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

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.

Caveolae optimize tissue factor-Factor VIIa inhibitory activity of cell-surface-associated tissue factor pathway inhibitor.

TFPI (tissue factor pathway inhibitor) is an anticoagulant protein that prevents intravascular coagulation through inhibition of fXa (Factor Xa) and the TF (tissue factor)-fVIIa (Factor VIIa) complex. Localization of TFPI within caveolae enhances its anticoagulant activity. To define further how caveolae contribute to TFPI anticoagulant activity, CHO (Chinese-hamster ovary) cells were co-transfected with TF and membrane-associated TFPI targeted to either caveolae [TFPI-GPI (TFPI-glycosylphosphatidylinositol anchor chimaera)] or to bulk plasma membrane [TFPI-TM (TFPI-transmembrane anchor chimaera)]. Stable clones had equal expression of surface TF and TFPI. TX-114 cellular lysis confirmed localization of TFPI-GPI to detergent-insoluble membrane fractions, whereas TFPI-TM localized to the aqueous phase. TFPI-GPI and TFPI-TM were equally effective direct inhibitors of fXa in amidolytic assays. However, TFPI-GPI was a significantly better inhibitor of TF-fVIIa than TFPI-TM, as measured in both amidolytic and plasma-clotting assays. Disrupting caveolae by removing membrane cholesterol from EA.hy926 cells, which make TFPIα, CHO cells transfected with TFPIß and HUVECs (human umbilical vein endothelial cells) did not affect their fXa inhibition, but significantly decreased their inhibition of TF-fVIIa. These studies confirm and quantify the enhanced anticoagulant activity of TFPI localized within caveolae, demonstrate that caveolae enhance the inhibitory activity of both TFPI isoforms and define the effect of caveolae as specifically enhancing the anti-TF activity of TFPI.

Murine hematopoietic cell tissue factor pathway inhibitor limits thrombus growth.

OBJECTIVE: Tissue factor (TF)-factor VIIa initiates blood coagulation and is found on microparticles that accumulate within intravascular thrombi. Tissue factor pathway inhibitor (TFPI), a factor Xa (fXa)-dependent inhibitor of TF-factor VIIa, is produced by megakaryocytes and is present in platelets. We sought to determine the role of platelet TFPI in regulation of thrombus growth. METHODS AND RESULTS: Western blot analyses demonstrated that murine platelets produce TFPIα, the most evolutionarily conserved alternatively spliced isoform of TFPI. A mouse model of hematopoietic cell TFPI deficiency was developed by transplanting irradiated TFPI(+/-) mice with TFPI(-/-) fetal liver cells. Platelets from transplanted mice totally lack TFPI inhibitory activity. An electrolytic vascular injury model was used to assess thrombus growth in the femoral vein and carotid artery. Mice lacking hematopoietic TFPI developed larger femoral vein and carotid artery thrombi than TFPI(+/-) mice transplanted with TFPI(+/+) hematopoietic cells, as evidenced by increased platelet accumulation. CONCLUSIONS: Hematopoietic TFPI limits thrombus growth following vascular injury. Because platelets are the primary hematopoietic cell accumulating within a growing thrombus, these findings suggest that TFPI present within platelets functions to limit intravascular thrombus growth, likely through inhibition of the procoagulant activity of blood borne TF.