The impact of factor Xa inhibition on axial dependent arterial thrombus formation triggered by a tissue factor rich surface

SummaryIt was the objective of this study to evaluate the anti-thrombotic potency of direct factor-Xa inhibition with ZK-807834 in stable coronary patients, using an ex-vivo model of arterial thrombus formation. Tissue factor pathway is important in atherothrom-bosis. Direct factor-Xa blockade may more potently reduce thrombosis and prevent coronary events. Badimon Perfusion Chamber 5-minute quantitative studies have shown 40-55% arterial thrombus reduction with abciximab, 23% with Clopidogrel, but none with heparin. Coronary patients (n= 18, 59 ± 9 years, 55% males) were blindly randomized to four groups receiving 24-hour infusion of a low, medium or high dose of direct factor-Xa inhibitor ZK-807834, or placebo. Arterial thrombus formation was measured in Badimon Chamber at baseline, end-of-infusion [Eol], and four hours and eight hours after Eol, and factor-X activity, prothrombin time [PT] ratio and plasma drug levels were measured simultaneously. For the low-, medium- and high-dose ZK-807834 groups, mean percent-reduction in thrombus size from baseline to Eol were 29%, 34% and 68%, respectively (p<0.00l), and at 8-h post Eol were 11%, 19% and 27%, respectively (p<0.01). Mean PT-ratio prolongation showed a strong linear relationship (Pearson’s r=0.93) with ZK-807834 plasma concentration. Mean percent-reduction in factor-X activity from baseline was 13%, 42% and 58%, respectively. Placebo had no effect on thrombus size or factor-X activity. In conclusion, direct factor-Xa inhibition with ZK-807834 markedly reduces ex-vivo arterial thrombus formation and factor-X activity in a dose-dependent manner. Plasma levels of ZK-807834 show a strong linear correlation with PT ratio. This direct factor-Xa inhibitor may reduce the need for additional potent glycoprotein llbllla inhibition.

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Effect of Depolymerized Holothurian Glycosaminoglycan (DHG) on Tissue Factor Pathway Inhibitor: In Vitro and In Vivo Studies

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657643 ◽

1997 ◽

Vol 78 (02) ◽

pp. 864-870 ◽

Cited By ~ 9

Author(s):

Hideki Nagase ◽

Kei-ichi Enjyoji ◽

Yu-ichi Kamikubo ◽

Keiko T Kitazato ◽

Kenji Kitazato ◽

...

Keyword(s):

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Factor Xa ◽

Free Form ◽

Initial Reaction ◽

Extrinsic Pathway ◽

Factor Xa Inhibition ◽

Tissue Factor Pathway

SummaryDepolymerized holothurian glycosaminoglycan (DHG) is a glycosaminoglycan extracted from the sea cucumber Stichopus japonicusSelenka. In previous studies, we demonstrated that DHG has antithrombotic and anticoagulant activities that are distinguishable from those of heparin and dermatan sulfate. In the present study, we examined the effect of DHG on the tissue factor pathway inhibitor (TFPI), which inhibits the initial reaction of the tissue factor (TF)-mediated coagulation pathway. We first examined the effect of DHG on factor Xa inhibition by TFPI and the inhibition of TF-factor Vila by TFPI-factor Xa in in vitro experiments using human purified proteins. DHG increased the rate of factor Xa inhibition by TFPI, which was abolished either with a synthetic C-terminal peptide or with a synthetic K3 domain peptide of TFPI. In contrast, DHG reduced the rate of TF-factor Vila inhibition by TFPI-factor Xa. Therefore, the effect of DHG on in vitroactivity of TFPI appears to be contradictory. We then examined the effect of DHG on TFPI in cynomolgus monkeys and compared it with that of unfractionated heparin. DHG induced an increase in the circulating level of free-form TFPI in plasma about 20-fold when administered i.v. at 1 mg/kg. The prothrombin time (PT) in monkey plasma after DHG administration was longer than that estimated from the plasma concentrations of DHG. Therefore, free-form TFPI released by DHG seems to play an additive role in the anticoagulant mechanisms of DHG through the extrinsic pathway in vivo. From the results shown in the present work and in previous studies, we conclude that DHG shows anticoagulant activity at various stages of coagulation reactions, i.e., by inhibiting the initial reaction of the extrinsic pathway, by inhibiting the intrinsic Xase, and by inhibiting thrombin.

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Dietary α-Linolenic Acid Inhibits Arterial Thrombus Formation, Tissue Factor Expression, and Platelet Activation

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvbaha.111.226118 ◽

2011 ◽

Vol 31 (8) ◽

pp. 1772-1780 ◽

Cited By ~ 45

Author(s):

Erik W. Holy ◽

Marc Forestier ◽

Eva K. Richter ◽

Alexander Akhmedov ◽

Florian Leiber ◽

...

Keyword(s):

Tissue Factor ◽

Linolenic Acid ◽

Platelet Activation ◽

Thrombus Formation ◽

Tissue Factor Expression ◽

Arterial Thrombus ◽

Factor Expression

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The impact of blood shear rate on arterial thrombus formation

Future Science OA ◽

10.4155/fso.15.28 ◽

2015 ◽

Vol 1 (4) ◽

Cited By ~ 44

Author(s):

Kjell S Sakariassen ◽

Lars Orning ◽

Vincent T Turitto

Keyword(s):

Shear Rate ◽

Thrombus Formation ◽

Arterial Thrombus ◽

The Impact

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Platelet Signaling in Primary Haemostasis and Arterial Thrombus Formation: Part 1

Hämostaseologie ◽

10.1055/s-0038-1675144 ◽

2018 ◽

Vol 38 (04) ◽

pp. 203-210 ◽

Cited By ~ 6

Author(s):

Rüdiger Scharf

Keyword(s):

Platelet Activation ◽

Thrombus Formation ◽

Vascular Lesions ◽

Arterial Thrombus ◽

Platelet Signaling ◽

Transduction Mechanisms ◽

Granule Secretion ◽

Von Willebrand ◽

The Impact

AbstractPlatelets react immediately in response to traumatic vascular injury by adhesion, activation, aggregation and subsequent haemostatic plug formation. While this reaction pattern is essential for haemostasis, platelet responses can also cause occlusive thrombi in diseased arteries, leading to myocardial infarction or stroke. Initially, flowing platelets are captured from the circulation to vascular lesions. This step is mediated by glycoprotein (GP) Ib-IX-V interacting with immobilized von Willebrand factor (VWF) on exposed subendothelial components. Tethered platelets can now bind to collagen through GPVI and integrin α2β1. Outside-in signals from the adhesion receptors act synergistically with inside-out signals from soluble stimuli and induce platelet activation. These mediators operate through G protein–coupled receptors and reinforce adhesion and activation. Typical manifestations of activated platelets include calcium mobilization, procoagulant activity, cytoskeletal reorganization, granule secretion and aggregation. This requires activation of integrin αIIbβ3 with shifting into a high-affinity state and is indispensable to bind soluble fibrinogen, VWF and fibronectin. The multiple interactions and the impact of thrombin result in firm adhesion and recruitment of circulating platelets into growing aggregates. A fibrin meshwork supports stabilization of haemostatic thrombi and prevents detachment by the flowing blood. This two-part review provides an overview of platelet activation and signal transduction mechanisms with a focus on αIIbβ3-mediated outside-in signaling in integrin variants. In the first part, a three-stage model of platelet recruitment and activation in vivo is presented. Along with that, platelet responses upon exposure to thrombogenic surfaces followed by platelet-to-platelet interactions and formation of haemostatic thrombi are discussed. Moreover, several determinants involved in pathological thrombosis will be reviewed.

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Factor Xa inhibition by immobilized recombinant tissue factor pathway inhibitor

Journal of Biomaterials Science Polymer Edition ◽

10.1163/156856206778366013 ◽

2006 ◽

Vol 17 (9) ◽

pp. 1025-1037 ◽

Cited By ~ 6

Author(s):

Amito Chandiwal ◽

Fowzia Shahreen Zaman ◽

Alan E. Mast ◽

Connie L. Hall

Keyword(s):

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Factor Xa ◽

Factor Xa Inhibition ◽

Tissue Factor Pathway

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Factor XI Deficient Mice Have Reduced Platelet Accumulation and Fibrin Deposition after Laser Injury.

Blood ◽

10.1182/blood.v104.11.218.218 ◽

2004 ◽

Vol 104 (11) ◽

pp. 218-218

Author(s):

T. Regan Baird ◽

David Gailani ◽

Bruce Furie ◽

Barbara C. Furie

Keyword(s):

Tissue Factor ◽

Factor Viia ◽

Thrombus Formation ◽

Factor Xa ◽

Fibrin Deposition ◽

Wild Type ◽

Factor Xi ◽

Factor Xia ◽

Tissue Factor Pathway ◽

Platelet Accumulation

Abstract Tissue factor exposure at sites of vascular injury results in the generation of factor Xa and thrombin. A current model of blood coagulation suggests that the amount of thrombin generated through this pathway is limited by the inhibition of the factor VIIa-tissue factor complex by tissue factor pathway inhibitor in the presence of factor Xa. The initial thrombin activates a number of hemostatic proteins including factor XI. Factor XIa then activates factor IX leading to generation of the tenase complex to maintain the thrombin flux. While in vitro studies support this hypothesis the importance of factor XI for thrombus formation in vivo remains unclear. We have examined thrombus formation upon laser injury to the arterioles (30–50 μm diameter) of the cremaster muscle in living mice lacking factor XI using digital multi-channel fluorescence intravital microscopy. Platelets were labeled with Alexa 488 conjugated murine CD41 Fab fragments by systemic infusion of the antibody. Maximum platelet accumulation in factor XI null mice (median of 35 thrombi in 4 mice) is only 25% of that of wild type mice (median of 40 thrombi in 4 mice) after injury (p<0.03). The time course of platelet accumulation is similar between both genotypes. Maximum platelet accumulation occurs in approximately 90 seconds (p<0.2). Fibrin deposition was observed simultaneously using an Alexa 660 conjugated anti-fibrin antibody that does not recognize fibrinogen. Maximum fibrin deposition in factor XI null mice is 50% that of wild type mice (p<0.001) and the rate of fibrin generation is slower in factor XI null mice. However, the time to achieve half maximal fibrin deposition is approximately the same in factor XI null mice (77 sec) compared to wild type mice (63.5 sec, p<0.09). These data suggest that the primary difference in response to laser induced injury between the factor XI null mice and wild type mice is the level of thrombin generated and supports the hypothesis that factor XI participates in maintaining thrombin flux after inhibition of the factor VII-tissue factor. The model above postulates a single source of tissue factor, the vessel wall, and further, that the tissue factor-factor VIIa complex formed from the exposed tissue factor is rapidly inactivated by tissue factor pathway inhibitor after the appearance of the initial factor Xa formed. In addition it has been suggested that a rapidly growing thrombus blocks access to vascular wall tissue factor. However we have recently observed that there is a P-selectin and P-selectin glycoprotein ligand 1 dependent pathway of blood coagulation that recruits blood borne tissue factor into a growing thrombus at sites of laser-induced vessel injury. Both vessel wall and blood borne tissue factor are required for normal thrombus formation. Our data suggest that although tissue factor is continuously recruited to the growing thrombus, factor XIa plays a significant role in thrombin generation.

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

Blood ◽

10.1182/blood.v79.8.2004.2004 ◽

1992 ◽

Vol 79 (8) ◽

pp. 2004-2010 ◽

Cited By ~ 108

Author(s):

R Wesselschmidt ◽

K Likert ◽

T Girard ◽

TC Wun ◽

GJ Jr Broze

Keyword(s):

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Factor Viia ◽

Factor Xa ◽

Reversible Inhibitor ◽

Normal Plasma ◽

Encounter Complex ◽

Factor Xa Inhibition ◽

Tissue Factor Pathway ◽

Inhibit Factor

Abstract Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type protease inhibitor that binds to and inactivates factor Xa directly, and in a factor Xa-dependent fashion inhibits the factor VIIa/tissue factor catalytic complex. TFPI is a slow, tight-binding, competitive, and reversible inhibitor of factor Xa, in which the formation of an initial encounter complex between TFPI and factor Xa is followed by slow isomerization to a final, tightened complex. Wild-type recombinant TFPI (rTFPI), expressed in mouse C127 cells, separates into two forms on heparin-agarose chromatography that elute at 0.3 mol/L and 0.6 mol/L NaCl. Western blot analysis shows that both forms contain the N- terminus of full-length TFPI, but only rTFPI(0.6) is recognized by an antibody directed against the C-terminus. rTFPI(0.3) and rTFPI(0.6) inhibit factor Xa with 1:1 stoichiometry and inhibit factor VIIa/tissue factor equally in an endpoint-type assay. However, rTFPI(0.6) is a more potent inhibitor than rTFPI(0.3) of coagulation in normal plasma induced by either factor Xa or tissue factor. The initial inhibition of factor Xa (less than 5 seconds) produced by rTFPI(0.6) is several-fold greater than that produced by rTFPI(0.3), presumably reflecting a lower Ki of the immediate encounter complex between factor Xa and TFPI. The differential effect of these forms of TFPI on tissue factor-induced coagulation in normal plasma appears to be directly related to their ability to inhibit factor Xa. To confirm the role of the C-terminal region of TFPI in optimal factor Xa inhibition, a carboxy-terminal mutant of rTFPI, which is truncated after leucine 252 and thus lacks the basic sequence K T K R K R K K Q R V K (residues 254–265), was expressed in C127 cells. This form of rTFPI elutes from heparin-agarose at 0.28 mol/L NaCl and inhibits factor Xa at a rate that is slower than rTFPI(0.3). The Ki(final)s for factor Xa inhibition by rTFPI(0.6), rTFPI(0.3), and rTFPI1–252 are 3.1 +/- 0.6, 19.6 +/- 0.8, and 19.6 +/- 3.0 pmol/L, respectively.

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Interleukin-1β Mediates Arterial Thrombus Formation via NET-Associated Tissue Factor

Journal of Clinical Medicine ◽

10.3390/jcm8122072 ◽

2019 ◽

Vol 8 (12) ◽

pp. 2072 ◽

Cited By ~ 14

Author(s):

Luca Liberale ◽

Erik W. Holy ◽

Alexander Akhmedov ◽

Nicole R. Bonetti ◽

Fabian Nietlispach ◽

...

Keyword(s):

Tissue Factor ◽

Plasma Levels ◽

Thrombus Formation ◽

Mechanistic Explanation ◽

Multicenter Trial ◽

Coagulation Cascade ◽

Reactive Protein ◽

Arterial Thrombus ◽

Activated Neutrophils ◽

Potential Link

CANTOS reported reduced secondary atherothrombotic events in patients with residual inflammatory risk treated with the inhibitory anti-IL-1β antibody, Canakinumab. Yet, mechanisms that underlie this benefit remain elusive. Recent work has implicated formation of neutrophil extracellular traps (NETosis) in arterial thrombosis. Hence, the present study explored the potential link between IL-1β, NETs, and tissue factor (TF)—the key trigger of the coagulation cascade—in atherothrombosis. To this end, ST-elevation myocardial infarction (STEMI) patients from the Swiss multicenter trial SPUM-ACS were retrospectively and randomly selected based on their CRP levels. In particular, 33 patients with STEMI and high C-reactive protein (CRP) levels (≥ 10 mg/L) and, 33 with STEMI and low CRP levels (≤ 4 mg/L) were investigated. High CRP patients displayed elevated circulating IL-1β, NETosis, and NET-associated TF plasma levels compared with low CRP ones. Additionally, analysis of patients stratified by circulating IL-1β levels yielded similar results. Moreover, NETosis and NET-associated TF plasma levels correlated positively in the whole population. In addition to the above, translational research experiments provided mechanistic confirmation for the clinical data identifying IL-1β as the initial trigger for the release of the pro-coagulant, NET-associated TF. In conclusion, blunted TF presentation by activated neutrophils undergoing NETosis may provide a mechanistic explanation to reduced secondary atherothrombotic events as observed in canakinumab-treated patients in CANTOS.

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