Response to TFPI-inhibition in severe factor v deficiency with a novel compound heterozygous mutation

Abstract Introduction Coagulation factor V (FV) plays a central role in blood clot formation. FV has both procoagulant and anticoagulant properties making it a key regulator of thrombin generation. Congenital deficiency of FV is rare (1 in 1 million births) and presents with mild to severe bleeding complications in the affected individuals. Low to undetectable antigen levels and low functional activity is a hallmark of homozygous or compound heterozygous mutations in the F5 gene. Heterozygous individuals have about 50% normal FV antigen expression and are asymptomatic. Prior studies have shown that plasma tissue factor pathway inhibitor-alpha (TFPIα) levels correlate with plasma FV. It is thought in FV deficiency reduced levels of TFPIα may blunt the bleeding phenotype making it an important and unique modifier of FV deficiency. Patient history Here, we describe a case of a 14 year old male with severe FV deficiency (<1% FV activity). He had intracranial hemorrhage during infancy and has been admitted multiple (8) times for bleeding episodes and treated with plasma transfusions. Bleeds were either from sports participation or spontaneous joint and muscle bleeds. His mother is clinically asymptomatic with no bleeds despite past caesarean births and has FV level of ~50%. Methods Genomic DNA from the consented patient was isolated from peripheral blood and then outsourced to PreventionGenetics (USA) for a F5 gene testing and analyses using Next-Gen sequencing. FV activity was determined with standard one-stage clotting assay. Plasma and platelet FV antigens were measured and visualized by immunoassays and western blotting analyses using FV-specific polyclonal and monoclonal (mAb) antibodies, respectively. Free plasma protein S was measured with a commercial ELISA kit (Asserachrom®, Stago). Thrombin generation assays (TGA) were triggered with low tissue-factor concentration and thrombin generation assessed with anti-TFPIα mAbs and in mixing studies with pooled normal plasma. Results Genetic analyses revealed compound heterozygous mutations in the F5 gene: one in exon 3 (c254T>G) resulting in Leu-57 to Arg (Leu57Arg; legacy numbering) change and a second in exon 13 (A>C/T) leading to a nonsense mutation (Leu906*; legacy numbering). Plasma prothrombin time was 46.6 (reference range: 11.1-13.5 sec) and activated partial thromboplastin time was 156.2 (reference range: 25.0-37.0 sec). Plasma FV antigen was 3.3% of normal measured by ELISA and undetectable via western blotting analyses. Since TFPI is a known modifier of FV, we observed that total plasma, and platelet TFPIα levels were 62%, and 46%, of normal, respectively, and free protein S was normal (130.3 ± 0.3 nM). It is established that inhibiting TFPI's major function enhances thrombin generation in the context of bleeding. To assess this, we first blocked TFPI function using an anti-K2 mAb in a TGA. We found that the effect on peak thrombin was undetectable and similar to the unmodified patient plasma. However, adding a cocktail of anti-TFPI mAbs targeting each of its domains (K1, K2, K3 and C-term domains) to the patient's plasma shortened the lag time and enhanced peak thrombin by ~30% of normal suggesting blocking all molecular interactions of TFPI in the context of reduced FV antigen has a detectable procoagulant effect. Furthermore, in mixing studies assessed by TGA, 10% pooled normal plasma was enough to restore both peak thrombin and lag time in the patient's plasma to levels seen in normal healthy control plasma. Conclusions We identified a compound heterozygous mutation in a severe FV deficiency patient including a previously reported Leu57Arg variant and a novel Leu906* nonsense variant that is not reported in the clinical F5 gene database. In silco analyses suggest that a hydrophobic to basic amino acid substitution at position 57 may alter protein folding and function. A termination at Leu906* leads to nonsense mediated decay of mRNA and no translation consistent with reduced FV antigen in the patient. Further, we demonstrated in this study that complete inhibition of TFPI function, an important modifier of FV, promotes thrombin generation at low plasma FV levels in severe FV deficiency.