Paul E. R. Ellery

Comparison of the inhibitory activities of human tissue factor pathway inhibitor (TFPI)α and TFPIβ

Tissue factor pathway inhibitor (TFPI) is an alternatively spliced protein with two isoforms, TFPIα and TFPIβ, which differ in their C‐terminal structure and cellular localization. Detailed characterization of their inhibitory activity is needed to define potentially unique inhibitory roles in tissue factor (TF)‐mediated thrombotic and inflammatory disease, and to understand how pharmaceuticals targeted to different structural regions of the TFPI isoforms alter hemostasis in hemophilia patients.

Alternatively spliced isoforms of tissue factor pathway inhibitor

Tissue factor pathway inhibitor (TFPI) is the major regulator of tissue factor (TF)-induced coagulation. It down regulates coagulation by binding to the TF/fVIIa complex in a fXa dependent manner. It is predominantly produced by microvascular endothelial cells, though it is also found in platelets, monocytes, smooth muscle cells, and plasma. Its physiological importance is demonstrated by the embryonic lethality observed in TFPI knockout mice and by the increase in thrombotic burden that occurs when heterozygous TFPI mice are bred with mice carrying genetic risk factors for thrombotic disease, such as factor V Leiden. Multiple TFPI isoforms, termed TFPIalpha, TFPIbeta, and TFPIdelta in humans and TFPIalpha, TFPIbeta, and TFPIgamma in mice, have been described, which differ in their domain structure and method for cell surface attachment. A significant functional difference between these isoforms has yet to be described in vivo. Both human and mouse tissues produce, on average, approximately 10 times more TFPIalpha message when compared to that of TFPIbeta. Consistent with this finding, several lines of evidence suggest that TFPIalpha is the predominant protein isoform in humans. In contrast, recent work from our laboratory demonstrates that TFPIbeta is the major protein isoform produced in adult mice, suggesting that TFPI isoform production is translationally regulated.

Protein S Is a Cofactor for Platelet and Endothelial Tissue Factor Pathway Inhibitor-α but Not for Cell Surface–Associated Tissue Factor Pathway Inhibitor

Objective— Tissue factor pathway inhibitor (TFPI) is produced in 2 isoforms: TFPI&agr;, a soluble protein in plasma, platelets, and endothelial cells, and TFPI&bgr;, a glycosylphosphatidylinositol-anchored protein on endothelium. Protein S (PS) functions as a cofactor for TFPI&agr;, enhancing the inhibition of factor Xa. However, PS does not alter the inhibition of prothrombinase by TFPI&agr;, and PS interactions with TFPI&bgr; are undescribed. Thus, the physiological role and scope of the PS–TFPI system remain unclear. Approach and Results— Here, the cofactor activity of PS toward platelet and endothelial TFPI&agr; and endothelial TFPI&bgr; was quantified. PS enhanced the inhibition of factor Xa by TFPI&agr; from platelets and endothelial cells and stabilized the TFPI&agr;/factor Xa inhibitory complex, delaying thrombin generation by prothrombinase. By contrast, PS did not enhance the inhibitory activity of TFPI&bgr; or a membrane-anchored form of TFPI containing the PS-binding third Kunitz domain (K1K2K3) although PS did function as a cofactor for K1K2K3 enzymatically released from the cell surface. Conclusions— The PS–TFPI anticoagulant system is limited to plasma TFPI&agr; and TFPI&agr; released from platelets and endothelial cells. PS likely functions to localize solution-phase TFPI&agr; to the cell surface, where factor Xa is bound. PS does not alter the activity of membrane-associated TFPI. Because activated platelets release TFPI&agr; and PS, the PS–TFPI&agr; anticoagulant system may act physiologically to dampen thrombin generation at the platelet surface.

Tissue Factor Pathway Inhibitor: Then and Now

Tissue factor pathway inhibitor (TFPI) is the major physiological regulator of tissue factor (TF)-induced blood coagulation. TFPI inhibits the TF-activated factor VII (FVIIa) complex in an activated factor X (FXa)-dependent manner, helping to control thrombin generation and ultimately fibrin formation. The importance of TFPI is demonstrated in models of hemophilia where lower levels of FVIII or FIX are insufficient to overcome its inhibitory effect, resulting in a bleeding phenotype. There are two major isoforms in vivo; TFPIα contains three Kunitz-type inhibitory domains (designated K1, K2, and K3), is secreted by endothelial cells and requires protein S to enhance its anticoagulant activity. In contrast, TFPIβ contains only the K1 and K2 domains, but it is attached to the endothelial surface via a glycosylphosphatidylinositol anchor. This review will initially provide a brief history of the major discoveries related to TFPI, and then discuss new insights into the physiology of TFPI, including updates on its association with protein S and FV, as well as the current understanding of its association with disease.

Caveolae optimize tissue factor-Factor VIIa inhibitory activity of cell-surface-associated tissue factor pathway inhibitor

TFPI (tissue factor pathway inhibitor) is an anticoagulant protein that prevents intravascular coagulation through inhibition of fXa (Factor Xa) and the TF (tissue factor)-fVIIa (Factor VIIa) complex. Localization of TFPI within caveolae enhances its anticoagulant activity. To define further how caveolae contribute to TFPI anticoagulant activity, CHO (Chinese-hamster ovary) cells were co-transfected with TF and membrane-associated TFPI targeted to either caveolae [TFPI-GPI (TFPI-glycosylphosphatidylinositol anchor chimaera)] or to bulk plasma membrane [TFPI-TM (TFPI-transmembrane anchor chimaera)]. Stable clones had equal expression of surface TF and TFPI. TX-114 cellular lysis confirmed localization of TFPI-GPI to detergent-insoluble membrane fractions, whereas TFPI-TM localized to the aqueous phase. TFPI-GPI and TFPI-TM were equally effective direct inhibitors of fXa in amidolytic assays. However, TFPI-GPI was a significantly better inhibitor of TF-fVIIa than TFPI-TM, as measured in both amidolytic and plasma-clotting assays. Disrupting caveolae by removing membrane cholesterol from EA.hy926 cells, which make TFPIα, CHO cells transfected with TFPIβ and HUVECs (human umbilical vein endothelial cells) did not affect their fXa inhibition, but significantly decreased their inhibition of TF-fVIIa. These studies confirm and quantify the enhanced anticoagulant activity of TFPI localized within caveolae, demonstrate that caveolae enhance the inhibitory activity of both TFPI isoforms and define the effect of caveolae as specifically enhancing the anti-TF activity of TFPI.

A balance between TFPI and thrombin-mediated platelet activation is required for murine embryonic development.

Tissue factor pathway inhibitor (TFPI) is a critical anticoagulant protein present in endothelium and platelets. Mice lacking TFPI (Tfpi(-/-)) die in utero from disseminated intravascular coagulation. They are rescued by concomitant tissue factor (TF) deficiency, demonstrating that TFPI modulates TF function in vivo. Recent studies have found TFPI inhibits prothrombinase activity during the initiation of coagulation and limits platelet accumulation during thrombus formation, implicating TFPI in modulating platelet procoagulant activity. To examine whether altered platelet function would compensate for the lack of TFPI and rescue TFPI-null embryonic lethality, Tfpi(+/-) mice lacking the platelet thrombin receptor, protease activated receptor 4 (PAR4; Par4(-/-)), or its coreceptor, PAR3, were mated. PAR3 deficiency did not rescue Tfpi(-/-) embryos, but >40% of expected Tfpi(-/-):Par4(-/-) offspring survived to adulthood. Adult Tfpi(-/-):Par4(-/-) mice did not exhibit overt thrombosis. However, they had focal sterile inflammation with fibrin(ogen) deposition in the liver and elevated plasma thrombin-antithrombin complexes, indicating activation of coagulation at baseline. Tfpi(-/-):Par4(-/-) mice have platelet and fibrin accumulation similar to Par4(-/-) mice following venous electrolytic injury but were more susceptible than Par4(-/-) mice to TF-induced pulmonary embolism. In addition, ∼30% of the Tfpi(-/-):Par4(-/-) mice were born with short tails. Tfpi(-/-):Par4(-/-) mice are the first adult mice described that lack TFPI with unaltered TF. They demonstrate that TFPI physiologically modulates thrombin-dependent platelet activation in a manner that is required for successful embryonic development and identify a role for TFPI in dampening intravascular procoagulant stimuli that lead to thrombin generation, even in the absence of thrombin-mediated platelet activation.

Translation of Human Tissue Factor Pathway Inhibitor-β mRNA Is Controlled by Alternative Splicing Within the 5′ Untranslated Region

Objective— Tissue factor pathway inhibitor (TFPI) blocks the initiation of coagulation by inhibiting TF-activated factor VII, activated factor X, and early prothrombinase. Humans produce two 3′ splice variants, TFPI&agr; and TFPI&bgr;, which are differentially expressed in endothelial cells and platelets and possess distinct structural features affecting their inhibitory function. TFPI also undergoes alternative splicing of exon 2 within its 5′ untranslated region. The role of exon 2 splicing in translational regulation of human TFPI isoform expression is investigated. Approach and Results— Exon 2 splicing occurs in TFPI&agr; and TFPI&bgr; transcripts. Human tissue mRNA analysis uncovered a wide variability of exon 2 expression. Polysome analysis revealed a repressive effect of exon 2 on TFPI&bgr; translation but not on TFPI&agr;. Luciferase reporter assays further exposed strong translational repression of TFPI&bgr; (90%) but not TFPI&agr;. Use of a Morpholino to remove exon 2 from TFPI mRNA increased cell surface expression of endogenous TFPI&bgr;. Exon 2 also repressed luciferase production (80% to 90%) when paired with the &bgr;-actin 3′ untranslated region, suggesting that it is a general translational negative element whose effects are overcome by the TFPI&agr; 3′ untranslated region. Conclusions— Exon 2 is a molecular switch that prevents translation of TFPI&bgr;. This is the first demonstration of a 5′ untranslated region alternative splicing event that alters translation of isoforms produced via independent 3′ splicing events within the same gene. Therefore, it represents a previously unrecognized mechanism for translational control of protein expression. Differential expression of exon 2 denotes a mechanism to provide temporal and tissue-specific regulation of TFPI&bgr;-mediated anticoagulant activity.

Reduced prothrombinase inhibition by tissue factor pathway inhibitor contributes to the factor V Leiden hypercoagulable state

Activated factor V (FVa) and factor X (FXa) form prothrombinase, which converts prothrombin to thrombin. The α isoform of tissue factor (TF) pathway inhibitor (TFPI) dampens early procoagulant events, partly by interacting with FV. FV Leiden (FVL) is the most common genetic thrombophilia in Caucasians. Thrombosis risk is particularly elevated in women with FVL taking oral contraceptives, which produce acquired TFPIα deficiency. In mice, FVL combined with 50% reduction in TFPI causes severe thrombosis and perinatal lethality. However, a possible interaction between FVL and TFPIα has not been defined in humans. Here, we examined this interaction using samples from patients with FVL in thrombin generation and fibrin formation assays. In dilute TF- or FXa-initiated reactions, these studies exposed a TFPI-dependent activation threshold for coagulation initiation that was greatly reduced by FVL. The reduced threshold was progressively overcome with higher concentrations of TF or FXa. Plasma assays using anti-TFPI antibodies or a TFPI peptide that binds and inhibits FVa demonstrated that the decreased activation threshold resulted from reduced TFPIα inhibition of prothrombinase. In assays using purified proteins, TFPIα was a 1.7-fold weaker inhibitor of prothrombinase assembled with FVL than with FV. Thus, FVL reduces the threshold for initiating coagulation, and this threshold is further reduced in situations of low TFPIα concentration. Individuals with FVL are likely prone to thrombosis in response to weak procoagulant stimuli that would not initiate blood clot formation in individuals with FV.

Further insight into the Heparin-Releasable and Glycosylphosphatidylinositol-lipid anchored forms of tissue factor pathway inhibitor

The release of tissue factor pathway inhibitor (TFPI) from human umbilical vein endothelial cells (HUVECs) was investigated using heparin and phospholipase C. The experiment included incubating HUVECs with 0, 1, or 10 U/mL heparin diluted in Dulbecco Modified Eagles Medium plus 5% fetal calf serum for 1 or 24 hours. A statistically significant increase in TFPI activity levels was seen at 1 hour, but not at 24 hours. A 20-fold increase in the release of TFPI after phospholipase C treatment of HUVECs was demonstrated, confirming that it is glycosylphosphatidylinositol-lipid (GPI) anchored. Sequential treatment of HUVECs with phospholipase C and heparin was performed, and a trend was observed where GPI-anchored TFPI levels were increased after 1 hour of pretreatment with heparin but were decreased after 24 hours. Serum is a requirement for the heparin-dependent release of TFPI from HUVECs. Heparin pretreatment of HUVECs may affect levels of GPI anchored TFPI in a time and dose-dependent manner.

Elevated Plasma Factor IXa Activity in Premenopausal Women on Hormonal Contraception

Objective— Combined oral contraceptives induce a reversible hypercoagulable state with an enhanced risk of venous thromboembolism, but the underlying mechanism(s) remain unclear. Subjects on combined oral contraceptives also demonstrate a characteristic resistance to APC (activated protein C) in the thrombin generation assay. Here, we report the potential role of plasma factor IXa (FIXa) as a mechanism for hormone-induced systemic hypercoagulability. Approach and Results— A novel assay was used to determine FIXa activity in plasma samples from volunteer blood donors. Plasma from 36 premenopausal females on hormonal contraception and 35 not on hormonal contraception, 35 postmenopausal females, and 10 males were analyzed for FIXa activity, total PS (protein S), total tissue factor pathway inhibitor (TFPI), and TFPI-&agr; antigen. Premenopausal females on hormonal contraception demonstrated significantly increased FIXa activity and decreased TFPI-&agr; compared with the other groups. Remarkably, FIXa values were not normally distributed in the hormonal contraception group, but skewed toward the high end. Plasma FIXa activity inversely correlated with both TFPI-&agr; and total PS antigen. Ex vivo determination of TF-dependent FIX activation in FV-deficient plasma demonstrated that inhibitory anti-TFPI antibodies enhanced FIXa generation by 2- to 3-fold, whereas addition of 75 nmol/L PS reduced FIXa generation by ≈2-fold. Further, increasing FIXa concentration enhanced APC resistance during TF-triggered plasma thrombin generation. Conclusions— Elevation of plasma FIXa activity in association with reductions in TFPI-&agr; and PS is a potential mechanism for systemic hypercoagulability and resistance to APC in premenopausal females on hormonal contraception.