Absence of hematopoietic tissue factor pathway inhibitor mitigates bleeding in mice with hemophilia

Abstract

Tissue factor pathway inhibitor (TFPI) blocks thrombin generation via the extrinsic blood coagulation pathway. Because the severe bleeding in patients with hemophilia occurs from deficiency of intrinsic blood coagulation pathway factor VIII or IX, pharmacological agents that inactivate TFPI and, therefore, restore thrombin generation via the extrinsic pathway, are being developed for treatment of hemophilia. Murine models of combined TFPI and factor VIII deficiency were used to examine the impact of TFPI deficiency on bleeding and clotting in hemophilia. In breeding studies, Factor VIII null (F8(-/-)) did not rescue the embryonic death of TFPI null (Tfpi(-/-)) mice. Tfpi(+/-) did not alter the bleeding phenotype of F8(-/-) mice. However, total inhibition of intravascular TFPI through injection of anti-TFPI antibody mitigated tail vein bleeding. Interestingly, tail blood loss progressively decreased at doses greater than needed to totally inhibit plasma TFPI, suggesting that inhibition of a sequestered pool of TFPI released at the injury site mitigates bleeding. Because TFPI is sequestered within platelets and released following their activation, the function of platelet TFPI was examined in F8(-/-) mice lacking hematopoietic cell TFPI that was generated by fetal liver transplantation. Blood loss after tail transection significantly decreased in Tfpi(+/-);F8(-/-) mice with hematopoietic Tfpi(-/-) cells compared with Tfpi(+/-);F8(-/-) mice with Tfpi(+/+) hematopoietic cells. Additionally, following femoral vein injury, Tfpi(+/-);F8(-/-) mice with Tfpi(-/-) hematopoietic cells had increased fibrin deposition compared with identical-genotype mice with Tfpi(+/+) hematopoietic cells. These findings implicate platelet TFPI as a primary physiological regulator of bleeding in hemophilia.

Fig. 1.

F8^−/− does not rescue the embryonic lethality of Tfpi^−/− mice. A total of 445 pups was genotyped from breeding of Tfpi^+/−;F8^−/− male and female mice, with a range of 21 to 67 pups on each embryonic day. The expected survival of Tfpi^−/− mice from this mating is 25%, if no embryonic death occurs. There is no difference in embryonic survival between Tfpi^−/−;F8^−/− mice (■) and Tfpi^−/−;F8^+/+ mice (●), originally published by Huang and coworkers (21).

Fig. 2.

Tfpi^+/− does not alter the bleeding phenotype of F8^−/− mice. (A) The TEG R value (time for clot initiation) was significantly prolonged in F8^−/− mice (▲) compared with F8^+/+ mice (◆) (P = 0.0026) or Tfpi^+/− mice (▼) (P = 0.018), but the presence of Tfpi^+/− in F8^−/− mice (■) did not decrease this prolongation (P = 0.59). (B) The TEG α angle (a measure of the kinetics of fibrin formation) was significantly decreased in Tfpi^+/−;F8^−/− mice (■) compared with Tfpi^+/− mice (▼) (P = 0.017), but Tfpi^+/−;F8^−/− mice (■) were not different from F8^−/− mice (▲) (P = 1). (C) In the 1-mm clip tail bleeding model, F8^−/− mice (▲) showed a trend to bleed more than F8^+/+ mice (◆) but it was not statistically significant (P = 0.23), whereas Tfpi^+/−;F8^−/− mice (■) had essentially identical blood loss as F8^−/− mice (▲) (P = 1). Similarly, there was no difference between Tfpi^+/− (▼) and Tfpi^+/+ (◆) mice.

Fig. 3.

Intravenous injection of a polyclonal anti-TFPI antibody mitigates bleeding in F8^−/− mice. (A) Plasma FXa activity in samples from F8^−/− mice treated with different i.v. doses of polyclonal anti-mouse TFPI antibody or a vehicle control shows essentially total inhibition of TFPI activity at all doses of antibody tested. Data are presented as mean ± SD. Max represents data for plasma samples obtained from vehicle control-treated mice that were spiked with 100 μg/mL of the anti-mouse TFPI antibody and is the maximum total inhibition of plasma TFPI activity. (B) Treatment of F8^−/− mice with increasing doses of a polyclonal anti-mouse TFPI antibody produces progressively smaller amounts of blood loss following 4-mm tail transection (test for linear trend, r² = 0.40, P = 0.000024).

Fig. 4.

Absence of hematopoietic cell TFPI (HC TFPI) mitigates bleeding in F8^−/− mice. Tfpi^+/−;F8^−/− mice transplanted with Tfpi^−/− hematopoietic cells (▲) lost significantly less blood than Tfpi^+/−;F8^−/− mice transplanted with Tfpi^+/+ hematopoietic cells (■) (P = 0.00015) following 1-mm tail transection. Tfpi^+/+;F8^−/− mice transplanted with Tfpi^−/− hematopoietic cells (▼) also bled significantly less than Tfpi^+/−;F8^−/− mice transplanted with Tfpi^+/+ hematopoietic cells (■) (P = 0.033). Finally, there was a trend for Tfpi^+/−;F8^−/− mice transplanted with Tfpi^−/− hematopoietic cells (▲) to bleed less than Tfpi^+/+;F8^−/− mice transplanted with Tfpi^−/− hematopoietic cells (▼) (P = 0.067). With 80% power and a common SD of 200, this experiment had the ability to detect a difference in means of 300 units or more.

Fig. 5.

Absence of hematopoietic cell TFPI causes increased thrombus volume in F8^−/− mice in a femoral vein electrolytic injury model. (A) Accumulation of platelets and (B) fibrin at the site of injury in mice in Tfpi^+/−;F8^−/− mice transplanted with Tfpi^+/+ hematopoietic cells (□; n = 9) or with Tfpi^−/− hematopoietic cells (X; n = 15). Data points were determined every 2 min, with error bars showing the SEM. The fibrin accumulation in mice lacking hematopoietic cell TFPI was significantly greater than in mice transplanted with Tfpi^+/+ hematopoietic cells at 10–60 min (P < 0.05).