseline VWF sample (no added protease) were assessed [3]. VWFCP activity was expressed as the negative logarithim of: