Comparative anticoagulant activity and influence on thrombin generation of dextran derivatives and of a fucoidan fraction

Factor V (FV) assembles with factor Xa (FXa) into prothrombinase, the enzymatic complex that converts prothrombin to thrombin. Tissue factor pathway inhibitor α (TFPIα) inhibits prothrombinase by high affinity interactions with FXa-activated FV and the FXa active site, thereby blocking the initiation of coagulation. FV Leiden (FVL) is strongly linked to venous thrombosis through its resistance to degradation by activated protein C (aPC), which enhances the propagation of coagulation. FVL combined with a 50% reduction in TFPI causes severe thrombosis and perinatal lethality in mice, suggesting that FVL also promotes the initiation of coagulation. To examine this possibility, thrombin generation assays initiated with limiting FXa were performed with control or FVL plasma and platelet-rich plasma (PRP). The activation threshold for thrombin generation was 10 to 20 pM FXa in 10 control plasmas, but was 5 pM in 4 of 10 homozygous FVL plasmas. FVL PRP had a similar decrease in the activation threshold. The differences in activation threshold were totally normalized by an anti-TFPI antibody, while exogenous TFPIα and a FV-binding peptide that mimics TFPIα had reduced anticoagulant activity in FVL plasma, revealing that the procoagulant effects of FVL in these assays rely on TFPIα. Next, FVL plasmas were studied in fibrin clot formation assays, as they are sensitive to small amounts of thrombin. In reactions activated with 0.5 pM FXa, 1 of 8 control plasmas, compared to 7 of 8 homozygous FVL plasmas, clotted within 60 minutes, with differences again normalized by the anti-TFPI antibody. In prothrombinase activity assays using purified proteins, TFPIα was a 1.7-fold weaker inhibitor of prothrombinase assembled with FVL compared to FV. Thus, in addition to its aPC-mediated effect on the propagation of coagulation, FVL is resistant to TFPIα inhibition, exerting a procoagulant effect on coagulation initiation. This is evident in responses to small stimuli, where TFPIα blocks clotting in plasmas with FV but not FVL. The TFPIα-mediated modulation of the procoagulant threshold may explain the severe perinatal thrombosis in FVL mice with decreased TFPI and be clinically relevant in the clotting associated with oral contraceptives, which cause acquired TFPI deficiency.

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Thrombin generation test with the calibrated automated thrombogram and anticoagulant activity of Mentha crispa

Blood Coagulation & Fibrinolysis ◽

10.1097/mbc.0000000000000859 ◽

2020 ◽

Vol 31 (1) ◽

pp. 101-106

Author(s):

Paula M. Leite ◽

Ana P.N. Miranda ◽

Juliana M. Amorim ◽

Rita C.F. Duarte ◽

André A.G. Faraco ◽

...

Keyword(s):

Thrombin Generation ◽

Anticoagulant Activity ◽

Mentha Crispa ◽

Generation Test

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Evidence that the Protein C Activation Pathway Amplifies the lnhibition of Thrombin Generation by Recombinant Human Thrombomodulin in Plasma

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649598 ◽

1993 ◽

Vol 70 (03) ◽

pp. 423-426 ◽

Cited By ~ 16

Author(s):

Rika ohishi ◽

Naoko watanabe ◽

Masaharu Aritomi ◽

Komakazu Gomi ◽

Takao Kiyota ◽

...

Keyword(s):

Protein C ◽

Inhibitory Action ◽

Thrombin Generation ◽

Anticoagulant Activity ◽

Activated Protein C ◽

Inhibitory Effect ◽

Phospholipid Vesicles ◽

Activation Pathway ◽

Activated Platelets ◽

Rapid Generation

SummaryThrombomodulin (TM) is a cofactor for the thrombin-catalyzed activation of anticoagulant protein C. However, we have no evidence that thrombomodulin actually activates protein C during blood coagulation processing, nor do we know whether this activated protein C acts as an anticoagulant. We studied the inhibitory action of recombinant human soluble TM (rhs-TM) on thrombin generation in whole plasma. Human plasma was activated with small amounts of tissue factor using phospholipid vesicles in place of activated platelets. Thrombin generation was observed. The addition of only 2 nM of rhs-TM prevented rapid generation of thrombin and reduced the total amount of thrombin generated. In order to study the influence of the protein C activation pathway on this inhibitory action of rhs-TM, protein C-depleted plasma was used. rhs-TM had little inhibitory effect on protein C-depleted plasma. However, the addition of protein C caused a delay in thrombin generation and a reduction of the maximum thrombin concentration. We concluded that the anticoagulant activity of rhs-TM was amplified by the protein C activation pathway.

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In vitro effects of human neutrophil cathepsin G on thrombin generation: Both acceleration and decreased potential

Thrombosis and Haemostasis ◽

10.1160/th09-10-0690 ◽

2010 ◽

Vol 104 (09) ◽

pp. 514-522 ◽

Cited By ~ 3

Author(s):

Thomas Lecompte ◽

Agnès Tournier ◽

Lise Morlon ◽

Monique Marchand-Arvier ◽

Claude Vigneron ◽

...

Keyword(s):

Tissue Factor ◽

Thrombin Generation ◽

Time Course ◽

Anticoagulant Activity ◽

Cathepsin G ◽

Coagulation Cascade ◽

Clotting Factor ◽

Factor V ◽

Clotting Factors

SummaryCathepsin G (Cath G), a serine-protease found in neutrophils, has been reported to have effects that could either facilitate or impede coagulation. Thrombin generation (CAT method) was chosen to study its overall effect on the process, at a plasma concentration (240 nM) observed after neutrophil activation. Coagulation was triggered by tissue factor in the presence of platelets or phospholipid vesicles. To help identify potential targets of Cath G, plasma depleted of clotting factors or of inhibitors was used. Cath G induced a puzzling combination of two diverging effects of varying intensities depending on the phospholipid surface provided: accelerating the process under the three conditions (shortened clotting time by up to 30%), and impeding the process during the same thrombin generation time-course since thrombin peak and ETP (total thrombin potential) were decreased, up to 45% and 12%, respectively, suggestive of deficient prothrombinase. This is consistent with Cath G working on at least two targets in the coagulation cascade. Our data indicate that coagulation acceleration can be attributed neither to platelet activation and nor to activation of a clotting factor. When TFPI (tissue factor pathway inhibitor) was absent, no effect on lag time was observed and the anticoagulant activity of TFPI was decreased in the presence of Cath G. Consistent with the literature and the hypothesis of deficient prothrombinase, experiments using Russel’s Viper Venom indicate that the anticoagulant effect can be attributed to a deleterious effect on factor V. The clinical relevance of these findings deserves to be studied.

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Comparative Anticoagulant and Thrombin Generation Inhibitory Profile of Heparin, Sulodexide and Its Components

Clinical and Applied Thrombosis/Hemostasis ◽

10.1177/1076029620954913 ◽

2020 ◽

Vol 26 ◽

pp. 107602962095491

Author(s):

Fakiha Siddiqui ◽

Debra Hoppensteadt ◽

Emily Bontekoe ◽

Ambar Farooqui ◽

Walter Jeske ◽

...

Keyword(s):

Thrombin Generation ◽

Dermatan Sulfate ◽

Medical Center ◽

Anticoagulant Activity ◽

Pharmaceutical Ingredients ◽

Thrombin Generation Assay ◽

Venous Diseases ◽

Individual Determination ◽

Normal Human ◽

The Individual

Introduction: Sulodexide represents a mixture of fast-moving heparin (FMH) and dermatan sulfate (DS) and has been used for the management of venous diseases such as DVT and related disorders. The purpose of this study is to compare sulodexide and its components with unfractionated heparin (UFH) to determine its suitability for the indications in which UFH is used. Materials and Method: Active pharmaceutical ingredients (API) versions of sulodexide, FMH and DS were obtained from Alfasigma. API versions of UFH were obtained from Medefil Inc. Normal human citrated plasma was obtained from blood bank of the Loyola University Medical Center. Each of the individual agents were supplemented in plasma at a graded concentration of 0.0-10 µg/mL. Clotting assays (PiCT, aPTT, PT and TT), anti-Xa and anti-IIa and thrombin generation studies were carried out. Results were compiled as mean ± SD of 3 individual determination. Result: In the clot based (PiCT, aPTT and TT), anti-Xa and IIa assays, both the UFH and FMH produced stronger activities in these assays followed by sulodexide. DS did not show any anticoagulant activity. In the thrombin generation assay, FMH and UFH produced comparable inhibition of thrombin generation as measured by various parameters. Sulodexide was slightly weaker in this assay, whereas DS produced relatively weaker effects. Conclusion: In comparison to sulodexide, both UFH and FMH exhibit comparable anticoagulant activity despite differences in their molecular weight. These results suggest that sulodexide can be developed as a parenteral anticoagulant for indications in which UFH is used.

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The Effect of BIBT 986, a Novel Small Molecule Dual Inhibitor of Thrombin and Factor Xa, on Coagulation in a Model of Endotoxin Induced, Tissue Factor Triggered Coagulation Activation in Humans.

Blood ◽

10.1182/blood.v108.11.912.912 ◽

2006 ◽

Vol 108 (11) ◽

pp. 912-912 ◽

Cited By ~ 2

Author(s):

Bernd Jilma ◽

Judith M. Leitner ◽

Francesco Cardona ◽

Florian B. Mayr ◽

Christa Firbas ◽

...

Keyword(s):

Tissue Factor ◽

Platelet Activation ◽

Thrombin Generation ◽

Anticoagulant Activity ◽

Thrombin Time ◽

Coagulation Activation ◽

Dual Inhibitor ◽

Activation Markers ◽

Markers Of Inflammation ◽

Prolonged Aptt

Abstract Background: BIBT 986 is a novel potent anticoagulant that dually inhibits Factors Xa and IIa. We hypothesized that BIBT 986 would dose-dependently decrease endotoxin-induced, tissue factor triggered coagulation activation. Hence it was the aim of the study to compare with placebo the anticoagulant activity of three dosages of BIBT 986 on parameters of coagulation, platelet activation and inflammation and to examine the safety of BIBT 986 in this setting. Methods: This study was a prospective, randomized, double-blind, placebo-controlled, parallel-group dose escalation trial in 48 healthy male volunteers. Participants were randomised to receive bolus primed continuous infusions of one of the three doses of BIBT 986 or placebo. All of them received a bolus infusion of 2ng/kg body weight lipopolysaccharide (LPS). Results: BIBT dose-dependently increased anti-Xa activity, activated partial thromboplastin time (APTT), ecarin clotting time (ECT), thrombin time (TT) and the international normalisation ratio (INR). Importantly, BIBT 986 dose-dependently blocked the LPS-induced coagulation as assessed by the in vivo markers of thrombin generation and action: BIBT 986 doses that prolonged APTT by 25% were already effective. The BIBT dose that prolonged APTT by 100%, completely suppressed the increase in prothrombin fragment (F1+2), thrombin-antithrombin complexes (TAT) and D-dimer. BIBT 986 had no influence on activation markers of inflammation, fibrinolysis, endothelial or platelet activation. Conclusion: Infusion of BIBT 986 was safe and well tolerated. BIBT 986 specifically and dose-dependently blocked LPS-induced, tissue factor trigger coagulation. When compared to different anticoagulants tested previously in this standardized model, BIBT 986 was more effective in suppressing thrombin generation (F1+2 levels) than standard doses of danaparoid, dalteparin or lepirudin. BIBT 986 represents the first drug of a new class of dual FXa and FIIa inhibitors, and displays high potency.

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Low Concentrations of rhFVIIa or FEIBA Significantly and Rapidly Reverse the Anticoagulant Effects of Supratherapeutic Edoxaban

Blood ◽

10.1182/blood.v118.21.1252.1252 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1252-1252 ◽

Cited By ~ 4

Author(s):

Abdel-Baset Halim ◽

Yan Li ◽

Evan Stein ◽

Jeanne Mendell

Keyword(s):

Human Plasma ◽

Research Funding ◽

Thrombin Generation ◽

Time Course ◽

Anticoagulant Activity ◽

Plasma Concentrations ◽

Reversal Agents ◽

Plasma Fraction ◽

D Dimer ◽

Control Samples

Abstract Abstract 1252 Introduction: Edoxaban is an oral, direct factor Xa (FXa) inhibitor currently in phase 3 clinical development for stroke prevention in nonvalvular atrial fibrillation (AF) and the treatment and secondary prevention of venous thromboembolism (VTE). Guidance for the emergency management of serious bleeding and strategies for reversing the anticoagulant effects edoxaban are needed. Both recombinant human FVIIa (rhFVIIa; NovoSeven®) and a human plasma fraction containing nonactivated forms of FII, FIX, and FX/proteins C and S and activated FVII (FEIBA; factor eight inhibitor bypassing activity) have been identified as potential reversal agents for edoxaban. The minimum effective doses and the time course required for either rhFVIIa or FEIBA to reverse supratherapeutic concentrations of edoxaban in an anticoagulated patient have yet to be established. This ex vivo study was conducted to assess the effects and establish the time course of various concentrations of rhFVIIa and FEIBA on the reversal of edoxaban's anticoagulant effects. Methods: Edoxaban at supratherapeutic concentrations of 500 and 1000 ng/mL (therapeutic level is ∼250 ng/mL for Cmax of a 60-mg dose) was added to fresh blood drawn from healthy subjects; control samples were diluent alone. After 60 min of incubation, rhFVIIa, FEIBA, or control was added to the samples. rhFVIIa was added to produce final concentrations of 0.8 and 1.8 μg/mL, which correspond to known maximal observed human plasma concentrations at doses of 40 and 90 μg/kg. FEIBA was added to produce concentrations of 0.75 and 1.4 U/mL, which correspond to human plasma concentrations at the therapeutic doses of FEIBA of 50 to 100 U/kg. In order to determine reversal of anticoagulant effects over time, coagulation assays (PT, aPTT, anti-Xa, intrinsic FXa activity, thrombin generation assay [TGA], D-dimer, and FVIIa activity) were measured at times 0, 0.25, 0.50, 1, 2, and 4 h after incubation with rhFVIIa, FEIBA, or control. Results: In edoxaban alone and control samples, PT, aPTT, and anti-Xa results reflected previously established anticoagulant activity. Both 500 and 1000 ng/mL of edoxaban completely inhibited intrinsic FXa and thrombin generation as measured by TGA (peak). Measures of FVIIa activity indicated that the concentrations of rhFVIIa utilized were consistent with previously reported responses. With the exception of thrombin levels, reversal of edoxaban anticoagulant activity by both rhFVIIa and FEIBA was observed for PT, aPTT, and anti-Xa beginning at 0.25 h and was maintained across the experimental period. At 1000 ng/mL edoxaban, the observed decrease at 0.25 h post-reversal of anticoagulant activity was 82% for anti-Xa, 72% for PT, and 58% for aPTT. Compared with baseline, the maximum reversal of intrinsic FXa activity for edoxaban 500 ng/mL was ∼30% and for 1000 ng/mL was 15% across all concentrations of rhFVIIa and FEIBA. The TGA assay indicated both rhFVIIa and FEIBA reversed ∼45% and 20% of the effect of edoxaban at 500 ng/mL and 1000 ng/mL, respectively, 4 h after adding the reversal agents. With the exception of 1 baseline sample, levels of D-dimer did not show significant changes with the addition of edoxaban or with its subsequent reversal by either rhFVIIa or FEIBA. Conclusion: Low therapeutic concentrations of rhFVIIa and FEIBA showed significant and rapid reversal of supratherapeutic concentrations of the anticoagulant activity induced by edoxaban based on PT, aPTT, and anti-Xa activity. Disclosures: Halim: Daiichi Sankyo Pharma Development: Employment. Li:Daiichi Sankyo Pharma Development: Employment. Stein:Daiichi Sankyo: Consultancy, Research Funding; AACC: Consultancy, Honoraria, Research Funding; Abbott: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding; FDA: Consultancy, Honoraria, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding; ISIS: Consultancy, Honoraria, Research Funding; Merck & Co: Consultancy, Honoraria, Research Funding; National Lipid Association: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Reliant: Consultancy, Honoraria, Research Funding; Regeneron: Consultancy, Honoraria, Research Funding; SanofiAventis: Consultancy, Honoraria, Research Funding; Schering-Plough: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Wyeth: Consultancy, Honoraria, Research Funding. Mendell:Daiichi Sankyo Pharma Development: Employment.

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Inhibition of Platelet-Prothrombianse by a Covalent Antithrombin-Heparin Complex.

Blood ◽

10.1182/blood.v120.21.2212.2212 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2212-2212

Author(s):

Ivan Stevic ◽

Howard H.W. Chan ◽

Ankush Chander ◽

Leslie R. Berry ◽

Anthony K.C. Chan

Keyword(s):

Thrombin Generation ◽

Conflicts Of Interest ◽

Anticoagulant Activity ◽

Calcium Ionophore ◽

Thrombin Inhibitor ◽

Ionophore A23187 ◽

Specific Substrate ◽

Prothrombinase Complex ◽

Activated Platelets

Abstract Abstract 2212 Introduction: Factor Xa is protected within the prothrombinase complex from inhibition by heparin. We have developed a covalent antithrombin-heparin complex (ATH) with enhanced anticoagulant activity. Previously, we have shown that ATH is able to inhibit coagulation enzymes much more efficiently than regular antithrombin+heparin (AT+UFH). For example, ATH inhibited TF/VIIa ∼30-fold faster compared to AT+UFH. Furthermore, we have also demonstrated that ATH is capable of inhibiting Xa within a prothrombinase complex assembled on synthetic phospholipid vesicles better than AT+UFH. However, ATH's effect on prothrombinase when the complex is formed on a more native system such as platelets has never been explored. Thus, the objective of the present study is to determine the ability of ATH vs AT+UFH to inhibit Xa within the prothrombinase complex when the enzyme complex is assembled on the platelet system. Methods: Discontinuous second order rate constant assays were performed to obtain k2-values for inhibition of free or prothrombinase-bound Xa by AT+UFH or ATH. Freshly prepared resting platelets were subjected to inhibition analysis by first incubating them with Xa, Va, Ca2+ and pefabloc®-TH (thrombin inhibitor) in different wells of a 96-well plate for 3 min at 37 °C. Prothrombin was then simultaneously added to all wells to initiate thrombin generation, followed by addition of AT+UFH or ATH inhibitors to each well at specific time intervals. Reactions were neutralized by simultaneous addition of polybrene, Na2EDTA and Xa-specific substrate S-2222™ in buffer. The remaining Xa enzyme activity was obtained and final k2-values calculated. For experiments requiring activated platelets, freshly isolated platelets were activated with 5 μM calcium ionophore A23187 + 4 mM CaCl2 for 15 min at room temperature. The activated platelets were then tested in inhibition assays as described above. To investigate the roles of individual components of the prothrombinase complex on the anticoagulant effects of AT+UFH and ATH, additional experiments were performed where components of the complex (prothrombin, activated platelets or Va) were omitted prior to reaction with inhibitors. Thrombin generation was used to assess functionality of the activated platelet-prothrombinase system in the presence of inhibitors using a thrombin-specific substrate S-2238™. Results: The k2-values (×108M−1min−1) for inhibition of free Xa or resting platelet-prothrombinase were similar for both inhibitors, although the overall inhibition rates achieved by the ATH were 2-fold faster than AT+UFH (p<0.001). Since activated platelets are required for enhanced prothrombinase function, we then compared inhibition of free vs activated platelet-prothrombinase by the two inhibitors (platelet activation was confirmed with flow cytometry using an anti CD-41 antibody). No differences were observed in the k2-values between free Xa (3.96±0.23) and activated platelet prothrombinase (3.83±0.39) for ATH reactions. However, the k2-values for inhibition of free Xa by AT+UFH was 2.37±0.32, and assembly of Xa within the activated platelet-prothrombinase resulted in a reduction in the k2-values to 0.99±0.22 (p<0.001), thus confirming a moderate 60% protection of Xa by the prothrombinase components. However, omitting the components (prothrombin, activated platelets or Va) from the complex resulted in higher k2-values (1.76±0.37, 2.29±0.26 and 2.52±0.32, respectively p<0.01) for AT+UFH, and as expected, no net effect was observed for ATH. Thrombin generation was inhibited significantly by both AT+UFH and ATH compared to the control (p<0.001), but further analysis of thrombin potential yielded greater inhibition by ATH compared to AT+UFH (p<0.05). Conclusion: In this study, we report inhibition of the prothrombinase complex on the surface of resting and activated platelets. Consistent with previous investigations, a moderate protection of Xa was observed when the activated platelet-prothrombinase was inhibited by AT+UFH. ATH on the other hand, targets and inhibits prothrombinase complexed-Xa as fast as free Xa, and at inhibition rates that were significantly faster than AT+UFH. Thus, overall the covalent conjugate enhances anticoagulation of surface-bound enzymes and offers advantages over conventional heparin for the treatment of cell-based coagulation in vivo. Disclosures: No relevant conflicts of interest to declare.

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Coagulation and fibrinolytic activities in 2 siblings with β2-glycoprotein I deficiency

Blood ◽

10.1182/blood.v96.4.1594.h8001594_1594_1595 ◽

2000 ◽

Vol 96 (4) ◽

pp. 1594-1595

Author(s):

Rie Takeuchi ◽

Tatsuya Atsumi ◽

Masahiro Ieko ◽

Hiroyuki Takeya ◽

Shinsuke Yasuda ◽

...

Keyword(s):

Protein C ◽

Thrombin Generation ◽

Anticoagulant Activity ◽

Activated Protein C ◽

Healthy Woman ◽

Contact Activation ◽

Glycoprotein I ◽

Normal Ranges

β2-Glycoprotein I (β2GPI) is a major antigen for antiphospholipid antibodies, and its multiple in vitro functions have been reported. This glycoprotein not only down-regulates thrombin formation by inhibiting contact activation or prothrombinase activity, but also up-regulates coagulation by reducing protein C anticoagulant activity. However, the in vivo roles of β2GPI remain obscure. Coagulation and fibrinolytic characteristics were investigated in individuals with β2GPI deficiency. An apparently healthy woman and her brother are homozygotes for β2GPI deficiency. In these patients, Russell viper venom time was shortened (40.4 seconds; normal range, 47.8 ± 4.95 seconds), but all markers of thrombin generation and fibrin turnover were within normal ranges. Exogenous activated protein C adequately prolonged the clotting time of the β2GPI-deficient plasma, and euglobulin lysis time was also normal. Thus, elevated thrombin generation, enhancement of activated protein C response, and an altered fibrinolytic system were not found in congenitally β2GPI-deficient plasma.

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