Regular Article THROMBOSIS AND HEMOSTASIS A von Willebrand factor fragment containing the D9D3 domains is sufficient to stabilize coagulation factor VIII in mice

Loss of procoagulant factor VIII (FVIII) function results in pathologic bleeding.Noncovalent binding to vonWillebrand factor (VWF) protects FVIII from degradation and rapid clearance from plasma. FVIII levels in patients with vonWillebrand disease (VWD) type 1 or type 3 are generally reduced concomitant with decreased levels of plasma VWF. Patients with VWF mutations interfering with VWF: FVIII binding (VWD type 2N) exhibit normal plasma VWF but reduced plasma FVIII levels. Infusion of plasma fractions from hemophilia A (HA) patients (containing VWF but no FVIII) into severeVWDpatients restores plasmaFVIII levels, demonstrating that exogenous VWF stabilizes endogenous FVIII. Similarly, treatment of HA patients with recombinant FVIII free of VWF is successful because of the stabilization of exogenous FVIII by endogenous VWF. In contrast, correction of low FVIII levels in patients with type 2N or type 3 VWD requires infusion of VWF-containing plasma concentrates. Secreted from endothelial cells as a multimeric glycoprotein, VWF is composed of a series of repeated domains (Figure 1). In the endoplasmic reticulum, proVWF subunits dimerize via intermolecular disulfide bonds at the C-terminal CKdomain. In the trans-Golgi and post-Golgi compartments, the VWF propeptide catalyzes the formation of VWF multimers via disulfide bonds near the VWF N terminus (C1099-C1099 and C1142-C1142) and is cleaved from proVWFby furin. The resultingmature VWFmultimers condense into long, helical structures that characterize the shape of Weibel– Palade bodies and are released into the circulation as long, linear polymers with multiple concatemerized subunits. Each VWF multimer is composed of 2 to .60 subunits (with each monomer containing a single FVIII-binding site). Although plasma FVIII binds to all sizes of multimeric VWF with similar affinities (estimated KD 5 ;0.2-0.3 nM), the FVIII:VWF monomer stoichiometry is only;1:50. FVIII is a heterodimer composed of a heavy chain (domains A1a1-A2-a2-B) and a light chain (a3-A3-C1-C2) noncovalently linked through divalent cations, which binds to VWF via the FVIII light chain. The acidic a3, C1, and C2 domains of the FVIII light chain are thought to form the noncontiguous interface with VWF. The FVIII binding region within VWF has been localized to a proteolytic VWF fragment that spans residues S764-R1035, which compose the N terminus of the D9D3 domains and excludes the cysteines that coordinate multimerization. Removal of the N-terminal S764L1039 segment by granzyme M cleavage disrupts FVIII binding to multimeric VWF. Multiple biochemical methods and the location of VWD type 2N mutations implicate the VWF D9 domain