TFPI

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Primarily, tissue factor pathway inhibitor (TFPI) binds to and inactivates FXa. In a second step TFPI/... 

FXa complexes bind to and neutralize tissue factor/ FVIIa complexes, the key starting point of the extrinsic clotting cascade (see earlier) (Fig. 7). Heparin is able to enhance this reaction by direct binding to the complex and by releasing TFPI from the unaltered vessel wall, which then can access the TF-exposing surface. 

Tissue factor pathway inhibitor (TFPI) is a major physiologic inhibitor of coagulation. It is a protein that circulates in the blood associated with lipoproteins. TFPI directly inhibits factor Xa by binding to the enzyme near its active site. This factor Xa-TFPI complex then inhibits the factor Vlla-tissue factor complex. 

FIGURE 7-4. Coagulation cascade. AT, antithrombin HCII, heparin cofactor II TFPI, tissue factor pathway inhibitor. (Reproduced from Haines ST, Zeolla M, Witt DM. Venous thromboembolism. In ... 

HCII heparin co-factor II TFPI tissue factor pathway inhibitor... 

Tissue factor pathway inhibitor (TFPI), a 42-kDa protein with three Kunitz domains, is a potent inhibitor of coagulation. It inhibits tissue factor-factor Vila complex upon binding to the active site of Kunitz domain one. Factor Xa is inhibited upon binding to the active site of the second Kunitz domain of TFPI (27). 

A major portion of TFPI is bound to the endothelial surface and can be released from the cells by heparin (28). Platelets contain very small amounts of TFPI, which are released when platelets are activated (29). Figure 6 illustrates the mechanism of inhibition by TFPI. 

Fig. 6. Mechanism of inhibition of tissue factor pathway inhibitor (TFPI). Kunitz domain 1 (Dl) inhibits TF-VIIa complex. Domain 2 (D2) inhibits Xa.

A specific immunoassay for measuring two-chain factor VIIa levels in plasma has been developed to identify activation of factor VII in patients with acute coronary syndromes suchs as myocardial infarction and unstable angina (12). Because regulation of factor VIIa is believed to be mediated by tissue factor pathway inhibitor (TFPI), its measurement is also useful in assessing thombotic and cardio-vasular disorders. Because TFPI is released by heparin, its measurement is also useful in assessing the efficacy of heparin and endothelial cell function (93). 

Predicted secondary structure of tissue factor pathway inhibitor (TFPI) showing the Factor Xa and Factor Vila inhibitory domains. The arrows point to the PI sites. 

Examination of models of BPTI-mutants bound to Factor Xa show the L-amino acids in the P3 position project into solvent. In the Factor Xa cleavage sites in thrombin these residues are polar and acidic (Glu, Asp) they are polar and basic in antistasin (Arg), and polar and neutral in Ecotin (Ser). The exception is TFPI- II, with this position occupied by lie. The BPTI random mutant results are consistent with the TFPI-II case and show a preference for aliphatics or aromatics in this position. There is a hydrophobic pocket in the enzyme, formed by Trp215, Tyr"5 and Phe174, that would be accessible to a D-residue in this position. 

The accessibility of the S2 pocket of the enzyme by P2 groups would be expected to be influenced by the orientation of Tyr". As the x-ray structure shows its position this residue puts severe limitations on the size of the P2 group. Consistent with this is the observation that Gly is the sole residue in the FXa thrombin cleavage sites. Little information is available from the BPTI mutants, TFPI-II, or antistasin, which all have a structural requirement for Cys at this position. Synthetic compounds show, however, that in potent inhibitors large bulky aromatics are, in fact, allowed at this position, a situation that requires Tyr99 to move out of the way [75]. 

In the P 1 position, the natural cleavage sites use Thr and lie while Ecotin has Met. In contrast to thrombin, FXa lacks the 60-insertion loop and can accommodate large groups at this position. The BPTI mutants, however, are forced to use a small residue (Ala) because of steric hindrance from the Cys58-Cys42 group residue 61 in the enzyme. The inhibitor TFPI-II has a Gly at PT. 

See color plate.) The primar/ hemostatic response. Following loss of vascular integrity platelets adhere to subendothelial wall matrix, which triggers their activation. Abbreviations HT, hydroxy tryptamine TFPI, tissue factor pathway inhibitor. 

See color plate.) The coagulation cascade. The formation of fibrin strands is the result of a sequential activation of a number of enzymes. The hemostatic balance is maintained by endogenous inhibitors such as AT, HC-II, and TFPI. Abbreviations AT, antithrombin TFPI, tissue factor pathway inhibitor. 

Release of nitric oxide, TFPI and other substances to mediate various functions... 

In normal tissues of the vasculature, TFPI is produced by megakaryocytes and the endothelium (102). Once produced, this TFPI is stored in three intravascular pools. These pools are located in the plasma, in platelets, and bound to the endothelium (103). The smallest pool of TFPI is found in the platelets, accounting for less than 2.5% of the intravascular total. This small pool of TFPI is released upon platelet activation (104). 10% to 50% of the intravascular TFPI is in the plasma. Most plasma-based TFPI is bound to plasma lipoproteins (104,105). Approximately 5% of the plasma pool of TFPI circulates in the free form (103,104,105). The lipoprotein-bound TFPI is reported to be of relatively low inhibitory activity (104). The largest pool of TFPI is found bound to the endothelial surface (103,104,106). This pool can account for 50% to 90% of the total intravascular TFPI,... 

The TFPI pool bound to the endothelium has been shown to be heparin releasable in a number of studies (104,106), Venous occlusion and agents such as I-deamino-8-D-arginine vasopressin (DDAVP) that induce exocytosis of endothelial granular proteins do not cause the release of TFPI (106). Repeated heparin administration is observed to release... 

TFPI, when administered to rabbits, has been shown to have an antithrombotic effect when thromboplastin was used as a thrombogenic challenge (I 10). TFPI was also shown to be an effective inhibitor when thrombosis was induced in rabbit jugular veins by endothelial destruction and restricted blood flow. The antithrombotic and antiprotease actions of TFPI have been tested in several other animal models. Warn-Cramer et al. investigated the effect of immunodepletion of TFPI in factor Vila and Xa induced coagulation in rabbits (III). These rabbits were observed to be sensitized to the procoagulant effects of factor Vila, but not factor Xa in the absence of factor Vila. Two studies have indicated that TFPI administration reduces the lethal effects of . coli administration in a septic shock model in baboons (I 12). These studies also indicated that TFPI may have an anti-inflammatory effect, as an attenuation of the IL-6 response was also observed. Administration of TFPI has been observed to prevent... 

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