Inhibitory Properties of a Novel Human Kunitz-Type Protease Inhibitor Homologous to Tissue Factor Pathway Inhibitor

Abstract

In a previous report, we described the molecular cloning, expression, and partial characterization of a second human tissue factor pathway inhibitor (TFPI), which we designated as TFPI-2 [Sprecher, C. A., et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 3353-3357]. Recombinant TFPI-2 inhibited the amidolytic activity of trypsin as well as that of factor VIIa in complex with tissue factor. TFPI-2 recently has been shown to be identical to placental protein 5 (PP5), a glycoprotein originally isolated from placenta that exhibits serine protease inhibitory activity. In the present study, we have examined TFPI-2/PP5 for its ability to inhibit a number of serine proteases involved in blood coagulation and fibrinolysis, inasmuch as TFPI-2/PP5 prolonged the coagulation time of human plasma induced by either tissue factor or contact activation in a dose-dependent manner. In addition to its ability to inhibit the amidolytic and proteolytic activities of the factor VIIa-tissue factor complex, TFPI-2/PP5 strongly inhibited the amidolytic activities of human factor XIa, human plasma kallikrein, and human plasmin with Ki values of 15, 25, and 3 nM, respectively. TFPI-2/PP5 was also a weak inhibitor of the activation of factor X by a complex of human factor IXa and poly(lysine) with an apparent Ki of 410 nM. Heparin markedly enhanced the ability of TFPI-2/PP5 to inhibit factor VIIa-tissue factor both in the solution phase and on cell surfaces. In addition, heparin augmented the inhibition of human factor Xa amidolytic activity at relatively high levels (10-100 nM) of TFPI-2/PP5. No significant inhibition of glandular kallikrein, urinary plasminogen activator, tissue plasminogen activator, human activated protein C, human factor Xa, human thrombin, or leukocyte elastase was observed when these proteases were incubated with TFPI-2 in the absence of heparin.

Platelets Contain Tissue Factor Pathway Inhibitor-2 Derived from Megakaryocytes and Inhibits Fibrinolysis

“…KD1-WT inhibited plasmin, FXIa, and pKLK with K i values of 6 Ϯ 1, 18 Ϯ 2, and 26 Ϯ 3 nM, respectively (data not shown). Our K i values for TFPI-2 and KD1-WT are in excellent agreement with previously reported K i values (5,8,11,58) for each protease, suggesting proper folding of the recombinant domains. Further, fulllength TFPI-1 reacted with the antibodies to the N-terminal and C-terminal peptides, whereas TFPI-1 161 reacted with only the antibody to the N-terminal peptide (Fig.…”

“…TFPI-2, also known as placental protein 5 (3) or matrix serine protease inhibitor (4), features a domain organization similar to that of TFPI-1 and contains three Kunitz-type domains in tandem with a short N terminus and a very basic C-terminal region. With respect to the vascular system, TFPI-2 inhibition of plasma kallikrein (pKLK), factor XIa (FXIa), and plasmin are of primary importance (5)(6)(7)(8)(9). Notably, TFPI-2 inhibits FVIIa/ tissue factor (TF) very poorly, with K i ϳ1 M (5, 10, 11), and has very little inhibitory activity toward tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), activated protein C, tissue kallikreins, leukocyte elastase, and thrombin (5,11,12).…”

“…In further studies, we examined binding of TFPI-2 to plasma-derived FV (pdFV), partially B-domain-deleted FV (FV-1033) 5 (57), and recombinant FVa (rFVa); these data were then compared with the data obtained using TFPI-1. We also examined whether TFPI-2 binds to FV in the plasma of pregnant women and whether or not platelets contain TFPI-2 that is associated with FV/Va.…”

Adenovirus-Mediated Expression of Tissue Factor Pathway Inhibitor-2 Inhibits Endothelial Cell Migration and Angiogenesis

“…1 Tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type serine proteinase inhibitor 2 synthesized by endothelial cells (ECs), smooth muscle cells (SMCs), and syncytiotrophoblasts, 3,4 which associates through ionic interactions 5 with the extracellular matrix (ECM), 6 and inhibits trypsin, plasmin, and plasma kallikrein among others. 7 The ECM is essential for the integrity of the cardiovascular system. ECs use the matrix as scaffolding during invasion, but they also degrade it to provide space for new capillaries.…”

A novel function of tissue factor pathway inhibitor-2 (TFPI-2) in human glioma invasion

“…This triplet is synthesized from a 25-kDa non-glycosylated protein by means of di erential glycosylation (Rao et al, 1996). TFPI-2 inhibits trypsin, plasmin, chymotrypsin, cathepsin G, plasma kallikrein and factor VIIa-tissue factor complex but not uPA, tissue plasminogen activator (tPA), or thrombin (Sprecher et al, 1994;Rao et al, 1995a,b,c;Petersen et al, 1996). The TFPI-2 gene has been mapped to chromosome 7q22 by¯uoresence in situ hybridization (Miyagi et al, 1996b).…”