A Spacer Sequence Inserted between the A1 and A2 Domains of Factor VIII Overcomes the Requirement for Proteolytic Cleavage at Arg 372 in the Generation of Cofactor Activity

Factor VIII (FVIII) with a multi-domain structure (A1-a1-A2-a2-B-a3-A3-C1-C2) is a procofactor and precursor for the anti-hemophilic cofactor protein, FVIIIa. Following the intracellular processing within the B domain, secreted FVIII circulates as a heterodimer with variably sized (90K-200K) heavy chain (A1-a1-A2-a2-B) and an 80K light chain (a3-A3-C1-C2). Proteolytic activation of FVIII by thrombin that yields heterotrimeric FVIIIa (A1-a1/A2-a2/A3-C1-C2), the cofactor for intrinsic tenase, involves cleavage of three peptide bonds between Arg³⁷²-Ser³⁷³, Arg⁷⁴⁰-Ser⁷⁴¹, and Arg¹⁶⁸⁹-Ser¹⁶⁹⁰. Cleavage at Arg⁷⁴⁰ removes the B-domain, and cleavage at Arg¹⁶⁸⁹ removes the a3-acidic region and releases FVIII from vWF, its carrier protein, and exposes membrane binding sites within the FVIII light chain. Cleavage at Arg³⁷² separates A1-a1 and A2-a2 domains and is implicated in the cofactor-dependent recognition and enhancement in the rate of factor X (FX) activation by intrinsic tenase. Subsequently, the separated A2-a2 domain dissociates spontaneously from the heterotrimeric FVIIIa resulting in the rapid loss of cofactor activity. We speculated that the requirement for cleavage at Arg³⁷² might be obviated by the insertion of an optimized linker sequence between A1-a1 and A2-a2 domains on an uncleavable Gln³⁷² backbone. To investigate this possibility, we prepared cDNA constructs of B-domain deleted FVIII variants; FVIII wild-type (FVIII_WT), FVIII_372Q, and FVIII_372Q followed by a rigid (Ala-Pro)₅ linker sequence (FVIII_372Q-AP₅). All three FVIII constructs were stably transfected into BHK cells and high expressing clones were selected by one stage aPTT and western blotting of expression media. Selected stable clones were further expanded to collect 15L of expression media over 5-day period, and recombinant FVIII variants were purified using a three-step chromatographic approach. These FVIII variants were studied using SDS-PAGE, western blotting, aPTT assays, thrombin generation assay (TGA) and purified assays to assess kinetics of FX activation and spontaneous loss of cofactor activity. In contrast to FVIII_WT, FVIII_372Q and FVIII_372Q-AP₅ were completely resistant to cleavage at Gln³⁷² by thrombin, yielding bands corresponding to A1-a1-A2-a2 (90K) and A3-C1-C2 (73K). In one stage aPTT assays, FVIII_372Q showed prolonged clotting times with specific activity in the range of 200-400 U/mg, while FVIII_WT and FVIII_372Q-AP₅ displayed comparable clotting times with specific activities ranging between 8000-10000 U/mg and 4500-5500 U/mg, respectively. In TGA initiated with either 0.1 pM tissue factor or 1 pM factor XIa, both FVIII_WT and FVIII_372Q-AP₅ displayed similar TGA profiles. In steady state kinetic studies of FX activation using limiting concentrations of factor IXa, saturating concentrations of FVIII variants pretreated with thrombin, membranes and increasing concentrations of FX, the cofactor function of thrombin-cleaved FVIII_372Q was severely impaired. However, despite lack of cleavage at Gln³⁷² in FVIII_372Q-AP₅, catalytic efficiency for FX activation by intrinsic tenase assembled by this variant was comparable to that seen with FVIIIa_WT. At the physiological concentration of FX, the initial velocity for Xa formation (v/E) for intrinsic tenase assembled with FVIIIa_372Q-AP₅ was within a factor of 2 of that observed with FVIIIa_WT while the rate observed with FVIIIa_372Q was >10-fold lower. Following rapid activation with thrombin, loss of cofactor function was significantly slower for FVIIIa_372Q-AP₅(t_1/2 ~ 10 min) compared to FVIIIa_WT (t_1/2 ~ 2 min). Our findings indicate that the requirement for cleavage at Arg³⁷² for the development of full FVIIIa cofactor function can be overcome by modulating the A1-A2 connector with an optimized linker sequence. Failure to yield an infinitely stable cofactor in the case of FVIIIa_372Q-AP₅ suggests that cleavage at Arg³⁷² does not solely explain the spontaneous loss of FVIIIa cofactor function.