Pharmacological Interruption of Acute Thrombus Formation with Minimal Hemorrhagic Complications by a Small Molecule Tissue Factor/Factor VIIa Inhibitor: Comparison to Factor Xa and Thrombin Inhibition in a Nonhuman Primate Thrombosis Model

SummaryEdoxaban, an oral, direct factor Xa inhibitor, has a similar or low incidence of bleeding events compared with other anticoagulants in clinical trials. Therefore, agents to reverse the anticoagulant effects of edoxaban could be desirable in emergency situations. In this study, the reversal effects of haemostatic agents were determined on prothrombin time (PT) prolongation in vitro and bleeding time prolongation in vivo by edoxaban. PT using human plasma was measured in the presence of edoxaban at therapeutic and excess concentrations with the haemostatic agents, prothrombin complex concentrate (PPSB-HT), activated prothrombin complex concentrate (Feiba), and recombinant factor VIIa (rFVIIa). In rats, rFVIIa and Feiba was given during intensive anticoagulation with edoxaban. The haemostatic effect was evaluated in a model of planta template bleeding and a potential prothrombotic effect was evaluated in a venous thrombosis model. PPSB-HT, Feiba, and rFVIIa concentration-dependently shortened PT prolonged by edoxaban. Among these, rFVIIa and Feiba showed potent activities in reversing the PT prolongation by edoxaban. rFVIIa (1 and 3 mg/kg, i.v.) and Feiba (100 U/kg, i.v.) significantly reversed edoxaban (1 mg/kg/h)-induced prolongation of bleeding time in rats. In a rat venous thrombosis model, no potentiation of thrombus formation was observed when the highest dose (3 mg/kg) of rFVIIa was added to edoxaban (0.3 and 1 mg/kg/h) compared with the control. The present study indicated that rFVIIa, Feiba, and PPSB-HT have the potential to be reversal agents for edoxaban.

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Effect of Selective Factor Xa Inhibition on Arterial Thrombus Formation Triggered by Tissue Factor/Factor VIIa or Collagen in an Ex Vivo Model of Shear-Dependent Human Thrombogenesis

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/01.atv.15.12.2188 ◽

1995 ◽

Vol 15 (12) ◽

pp. 2188-2194 ◽

Cited By ~ 31

Author(s):

Una Ørvim ◽

R. Marius Barstad ◽

George P. Vlasuk ◽

Kjell S. Sakariassen

Keyword(s):

Tissue Factor ◽

Factor Viia ◽

Ex Vivo ◽

Thrombus Formation ◽

Factor Xa ◽

Ex Vivo Model ◽

Arterial Thrombus ◽

Factor Xa Inhibition

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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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Subcutaneous Dosing of CRA-027483, a Small Molecule FVIIa Inhibitor, Prevents Arterial Thrombosis in a Baboon Model.

Blood ◽

10.1182/blood.v106.11.1867.1867 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1867-1867

Author(s):

Ulla M. Marzec ◽

Juthamas Sukbuntherng ◽

Stacie A. Dalrymple ◽

Wendy B. Young ◽

Dange Vijaykumar ◽

...

Keyword(s):

Tissue Factor ◽

Small Molecule ◽

Bleeding Time ◽

Thrombus Formation ◽

Reversible Inhibitor ◽

Thrombosis Model ◽

Shunt Thrombosis ◽

Venous Shunt ◽

Fixed Concentration ◽

Indium 111

Abstract Vessel injury may expose tissue factor leading to the activation of FVII, initiation of coagulation, and thrombosis. A small reversible inhibitor of FVIIa (CRA-027483), dosed by subcutaneous (SC) injection, was investigated in an arterio-venous shunt thrombosis model in non-anticoagulated awake baboons. Drug pharmacokinetics (PK) and pharmacodynamics (PD) were studied in non-shunted baboons; PK and PD results were closely correlated with ~ 100% bioavailability following SC drug administration. Thrombosis was initiated by interposing within the shunt a segment of porous expanded (poly)tetrafluoroethylene vascular graft (ePTFE, 4mm ID), filled with relipidated tissue factor. Upon initiation of blood flow at 100mL/min (wall shear rate =256/sec) thrombus growth was monitored by gamma camera imaging of autologous Indium-111 labeled platelets for 1 hour. Fibrin accumulation was quantified using trace amounts of homologous fibrinogen labeled with I-125. Both bleeding time and prothrombin time (PT), the PD marker, were monitored throughout. Animals were dosed SC 90min prior to the initiation of thrombus formation, a regimen that was predicted from PK/PD to result in a fixed concentration of CRA-027483 during the thrombosis phase. Dosing of baboons with CRA 027483 at 1mg/kg, 2mg/kg and 4mg/kg SC (4–5 animals in each study group) inhibited total platelet deposition on the tissue factor surface by 13±4%, 43±30% and 67±26% respectively, vs. control results. Fibrin accumulation was reduced by 22±9%, 50±34%, and 76±19% respectively for these doses. PT values remained stable throughout the thrombosis phase and increased 1.3-, 1.6- and 2.1-fold over baseline with the escalating doses. Bleeding time measurements were slightly prolonged at the higher doses (7.3±1.6min and 7.1±2.5min for the 2mg/kg and 4mg/kg doses, respectively) as compared to the pre-drug measurement of 3.8±0.7min. We conclude that SC administration of CRA-027483, a reversible small molecule inhibitor of FVIIa, effectively reduces tissue factor-initiated thrombus formation in baboons with minimal hemostatic impairment.

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Characterisation of a Novel Series of Aprotinin-derived Anticoagulants

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649793 ◽

1995 ◽

Vol 74 (02) ◽

pp. 655-659 ◽

Cited By ~ 6

Author(s):

Jean Marie Stassen ◽

Anne-Marie Lambeir ◽

Ingrid Vreys ◽

Hans Deckmyn ◽

Gaston Matthyssens ◽

...

Keyword(s):

Blood Coagulation ◽

Initial Level ◽

Factor Viia ◽

Thrombus Formation ◽

Femoral Vein ◽

Factor Xa ◽

Vascular Trauma ◽

Coagulation Cascade ◽

Thrombosis Model

SummaryUpon vascular damage platelet activation and blood coagulation are initiated. Interference at the initial level of the activation of the coagulation cascade can result in effective inhibition of thrombus formation. The in vivo antithrombotic properties of a series of bovine pancreatic trypsin inhibitor mutants (BPTI, aprotinin) 4C2, 7L22, 5L15, 5L15-PEG, 6L15 and 5L84, as described in the accompanying paper, with a combined inhibitory activity on factor Xa, factor VIIa-tissue factor complex, factor XIa and plasma kallikrein were compared to rTAP, r-hirudin, heparin and enoxaparin in a platelet rich thrombosis model in hamsters.Platelet dependent thrombus deposition was quantified by dedicated image analysis after transillumination of the femoral vein to which a standardised vascular trauma was applied. After increasing intravenous bolus injections all tested agents, except for aprotinin, induced a dose dependent decrease of thrombus formation and a concomitant prolongation of the aPTT. From the linear correlation between these two parameters it was found that 5 out of the 6 tested aprotinin analogues, rTAP and r-hirudin completely inhibited thrombus formation at a therapeutical (2- to 3-fold) aPTT prolongation while 4C2, heparin and enoxaparin only inhibited thrombus formation for 40 to 50 percent at a 2-fold aPTT prolongation. Based on the calculated IC50 values for thrombus formation rTAP was found to be the most active compound in this model.It is concluded that acceptable interference at the initial level of the blood coagulation, e. g. within a therapeutical aPTT prolongation, can significantly inhibit platelet deposition at a site of vascular injury.

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Factors Affecting the lnteraction of Tissue Factor/Factor Vll with Factor X in a Heterogeneous Tubular Reactor

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647472 ◽

1991 ◽

Vol 65 (02) ◽

pp. 139-143 ◽

Cited By ~ 7

Author(s):

Cynthia H Gemmell ◽

Vincet T Turitto ◽

Yale Nemerson

Keyword(s):

Steady State ◽

Tissue Factor ◽

Factor Viia ◽

Transmembrane Protein ◽

Strong Association ◽

Tubular Reactor ◽

Factor Xa ◽

Phospholipid Bilayer ◽

Factor Vii ◽

Factor X

SummaryA novel reactor recently described for studying phospholipiddependent blood coagulation reactions under flow conditions similar to those occurring in the vasculature has been further charactenzed. The reactor is a capitlary whose inner wall is coated with a stable phospholipid bilayer (or two bilayers) containing tissue factor, a transmembrane protein that is required for the enzymatic activation of factor X by factor VIIa. Perfusion of the capillary at wall shear rates ranging from 25 s−1 to 1,200 s−1 with purified bovine factors X and VIIa led to steady state factor Xa levels at the outlet. Assay were performed using a chromogenic substrate, SpectrozymeTMFXa, or by using a radiometric technique. In the absence of Ca2+ or factor VIIa there was no product formation. No difference was noted in the levels of factor Xa achieved when non-activated factor VII was perfused. Once steady state was achieved further factor Xa production continued in the absence of factor VIIa implying a very strong association of factor VIIa with the tissue factor in the phospholipid membrane. In agreement with static vesicle-type studies the reactor was sensitive to wall tissue factor concentration, temperature and the presence of phosphatidylserine in the bilayer.

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The Factor VIII Bypassing Activity of Prothrombin Complex Concentrates: The Roles of Factor VIla and of Endothelial Cell Tissue Factor

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646459 ◽

1991 ◽

Vol 66 (05) ◽

pp. 559-564 ◽

Cited By ~ 4

Author(s):

Jerome M Teitel

Keyword(s):

Factor Viii ◽

Tissue Factor ◽

Factor Viia ◽

Factor Xa ◽

Procoagulant Activity ◽

Tissue Factor Expression ◽

Prothrombin Complex Concentrates ◽

Cell Tissue ◽

Factor Expression ◽

Necrosis Factor

SummaryAn experimental model incorporating cultured endothelial cells (EC) was used to study the "factor VIII bypassing" activity of prothrombin complex concentrates (PCC), a property exploited in the treatment of hemophiliacs with alloantibodies to factor VIII. Two PCC preparations were ineffective as stimuli of tissue factor expression by EC. However, incubation with a combination of PCC plus endotoxin (lipopolysaccharide, LPS) or tumor necrosis factor (TNF) induced much greater tissue factor expression than was seen in response to either substance alone. PCC expressed an additional direct procoagulant activity at the EC surface, which could not be attributed to either thrombin or factor Xa, and which was diminished by an anti-tissue factor antibody. Therefore factor VIIa, which was detectable in both PCC preparations, likely provided this additional direct procoagulant activity at the EC surface. We also excluded the possibility that coagulation proteases contained in or generated in the presence of PCC are protected from inactivation by AT III. Therefore, PCC can indirectly bypass factor VIII by enhancing induced endothelial tissue factor expression, and also possess direct procoagulant activity, probably mediated by factor VIIa.

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Comparative Studies of an Orally-active Factor Xa Inhibitor, YM-60828, with other Antithrombotic Agents in a Rat Model of Arterial Thrombosis

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615007 ◽

1998 ◽

Vol 79 (02) ◽

pp. 410-416 ◽

Cited By ~ 32

Author(s):

Kazuo Sato ◽

Yumiko Sakai ◽

Fukushi Hirayama ◽

Hiroyuki Koshio ◽

Yuta Taniuchi ◽

...

Keyword(s):

Electrical Stimulation ◽

Arterial Thrombosis ◽

Thrombus Formation ◽

Factor Xa ◽

Test Drug ◽

Antithrombotic Agent ◽

Antithrombotic Activity ◽

Thrombosis Model ◽

Treatment And Prevention ◽

Orally Active

SummaryWe examined the antithrombotic activity of a novel synthetic inhibitor of factor Xa, YM-60828, in an electrically-induced carotid artery thrombosis model in rats. In the first experiment, the antithrombotic activity of YM-60828 after i.v. infusion was compared with those of heparin, darteparin and argatroban. Test drug was administered by i.v. infusion from 30 min before electrical stimulation to the end of the experiment. YM-60828 at 1 mg/kg/h significantly improved patency status, prolonged the time to occlusive thrombus formation and duration of patency. Heparin at 300 U/kg/h also improved these parameters, but were accompanied by a marked increase in systemic coagulation time. In the second experiment, the antithrombotic activity of YM-60828 after oral administration was compared with those of ticlopidine, cilostazol, aspirin, beraprost, ethyl icosapentate and warfarin. Test drug was orally administered to fasted rats 60 min before electrical stimulation. YM-60828 at 30 mg/kg p.o., but not ticlopidine, cilostazol, aspirin, beraprost, ethyl icosapentate or warfarin, significantly reduced the incidence of occlusion and improved carotid arterial patency. These results suggest that YM-60828 may be a promising antithrombotic agent for the treatment and prevention of arterial thrombosis which can be given by oral as well as intravenous administration.

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TFPIβ is the GPI-anchored TFPI isoform on human endothelial cells and placental microsomes

Blood ◽

10.1182/blood-2011-10-388512 ◽

2012 ◽

Vol 119 (5) ◽

pp. 1256-1262 ◽

Cited By ~ 37

Author(s):

Thomas J. Girard ◽

Elodee Tuley ◽

George J. Broze

Keyword(s):

Endothelial Cells ◽

Tissue Factor ◽

Feedback Inhibition ◽

Culture Media ◽

Factor Viia ◽

Factor Xa ◽

Gpi Anchor ◽

C Terminus ◽

Before And After ◽

Tissue Factor Pathway

Abstract Tissue factor pathway inhibitor (TFPI) produces factor Xa-dependent feedback inhibition of factor VIIa/tissue factor-induced coagulation. Messages for 2 isoforms of TFPI have been identified. TFPIα mRNA encodes a protein with an acidic N-terminus, 3 Kunitz-type protease inhibitor domains and a basic C-terminus that has been purified from plasma and culture media. TFPIβ mRNA encodes a form in which the Kunitz-3 and C-terminal domains of TFPIα are replaced with an alternative C-terminus that directs the attachment of a glycosylphosphatidylinositol (GPI) anchor, but whether TFPIβ protein is actually expressed is not clear. Moreover, previous studies have suggested that the predominant form of TFPI released from cells by phosphatidylinositol-specific phospholipase C (PIPLC) treatment is TFPIα, implying it is bound at cell surfaces to a separate GPI-anchored coreceptor. Our studies show that the form of TFPI released by PIPLC treatment of cultured endothelial cells and placental microsomes is actually TFPIβ based on (1) migration on SDS-PAGE before and after deglycosylation, (2) the lack of a Kunitz-3 domain, and (3) it contains a GPI anchor. Immunoassays demonstrate that, although endothelial cells secrete TFPIα, greater than 95% of the TFPI released by PIPLC treatment from the surface of endothelial cells and from placental microsomes is TFPIβ.

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