Characterization of the association of tissue factor pathway inhibitor with human placenta

Abstract

Objective - Tissue factor pathway inhibitor (TFPI) is an endothelial-associated inhibitor of blood coagulation. Because the mechanism for attachment of TFPI to endothelium is not clear, we investigated its association with human placenta. Methods and Results - Western blots demonstrate that treatment with phosphatidylinositol-specific phospholipase C (PIPLC) removes more placental TFPI than either PBS or heparin, a finding confirmed by immunohistochemistry. The amounts of heparin-releasable and PIPLC - releasable TFPI activity on placental endothelium were measured in placentas from 5 individuals. PIPLC removes >10-fold more TFPI activity from the placental fragments than 10 U/mL heparin and gt;100-fold more than 1 U/mL heparin. Pretreatment of the placental fragments with PIPLC increases the amount of heparin-releasable TFPI by 3-fold. An antibody specific for the C-terminal region of TFPI recognizes PIPLC-releasable TFPI in Western blots. Conclusions - GPI-anchored TFPI is the predominant form on placental endothelium. Heparin -releasable TFPI likely represents only a small portion of the total TFPI on endothelium that remains attached to cell-surface glycosaminoglycans after cleavage of the GPI anchor by endogenous enzymes. The predominance of GPI-anchored TFPI suggests that heparin infusion does not significantly redistribute TFPI within the vasculature. The intact C-terminus in GPI-anchored TFPI indicates it is not directly attached to a GPI anchor. Rather, it most likely associates with endothelium by binding to a GPI-anchored protein.