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.     

Concentration of vascular endothelial growth factor in patients with acute coronary syndrome

Vascular endothelial growth factor (VEGF) is a regulator of vascular formation in physiological and pathological conditions. The aim of our study was to evaluate the value of VEGF as a surrogate marker of myocardial injury in acute ischemic conditions.Materials and methodsIn 104 consecutive patients with acute coronary syndrome (ACS) with and without ST segment elevation (STEMI and NSTEMI) the plasma and serum human VEGF (hVEGF) concentration was measured two times i.e. immediately after admission due to ACS and 24 h later. According to ECG findings and coronary angiography results, patients were divided into three groups. Group A represented major myocardial injury due to ST-segment elevation in precordial leads and/or in I and aVL leads and with left anterior descending (LAD) artery responsible for STEMI symptoms or additionally with significant atherosclerotic lesions (lumen vessel narrowed >50%) in other than LAD coronary arteries. Group B (medium myocardial injury) consisted of patients with ST-segment elevation in II, III and aVF leads and/or ST-segment depression in V2-V3 leads with one-vessel disease and the culprit artery was not LAD. Group C included patients with changes in ECG other than ST-segment elevation independently of the site of atherosclerotic lesions in coronary arteries.ResultsIn all 104 patients with ACS the highest values of serum hVEGF were observed in second measurement (357.9 ± 346 pg/ml, p < 0.01). Although in the first measurement, plasma and serum hVEGF concentration did not differentiate groups, the difference between deltas for serum hVEGF was observed (p < 0.05). Increased number of neutrophils in the first measurement increased the OR of the high serum hVEGF concentration in the first measurement (OR = 1.155; 95%CI: 1.011; 1.32) (p < 0.05). The number of neutrophils in the second measurement also revealed significant relationship with high serum hVEGF in the first assessment (OR = 1.318, 95%CI: 1.097; 1.583) (p < 0.01). Increased values of triglycerides (exceeding the upper limit) were connected with decreased OR of high serum hVEGF concentrations in the first measurement (OR = 0.152, 95%CI: 0.033; 0.695, p < 0.05).ConclusionsIn acute coronary syndrome, serum VEGF concentrations are elevated and can serve as a surrogate marker of myocardial injury. The elevated number of neutrophils increases odds ratio of high VEGF concentrations in ACS. In patients with high concentrations of triglycerides, odds ratio of low level of hVEGF is expected.     

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.     

Tissue factor pathway inhibitor binds to platelet thrombospondin-1

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type serine proteinase inhibitor that down-regulates tissue factor-initiated blood coagulation. The most biologically active pool of TFPI is associated with the vascular endothelium, however, the biochemical mechanisms responsible for its cellular binding are not entirely defined. Proposed cellular binding sites for TFPI include nonspecific association with cell surface glycosaminoglycans and binding to glycosyl phosphatidylinositol-anchored proteins. Here, we report that TFPI binds specifically and saturably to thrombospondin-1 (TSP-1) purified from platelet alpha-granules with an apparent K(D) of approximately 7.5 nm. Binding is inhibited by polyclonal antibodies against TFPI and partially inhibited by the B-7 monoclonal anti-TSP-1 antibody. TFPI bound to immobilized TSP-1 remains an active proteinase inhibitor. Additionally, in solution phase assays measuring TFPI inhibition of factor VIIa/tissue factor catalytic activity, the rate of factor Xa generation was decreased 55% in the presence of TSP-1 compared with TFPI alone. Binding experiments done in the presence of heparin and with altered forms of TFPI suggest that the basic C-terminal region of TFPI is required for TSP-1 binding. The data provide a mechanism for the recruitment and localization of TFPI to extravascular surfaces within a bleeding wound, where it can efficiently down-regulate the procoagulant activity of tissue factor and allow subsequent aspects of platelet-mediated healing to proceed.     

Plasminogen activator inhibitor-1 is an inhibitor of factor VII-activating protease in patients with acute respiratory distress syndrome

Factor VII-activating protease (FSAP) is a novel plasma-derived serine protease structurally homologous to tissue-type and urokinase-type plasminogen activators. We demonstrate that plasminogen activator inhibitor-1 (PAI-1), the predominant inhibitor of tissue-type and urokinase-type plasminogen activators in plasma and tissues, is an inhibitor of FSAP as well. We detected PAI-1.FSAP complexes in addition to high levels of extracellular RNA, an important FSAP cofactor, in bronchoalveolar lavage fluids from patients with acute respiratory distress syndrome. Hydrolytic activity of FSAP was inhibited by PAI-1 with a second-order inhibition rate constant (K(a)) of 3.38 +/- 1.12 x 10(5) m(-1).s(-1). Residue Arg(346) was a critical recognition element on PAI-1 for interaction with FSAP. RNA, but not DNA, fragments (>400 nucleotides in length) dramatically enhanced the reactivity of PAI-1 with FSAP, and 4 microg.ml(-1) RNA increased the K(a) to 1.61 +/- 0.94 x 10(6) m(-1).s(-1). RNA also stabilized the active conformation of PAI-1, increasing the half-life for spontaneous conversion of active to latent PAI-1 from 48.4 +/- 8 min to 114.6 +/- 5 min. In contrast, little effect of DNA on PAI-1 stability was apparent. Residues Arg(76) and Lys(80) in PAI-1 were key elements mediating binding of nucleic acids to PAI-1. FSAP-driven inhibition of vascular smooth muscle cell proliferation was antagonized by PAI-1, suggesting functional consequences for the FSAP-PAI-1 interaction. These data indicate that extracellular RNA and PAI-1 can regulate FSAP activity, thereby playing a potentially important role in hemostasis and cell functions under various pathophysiological conditions, such as acute respiratory distress syndrome.     

Lymphocyte DNA damage in patients with acute coronary syndrome and its relationship with severity of acute coronary syndrome

OBJECTIVE: The aim of this study was to investigate the association between lymphocyte DNA damage and acute coronary syndromes (ACS). METHODS: The study population contained 53 patients with ACS, 48 patients with stable angina and 35 voluntary healty subjects. DNA damage was assessed by alkaline comed assay in peripheral lymphocyte and plasma levels of total antioxidant capacity (TAC) were determined using a novel automated measurement method. RESULTS: In ACS patients, DNA damage was significantly higher than in patients with stable angina and control subjects (144+/-52 AU, 116+/-37, 68+/-34 AU; for three p<0.001, respectively). The TAC levels in patients with ACS were lower than the other groups (1.24+/-0.31 mmol Trolox equiv./l, 1.46+/-0.29 mmol Trolox equiv./l, p<0.05, respectively). DNA damage values in patients with acute miyocardial infarction were significantly higher than in patients with unstable angina (159.8+/-53.0 AU versus 131.8+/-48.4 AU; p<0.05, respectively). Lymphocyte DNA damage values in patients with ACS showed positive correlation with d-dimer (r=0.880, p<0.001) troponin I (r=538, p<0.001) and C-reactive protein (r=0.544, p<0.001) and negative correlation with TAC (r=-0.346, p=0.011). In multiple linear regression analysis, TAC (beta=-0.213, p=0.001) and d-dimer (beta=0.697, p<0.001) were independent predictors of DNA damage in patients with ACS. CONCLUSIONS:These findings indicate that lymphocyte DNA damage level increases in patients with ACS. Elevated DNA damage may be related with plaque instability and be useful for the identification of patients with acute coronary syndromes.     

Tissue factor pathway inhibitor is highly susceptible to chymase-mediated proteolysis

Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type protease inhibitor that primarily inhibits the extrinsic pathway of blood coagulation. It is synthesized by various cells and its expression level increases in inflammatory environments. Mast cells and neutrophils accumulate at sites of inflammation and vascular disease where they release proteinases as well as chemical mediators of these conditions. In this study, the interactions between TFPI and serine proteinases secreted from human mast cells and neutrophils were examined. TFPI inactivated human lung tryptase, and its inhibitory activity was stronger than that of antithrombin. In contrast, mast cell chymase rapidly cleaved TFPI even at an enzyme to substrate molar ratio of 1:500, resulting in markedly decreased TFPI anticoagulant and anti-(factor Xa) activities. N-terminal amino-acid sequencing and MS analyses of the proteolytic fragments revealed that chymase preferentially cleaved TFPI at Tyr159-Gly160, Phe181-Glu182, Leu89-Gln90, and Tyr268-Glu269, in that order, resulting in the separation of the three individual Kunitz domains. Neutrophil-derived proteinase 3 also cleaved TFPI, but the reaction was much slower than the chymase reaction. In contrast, alpha-chymotrypsin, which shows similar substrate specificities to those of chymase, resulted in a markedly lower level of TFPI degradation. These data indicate that TFPI is a novel and highly susceptible substrate of chymase. We propose that chymase-mediated proteolysis of TFPI may induce a thrombosis-prone state at inflammatory sites.     

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.     

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.     

Tissue factor pathway inhibitor 2 is induced by thrombin in human macrophages

Tissue factor pathway inhibitor 2 (TFPI2) is a serine protease inhibitor critical for the regulation of extracellular matrix remodeling and atherosclerotic plaque stability. Previously, we demonstrated that TFPI2 expression is increased in monocytes from patients with familial combined hyperlipidemia (FCH). To gain insight into the molecular mechanisms responsible for this upregulation, we examined TFPI2 expression in THP-1 macrophages exposed to lipoproteins and thrombin. Our results showed that TFPI2 expression was not affected by treatment with very low density lipoproteins (VLDL), but was induced by thrombin (10 U/ml) in THP-1 (1.9-fold increase, p < 0.001) and human monocyte-derived macrophages (2.3-fold increase, p < 0.005). The specificity of the inductive effect was demonstrated by preincubation with the thrombin inhibitors hirudin and PPACK, which ablated thrombin effects. TFPI2 induction was prevented by pre-incubation with MEK1/2 and JNK inhibitors, but not by the EGF receptor antagonist AG1478. In the presence of parthenolide, an inhibitor of NFκB, but not of SR-11302, a selective AP-1 inhibitor, thrombin-mediated TFPI2 induction was blunted. Our results also show that thrombin treatment increased ERK1/2, JNK and IκBα phosphorylation. Finally, we ruled out the possibility that TFPI2 induction by thrombin was mediated by COX-2, as preincubation with a selective COX-2 inhibitor did not prevent the inductive effect. In conclusion, thrombin induces TFPI2 expression by a mechanism involving ERK1/2 and JNK phosphorylation, leading finally to NFkB activation. In the context of atherosclerosis, thrombin-induced macrophage TFPI2 expression could represent a means of avoiding excessive activation of matrix metalloproteases at sites of inflammation.     

Primary percutaneous coronary angioplasty in patients with acute coronary syndrome and concomitant cancer

The paper gives the results of X-ray surgical treatment in patients with acute coronary syndrome who have been ascertained to have concomitant cancer during their examination. Cancer was found in 11 patients in their medical history and diagnosed in 2 patients during examination after surgical treatment and 1 patient one year after his hospital discharge. The results of combination treatment showed the high efficiency of X-ray surgical treatment for acute coronary syndrome. Recovery of TIMI-III blood flow through the infarct-related coronary artery was achieved in 100% of cases; immediate clinical efficiency was 97.4%. In the concomitant cancer group, the therapeutic efficacy was 100%; there were no complications during X-ray surgery. All the patients from this group were discharged from hospital in a satisfactory state to be followed up by a cardiologist and oncologist for further treatment. The study performed suggests that concomitant cancer is not a contraindication to primary coronary angioplasty in patients with acute coronary syndrome. Primary coronary angioplasty with stenting is a safe effective treatment for acute coronary syndrome in this category of patients.     

Tissue factor pathway inhibitor induces expression of JUNB and GADD45B mRNAs

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor that regulates tissue factor-triggered blood coagulation. It has previously been reported that TFPI inhibits the proliferation of human umbilical vein endothelial cells (HUVECs), suggesting that TFPI may act as more than just a mediator of coagulation through changes in gene expression. By using DNA-array techniques and Northern blot analysis, we here revealed that TFPI transiently induced the mRNA expression of JUNB and GADD45B. The inducible effects were not observed in TFPIdeltaC (lacking the C-terminal basic region) or antithrombin (heparin-binding anticoagulant protease inhibitor). Moreover, the TFPI-induced expression of GADD45B was blocked by receptor-associated protein, which masks the ligand-binding domain of very low density lipoprotein receptor (VLDL-R). In conclusion, this is the first report to show an effect of TFPI on mRNA expression, and suggests that TFPI modulates cellular functions by inducing JUNB and GADD45B expression through binding to VLDL-R.     

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.     

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.     

IL-17A facilitates platelet function through the ERK2 signaling pathway in patients with acute coronary syndrome

Background

Platelet aggregation mediated by inflammation played a critical role in the development of coronary heart diseases (CHD). Our previous clinical researches showed that Th17 cells and their characteristic cytokine IL-17A were associated with the plaque destabilization in patients with acute coronary syndrome (ACS). However, the potent effect of IL-17A on platelets-induced atherothrombosis remains unknown.

Methods and Results

In this study, we detected the plasma IL-17A levels and platelet aggregation in patients with stable angina (SA), unstable angina (UA), acute myocardial infarction (AMI) and chest pain syndrome (CPS). In addition, the markers of platelet activation (CD62P/PAC-1) and the mitogen-activated protein kinases (MAPKs) pathway were detected in platelets from ACS patients. We found that plasma IL-17A levels and platelet aggregation in patients with ACS (UA and AMI) were significantly higher than patients with SA and CPS, and the plasma IL-17A levels were positively correlated with the platelet aggregation (R = 0.47, P<0.01). In addition, in patients with ACS, the platelet aggregation, CD62P/PAC-1 and the phosphorylation of ERK2 signaling pathway were obviously elevated in platelets pre-stimulated with IL-17A in vitro. Furthermore, the specific inhibitor of ERK2 could attenuate platelet aggregation and activation triggered by IL-17A.

Conclusion

Our experiment firstly proved that IL-17A could promote platelet function in patients with ACS via activating platelets ERK2 signaling pathway and may provide a novel target for antiplatelet therapies in CHD.     

Matrix metalloproteinases cleave tissue factor pathway inhibitor. Effects on coagulation

The capacity of inflammatory cell-derived matrix metalloproteinases (MMPs) to cleave tissue factor pathway inhibitor (TFPI) and alter its activity was investigated. MMP-7 (matrilysin) rapidly cleaved TFPI to a major 35-kDa product. In contrast, MMP-1 (collagenase-1), MMP-9 (gelatinase B), and MMP-12 (macrophage elastase) cleaved TFPI into several fragments including the 35-kDa band. However, rates of cleavage were most rapid for MMP-7 and MMP-9. NH(2)-terminal amino acid sequencing revealed that MMP-12 cleaved TFPI at Lys(20)-Leu(21)(close to Kunitz I domain and producing a 35-kDa band), Arg(83)-Ile(84) (between Kunitz I and II domains), and Ser(174)-Thr(175) (between Kunitz II and III domains). MMP-7 and MMP-9 cleaved TFPI at Lys(20)-Leu(21) with additional COOH-terminal processing. These MMPs did not cleave tissue factor (TF), factor VII, and factor Xa. Proteolytic cleavage by MMP-1, MMP-7, MMP-9, and MMP-12 resulted in considerable loss of TFPI activity. These observations indicate specific cleavage of TFPI by MMPs, which broadens their substrate profile. Co-localization of MMPs, TF, and TFPI in atherosclerotic tissues suggests that release of MMPs from inflammatory cell leukocytes may effect TF-mediated coagulation.     

Detection of methylated tissue factor pathway inhibitor 2 and human long DNA in fecal samples of patients with colorectal cancer in China

Background: To investigate the feasibility of detecting methylated tissue factor pathway inhibitor (TFPI2) and quantifying human long DNA with fluorescent quantitative Alu PCR in fecal DNA as a non-invasive screening tool for colorectal cancer (CRC). Materials and Methods: Methylation-specific PCR (MSP) was performed to analyze TFPI2 gene promoter methylation status in a blinded fashion in stool samples taken from 30 endoscopically diagnosed healthy controls, 20 patients with adenomas, and 60 patients with colorectal cancer. Real-time Alu PCR was used to quantify human long DNA. Results: The specificity of fecal TFPI2 MSP assay and long DNA assay was 100% and 83.3%, respectively. The sensitivity of fecal TFPI2 MSP assay and long DNA assay was 68.3% and 53.3%, respectively. The sensitivity of fecal DNA assay (either marker being positive) was 86.7%, which was high for CRC. Conclusions: Our results have demonstrated the feasibility of using TFPI2 methylation and quantify human long DNA with fluorescent quantitative Alu PCR in fecal samples as a new noninvasive test for CRC.