Dual Inhibition of Blood Coagulation Factors Xa and Iia Synergize To Reduce Thrombus Weight and Thrombin Generation in a Rabbit Arteriovenous Shunt Model of Thrombosis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1866-1866
Author(s):  
Thomas B. McClanahan ◽  
Sangita M. Baxi ◽  
Liguo Chi ◽  
Tawny Dahring ◽  
Weston R. Gould ◽  
...  
Keyword(s):  
Blood Coagulation ◽  
Single Molecule ◽  
Thrombin Generation ◽  
Ex Vivo ◽  
Factor Xa ◽  
Coagulation Cascade ◽  
Vehicle Group ◽  
Thrombin Inhibitor ◽  
Antithrombotic Effect ◽  
Dual Inhibitor

Abstract Several compounds currently in development for the treatment of thrombotic disorders demonstrate high levels of specificity for single targets of the blood coagulation cascade such as factor Xa and thrombin. However, development of a single molecule dual inhibitor against factor Xa and thrombin may expand the efficacy to safety ratio of treatment options for arterial and venous thrombosis. The objective of this study was to determine if simultaneous administration of PD 0313052, a selective Xa inhibitor and argatroban, a direct thrombin inhibitor, would lead to a synergistic antithrombotic effect in a rabbit AV shunt model of thrombosis. Intravenous administration of PD 0313052 alone at doses of 0.1, 0.3, and 1.0 mg/kg/min resulted in thrombus weight (TW) reductions of 11±3, 25±10 and 67±7 % compared to the vehicle group. Argatroban at 1, 3 and 10 mg/kg/min reduced TW 16±13, 47±10 and 75±6 %. When PD 0313052 was administered at 0.1 mg/kg/min in combination with argatroban at 1, 3 or 10 mg/kg/min TW was reduced 50±7, 60±7 and 82±9 %. Likewise, argatroban at 1 mg/kg/min combined with 0.1, 0.3 or 1mg/kg/min of PD 0313052 resulted in TW reductions of 56±9, 60±9 and 84±5 %, respectively. At the lowest combined doses of PD 0313052 and argatroban there was no change in bleeding time relative to the additive fold-increases from each drug alone. The EC50 of intravenously administered PD 0313052 and argatroban was 67±23 and 178±58 ng/ml, respectively. When the drugs were combined the EC50 was reduced to 12±6 ng/ml with the PD 0313052/argatroban combination and to 83±29 ng/ml with the argatroban/PD 0313052 combination. A synergistic effect was also observed in an ex vivo assay of thrombin generation (TG). Predicted additive inhibition of TG based on the individual effects of each compound was −9±7, 9±2 and 29±7 % compared to 10±5, 32±5 and 55±3 % with the 313052/argatroban combination. The predicted effects of the argatroban/PD 0313052 combination was −9±7, 1±7 and 16±9 % compared to the actual inhibition of 5±3, 14±5 and 31±7 %. These results demonstrate a significant synergistic antithrombotic effect by combining low doses of a factor Xa and a thrombin inhibitor and support the hypothesis that development of a single molecule inhibitor against different hemostatic targets may offer greater efficacy in the prevention and treatment of venous and arterial thrombosis.

A Zymogen-like Factor Xa Improves Hemostasis in a Murine Bleeding Model

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1476-1476 ◽  
Author(s):  
Jasuja Reema ◽  
Sunita Patel-Hett ◽  
Rodney M. Camire ◽  
Joachim Fruebis ◽  
Debra Pittman
Keyword(s):  
Blood Loss ◽  
Whole Blood ◽  
Thrombin Generation ◽  
Ex Vivo ◽  
Factor Xa ◽  
Coagulation Cascade ◽  
Effective Control ◽  
Normal Male ◽  
Severe Bleeding ◽  
K Value

Abstract In many clinical indications, effective control of bleeding is needed. Factor Xa (FXa) is a vitamin K-dependent trypsin-like serine protease that interacts with non-enzymatic coagulation factor Va (FVa) on negatively charged membrane surfaces to generate thrombin during hemostasis. Based on its central role in the coagulation cascade at the intersection of both intrinsic and extrinsic pathways, direct administration of FXa is an attractive approach to restoring hemostasis in bleeding disorders by leading to direct thrombin generation and fibrin formation. However, the short plasma half-life of the activated FXa protease renders it inadequate as a therapeutic for acute bleeding. Here, we investigate FXaI16L,a recently described variant of coagulation FXa engineered to overcome these limitations. The FXaI16L variant has an isoleucine (I) to leucine (L) substitution at amino acid 16 (based on chymotrypsin numbering). FXaI16L exhibits zymogen-like properties with both reduced activity and sensitivity toward plasma inhibitors. In the presence of its cofactor, FVa, FXaI16L activity is restored. We assessed the hemostatic activity of FXaI16L in an acute tail bleeding model that results in severe bleeding in normal mice. Ex vivo pharmacodynamic parameters in plasma and whole blood were also measured. FXaI16L was administrated intravenously to normal male C57BL/6J mice at doses of 1, 10, 25, 50, 100, or 200 μg/kg. Control mice received vehicle only. Two minutes post administration, a 3 mm tail transection was made. Tails were immediately immersed in tubes containing pre-warmed phosphate buffered saline for blood collection over a ten minute period. Bleeding times were recorded and volume of blood loss was determined by measurement of the hemoglobin content in the collected blood. Following administration of FXaI16L, a dose dependent reduction in bleeding was observed. Mice dosed with FXaI16Lshowed a decrease in blood loss of 12% (1 μg/kg), 16.6% (10 μg/kg), 26.7% (25 μg/kg), 45.3% (50 μg/kg), 62.9% (100 μg/kg), and 69.6% (200 μg/kg) compared to vehicle-dosed mice. The estimated ED50 was 46 μg/kg. Following infusion of FXaI16L (25 μg/kg) or vehicle into normal male CD-1 mice, we measured the ex vivo activity in plasma using an activated partial thromboplastin time (aPTT) clotting assay and a thrombin generation assay (TGA). Plasma collected from FXaI16L-dosed animals at 2 minutes post-injection displayed a 67% reduction in aPTT compared to vehicle-dosed mice. Dosing of FXaI16L at 25 μg/kg also enhanced thrombin generation, as reflected by a shortened lag phase, increased peak thrombin, increased endogenous thrombin potential and higher velocity index compared to vehicle treated mice. We also measured thromboelastography (TEG) parameters of whole blood collected from mice infused with FXaI16L. At a 10 μg/kg intravenous dose of FXaI16L, the TEG R-value and K-value measures of clotting time decreased, while TEG alpha angle and maximum amplitude increased compared to vehicle treated mice. We conclude that administration of FXaI16L in normal mice enhances hemostasis, decreasing bleeding in an injury model. Together, these studies suggest that FXaI16L may provide a new and unique way to achieve hemostasis in clinical situations of uncontrolled bleeding. Disclosures Reema: Pfizer: Employment. Patel-Hett:Pfizer: Employment. Camire:Pfizer: Consultancy, Patents & Royalties, Research Funding. Fruebis:Pfizer: Employment. Pittman:Pfizer: Employment.

2348Novel direct thrombin inhibitor achieves superior antithrombotic effect with lower bleeding risk than heparin or bivalirudin

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
C Y Koh ◽  
N Shih ◽  
E J E Leong ◽  
F S Amran ◽  
A W L Li ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Low Dose ◽  
Ex Vivo ◽  
Bleeding Risk ◽  
Direct Thrombin Inhibitor ◽  
Time Response ◽  
Waveform Analysis ◽  
Thrombin Inhibitor ◽  
Response Model ◽  
Antithrombotic Effect

Abstract Background We have isolated, variegin, a unique direct thrombin inhibitor (DTI) from tropical bont tick Amblyomma variegatum. Variegin inhibits thrombin active site and exosite-1 with an inhibitory constant of 0.3 nM (9-fold better than bivalirudin). It is also >5 orders of magnitude more selective for thrombin than other blood coagulation serine proteases. Variegin has a plasma half-life of 50 minutes (compared with bivalirudin 25 minutes and heparin ∼ hours). Purpose We aimed to develop variegin into a parenteral anticoagulant for percutaneous coronary intervention (PCI) and tested variegin in several pre-clinical models. Methods In rats, variegin was tested for efficacy (anticoagulation intensity) in a FeCl3-induced carotid artery thrombosis model while safety (bleeding risk) was tested in a tail incision model that recapitulated the time-frame of PCI (∼1 hour) in humans (time-response model). In pigs, an extracorporeal circuit with modified Badimon chambers containing coronary stents was used to assess efficacy, while bleeding risk was evaluated through needle-induced injury on a superficial ear vein, with or without concurrent administration of aspirin and ticagrelor (DAPT). Unfractionated heparin (UFH) and bivalirudin at dosages recommended for PCI were used as references. Ex vivo clotting analyses including thrombin generation test, rotational thromboelastometry, activated partial thromboplastin time and clot waveform analysis were performed in human blood spiked with DAPT and the three anticoagulants. Results In the rat time-response model, a single variegin bolus conferred better antithrombotic effect than a continuous infusion of bivalirudin and more rapid recovery of haemostasis than a single bolus of heparin. In the porcine ex vivo model, without DAPT, UFH, bivalirudin and 1 mg/kg variegin reduced stent thrombus by 35% (P<0.001), 60% (P<0.0001), and 80% (P<0.0001), compared with saline, respectively. In the presence of DAPT, UFH, bivalirudin and only 0.1 mg/kg of variegin (10-fold lowered dose) reduced stent thrombus by 65% (P<0.01), 75% (P<0.001), and 87% (P<0.0001), respectively (Fig. 1A). However, in the presence of DAPT, standard-dose UFH and bivalirudin prolonged bleeding times far longer than low-dose variegin (Fig. 1B). In human platelet rich plasma treated with DAPT, UFH showed a much more precipitous decline in thrombin generation potential than variegin (Fig. 1C). Dose response curves for inhibition of thrombin generation are also steeper in UFH and bivalirudin than in variegin, suggesting larger safety dose margin for variegin (Fig. 1D). These observations potentially account for the better preservation of haemostasis with low-dose variegin in combination with DAPT. Figure 1 Conclusion In the presence of aspirin and ticagrelor, a low dose of variegin, a novel direct thrombin inhibitor, achieved superior antithrombotic effect with significantly lower bleeding risk than heparin or bivalirudin in pre-clinical PCI models.

scholarly journals Specificity and selectivity profile of EP217609: a new neutralizable dual-action anticoagulant that targets thrombin and factor Xa

Blood ◽  
2012 ◽  
Vol 119 (10) ◽  
pp. 2187-2195 ◽  
Author(s):  
Steven T. Olson ◽  
Richard Swanson ◽  
Maurice Petitou
Keyword(s):  
Single Molecule ◽  
Factor Xa ◽  
Thrombin Inhibitors ◽  
Thrombin Inhibitor ◽  
Direct Thrombin Inhibitors ◽  
Dual Inhibitor ◽  
High Affinity ◽  
Dual Action ◽  
Rapid Kinetics ◽  
Linkage Effects

Abstract EP217609 is a new dual-action parenteral anticoagulant that combines an indirect factor Xa inhibitor (fondaparinux analog) and a direct thrombin inhibitor (α-NAPAP analog) in a single molecule together with a biotin tag to allow avidin neutralization. EP217609 exhibits an unprecedented pharmacologic profile in showing high bioavailability, long plasma half-life, and potent antithrombotic activity in animals without the complications of thrombin rebound. Here we report the exceptional specificity and selectivity profile of EP217609. EP217609 inhibited thrombin with rapid kinetics (kon > 107M−1s−1), a high affinity (KI = 30-40pM), and more than 1000-fold selectivity over other coagulation and fibrinolytic protease targets, comparing favorably with the best direct thrombin inhibitors known. EP217609 bound antithrombin with high affinity (KD = 30nM) and activated the serpin to rapidly (kass ∼ 106M−1s−1) and selectively (> 20-fold) inhibit factor Xa. The dual inhibitor moieties of EP217609 acted largely independently with only modest linkage effects of ligand occupancy of one inhibitor moiety on the potency of the other (∼ 5-fold). In contrast, avidin binding effectively neutralized the potency of both inhibitor moieties (20- to 100-fold). These findings demonstrate the superior anticoagulant efficacy and rapid avidin neutralizability of EP217609 compared with anticoagulants that target thrombin or factor Xa alone.

Inhibition of Thrombin Generation in Plasma by Inhibitors of Factor Xa

1997 ◽  
Vol 78 (04) ◽  
pp. 1215-1220 ◽  
Author(s):  
D Prasa ◽  
L Svendsen ◽  
J Stürzebecher
Keyword(s):  
Thrombin Generation ◽  
Factor Xa ◽  
Thrombin Inhibitors ◽  
Thrombin Inhibitor ◽  
Thrombin Generation Assay ◽  
Ic50 Values ◽  
Fxa Inhibitor ◽  
20 Nm ◽  
Tick Anticoagulant Peptide ◽  
Inhibition Of Thrombin

SummaryA series of inhibitors of factor Xa (FXa) were investigated using the thrombin generation assay to evaluate the potency and specificity needed to efficiently block thrombin generation in activated human plasma. By inhibiting FXa the generation of thrombin in plasma is delayed and decreased. Inhibitor concentrations which cause 50 percent inhibition of thrombin generation (IC50) correlate in principle with the Ki values for inhibition of free FXa. Recombinant tick anticoagulant peptide (r-TAP) is able to inhibit thrombin generation with considerably low IC50 values of 49 nM and 37 nM for extrinsic and intrinsic activation, respectively. However, the potent synthetic, low molecular weight inhibitors of FXa (Ki values of about 20 nM) are less effective in inhibiting the generation of thrombin with IC50 values at micromolar concentrations.The overall effect of inhibitors of FXa in the thrombin generation assay was compared to that of thrombin inhibitors. On the basis of similar Ki values for the inhibition of the respective enzyme, synthetic FXa inhibitors are less effective than thrombin inhibitors. In contrast, the highly potent FXa inhibitor r-TAP causes a stronger reduction of the thrombin activity in plasma than the most potent thrombin inhibitor hirudin.

Biochemical and Pharmacological Properties of SANORG 34006, a Potent and Long-Acting Synthetic Pentasaccharide

Blood ◽  
1998 ◽  
Vol 91 (11) ◽  
pp. 4197-4205 ◽  
Author(s):  
J.M. Herbert ◽  
J.P. Hérault ◽  
A. Bernat ◽  
R.G.M. van Amsterdam ◽  
J.C. Lormeau ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Factor Xa ◽  
Therapeutic Index ◽  
Inhibitory Effect ◽  
Experimental Models ◽  
Treatment And Prevention ◽  
Long Acting

Abstract SANORG 34006 is a new sulfated pentasaccharide obtained by chemical synthesis. It is an analog of the “synthetic pentasaccharide” (SR 90107/ ORG 31540) which represents the antithrombin (AT) binding site of heparin. SANORG 34006 showed a higher affinity to human AT than SR 90107/ORG 31540 (kd = 1.4 ± 0.3 v 48 ± 11 nmol/L), and it is a potent and selective catalyst of the inhibitory effect of AT on factor Xa (1,240 ± 15 anti–factor Xa U/mg v850 ± 27 anti-factor Xa U/mg for SR 90107/ORG 31540). In vitro, SANORG 34006 inhibited thrombin generation occurring via both the extrinsic and intrinsic pathway. After intravenous (IV) or subcutaneous (SC) administration to rabbits, SANORG 34006 displayed a long-lasting anti–factor Xa activity and inhibition of thrombin generation (TG) ex vivo. SANORG 34006 was slowly eliminated after IV or SC administration to rats, rabbits, and baboons, showed exceptionally long half-lives (between 9.2 hours in rats and 61.9 hours in baboons), and revealed an SC bioavailability near 100%. SANORG 34006 displayed antithrombotic activity by virtue of its potentiation of the anti–factor Xa activity of AT. It strongly inhibited thrombus formation in experimental models of thromboplastin/stasis-induced venous thrombosis in rats (IV) and rabbits (SC) (ED50values = 40.0 ± 3.4 and 105.0 ± 9.4 nmol/kg, respectively). The duration of its antithrombotic effects closely paralleled the ex vivo anti–factor Xa activity. SANORG 34006 enhanced rt-PA–induced thrombolysis and inhibited accretion of125I-fibrinogen onto a preformed thrombus in the rabbit jugular vein suggesting that concomitant use of SANORG 34006 during rt-PA therapy might be helpful in facilitating thrombolysis and preventing fibrin accretion onto the thrombus under lysis. Contrary to standard heparin, SANORG 34006 did not enhance bleeding in a rabbit ear incision model at a dose that equals 10 times the antithrombotic ED50 in this species and, therefore, exhibited a favorable therapeutic index. We suggest that SANORG 34006 is a promising compound in the treatment and prevention of various thrombotic diseases.

Effects of Tannins fromGeumjaponicumon the Catalytic Activity of Thrombin and Factor Xa of Blood Coagulation Cascade

1998 ◽  
Vol 61 (11) ◽  
pp. 1356-1360 ◽  
Author(s):  
Hui Dong ◽  
Shao-Xing Chen ◽  
R. Manjunatha Kini ◽  
Hong-Xi Xu
Keyword(s):  
Catalytic Activity ◽  
Blood Coagulation ◽  
Factor Xa ◽  
Coagulation Cascade

scholarly journals Role of the blood coagulation cascade in hepatic fibrosis

2018 ◽  
Vol 315 (2) ◽  
pp. G171-G176 ◽  
Author(s):  
Asmita Pant ◽  
Anna K. Kopec ◽  
James P. Luyendyk
Keyword(s):  
Blood Coagulation ◽  
Hepatic Fibrosis ◽  
Animal Studies ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Primary Source ◽  
Bleeding Risk ◽  
Normal Function ◽  
Coagulation Cascade ◽  
Rapid Progression

Liver is the primary source of numerous proteins that are critical for normal function of the blood coagulation cascade. Because of this, diseases of the liver, particularly when affiliated with severe complications like cirrhosis, are associated with abnormalities of blood clotting. Although conventional interpretation has inferred cirrhosis as a disorder of uniform bleeding risk, it is now increasingly appreciated as a disease wherein the coagulation cascade is precariously rebalanced. Moreover, prothrombotic risk factors are also associated with a more rapid progression of fibrosis in humans, suggesting that coagulation proteases participate in disease pathogenesis. Indeed, strong evidence drawn from experimental animal studies indicates that components of the coagulation cascade, particularly coagulation factor Xa and thrombin, drive profibrogenic events, leading to hepatic fibrosis. Here, we concisely review the evidence supporting a pathologic role for coagulation in the development of liver fibrosis and the potential mechanisms involved. Further, we highlight how studies in experimental animals may shed light on emerging clinical evidence, suggesting that beneficial effects of anticoagulation could extend beyond preventing thrombotic complications to include reducing pathologies like fibrosis.

Animal Models of Thrombosis Help Predict the Human Therapeutic Concentration of PRT54021, a Potent Oral Factor Xa Inhibitor.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 901-901 ◽  
Author(s):  
Keith Abe ◽  
Gail Siu ◽  
Susan Edwards ◽  
Pei Hua Lin ◽  
Bing Yan Zhu ◽  
...  
Keyword(s):  
Animal Models ◽  
Venous Thrombosis ◽  
Thrombin Generation ◽  
Ex Vivo ◽  
Vena Cava ◽  
Factor Xa ◽  
Fold Change ◽  
In Vivo Models ◽  
Antithrombotic Activity ◽  
Fxa Inhibitor

Abstract Factor Xa (fXa) inhibition has resulted in the emergence of a new class of antithrombotics. Pharmacodynamic monitoring of these agents has proven problematic. The present study was designed to determine the target concentration of an oral fXa inhibitor required for clinical trials using both thrombin generation assays and three in vivo models and determine whether clotting assays such as activated partial thromboplastin time (aPTT) and prothrombin time (PT) would be suitable for monitoring human dosing. PRT54021 (PRT021) is a potent inhibitor of human fXa (Ki=117pM). PRT021 and fondaparinux, an indirect fXa inhibitor, both significantly inhibited TAT and F1.2 generation in human whole blood. Compared to a therapeutic level of fondaparinux (200nM), PRT021 (200nM) was more potent in suppressing both markers. Multiple doses of PRT021 were evaluated in three animal models. The first model, which measured clot accretion on cotton threads placed in rabbit abdominal vena cava, compared inhibition of thrombus mass by PRT021 to that of supratherapeutic doses of enoxaparin (a LMW heparin). The second model compared the ability of PRT021 to maintain vessel patency under arterial flow conditions in FeCl3 induced thrombosis in rat carotid artery to that achieved by enoxaparin or clopidogrel (an antiplatelet agent). The third model investigated inhibition of 111In labeled platelet deposition on dacron grafts and expansion chambers placed in femoral arteriovenous shunts in baboons. PRT021 and enoxaparin were administered as IV infusions and clopidogrel was dosed orally for three days. Ex vivo PT and aPTT were measured in all models. The models encompass stringent criteria of arterial and venous thrombosis and PRT021 produced dose-responsive antithrombotic activity in each of the three models. The efficacy of PRT021 compared favorably to supratherapeutic levels of enoxaparin and clopidogrel. Unlike in the rodent models, efficacy in primates was attained at a much lower dose with minimal prolongation of PT. Species specificity was also demonstrated by in vitro extensions of PT and aPTT in rat, rabbit, baboon and human plasma. A 2X change of PT was attained at concentrations of 8.9, 1.6, 1 and 0.4μM respectively. The data indicate that doses of PRT021 that inhibit thrombin generation in human blood and that provide anticoagulation similar to baboon dosed at 0.49mg/kg may be sufficient to prevent venous thrombosis in humans. Comparative modeling of extents of change in PT to levels of antithrombotic efficacy also leads us to predict that human therapeutic activity for PRT021 may be attained without concurrent changes in ex vivo clotting parameters. The targeted concentration is currently being tested in Phase II trials for its ability to prevent venous thromboembolism in orthopedic surgery patients. Model of Thrombosis Agent, Dose Antithrombotic Activity aPTT fold change PT fold change Rabbit vena cava PRT021,3mg/kg 76% inhibition 2.22 2.34 Rabbit vena cava Enoxaparin, 1.6mg/kg 96% inhibition 2.06 2.01 Rat carotid PRT021,19.1mg/kg 90% patency 1.69 2.20 Rat carotid Enoxaparin, 7.6mg/kg 70% patency 3.49 1.19 Rat carotid Clopidogrel, 3mg/kg/day 80% patency 1.03 1.01 Baboon arteriovenous PRT021,0.49mg/kg 90% inhibition (venous), 32% inhibition (arterial) 1.29 1.17

Differential effects of TAK-442, a novel orally active direct factor Xa inhibitor, and ximelagatran, a thrombin inhibitor, on factor V-mediated feedback on coagulation cascade and bleeding

2010 ◽  
Vol 104 (09) ◽  
pp. 504-513 ◽  
Author(s):  
Noriko Konishi ◽  
Katsuhiko Hiroe ◽  
Masaki Kawamura
Keyword(s):  
Venous Thrombosis ◽  
Positive Feedback ◽  
Response Curve ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Therapeutic Window ◽  
Thrombin Inhibitor ◽  
Factor V ◽  
Differential Effects ◽  
Concentration Response Curve

SummaryThrombin amplifies the blood coagulation via factor V (FV)-mediated positive feedback loop. We hypothesised that factor Xa (FXa) inhibitors would interfere more gradually with this feedback activation loop than thrombin inhibitors, thereby achieving a better balance between haemostasis and prevention of thrombosis. In this study, we compared the effects of TAK-442, a novel FXa inhibitor, versus ximelagatran, a thrombin inhibitor, on FV-mediated positive feedback, venous thrombosis and bleeding. In normal plasma, TAK-442 delayed the onset of tissue factor-induced thrombin generation and prolonged prothrombin time (PT) with more gradual concentration-response curve than melagatran, the active form of ximelagatran. The effect of melagatran on the onset of thrombin generation decreased in an FVa-concentration-dependent manner in FV-deficient plasma supplemented with FVa. Furthermore, in FV-deficient plasma, the PT-prolonging potency of melagatran was markedly increased with a change in its concentration-response curve from steep to gradual. In the rat venous thrombosis model, TAK-442 (10 mg/kg, p.o.) prevented thrombus formation by 55% with 1.2 times prolongation of PT; a similar effect was observed in ximelagatran-treated (3 mg/kg, p.o.) rats. TAK-442 at 100 mg/kg prolonged PT by only 2.1 times with no change in bleeding time (BT), whereas ximelagatran at 10 mg/kg prolonged PT by 3.9 times and significantly increased BT. These results suggest that the differential effects of the two agents on FV-mediated amplification of thrombin generation may underlie the observation of a wider therapeutic window for TAK-442 than for ximelagatran.

scholarly journals Highly Procoagulant Extracellular Vesicles in Amniotic Fluid

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4961-4961
Author(s):  
Johannes Thaler ◽  
Lena Hell ◽  
Lukas Wisgrill ◽  
Andreas Spittler ◽  
Michael Schwameis ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Amniotic Fluid ◽  
Blood Coagulation ◽  
Extracellular Vesicles ◽  
Whole Blood ◽  
Blood Clotting ◽  
Factor Xa ◽  
Coagulation Cascade ◽  
Contact Activation ◽  
Plasma Factor

Abstract Background: The pathomechanisms underlying disseminated intravascular coagulation (DIC) following amniotic fluid (AF) embolism remain to be fully elucidated. Highly procoagulant phosphatidylserine (PS)- and tissue factor (TF) expressing extracellular vesicles (EVs) might play a central role. Objective: To perform extensive analyses of the procoagulant properties of AF with a panel of functional coagulation assays and flow cytometry to investigate the pathogenesis of AF induced DIC. Methods: A prothrombinase assay, an EV-TF dependent factor Xa (FXa) generation assay, a modified thrombin- and fibrin-generation assay, a whole blood clotting model and flow cytometry were applied in AF- and control plasma. Results: Phosphatidylserine expression was 21-fold increased in AF compared to plasma. Factor Xa generation was extremely high when TF-expressing EVs from AF were co-incubated with recombinant FVIIa. In the thrombin- and fibrin generation assay AF-derived EVs strongly activated the blood coagulation cascade via PS and TF. In a whole blood clotting model AF-derived TF-expressing EVs significantly shortened the clotting time from 734 ± 139 seconds in the presence- to 232 ± 139 seconds in the absence of an anti-TF antibody. The contact activation pathway via factor FXII was not affected. Applying flow cytometry, a sub-population of PS- and TF co-expressing EVs was clearly identified in AF. Conclusions: We thoroughly investigated the effect of AF on blood coagulation and found that PS+ and TF+ EVs determine its procoagulant potential. Taken together our data further delineate the pathomechanisms underlying AF-induced coagulopathy, which could improve diagnostic- and treatment modalities. Disclosures No relevant conflicts of interest to declare.

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