CHARACTERIZATION OF THE BINDING BETWEEN TISSUE FACTOR PATHWAY INHIBITOR AND GLYCOSAMINOGLYCANS

Tissue Factor Pathway Inhibitor (TFPI) is a heparin binding protein an d injection of heparin causes a release of TFPI to plasma. In order to understand the binding between TFPI and heparin in more detail we hav e in this study looked into some of the heparin characteristics and th eir 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 gly cosaminoglycans on a weight basis for their TFPI binding. in this assa y a glycosaminoglycan in solution compete with heparin-Sepharose for T FPI binding. Size fractionated heparin was analyzed for binding to TFP I, and a clear dependency on the molecular weight was observed. The hi ghest TFPI binding capacity was found for fractions with a molecular w eight above 10,000 Da, while no binding was measured below 2,000 Da. N o difference in TFPI binding appeared after fractionation of heparin a ccording to its affinity towards antithrombin, thus indicating that TF PI binding does not require the specific antithrombin binding site. A heparin fraction of 10,000 Da was fractionated on a mono Q column, res ulting in four fractions with different charge densities. The charge d ensity turned out to be a very important parameter for the binding of TFPI. A number of different glycosaminoglycans were tested and the fol lowing order of TFPI affinity was found: heparin much greater than der matan sulphate > heparan sulphate > chondroitin sulphate C. No binding was observed for chondroitin sulphate A or hyaluronic acid.