Factor Xa Enhances the Binding of Tissue Factor Pathway Inhibitor to Acidic Phospholipids

1996 ◽  
Vol 75 (05) ◽  
pp. 796-800 ◽  
Author(s):  
Sanne Valentin ◽  
Inger Schousboe
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Xa ◽  
Microtitre Plate ◽  
C Terminus ◽  
Microtitre Plate Assay ◽  
Kunitz Domain ◽  
Tissue Factor Pathway ◽  
Acidic Phospholipids

SummaryIn the present study, the interaction between tissue factor pathway inhibitor (TFPI) and phospholipids has been characterized using a microtitre plate assay. TFPI was shown to bind calcium-independently to an acidic phospholipid surface composed of phosphatidylserine, but not a surface composed of the neutral phosphatidylcholine. The interaction was demonstrated to be dependent on the presence of the TFPI C-terminus. The presence of heparin (1 U/ml, unfractionated) was able to significantly reduce the binding of TFPI to phospholipid. The interaction of TFPI with phosphatidylserine was significantly decreased in the presence of calcium, but this was counteracted, and even enhanced, following complex formation of TFPI with factor Xa prior to incubation with the phospholipid surface. Moreover, a TFPI variant, not containing the third Kunitz domain and the C-terminus, was unable to bind to phospholipid. However, following the formation of a TFPI/factor Xa-complex this TFPI variant was capable of interacting with the phospholipid surface. This indicates that the role of factor Xa as a TFPI cofactor, at least in part, is to mediate the binding of TFPI to the phospholipid surface.

scholarly journals Structural and functional characterization of tissue factor pathway inhibitor following degradation by matrix metalloproteinase-8

2002 ◽  
Vol 367 (2) ◽  
pp. 451-458 ◽  
Author(s):  
Anna C. CUNNINGHAM ◽  
Karen A. HASTY ◽  
Jan J. ENGHILD ◽  
Alan E. MAST
Keyword(s):  
Matrix Metalloproteinase ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Proteolytic Enzymes ◽  
Functional Characterization ◽  
Factor Xa ◽  
Terminal Region ◽  
The Third ◽  
Kunitz Domain ◽  
Tissue Factor Pathway

Vascular injury results in the activation of coagulation and the release of proteolytic enzymes from neutrophils and connective- tissue cells. High concentrations of these inflammatory proteinases may destroy blood coagulation proteins, contributing to coagulation and bleeding disorders associated with severe inflammation. Matrix metalloproteinase-8 (MMP-8) is released from neutrophils at sites of inflammation and vascular disease. We have investigated the effect of MMP-8 degradation on the anticoagulant function of tissue factor pathway inhibitor (TFPI) as a potential pathological mechanism contributing to coagulation disorders. MMP-8 cleaves TFPI following Ser174 within the connecting region between the second and third Kunitz domains (kcat/Km75M-1·s-1) as well as following Lys20 within the NH2-terminal region. MMP-8 cleavage of TFPI decreases the anticoagulant activity of TFPI in factor Xa initiated clotting assays as well as the ability of TFPI to inhibit factor Xa in amidolytic assays. Yet, MMP-8 cleavage does not alter the ability of TFPI to inhibit trypsin. Since the inhibition of both factor Xa and trypsin is mediated by binding to the second Kunitz domain, these results suggest that regions of TFPI other than the second Kunitz domain may directly interact with factor Xa. 125I-factor Xa ligand blots of TFPI fragments generated following MMP-8 degradation were used for probing binding interactions between factor Xa and regions of TFPI, other than the second Kunitz domain. In experiments performed under reducing conditions that disrupt the Kunitz domain structure, 125I-factor Xa binds to the C-terminal fragment of MMP-8-degraded TFPI. This fragment contains portions of TFPI distal to Ser174, which include the third Kunitz domain and the basic C-terminal region. An altered form of TFPI lacking the third Kunitz domain, but containing the C-terminal region, was used to demonstrate that the C-terminal region directly interacts with factor Xa.

Plasmatic tissue factor pathway inhibitor is a major determinant of clotting in factor VIII inhibited plasma or blood

2013 ◽  
Vol 109 (03) ◽  
pp. 450-457 ◽  
Author(s):  
Sabine Knappe ◽  
Bernd Jilma ◽  
Ulla Derhaschnig ◽  
Rudolf Hartmann ◽  
Michael Palige ◽  
...  
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Ex Vivo ◽  
Major Determinant ◽  
Prospective Clinical Study ◽  
Acquired Haemophilia ◽  
C Terminus ◽  
Kunitz Domain ◽  
Tissue Factor Pathway

SummaryTissue factor pathway inhibitor (TFPI) is a major inhibitor of coagulation. We therefore hypothesised that high plasmatic TFPI levels are associated with impaired ex vivo clotting in a model of acquired haemophilia. Blood samples were collected in a prospective clinical study from 30 healthy volunteers. Coagulation in normal or factor VIII (FVIII)-inhibited human blood or plasma was measured by the calibrated automated thrombogram (CAT) and rotational thromboelastometry (ROTEM). Both methods are global haemostatic assays that provide insight into the whole coagulation process. Monoclonal mouse antibodies raised against either the C-terminus or the Kunitz domain 2 of TFPI were used to determine full-length (fl-) and total TFPI by an enzyme-immunoassay. Clotting times and parameters of thrombin generation correlated with TFPI levels. Subjects with low fl-TFPI levels had significantly shorter clotting times and a higher endogenous thrombin potential (ETP) compared to those with high fl-TFPI levels (p≤0.005 for all). An even stronger effect was seen in FVIII-inhibited blood/plasma: ROTEM clotting time was 26% shorter (p=0.01) and the ETP assessed by CAT was >2-fold higher in subjects with low fl-TFPI levels (p≤0.0001). Plasmatic TFPI is a major determinant of coagulation in global haemostatic tests particularly when FVIII is missing. Thus, inhibition of TFPI might be a promising novel treatment approach, especially in haemophilia patients with FVIII inhibitors.

scholarly journals Tissue Factor Pathway Inhibitor Gene Disruption Produces Intrauterine Lethality in Mice

Blood ◽  
1997 ◽  
Vol 90 (3) ◽  
pp. 944-951 ◽  
Author(s):  
Zhong-Fu Huang ◽  
Darryl Higuchi ◽  
Nina Lasky ◽  
George J. Broze
Keyword(s):  
Tissue Factor ◽  
Gene Disruption ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Factor Xa ◽  
Yolk Sac ◽  
Functional Assay ◽  
Wild Type ◽  
Kunitz Domain ◽  
Tissue Factor Pathway

Abstract Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type proteinase inhibitor that directly inhibits factor Xa and, in a factor Xa–dependent fashion, produces feedback inhibition of the factor VIIa/TF catalytic complex responsible for the initiation of coagulation. To further define the physiologic role of TFPI, gene-targeting techniques were used to disrupt exon 4 of the TFPI gene in mice. This exon encodes Kunitz domain-1 of TFPI, which is required for factor VIIa/TF inhibition. In mice heterozygous for TFPI gene-disruption, TFPIK1(+/−), an altered form of TFPI lacking Kunitz domain-1, circulates in plasma at a concentration ∼40% that of wild-type TFPI. TFPIK1(+/−) animals have plasma TFPI activity ∼50% that of wild-type mice, based on a functional assay that measures factor VIIa/TF inhibition, and have a normal phenotype. Sixty percent of TFPIK1(−/−) mice die between embryonic days E9.5 and E11.5 with signs of yolk sac hemorrhage. The extent of structural abnormalities within the yolk sac vascular system appears to mirror the condition of the embryo, suggesting that the embryonic and extra-embryonic tissues are both responding to same insult, presumably circulatory insufficiency. Organogenesis is normal in TFPIK1 null animals that progress beyond E11.5, but hemorrhage, particularly in the central nervous system and tail, is evident during later gestation and none of the TFPIK1(−/−) mice survive to the neonatal period. The presence of immunoreactive fibrin(ogen) in the liver and intravascular thrombi is consistent with the notion that unregulated factor VIIa/TF action and a consequent consumptive coagulopathy underlies the bleeding diathesis in these older embryos. Human TFPI-deficient embryos may suffer a similar fate because an individual with TFPI deficiency has not been identified.

The second Kunitz domain of human tissue factor pathway inhibitor: cloning, structure determination and interaction with factor Xa 1 1Edited by T. Richmond

1997 ◽  
Vol 269 (3) ◽  
pp. 395-407 ◽  
Author(s):  
Maurits J.M Burgering ◽  
Leon P.M Orbons ◽  
Antoon van der Doelen ◽  
John Mulders ◽  
Henri J.M Theunissen ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Structure Determination ◽  
Human Tissue ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Xa ◽  
Kunitz Domain ◽  
Tissue Factor Pathway

scholarly journals Prothrombinase is protected from inactivation by tissue factor pathway inhibitor: competition between prothrombin and inhibitor*

1997 ◽  
Vol 323 (1) ◽  
pp. 33-37 ◽  
Author(s):  
Jo FRANSSEN ◽  
Irene SALEMINK ◽  
George M. WILLEMS ◽  
Tze-Chein WUN ◽  
H. Coenraad HEMKER ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Rate Constant ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Xa ◽  
Full Length ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
C Terminus ◽  
Length Variant ◽  
Tissue Factor Pathway

The inhibition of prothrombinase by tissue factor pathway inhibitor (TFPI) has been studied in the presence and absence of prothrombin. The rate constant of association of prothrombinase with full-length TFPI was 2.1×107 M-1ċs-1 and 0.05×107 M-1ċs-1 for the reaction with C-terminus truncated TFPI (TFPI1-161). The rate constant of dissociation was 0.65×10-4 s-1 in both cases. The rate constant of inhibition of prothrombinase by TFPI1-161 was similar to that of solution-phase factor Xa. In contrast, phospholipids and factor Va enhanced the association rate of the reaction between factor Xa and full-length TFPI by approx. 20-fold. Although TFPI, and in particular the full-length variant of the molecule, is a potent inhibitor of prothrombinase (overall inhibition constant of 3 pM), we also found that prothrombin competed very effectively with TFPI for the active site of factor Xa in the prothrombinase complex. A 50% reduction of the rate constant of inhibition was measured in the presence of 4 nM prothrombin, i.e. 0.2% of the plasma concentration of prothrombin. The physiological significance of TFPI as an inhibitor of prothrombinase activity is thus questionable.

scholarly journals Tissue Factor Pathway Inhibitor Gene Disruption Produces Intrauterine Lethality in Mice

Blood ◽  
1997 ◽  
Vol 90 (3) ◽  
pp. 944-951 ◽  
Author(s):  
Zhong-Fu Huang ◽  
Darryl Higuchi ◽  
Nina Lasky ◽  
George J. Broze
Keyword(s):  
Tissue Factor ◽  
Gene Disruption ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Factor Xa ◽  
Yolk Sac ◽  
Functional Assay ◽  
Wild Type ◽  
Kunitz Domain ◽  
Tissue Factor Pathway

Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type proteinase inhibitor that directly inhibits factor Xa and, in a factor Xa–dependent fashion, produces feedback inhibition of the factor VIIa/TF catalytic complex responsible for the initiation of coagulation. To further define the physiologic role of TFPI, gene-targeting techniques were used to disrupt exon 4 of the TFPI gene in mice. This exon encodes Kunitz domain-1 of TFPI, which is required for factor VIIa/TF inhibition. In mice heterozygous for TFPI gene-disruption, TFPIK1(+/−), an altered form of TFPI lacking Kunitz domain-1, circulates in plasma at a concentration ∼40% that of wild-type TFPI. TFPIK1(+/−) animals have plasma TFPI activity ∼50% that of wild-type mice, based on a functional assay that measures factor VIIa/TF inhibition, and have a normal phenotype. Sixty percent of TFPIK1(−/−) mice die between embryonic days E9.5 and E11.5 with signs of yolk sac hemorrhage. The extent of structural abnormalities within the yolk sac vascular system appears to mirror the condition of the embryo, suggesting that the embryonic and extra-embryonic tissues are both responding to same insult, presumably circulatory insufficiency. Organogenesis is normal in TFPIK1 null animals that progress beyond E11.5, but hemorrhage, particularly in the central nervous system and tail, is evident during later gestation and none of the TFPIK1(−/−) mice survive to the neonatal period. The presence of immunoreactive fibrin(ogen) in the liver and intravascular thrombi is consistent with the notion that unregulated factor VIIa/TF action and a consequent consumptive coagulopathy underlies the bleeding diathesis in these older embryos. Human TFPI-deficient embryos may suffer a similar fate because an individual with TFPI deficiency has not been identified.

scholarly journals TFPIβ, a Second Product from the Mouse Tissue Factor Pathway Inhibitor (TFPI) Gene

1999 ◽  
Vol 81 (01) ◽  
pp. 45-49 ◽  
Author(s):  
Dougald Monroe ◽  
Julie Oliver ◽  
Harold Roberts ◽  
Jen-Yea Chang
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Enzyme Inhibitor ◽  
Factor Xa ◽  
Kinetic Studies ◽  
Apparent Molecular Weight ◽  
Mouse Tissue ◽  
Mouse Lung ◽  
C Terminus ◽  
Tissue Factor Pathway

SummaryTissue factor pathway inhibitor (TFPI) contains three Kunitz domains separated by two connecting regions. We have cloned another naturally occurring TFPI gene product from a mouse lung cDNA library which we have called TFPI β. TFPIβ is derived from alternative splicing of the TFPI gene. Analysis of the cDNA shows that mouse TFPIβ protein is identical to TFPI from the N’-terminus through the second connecting region. However, mouse TFPIβ possesses neither a third Kunitz domain nor an Arg, Lys-rich C’-terminus but instead has a completely different C’-terminal (β-domain) sequence which is not homologous to any known protein. Northern blot analyses show that the tissues for mouse TFPIβ synthesis are heart and lung; in contrast, TFPI appears in Northern blots of heart and spleen. Both TFPIβ and TFPI messages first appear in 7-day-old mouse embryos, but only the TFPI mRNA persists until 17 days. Purified recombinant TFPIβ shows an apparent molecular weight of 38 kDa. Kinetic studies indicate that mouse TFPIβ is a slow-binding enzyme inhibitor for human factor Xa. In addition, heparin does not enhance the inhibition of factor Xa by mouse TFPIβ although it does accelerate factor Xa inhibition by TFPI.

The Kunitz 1 and Kunitz 3 domains of tissue factor pathway inhibitor are required for efficient inhibition of factor Xa

2012 ◽  
Vol 108 (08) ◽  
pp. 266-276 ◽  
Author(s):  
Sameera Peraramelli ◽  
Dennis P. L. Suylen ◽  
Jan Rosing ◽  
Tilman M. Hackeng
Keyword(s):  
Complex Formation ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Tight Binding ◽  
Factor Xa ◽  
C Terminus ◽  
Encounter Complex ◽  
Rapid Formation ◽  
Tissue Factor Pathway ◽  
Gla Domain

SummaryTissue factor pathway inhibitor (TFPI) is a slow tight-binding inhibitor that inhibits factor (F)Xa in a biphasic fashion: a rapid formation of loose FXa•TFPI encounter complex is followed by slow rearrangement into a tight FXa•TFPI* complex in which the Kunitz-2 (K2) domain of TFPI binds and inhibits FXa. In the current study, full-length TFPI (TFPIfl) and various truncated TFPI constructs were used to assess the importance of TFPI domains other than K2 in the inhibition of FXa. In the absence of Ca2+ ions, FXa was more effectively inhibited by TFPIfl than Gladomain less FXa. In turn, Ca2+ ions impaired FXa inhibition by TFPIfl but not by TFPI constructs that lack the C-terminus. This suggests that, in absence of Ca2+ ions, interactions between the C-terminus of TFPI and the Gla-domain of FXa promote FXa-inhibition. TFPIfl and K2K3 had similar efficiencies for encounter complex formation. However, K2K3 showed monophasic inhibition instead of biphasic inhibition, indicating absence of rearrangement into a tight complex. K1K2 and TFPI1–161 showed biphasic inhibition, but had less efficient encounter complex formation than TFPIfl. Finally, K2K3 was a 10-fold more efficient FXainhibitor than K2. These results indicate that K3-C-terminus enhances the formation of encounter complex and that K1 is required for isomerisation of the encounter- into tight complex. Since TFPIfl has a 10-fold higher Ki than K2K3-C-terminus, we propose that K1 is not only required for the transition of the loose to the tight FXa•TFPI* complex, but also inhibits FXa•TFPI encounter complex formation. This inhibitory activity is counteracted by K3 and C-terminus.

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