Mechanism of the Potent Activated Protein C Resistance of Novel Factor V Mutation with W1920R (FV Nara) Relative to R506Q (FV Leiden)

Factor V (FV) functions both as procoagulant and anticoagulant factors. FV R506Q (FV Leiden) showing activated protein C resistance (APCR) is popular among Caucasians, whilst it has not been reported in Asians. Recently, we have reported a Japanese boy with deep venous thrombosis (DVT) who has a novel FV W1920R mutation (FV Nara, ISTH 2009, 2011). Although he showed APCR in activated partial thromboplastin time (APTT)-based assay, low levels of FV activity (10%) and antigen (40%) made it difficult to explain his severe DVT (both lower extremities and inferior vena cava). Here, we show the detailed mechanisms of his thrombotic diathesis via APCR relative to FV Leiden. Methods: We performed the detection of PC pathway inhibition with Thrombopath^® (Protac-induced coagulation inhibition %; PiCi%) or the detection of APCR with calibrated automated thrombogram (CAT) in patient's plasma. FV-deficient plasmas containing varying concentrations of FV wild-type (WT) or W1920R were evaluated by an APCR-assay that specifically can measure the APC cofactor activity of FV in activated FVIII (FVIIIa) inactivation and by the APTT-based assay that probes both the susceptibility and APC cofactor components (Castoldi E, 2004). Recombinant FV proteins (FV-WT, -R506Q, and -W1920R) under pMT2/FV vector were expressed in HEK293 cells. In purified assays, activated FV (FVa) was inactivated by APC in the presence of protein S (PS). FVIIIa were also inactivated by APC/PS in the presence of FV. Cleavages of FV heavy chain were observed in SDS-PAGE and Western blotting. Results: PiCi%, a decrease ratio of thrombin generation by the addition of PC activator (protac) was low in patient's plasma (protac absent/present 794/221 mOD/min; PiCi% 72.2%) (v.s. normal plasma absent/present 834/76.1 mOD/min; PiCi% 90.9%). In CAT using platelet-rich plasmas, peak thrombin level of patient was lower than control (169 and 191 nM), but was greater in the presence of 40 nM APC (103 and 71 nM). In FV-deficient plasma containing FV WT or W1920R, W1920R exhibited no cofactor activity in FVIIIa inactivation, likely supporting the contribution to W1920R-associated APCR more significantly rather than poor susceptibility to APC. In purified assays, FVa (8 nM) activity in FV-WT, -R506Q and -W1920R after 5-min reaction with APC (25 pM), PS (30 nM), PL (20 μM) was decreased to 2.6%, 16.7%, and 62.8%, respectively. SDS-PAGE analysis for FVa heavy chain revealed little cleavage of Gln⁵⁰⁶ in FV-R506Q and markedly delayed cleavage of Arg⁵⁰⁶ in FV-W1920R. Of surprise, cleavage of Arg³⁰⁶ was little observed in FV-W1920R. In addition, FVIIIa (10 nM) activity after 20-min reaction with APC (0.5 nM), PS (5 nM), PL (20 μM) and FV (0.5 nM) in FV-WT, -R506Q, and -W1920R was diminished to 18.0%, 52.8% and >95% of those without FV, respectively. Although Trp¹⁹²⁰ in FV is close to PL-binding site of the C1 domain, the binding ability of FV-W1920R to immobilized PL retained ∼70-80% of that of FV-WT. Conclusion: The novel FV mutant, W1920R possessed the potent APCR relative to FV Leiden, by reducing the susceptibility of FVa to APC-mediated inactivation and/or by interfering with the FV cofactor activity in APC-catalyzed FVIIIa inactivation. This mechanism might be associated with the impaired cleavages of Arg⁵⁰⁶ and Arg³⁰⁶ due to the conformational change of this FV mutant.

Nogami:Bayer Award 2009: Research Funding.