Local application of recombinant active-site inhibited human clotting factor VIIa reduces thrombus weight and improves patency in a rabbit venous thrombosis model.

OBJECTIVE: To study whether locally administered recombinant inactivated human coagulation factor VIIa (FFR-rFVIIa) would reduce the thrombus formation and improve patency in an experimental venous thrombosis model without inducing systemic changes in the coagulation. DESIGN: Experimental double-dummy randomised study. MATERIALS: In 20 healthy New Zealand White rabbits both jugular veins were exposed under general anaesthesia. METHODS: The thrombi were induced in a 10 mm long jugular vein segment with a combination of chemical destruction of the intima and a restriction of the bloodflow. Each segment was treated with either FFR-rFVIIa or placebo injected directly into the vein. RESULTS: 1.5 mg topically applied FFR-rFVIIa significantly reduced the thrombus weight (p < 0.001). The 30 and the 120 min patency tests were significantly improved (p < 0.05 and p < 0.001, respectively) Plasma analyses (APTT, dilute-TF time, FVII protein) were evaluated as baseline, 3 min after declamping and at sacrifice. No prolongation of the clotting times were seen. FFR-rFVIIa protein was detected in minute amounts (ng/ml); however, this was not enough to prolong the dilute-TF time. CONCLUSIONS: Local application of recombinant active-site inhibited human FVIIa reduced both thrombus weight and improved patency significantly in an experimental venous thrombosis model without affecting the systemic clotting times.

The significance of TFPI in clotting assays--comparison and combination with other anticoagulants.

The anticoagulant activities of Tissue Factor Pathway Inhibitor (TFPI), heparin and hirudin were compared in intrinsic (APTT) and extrinsic (PT) activated clotting assays. In contrast to the thrombin inhibitor hirudin, heparin was 10 fold more potent in the APTT assay than in the PT assay, indicating that inhibition of intrinsic activation is important for the anticoagulant activity of heparin as measured in an APTT assay. TFPI was most potent in the PT assay and the effect of TFPI was most pronounced in the presence of other anticoagulants (heparin and hirudin). The activities of the two natural anticoagulants antithrombin III (ATIII) and TFPI were compared in a PT assay with very dilute tissue factor. In this assay system TFPI in normal plasma affected the clotting time more than ATIII in the plasma. However, when heparin was added ATIII was the major anticoagulant, but profound prolongation of the clotting time was only seen when TFPI was also added. In an ATIII deficient plasma heparin did not augment the effect of TFPI, showing that the increased effect of TFPI in the presence of heparin is dependent on the anticoagulant activity of ATIII/heparin. The effect of TFPI at prolonged clotting times was also illustrated by the significant effect of blocking TFPI in the plasma from warfarin-treated patients. Thus TFPI is a major anticoagulant in normal plasma and the effect of TFPI is especially seen at prolonged clotting times.

Effect of tissue factor pathway inhibitor (TFPI) in the HEPTEST assay and in an amidolytic anti factor Xa assay for LMW heparin.

Both the HEPTEST and amidolytic anti factor Xa assays are currently being used for heparin activity detection in plasma from patients receiving standard heparin or low molecular weight heparin (LMWH). In this study we have investigated the influence of recombinant and endogenous Tissue Factor Pathway Inhibitor (TFPI) on these assays. The HEPTEST determinations were performed on an ACL 300 R Clottimer using the APTT program which resulted in a longer incubation time with factor Xa than recommended by the manufacturer. rTFPI added to plasma prolonged the HEPTEST clotting time markedly, but had only a little effect in the amidolytic assay. Antibodies against TFPI (anti-TFPI) abolished these effects. The effect of adding rTFPI and Logiparin was additive. When anti-TFPI IgG was added to samples of normal plasma, a statistically significant shortening of the HEPTEST clotting time was seen. When anti-TFPI was added to plasma samples from volunteers who had received Logiparin by subcutaneous or intravenous injection, then the HEPTEST clotting time was shortened considerably. For some samples the clotting time was halved. These experiments show that the HEPTEST clotting time is prolonged not only by heparin-antithrombin III, but also by TFPI released by heparin injection.

Development and validation of a size exclusion chromatography method for determination of molecular masses and molecular mass distribution in low molecular weight heparin.

A precise and rugged high performance size exclusion chromatography (HPSEC) method for determination of peak maximum molecular mass (Mp), weight average molecular mass (Mw), number average molecular mass (Mn), and molecular mass distribution (MMD) in Low Molecular Weight heparin (LMW heparin) has been developed and validated on two Ultrahydrogel 250 (ID 7, 8 mm, length 30 cm) columns in series at three laboratories using different equipment. The calibration is based on four local laboratory heparin standards with relatively narrow molecular mass distribution and Mp covering the range from 3,000 to 17,000 Da. The calibration curve describing the logarithm of the molecular mass versus retention time is linear in the range 3,000 to 17,000 Da. The precision (relative standard deviation) within-run and between-run is better than 2%. The between-laboratory variation is below 5%, 3% and 8% for Mp, Mw and Mn, respectively. This method is reproducible, rugged, and fast and is suited for the determination of average molecular masses and molecular mass distribution of LMW heparins on a routine basis.