Ole Nordfang

Characterization of the binding between tissue factor pathway inhibitor and glycosaminoglycans

Tissue Factor Pathway Inhibitor (TFPI) is a heparin binding protein and injection of heparin causes a release of TFPI to plasma. In order to understand the binding between TFPI and heparin in more detail we have in this study looked into some of the heparin characteristics and their importance for the TFPI-heparin interaction. We have developed an assay based on the use of heparin-Sepharose micro columns in order to compare small quantities of heparin fractions as well as different glycosaminoglycans on a weight basis for their TFPI binding. In this assay a glycosaminoglycan in solution compete with heparin-Sepharose for TFPI binding. Size fractionated heparin was analyzed for binding to TFPI, and a clear dependency on the molecular weight was observed. The highest TFPI binding capacity was found for fractions with a molecular weight above 10,000 Da, while no binding was measured below 2,000 Da. No difference in TFPI binding appeared after fractionation of heparin according to its affinity towards antithrombin, thus indicating that TFPI binding does not require the specific antithrombin binding site. A heparin fraction of 10,000 Da was fractionated on a mono Q column, resulting in four fractions with different charge densities. The charge density turned out to be a very important parameter for the binding of TFPI. A number of different glycosaminoglycans were tested and the following order of TFPI affinity was found: heparin >> dermatan sulphate > heparan sulphate > chondroitin sulphate C. No binding was observed for chondroitin sulphate A or hyaluronic acid.

AN ENZYME LINKED IMMUNOSORPTION ASSAY FOR TISSUE FACTOR PATHWAY INHIBITOR

An assay for the quantification of full-length and carboxy-terminus truncated tissue factor pathway inhibitor (TFPI) has been developed. The assay is a classical two-antibody sandwich assay with a monoclonal capture antibody directed against the third Kunitz-type domain of human TFPI and a polyclonal rabbit peroxidase-labelled anti-human TFPI detecting antibody. The assay is sensitive to full-length and carboxy-terminus truncated TFPI with intact third Kunitz-type domain, but not to two-domain TFPI. TFPI associated with lipoproteins is not or only sparsely detected and TFPI in complex with factor Xa only partially measured. The assays gives linear reference curves in the dose range of 5 to 100 ng/ml in a double logarithmic plot. The normal range assessed from analyses on citrated plasma from 81 normal human donors is 7.8 to 26.0 ng/ml (average +/- 2 SD, log-normal distribution). There is no statistically significant difference between TFPI levels measured in 10 fasting and 10 non-fasting individuals. The reproducibility of the assay is about 5.6-5.9% (relative standard error) and the within-days and between-day reproducibilities are 4.7-5.1% and 5.9-8.5%, respectively. The assay is in very good agreement with a commercial ELISA assay recently marketed. A robust, reproducible and convenient ELISA assay for the determination of full-length and three-domain TFPI has been developed.

Antithrombotic Properties of a Truncated Recombinant Tissue Factor Pathway Inhibitor in an Experimental Venous Thrombosis Model

The aim of this study was to investigate whether a truncated recombinant tissue factor pathway inhibitor (rTFPI1-161) had an antithrombotic effect comparable to low-molecular-weight (LMW) heparin. A randomized double-dummy study was conducted with 40 rabbits in 6 groups. An experimental thrombosis was induced in the jugular veins by a combination of destroyed endothelium and restricted blood flow. Group 1 was given placebo; group 2, LMW heparin 60 anti-factor Xa units/kg; group 3, rTFPI1-161 0.1 mg/kg; group 4, rTFPI1-161 1.0 mg/kg and group 5, rTFPI1-161 10.0 mg/kg. rTFPI1-161 reduced the thrombus weights in all treated groups, with a significant effect in doses between 1.0 and 10.0 mg/kg compared to placebo. The frequency of thrombosis was significantly reduced in all treated groups. No hemorrhagic side effects were noted. In conclusion, rTFPI1-161 (1.0-10.0 mg/kg) has an antithrombotic effect comparable to that of LMW heparin.

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.

The effect of protamine sulphate on plasma tissue factor pathway inhibitor released by intravenous and subcutaneous unfractionated and low molecular weight heparin in man.

Heparin, a negatively charged sulphated glycosaminoglycan, is clinically the most important antithrombotic drug. Heparin augments the inhibitory activity of antithrombin (AT) towards thrombin, factor Xa (FXa) and other activated clotting enzymes. Tissue factor pathway inhibitor (TFPI) is an endogenous heparin releasable three domain Kunitz-type coagulation inhibitor which inhibits the crucial tissue factor-factor VIIa (TF-FVIIa) dependent coagulation pathway in the presence of FXa. The importance of the TF-FVIIa pathway and TFPI has recently been reviewed (1). TFPI is located to different vascular pools, the largest being the vascular endothelium from where TFPI can be released dose-dependently to the blood by heparins (2). TFPI is speculated to contribute to the anticoagulant properties of heparins, but to which degree is not yet fully understood. In recent years low molecular weight heparins (LMWH) have proven to be effective and safe both for prophylactic (3) and therapeutic treatment (4) of deep vein thrombosis (DVT). Protamine is the least toxic and clinically most commonly used antidote to heparin. However, in vitro and in vivo LMW heparinized blood is not fully neutralized by protamine, as substantial anti-Xa activity remains following neutralization (5). This post-protamine effect has been shown to be partly TFPI dependent when measured in a dilute TF-dependent assay (6,7). We undertook this in vivo study on healthy volunteers in order to investigate whether TFPI released by UH or LMWH (intravenous (iv) or subcutaneous (sc)) remains in the circulation following neutralization of the heparin activity with protamine sulphate (PS). We measured TFPI by three different methods-chromogenic activity, anticlotting activity and a new antigen assay specific for full-length and three-domain TFPI.

Is Tissue Factor Pathway Inhibitor Involved in the Antithrombotic Effect of Heparins

Tissue factor pathway inhibitor (TFPI) is released into the circulation after intravenous or subcutaneous injection of heparin or low-molecular-weight heparin (Logiparin®) in humans. The plasma concen

Pharmacokinetics and delayed experimental anti-thrombotic effect of two domain non-glycosylated tissue factor pathway inhibitor

Tissue Factor Pathway Inhibitor (TFPI) is a naturally occurring inhibitor of the TF-FVIIa induced coagulation in the presence of FXa. Recombinant two domain TFPI, where Asn 117 on the FXa-inhibitory domain was exchanged to a Gln yielding non-glycosylated TFPI (117QTFPI1-161), was evaluated regarding pharmacokinetics and delayed antithrombotic potential in the rabbit. Pharmacokinetic study; 117QTFPI1-161 vs glycosylated TFPI1-161. Three rabbits/group were used and received 1,0 mg/kg a bolus iv injection. Plasma-TFPI was measured for three hours. The alpha-phase half-life was similar, the beta-phase half-life was close to four times longer for 117QTFPI1-161 (37 vs 10 min). Clearance of 117QTFPI1-161 was nearly two times lower (45 vs 21 ml/kg/min). Delayed anti-thrombotic study; 10 rabbits/group were used. 5 Groups; placebo + placebo, placebo + LMWH60 anti-Xa IU/kg, placebo + 117QTFPI1-161 0,25 mg/kg, 117QTFPI1-161 1,0 and 4,0 mg/kg + placebo. First injection 60 min prior to the second one, which coincided with the thrombus induction. The experimental thrombosis used combines a chemical destruction of the endothelium with a partial restriction of the bloodflow in the jugular veins. The thrombusweight was significantly reduced in LMWH and 117QTFPI1-161 1,0 and 4,0 mg/kg groups (0,6-2,6 vs 11,8 mg). Frequency of occlusive thrombosis was significantly reduced in the LMWH and 117QTFPI1-161 4,0 mg groups. All groups significantly effected the aXa-assay, the LMWH-group the most (0,85 IU/ml). LMWH was the only substance to prolong the dilute-PT-assay at the different timepoints. Absence of glycosylation increases the beta-phase half-life and decreases clearance of two domain TFPI. 117QTFPI1-161 (1,0 and 4,0 mg) has an antithrombotic effect indistinguishable from LMWH even though given 60 min before the thrombusinduction but with a considerable less effect on anti-Xa, APTT and no effect on dilute-PT. Glycosylation of TFPI influences the pharmacokinetics but not the antithrombotic capacity in this experimental setting.

Does glycosylation influence the experimental antithrombotic effect of a two-domain tissue factor pathway inhibitor?

We have earlier shown that both full-length and truncated glycosylated tissue factor pathway inhibitor (TFPI) lacking the third Kunitz domain and the c-terminal region has an antithrombotic effect comparable to lowmolecular-weight heparin (LMWH) in an experimental venous thrombosis model. The aim of this study was to investigate whether a recombinant truncated non-glycosylated TFPI (117QTFPI1-161) had an antithrombotic effect similar to the glycosylated TFPI1-161 and LMWH. We also followed the coagulation parameters. The thrombi were induced in rabbit jugular veins with a combination of endothelium destruction and restricted blood flow. Group 1: placebo; group 2: LMWH 60 anti-Xa IU/kg, i.v.; groups 3 and 4: TFPI1-161 0.8 and 0.2 mg/kg, i.v., respectively; groups 5 and 6: 0.8 and 0.2 mg/kg 117QTFPI1-161, i.v., respectively, in a randomized double-dummy fashion. Twelve animals were included in the placebo group and 6 in each of the other groups. The frequency of thrombosis and also of occlusive thrombosis was reduced in all groups compared to placebo. The thrombus weight was reduced (0-9.9 mg) in all groups, significantly in groups 2, 4 and 5 (p = 0.004-0.02) compared to placebo (21.1 mg). In group 3, a borderline p value was achieved (0.06 likely a beta-error). The two forms of TFPI1-161 given in the higher doses showed a significantly greater increase of anti-Xa activity, but with a shorter duration compared to LMWH (1.7-1.9 vs. 0.9 anti-Xa IU/ml). Activated partial thromboplastin time (aPTT)-analysis revealed that only LMWH (52 s) caused a significant transient elevation 2 min after injection. In the other groups, a temporary but insignificant elevation of aPTT (27-37 s) was seen. No detectable effect on anti-IIa activity and prothrombin time (PT) was seen in any TFPI group. The glycosylation of the second domain on TFPI does not substantially contribute to the antithrombotic effect of TFPI. Regardless of the glycosylation of TFPI1-161, it has a dose-dependent effect on anti-Xa, a small effect on the aPTT, but no effect on anti-Ila and PT. LMWH has a more pronounced and sustained impact on these parameters.