Contribution of Regions Distal to Glycine-160 to the Anticoagulant Activity of Tissue Factor Pathway Inhibitor

Abstract

The functions of the first two Kunitz domains of tissue factor pathway inhibitor (TFPI) are well defined as active site-directed inhibitors of factor VIIa and factor Xa. The anticoagulant properties of the third Kunitz domain and C-terminal region were probed using altered forms of TFPI. TFPI-160 contains the first two Kunitz domains. K1K2C contains the first two Kunitz domains and the basic C-terminus. Neither TFPI-160 nor K1K2C contains the third Kunitz domain. In amidolytic assays containing calcium, TFPI-160 is a less potent inhibitor of factor Xa than TFPI. However, addition of the C-terminus in K1K2C nearly restores inhibitory activity to that of TFPI, indicating that the third Kunitz domain is not required for direct inhibition of factor Xa. When compared in assays containing phospholipids and factor Va, K1K2C and TFPI-160 are poor inhibitors compared to TFPI, demonstrating that the third Kunitz domain is required for the full anticoagulant activity of TFPI. TFPI was further characterized in amidolytic assays performed with Gla-domainless factor Xa and in prothrombin activation assays using submicellar concentrations of short-chain phospholipids (C6PS). TFPI and K1K2C are worse inhibitors of Gla-domainless factor Xa, compared to wild-type factor Xa, while TFPI-160 inhibits both forms of factor Xa equally, suggesting a C-terminus/Gla domain interaction. TFPI is a potent inhibitor of thrombin generation by prothrombinase assembled with C6PS, while TFPI-160 and K1K2C are not. Conversely, TFPI does not inhibit prothrombin activation by prothrombinase assembled on a two-dimensional lipid bilayer. Together, the data indicate that the region between Gly-160 and the end of the third Kunitz domain contributes to TFPI function by orienting the second Kunitz domain so that it can bind the active site of phospholipid-associated factor Xa prior to prothrombinase assembly and/or by slowing formation of the prothrombinase complex.

Gla-domainless factor Xa: molecular bait to bypass a blocked tenase complex

“…This may be explained by persistence of the GDXa-TFPI complex, which therefore sustains the correction of thrombin generation. Moreover, as the affinity of GDXa for TFPI is lower than that of FXa ( Figure 3D), as previously demonstrated, 28 the generation of FXa in the presence of GDXa would result in the accumulation of sufficient FXa to reverse the formation of the GDXa-TFPI complex and ultimately restore the inhibition of coagulation activation.…”

“…TFPI is a slow, tight binding inhibitor of FXa 26,27 and, at a low concentration, a weak inhibitor of GDXa. [28][29][30] We, therefore, compared TFPI inhibition of FXa and GDXa (Table 1). GDXa showed a lower affinity for TFPI (Ki* = 0.31±0.04 nM, Figure 3D) compared to FXa (Ki* = 0.17±0.03 nM, Figure 3C).…”

The Factor IX γ-Carboxyglutamic Acid (Gla) Domain Is Involved in Interactions between Factor IX and Factor XIa

“…A recombinant prothrombin molecule containing the protein C Gla domain bound specifically to cells expressing the protein C receptor. Along similar lines, Lockett and Mast (61) presented data suggesting that an interaction between the C terminus of TF pathway inhibitor and the factor Xa Gla domain is required for proper TF pathway inhibitor mediated-inhibition of factor Xa. Thus, Gla domain involvement in proteinprotein interactions may be common.…”

Proteolytic inactivation of tissue factor pathway inhibitor by bacterial omptins

“…Separation of the C-terminal tail region from the inhibitory Kunitz-1 and -2 domains of TFPI profoundly depresses TFPI's anticoagulant function by preventing its association with FXa. 41 And since formation of a FXa-TFPI inhibitory complex is a prerequisite for TFPI's inhibition of TF:FVIIa, loss of the tail region would nullify inhibition of both FXa and TF:FVIIa. Thus, the K249-G250 scissile bond is ideally situated to exert a maximal impact on TFPI function upon its hydrolysis.…”