The incorporation of blood-borne forms of tissue factor (TF) into a growing blood clot is necessary for normal fibrin generation and stabilization of the blood clot. Tissue factor pathway inhibitor (TFPI) is the primary physiologic inhibitor of tissue factor and is present within platelets. Expression of TFPI on the platelet surface may be the optimal location for it to abrogate blood-borne TF activity that incorporates within the blood clot, balancing the need for adequate hemostasis while preventing development of occlusive thrombosis. TFPI is produced by megakaryocytes but is not expressed on the platelet surface. Activation of platelets with thrombin receptor activation peptide does not cause release or surface expression of TFPI, demonstrating that TFPI is not stored within platelet ␣ granules. TFPI is expressed on the platelet surface following dual-agonist activation with convulxin plus thrombin to produce coated platelets. In association with its expression on the surface of coated platelets TFPI is also released in microvesicles or as a soluble protein. IntroductionVascular injury results in the exposure of collagen and tissue factor, present within the vessel wall, to flowing blood. Platelets rapidly adhere to the collagen within the subendothelial tissue of the injured vessel wall where they are activated; subsequently, platelet aggregation provides an initial barrier against blood loss. Tissue factor within the subendothelial tissue binds to factor VIIa, leading to the production of thrombin, which further activates platelets and generates fibrin. Platelets exposed to dual-agonist stimulation with collagen plus thrombin form a distinct subpopulation of platelets, called coated platelets, that express high levels of procoagulant proteins, including factor V, fibrinogen, fibronectin, and VWF, providing a procoagulant surface that supports further fibrin generation. [1][2][3] In this manner, collagen and tissue factor work synergistically at the site of vascular injury to produce a plateletfibrin plug that closes the wound and prevents severe hemorrhage.In addition to its presence within the subendothelial tissues of the vessel wall, tissue factor is also present on circulating microvesicles released from activated leukocytes or endothelial cells. Incorporation of these tissue factor-bearing microvesicles into the blood clot is thought to be necessary for effective stabilization within the vasculature. [4][5][6] The recruitment of tissue factor-bearing microvesicles into growing thrombi is mediated by interactions between P-selectin glycoprotein ligand-1 present on the microvesicles and P-selectin expressed on the surface of activated platelets within the blood clot. 7 It has been demonstrated that the tissue factor-bearing microvesicles can fuse with activated platelets. 8 In addition, splicing of tissue factor pre-mRNA within activated platelets is another potential mechanism for expression of tissue factor on the platelet surface. 9 However, overexpression of tissue factor or inadequate regulation o...

Summary. Background: Mouse tissue factor pathway inhibitor (TFPI) is produced in three alternatively spliced isoforms that differ in domain structure and mechanism for cell surface binding. Tissue expression of TFPI isoforms in mice was characterized as an initial step for identification of their physiological functions. Methods and Results: Sequence homology demonstrates that TFPIa existed over 430 Ma while TFPIb and TFPIc evolved more recently. In situ hybridization studies of heart and lung did not reveal any cells exclusively expressing a single isoform. Although our previous studies have demonstrated that TFPIa mRNA is more prevalent than TFPIb or TFPIc mRNA in mouse tissues, western blot studies demonstrated that TFPIb is the primary protein isoform produced in adult tissues, while TFPIa is expressed during embryonic development and in placenta. Consistent with TFPIb as the primary isoform produced within adult vascular beds, the TFPI isoform in mouse plasma migrates like TFPIb in SDS-PAGE and mice have a much smaller heparin-releasable pool of plasma TFPIa than humans. Conclusions: The data demonstrate that alternatively spliced isoforms of TFPI are temporally expressed in mouse tissues at the level of protein production. TFPIa and TFPIb are produced in embryonic tissues and in placenta while adult tissues produce almost exclusively TFPIb.

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.hemostasis | Kunitz | coagulopathy

To cite this article: Maroney SA, Cunningham AC, Ferrel J, Hu R, Haberichter S, Mansbach CM, Brodsky RA, Dietzen DJ, Mast AE. A GPI-anchored co-receptor for tissue factor pathway inhibitor controls its intracellular trafficking and cell surface expression. J Thromb Haemost 2006; 4: 1114-24.Summary. Background: Tissue factor pathway inhibitor (TFPI) lacks a membrane attachment signal but it remains associated with the endothelial surface via its association with an, as yet, unidentified glycosyl phosphatidylinositol (GPI)-anchored coreceptor. Objectives/methods: Cellular trafficking of TFPI within aerolysin-resistant ECV304 and EA.hy926 cells, which do not express GPI-anchored proteins on their surface, was compared with their wild-type counterparts. Results and conclusions: Although aerolysin-resistant cells produce normal amounts of TFPI mRNA, TFPI is not expressed on the cell surface and total cellular TFPI is greatly decreased compared with wild-type cells. Additionally, normal, not increased, amounts of TFPI are secreted into conditioned media indicating that TFPI is degraded within the aerolysin-resistant cells. Confocal microscopy and studies using metabolic inhibitors demonstrate that aerolysin-resistant cells produce TFPI and transport it into the Golgi with subsequent degradation in lysosomes. The experimental results provide no evidence that cell surface TFPI originates from secreted TFPI that binds back to a GPI-anchored protein. Instead, the data suggest that TFPI tightly, but reversibly, binds to a GPI anchored co-receptor in the ER/Golgi. The co-receptor then acts as a molecular chaperone for TFPI by trafficking it to the cell surface of wild-type cells or to lysosomes of aerolysin-resistant cells. TFPI that escapes co-receptor binding is secreted through the same pathway in both wild-type and aerolysin-resistant cells. The data provide a framework for understanding how TFPI is expressed on endothelium.

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.