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

Abstract

native C-terminus that directs the attachment of a glycosylphosphatidylinositol (GPI) anchor, but whether TFPI␤ protein is actually expressed is not clear. Moreover, previous studies have suggested that the predominant form of TFPI released from cells by phosphatidylinositolspecific phospholipase C (PIPLC) treatment is TFPI␣, implying it is bound at cell surfaces to a separate GPI-anchored coreceptor. Our studies show that the form of TFPI released by PIPLC treatment of cultured endothelial cells and placental microsomes is actually TFPI␤ based on (1) migration on SDS-PAGE before and after deglycosylation, (2) the lack of a Kunitz-3 domain, and ( IntroductionTissue factor pathway inhibitor (TFPI) is the key endogenous regulator of TF-initiated coagulation. TFPI inhibits factor Xa (FXa) directly, and, in a FXa-dependent manner, inhibits factor VIIa/tissue factor (FVIIa/ TF) activity. 1 The human TFPI gene encodes for 2 dominant messages that are generated by alternative intron/exon splicing (GenBank NM_006287.4, GenBank NM_001032281.2). TFPI␣, the originally described message, encodes a signal peptide that is removed by processing, followed by a 276-amino acid protein that consists of an acidic N-terminal region, 3 tandem Kunitz-type protease inhibitor domains, and a basic C-terminal region. 2 Functional studies reveal that the Kunitz-2 domain mediates binding to and inhibition of FXa, whereas the Kunitz-1 domain is responsible for recognition and inhibition of the TF/FVIIa complex. 3 The Kunitz-3 domain lacks protease inhibitor activity; however, it and the C-terminal basic region of TFPI␣ are required for protein S enhancement of inhibition of FXa. 4,5 The TFPI␤ message lacks the Kunitz-3 and C-terminal exons of TFPI␣, and in their place is an exon that encodes a 42-amino acid C-terminal sequence following residue 181 of TFPI␣. This new C-terminal sequence is predicted to direct the proteolytic cleavage of the peptide following N193 with the attachment of a GPI anchor. The protein mass of TFPI␤ is less than that of TFPI␣, but it migrates on SDS-PAGE at a molecular weight similar to TFPI␣ (46 kDa) apparently because of greater sialylation of its O-linked carbohydrate. 6 In humans, TFPI circulates in plasma at approximately 70 ng/mL (1.6nM), with 80% being a C-terminal truncated form that is lipoprotein associated and the remaining 20% being full-length and near full-length TFPI␣ forms. 7,8 Platelets contain TFPI␣ and, at the site of injury TFPI␣ levels, increase dramatically. 9,10 The parenteral administration of heparin to humans increases the circulating levels of total TFPI in plasma to approximately 2.5-fold baseline levels. 11,12 The form of TFPI that is released is full-length TFPI␣. 13 Heparin also increases the release of TFPI from endothelial cells in culture, 6,14 even though treatment with heparin does not perceptibly reduce surface TFPI. 6,15,16 The endothelium appears to be the major source of TFPI in vivo. In human endothelial cell cultures, the ratio of TFPI␣/TFPI␤ mRNA varies between 5 and 10. 1...

New Fundamentals in Hemostasis

“…This isoform is expressed in endothelial cells, albeit at lower levels than TFPI␣, and was identified as the major endothelial cell surface-associated form of TFPI (104). Given that it is easily cleaved from the phospholipid anchor, TFPI␤ may well be the predominant TFPI pool available in the body (104). A third TFPI isoform containing only Kunitz domains 1 and 2, TFPI␦, has only been identified at the mRNA level.…”

“…In the second isoform, TFPI␤, the third Kunitz domain and COOH terminus are replaced by a neo COOH terminus containing a glycophosphatidylinositol anchor. This isoform is expressed in endothelial cells, albeit at lower levels than TFPI␣, and was identified as the major endothelial cell surface-associated form of TFPI (104). Given that it is easily cleaved from the phospholipid anchor, TFPI␤ may well be the predominant TFPI pool available in the body (104).…”

Coagulation factor VA2440G causes east Texas bleeding disorder via TFPIα

“…Presumably the binding of TFPIα to FV-short in affected family members' plasma results in its retention in circulation, thus causing the higher TFPIα plasma concentrations. Even though the concentration of TFPIα in plasma is normally very low, significant amounts are present in the vasculature associated with the endothelium (25,26). Infusion of heparin releases part of the TFPIα from the endothelium to the circulation.…”

“…It circulates bound to the lipoproteins, mainly to LDL (25). Another truncated isoform denoted TFPIβ is present on endothelium, where it is bound with a GPI anchor (26). Recently, Ndonwi et al demonstrated that the positively charged C terminus of TFPIα contains the binding site for FV (27).…”

The prothrombotic state in paroxysmal nocturnal hemoglobinuria: a multifaceted source

“…72 It is still disputed which TFPI isoform is the most abundantly expressed isoform, however TFPI-β, a GPI-AP expressed on ECs, is thought to exert 80% of anticoagulant activity. 72,73 Possible deficiency of the GPI anchor of TFPI-β in PNH may therefore reduce the anticoagulant properties of the endothelium in PNH and contribute to thrombus formation. 74,75 Anti-thrombin is enhanced by binding to the heparan sulphate receptor, a GPI-linked protein expressed on endothelial cells and hypothesized to be lost in patients with PNH.…”