Tissue factor pathway inhibitor: the carboxy-terminus is required for optimal inhibition of factor Xa

Wesselschmidt, Robin L.; Likert, Karen M.; Girard, TJ; Wun, TC; Broze, GJ Jr

doi:10.1182/blood.v79.8.2004.bloodjournal7982004

Cited by 60 publications

(98 citation statements)

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“…Activated coagulation factor V (FVa) is an essential component of the prothrombinase complex [1]. In humans, 80% of FV is found in plasma, and the remainder is stored in platelets, complexed to multimerin 1 (MMRN1), after FV is endocytosed from plasma [2][3][4]. Acquired FV inhibitors, although rare, can cause serious bleeding [5][6][7][8][9].…”

Section: Disclosure Of Conflict Of Interestsmentioning

confidence: 99%

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The association between protein S levels and anticoagulant activity of tissue factor pathway inhibitor type 1

2008

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To cite this article: Dahm AEA, Sandset PM, Rosendaal FR. The association between protein S levels and anticoagulant activity of tissue factor pathway inhibitor type 1. J Thromb Haemost 2008; 6: 393-5.Protein S (PS) is a vitamin K-dependent coagulation inhibitor of which 40% circulates in a free form and 60% circulates bound to C4b-binding protein (C4BP). PS exerts its anticoagulant activity mainly by being a cofactor for activated protein C (APC) in the inactivation of factor (F) Va and FVIIIa [1].Tissue factor (TF) pathway inhibitor type 1 (TFPI) is the physiological inhibitor of the TF pathway of blood coagulation. Approximately 80% of circulating TFPI in blood is Cterminal truncated and bound to lipoproteins, while 20% is carrier free full-length TFPI [2]. The TFPI-mediated inhibition of the TF/FVIIa complex occurs in two steps. Firstly, TFPI binds and inhibits FXa. Secondly, the TFPI/FXa complex binds to the complex of TF and FVIIa to form an inactive quaternary TFPI/FXa/TF/FVIIa complex:The first step of this reaction is rate limiting [3], and it is much faster with full-length TFPI than with truncated TFPI [4,5]. Recently, Hackeng et al. showed in an in vitro model in the absence of APC that PS increases the reaction rate between full-length TFPI and FXa. Moreover, this increase was not observed for truncated TFPI [6]. Hence, PS enhances the inhibition of coagulation by full-length TFPI.We have recently reported a TFPI anticoagulant activity assay based on the ability of TFPI to inhibit clotting in a modified diluted prothrombin time assay. This assay measures the activity of full-length TFPI, but not of truncated TFPI [7]. We suggested that the assay discriminated between full-length and truncated TFPI in relation to differences in reaction kinetics with FXa. Based on the findings of Hackeng et al., we hypothesized that the anticoagulant activity of fulllength TFPI was associated with the concentration of PS in plasma, and that the concentration of PS would influence the risk for venous thrombosis (VT) associated with low TFPI anticoagulant activity and vice versa. Although the results by Hackeng et al. are primarily applicable to free PS and fulllength TFPI, they also found some anticoagulant effect of PS bound to C4BP. We therefore decided to investigate both free and bound PS.To test these hypotheses we used data collected in the Leiden Thrombophilia Study (LETS) that included free and total PS [8], TFPI free and total antigen, and TFPI anticoagulant activity [7]. LETS is a population-based case-control study of 474 cases with deep vein thrombosis (DVT) and 474 healthy controls matched for sex and age, excluding individuals with cancer. The study has been described in detail previously [9]. The association between free PS and TFPI anticoagulant activity was studied in the 422 controls in whom both free PS and TFPI anticoagulant activity had been assayed. Correspondingly, the association between total PS and TFPI anticoagulant activity was studied in 436 controls. For the calculation of risk for DVT, u...

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Section: Disclosure Of Conflict Of Interestsmentioning

confidence: 99%

“…The first step of this reaction is rate limiting [3], and it is much faster with full-length TFPI than with truncated TFPI [4,5].…”

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The association between protein S levels and anticoagulant activity of tissue factor pathway inhibitor type 1

2008

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“…1). As a result of its intact positively charged C-terminal tail that interacts with anionic membrane surfaces, full-length TFPI is a more effective inhibitor than truncated TFPI [6,7].…”

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Regulation of TFPI function by protein S

Hackeng

Maurissen

Castoldi

et al. 2009

Journal of Thrombosis and Haemostasis

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Summary. Protein S is an anticoagulant cofactor of full-length tissue factor pathway inhibitor (TFPI) that facilitates optimal factor Xa-inhibition and efficient down-regulation of thrombin generation in plasma. Protein S and TFPI are constitutively active in plasma and therefore provide an effective anticoagulant barrier against unwanted procoagulant activity in the circulation. In this review, we describe the current status on how TFPI-activity depends on protein S, and show that TFPI and protein S are major regulators of thrombin generation both in the absence and presence of activated protein C (APC). As there is covariation of plasma TFPI and protein S levels both in health and in disease, these findings suggest that the risk of venous thrombosis associated with protein S deficiency states might be in part explained by the accompanying low plasma TFPI levels.Keywords: APC, protein S, TFPI, thrombosis. TFPIThe natural anticoagulant tissue factor pathway inhibitor (TFPI) regulates the initiation of coagulation by inhibiting both factor Xa (FXa) and the phospholipid-bound complex of tissue factor and factor VIIa (TF-FVIIa) [1,2]. TFPI is a Kunitz-type protease inhibitor comprising an acidic N-terminus, 3 Kunitz domains and a basic C-terminus [2]. TFPI is synthesized by endothelial cells and about 80% of the same associates with the vessel wall ( Fig. 1), whereas the remainder circulates in plasma at a concentration of approximately 2.5 nM [3,4]. The majority of TFPI in plasma (70-80%) is truncated and bound to low-density lipoproteins through disulfide bonds with . Only 10% of plasma TFPI (i.e. about 2% of total TFPI) is considered to be free full-length TFPI (Fig. 1). As a result of its intact positively charged C-terminal tail that interacts with anionic membrane surfaces, full-length TFPI is a more effective inhibitor than truncated TFPI [6,7]. Protein SProtein S is a vitamin K-dependent plasma protein that is mainly synthesized in hepatocytes and endothelial cells and circulates in plasma at a concentration of 350 nM [8]. More than half of the total protein S exists in a high-affinity complex (Kd approximately 0.1 nM) with C4b-binding protein (C4BP) and the molar excess of total protein S over C4BP circulates free [9]. Protein S, which lacks enzymatic activity, acts as a cofactor for activated protein C (APC) in the proteolytic inactivation of coagulation factors (F) Va and VIIIa [10] However, protein S was also shown to effectively reduce thrombin generation in plasma triggered at low TF concentrations (approximately 1 pM) in the absence of APC [14]. As no APC-independent anticoagulant activity of protein S was observed when coagulation was triggered via the intrinsic pathway, it was concluded that protein S participated in the regulation of the extrinsic pathway. These observations led us to discover that protein S is a cofactor for TFPI in the inhibition of FXa [15]. The in vitro-generated protein S multimers [16,17] are not involved in this anticoagulant activity of protein S. TFPI-protein S anticoagul...

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“…The 34 kDa form of TFPI is truncated within the C-terminal region [9]. Therefore, it is a slow inhibitor of FXa compared with the 41 kDa form [27] The 38 kDa form of TFPI is truncated within the C-terminal region Figure 5 shows Western blots of plasma from the postprotamine time-point using a polyclonal antibody to the entire TFPI molecule or an antibody specific for the C-terminal region of TFPI. As expected, both antibodies recognize the 41 kDa form of TFPI, while the C-terminal antibody does not recognize the 38 or 34 kDa species indicating they have undergone Cterminal degradation.…”

Section: Resultsmentioning

confidence: 99%

“…The lack of reactivity of the 38 kDa form with an antibody directed against the final 12 amino acids of TFPI indicates that it has undergone degradation in the C-terminal region. Cleavage of TFPI within this region has been shown to significantly slow the rate at which it inhibits FXa in progress curve amidolytic assays and decreases its ability to inhibit clot formation in FXa and TF initiated plasma clotting assays [27]. Both plasmin and thrombin, but not the other proteases listed above, have been shown to cleave TFPI in this region.…”

Section: Discussionmentioning

confidence: 99%

Disposition of tissue factor pathway inhibitor during cardiopulmonary bypass

Donahue

Gailani

Mast

2006

Journal of Thrombosis and Haemostasis

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Summary. Background: The tissue factor (TF) factor (F) VIIa complex activates coagulation FIX and FX to initiate coagulation, and also cleaves protease activated receptors (PARs) to initiate inflammatory processes in vascular cells. Tissue factor pathway inhibitor (TFPI) is the only specific inhibitor of the TF-FVIIa complex, regulating both its procoagulant and pro-inflammatory properties. Upon heparin infusion during cardiopulmonary bypass (CPB), a heparin releasable pool of endothelial associated TFPI circulates in plasma. Following protamine neutralization of heparin, the plasma TFPI level decreases, but does not return completely to baseline, suggesting that during CPB a fraction of the plasma TFPI becomes heparin-independent. We have investigated the structural and functional properties of plasma TFPI during CPB to further characterize how TFPI is altered during this procedure. Methods: We enrolled 17 patients undergoing firsttime cardiac surgery involving CPB. Plasma samples were obtained at baseline, 5 min and 1 h after start of CPB (receiving heparin), 10 min after protamine administration (off CPB) and 24 h following surgery. Samples were analyzed for full-length and free (non-lipoprotein bound) TFPI antigen by enzymelinked immunosorbent assay (ELISA) and for TFPI anticoagulant activity using an amidolytic assay. Western blot analysis was used to identify TFPI species of varying molecular weights in three additional patients. Dunnett's test for post hoc comparisons was used for statistical analysis. Results: The ELISA and Western blot data indicated that an increase in fulllength TFPI accounted for most of the heparin releasable TFPI. Following heparin neutralization with protamine, the fulllength TFPI antigen returned to baseline levels while the free TFPI antigen and the total plasma TFPI activity remained elevated. This was associated with the appearance of a new 38 kDa form of plasma TFPI identified by Western blot analysis. The 38 kDa form of TFPI did not react with an antibody directed against the C-terminal region of TFPI indicating it has undergone proteolysis within this region. All TFPI measurements returned to baseline 24 h following CPB. Conclusions: During CPB the full-length form of TFPI is the predominant form in plasma because of its prompt release from the endothelial surface following heparin administration. Upon heparin neutralization with protamine, full-length TFPI redistributes back to the endothelial surface. However, a new 38 kDa TFPI fragment is generated during CPB and remains circulating in plasma, indicating that TFPI undergoes proteolytic degradation during CPB. This degradation may result in a decrease in endothelium-associated TFPI immediately post-CPB, and may contribute to the procoagulant and proinflammatory state that often complicates CPB.

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Tissue factor pathway inhibitor: the carboxy-terminus is required for optimal inhibition of factor Xa

Cited by 60 publications

References 0 publications

The association between protein S levels and anticoagulant activity of tissue factor pathway inhibitor type 1

The association between protein S levels and anticoagulant activity of tissue factor pathway inhibitor type 1

Regulation of TFPI function by protein S

Disposition of tissue factor pathway inhibitor during cardiopulmonary bypass

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