Factor VIII has to be activated before it can serve efficiently as a cofactor in the intrinsic pathway of blood coagulation. This activation occurs through specific proteolytic cleavages in the molecule by either thrombin or factor Xa. In this study, we show that von Willebrand factor inhibits the activation of factor VIII by factor Xa. Incubation of factor VIII (30 Ujml) with 0.1 pg/ml factor Xa resulted in a 1.6-fold activation followed by a decay of coagulant activity. In the presence of 10 pg/ml von Willebrand factor, activation and inactivation of factor VIII was completely inhibited. In contrast, the activation of factor VIII by thrombin was not influenced by von Willebrand factor. At high concentrations of factor Xa (10 pg/ml), von-Willebrand-factor-bound factor VIII could be cleaved and activated. The generated proteolytic fragments were identical to the fragments produced in the absence of von Willebrand factor and all fragments were released from von Willebrand factor. The major products were light-chain-derived fragments of molecular mass 66/68 kDa and 60 kDa and heavy-chain-derived fragments of 40 and 42 kDa. Also minor products of 12,20/21,23,27 and 30 kDa were observed, most of which were specific for cleavage of factor VIII by factor Xa.Factor VIII is an important plasma protein which acts as a cofactor in the intrinsic pathway of coagulation. It does so by dramatically increasing the V,,, for the activation of factor X by the enzyme factor IXa in the presence of calcium and a phospholipid surface [I]. The critical role of factor VIII in hemostasis is illustrated by the severe bleeding tendency (known as hemophilia A) in its absence [2]. It is a key protein in the regulation of the coagulation cascade, since its cofactor activity can be enhanced through feed-back mechanisms by thrombin and factor Xa. Factor VIII activity is subsequently destroyed by the regulatory-enzyme-activated protein C. Both activation and inactivation of factor VIII occur through specific proteolytic cleavages within the molecule [3 -51.Factor VIII is synthesized as a single-chain precursor molecule of 2332 amino acids [6, 71 and purified from plasma as a carboxy-terminal light chain of 80 kDa which is associated with an amino-terminal heavy chain ranging in molecular mass between 90 -210 kDa [4, 8 -111. In many studies, the kinetics of factor-VIII and factor-X activation [I, 12-181, as well as the changes that occur within the factor VIII molecule upon activation by thrombin or factor Xa [3,4, 17, 191, have been described. These studies have ignored, however, that factor VIII circulates as a non-covalent complex with von Willebrand factor [20]. Formation of this complex is of great physiological importance for factor VIII, since it prolongs its half-life in the circulation [21,22]. Since von Willebrand factor is a very large glycoprotein which represents 99% of the mass of the factor-VIII -von-Willebrand-factor complex, it is conceivable that it modulates the activation and inactivation of factor VIII.In a previous paper ...

The formation of a complex between factor VIII (FVIII) and von Willebrand factor (vWF) was studied using purified radiolabeled human FVIII and purified human vWF. A binding assay was developed in which vWF was coated on microtiter wells. FVIII was shown to bind specifically and reversibly to the immobilized vWF. At a coating of 70 pg vWF/well, binding was half-maximal at a FVIII concentration of 70In order to ascertain which part of FVIII interacted with vWF, eight monoclonal antibodies, directed against FVIII, were tested for their ability to inhibit FVIII-vWF interaction. One of the eight antibodies, CLB-CAg : 58, inhibited binding completely. This antibody was demonstrated to react with the Mr-80000 light chain of FVIII. Direct evidence for the involvement of this chain in vWF binding was obtained by studying the binding of isolated, radiolabeled FVIII heavy and light chains. In a typical experiment 23 -30% of the radioactivity bound when the FVIII light chain was added and less than 1 %O when the FVIII heavy chain was added. The interaction of FVIII and vWF has been studied extensively by several investigators [8 ~ 1 1, 131. From the detailed study of Owen and Wagner [ll] it was clear that both hydrophobic and electrostatic forces are involved in the binding of FVIII to vWF. The FVIII-vWF complex can be dissociated at 1.0 M NaCl or at 0.24 M CaClz whereas at higher concentrations of these reagents the complex is not dissociated. Mikaelsson [ 121 and Tran [13] suggested that calcium ions play a role in the association of the two proteins. Tran demonstrated that the complex can be dissociated with the calcium-chelating agent EDTA. Direct evidence for the involvement of calcium was however not presented. Furthermore, it is not known which domains of the two proteins interact with each other.In the present study a system is presented which is particularly suitable to study the interaction of FVIII with vWF. Data will be presented on the characterization of the binding of radiolabeled FVIII to vWF and the part of FVIII responsible for the interaction with vWF.

No abstract

Purified human factor FVIII (FVIII; 6000-8000 U/mg) was radiolabeled and bound to immobilized von Willebrand factor (vWF). The complex was incubated with human thrombin. Thrombin induced a release of 65% of the radioactivity initially bound. Released FVIII fragments and fragments remaining bound during incubation with thrombin were analyzed using gel electrophoresis. This led to the following observations. Released fragments largely consisted of Mr-70000 and Mr-50000 fragments; Mr-90000 and Mr-80000 fragments were only found in the fractions remaining bound to vWF and decreased with time. In contrast to these digestion products of FVIII, the Mr-42 000 heavy-chain fragment remained bound to vWF, comprising the larger part of the radioactivity after a 2-h incubation. No thrombin-induced cleavages were observed in vWF. Furthermore, vWF-coated wells preincubated with thrombin were still able to bind '"I-FVIII.These results implicate a new concept for the activation of vWF-bound FVIII. Activation is a multistep process in which several cleavages are necessary to produce and release a coagulant-active FVIII molecule (FVIIIa), which is probably an Mr-50000/70000 heterodimer. Inactivation of FVIIIa is likely to be the result of a nonproteolytic dissociation due to loss of the joining divalent cation(s).A deficiency or abnormality of factor VIII (FVIII) causes a severe bleeding disorder. This suggests that FVIII plays a key role in the series of enzymatic reactions leading to the formation of thrombin. FVIII acts in the intrinsic pathway of blood coagulation and, together with phospholipid, calcium, and activated factor IX (FIXa), promotes the conversion of factor X (FX) into the active enzyme FXa [l-31. FVIII functions non-enzymatically in this reaction by increasing the effectiveness of the enzyme FIXa. Reports differ on the extent of amplification due to FVIII; in one report an amplification of 200000-fold has been demonstrated for bovine FVIII [4]. Until recently, it was not known in which form FVIII participates in this reaction, but experimental evidence has accumulated that proteolysis of FVIII is a prerequisite [5 -71. Thrombin is a strong activator of FVIII [7-91 and has been implicated in the physiological activation of FVIII [9]. In most studies on the activation of FVIII by thrombin, the fact that FVIII forms a high-affinity complex with von Willebrand factor (vWF) [lo] was ignored. In plasma, FVIII presumably only exists in complex with vWF. Thus far, this unique property of FVIII has only been connected with stability in terms of survival in plasma [ll, 121. Recent data, obtained in our laboratory, indicated that vWF protects FVIII against inactivation by activated protein C.In a previous paper [lo], we reported a microtiter well binding system with which we studied the interaction between FVIII and vWF. In the present study, we have used the same Correspondence to J. J.

The relationship between the multimeric size of factor VIII-von Willebrand factor (FVIII-vWF) and the support of platelet adhesion to subendothelium was studied in an annular perfusion chamber, employing human renal and umbilical arteries. Commercial factor VIII concentrates containing multimers of low molecular weight that had been shown not to correct the bleeding time upon infusion into patients with von Willebrand's disease did not support platelet adhesion in the perfusion chamber. Cryoprecipitate and two experimental FVIII-vWF concentrates containing multimers of high molecular weight supported platelet adhesion. Factor VIII-vWF purified from cryoprecipitate was subdivided into three fractions of different molecular weights (6.0–14.0, 4.0–9.0, and 3.0–7.5 X 10(6) dalton). These fractions appeared to bind equally well and to be equally effective in supporting platelet adhesion. Factor VIII-vWF with multimers of low molecular weight (0.5–1.5 X 10(6) dalton) were prepared by partial reduction. Binding of FVIII-vWF to subendothelium was not impaired, and the support of platelet adhesion appeared to be more resistant to the effect of reduction than the ristocetin cofactor activity. At high shear rate (2,500 sec-1), increased platelet adhesion was observed with partially reduced FVIII- vWF. These data indicate that the ability of FVIII-vWF preparations to correct the bleeding time is reflected in enhanced platelet adhesion to subendothelium in a perfusion chamber. These data also emphasize that multimeric size is not the only factor determining whether FVIII-vWF will support platelet adhesion.