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.