Human TFPI blocking peptide

Factor V Leiden (F5(L)) is a common genetic risk factor for venous thromboembolism in humans. We conducted a sensitized N-ethyl-N-nitrosourea (ENU) mutagenesis screen for dominant thrombosuppressor genes based on perinatal lethal thrombosis in mice homozygous for F5(L) (F5(L/L)) and haploinsufficient for tissue factor pathway inhibitor (Tfpi(+/-)). F8 deficiency enhanced the survival of F5(L/L) Tfpi(+/-) mice, demonstrating that F5(L/L) Tfpi(+/-) lethality is genetically suppressible. ENU-mutagenized F5(L/L) males and F5(L/+) Tfpi(+/-) females were crossed to generate 6,729 progeny, with 98 F5(L/L) Tfpi(+/-) offspring surviving until weaning. Sixteen lines, referred to as "modifier of Factor 5 Leiden (MF5L1-16)," exhibited transmission of a putative thrombosuppressor to subsequent generations. Linkage analysis in MF5L6 identified a chromosome 3 locus containing the tissue factor gene (F3). Although no ENU-induced F3 mutation was identified, haploinsufficiency for F3 (F3(+/-)) suppressed F5(L/L) Tfpi(+/)- lethality. Whole-exome sequencing in MF5L12 identified an Actr2 gene point mutation (p.R258G) as the sole candidate. Inheritance of this variant is associated with suppression of F5(L/L) Tfpi(+/-) lethality (P = 1.7 x 10(-6)), suggesting that Actr2(p.R258G) is thrombosuppressive. CRISPR/Cas9 experiments to generate an independent Actr2 knockin/knockout demonstrated that Actr2 haploinsufficiency is lethal, supporting a hypomorphic or gain-of-function mechanism of action for Actr2(p.R258G). Our findings identify F8 and the Tfpi/F3 axis as key regulators in determining thrombosis balance in the setting of F5L and also suggest a role for Actr2 in this process.

Replacement therapy with missing factor (F) VIII or IX in haemophilia patients for bleed management and preventative treatment or prophylaxis is standard of care. Restoration of thrombin generation through novel mechanisms has become the focus of innovation to overcome limitations imposed by protein replacement therapy. Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type serine protease inhibitor that regulates tissue factor (TF)-induced coagulation through a FXa-dependent feedback inhibition of the TF.FVIIa complex in plasma and on endothelial surfaces. Concizumab is a monoclonal, humanised antibody, specific for the second Kunitz domain of TFPI that binds and inhibits FXa, abolishing the inhibitory effect of TFPI. Concizumab restored thrombin generation in FVIII and FIX deficient plasmas and decreased blood loss in a rabbit haemophilia model. Phase 1 single and multiple dose escalation studies in haemophilia patients demonstrated a dose dependent decrease in TFPI levels and a pro-coagulant effect with increasing d-dimers and prothrombin fragment 1 + 2. A dose dependent increase in peak thrombin and endogenous thrombin potential was observed with values in the normal range when plasma TFPI levels were nearly undetectable. A few haemophilia patients in the highest dose cohorts with complete inhibition of plasma TFPI showed a decreased fibrinogen concentration with normal levels of anti-thrombin and platelets and no evidence of thrombosis. Pharmacokinetic parameters were influenced by binding to the target (TFPI), demonstrating target mediated drug disposition. A trend towards decreasing bleeding tendency was observed and this preventative effect is being studied in Phase 2 studies with additional data gathered to improve our understanding of the therapeutic window and potential for thrombosis.