Sanne Valentin

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.

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

Tissue factor pathway inhibitor: regulation of its inhibitory activity by phospholipid surfaces.

The basic C-terminus of Tissue Factor Pathway Inhibitor (TFPI) appears to be essential for its anticoagulant activity when tested in a diluted thromboplastin prothrombin time assay. Although the data reported so far have increased our knowledge about the C-terminus as a major binding site for heparin, lipoproteins and phospholipids, it is still unclear how this region of TFPI plays a role in its anticoagulant mode of action. We earlier reported that in the presence of phospholipid the rate of association of factor Xa with full length TFPI (FL-TFPI) is about 10-fold faster than with C-terminus truncated TFPI. This in turn makes that, in vitro, full length TFPI is a more potent inhibitor of tissue factor-factor VIIa catalyzed factor X activation than truncated TFPI. Binding studies, utilizing an ellipsometer, revealed that in contrast to the complex of C-terminus truncated TFPI (TFPI1-161) and factor Xa, the FL-TFPI.factor Xa complex has a high affinity for negatively charged phospholipids. However, when examined in a tubular flow reactor containing tissue factor embedded in a phospholipid bilayer composed of 25 mol% phosphatidyl-serine/75 mol% phosphatidylcholine, no differences in the potency of FL-TFPI and TFPI1-161 to inhibit factor X activation were found. The two variants of TFPI did show an interesting difference though. We found that the quaternary complex of TF.factor VIIa.FL-TFPI.factor Xa was much more stable than the complex containing TFPI1-161. This difference could not be attributed to their different phospholipid-binding properties since the same difference in stability was found on membranes that contained only DOPC.