Release Of Tissue Factor Pathway Inhibitor Research Articles

Inhibition of factor VIIa generation and prothrombin activation by treatment with enoxaparin in patients with unstable angina.

Factor VIIa (FVIIa) and thrombin generation occur in patients suffering an acute coronary event. We studied the effect of treatment with enoxaparin on FVIIa and prothrombin activation in patients with unstable angina. Anti-Xa activity, FVIIa, FVII coagulant activity (FVII:C) and FVII antigen (FVII:Ag), free tissue factor pathway inhibitor (TFPI), and prothrombin fragments 1 + 2 (F1+2) were measured in patients' plasma, over a 24-h treatment period with enoxaparin. All 14 patients recruited in the study (mean age 68 years) were treated with a subcutaneous injection of enoxaparin, 1 mg/kg twice daily. Blood was drawn just before, and at different time intervals after, the first injection. Before enoxaparin administration, the levels of FVIIa (4.02 +/- 0.8 ng/ml) and F1+2 (2.68 +/- 0.2 nmol/l) were significantly increased as compared with control subjects (2.3 +/- 0.3 ng/ml and 0.9 +/- 0.1 nmol/l respectively, P < 0.05). Free TFPI, FVII:C and FVII:Ag were within normal ranges. One hour after the first injection of enoxaparin, FVIIa and F1+2 levels decreased by 65% and 50%, respectively, and no significant fluctuations were noted throughout the observation period. The concentrations of FVII:C and FVII:Ag were not modified as compared with baseline values. After each injection, the peak concentrations of free TFPI and anti-Xa activity were observed at 2 and 4 h respectively. The kinetics of FVIIa and F1+2 inhibition did not follow those of anti-Xa activity and TFPI release.

Tissue factor pathway inhibitor (TFPI) release after heparin stimulation is increased in Type 1 diabetic patients with albuminuria.

To study heparin-stimulated TFPI release in relation to complications in Type 1 diabetic patients. Nineteen uncomplicated Type 1 diabetic patients (group I) were compared with 18 patients with retinopathy (group II), and nine patients with retinopathy and albuminuria (group III). Blood samples were taken before (basal) and till 30 min after 5000 IU of heparin i.v. (post-heparin). TFPI activity was measured chromogenically. Von Willebrand factor, tissue plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1) and thrombomodulin were also measured. Basal TFPI activity was higher in group III (121 +/- 10%) compared with group II (111 +/- 8%) or group I (110 +/- 13%) (P < 0.05), and strongly correlated with albuminuria (r = 0.66, P < 0.05). At all time points after heparin administration, TFPI activity in group III was significantly higher than in group I. TFPI activity was also higher in group III than in group II 5-30 min post-heparin. The increase in post-heparin TFPI activity, measured as the incremental area under the curve, was higher in group III compared with group I (65 +/- 7 vs. 59 +/- 4; P < 0.05). Of the other parameters, only thrombomodulin was higher in group III (44 +/- 24 vs. 26 +/- 7 (group II) and 28 +/- 9 ng/ml (group I); P < 0.01). We conclude that basal and post-heparin TFPI activity is increased in albuminuric patients. The increase in heparin-stimulated TFPI release in patients with albuminuria is higher than in patients with retinopathy or without complications. This could be the result of altered endothelial glycosaminoglycan characteristics.

Plasmin-mediated proteolysis of vascular endothelial cell heparin releasable tissue factor pathway inhibitor.

Fibrinolytic therapy represents a widely available and effective treatment modality for ST segment elevation myocardial infarction (MI). Its overall benefit is attenuated by a high incidence of coronary arterial reocclusion. Human umbilical vein endothelial cells (HUVEC) were incubated with unfractionated heparin (1.5 U/ml) (to provoke tissue factor pathway inhibitor [TFPI] release) followed by the addition of varying concentrations of alteplase (recombinant tissue plasminogen activator), plasminogen, their combination or plasmin alone. In the presence of 20% TFPI-depleted human plasma, there was a concentration-dependent decrease in TFPI levels following incubation with alteplase (28% reduction at 200 ng/ml; P < 0.01); 37% reduction at 1000 ng/ml (P < 0.001). Similar effects were observed for alteplase combined with plasminogen (P < 0.001), plasmin alone (P < 0.001) and with HUVEC incubated with low concentrations of plasmin (10 ng/ml) prior to heparin exposure. Plasmin, a non-specific protease, degrades vascular endothelial cell (constitutive) TFPI and heparin-releasable TFPI, attenuating an important pathway of vascular surface thromboresistance and potentially contributing to coronary arterial reocclusion after fibrinolytic therapy.

Local tissue factor pathway inhibitor release in the human forearm

Nineteen healthy men received unilateral brachial artery infusions of either unfractioned heparin (0.3-100 IU/min), saline or the endothelium-dependent vasodilators substance P (2-8 pmol/min) and bradykinin (100-1000 pmol/min), and the endothelium-independent vasodilator sodium nitroprusside (2-8 micro g/min). Heparin caused a dose-dependent increase in plasma TFPI concentrations in both arms (ANOVA, p <0.0001). Estimated net forearm TFPI release was 7 +/- 16, 29 +/- 20 and 138 +/- 72 ng/100 mL tissue/min during 10, 30 and 100 IU/min of heparin respectively (ANOVA, p <0.0001). Compared to the systemic circulation, the forearm sensitivity to heparin induced TFPI release was 3.6-fold lower (166 +/- 67 ng/IU vs. 596 +/- 252 ng/IU: t-test, p = 0.004). Substance P, bradykinin and sodium nitroprusside all caused substantial dose-dependent increases in blood flow (ANOVA, p <0.001 for all) without affecting plasma TFPI concentrations. There are important regional differences in endothelial TFPI release, with the forearm circulation being relatively insensitive to heparin.

Adrenomedullin augments the release and production of tissue factor pathway inhibitor in human aortic endothelial cells.

Adrenomedullin (AM) is a hypotensive and vasodilative peptide. It has been reported that AM plays several biological roles in cardiovascular and endocrine systems, however, effects on the blood coagulation system have not been examined. In this study, we examined its effect on tissue factor pathway inhibitor (TFPI), which is a potent inhibitor of tissue factor/factor VIIa complex-induced coagulation cascade, and is synthesized and constitutively secreted by endothelial cells (ECs). The aim of this study was to elucidate the effects of AM on release and production of TFPI in ECs. Cultured human aortic ECs were incubated with AM (10(-14)-10(-6) M), and the antigen levels and activity of the free and total form of TFPI after AM exposure were measured in culture media using ELISA. Furthermore, receptor interaction and cellular signaling of AM were investigated. AM augmented TFPI release and production in a dose-dependent manner. The effect on TFPI production was inhibited by AM receptor antagonist (AM22-52), the monoclonal antibody against C-terminal region of AM, MAPKK inhibitor, and cAMP antagonist. These findings indicate that AM might play an important role in the modulation of anticoagulant properties in blood circulation.

Quantification of heparin-induced TFPI release: a maximum release at low heparin dose.

Heparin releases tissue factor pathway inhibitor (TFPI) from the endothelium and this release may decrease after repeated high dose heparin administration. The primary aim was to investigate and quantify this phenomenon during a short low dose heparin infusion. Also, the effects of heparin on tissue plasminogen activator (t-PA) were studied. Nine healthy, nonsmoking, male volunteers (range 19-23 years) received a continuous heparin infusion (2000 IU) over 40 min. The endothelial TFPI release rate was estimated from the total TFPI concentration profile using a pharmacokinetic model. Mean +/- s.d. total and free TFPI increased from 62.9 +/- 9.4/8.3 +/- 2.1 ng ml-1 at baseline to 237.2 +/- 40.9/111.0 +/- 19.9 ng ml-1 after 40 min infusion. The relationship between heparin concentration (anti-IIa activity) and TFPI concentration followed a maximum effect model and a clockwise loop (proteresis) was observed. The TFPI release rate rapidly increased to maximum of 200 +/- 45 micro g min-1 after 17.5 min heparin infusion but did not increase further although heparin concentrations further doubled. In contrast to TFPI, t-PA antigen decreased from 5.6 +/- 1.0 at baseline to 4.5 +/- 1.0 ng ml-1 at the end of infusion (t = 40 min) (difference of 1.1 ng ml-1 (95% confidence interval; 0.9, 1.3). Our application of concentration-effect models and pharmacokinetic principles to these haemostatic variables showed that endothelial TFPI release has a maximum that is already reached at low heparin dose, corresponding with an anti-IIa activity of 0.08 IU ml-1. The relationship between anti-IIa activity and TFPI release rate showed signs of acute tolerance (clockwise loop) indicating exhaustion of endothelial TFPI pools. These findings may be of importance for the heparin dose used in conditions such as unstable angina, in which the favourable effects of heparin have been ascribed to its ability to release TFPI.

Randomized trial of different regimens of heparins and in vivo thrombin generation in acute deep vein thrombosis

AbstractLow-molecular-weight and unfractionated heparins are frequently used to treat venous thromboembolism, but it is not known whether they are equally effective in inhibiting in vivo generation of thrombin. In this multicenter trial, 1048 patients were randomized to intravenous unfractionated heparin (group A), twice daily low-molecular-weight heparin (reviparin) for 1 week (group B), or once daily reviparin for 4 weeks (group C). All patients received vitamin K antagonists. Blood samples withdrawn at the baseline and at weeks 1 and 3 were analyzed using markers of in vivo thrombin generation and other coagulation parameters. During the first 3 weeks symptomatic recurrent deep vein thrombosis–pulmonary embolism (DVT/PE) occurred in 17 (4.5%) of 375 patients in group A compared with 4 (1.0%) of 388 patients in group B, and 9 (2.4%) of 374 patients in group C. Forty percent of patients in group A, 53.4% in group B, and 53.5% in group C showed 30% or greater reduction in thrombus size assessed by venography. Patients in group B had significantly greater reduction in D-dimer, prothrombin fragments 1 and 2 (F1 + 2), endogenous thrombin potential (ETP), and thrombin-antithrombin (TAT) complexes compared to groups A and C. Greater release of tissue factor pathway inhibitor (TFPI) and reduction in levels of thrombin activatable fibrinolysis inhibitor (TAFI) and fibrinogen were significantly more pronounced in group C patients. Reviparin administered twice daily plus vitamin K antagonist is more effective in inhibiting in vivo thrombin generation compared to intravenous unfractionated heparin plus vitamin K antagonist, and reviparin once daily produced significantly higher TFPI release and greater reduction in TAFI and fibrinogen levels.

Anticoagulant and antiprotease profiles of a novel natural heparinomimetic mannopentaose phosphate sulfate (PI-88).

Heparinomimetic mannopentaose phosphate sulfate (PI-88) (Progen Industries Ltd. Brisbane, Australia), currently developed as an anticoagulant and antiproliferative agent, mainly is composed of a pentomannan. However, tetrasaccharide and disaccharide components are also present. The molecular profile and the anticoagulant potency of PI-88 are investigated in this study. Gel permeation chromatography and polyacrylamide gel electrophoresis analyses were carried out to determine the molecular profile and separation of components of PI-88, respectively. Potentiation of antithrombin III (ATIII) and heparin cofactor-II (HC-II) activity were measured using chromogenic substrate assay. In order to determine anticoagulant and antiprotease effects of PI-88, various global anticoagulant tests, such as the prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), Hep-test (Haemachem Inc., St. Louis), ecarin clotting time (ECT), activated clotting time (ACT), and thromboelastography (TEG) were used. Anti-Xa and anti-IIa activities also were measured. The effect of PI-88 on the release of tissue factor pathway inhibitor (TFPI) was performed in nonhuman primates who received PI-88 and in endothelial cell culture systems. The relative susceptibility of PI-88 to heparinase I, protamine sulfate (PS), and platelet factor 4 (PF4) also was evaluated. The high-performance liquid chromatography profiles of PI-88 showed that its average molecular weight is approximately 2300 Da. Separation and gradient electrophoretic patterns of PI-88 showed that it is composed of five different fractions. This agent activates HC-II through inhibiting the thrombin generation but not inhibiting ATIII. Although PI-88 produced a concentration-dependent prolongation of all of the clotting tests, ECT gave the best correlation in the dose-response curve (ECT, r2 = 0.94; TT, r2 = 0.84; APTT, r2 = 0.69). Heparinomimetic mannopentaose phosphate sulfate (PI-88) exhibited marked inhibition of FIIa, but not of FXa. Heparinase I failed to produce significant neutralization of PI-88 in all the assays used, whereas PS and PF4 partially neutralized the effects of this compound. Heparinomimetic mannopentaose phosphate sulfate (PI-88) produced fivefold increase in the TFPI levels at 15 minutes after intravenous (IV) injection to primates. The incubation of PI-88 in endothelial cell culture system also showed a strong effect on TFPI release. These results suggest that PI-88 exhibited strong antithrombotic and anticoagulant activity in addition to its known antiproliferative properties. Because of the molecular characteristics and the dual nature of the pharmacologic action of PI-88, this agent represents an attractive pharmacologic agent for the control of thrombotic and proliferative disorders.

Another point of view on the mechanism of thrombin generation during cardiopulmonary bypass: Role of tissue factor pathway inhibitor

Objective: To determine the role of tissue factor and tissue factor pathway inhibitor (TFPI) in coagulation activation during cardiopulmonary bypass (CPB). Design: Prospective, observational study. Setting: Operating room in a city hospital. Participants: Thirty-one patients undergoing cardiac surgery. Measurements and Main Results: The plasma levels of tissue factor antigen (tissue factor), total and free TFPI, several markers of thrombin generation (prothrombin fragment F1+2, thrombin antithrombin complex, and fibrinopeptide A), and heparin concentration were measured. Blood samples were obtained after induction of anesthesia (baseline level), before and after CPB, and at the end of the surgery. Despite an average heparin concentration of 2.9 ± 0.2 IU/mL, markers of thrombin generation, fibrin formation and its degradation (D-dimer) were observed during CPB. Significant increases of total and free TFPI levels (p < 0.0001) were found during CPB associated with lower tissue factor concentration (p < 0.0001) compared with the baseline values. Heparin concentration correlated with levels of total TFPI (r2 = 0.613, p < 0.0001) and free TFPI (r2 = 0.689, p < 0.0001). Tissue factor concentration showed significant negative correlations with levels of total TFPI (r2 = 0.128, p = 0.0003) and free TFPI (r2 = 0.070, p = 0.0078). Conclusion: These data indicate that TFPI release by heparin probably has an important role in the suppression of the tissue factorNdependent coagulation pathway during CPB. These changes occur along with ongoing thrombin generation and its activation. Either insufficient prevention of thrombin generation by TFPI or indirect activation of the intrinsic coagulation pathway occurs during CPB. Copyright © 2001 by W.B. Saunders Company

Bemiparin and Fluid Flow Modulate the Expression, Activity and Release of Tissue Factor Pathway Inhibitor in Human Endothelial Cells In Vitro

We investigated the localisation, gene expression, and activity of tissue factor pathway inhibitor (TFPI) in endothelial cells (EC) grown in static conditions or under shear stress, in the presence of unfractionated heparin (UFH) and two low-molecular-weight heparins (LMWHs). dalteparin and bemiparin (a second generation of LMWHs). All three preparations induced increased release, cellular redistribution, and enhanced activity of TFPI on the cell surface in static EC. In EC grown under shear stress (0.27, 4.1 and 19 dyne/cm2) and incubated with each heparin for 24 h, the release of TFPI was significantly correlated with the level of flow for bemiparin and dalteparin, but not for UFH. For all three levels of flow tested, bemiparin induced the highest secretion and increase of both cellular TFPI and cell surface activity of the inhibitor. The expression of TFPI mRNA, determined by Northern blotting, was specifically modulated by heparins. All three preparations increased the expression of TFPI by 60 to 120% in EC under minimal flow, but only bemiparin enhanced TFPI mRNA in EC under the arterial flow. Immunogold electron microscopy revealed that EC exhibited strong cellular labelling for TFPI when grown under arterial flow in the presence of bemiparin. We conclude that in EC subjected to shear stress in vitro bemiparin is more efficient than UFH or dalteparin in modulating the expression. release and activity of TFPI. We therefore suggest that bemiparin may be superior over the conventional heparins in maintaining the anticoagulant properties of the endothelium.

Plasma Levels of Total and Free Tissue Factor Pathway Inhibitor (TFPI) as Individual Pharmacological Parameters of Various Heparins

The release of circulating tissue factor pathway inhibitor (TFPI) into plasma by heparins is thought to contribute to their overall antithrombotic activity. In the presented study in healthy volunteers, we measured the heparin-induced increase of circulating total and free TFPI antigen and the aXa- and aIIa activity after subcutaneous (s.c.) injection of 9000 aXa-U of four different heparins: unfractionated heparin (UFH) (13.0 kDa), a medium molecular weight (MW) heparin with a narrow MW range (HF) (10.5 kDa), certoparin (6.0 kDa) and enoxaparin (4.5 kDa). Based on the administration of equi-active aXa doses, certoparin induced the highest increase in total TFPI determined as AUC (p <0.01). The lowest effect was observed for UFH (p <0.0001). However, the AUC of released free TFPI significantly increased in the order: enoxaparin < UFH < certoparin < HF, showing MW dependency with the exception of UFH. Comparing the effects of equi-gravimetric heparin doses, the MW dependency becomes even more pronounced. The mismatch of UFH may be due to its poor bioavailability, which becomes obvious from its low ex vivo aXa activity. In contrast to the TFPI releasing potency, the ex vivo aXa activity continuously decreased with increasing MW. Although the ex vivo aIIa activity of the heparins increased in the same order like the release of free TFPI, there was no clear correlation. This is attributed to the fact that the aIIa activity of heparin is not only dependent on the MW, but, in contrast to its TFPI releasing effect, also on the percentage of material with high affinity to AT. In conclusion, besides the aXa- and aIIa activity, the TFPI releasing effect of heparins is an additional parameter of their individual pharmacological profile.

Fluid flow induces upregulation of synthesis and release of tissue factor pathway inhibitor in vitro.

Fluid flow modulates the synthesis and secretion by endothelial cells (ECs) of several proteins that control the hemostatic properties of the vessel wall. Tissue factor pathway inhibitor (TFPI), also synthesized by ECs, is the main downregulator of tissue factor-dependent procoagulant activity. In the present study, we investigated the effect of physiological shear stress on the expression, distribution, and release of TFPI in cultured ECs. The EA.hy926 cell line was grown in a hollow-fiber perfusion system and exposed for variable times to different shear values: 0.27 dyne/cm(2) (minimal flow), 4.1 dyne/cm(2) (venous flow), and 19 dyne/cm(2) (moderate arterial flow). Step increase of the shear stress from 0.27 to 19 dyne/cm(2) induced a sharp increase of TFPI released into the medium and a parallel decrease and redistribution of cell-associated TFPI, which suggests that an acute release of TFPI occurred from the cellular pools. During 24 hours of high shear stress, cell-associated TFPI antigen and mRNA increased time-dependently. Subjecting ECs to steady shear stress for 72 hours also upregulated the expression and production of TFPI, in direct correlation with the degree of the shear. The secretion of TFPI was enhanced 1.9-fold under venous flow and 2.4-fold under arterial flow compared with minimal flow. Equally, cell-associated TFPI antigen and cell surface TFPI activity increased proportionally with the shear stress. The expression of TFPI mRNA, as determined by Northern blotting, increased up to 2-fold in ECs under venous flow and up to 3-fold under arterial flow. These results suggest that shear forces regulate TFPI by modulating its release and gene expression in ECs in vitro.

Long-Term Effect of 17β-Estradiol and Thrombin on Tissue Factor Pathway Inhibitor Release from HUVEC ☆

In spite of the increasing evidence that estrogens have protective effects on the vascular system, the evidence that estrogens may contribute to the risk of thrombosis is still being debated. We investigated the effect of 17β-estradiol (E2) on tissue factor pathway inhibitor (TFPI) release from of cultured human umbilical vein endothelial cells (HUVEC). In this study HUVEC were harvested by collegenase treatment and cultured in multiwelled plates with medium 199 supplemented with 10% fetal calf serum and antibiotics. The cells were incubated in the presence or absence of E2 (1 and 100 nM) with/without thrombin (4 U/mL) for 6 or 24 hours. After the incubations TFPI level of media were measured by IMUBIND Total Eliza kit. Our results demonstrates that E2 at physiological concentrations decreases the release of TFPI from HUVEC significantly. Thrombin also decreases TFPI antigen levels detected in culture media. When combined with thrombin the effect of estrogen is not visible due the much higher effectivity of thrombin in diminishing TFPI levels. These results show that E2 shifts the hemostatic balance towards the procoagulant phase through lowering the TFPI levels secreted by the endothelium.

Dose-dependent release of endogenous tissue factor pathway inhibitor by different low molecular weight heparins.

Tissue factor pathway inhibitor (TFPI) is released to circulating blood after intravenous and subcutaneous injections of heparins, and may thus contribute to the antithrombotic effect of heparins. A previous study suggested different abilities of various low molecular weight heparins (LMWH) to release endogenous TFPI, but the dose-response relationship was not determined. In the present study, the dose-response relationship for escalating doses of two LMWHs, dalteparin and enoxaparin, on the release of endogenous TFPI was investigated. Six healthy male participants were given 50, 100 and 200 U/kg dalteparin and 0.5, 1.0 and 2.0 mg/kg enoxaparin as a single subcutaneous injection. The study was a randomized, cross-over design with a 1-week wash-out period between each injection. Peak free TFPI antigen and TFPI activity were detected after only 1 h, whereas anti-activated factor X (anti-FXa) and anti-activated factor II (anti-FIIa) activities were detected after 2-6 h. Putative therapeutic equivalent doses of dalteparin and enoxaparin gave similar release of endogenous TFPI, but dissimilar effects on anti-FXa and anti-FIIa activities.

Modulation of Vascular Human Endothelial and Rat Smooth Muscle Cell Growth by a Fucosylated Chondroitin Sulfate from Echinoderm

Fucosylated chondroitin sulfate is a glycosaminoglycan extracted from the sea cucumber Ludwigothurea grisea. This polysaccharide has the same structure as a mammalian chondroitin sulfate but some of the glucuronic acid residues display sulfated fucose branches. Anticoagulant and antithrombotic properties of fucosylated chondroitin sulfate have already been described. In order to further investigate its potential therapeutic use as an antithrombotic agent, we studied its effect on vascular smooth muscle cell (SMC) proliferation and endothelial cell proliferation, migration and Tissue Factor Pathway Inhibitor (TFPI) release. The experiments were performed on SMC from rat thoracic aorta and on human umbilical vein endothelial cell (HUVEC) in culture with or without added fibroblast growth factors (FGF-1 and FGF-2). Our results showed that: (i) fucosylated chondroitin sulfate had a strong inhibitory effect on SMC proliferation (IC50 =10 +/- 5 microg/ml) and (ii) no effect on HUVEC proliferation and migration assays, in the absence of exogenous FGF, while heparin had inhibitory effects; (iii) fucosylated chondroitin sulfate (10 microg/ml) enhanced FGF-1 and FGF-2 induced HUVEC proliferation by 45% (145.4 +/- 7.2%) and 27% (126.9 +/- 4.2%), respectively; (iv) on FGF-induced HUVEC migration, fucosylated chondroitin sulfate (10 microg/ml) had a strong enhancing effect with FGF-1, +122% (222.2 +/- 15.8%), three times higher than that of heparin, and a lower enhancing effect with FGF-2, +43% (142.7 +/- 4.6%), whereas heparin had no effect; (v) fucosylated chondroitin sulfate stimulated TFPI release, mainly on the free form. +98% (198.2 +/- 25%). In addition, the structural features of the polysaccharide associated with its biological activity were resolved using chemically modified fucosylated chondroitin sulfates. Sulfated fucose branches groups are essential to the potentiating effect of the polysaccharide on HUVEC proliferation and migration. Surprisingly, removal of fucose branches from the fucosylated chondroitin sulfate did not abolish TFPI release. Finally, partial reduction of the glucuronic acid carboxyl groups limited the potentiating effect on HUVEC proliferation and migration but did not affect TFPI release. In conclusion, this fucosylated chondroitin sulfate from invertebrate origin reveals useful properties for an antithrombotic agent: inhibition of SMC proliferation, enhancement of endothelium wound repair and TFPI release. These properties on vascular cells, associated with a low bleeding tendency and an antithrombotic activity, strongly suggest its potential use as a new therapeutic agent in arterial thrombosis and restenosis, with a more favorable effect than heparin.

Cellular effects of heparin on the production and release of tissue factor pathway inhibitor in human endothelial cells in culture.

Tissue factor pathway inhibitor (TFPI), the major downregulator of procoagulant activity of the tissue factor-factor VIIa complex (TF. FVIIa), is synthesized and constitutively secreted by endothelial cells (ECs). Here we describe the in vitro effects of heparin on the cellular localization, gene expression, and release of TFPI in human ECs in culture. Both unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH; Fragmin) time-dependently induced a significant enhanced secretion of TFPI, paralleled by a redistribution and increase of TFPI on the cell surface and a decrease of intracellular TFPI. Immunogold electron microscopy showed the presence of clusters of TFPI, both on the plasmalemma proper and within cell-surface opened caveolae/enlarged caveolar profiles. Activation of FX by TF. FVIIa on ECs treated with endotoxin was inhibited by both heparins but to a higher extent by LMWH. Inhibition of protein synthesis by cycloheximide did not reduce the release of TFPI induced by heparin. Long-term incubation (48 hours) resulted in a time-dependent enhanced production of TFPI. After the first 4 to 8 hours, depletion of intracellular TFPI was observed, more significantly with UFH. Northern blot analysis of TFPI mRNA also showed a decrease of the 1.4-kb transcript after 4 hours of incubation with UFH, followed by recovery and an increase over the control level after 24 hours. Incubation of ECs with phorbol ester (PMA) significantly enhanced the secretion of TFPI and increased its activity on the cell surface, probably by preventing invagination of caveolae. Heparin-stimulated release of TFPI decreased significantly in the presence of PMA to a level that was 2. 4 times lower than the expected additive value for PMA and UFH separately. Pretreatment of ECs with PMA suppressed a subsequent response to heparin. Altogether, our results suggest that the heparin-induced release of TFPI might involve a more specific mechanism(s) than the previously hypothesized simple displacement of TFPI from the cell surface glycocalyx. We assume that the increased secretion and redistribution of cellular TFPI induced by heparins in ECs in culture can play an important role in the modulation of the anticoagulant properties of the endothelium.

Do Antiphospholipid Antibodies Interfere with Tissue Factor Pathway Inhibitor?

This study was conducted to investigate whether antiphospholipid antibodies (APA) can interfere with the phospholipid-dependent inhibition of coagulation exerted by tissue factor pathway inhibitor (TFPI). Eleven patients with APA and eleven healthy controls matched for age and gender were enrolled. Blood samples were drawn before and 5 minutes after an intravenous injection of unfractionated heparin 5000 IE, which is known to cause TFPI release in healthy individuals. The preheparin samples showed significantly higher TFPI free antigen levels in the APA positive patients than in the controls (21.7 vs. 14.2 ng/ml, p=0.03). TFPI activity as measured in a chromogenic substrate assay also was higher in patients, but this difference was not statistically significant (1.13 vs. 1.01 U/ml, p=0.2). The TFPI levels showed a considerable rise in both patients and controls after heparin injection. In both assays, the postheparin levels were significantly higher in patients than in controls (TFPI antigen: 179 vs. 153 ng/ml, p=0.05; TFPI activity: 3.26 vs. 2.51 U/ml, p=0.03). A modified diluted prothrombin time assay (dPT) was used to measure TFPI anticoagulant activity. In this assay, samples from the patients with the strongest effect of lupus anticoagulants (LAs) on preheparin coagulation times showed little or no increase after heparin injection. This result may reflect an inhibition of TFPI anticoagulant activity by strong LAs. In conclusion, we have found that patients with APA have higher TFPI amidolytic activity/antigen level both before and after heparin stimulation of TFPI release. These observations do not explain the higher thrombotic risk in these patients but may reflect an upregulated tissue factor activity, which has been demonstrated in these patients. TFPI anticoagulant activity, however, as measured in a dPT assay, may be inhibited by strong LAs.

The Effects of Intermittent Pneumatic Compression on Systemic and Local Fibrinolysis

Intermittent pneumatic compression (IPC) is effective in deep venous thrombosis prophylaxis. IPC prevents venous stasis by collapsing the peripheral venous plexus in an extremity leading to increased venous return. It has been suggested that IPC has an additional effect of enhancing fibrinolysis. This study was designed to evaluate the effect of IPC on both systemic and local fibrinolysis in normal volunteers by measuring the activity of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-type 1 (PAI-1). In addition, tissue factor pathway inhibitor (TFPI) was measured to assess changes in the extrinsic coagulation cascade. IPC was applied in the foot and calf of 10 healthy subjects and blood was drawn from the antecubital fossa to determine systemic fibrinolytic activity. Local fibrinolysis was assessed in 15 healthy subjects by placing an IPC cuff on the forearm and drawing blood 2 cm above the cuff The IPC maximum inflation pressure was 120 mm Hg lasting for 3 seconds at three cycles per minute. Blood samples were taken at rest, on the 10th minute of active IPC, and 5 minutes after cessation of IPC for both systemic and local measurements. The plasma was analyzed for TFPI, t-PA, and PAI-1 antigen by use of enzyme-linked immunosorbent assays. There were no significant changes in systemic or local fibrinolytic activity before, during, or after application of IPC. TFPI systemic activity before, during, and after was 111 ±24, 118 + 18, and 116 ±22, respectively. Local TFPI activity was 91 ±32, 93 +36, and 91 ± 24, respectively. The t-PA systemic activity before, during, and after was 4.1 ± 1.9, 4.7 ± 2.3, and 5 + 2.8, respectively. Local t-PA activity was 4.5 ± 1.3, 4.5 ± 1.4, and 4.2 ± 1.4, respectively. Systemic PAI-1 activity was 11 ± 9.2, 17 ± 19, and 17 ±31, respectively. Local PAI-I activity was 3.7 ± 3.1, 3 ± 1, and 2.8 + 1, respectively, p &gt; 0.38 for all comparisons in both groups. No evidence was found that IPC enhances systemic and local fibrinolysis or TFPI release. Irrespective of the length of IPC application or the inflation pressure, several studies have reported increased fibrinolysis, whereas others have not found any changes. Although, according to the literature, there is a trend toward increased fibrinolytic activity, further controlled studies with adequate sample size should be performed to provide an answer to this controversial topic.

Tissue Factor Reduction and Tissue Factor Pathway Inhibitor Release after Heparin Administration

Elevated plasma levels of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) and large amounts of monocyte procoagulant activity (PCA) have been documented in unstable angina (UA) patients. In in vitro experiments heparin is able to blunt monocyte TF production by inhibiting TF and cytokine gene expression by stimulated cells and after in vivo administration it reduces adverse ischemic outcomes in UA patients. TF and TFPI plasma levels and monocyte PCA have been investigated in 28 refractory UA patients before and during anticoagulant subcutaneous heparin administration (thrice daily weight- and PTT-adjusted for 3 days) followed by 5000 IU X 3 for 5 days. After 2-day treatment, immediately prior to the heparin injection, TF and TFPI plasma levels [(median and range): 239 pg/ml, 130-385 pg/ ml and 120 ng/ml, 80-287 ng/ml] were lower in comparison to baseline samples (254.5 pg/ml, 134.6-380 pg/ml and 135.5 ng/ml, 74-306 ng/ml). Four h after the heparin injection TF furtherly decreased (176.5 pg/ml, 87.5-321 pg/ml; -32.5%. p<0.001) and TFPI increased (240.5 ng/ml, 140-450 ng/ml; +67%, p<0.0001). After 7-day treatment, before the injection of heparin, TF and TFPI plasma levels (200 pg/ml, 128-325 pg/ml and 115 ng/ml, 70-252 ng/ml) significantly decreased (p<0.05) in comparison to the pre-treatment values. On the morning of the 8th day, 4 h after the injection of heparin TF plasma levels and monocytes PCA significantly decreased (156.5 pg/ml, 74-259 pg/ml and from 180 U/105 monocytes, 109-582 U/10(5) monocytes to 86.1 U/10(5) monocytes, 28-320 U/10(5) monocytes; - 38% and -55% respectively) and TFPI increased (235.6 ng/ml, 152-423 ng/ ml; +70%, p<0.001). In conclusion, heparin treatment is associated with a decrease of high TF plasma levels and monocyte procoagulant activity in UA patients. These actions of heparin may play a role in determining the antithrombotic and antiinflammatory properties of this drug.

Acute Release of Tissue Factor Pathway Inhibitor after In Vivo Thrombin Generation in Baboons

Tissue factor pathway inhibitor (TFPI), the major downregulator of the procoagulant activity of tissue factor (TF), is synthesised by endothelial cells (EC) and acutely released in vitro after thrombin stimulation. Expression of TF on EC and subsequent thrombin generation occurs in vivo during sepsis or malignancy, inducing disseminated intravascular coagulation (DIC). The present study investigates the changes in plasma TFPI in relation to markers of in vivo thrombin generation induced by injection of factor Xa (FXa)/phospholipids in baboons at dosages leading to partial (48%) or complete fibrinogen depletion. The plasma concentrations of thrombin-antithrombin III (TAT) and fibrinopeptide A (FpA), as markers of in vivo generation of thrombin, were strongly enhanced after injection of FXa/phospholipids. TFPI levels, whether measured as antigen or activity, increased significantly in both treatment groups within few minutes, and were dependent on the dose of FXa/phospholipids. Significant positive correlations between plasma levels of TFPI and of TAT or FpA were observed. Altogether, our results indicate that experimentally induced in vivo generation of thrombin causes the acute release of TFPI, high-lighting a possible novel function of thrombin in downregulation of the coagulation process, potentially relevant for the outcome of DIC.