EVIDENCE THAT THE C-TERMINUS OF TISSUE FACTOR PATHWAY INHIBITOR (TFPI) IS ESSENTIAL FOR ITS IN-VITRO AND IN-VIVO INTERACTION WITH LIPOPROTEINS

We have previously shown that the C-terminus of TFPI is essential for its anticoagulant activity. In the present study we have assessed the role of this region in the binding of TFPI to lipoproteins. We found t hat full length TFPI, but not C-terminal degraded TFPI, was capable of coeluting with the plasma lipoprotein fraction on a Superose-6 column . The importance of the TFPI C-terminus in lipoprotein interactions wa s also assessed using a microtitre plate binding assay. We found that full-length TFPI was capable of binding to VLDL or LDL coated microtit re plates. C-terminal degraded TFPI also bound to VLDL, but with a ten -fold lower affinity than full length TFPI. Interestingly, removal of the C-terminus along with the third Kunitz-type domain resulted in a T FPI form incapable of lipoprotein binding. Since heparin shows strong binding to the C-terminus of TFPI, we also tested its effect on the bi nding of full length TFPI to VLDL. We found that co-incubation of TFPI with heparin inhibited this binding in a dose-dependent manner. Hepar in was also capable of releasing TFPI from a preformed TFPI:VLDL compl ex, although this reaction required unphysiological amounts of heparin . To assess the physiological function of heparin on FL-TFPI:lipoprote in interactions we also performed gel filtration chromatography of rab bit plasma immediately following i.v. administration of FL-TFPI with a nd without heparin. Previous experiments indicated that heparin has a protective effect on exogenously added FL-TFPI, increasing its recover y by ten-fold. We found that FL-TFPI remaining in circulation 5 min af ter administration migrates solely in high molecular weight fractions, whereas the TFPI recovered after co-injection of heparin migrates sol ely in the low molecular weight fractions. Collectively our results in dicate that the C-terminus of TFPI is involved in lipoprotein binding, and that binding of TFPI to lipoproteins and glycosaminoglycans may b e related phenomena.