Recombinant Thrombomodulin Inhibits Tissue Factor Mediated Thrombin Generation in Blood Plasma and Is Modulated By Prothrombin Complex Concentrates

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2303-2303
Author(s):  
Jawed Fareed ◽  
Debra Hoppensteadt ◽  
Daneyal Syed ◽  
Daniel Kahn ◽  
Michael Mosier ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Anticoagulant Effect ◽  
Dependent Manner ◽  
Prothrombin Complex Concentrates ◽  
Complex Disorders ◽  
Fibrin Formation ◽  
Recombinant Thrombomodulin ◽  
Inhibition Of Thrombin

Abstract Introduction: Thrombomodulin plays an important role in the regulation of serine proteases and endothelial function. Because of its multiple modulatory action it has a central role in inhibiting thrombogenesis and inflammatory processes in such complex disorders as sepsis associated coagulopathies. A recombinant thrombomodulin (Recomodulin, Asahai Kasei, Tokyo, Japan) is currently used for the management of disseminated intravascular coagulation (DIC) and related disorders in Japan and is currently undergoing a global phase 3 trial in sepsis-induced coagulopathy. Recomodulin (RM) is capable of forming complexes with circulating thrombin generated in sepsis and not only inhibits its coagulant function but also inhibits thrombin generation. The purpose of this study is to determine the effect of RM on thrombin generation in plasma systems and its modulation by both activated and non-activated prothrombin complex concentrates (PCCs). PCCs may have the potential to overcome the anticoagulant effects of RM and may be useful as an antidote. Materials and Methods: RM 12,800 IU (2.02 mg) ampules were commercially obtained. Working solutions of buffered RM were prepared at 100 µg/ml. Tissue factor mediated generation of thrombin and its inhibition in normal human plasma (NHP) (n=50), platelet rich plasma (PRP) (n=50), plasma obtained from patients with DIC (n=25) and various PCCs such as Beriplex, Cofact, Feiba, Konyne, Octaplex, Preconativ, Profilnine, Prothromplex at 1 - 5 IU/ml were investigated. A fluorometric thrombokinetics method (Technoclone, Vienna, Austria) was used to determine thrombin generation and its modulation by RM. In addition such thrombin generation markers as prothrombin fragment (F1.2) and thrombin-antithrombin complex (TAT) were measured. The effect of RM on thrombin mediated fibrinokinetics was also measured using an optical method. Results: RM produced a concentration dependent inhibition of thrombin generation in the plasma based systems. In NHP the IC50 was 0.29±0.06 µg/ml in contrast to PRP where the IC50 was 1.99±0.31 µg/ml. The inhibition of thrombin generation in various PCCs was also concentration and product dependent and only four factor concentrates were found to generate thrombin.. At concentrations of 1 IU/ml marked inhibition of thrombin generation was noted in Preconativ, Prothromplex and Beriplex. RM decreased the generation of F1.2 and TAT in a concentration dependent manner. However, at concentrations >2.5 IU/ml the thrombin generated in such PCCs as Prothromplex and Beriplex, markedly higher thrombin was generated overcoming the anticoagulant effect of RM. Proportionately higher levels of thrombin generation markers were formed and were dependant on PCC concentrations. RM also inhibited the thrombin induced formation of fibrin in both PRP and PPP systems in a differential manner. However, at higher concentration of thrombin the inhibitory effects of RM on fibrin formation were overcome. Conclusions: These results suggest that in plasma based systems and in the PCCs at concentrations <1 IU/ml, RM is capable of inhibiting tissue factor mediated thrombin generation in addition to the direct inhibition of pre-formed thrombin. However, such PCCs as Beriplex and Prothromplex are capable of overcoming the thrombin generation inhibitory actions of RM in a concentration dependent fashion. Moreover, RM is also capable of modulating fibrin formation as evident by fibrinokinetic studies where increased thrombin levels may also overcome its effect on fibrin formation. These observations suggest that PCCs may have the potential to reverse the anticoagulant effect of RM and may be considered as a potential antidote for this agent. Disclosures Williams: Asahi Kasei Pharma America: Employment.

Oral Anti-Factor Xa and Factor IIa Agent Mediated Inhibition Of Tissue-Factor Mediated Generation Of Thrombin In Prothrombin Complex Concentrates

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4810-4810
Author(s):  
Daneyal Syed ◽  
Debra Hoppensteadt ◽  
Daniel Kahn ◽  
Job Harenberg ◽  
Jawed Fareed
Keyword(s):  
Tissue Factor ◽  
Oral Anticoagulant ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Factor Xa ◽  
Onset Time ◽  
Thrombin Inhibitors ◽  
Bleeding Complications ◽  
Prothrombin Complex Concentrates ◽  
Inhibition Of Thrombin

Introduction Several oral anti-factor IIa and factor Xa agents have recently been developed. These include the thrombin inhibitors Ximelagatran/Melagatran (M) and Dabigatran Etexilate/Dabigatran (D), which require endogenous conversion to the active agents D and M. The factor Xa inhibitors, Rivaroxaban (R) and Apixaban (A), are anti-Xa agents that do not require any endogenous activation. Ximelagatran was withdrawn from the market due to adverse reactions. Dabigatran, Rivaroxaban, and Apixaban are approved for various clinical indications. Antagonism of the anticoagulant effect may be required in bleeding complications. Contradictory results were reported for the efficacy of various prothrombin complex concentrates (PCCs) with these new oral anticoagulants (NOACs). The purpose of this study was to determine the differences in the thrombin generation inhibitory profiles of the newer oral anticoagulant agents. Methods Commercially available PCCs namely Octaplex and Beriplex, were used as a source of Factors II, VII, IX and X. To investigate the effect of each of these agents, a working solution of 1U/ml of both PCCs were supplemented in a graded concentration of 0-1250ng/ml with M, D, R and A. Thrombin generation studies were carried out using a thromboplastin activator (RC High, Technoclone Vienna, Austria). Total thrombin generated was measured in terms of nM’s. The IC-50 for each agent was calculated individually. The time course of thrombin generation was also measured following the kinetic profiles and AUC. Results Dabigatran and Melagatran produced relatively weaker inhibition of thrombin generation with the IC-50 values ranging from 410-110ng/ml in Beriplex and 350-1120ng/ml in Octaplex. Both Rivaroxaban and Apixaban produced strong inhibition of thrombin generation, with the IC-50 ranging from 58-62ng/ml in Octaplex; whereas, in Beriplex these values ranged from 48-50ng/ml. The onset time for thrombin generation and total thrombin formation was concentration dependent. The kinetics of thrombin generation with A and R were distinct from D and M. At concentrations below 310ng/ml the total amount of thrombin generated was comparable to the control; however, its formation was delayed. In both systems, D exhibited the weakest thrombin generation inhibitory potential. While the onset time of thrombin generation was delayed at concentrations below 310ng/ml the levels were comparable to or higher than the control. Discussion This data suggests that PCC’s such as Octaplex and Beriplex can be used to generate thrombin and it’s inhibition by new oral anticoagulant drugs. Octaplex generates much higher amount of thrombin than Beriplex at equivalent units. These results also show that in comparison to the oral anti-Xa agents, the oral anti-IIa agents are relatively weaker inhibitors of thrombin generation. These studies also suggest that the differential inhibition of the generation of thrombin through tissue factor by the anti-Xa and IIa agents may contribute to the potential neutralization profile of PCC’s for these drugs. Disclosures: No relevant conflicts of interest to declare.

The Effect of Active Site-inhibited Factor VIIa on Tissue Factor-initiated Coagulation Using Platelets before and after Aspirin Administration

1997 ◽  
Vol 78 (04) ◽  
pp. 1202-1208 ◽  
Author(s):  
Marianne Kjalke ◽  
Julie A Oliver ◽  
Dougald M Monroe ◽  
Maureane Hoffman ◽  
Mirella Ezban ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Active Site ◽  
Large Scale ◽  
Thrombin Generation ◽  
Factor Viia ◽  
Dependent Manner ◽  
Antithrombotic Agent ◽  
Before And After ◽  
Ic50 Values

SummaryActive site-inactivated factor VIIa has potential as an antithrombotic agent. The effects of D-Phe-L-Phe-L-Arg-chloromethyl ketone-treated factor VIla (FFR-FVIIa) were evaluated in a cell-based system mimicking in vivo initiation of coagulation. FFR-FVIIa inhibited platelet activation (as measured by expression of P-selectin) and subsequent large-scale thrombin generation in a dose-dependent manner with IC50 values of 1.4 ± 0.8 nM (n = 8) and 0.9 ± 0.7 nM (n = 7), respectively. Kd for factor VIIa binding to monocytes ki for FFR-FVIIa competing with factor VIIa were similar (11.4 ± 0.8 pM and 10.6 ± 1.1 pM, respectively), showing that FFR-FVIIa binds to tissue factor in the tenase complex with the same affinity as factor VIIa. Using platelets from volunteers before and after ingestion of aspirin (1.3 g), there were no significant differences in the IC50 values of FFR-FVIIa [after aspirin ingestion, the IC50 values were 1.7 ± 0.9 nM (n = 8) for P-selectin expression, p = 0.37, and 1.4 ± 1.3 nM (n = 7) for thrombin generation, p = 0.38]. This shows that aspirin treatment of platelets does not influence the inhibition of tissue factor-initiated coagulation by FFR-FVIIa, probably because thrombin activation of platelets is not entirely dependent upon expression of thromboxane A2.

Abstract 43: Prothrombinase Assembled with Factor V Leiden is Resistant to Inhibition by Tissue Factor Pathway Inhibitor α Lowering the Procoagulant Threshold for Initiation of Coagulation

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Jeremy P Wood ◽  
Lisa M Baumann Kreuziger ◽  
Susan A Maroney ◽  
Rodney M Camire ◽  
Alan E Mast
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Tissue Factor Pathway Inhibitor ◽  
Platelet Rich Plasma ◽  
Anticoagulant Activity ◽  
Factor V Leiden ◽  
Factor Xa ◽  
Factor V ◽  
Activation Threshold ◽  
Tissue Factor Pathway

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.

In Vitro Effect of Danaparoid Sodium (Orgaran®) on Thrombin Generation after Minimal Tissue Factor Pathway Activation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4151-4151
Author(s):  
Ismail Elalamy ◽  
Anna D. Petropoulou ◽  
Mohamed Hatmi ◽  
Meyer M. Samama ◽  
Grigoris T. Gerotziafas
Keyword(s):  
Molecular Weight ◽  
Tissue Factor ◽  
Thrombin Generation ◽  
Lag Time ◽  
Dependent Manner ◽  
Pathway Activation ◽  
Coagulation Tests ◽  
Vitro Effect ◽  
Routine Coagulation

Abstract Introduction: Orgaran® (Org 10172) is a low molecular weight heparinoid which consists of natural sulphated glycosaminoglycans (heparan, dermatan, chondroitin sulphate). It has a mean molecular weight of approximately 6 kDa (4–10 kDa), an excellent bioavailability following subcutaneous administration and an anti-Xa/anti-IIa activity ratio superior to 22. It is the anticoagulant of choice in patients developping Heparin-Induced Thrombocytopenia (HIT), whereas its’ use is also proposed for surgical thromboprophylaxis. Orgaran® has no effect on routine coagulation tests (aPTT, PT, TT). Thrombin generation test(TG) is a global clotting assay proven to be sensitive to the anticoagulant effect of LMWHs and specific FXa inhibitors (i.e. fondaparinux and BAY-597939). In this in vitro study, we determined the tissue factor (TF)-induced TG inhibition potency of Orgaran® using the Thrombogram-Thrombinoscope® assay. Materials and Methods: TG was assessed after TF pathway activation in Platelet Rich Plasma (PRP) (1.5x105 platelets/μl) using diluted thromboplastin (Dade Innovin®, 1:1000 final dilution). The clotting process is provoked by a physiologically relevant TF concentration. Orgaran® was added to control plasma from 8 healthy volunteers at five different final concentrations (0.2, 0.4, 0.6, 0.8 and 1IU anti-Xa/ml). TG was initiated by adding the triggering solution containing CaCl2 and the fluorogenic substrate. The analyzed TG parameters are the lag time, the maximal concentration of thrombin (Cmax), the time to reach Cmax (Tmax), the TG velocity and the endogenous thrombin potential (ETP). Results: Orgaran® prolonged significantly the lag time and the Tmax at a concentration over 0.40 IU anti-Xa/ml (p<0.05). At the lowest studied concentration (0.20 IU anti-Xa/ml), lag time and Tmax were only prolonged by 12%, whereas their maximal prolongation (around 50%) was observed at 1IU anti-Xa/ml. Furthermore, Orgaran® inhibited ETP, Cmax and TG velocity in an almost linear dose dependent manner. A significant inhibition of ETP, Cmax and TG velocity was obtained at concentrations superior to 0.20 IU anti-Xa/ml. (p<0.05). At the highest studied concentration (1IU anti-Xa/ml) Orgaran® suppressed all TG parameters by about 80% (Table 1). Conclusion: Orgaran® exhibited a significant inhibitory activity of in vitro TG. At concentrations achieved in clinical practice (prophylactic or therapeutic dose), Orgaran® modified in vitro TG profile while it has no effect on routine coagulation tests. Thus, TG assay is a sensitive method for monitoring Orgaran® and this test requires a clinical prospective evaluation. Table 1. Determination of IC20 and IC50 anti-Xa inhibitory concentrations of Orgaran® on TG parameters Lag Time Tmax ETP Cmax Velocity IC: Inhibitory Concentration * or Concentration increasing 20% and 50% the lag time and the Tmax respectively IC 20 (IU/ml) 0.30 0.30 0.18 0.18 0.15 IC 50 (IU/ml) 0.83 >1 0.30 0.50 0.35 1IU anti-Xa/ml 53% 47% 68% 76% 84%

Tissue Factor Mediated Activation of Prothrombin Complex Concentrates (PCCs) Is Differently Inhibited by Dabigatran, Rivaroxaban and Apixaban. Potential Clinical Implications

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3410-3410
Author(s):  
Jawed Fareed ◽  
Nasir Sadeghi ◽  
Daniel Kahn ◽  
Josephine Cunanan ◽  
Kimberly Bartosiak ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Oral Anticoagulants ◽  
Protein G ◽  
Prothrombin Complex Concentrates ◽  
Sds Page ◽  
Activation Products ◽  
Tissue Factors

Abstract Abstract 3410 The newly developed oral anticoagulants represent specific antithrombin (dabigatran, Boehinger, Ingelheim) and antifactor Xa agents (rivaroxaban, Bayer Health Care/Jhonsen) and apixaban, Bristol Myers Squibb/Pfizer). Prothrombin Complex Concentrates (PCCs) such as profilnine® and beriplex® are reported to partially neutralize the anticoagulant effects of these agents. Since these PCCs are capable of generating factor Xa and thrombin, the newer anticoagulants may be neutralized differentially by the proteases generated by PCCs. Coagulation and thrombosis are activated substantially by tissue factor in vivo. The purpose of this study is to compare the inhibitory effects of dabigatran, rivaroxaban and apixaban in tissue factor mediated thrombin generation using profilnine, by utilizing various approaches to characterize activation products including thrombin. Materials and Methods Dabigatran, rivaroxaban, and apixaban were synthesized and/or commercially obtained. Profilnine (Grifols Biologicals Inc.) was also commercially obtained. One commercial lot of a recombinant thrombin preparation Recothrom® was obtained from ZymoGenetics Inc for the development of polyclonal antibodies. To generate specific antisera, individual groups of rabbits (n = 3–6) were challenged repeatedly with human recombinant thrombin, over a 9-month period. At the end of this time the antisera from each rabbit was collected and pooled. Immunglobulin (IgGs) were isolated using a protein G column (HiTrap Protein G HP – GE Helathcare Bio-Science Crop). Buffered profilnine (2.5 u/ml) was activated with routinely used tissue factor reagents by adding commercially available PT reagents such as thromboplastin C, neoplastinPlus, and simplastin at a 1:4 ratio and incubated for 30 minutes. The activation of profilnine was measured by using thrombin generation utilizing a fluorogenic substrate method (Technoclone) and the protease generation profile was evaluated using mass spectrometry method (SELDI), SDS-PAGE analysis and immunoblotting using a specific antithrombin (Recothrombin) antibody to profile the activation products. Similar studies were carried out in profilnine supplemented with graded amount of various oral anticoagulants in the concentration range of 0–2.5ug/ml. Results All tissue factors produce varying degrees of time dependent activation of profilnine as measured by consumption of prothrombin peak at 71 KDa and generation of thrombin peaks at 3l–37 KDa as observed in the SELDI. Varying amounts of prothrombin generation at 52 KDa was also evident. Distinct immunoblot for thrombin in western blotting analysis was consistent with SDS-PAGE and SELDI analysis showing the generation of thrombin. The anti-Xa agents blocked the generation of thrombin whereas dabigatran failed to produce this effect. This phenomenon was also observed in all three methods used to study generation of the thrombin when using other PCCs such as octaplex and thromboplex activated by various tissue factors. In the fluorometric thrombin generation assays both apixaban and rivaroxaban produced a relatively stronger inhibition of thrombin generation (IC50= 20–200ng/ml) wheras > 500ng/ml for dabigatran in various PCCs. Conclusion These results suggest that in contrast to dabigatran both rivaroxaban and apixaban produce a much stronger inhibition of tissue factor mediated generation of the thrombin in PCCs. Inhibition of the functional generation of thrombin was weaker with dabigatran in contrast to apixiban and rivaroxiban. The observed ex-vivo neutralization profile of these agents by PCCs may be due to the differential interactions with the protease generated during their activation. These differences along with the compositional variations in the PCCs should be taken into account while considering prothrombin complexes for the neutralization of new oral anticoagulants. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Andexanet Alpha Differentially Neutralizes the Anticoagulant, Antiprotease and Thrombin Generation Inhibitory Effects of Unfractionated Heparin, Enoxaparin and Fondaparinux

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1158-1158
Author(s):  
Fakiha Siddiqui ◽  
Alfonso J Tafur ◽  
Debra Hoppensteadt ◽  
Jeanine Walenga ◽  
Walter Jeske ◽  
...  
Keyword(s):  
Research Funding ◽  
Thrombin Generation ◽  
Surrogate Marker ◽  
Factor Xa ◽  
Bleeding Complications ◽  
Dependent Manner ◽  
Thrombin Time ◽  
Inhibitory Effects ◽  
Biologic Effects ◽  
Inhibition Of Thrombin

Introduction: Andexanet Alpha (Coagulation factor Xa recombinant, inactivated Zh-zo; AA, Portola Pharmaceuticals) is a recombinant factor Xa decoy protein which is designed to reverse the effects of apixaban and rivaroxaban and is approved for the control of bleeding complications associated with their use. The molecular modification in this recombinant protein involves the substitution of serine active site by alanine and the removal of the gamma-carboxyglutamic acid (GLA) domain to restrict its assemblage into prothrombinase complex. Beside the reversal of the effects of anti-Xa agents AA is also reported to neutralize the biologic effects of heparin and related drugs. Assay dependent variations in the neutralization profile of various factor Xa inhibitors by andexanet has been recently reported https://doi.org/10.1177/1076029619847524. Since heparin and related drugs also mediate their biologic actions by inhibiting factor Xa via AT complexation, it is hypothesized that AA may also inhibit their biologic effects as measured in various laboratory assays. It is the purpose of this study is to compare the relative neutralization profile of heparin (UFH), a low molecular weight heparin, enoxaparin (E) and a chemically synthetic pentasaccharide, Fondaparinux (F) by AA. Materials and Methods: API versions of UFH, E and F were commercially obtained in powdered forms and dissolved in saline at a working dilution of 1mg/ml. AA was dissolved in saline to obtain a 10mg/ml working solution. The anticoagulant profile of UFH, E and F was studied using the activated partial thromboplastin time (APTT) and thrombin time (TT) in a concentration range of 0 - 10 ug/ml in pooled human plasma. The anti-Xa and anti-IIa studies were carried out in amidolytic assays in the same concentration range. The thrombin generation inhibition was studied using calibrated automated thrombin generation systems (CAT, Diagnostica Stago). The effect of AA on the reversal of the anticoagulant and anti-protease and thrombin generation effects of each of these agents were studied by supplementing this agent at 100 ug/ml. The results are compared to determine the difference between pre and post AA neutralization settings. Results: All agents produce a concentration dependent effect in the anticoagulant and anti-protease assays with the exception of F which showed mild anticoagulant effects, and very weak anti-IIa actions and strong anti-Xa activity. In the anti-Xa assay the IC-50 for UFH was 2.1ug/ml (0.13 um), E 4.3 ug/ml (0.95 um) and F 0.7 ug/ml (0.41 um) upon supplementation of AA the IC50s for UFH was increased to 5 ug/ml (0.31 um) and for E 5 ug/ml (1.11 um). However, there was no neutralization of the anti-Xa effects of the F by AA and the IC50 remained the same for both pre and post andexxa studies. The anticoagulant effects of UFH as measured by aPTT and TT was strongly neutralized whereas E was only partially neutralized in the aPTT assay and almost completely neutralized in the thrombin time assay. At concentrations of up to 10 ug/ml F did not produced any significant anticoagulant effects, both in the presence and absence of AA. In the thrombin generation inhibition assays, UFH produced a complete inhibition of thrombin generation which was completely reversed by AA. Although both E and F produced strong inhibition of thrombin generation, AA did not completely neutralize these effects. The results are tabulated on table 1 for the studies carried out at 10 ug/ml of UFH, E and F. Conclusion: These results indicate that AA is capable of differentially neutralizing anticoagulant and anti-protease effects of UFH in an assay dependent manner. However, AA is incapable of neutralizing the anti-Xa effects of E and F. This may be due to the relatively differential affinities of enoxaparin and fondaparinux AT complex to factor Xa rendering it inhibited in the presence of AA. These studies also demonstrate that the primary surrogate marker anti-Xa activity for measuring the activities of anti-Xa agents is not proportional to the anticoagulant and thrombin generation inhibitory effects of these agents. A global clotting assay may be a better indication of the biologic effects of these agents and their reversal by AA. Disclosures Tafur: Recovery Force: Consultancy; Janssen: Other: Educational Grants, Research Funding; BMS: Research Funding; Idorsia: Research Funding; Daichi Sanyo: Research Funding; Stago: Research Funding; Doasense: Research Funding.

scholarly journals Monitoring Compound-Related Effects on Coagulability in Rats and Cynomolgus and Rhesus Monkeys by Thrombin Generation Kinetic Measurement

2020 ◽  
Vol 39 (3) ◽  
pp. 207-217
Author(s):  
F. Poitout-Belissent ◽  
D. Culang ◽  
D. Poulin ◽  
R. Samadfan ◽  
S. Cotton ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
In Vivo Studies ◽  
Coagulation Cascade ◽  
Dependent Manner ◽  
Kinetic Assay ◽  
Thrombin Generation Assay ◽  
Coefficients Of Variation

Thrombin generation assay (TGA) is a sensitive method for the assessment of the global clotting potential of plasma. This kinetic assay can detect both hypocoagulable and hypercoagulable conditions: delayed or reduced thrombin generation leading to a prolonged clotting time, or induced thrombin activity, shifting the coagulation cascade toward thrombosis. The purpose of this study is to qualify the TGA in nonhuman primates (NHP) and rats for its use during nonclinical in vivo and in vitro studies. Blood was drawn from nonanesthetized animals, and platelet-poor plasma was obtained after double centrifugation; coefficients of variation were <10% for all derived parameters of thrombin generation assessed with 5 pM of tissue factor. Thrombin generation was evaluated in vitro in rat and NHP plasmas with ascending doses of unfractionated heparin (UFH), recombinant tissue factor, and anticoagulant compounds. Thrombin generation was decreased with UFH and anticoagulant compounds, but was increased in the presence of tissue factor, in a dose-dependent manner. In a rat model of inflammation, animals were administered a low dose of lipopolysaccharides. Thrombin generation measurements were decreased 3 hours post-LPS administration with a nadir at 24 hours, while thrombin–antithrombin complexes reached a peak at 8 hours, supporting an earlier production of thrombin. In conclusion, these data demonstrated that TGA can be performed in vitro for screening of compounds expected to have effects on coagulation cascade, and thrombin generation can be measured at interim time points during nonclinical in vivo studies in rats and NHP.

Novel Initiation Mechanism of Blood Coagulation by Intravascular Tissue Factor.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3967-3967
Author(s):  
Bernd Engelmann
Keyword(s):  
Plasma Membrane ◽  
Tissue Factor ◽  
Whole Blood ◽  
Thrombin Generation ◽  
Clinical Chemistry ◽  
Sandwich Elisa ◽  
Blood Component ◽  
Potential Contribution ◽  
Fibrin Formation ◽  
Activated Platelets

Abstract Bernd Engelmann Vascular Biology and Hemostasis, Inst. of Clinical Chemistry, Ludwig-Maximilians-Universität, Munich, Germany Following addition of fibrillar collagen to whole blood in order to mimick the starting process of hemostasis, we unexpectedly observed that tissue factor (TF), the central initiator of coagulation, was exposed within 3–5 min in association with CD15 and CD14 positive blood cells. A series of experiments revealed that the TF presentation was restricted to conjugates of neutrophils (and monocytes) with platelets. To verify the source of the TF, isolated neutrophils and platelets were evaluated for the presence of TF. Using a double sandwich Elisa, the washed platelets were found to contain TF. Conversely, TF was undetectable in the neutrophils. When searching for the intraplatelet location of TF by immunoelectron microscopy (IEM), TF was observed to reside in the alpha-granules and in the surface connected system. No TF was present in the cytoplasma and the dense granules. In response to activation, platelet TF was translocated to the cell surface by fusion of the alpha-granules with the plasma membrane. The externalized TF was found to cluster on platelet filopodia. Inspection of rapidly isolated buffy coat preparations confirmed the absence of TF from the neutrophils. Stimulation of TF-dependent factor Xa formation by the activated platelets was markedly amplified by the isolated neutrophils. This required neutrophil-platelet conjugate formation, as evident from inhibition by antibodies targeting PSGL-1 and CD18. To assess whether the TF triggered coagulation was connected to the platelet recruitment, we evaluated the participation of the ADP system. Disrupting the interaction of ADP with its platelet receptors P2Y12 and P2Y1 suppressed the TF activity in the neutrophil-platelet conjugates. Since the TF exposing filopodia represent preferential sites for the formation of microparticles (MP), we isolated the total pool of circulating MP from whole blood, known to be mainly derived from the platelets. Then, the MP were separated by cell sorting. In MP positive for the platelet specific CD42b, TF could be detected and quantified by western blotting and Elisa. Moderate increases in MP number excessively stimulated blood based TF activity in the presence of platelets and in whole blood. Since activated platelets are known to secrete tissue factor pathway inhibitor (TFPI), an anti-TFPI antibody targeting the Kunitz-2 domain of TFPI was included into the suspensions of the activated platelets. Thereby the TF activity of the isolated platelets was enforced, while the activity in the presence of the neutrophils remained unaffected, suggesting that TFPI partially masks the functional competence of the platelet TF. The potential contribution of platelet-collagen interactions for the activation of coagulation in vivo was analyzed by injecting collagen into the venous blood of mice. Local fibrin formation was documented in pulmonary vessels by EM, and systemic thrombin generation was revealed by increased thrombin-antithrombin complexes. In mice deficient for the P2Y1 ADP receptor, the thrombin generation was markedly reduced, indicating a basic role for the platelet-triggered coagulation during thrombus growth. In conclusion, the intravascular tissue factor enables the entire coagulation system to proceed on the plasma membrane of a single blood component, the surface of the activated platelets. Consequently the coagulation start can be regulated within the platelet aggregate, allowing fibrin formation to be flexibly adjusted to the size of the thrombus and the duration of its development.

Differential Procoagulant Phenotype of Pancreatic and Breast Cancer Cells Related to Different Tissue Factor Activity.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3992-3992
Author(s):  
Grigoris T. Gerotziafas ◽  
Ismail Elalamy ◽  
Marie-Paule Roman ◽  
Claudine Prengel ◽  
Elisabeth Verdy ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Tissue Factor ◽  
Breast Cancer Cells ◽  
Thrombin Generation ◽  
Human Platelet ◽  
Procoagulant Activity ◽  
Dependent Manner ◽  
Mcf7 Cells ◽  
Platelet Poor Plasma

Abstract Tissue factor (TF) expressed by some cancer cells is implicated in metastasis and angiogenesis. The influence of cancer cells on blood coagulation has not been adequately studied. We evaluated the procoagulant potential of pancreatic and breast cancer cells (BXPC3 and MCF7 cell lines respectively) when they are in contact with human platelet-poor plasma (PPP). At 40% and 90% confluence, adhesive cultures of BXPC3 and MCF7 cells were treated with trypsine according to standardized procedure and cancer cells were suspended in normal human platelet poor plasma (PPP) at increasing concentrations. Coagulation was triggered by CaCl2 addition and thrombin generation (TG) was monitored using the Calibrated Automated Thrombogram-Thrombinoscope® (Biodis-France). In some experiments, cancer cells were incubated for 30 min with a polyclonal specific anti-TF antibody (American Diagnostics). Cancer cells accelerated TG by decreasing significantly lag-time, and time to Peak of thrombin (ttPeak) but they did not significantly influence the endogenous thrombin potential. BXPC3 had significantly more potent procoagulant activity compared to MCF7 cells. Incubation of cancer cells with anti-TF antibody resulted in a concentration dependent inhibition of their procoagulant effect. The IC50 of the anti-TF antibody for TG induced by BXPC3 was about 10-fold higher to that for MCF7. Pancreatic cancer cells (BXPC3) and breast cancer cells (MCF7) accelerate thrombin generation of human plasma in a TF-dependent manner. BXPC3 have more potent procoagulant activity than MCF7 probably due to increased TF expression.

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