The Interaction Between Factor Viii and Von Willebrand Factor

Abstract

Factor VIII (FVIII) circulates in plasma as a non-covalent complex with von Willebrand factor (VWF), a large multimeric adhesive glycoprotein. VWF serves as a carrier for FVIII and is thought to stabilize FVIII. The interaction between the two proteins was studied by binding purified human 125I-FVIII to VWF which was coated on a solid matrix. Experiments employing isolated heavy and light chains of FVIII and monoclonal antibodies indicated that binding occurred through the carboxyterminal 80kDa light chain of factor VIII. Treatment of VWF-bound 125I-FVIII with thrombin resulted in the release of a light chain-derived 70kDa fragment and a heavy chain-derived 50kDa fragment. A 42kDa heavy chain-derived fragment was found in the fraction which remained bound to VWF. Treatment with factor Xa (FXa) resulted in the release of 63, 50, 45, and 42kDa fragments. No phospholipids were required for proteolysis of FVIII by either of these enzymes. In solution, the activation of FVIII by FXa, but not by thrombin, was inhibited by VWF. Neither activation, nor cleavage or release from VWF were observed when FVIII was incubated with factor IXa. Activation of FVIII was parallelled by its release from VWF. We conclude that the thrombin-activated form of FVIII consists of a complex between the 70kDa and 50kDa fragments. Inactivation of FVIII by activated protein C (APC) was inhibited when FVIII was complexed to VWF. This protective effect of VWF was abolished upon activation of FVIII and its subsequent release from VWF.In order to locate the binding site for FVIII on the VWF molecule, we digested VWF with Staphylococcal V8 protease (Sp). Digestion products were isolated with Mono Q ion-exchange chromatography and identified as Spl (39 kDa), SpII dimers (220 kDa) and Spill dimers (a triplet ranging from 210-280 kDa) by their molecular weight and chromatographic behaviour (J.-P. Girma et al.. Biochemistry 1986, 25:3156-3163). Purified VWF or digestion products were spotted on nitrocellulose paper, followed by blocking with an albumin solution. Binding of FVIII was studied by incubating the filters with 125I-FVIII, followed by autoradiography. Fifty ng of VWF was sufficient in order to detect FVIII binding. No binding was observed to partially reduced dimeric undigested VWF. Of the isolated digestion products, only the SpIII dimer was able to bind 125I-FVIII. After Western blotting of VWF-fragments from SDS-polyacrylamide gels, 125I-FVIII bound only to the bands which represented SpIII. Therefore, the domain on VWF responsible for the binding of FVIII seems to be located on its aminoterminal SpIII fragment. The integrity of internal disulfide bonds and dimerisation of VWF are required for FVIII binding.

Assay Discrepancies with Highly Purified Factor VIII Concentrates

“…The addi tion of purified vWF appeared to increase the potency of the purified concentrates when assayed with the Diagen immunode pleted plasma (fig. 2); this could be due to enhanced stability of factor VIII:C in the presence of excess vWF, since it has been shown that vWF protects factor VIII:C from degradation by activated protein C [14]. These results, if confirmed, suggest that plas mas which have been immunodepleted of vWF may be unsuitable for assay of highly purified factor VIII.…”

Intranasal DDAVP induced increases in plasma von Willebrand factor alter the pharmacokinetics of high‐purity factor VIII concentrates in severe haemophilia A patients

“…Plasma FVIII is maximally stabilized when it circulates in a noncovalent complex with vWf [11, 12]. In vitro , vWf appears to protect FVIII from activation by factor Xa [13], from inactivation by activated protein C [14], and to prevent FVIII from binding to phospholipids [15] and activated platelets [16]. In vivo , FVIII has a normal intravascular survival only when complexed to vWf, which not only preserves FVIII integrity but also may selectively concentrate FVIII at sites of vascular injury [16].…”

Biosynthesis of Human von Willebrand Factor

“…Its function in plasma remains to be elucidated. The presence of the RGD tripeptide indi cates that it may interact with other proteins, such as cell surface receptors [48], Further more, it has been suggested that pro-poly peptide in plasma, as part of pro-vWF, stabi lizes factor VIII [49], Although a factor VIII binding domain on mature vWF is localized within the First N-terminal 272 amino acid residues (D' domain and part of D3), this suggestion can formally not be excluded as yet [93][94][95].…”