Tissue Factor Pathway Inhibitor (TFPI) Beta and Gamma Isoforms Contribute to Bleeding in Hemophilia Mice

Key Points:

1. Expression of any single TFPI isoform (α, β, or γ) is sufficient for mouse survival.
2. Two-Kunitz domain TFPI isoforms, anchored β and circulating γ, each contribute to hemophilia bleeding.
3. In vivo, TFPIβ is a more potent anticoagulant than TFPIα.

Upon injury, tissue factor (TF) triggers coagulation by complexing with factor (F)VIIa to activate FIX and FX, leading to thrombin generation and a fibrin clot. Tissue factor pathway inhibitor limits TF-triggered coagulation. Humans and mice express transcripts encoding for 3-Kunitz domain TFPIα and membrane-anchored 2-Kunitz TFPIβ. Mice also express transcripts encoding untethered, 2-Kunitz TFPIγ. In humans, proteolysis of TFPIα and/or β produces “γ-like” 2-Kunitz TFPI.

Hemophilia A and B, caused by deficiency in factors VIII and IX, respectively, are congenital X-linked recessive bleeding disorders that affect an estimated 1.1 million males worldwide. Hemophilia can cause excessive bleeding spontaneously or in response to trauma that can be life-threatening or significantly decrease the quality of life. Approved treatments for hemophilia include factor replacement, bypassing agents, the bispecific antibody Emicizumab, and recently approved gene therapies. Also in development are agents that reduce endogenous anticoagulant activity to counter the procoagulant deficiency of hemophiliacs, including monoclonal antibodies (Mab) to TFPI. These Mab target all TFPI isoforms to improve coagulation and correct bleeding in hemophilia patients, but with some demonstrated thrombosis risk.

We investigated if TFPI-isoform-specific inhibition could provide equivalent efficacy which might reduce thrombotic risk. We generated TFPI-isoform-specific exon deletions in mice bred into hemophilia (FVIII-null) background. We find mice expressing any single TFPI isoform (α, β, or γ) appear healthy and reproduce. Our in vivo tail-vein re-bleeding assay is based on serial clot disruptions over 15 minutes, which we have shown is sensitive to anticoagulant and antiplatelet treatment, as well as factor replacement therapy. In this assay, we find C57Bl/6 WT mice clot 25.6 + 0.8 times (mean + SEM), whereas FVIII-null hemophilia mice clot 5.3 +0.4 times. In hemophilia mice, TFPIα-specific deletion is without impact, forming clots 5.1 + 0.8 times, whereas TFPIβ-specific or TFPIγ-specific deletion improves clot formation to 16.8 + 2.0 and 15.2 + 1.5 times, respectively (p<0001). Even hemophilia mice with heterozygotic deletion of the β exon form clots 11.8 + 0.8 times (p<0001). Thus, in vivo 2-Kunitz TFPI isoforms, anchored β and untethered γ, each provides greater anticoagulant activity and impact on bleeding than 3-Kunitz TFPIα.

Ex vivo plasma-based thrombin generation assays show that TFPIγ provides more anticoagulant activity than TFPIα. As shown in the figure, the deletion of TFPIγ increases thrombin generation to a greater extent than the deletion of TFPIα. The addition of an anti-TFPI antibody that effectively blocks TFPI activity shows a significant increase in endogenous thrombin potential in the plasma of TFPIαβ-deleted (γ-only) mice (370 nM.min), versus TFPIβγ-deleted (α-only) plasma (70 nM.min) and TFPIαγ-deleted (β-only) mice plasma (22 nM.min), (p<0.001). As expected, membrane-anchored TFPIβ is not detected in plasma.

It is well established that 3-Kunitz TFPIα is a potent anticoagulant in plasma-based clotting time assays, while truncated 2-Kunitz TFPI, like mouse TFPIγ, appears inactive. Based on such assays, it was unexpected that TFPIγ-only expressing mice thrive, and in vivo, TFPIα is not the dominant endogenous anticoagulant. If mouse models are to be useful in providing insights into human hemostasis and TF-associated diseases, then it is critical to understand the similarities and differences between mice and humans. As humans do not express TFPIγ, the observed impact of depleting circulating 2-Kunitz TFPI on bleeding and thrombin generation in hemophilic mice is unlikely to translate to man. By contrast, our results demonstrating that, in vivo, TFPIβ is a more potent anticoagulant than TFPIα in mice has potential therapeutic implications in humans.