Tissue factor pathway inhibitor: structure-function

Abstract

TFPI is a multivalent, Kunitz-type proteinase inhibitor, which, due to alternative mRNA splicing, is transcribed in three isoforms: TFPIalpha, TFPIdelta, and glycosyl phosphatidyl inositol (GPI)-anchored TFPIbeta. The microvascular endothelium is thought to be the principal source of TFPI and TFPIalpha is the predominant isoform expressed in humans. TFPIalpha, apparently attached to the surface of the endothelium in an indirect GPI-anchor-dependent fashion, represents the greatest in vivo reservoir of TFPI. The Kunitz-2 domain of TFPI is responsible for factor Xa inhibition and the Kunitz-1 domain is responsible for factor Xa-dependent inhibition of the factor VIIa/tissue factor catalytic complex. The anticoagulant activity of TFPI in one-stage coagulation assays is due mainly to its inhibition of factor Xa through a process that is enhanced by protein S and dependent upon the Kunitz-3 and carboxyterminal domains of full-length TFPIalpha. Carboxyterminal truncated forms of TFPI as well as TFPIalpha in plasma, however, inhibit factor VIIa/tissue factor in two-stage assay systems. Studies in gene-disrupted mice demonstrate the physiological importance of TFPI.

Figure 1. Structure of the human TFPI Gene

The TFPI gene spans 90 kb and contains 10 exons (vertical boxes) and 9 introns that encode for three isoforms. Exons are labeled numerically (top), introns alphabetically (bottom). Translated exons encoding TFPIα are filled in blue. Alternatively spliced exons leading to the generation of TFPIδ and TFPIβ are filled in magenta and red, respectively. Exons 1 and 2 encode 5′ untranslated (5′UT) sequences with alternative splicing resulting in the absence of exon 2 in some messages. Exon 3 encodes the signal peptide and amino terminal peptide (SP+NT). Exons 4, 6 and 9 encode Kunitz domains 1 (K1), 2 (K2) and 3 (K3), respectively and intervening peptide sequences that link the Kunitz domains are encoded by exons 5 (IP1) and 7 (IP2). A run-on of exon 6 into intron F (magenta) encodes the short TFPIδ variant carboxyl terminus (δCT). The TFPIβ carboxy-terminal peptide (βCT) is encoded by exon 8 (red) and splicing of exon 7 to exon 8 occurs to generate the TFPIβ message. In the TFPIα message exon 9 is directly spliced to exon 7 (K3) and to exon 10 (αCT), thereby encoding the Kunitz-3 and carboxyterminus of the TFPIα protein.

Figure 2. Structure of TFPIα

Amino acids are identified by the single letter code (encircled). Positively charged amino acids are shown in red, negatively charge amino acids in blue and neutral amino acids in white (histidine residues are considered uncharged). The Kunitz domains are labeled and the basic P₁ residues at the active site cleft for each domain is shown in black. N-linked glycosylation sites are denoted at N117 and N167 by and O-linked glycosylation sites are denoted at T14, S174 and T175 by . Partial phosphorylation at Ser2 is also shown . The sites of introns in the TFPI gene are labeled with dotted lines and capital letters.

Figure 3. Structure of TFPIβ

Symbols as denoted in the legend of Figure 2. The GPI-anchor is shown attached to mature TFPIβ along with the released carboxyterminal peptide.

Figure 4. Structure of TFPIδ

Symbols as denoted in the legend of Figure 2.

Figure 5. TFPIδ mRNA in human tissues

Tissue northern blot of oligo (dT) isolated mRNA hybridized with a labeled probe specific for TFPIδ mRNA.s

Figure 6. Distribution of TFPI

In plasma, carboxyterminal truncated forms of TFPI are bound to lipoproteins and comprise ~80% of circulating TFPI (blue). Whether these forms represent extensively truncated forms of TFPIα, TFPIβ or TFPIδ has not been determined. “Free” forms of TFPI (~20%) contain the Kunitz-3 domain and include carboxyterminal truncated forms of TFPIα (~10%) (yellow) and full-length TFPIα (~10%) (green). Following heparin treatment in vivo, the level of total TFPI in plasma increases 1.5–3.0-fold and the released TFPI is full-length TFPIα. Platelets carry a level of full-length TFPIα that is equivalent to that present in plasma. At sites where platelets aggregrate, the contribution of platelets to the local concentration of full-length TFPIα could exceed the level of full-length TFPIα in plasma by >30-fold. Based on studies of cultured endothelial cells, the quantity of surface TFPI released by PIPLC greatly exceeds (>5-fold) that released by heparin. The PIPLC-releasable TFPI appears predominantly to be full-length TFPIα; PIPLC-induced release of TFPIβ has not yet been documented in endothelial cells.

Figure 7. Cell Surface TFPI

Left: TFPIα appears to attach to the cell membrane by binding to a GPI-anchored co-receptor. The Kunitz-3 and carboxyterminal domains of TFPIα are involved in its interaction with the co-receptor. Right: TFPIβ attaches to the cell membrane through an intrinsic GPI-anchor. The Kunitz-1, Kunitz-2, and Kunitz-3 domains of TFPI are labeled K1, K2, and K3, respectively.

Figure 8. Functions of TFPI

The factor VIIa/tissue factor (TF) catalytic complex on a phospholipid surface activates factor X and factor IX (top). TFPI (center) binds to the transient tertiary factor VIIa/tissue factor/factor Xa complex (left) produced during the activation of factor X, forming a quaternary factor VIIa/tissue factor/factor Xa/TFPI inhibitory complex (bottom) in which the Kunitz-1 and Kunitz-2 domains of TFPI bind and inhibit factor VIIa and factor Xa, respectively. On the right, factor Xa, protein S and TFPI are shown forming an inhibitory complex at a phospholipid surface in which the Kunitz-2 domain binds and inhibits factor Xa and the Kunitz-3 and carboxyterminal domains of TFPIα are required for the interaction with protein S. Direct inhibition of factor Xa by TFPI and an alternative two-step pathway for factor VIIa/tissue factor inhibition in which a factor Xa/TFPI complex binds to factor VIIa/tissue factor producing the final quaternary inhibitory complex are not shown. Also not depicted are the factor VIIa/tissue factor and factor Xa inhibition produced by cell membrane-bound forms of TFPI. The Kunitz-1, Kunitz-2 and Kunitz-3 domains of TFPI are labeled K1, K2 and K3, respectively.