Tissue factor (TF) pathway inhibitor (TFPI) is a well-characterized activated factor X (FXa)-dependent inhibitor of TF-initiated coagulation produced in two alternatively spliced isoforms, TFPIα and TFPIβ. The TFPIα C terminus has a basic sequence nearly identical to a portion of the factor V (FV) B domain necessary for maintaining FV in an inactive conformation via interaction with an acidic region of the B domain. We demonstrate rapid inhibition of prothrombinase by TFPIα mediated through a high-affinity exosite interaction between the basic region of TFPIα and the FV acidic region, which is retained in FXa-activated FVa and platelet FVa. This inhibitory activity is not mediated by TFPIβ and is lost upon removal of the acidic region of FVa by thrombin. The data identify a previously undescribed, isoform-specific anticoagulant function for TFPIα and are a unique description of physiologically relevant inhibition of prothrombinase. These findings, combined with previous descriptions of differential expression patterns of TFPIα and TFPIβ in platelets and endothelial cells, suggest that the TFPI isoforms may act through distinct mechanisms to inhibit the initial stages of intravascular coagulation, with TFPIβ acting to dampen TF expressed on the surface of vascular cells, whereas TFPIα dampens the initial prothrombinase formed on the activated platelet surface.hemophilia | bleeding | thrombosis

Recent studies of the anticoagulant activities of the tissue factor (TF) pathway inhibitor (TFPI) isoforms, TFPIα and TFPIβ, have provided new insight into the biochemical and physiological mechanisms that underlie bleeding and clotting disorders. TFPIα and TFPIβ have tissue-specific expression patterns and anticoagulant activities. An alternative splicing event in the 5′ untranslated region allows for translational regulation of TFPIβ expression. TFPIα has 3 Kunitz-type inhibitor domains (K1, K2, K3) and a basic C terminus, whereas TFPIβ has the K1 and K2 domains attached to a glycosylphosphatidyl inositol–anchored C terminus. TFPIα is the only isoform present in platelets, whereas endothelial cells produce both isoforms, secreting TFPIα and expressing TFPIβ on the cell surface. TFPIα and TFPIβ inhibit both TF–factor VIIa–dependent factor Xa (FXa) generation and free FXa. Protein S enhances FXa inhibition by TFPIα. TFPIα produces isoform-specific inhibition of prothrombinase during the initiation of coagulation, an anticoagulant activity that requires an exosite interaction between its basic C terminus and an acidic region in the factor Va B domain. Platelet TFPIα may be optimally localized to dampen initial thrombin generation. Similarly, endothelial TFPIβ may be optimally localized to inhibit processes that occur when endothelial TF is present, such as during the inflammatory response.

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 Tissue factor pathway inhibitor (TFPI) dampens the initiation of blood coagulation by inhibiting two potent procoagulant complexes, tissue factor–factor VIIa (TF–FVIIa) and early forms of prothrombinase. TFPI isoforms, TFPIα and TFPIβ, result from alternative splicing of mRNA, producing distinct C-terminal ends of the two proteins. Both isoforms inhibit TF–FVIIa, but only TFPIα can inhibit early forms of prothrombinase by binding of its positively charged C-terminus with high affinity to the acidic B-domain exosite of FVa, which is generated upon activation by FXa. TFPIα and TFPIβ are produced in cultured human endothelial cells, while platelets contain only TFPIα. Knowledge of the anticoagulant mechanisms and tissue expression patterns of TFPIα and TFPIβ have improved our understanding of the phenotypes observed in different mouse models of TFPI deficiency, the east Texas bleeding disorder, and the development of pharmaceutical agents that block TFPI function to treat hemophilia.

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.