Caveolae optimize tissue factor-Factor VIIa inhibitory activity of cell-surface-associated tissue factor pathway inhibitor

TFPI (tissue factor pathway inhibitor) is an anticoagulant protein that prevents intravascular coagulation through inhibition of fXa (Factor Xa) and the TF (tissue factor)-fVIIa (Factor VIIa) complex. Localization of TFPI within caveolae enhances its anticoagulant activity. To define further how caveolae contribute to TFPI anticoagulant activity, CHO (Chinese-hamster ovary) cells were co-transfected with TF and membrane-associated TFPI targeted to either caveolae [TFPI-GPI (TFPI-glycosylphosphatidylinositol anchor chimaera)] or to bulk plasma membrane [TFPI-TM (TFPI-transmembrane anchor chimaera)]. Stable clones had equal expression of surface TF and TFPI. TX-114 cellular lysis confirmed localization of TFPI-GPI to detergent-insoluble membrane fractions, whereas TFPI-TM localized to the aqueous phase. TFPI-GPI and TFPI-TM were equally effective direct inhibitors of fXa in amidolytic assays. However, TFPI-GPI was a significantly better inhibitor of TF-fVIIa than TFPI-TM, as measured in both amidolytic and plasma-clotting assays. Disrupting caveolae by removing membrane cholesterol from EA.hy926 cells, which make TFPIα, CHO cells transfected with TFPIβ and HUVECs (human umbilical vein endothelial cells) did not affect their fXa inhibition, but significantly decreased their inhibition of TF-fVIIa. These studies confirm and quantify the enhanced anticoagulant activity of TFPI localized within caveolae, demonstrate that caveolae enhance the inhibitory activity of both TFPI isoforms and define the effect of caveolae as specifically enhancing the anti-TF activity of TFPI.     

Estrogenic regulation of tissue factor and tissue factor pathway inhibitor in platelets

Oral estrogen treatment increases thrombotic risk. Tissue factor (TF), tissue factor pathway inhibitor (TFPI), and platelet interaction with leukocytes are important determinants of thrombogenesis. Therefore, the present study was designed to define and compare platelet TF and TFPI mRNA and adhesion protein expression in platelets derived from animals treated with different types of oral estrogens. Ovariectomized pigs were treated with 17beta-estradiol (2 mg/day), conjugated equine estrogen (CEE; 0.625 mg/day), or raloxifene (60 mg/day) for 4 wk. Compared with intact animals, ovariectomy and treatment differentially affected populations of leukocytes: neutrophils decreased whereas lymphocytes increased significantly 4 wk after ovariectomy and with 17beta-estradiol and CEE treatments; eosinophils increased only with 17beta-estradiol treatment. Content of TF protein increased in platelets from 17beta-estradiol- and raloxifene-treated pigs, whereas TF mRNA was detected only in platelets from 17beta-estradiol- and CEE treated pigs. TFPI mRNA increased in platelets after ovariectomy and estrogen treatment. Only a trace of TFPI protein was detected, but a higher-molecular-mass protein was observed in all treatment groups. Expression of CD40 and CD40 ligand increased with ovariectomy and decreased with 17beta-estradiol and CEE treatments more than with raloxifene. The ratio of activated to basal P-selectin expression decreased with ovariectomy and increased with raloxifene treatments. These results suggest that estrogenic formulations may affect individual thrombotic risk by different mechanisms that regulate TF and platelet-leukocytic interactions. These studies provide the rationale for evaluation of interactions among platelets and TF and TFPI expression on thrombin generation during estrogen treatment in humans.     

The N-terminal domain of tissue inhibitor of metalloproteinases retains metalloproteinase inhibitory activity

Recombinant tissue inhibitor of metalloproteinases (TIMP-1) and a truncated version containing only the three N-terminal loops, delta 127-184TIMP, have been expressed in myeloma cells and purified by affinity chromatography and gel filtration. delta 127-184TIMP was found to exist as two main glycosylation variants of molecular mass 24 kD and 19.5 kDa and an unglycosylated form of 13 kDa. All forms of the truncated inhibitor were able to inhibit and form complexes with active forms of the matrix metalloproteinases, indicating that the major structural features for specific interaction with these enzymes resides in these three loops. Stable binding of delta 127-184TIMP to pro 95-kDa gelatinase was not demonstrable under the conditions for binding of full-length TIMP-1.     

Expression and characterization of Kunitz domain 3 and C-terminal of human tissue factor pathway inhibitor-2

Human tissue factor pathway inhibitor-2 （hTFPI-2） is a serine protease inhibitor and its inhibitory activity is enhanced by heparin. The Kunitz domain 3 and C- terminal of hTFPI-2 （hTFPI-2/KD3C）, which has the activity toward heparin calcium, have been successfully expressed in Pichia pastoris and purified by SP- Sepharose and heparin-Sepharose chromatography. The Fourier transformed infrared spectroscopy （FTIR）, Raman spectroscopy, and circular dichroism （CD） experiment results implied that hTFPI-2/KD3C contained small contents of α-helix and β-strand, but large amounts of random coil and two kinds of disulfide bonds, gauche-gauche-gauche （ggg） and trans-gauchetrans （tgt）. The interaction of hTFPI-2/KD3C with heparin calcium was investigated by CD. It was found that heparin calcium induced β-strands in hTFPI-2/ KD3C to different extents depending on the ratio of hTFPI-2/KD3C and heparin calcium.     

Recombinant tissue factor pathway inhibitor induces apoptosis in cultured rat mesangial cells via its Kunitz-3 domain and C-terminal through inhibiting PI3-kinase/Akt pathway

Tissue factor pathway inhibitor (TFPI) is an endogenous inhibitor of tissue factor (TF) induced coagulation. In addition to its anticoagulation activity, TFPI has other functions such as antiproliferation and inducing apoptosis. In the present study, we investigated whether or not TFPI induced apoptosis in cultured rat mesangial cells (MsCs) and the possible signal pathway that involved in the apoptotic process. We demonstrated that recombinant TFPI (rTFPI) induced apoptosis in cultured MsCs via its Kunitz-3 domain and C-terminal in a dose- and time-dependent manner by Hoechst 33258 assay, flow cytometry, nucleosomal laddering of DNA, caspase 3 assay. Because the serine/threonine protein kinase Akt has attracted attention as a mediator of survival (anti-apoptotic) signal in MsCs, we investigated the expression of phosphospecific-Akt and its downstream signal phospho-IκB-α and some other signal molecules like Fas and bcl-2. The results indicated that the process of apoptosis triggered by rTFPI is, at least in part, actively conducted by rat MsCs possibly through PI3-Kinase-Akt signal pathway not by binding to tissue factor. Our findings suggest that rTFPI has the potential usefulness in inducing apoptosis of MsCs under inflammatory conditions.     

Structural mechanism for heparin-binding of the third Kunitz domain of human tissue factor pathway inhibitor.

Tissue factor pathway inhibitor (TFPI) inhibits the activity of coagulation factor VIIa and Xa through its K1 and K2 domain, respectively, and the inhibitory activity is enhanced by heparin. The function of the K3 domain of TFPI has not been established, but the domain probably harbors a heparin binding site (HBS-2). We determined the three-dimensional solution structure of the TFPI K3 domain (Glu182-Gly242) by heteronuclear multidimensional NMR. The results showed that the molecule is composed of one antiparallel beta-sheet and one alpha-helix, and in overall structure is very similar to the K2 domain, with the rms deviation of 1.55 A for the 58 defined C(alpha) positions. However, the surface electrostatic properties of both domains are different each other. The lack of inhibitory activity of the K3 domain is explained by the absence of electrostatic interaction with factor Xa over a large surface area. A titration experiment with size-fractionated heparin showed that a heparin binding site was located in the vicinity of the alpha-helix. In this region, a positively charged cluster is formed by Lys213, Lys232, and Lys240, and the negatively charged sulfate groups of heparin bind there. The enhancement of inhibitory activity by heparin probably was not due to a conformational change to TFPI itself. It is likely that heparin simply increases the local concentration of TFPI on the cell surface and stabilizes the initial complex that forms.     

Differential expression of tissue factor and tissue factor pathway inhibitor in metastatic melanoma lesions

Tissue factor (TF) is involved in tumor progression and metastatic potency in some malignant tumors and its function is regulated by tissue factor pathway inhibitor (TFPI) therefore the interaction of both molecules is crucial for their functional role. We evaluated the clinical relevance of TF and TFPI expression in benign and malignant melanocytic lesions. Expression of both was examined by immunoperoxidase staining using serial tissue sections in 16 nevi, 34 primary and 15 metastatic melanoma lesions. TF and TFPI were ubiquitously expressed in benign and malignant melanocytic lesions. This finding was confirmed by Western blot analysis using cultured human melanocytes, nevi cells (NCN) and melanoma cell lines. Although TF expression was not associated with malignant transformation and disease progression, TFPI expression in primary and metastatic melanoma lesions was significantly lower and weaker than that in nevi lesions in terms of intensity and percentage of stained cells. In addition, TFPI expression in metastatic lesions was significantly lower and weaker than that of TF. These results suggest that the relative expression of TF to TFPI may play a crucial role in the malignant transformation and metastatic potency in melanocytic cells.     

An inhibitory role of the phosphatidylinositol 3-kinase-signaling pathway in vascular endothelial growth factor-induced tissue factor expression.

Vascular endothelial growth factor (VEGF) is not only essential for vasculogenesis and angiogenesis but is also capable of inducing tissue factor, the prime initiator of coagulation, in endothelial cells. In this study we have analyzed the VEGF-elicited pathways involved in the induction of tissue factor in human umbilical cord vein endothelial cells. Using specific low molecular weight inhibitors we could demonstrate a crucial role of the p38 and Erk-1/2 mitogen-activated protein (MAP) kinases. In contrast, treatment with wortmannin or LY294002, inhibitors of phosphatidylinositol 3 (PI3)-kinase, resulted in a strong enhancement of the VEGF-induced tissue factor production, indicating a negative regulatory role of the PI3-kinase on tissue factor-inducing pathways. Accordingly, transduction with constitutively active Akt led to a reduction of VEGF-induced tissue factor production. Western blot analyses using antibodies specific for phosphorylated p38 showed an enhanced activation of this MAP kinase in human umbilical cord vein endothelial cells when stimulated with VEGF in the presence of wortmannin in comparison to either agent alone. Thus, the negative regulation of the PI3-kinase pathway on endothelial tissue factor activity can be explained at least in part by a suppression of this MAP kinase-signaling pathway. This is the first demonstration of a reciprocal relationship between procoagulant activity and the PI3-kinase-Akt signaling pathway, and it reveals a novel mechanism by which tissue factor expression can be controlled in endothelial cells.     

The regulatory role of the C-terminal domain of connexin43

The C-terminal (CT) domain of connexin43 (Cx43) is thought to be important in the control of gap junction function via: a.) CT phosphorylation-dependent control of gap junction assembly and gating, b.) interactions of CT with key regulatory binding partners. To more closely examine CT-dependent regulation, we have expressed a hemagglutinin-Cx43CT (amino acids 235-382) fusion protein in Normal Rat Kidney (NRK) cells under a tetracycline-responsive inducible promoter. Western blot analysis shows that Cx43CT expression is markedly induced by at least 48 h oftreatment with the tetracycline analogue, doxycycline. Furthermore, Cx43CT is modified within the cell, as several treatments/conditions that increase endogenous Cx43 phosphorylation induced a mobility shift in Cx43CT. Treatment with kinase activators, including epidermal growth factor (EGF) and the tumor promoting phorbol ester 12-O-tetradecanylphorbol-13-acetate (TPA), caused a shift in the mobility of the Cx43CT in a manner consistent with the mobility shift observed upon increased phosphorylation of endogenous Cx43. Similarly, Cx43CT in mitotic cells is extensively shifted, consistent with reports which show that Cx43 is phosphorylated to a unique phosphoisoform in mitotic cells. These results indicate that the Cx43CT can interact with at least some of the kinases that phosphorylate endogenous Cx43 in cells and possibly modulate the effects of kinase activation on gap junctional communication.     

Catabolism of factor VIIa bound to tissue factor in fibroblasts in the presence and absence of tissue factor pathway inhibitor

Vascular injury leads to the exposure of blood to fibroblasts and smooth muscle cells within the vessel wall. These cells constitutively express tissue factor (TF), the cellular receptor for plasma clotting factor VIIa (FVIIa). Formation of TF.FVIIa complexes on cell surfaces triggers the blood coagulation cascade. In the present study, we have investigated the fate of TF.FVIIa complexes formed on the cell surface of fibroblasts in the presence and absence of plasma inhibitor, tissue factor pathway inhibitor (TFPI). FVIIa bound to TF on the cell surface was internalized and degraded without depleting the cell surface TF antigen and activity. TFPI significantly enhanced the TF-specific internalization and degradation of FVIIa. TFPI-enhanced internalization and degradation of FVIIa requires the C-terminal domain of TFPI and factor Xa. TFPI. Xa-mediated internalization of FVIIa was associated with the depletion of TF from the cell surface. A majority of the internalized FVIIa was degraded, but a small portion of the internalized FVIIa recycles back to the cell surface as an intact protein. In addition to TF, other cell surface components, such as low density lipoprotein receptor-related protein (LRP) and heparan sulfates, are essential for TFPI.Xa-induced internalization of FVIIa. Acidification of cytosol, which selectively inhibits the endocytotic pathway via coated pits, inhibited TFPI.Xa-mediated internalization but not the basal internalization of FVIIa. Overall, our data support the concept that FVIIa bound to cell surface TF was endocytosed by two different pathways. FVIIa complexed with TF in the absence of the inhibitor was internalized via a LRP-independent and probably noncoated pit pathway, whereas FVIIa complexed with TF along with the inhibitor was internalized via LRP-dependent coated pit pathway.     

Synthesis of the C-terminal domain of the tissue inhibitor of metalloproteinases-1 (TIMP-1).

According to recent investigations, the C-terminal domain of the tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) is responsible for some biological effects that are independent of the enzyme-inhibiting effect of the N-terminal domain of the molecule. The C-terminal domain has been prepared for structure-biological activity investigations. After the chemical synthesis and the folding of the linear peptide. LC-MS and MALDI-MS analysis revealed that two isomers with different disulphide bond arrangements were formed. Since more than 30 folding experiments resulted in products with a very similar HPLC-profile, it was concluded that in the absence of the TIMP-1 N-terminal domain no entirely correct folding of the C-terminal domain occurred. Furthermore, it was observed that, in spite of several purification steps, mercury(II) ions were bound to the 6SH-linear peptide; it was demonstrated--using disulphide bonded TIMP-1(Cys145-Cys166) as a model--that mercury(II) ions can cause peptide degradation at pH 7.8 as well as in 0.1% trifluoroacetic acid.     

Homology modeling and structural validation of tissue factor pathway inhibitor

Blood coagulation is a cascade of complex enzymatic reactions which involves specific proteins and cellular components to interact and prevent blood loss. The coagulation process begins by either “Tissue Dependent Pathway” (also known as extrinsic pathway) or by “contact activation pathway” (also known as intrinsic pathway). TFPI is an endogenous multivalent Kunitz type protease inhibitor which inhibits Tissue factor dependent pathway by inhibiting Tissue Factor:Factor VIIa (TF:FVIIa) complex and Factor Xa. TFPI is one of the most studied coagulation pathway inhibitor which has various clinical and potential therapeutic applications, however, its exact mechanism of inhibition is still unknown. Structure based mechanism elucidation is commonly employed technique in such cases. Therefore, in the current study the generated a complete TFPI structural model so as to understand the mechanistic details of it''s functioning. The model was checked for stereochemical quality by PROCHECK-NMR, WHATIF, ProSA, and QMEAN servers. The model was selected, energy minimized and simulated for 1.5ns. The result of the study may be a guiding point for further investigations on TFPI and its role in coagulation mechanism.     

The role of the C-terminal domain in collagenase and stromelysin specificity.

Recombinant human interstitial collagenase, an N-terminal truncated form, delta 243-450 collagenase, recombinant human stromelysin-1, and an N-terminal truncated form, delta 248-460 stromelysin, have been stably expressed in myeloma cells and purified. The truncated enzymes were similar in properties to their wild-type counterparts with respect to activation requirements and the ability to degrade casein, gelatin, and a peptide substrate, but truncated collagenase failed to cleave native collagen. Removal of the C-terminal domain from collagenase also modified its interaction with tissue inhibitor of metalloproteinases-1. Hybrid enzymes consisting of N-terminal (1-242) collagenase.C-terminal (248-460) stromelysin and N-terminal (1-233) stromelysin.C-terminal (229-450) collagenase, representing an exchange of the complete catalytic and C-terminal domains of the two enzymes, were expressed in a transient system using Chinese hamster ovary cells and purified. Both proteins showed similar activity to their N-terminal parent and neither was able to degrade collagen. Analysis of the ability of the different forms of recombinant enzyme to bind to collagen by ELISA showed that both pro and active stromelysin and N-terminal collagenase.C-terminal stromelysin bound to collagen equally well. In contrast, only the active forms of collagenase and N-terminal stromelysin.C-terminal collagenase bound well to collagen, as compared with their pro forms.     

The C-terminus of tissue factor pathway inhibitor is essential to its anticoagulant activity

Tissue factor pathway inhibitor (TFPI) from different cell lines shows up to 15-fold differences in the ratio of anticoagulant to chromogenic activity. The anticoagulant activity was dependent on the purification procedure used and it was possible to isolate two fractions of recombinant TFPI. Only one of these fractions showed anticoagulant activity comparable with TFPI from normal human plasma, and Western blotting showed that the low-activity fraction did not react with an antibody raised against a peptide of TFPI located near the C-terminal. Analysis by mass spectroscopy of peptides from V8 protease digests showed that C-terminal amino acids could only be identified from the high-activity form, while heterologous fragmentation had taken place in the form with low anticoagulant activity. Previously published studies on TFPI have been performed using material of low anticoagulant activity compared with plasma TFPI, and we suggest that these studies have been performed with material degraded in the C-terminus.     

Probing the interface between factor Xa and tissue factor in the quaternary complex tissue factor-factor VIIa-factor Xa-tissue factor pathway inhibitor.

Blood coagulation is triggered by the formation of a complex between factor VIIa (FVIIa) and its cofactor, tissue factor (TF). TF-FVIIa is inhibited by tissue factor pathway inhibitor (TFPI) in two steps: first TFPI is bound to the active site of factor Xa (FXa), and subsequently FXa-TFPI exerts feedback inhibition of TF-FVIIa. The FXa-dependent inhibition of TF-FVIIa activity by TFPI leads to formation of the quaternary complex TF-FVIIa-FXa-TFPI. We used site-directed fluorescence probing to map part of the region of soluble TF (sTF) that interacts with FXa in sTF-FVIIa-FXa-TFPI. We found that the C-terminal region of sTF, including positions 163, 166, 200 and 201, is involved in binding to FXa in the complex, and FXa, most likely via its Gla domain, is also in contact with the Gla domain of FVIIa in this part of the binding region. Furthermore, a region that includes the N-terminal part of the TF2 domain and the C-terminal part of the TF1 domain, i.e. the residues 104 and 197, participates in the interaction with FXa in the quaternary complex. Moreover, comparisons of the interaction areas between sTF and FX(a) in the quaternary complex sTF-FVIIa-FXa-TFPI and in the ternary complexes sTF-FVII-FXa or sTF-FVIIa-FX demonstrated large similarities.     

Intratracheal gene transfer of tissue factor pathway inhibitor attenuates pulmonary fibrosis

Activation of the coagulation system and increased expression of tissue factor (TF) in pulmonary fibrosis associated with acute and chronic lung injury have been previously documented. In the present study, we evaluated the effect of TF inhibition with intratracheal gene transfer of tissue factor pathway inhibitor (TFPI), a potent and highly specific endogenous inhibitor of TF-dependent coagulation activation, in a rat model of bleomycin-induced lung fibrosis. Significant lung fibrotic changes as assessed by histologic findings and hydroxyproline content, and increased procoagulant activity and thrombin generation in bronchoalveolar lavage fluid were detected in rats after intratracheal injection of bleomycin. Intratracheal administration of an adenovirus vector expressing TFPI significantly decreased bleomycin-induced procoagulant and thrombin generation resulting in a strong inhibition of pulmonary fibrosis. TFPI-overexpression in the lung was associated with a significant reduction in gene expression of the connective tissue growth factor, a potent profibrotic growth factor. This is the first report showing that direct inhibition of TF-mediated coagulation activation abrogates bleomycin-induced pulmonary fibrosis.     

Crystal structure of the catalytic domain of matrix metalloproteinase-1 in complex with the inhibitory domain of tissue inhibitor of metalloproteinase-1

The mammalian collagenases are a subgroup of the matrix metalloproteinases (MMPs) that are uniquely able to cleave triple helical fibrillar collagens. Collagen breakdown is an essential part of extracellular matrix turnover in key physiological processes including morphogenesis and wound healing; however, unregulated collagenolysis is linked to important diseases such as arthritis and cancer. The tissue inhibitors of metalloproteinases (TIMPs) function in controlling the activity of MMPs, including collagenases. We report here the structure of a complex of the catalytic domain of fibroblast collagenase (MMP-1) with the N-terminal inhibitory domain of human TIMP-1 (N-TIMP-1) at 2.54 A resolution. Comparison with the previously reported structure of the TIMP-1/stromelysin-1 (MMP-3) complex shows that the mechanisms of inhibition of both MMPs are generally similar, yet there are significant differences in the protein-protein interfaces in the two complexes. Specifically, the loop between beta-strands A and B of TIMP-1 makes contact with MMP-3 but not with MMP-1, and there are marked differences in the roles of individual residues in the C-D connector of TIMP-1 in binding to the two MMPs. Structural rearrangements in the bound MMPs are also strikingly different. This is the first crystallographic structure that contains the truncated N-terminal domain of a TIMP, which shows only minor differences from the corresponding region of the full-length protein. Differences in the interactions in the two TIMP-1 complexes provide a structural explanation for the results of previous mutational studies and a basis for designing new N-TIMP-1 variants with restricted specificity.