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 Ͼ10-fold more TFPI activity from the placental fragments than 10 U/mL heparin and Ͼ100-fold more than 1 U/mL heparin. Pretreatment of the placental fragments with PIPLC increases the amount of heparin-releasable TFPI by Ϸ3-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.

TFPIβ is the GPI-anchored TFPI isoform on human endothelial cells and placental microsomes

“…22 It has been reported that TFPI␤ is not produced by human endothelial EAhy926 cells. 23 The cell surface association of TFPI␣ was thought to involve its interaction with a separate, as yet unidentified, GPI-anchored coreceptor(s) [15][16][17][20][21][22][23] that may control its cellular trafficking and surface expression. 21 As we initiated studies intended to identify the putative GPI-anchored coreceptor(s) for TFPI␣¸our results led to the realization that TFPI␤, rather than TFPI␣, is the dominant PIPLC-releasable isoform on endothelial cells and placental microsomes.…”

Alternatively spliced tissue factor pathway inhibitor Functional Implications

“…TFPIalpha associates with the endothelium surface in two ways. About 90% is indirectly bound through an, as yet, unident4ified GPI-anchored protein 16,17 . The association of TFPIalpha with endothelium through a GPI-anchored protein localizes TFPI to caveolae, where it may interact with caveolin-1 which increases its surface expression and anticoagulant activity 18 .…”

“…The association of TFPIalpha with endothelium through a GPI-anchored protein localizes TFPI to caveolae, where it may interact with caveolin-1 which increases its surface expression and anticoagulant activity 18 . The remaining 10% of TFPIalpha is nonspecifically bound to cell surface glycosaminoglycans 17 . In humans, the glycosaminoglycan bound TFPI alpha is released into plasma following heparin infusion causing the plasma TFPI concentration to increase 2-to 4-fold 19,20 .…”

“…At the level of protein production, it appears that TFPIalpha is the major TFPI isoform produced in humans. TFPIalpha protein has been identified in human platelets 3 and placenta 17 . As mentioned, human plasma contains TFPIalpha and the heparin-releasable form of TFPI is TFPIalpha 20 .…”

Effects of Melatonin and Luzindole on plasma levels of tissue factor, tissue factor Pathway inhibitor and Von Willebrand factor in rats

“…Its biosynthesis is associated primarily with the microvascular endothelium (10,11). The TFPI molecule contains three consecutive Kunitztype protease inhibitor domains, and the second domain (K2) connects and inhibits the Xa factor, and the first (K1) -with the tenase complex (TF/FVIIa) (12,13).…”