B Domain of Coagulation Factor VIII Regulates Exposure of Its Heparin-Binding Site.

Abstract

B domain of coagulation factor VIII (fVIII) was previously considered to be dispensable for fVIII function. Recently, it was found that the B domain is important for fVIII intracellular interaction with its chaperon and likely involved in fVIII clearance via asialoglycoprotein receptor. At the same time, the major clearance mechanism of fVIII involves initial interaction with heparan sulfate proteoglycans (HSPGs) followed by internalization via low-density lipoprotein receptor-related protein (LRP), member of low-density lipoprotein receptor (LDLR) family (Saenko et al, 1999; Sarafanov et al, 2001). It is possible that recently discovered clearance of fVIII via LDLR (Bovenschen et al, 2005) occurs in the same way. Since it was previously shown that fVIII binding sites for LRP are not located within B domain, we investigated if the latter regulates fVIII interaction with HSPGs. To explore this role of B domain, we compared the binding of plasma-derived fVIII (pd-fVIII) and recombinant B domain-deleted fVIII (BDD-fVIII) to immobilized LRP and heparin (a model of HSPGs) in surface plasmon resonance-based assay. The corresponding affinities were assessed by processing the binding signals obtained for five different concentrations of each analyte. Both pd-fVIII and BDD-fVIII showed similar affinities for LRP (KD 42–60 nM). The LRP-binding site of BDD-fVIII was partially blocked by pre-incubation with its carrier protein von Willebrand factor (vWf) indicating that it is only partially accessible within fVIII/vWf complex. This was further confirmed by the finding that monoclonal antibody 413, which recognizes a high-affinity LRP-binding site within the fVIII A2 domain, interacted with ~25% of BDD-fVIII molecules bound to immobilized vWf. The affinities of pd-fVIII and recombinant BDD-fVIII for immobilized heparin were similar (KD ~20 nM) and 2-fold higher than that for purified A2 domain (KD ~46 nM). Noteworthy, the maximal binding level (Rmax) proved to be 10-fold lower for pd-fVIII in comparison with BDD-fVIII indicating that in the circulation intact fVIII may have limited interaction with HSPGs. Importantly, pre-incubation with vWf did not interfere with the interaction between BDD-fVIII and heparin (KD ~ 19.5 and 21.8 nM, Rmax ~ 194 and 354 RU for BDD-fVIII and BDD-fVIII/vWf, respectively) thus revealing that heparin-binding site of fVIII is completely exposed in BDD-fVIII/vWf complex. These findings suggest that the presence of B domain in circulating fVIII/vWf complex may regulate fVIII clearance by preventing its interaction with HSPGs. The absence of B domain leads to exposure of heparin-binding site within fVIII and binding of fVIII/vWf complex to HSPGs. This binding may be a driving force in fVIII clearance which involves subsequent exposure of LRP (LDLR)-binding site(s) and internalization of fVIII from its complex with vWf via these receptors.