The effect of heparin on tissue factor and tissue factor pathway inhibitor in patients with acute myocardial infarction.

We examined plasma TF and free TFPI levels in 26 consecutive patients with AMI, 26 patients with stable exertional angina, and 25 patients with chest pain syndrome. In patients with AMI, blood samples were obtained immediately after admission and at 4, 8, 16, 24, and 48 h, and the third, fifth, seventh, and fourteenth day after initiation of reperfusion therapy. Plasma TF levels in patients with AMI on admission were significantly higher than in the chest pain syndrome and stable exertional angina groups (248.0+/-117. 4 vs. 179.5+/-29.2 vs. 189.5+/-29.6 pg/ml, P<0.01). In patients with AMI, the level subsequently decreased after heparin administration and was maintained at significantly lower levels compared to those on admission. Plasma free TFPI levels in patients with AMI on admission were significantly higher than in the chest pain syndrome and stable exertional angina groups [33.5+/-12.4 vs. 26.0+/-7.6 ng/ml (P<0.01) vs. 27.5+/-6.3 ng/ml, P<0.05]. In patients with AMI, it reached the maximum level at 4 h after the administration of heparin, and gradually decreased over the time course. These data indicated that continuous administration of a low dose of heparin was effective in decreasing TF levels without affecting TFPI levels. Our results elucidate one of the mechanisms by which the administration of heparin is beneficial in AMI patients undergoing percutaneous revascularization.

Changes of hemostatic molecular markers after gynecological surgery.

The authors evaluated the hemostatic abnormalities occurring in the postoperative period of eight patients with malignant tumors and compared them with those occurring in the postoperative period of eight patients with benign tumors. Two of the patients with malignant tumor presented pulmonary embolism after operation. Plasma fibrinogen and fibrin degradation product levels in patients with malignant tumors were already high before operation and further increased significantly after operation. The plasma levels of D-dimer, thrombin-antithrombin complex, and free-tissue factor pathway inhibitor were increased in both groups after operation, but they were higher in patients with malignant tumors than in patients with benign tumors. The plasma levels of protein C and antithrombin were significantly decreased in both groups after operation. but they were significantly lower in patients with malignant tumors than in those with benign tumors. The decreased activity of protein C or antithrombin may be not only a risk factor of thrombotic disease, such as pulmonary embolism, but also the cause of thrombosis. In patients with malignant tumors, the operation time was significantly longer than that in patients with benign tumors. This long operative period might cause vascular endothelial cell injury which is reflected by the plasma levels of free-tissue factor pathway inhibitor, antithrombin, and protein C.

Decreased tissue factor and tissue-plasminogen activator antigen in relapsed acute promyelocytic leukemia.

This study evaluated hemostatic data in 28 patients with newly diagnosed acute promyelocytic leukemia (APL) and 15 patients with relapsed APL. Activated partial thromboplastin time and prothrombin time were prolonged at initial onset of APL. Plasma level of fibrinogen was significantly decreased in patients with initial disease of APL, but it was not decreased significantly during the relapse of APL. Plasma fibrin and fibrinogen degradation products levels were significantly increased and platelet counts significantly decreased in both groups. Plasma levels of antiplasmin significantly decreased at initial onset but not during relapse. Plasma levels of antithrombin were within normal range in patients with initial disease but significantly decreased in those with relapse. Plasma levels of D-dimer, soluble fibrin monomer (sFM), plasmin-plasmin inhibitor complex (PPIC), and thrombin antithrombin complex (TAT) levels were significantly high in both groups. Plasma levels of PPIC, sFM, and D-dimer were significantly higher at initial onset of APL than during relapse. However, there was no significant difference in DIC score between patients with initial onset and those with relapse; plasma levels of tissue factor (TF) significantly increased in both groups, but they were significantly higher at initial onset of APL than during relapse. TF and tissue type plasminogen activator (t-PA) antigen levels in leukemic cell lysate were significantly increased in both groups, and they were significantly lower during relapse than at initial onset. Hemostatic abnormalities occurring in patients with relapsed APL might be the result of the decrease of TF and t-PA in leukemic cells. These findings suggest that DIC in APL patients with relapse might not be caused only by TF and t-PA and thus should be treated with different therapy from patients with initial onset of APL.

Intravascular free tissue factor pathway inhibitor is inversely correlated with HDL cholesterol and postheparin lipoprotein lipase but proportional to apolipoprotein A-II.

To elucidate the distribution and clinical implications of tissue factor pathway inhibitor (TFPI) concentrations, we measured TFPI levels consisting of preheparin free, lipoprotein-bound (Lp-bound), and endothelial cell-anchor pools in 156 patients with coronary artery disease (average age, 61.2+/-9.1 years; range, 32 to 78 years) by heparin infusion (50 IU/kg) and compared them with the preheparin TFPI levels of 229 healthy subjects (average age, 59. 6+/-9.4 years; range, 41 to 80 years). The patients had lower preheparin free TFPI and lower HDL cholesterol (HDL-C) levels than the healthy subjects with equivalent Lp-bound forms (free TFPI, 15. 9+/-6.5 versus 19.2+/-8.1 ng/mL). In a partial correlation analysis, the preheparin free TFPI levels were shown to be inversely correlated with the HDL-C concentrations in both the patients (r=-0. 454, P<0.001) and the healthy subjects (r=-0.136, P<0.05). As determined by comparison of preheparin and postheparin plasma, the patients generally showed preheparin free TFPI <10%, Lp-bound TFPI at 30%, and endothelial cell-anchor TFPI at 60%. When the patients were divided into 4 categories by their LDL cholesterol (LDL-C, 130 mg/dL) and HDL-C (40 mg/dL) levels to specify their coronary risks, the low-HDL-C groups had significantly increased preheparin and postheparin free TFPI levels and decreased postheparin LPL levels, whereas the high-LDL-C groups showed increased levels of Lp-bound TFPI. In a partial correlation analysis, we found a proportional relation between postheparin free TFPI and apolipoprotein A-II (r=0. 5327) and between HDL-C and LPL (r=0.4906), whereas postheparin free TFPI was inversely correlated with HDL-C (r=-0.4280) and postheparin LPL (r=-0.4791). The inverse relationship between TFPI and LPL suggests that increased free TFPI concentrations as a compensatory response of the endothelium to prevent atherothrombotic processes compete with and displace LPL on endothelial surface, resulting in reduced LPL and low HDL-C.

Monoclonal antibody specific for tissue factor pathway inhibitor-factor Xa complex: its characterization and application to plasmas from patients with disseminated intravascular coagulation and pre-disseminated intravascular coagulation.

Tissue factor pathway inhibitor (TFPI), a Kunitz-type protease inhibitor with three tandem inhibitory domains (K1, K2 and K3), inhibits the initial reactions of the extrinsic blood coagulation pathway through its K1 and K2 domains. We prepared and characterized a monoclonal antibody (Mab8-1) against TFPI-factor Xa (TFPI-Xa) complex. The reactivities of Mab8-1 toward TFPI-Xa complex, TFPI without C-terminal (TFPI-C)-Xa complex, K1K2-Xa complex and K2K3-Xa complex were examined using a surface plasmon resonance analysis (Biacore). The Biacore system allowed a quantitative analysis of antibody-antigen interaction, in real time, from which the association and dissociation rate constants could readily be obtained. The bindings of Mab8-1 to TFPI-Xa complex, TFPI-C-Xa complex and K2K3-Xa complex were each concentration-dependent. However, no binding of Mab8-1 to the K1K2-Xa complex was observed. The binding of Mab8-1 to TFPI or Xa was also not observed. These results suggested that the epitope for Mab8-1 was exposed in the K3 domain of TFPI, which was generated by the conformational change after the formation of TFPI-Xa complex. We then developed an enzyme-linked immunosorbent assay method specific for TFPI-Xa complex using Mab8-1, and we used this assay to measure plasma levels of TFPI-Xa. The normal range assessed from analyses of plasma from 30 normal healthy volunteers was 17.7-66.7 with a mean of 35.5 +/- 11.7 pmol/l. In order to asses the clinical implication of TFPI-Xa complex in the plasma of patients with thrombotic disorders, plasma concentrations were measured in 37 patients with disseminated intravascular coagulation (DIC) caused by a variety of underlying diseases. The TFPI-Xa antigen levels were significantly higher in the patients with DIC (51.9 +/- 21.6 pmol/l) and the 36 patients with pre-DIC (55.1 +/- 20.2 pmol/l) than in the 137 non-DIC patients (37.9 +/- 13.1 pmol/l). In the patients with DIC or pre-DIC, there was no significant correlation between TFPI-Xa complex and the elevated levels of thrombin-antithrombin complex, plasmin-alpha2 plasmin inhibitor complex, D-dimer, soluble fibrin monomer, soluble thrombomodulin or tissue factor. These data indicate that the plasma level of TFPI-Xa seems to be a novel independent molecular marker of DIC and pre-DIC.

Changes of plasma hemostatic markers during percutaneous transluminal coronary angioplasty in patients with chronic coronary artery disease.

Changes of hemostatic parameters during percutaneous transluminal coronary angioplasty (PTCA) in 75 patients with chronic coronary artery disease were evaluated. Plasma levels of D-dimer, soluble fibrin monomer, plasmin-alpha2 antiplasmin inhibitor complex, and tissue factor (TF) were significantly increased in all patients with chronic coronary artery disease. The activity of antithrombin and protein C and the levels of protein C antigen were significantly decreased 1 hr after PTCA, but they returned to normal range 1 day after PTCA. There was no significant difference in the level of plasma APC-PCI complex before and 1 hr after PTCA. The plasma levels of D-dimer, soluble fibrin monomer, thrombomodulin, TF and PPIC were significantly decreased 1 hr, and the plasma levels of plasmin-alpha2 antiplasmin inhibitor complex 1 day after PTCA. These findings suggest that the decrease of protein C and antithrombin resulted in activation of the coagulation system. One hour after PTCA, the plasma levels of (total-free) TF pathway inhibitor (TFPI) were significantly decreased, but the plasma levels of total and free-TFPI were significantly increased, suggesting that consumption of (total-free) TFPI occurs during PTCA. Overall, these findings suggest that the hypercoagulable state improves during PTCA and that transient decrease of antithrombin, protein C, (total-free) TFPI or plasmin-alpha2 antiplasmin inhibitor complex may cause restenosis of coronary artery.

Heightened tissue factor associated with tissue factor pathway inhibitor and prognosis in patients with unstable angina.

BACKGROUND: This study was designed to evaluate the plasma levels of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) in patients with unstable angina and investigate whether there is a relationship between these levels and unfavorable outcome. METHODS AND RESULTS: The plasma TF and free TFPI antigen levels were determined in plasma samples taken from 51 patients with unstable angina, 56 with stable exertional angina, and 55 with chest pain syndrome. The plasma TF and free TFPI antigen levels were higher in the unstable angina group than in the stable exertional angina and chest pain syndrome group. There was a good correlation between TF and TFPI. We established borderline as maximum level in the patients with chest pain syndrome. Seven patients (of the 22 in the high TF group) required revascularization to control their unstable angina during in-hospital stay. On the other hand, only 1 of the 29 patients in the low TF group required myocardial revascularization. Four patients of the 14 patients in the high free TFPI group required myocardial revascularization during in-hospital stay, and 4 of the 37 patients in the low free TFPI group required myocardial revascularization. We compared the TF and free TFPI levels between the cardiac event (+) group and cardiac event (-) group. TF levels were significantly higher in the cardiac event (+) group than in the cardiac event (-) group. CONCLUSIONS: We have demonstrated that not only the plasma TF levels but also the plasma-free TFPI levels are elevated in patients with unstable angina. Patients with unstable angina and heightened TF and free TFPI are at increased risk for unfavorable outcomes. The heightened TF level was a more important predictor in patients with unstable angina.

Plasma tissue factor pathway inhibitor and tissue factor antigen levels after administration of heparin in patients with angina pectoris.

The hypercoagulability is associated with expression of tissue factor in patients with angina. Tissue factor pathway inhibitor regulates the extrinsic coagulation pathway mediated by tissue factor. Plasma samples were obtained from 14 patients with angina pectoris and 9 with chest pain syndrome before and 5, 30, 60, and 120 minutes after administration of heparin (50 IU/kg). The tissue factor and prothrombin fragment 1+2 levels before administration were elevated in patients with angina pectoris and were reduced to the levels of chest pain syndrome after the administration. The free tissue factor pathway inhibitor levels after the administration were higher in patients with angina pectoris than in patients with chest pain syndrome. Plasma tissue factor pathway inhibitor levels correlated positively with plasma tissue factor and prothrombin fragment 1+2 levels. We showed that plasma-free TFPI levels after administration of heparin, which may indicate endothelial cell associated TFPI levels, increased in patients with angina pectoris compared with patients with chest pain syndrome. Increased endothelial cell associated TFPI was associated with hypercoagulability in patients with angina pectoris. These may help to explain the reduction in thrombotic risk associated with the use of heparin.

Changes in plasma tissue factor pathway inhibitor levels during the clinical course of disseminated intravascular coagulation.

In healthy volunteers, the plasma total tissue factor pathway inhibitor (TFPI) level was 68.7+/-14.1 ng/ml; the plasma free TFPI level, 17.7+/-5.4 ng/ml; the lipoprotein-associated TFPI (LP-TFPI), 51.1+/-12.0 ng/ml; the free TFPI/total TFPI ratio 0.26+/-0.07; and the plasma tissue factor levels were 149+/-46 pg/ml. Plasma tissue factor levels in patients with disseminated intravascular coagulation (DIC) were significantly higher than those in pre-DIC patients or in non-DIC patients. Plasma total-TFPI, free-TFPI and LP-TFPI levels were significantly higher in DIC patients than those in pre-DIC patients or in non-DIC patients. Before the onset of DIC, the plasma levels of tissue factor gradually increased, and 3 days before the onset of DIC they were significantly higher than those in non-DIC patients. The plasma levels of tissue factor reached their highest level 1 day before the onset of DIC and gradually decreased after the onset of DIC. Plasma levels of total-TFPI, free-TFPI, and LP-TFPI gradually increased before the onset of DIC, and the total-TFPI and LP-TFPI reached their highest levels at the onset of DIC. Plasma free-TFPI reached highest level one day after the onset of DIC. During the clinical course of DIC, the plasma level of tissue factor was the first to increase, then that of LP-TFPI and finally the free-TFPI plasma levels. These differences in the peak plasma levels of tissue factor, free-TFPI, and LP-TFPI might be related to the clinical course of DIC.

Simultaneous elevation of the levels of circulating monocyte chemoattractant protein-1 and tissue factor in acute coronary syndromes.

The levels of circulating monocyte chemoattractant protein-1 (MCP-1) and tissue factor (TF) were examined on admission in 46 consecutive patients with acute coronary syndromes (ACS) and 30 patients with stable exertional angina (SEA). The plasma levels of both MCP-1 and TF were higher in the ACS patients than in the SEA patients (MCP-1: p<0.001; TF: p<0.001). Only the circulating TF level related to the number of diseased vessels. A positive correlation between plasma MCP-1 and TF levels was found (r=0.476, p<0.001). These results suggest that circulating MCP-1 plays an important role in the pathogenesis and/or development of ACS.

Circadian variation in plasma levels of free-form tissue factor pathway inhibitor antigen in patients with coronary spastic angina.

Tissue factor pathway inhibitor (TFPI) is known to inhibit the initial reaction in the tissue factor-mediated coagulation pathway. We measured plasma free-form TFPI antigen levels and monitored 24-h Holter recordings at 06.00, 14.00 and 22.00 h in 15 patients with coronary spastic angina, 13 patients with stable exertional angina, and 11 control subjects. There was a significant circadian variation in plasma free-form TFPI antigen levels in patients with coronary spastic angina (25.8+/-2.0 ng/ml at 06.00 h, 21.1+/-1.6 ng/ml at 14.00 h, and 20.2+/-1.4 ng/ml at 22.00 h; p<0.01). Furthermore, free-form TFPI antigen levels at 06.00 h were significantly higher in coronary spastic angina patients than in patients with stable exertional angina or control subjects (p<0.01). Free-form TFPI antigen levels increased after the ischemic attacks in coronary spastic angina (p<0.01). This circadian variation correlated with the frequency of attacks, with the peak level occurring between midnight to early morning in patients with coronary spastic angina.

Structural requirements of human tissue factor pathway inhibitor (TFPI) and heparin for TFPI-heparin interaction.

Heparin affinity chromatography of synthetic peptide fragments mimicking tissue factor pathway inhibitor (TFPI) indicated that the minimal heparin binding sequence consists of 12 amino acid residues located at the C-terminal tail. Within this minimal sequence, Arg-257 and Arg-259 appeared to contribute most significantly to interaction with heparin. Affinity chromatography of TFPI using immobilized heparin derivatives regiospecifically desulfated at O-6 of the glucosamine residue, N-2 of the glucosamine residue, and/or O-2 of the iduronic acid residue indicated that all the sulfate groups in heparin appeared to be required for TFPI-heparin interaction. Among them, however, the 6-O-sulfate groups appeared to make the largest contribution to the interaction, while the 2-O-sulfate groups contributed the least. In vitro experiments on the inhibition of factor Xa by TFPI enhanced with native and chemically modified heparins afforded similar results.

Poor outcome in disseminated intravascular coagulation or thrombotic thrombocytopenic purpura patients with severe vascular endothelial cell injuries.

Various hemostatic and vascular endothelial cell markers were measured in patients with disseminated intravascular coagulation (DIC), non-DIC, or thrombotic thrombocytopenic purpura (TTP) and in healthy volunteers to examine the relationships between the hemostatic abnormalities or vascular endothelial cell injuries and the patients' outcomes. Although the plasma levels of soluble fibrin monomer, thrombin-antithrombin complex, plasmin-plasmin inhibitor complex, and D-dimer were significantly increased in the DIC patients, there were no significant differences in these markers between the DIC patients who survived and those who died, suggesting that these markers might not be directly related to the patient outcome. The plasma thrombomodulin (TM) levels in the DIC and TTP patients were significantly higher than those in the healthy volunteers, and the plasma TM levels in the patients who died were significantly higher than those in the patients who survived. These findings showed that the TM level reflected the outcome, and that the outcome of the diseases underlying DIC and TTP might depend on vascular endothelial cell injuries. The plasma protein C and antithrombin activities were markedly reduced in the DIC, non-DIC, and TTP patients who died compared to those who survived. These findings suggest that reduced plasma antithrombin and protein C activities are useful markers of systemic vascular endothelial injuries. Although the plasma tissue factor (TF) levels were significantly increased in the DIC patients, there was no significant difference in the plasma TF levels between the DIC patients who died and those who survived. In conclusion, we found that the outcome of the diseases underlying DIC and TTP is related to vascular endothelial cells, and that plasma TM, antithrombin, and protein C are useful markers for systemic vascular endothelial cell injury.

Heparin-releasable endothelial cell-associated tissue factor pathway inhibitor (TFPI) is increased in the coronary circulation after coronary spasm in patients with coronary spastic angina.

Tissue factor pathway inhibitor (TFPI) is a physiological regulator of the extrinsic coagulation cascade. Coronary spasm can alter endothelial cell properties in the coronary artery with resultant thrombosis. To determine whether coronary spasm affects plasma TFPI level, we measured the heparin-releasable endothelial cell-associated TFPI (heparin-releasable TFPI) (ng/ml) in the coronary sinus and the aortic root before and after coronary spasm induced by an injection of acetylcholine in 18 patients with coronary spastic angina, and before and after myocardial ischemia induced by rapid atrial pacing in 18 patients with stable exertional angina, and in 17 control subjects with normal coronary arteries and no coronary spasm. Heparin-releasable TFPI level in the coronary spastic angina group significantly increased in the coronary sinus (1 22+/-46 to 147+/-63, p<0.001) after the ischemic event but not in the aortic root (113+/-44 to 121+/-58). The level in the coronary sinus and the aortic root remained unchanged after the ischemic event in the stable exertional angina group and after the injection of acetylcholine in the control group. The coronary sinus-arterial difference in the amount of the heparin-releasable TFPI significantly increased after the ischemic event only in the coronary spastic angina group (10+/-18 to 26+/-18, p<0.002). Our result suggested that heparin-releasable TFPI is increased in the coronary circulation after coronary spasm.

Comparison of plasma tissue factor levels in unstable and stable angina pectoris.

We have reported that the plasma levels of plasma fibrinopeptide A and plasminogen activator inhibitor activity increase in patients with unstable angina and acute myocardial infarction. Tissue factor (TF) is a low-molecular-weight glycoprotein that binds to and acts on essential cofactor VII, and the resulting complex activates factors IX and X, initiating the coagulation cascade. We measured plasma TF antigen levels in 21 patients with unstable angina (on admission and after treatment), 27 patients with stable exertional angina, and 27 control subjects. The 3 groups were matched for age, gender, and other clinical variables. The plasma TF antigen levels were higher in the unstable angina group than in the stable exertional angina and control groups (240 +/- 75 vs 184 +/- 46 and 177 +/- 37 pg/ml, p < 0.01). There were no significant differences in the plasma TF antigen levels between the stable exertional angina and the control groups. Furthermore, the plasma TF antigen levels were reexamined after treatment in the 21 patients with unstable angina. The mean level in these 21 patients decreased after 2 weeks of treatment (from 240 +/- 75 to 206 +/- 57 pg/ml, p < 0.01). This study suggests that the plasma TF antigen levels correlate with disease activity in patients with unstable angina. The increased plasma TF antigen levels in patients with unstable angina may reflect intravascular procoagulant activity.

Increased tissue factor pathway inhibitor in patients with acute myocardial infarction.

We examined hemostatic abnormalities in 23 patients with acute myocardial infarction (AMI), 10 with pulmonary embolism (PE), and 10 with deep vein thrombosis (DVT). At the onset of AMI, plasma levels of tissue-type plasminogen activator (t-PA), PA inhibitor-I (PAI-I), fibrin-D-dimer, thrombin-antithrombin complex (TAT), and plasmin-plasmin inhibitor complex (PPIC) were significantly increased. Both the plasma total TFPI and free-TFPI levels in the AMI patients were significantly higher than those in the healthy volunteers, PE patients, and DVT patients. There was no significant difference in total TFPI or free-TFPI among patients with PE, those with DVT, and healthy volunteers. One hour after percutaneous transluminal coronary angioplasty (PTCA) in the AMI group, the total TFPI level was further increased, and it was significantly reduced 24 hr after PTCA, to a level similar to that in healthy volunteers. Free-TFPI showed a pattern similar to that of total TFPI. The ratio of free-TFPI/total TFPI was highest 1 hr after PTCA. Increased TFPI in AMI patients might be released from ischemic tissues.

Increased plasma tissue factor levels in acute myocardial infarction.

BACKGROUND: Tissue factor (TF) is a low molecular weight glycoprotein that initiates the clotting cascade and is considered to be a major regulator of coagulation, hemostasis, and thrombosis. METHODS AND RESULTS: We examined plasma TF levels in 31 consecutive patients with acute myocardial infarction (AMI) (within 6 hours after the onset of symptoms), 27 patients with stable exertional angina, and 27 control subjects. Ten patients with AMI had a history of unstable angina before infarction, and 21 had a sudden onset of infarction. The plasma TF level was higher in the AMI group than in the stable exertional angina and control groups (240 +/- 112 vs 184 +/- 46 pg/ml [p < 0.05] vs 177 +/- 37 pg/ml, p < 0.01, respectively). TF levels were decreased in the chronic phase (2 weeks after admission) compared with the acute phase of infarction (from 240 +/- 112 pg/ml to 222 +/- 97 pg/ml, p < 0.05). In addition, plasma TF levels were higher in patients with AMI with prodromal unstable angina than in patients with a sudden onset of infarction (300 +/- 169 pg/ml vs 212 +/- 57 pg/ml, p < 0.05). TF levels were similar in the acute and chronic phases in the patients with AMI with prodromal unstable angina (300 +/- 169 pg/ml vs 290 +/- 136 pg/ml, p = not significant) but were decreased in the chronic phase in the patients with AMI with sudden onset (from 212 +/- 57 pg/ml to 190 +/- 49 pg/ml, p < 0.05). CONCLUSION: Increased plasma TF levels in patients with AMI may reflect enhanced intravascular procoagulant activity. The higher TF levels in patients with AMI with prodramol unstable angina may be associated with repeated episodes of myocardial ischemia and reperfusion.

Effects of enalapril on tissue factor in patients with uncomplicated acute myocardial infarction.

In a randomized, double-blind, placebo-controlled study beginning 4 weeks after uncomplicated acute myocardial infarction, it was established that the baseline plasma tissue factor antigen level was significantly higher in patients with myocardial infarction than in control subjects, and enalapril therapy significantly reduced the elevated plasma tissue factor antigen level. This may be associated with the reduction in the risk of coronary thrombosis seen with the use of angiotensin-converting enzyme inhibitors.

Measurement of the free form of TFPI antigen in hyperlipidemia. Relationship between free and endothelial cell-associated forms of TFPI.

Tissue factor pathway inhibitor (TFPI), a protease with three tandem Kunitz-type (K1, K2, and K3) domains, inhibits the initial reaction of the TF-mediated coagulation pathway. TFPI occurs in a free and a lipoprotein-associated form in plasma as well as an endothelial cell-associated form on vascular walls. In a previous study we had demonstrated that free-form TFPI activity was lower in hyperlipidemic patients. In the present study we established a new enzyme immunoassay method for measuring free-form TFPI antigen; this new method uses a monoclonal antibody that recognizes the K3 domain of free-form TFPI but not lipoprotein-associated TFPI. Free-form TFPI antigen was significantly lower in hyperlipidemic patients compared with those in normolipidemic individuals. We applied this new method to measure the amount of endothelial cell-associated TFPI, which can be released by heparin injection, as "free-form TFPI." We found that free-form TFPI antigen in plasma was positively correlated with the endothelial cell-associated form. These results indicate that both of these forms of TFPI are in equilibrium in vivo and that our new method can be used for assessing changes in the levels of endothelial cell-associated TFPI antigen and, hence, for assessing thrombotic tendencies in various disease states.