Abstract
Factor VIII (FVIII) has a multi-domain structure (A1-a1-A2-a2-B-a3-A3-C1-C2), and intracellular processing within the B domain results in its secretion as a heterodimeric procofactor consisting of a variably sized heavy chain (A1-a1-A2-a2-B) and a light chain (a3-A3-C1-C2). Proteolytic cleavage by thrombin at R372, R740, and R1689 removes the B-domain, releases it from vWF by cleaving the a3-acidic region and activates FVIII to the heterotrimeric cofactor, FVIIIa (A1-a1/A2-a2/A3-C1-C2). The requirement for cleavage following a1 to generate active cofactor sets FVIII/FVIIIa apart from factor V, its structural and functional homolog. FVIII binds its cognate protease, factor IXa (FIXa) with high affinity in a membrane-dependent way. However, this complex does not efficiently activate factor X (FX) until R372 is cleaved, indicating that this cleavage reaction is important in facilitating FX recognition and its enhanced activation by intrinsic tenase complex. We speculated that separation of A1-a1 and A2-a2 domains through cleavage at R372 might be replicated by inserting a linker between A1-a1 and A2-a2 domains even when the 372 site was rendered uncleavable. We chose canine FVIII (cFVIII) to test our ideas due to its higher expression levels in cell culture compared to human FVIII. We made cDNA constructs encoding cFVIII variants with flexible (GGGGS, [GGGGS]3, GGGGGG) or rigid (EAAAK, [EAAAK]3, PAPAP) linkers between A1-a1 and A2-a2 domains on the R366Q backbone (corresponding to R372 in human FVIII). All constructs were stably transfected into BHK cells and high expressing clones were selected by one stage aPTT and western blotting of expression media. Clotting times of cFVIII variants with flexible linkers were very similar to cFVIII R366Q without linker (~64 sec). In contrast, clotting times of cFVIII variants with rigid linkers were consistently lower (16 - 26 sec). cFVIII variants (R366Q, R366Q-EAAAK, R366Q-PAPAP) were purified from 15L of expression media each. In one stage aPTT assays purified cFVIII variants (10 nM) showed clotting times (R366Q-EAAAK, 33.3 s, R366Q-PAPAP, 26 s) that were considerably shorter than the R366Q variant (52.2 s) and more in line with wild type cFVIII (cFVIII-WT,19.3 s). To assess possible cleavage at the 366 site, purified cFVIII variants were analyzed by SDS-PAGE following treatment with a high concentration of thrombin. As expected, cFVIII-WT was quantitatively cleaved at R366, R734 and R1689 to produce bands corresponding to A1-a1, A2-a2 and A3-C1-C2. In contrast, both R366Q and R366Q-PAPAP variants were resistant to cleavage at 366, yielding bands corresponding to A1-a1-A2-a2 and A3-C1-C2. The cFVIII-R366Q-EAAAK variant was susceptible to some cleavage, likely at the lysine present within linker. We also performed steady state kinetic studies of FX activation using limiting concentrations of IXa, saturating concentrations of cFVIII variants pretreated with thrombin, membranes and increasing concentrations of FX. Vmax/[E] for FXa formation was 140 ± 7 min-1 with cFVIIIa-WT, 4 ± 0.3 min-1 with cFVIIIa-R366Q, and 42 ± 4 min-1 with cFVIIIa-R366Q-PAPAP. The Km for FX remained unaffected for all variants. The results indicate that the impaired cofactor activity of the FVIII variant that is not cleaved at the 366 site can be substantially rescued by insertion of the PAPAP linker after 366Q. Further optimization of the linker might completely correct defective FX activation associated with the lack of proteolytic cleavage between A1-a1 and A2-a2 domains. Our study indicates that cleavage at the 366 site is not an absolute determinant of FVIII activation and provides new insights into the function of FVIIIa as a cofactor within the intrinsic tenase complex.
Disclosures
Goolyam Basavaraj: Bayer: Research Funding; Grifols: Research Funding. Krishnaswamy:Portola: Research Funding; Baxalta: Consultancy; Janssen Research & Development: Research Funding.
Occlusion of anion-binding exosite 2 in meizothrombin explains its impaired ability to activate factor V