Anne Karin Lindahl

THE PRESENT STATUS OF TISSUE FACTOR PATHWAY INHIBITOR

Tissue factor pathway inhibitor (TFPI) is the factor Xa-dependent inhibitor of the factor VIIa/tissue factor complex. The plasma concentration of this 276 amino acid, 40 kDa glycoprotein is normally about 100 ng/ml. There are three intravascular pools of TFPI: 50-90% is on the endothelium, 10-50% is in plasma and less than 2.5% is in platelets. The TFPI in plasma is mainly associated with lipoproteins-only about 5% is free TFPI. The lipoprotein-associated TFPI seems to be of less anticoagulant effect than the free TFPI. Both unfractionated heparin, low-molecular-weight heparins and pentosan polysulphate induce release of TFPI after intravenous injection, whereas dermatan sulphate does not. The interactions with TFPI account for a considerable amount of the anticoagulant effect of heparin. Studies have shown increased TFPI levels in plasma from patients with advanced malignancy and in subjects with fatal DIC or septicaemia. The reason for this is unknown. For measuring the anticoagulant activity of TFPI in plasma, end-point or antigen assays may be less useful than the clotting assay with dilute tissue factor. Animal studies indicate that the main physiological role of TFPI is the inhibition of small amounts of tissue factor. TFPI is probably essential for a normal haemostatic balance.

Chromogenic substrate assay of extrinsic pathway inhibitor (EPI): levels in the normal population and relation to cholesterol.

A two-stage chromogenic substrate assay was standardized to measure extrinsic pathway inhibitor (EPI) activity in plasma and serum samples. In the first stage, diluted plasma or serum (0–0.8%) was incubated with factor VHa (25 pM), tissue thromboplastin (tissue factor, TF, 1% v/v) with excess binding sites for factor VIIa, and factor Xa (0.8 nM). In the second stage, excess factor X and chromogenic substrate were added as substrate for residual TF/factar VIIa catalytic activity. Heating the samples at 56±C for 15 min before assay removed ≥95% of the factor VII amidolytic activity of the samples, defibrinated the plasma, and produced only slight reduction of EPI activity. The coefficient of variation for the same sample assayed on different days was 8.7–10.6% and the intra-assay coefficient of variation was 5.0%. Addition of anti-EPI immunoglobulin to normal plasma completely abolished the EPI activity of the sample. EPI activity was stable in plasma samples stored at – 20± C, but in serum, some samples lost > 50% activity after 3 months at – 70± C. Median EPI activity of umbilical cord blood was 45% (range 33 – 93%). In a cohort of healthy blood donors (n = 176) EPI activity was significantly correlated with age; the regression line was y = 68% + 0.60x (r = 0.39). The approximated standard deviation for the regression line was 17.9% and the age-adjusted reference limits were determined. Equal levels were seen in maies and females. EPI activity was significantly correlated with total cholesterol (r = 0.58), and multiple regression showed that cholesterol was a stronger predictor of EPI activity than age.

Tissue factor pathway inhibitor with high anticoagulant activity is increased in post-heparin plasma and in plasma from cancer patients.

This study was performed in order to separate plasma fractions of tissue factor pathway inhibitor (TFPI) on the basis of TFPIs heparin binding properties. A main goal was to look for differences in anticoagulant effect of the TFPI fractions from plasma. Normal plasma and plasma with increased amounts of TFPI were used; plasma from cancer patients and post-heparin plasma (plasma drawn 5 min after heparin injection). Heparin affinity chromatography separated plasma TFPI into four fractions with increasing heparin affinity: the flow-through fraction, a low affinity fraction eluting at < 0.3 M NaCl, an intermediate affinity fraction eluting at 0.3–0.55 M NaCl and a high affinity fraction eluting at 0.55 −1.0 M NaCl. These fractions corresponded partly to the three TFPI activity fractions obtained by gel filtration. In plasma from cancer patients, the two fractions with intermediate and high heparin affinity were increased three- to fourfold, compared to normal plasma. The TFPI activity in these two fractions eluted with low-molecular-weight (35–60 kD) on gel filtration. In post-heparin plasma an even larger increase in the fraction with high heparin affinity was found; compared to that in normal plasma it was increased 14-fold. TFPI was purified to 0.72 U/mg protein in this fraction (about 43-fold compared to normal plasma TFPI). The anticoagulant effect of TFPI, relative to the chromogenic substrate TFPI activity, was greater in plasma fractions with high heparin affinity than in the other plasma TFPI fractions, and it was five-fold greater than the anticoagulant effect of recombinant TFPI. Thus, plasma TFPI is heterogenous in heparin affinity and in anticoagulant potency. Free TFPI with high heparin affinity is a more potent coagulation inhibitor than the other plasma TFPI fractions.

Markers of vascular inflammation are associated with the extent of atherosclerosis assessed as angiographic score and treadmill walking distances in patients with peripheral arterial occlusive disease

The importance of inflammation in atherosclerosis is well established in cardiovascular disease. However, limited data exist on the relationship between vascular inflammation and the severity of peripheral arterial occlusive disease (PAD). We investigated the relationship between biochemical markers of vascular inflammation and the diagnostic measures of PAD: ankle-brachial pressure index (ABI), maximum treadmill walking distance and angiographic score. In 127 patients (mean age 66 years; 64% males) with angiographically verified PAD, fasting blood samples were drawn for determination of selected soluble cell adhesion molecules, cytokines and chemokines. Tumor necrosis factor-α (TNFα), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1) and CD40 ligand (CD40L) were all significantly correlated with the angiographic score (p < 0.05 for all). After adjustment for relevant co-variates, MCP-1 and CD40L remained statistically significant (p < 0.01 for both). IL-6 was, independent of other risk factors, inversely correlated with the maximum treadmill walking distance (p < 0.01). Our cross-sectional study in PAD patients showed that the vascular inflammatory markers MCP-1, CD40L and IL-6 were significantly associated with the extent of atherosclerosis, assessed by angiographic score and maximum treadmill walking distance. These findings indicate that vascular inflammation is implicated in PAD, which might be of importance in future diagnosis and treatment of the disease.

The anticoagulant effect in heparinized blood and plasma resulting from interactions with extrinsic pathway inhibitor.

The influence of Extrinsic pathway inhibitor (EPI) on global clotting times of plasma was studied using activity-blocking IgG antibodies. Dilute tissue thromboplastin (TP) clotting times in plasma collected after intravenous injection of heparin were dramatically shortened by the addition of anti-EPI IgG. Anti-EPI IgG shortened the TP times to a lesser degree in plasma heparinized in vitro. Compared to plasma heparinized in vitro, the TP clotting times were markedly prolonged in post-heparin plasma of equal heparin concentration. Addition of anti-antithrombin IgG reduced the clotting times somewhat more than did anti-EPI IgG, particularly in normal plasma. In plasma from patients with cancer, about equal effect was obtained by blocking either EPI or antithrombin. These clotting time studies suggested that much of the anticoagulant effect caused by injection of heparin depended on EPI. This was confirmed by recording the release of fibrinopeptide A (FPA), as marker of thrombin generation, following addition of TP and CaCl2 to citrated blood. Thrombin generation was delayed and markedly reduced in post-heparin blood compared to that in normal blood. After incubating post-heparin citrated blood with anti-EPI IgG, the generation of FPA was more rapid; the amounts released 30 seconds after addition of TP were 6 times greater (36 vs 6 ng/ml) than in post-heparin blood without anti-EPI IgG. The subsequent FPA values were midway between pre-injection and post-heparin values. In conclusion, between one third and one half of the inhibition of TP-initiated coagulation in post-heparin plasma depends on EPI. This inhibition is mainly due to inactivation of the factor VIIa-TP complex. A small, but distinct contributing effect observed in the APTT assay (and hence no TP) indicates that even increased inactivation of activated factor X contributes. In cancer patients, these EPI-heparin interactions contribute even more to the anticoagulant effects of heparin.

Elevated plasma levels of the factor Xa-TFPI complex in cancer patients

We have previously shown that cancer patients with solid tumour disease have increased plasma levels of both the free and total forms of the coagulation inhibitor, tissue factor pathway inhibitor (TFPI), whereas patients with leukemia and related blood malignancies have levels within the normal range. We now report that also the median plasma levels of the Factor Xa (FXa)-TFPI complex were significantly higher in patients with solid tumours, compared to patients with haematological malignancy and healthy controls. There were significant positive correlations between the FXa-TFPI complex and total TFPI antigen (r=.47, P=.001) and TFPI activity (r=.33, P<.023). In plasma samples from patients with solid tumours, the ratio between the FXa-TFPI complex and free TFPI was 3.4 times higher than in patients with haematological malignancies. Increased levels of the FXa-TFPI complex in solid tumour disease may reflect both increased FXa generation and the increased TFPI concentration in the patients. It is speculated that high levels of the inhibitory FXa-TFPI complex in cancer patients may protect against microthrombosis and organ failure, which are relatively rare in cancer despite long-lasting hypercoagulation.

Tissue factor pathway inhibitor: from unknown coagulation inhibitor to major antithrombotic principle

Time for primary review 20 days. Acute thrombosis is the major immediate cause of cardiovascular death. The thrombotic process is also a stimulus for developing and continuing the atherosclerotic process. To control thrombus formation has been the aim of scientists during several decades. Exposure of tissue factor (TF) to blood, for instance on rupture of atherosclerotic plaques or on endothelial cell damage, may cause acute obstruction of a vessel due to thrombus formation. Thus, the physiological TF inhibitor, tissue factor pathway inhibitor (TFPI), is of interest both pathophysiologically and for its pharmacological potential in inhibiting TF-induced thrombus formation. Exposure of TF to circulating blood starts the coagulation cascade by binding to Factor VII(a) (FVIIa), and the complex of FVIIa and TF activates Factor X (FX) and Factor IX (FIX). TFPI is dependent on some degree of coagulation activation and formation of activated FX (FXa) in order to inhibit the FVIIa/TF complex. Physiologically, TFPIs main role is inhibition of small amounts of tissue factor, and this inhibition is probably essential for maintaining a normal hemostatic balance. It is not known whether total lack of this inhibitor is compatible with life, since no such deficiency has been found. Serial measurements of increasing plasma TFPI during the course of a disease may signal poor prognosis. Heparin accelerates TFPIs inhibitory effect and free TFPI has great heparin affinity. TFPI probably contributes significantly to the anticoagulant properties of the endothelium, and may be particularly important for the outcome of vascular injury. The discovery of the inhibitory principle of TFPI has opened a new arena for pharmacological intervention in the thrombotic process. This may well be a major breakthrough for the control of acute thrombosis in cardiovascular disease. Recombinant TFPI has proved effective in the treatment of experimental disseminated intravascular coagulation, sepsis and thrombosis. Whether TFPI …

Heparin requires both antithrombin and extrinsic pathway inhibitor for its anticoagulant effect in human blood

Heparinization of blood inhibited the generation of fibrinopeptide A (FPA) after addition of thromboplastin (TP). Heparinization was more effective when performed in vivo than in vitro; the amounts of FPA at 60 s incubation were 8% and 32%, respectively, of control values in nonheparinized blood. When monospecific, neutralizing IgG against extrinsic pathway inhibitor (anti-EPI) were added to heparinized blood prior to TP, the amount of FPA increased to 65%. When monospecific IgG blocking antithrombin (anti-AT) was used, the amount of FPA increased to values similar to those in nonheparinized blood. When anti-AT and anti-EPI were both added to heparinized blood, FPA was generated about 25% faster than in normal blood. These results show that EPI contributes significantly to the anticoagulant effect of heparin in human blood.

Extrinsic pathway inhibitor (EPI) and the post-heparin anticoagulant effect in tissue thromboplastin induced coagulation

It is known that the anticoagulant effect of blood or plasma is greater when heparin is given in vivo than when added in similar heparin concentrations in vitro. In this study, we neutralized heparin in citrated blood with polybrene, and then triggered coagulation with dilute tissue thromboplastin (TTP) and CaCl2. The clotting time was longer and the release of fibrinopeptide A (FPA) was retarded in the post injection samples compared to samples spiked with heparin in vitro. We have earlier reported that the extrinsic pathway inhibitor (EPI) is released to the blood after heparin injection. This was demonstrated here also for LMW heparin Enoxaparine both after intravenous and subcutaneous administration. Polyclonal blocking antibodies to EPI were added to blood or plasma heparinized in vivo or in vitro, and the direct heparin effect was neutralized with polybrene. When TTP and CaCl2 now were added and clotting time and the release of FPA recorded, the postheparin effect was greatly reduced by the antibodies. Addition of EPI antibodies to post-heparin plasma samples from cancer patients caused a marked reduction in the thromboplastin clotting times. We conclude that the release of EPI to the blood contributes significantly to the anticoagulant effect of heparin ex vivo.

Extrinsic pathway inhibitor (EPI) released to the blood by heparin is a more powerful coagulation inhibitor than is recombinant EPI

EPI released to the blood after injection of heparin, as well as recombinant EPI (r-EPI) added to normal plasma prolonged both the dilute Tissue Thromboplastin (TTP) time and the Activated Partial Thromboplastin Time (APTT). It is known that EPI inhibits both factor Xa and the factor VIIa-TTP complex. The prolongation of the APTT by EPI reflects only its inhibition of factor Xa. Addition of anti-EPI immunoglobulins (IgG) to normal plasma shortened the dilute TTP time 7.3 seconds (p less than 0.001) and the APTT by 0.7 seconds (p less than 0.001). In postheparin plasma, with polybrene added to neutralize the direct effect of heparin, the TTP was about 26 seconds longer and the APTT about 9 seconds longer than baseline values. These effects were completely abolished by anti-EPI IgG, as were the effects of r-EPI. The EPI activity (chromogenic substrate-assay) of this postheparin plasma was 1.7 U/ml. The EPI activity of the plasma spiked with r-EPI to obtain comparable effects on clotting were much higher; about 22 U/ml for the TTP effect and about 5 U/ml for the APTT effect. The findings indicate that r-EPI is considerably less potent than postheparin EPI as inhibitor of plasma coagulation. This is most striking when coagulation is initiated through the extrinsic pathway. Possibly, the anticoagulant effect of r-EPI mainly depends on its Xa inhibitory effect.