Impact Of Thrombin Generation, Tissue Factor Activity and Thrombomodulin Activity On The Positivity Of Assisted Reproductive Technique In Infertile Women

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3630-3630
Author(s):  
Emmanuelle Mathieu d'Argent ◽  
Patrick Van Dreden ◽  
Marjorie Comtet ◽  
Vassiliki Galea ◽  
Hela Ketatni ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Factor Xa ◽  
Hormone Treatment ◽  
Control Group ◽  
Significant Finding ◽  
Assisted Reproductive Technique ◽  
Infertile Women ◽  
Reproductive Techniques

Abstract Introduction Women undergoing assisted reproductive techniques (ART) are given gonadotrophins to promote the development of multiple follicles within their ovaries. This treatment may be associated with a risk of ovarian hyperstimulation syndrome and venous or arterial thrombosis. The association of clinical and biological criteria of hypercoagulability might contribute to the identification of patients at risk and probably could predict success of ART. The aim of this study was to evaluate thrombin generation, thrombomodulin activity, tissue factor (TF) activity, and procoagulant phospholipids in selected women undergoing ART. We also assessed the potential correlation between the levels of the studied biomarkers and the outcome of the ART. Material and Method A cohort of 27 infertile women eligible for ART and 30 healthy age matched women was studied. All patients were Caucasian aged 33.75 ± 3.52 years and weight 61.08 ± 8.10 kg. Women included in the study did not present any personal or family history of VTE or known thrombophilia. They did not present any signs of hemorrhagic syndrome and did not suffer from any known autoimmune disease. Blood samples were taken under fasting conditions at the following time-points: at the inclusion (T0), between the 5th and 8th day of ovarian stimulation with gonadotrophines (T1) and at the day of HCG administration (T2). Thrombin generation (TG) in plasma was assessed using the Calibrated Automated Thrombogram assay (using, PPP-Reagent-5pM TF from Diagnostica Stago, France), Plasma levels of thrombomodulin activity (TMa), and TF activity (TFa) were measured by home-made tests, Procoagulant phospholipids (PPL) dependent clotting time was measured using a factor Xa-based assay (STA-R Procoag-PPL, Diagnostica Stago, France). Results The endogenous thrombin potential (ETP), PPL, TMa and TFa were significantly higher in the ART-T0 group as compared to the control group. At ART-T2 a significant increase of TG was observed as compared to ART-T0. At ART-T0 44.5%, 44.4 % and 33.3 % of women had ETP, TFa and TMa higher than the Upper Normal Limit respectively (UNL = mean+2 S.D.). Among with negative ART 89% and 91.7% showed TMa and TFa levels > UNL at ART-T0. At T1 50% of women had a least one parameter of TG higher than the UNL. At ART T2 65.2 % of women had TG > UNL. At the same time, 87.5% and 83.4% of women with negative ART had levels of TMa and TFa > UNL. Conclusion This study analyzed the profile of thrombin generation in infertile women eligible for ART and investigated the influence of hormone treatment with gonadotropins and HCG on TG and levels of TMa and TFa. Hypercoagulability, in terms of increased ETP is present in 46% of infertile women eligible for ART. These women remain in a hypercoagulable state throughout the entire period of hormone treatment. The most significant finding of this study was that 33% of patients show a value superior to the UNL for thrombomodulin and 45% for tissue factor. Interestingly 89%of women with negative ART had TMa higher than the UNL. Respectively 91% of women with negative ART had TFa levels higher than the UNL Disclosures: No relevant conflicts of interest to declare.

P-093 Impact of hormone treatment on thrombin generation in infertile women undergoing artificial reproductive techniques

2013 ◽  
Vol 131 ◽  
pp. S100-S101
Author(s):  
E.M. d'Argent ◽  
M. Comtet ◽  
H. Ketatni ◽  
V. Galea ◽  
S. Bouffard ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Hormone Treatment ◽  
Infertile Women ◽  
Reproductive Techniques

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.

scholarly journals Assay Dependent Reversal of the Oral and Parenteral Anti-Xa Agents By Andexanet Alfa

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 39-40
Author(s):  
Fakiha Siddiqui ◽  
Debra Hoppensteadt ◽  
Jeanine Walenga ◽  
Walter Jeske ◽  
Alfonso J Tafur ◽  
...  
Keyword(s):  
Prothrombin Time ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Factor Xa ◽  
Control Group ◽  
Inhibitory Effects ◽  
Strong Concentration ◽  
Dependent Inhibition ◽  
Ic50 Values ◽  
Andexanet Alfa

Introduction: Andexanet alfa (AA, Portola Pharmaceuticals, San Francisco, USA) is an approved reversal agent for the control of potential bleeding associated with apixaban and rivaroxaban. Beside the oral anti-Xa agents, parenteral forms of the inhibitors of factor Xa such as otamixaban (Sanofi Aventis, Paris, France) and DX9065a (Mitsubishi Pharmaceuticals, Tokyo, Japan) have also been developed. These agents represent synthetic organo-mimerics with comparable selectivity and inhibitory profile to the currently available oral anti-Xa agents. Parenteral anti-Xa agents are considered for clinical development. Andexanet alfa is a broad-spectrum neutralizing agent for anti-Xa drugs including heparin and heparino-mimerics. We hypothesized that andexanet alfa may also reverse the effects of such parenteral anti-Xa agents as otamixaban and DX9065a. This study is designed to compare the neutralization profile of andexanet alfa for apixaban and rivaroxaban with otamixaban and DX9065a in various laboratory assays. Materials and Method: Apixaban, rivaroxaban, otamixaban and DX9065a were commercially obtained in powdered form and diluted in 0.9 % sodium chloride to make stock solution of 1.0 mg/ml. Andexanet alfa was obtained from the hospital pharmacy. Drugs were supplemented in plasma in the concentration range of 0.0 - 1.0 ug/ml. Individual aliquots of samples were supplemented with either saline or andexanet alfa at a final concentration of 100 ug/ml. Factor Xa activity was measured by using an amidolytic method. For clotting profile, prothrombin time (PT) and activated partial thromboplastin time were measured. Thrombin generation studies were carried out using a calibrated automated thrombogram (CAT, Diagnostica Stago, Paris, France). Such parameters as peak thrombin (PT), area under the curve (AUC) and lag time (LT) were measured. The inhibitory effects of each of these agents towards factor Xa were calculated and their reversal by andexanet alfa was determined. Results were compiled as mean SD of 3 individual determination. Result: Both the oral and parenteral anti-Xa agents produced a concentration dependent inhibition of factor-Xa with the IC50 values ranging from 0.17 - 1.1 ug/ml in control group. Supplementation of andexanet alfa at 100 ug/ml resulted in the neutralization of the anti-Xa activities of these agents with the IC50 values ranging from 0.22 - 1.1 ug/ml. Andexanet alfa did not produce any reversal of the anti-Xa activities of DX9065a. In the thrombin generation studies, Apixaban, rivaroxaban and otamixaban produced strong concentration dependent inhibition of thrombin formation. However, DX9065a produced relatively weaker anti-Xa effects. The IC50 values varied with apixaban (0.08 ug/ml), rivaroxaban (0.22 ug/ml), otamixaban (0.6 ug/ml) and DX9065a (>2.5 ug/ml). In the clot-based prothrombin time assay all agents produced a concentration dependent prolongation of PT in the range of 0 - 1 ug/ml. Andexanet alfa at 100 ug/ml produced a complete neutralization of apixaban, rivaroxaban and otamixaban, whereas it partially neutralized the anticoagulant effects of DX9065a in this assay. The parenteral anticoagulants otamixaban and DX9065a produced a much stronger anticoagulant effects in the aPTT assay in comparison to both apixaban and rivaroxaban. Andexanet alfa at 100 ug/ml effectively neutralized the anticoagulant effects of otamixaban in comparison to Apixaban and rivaroxaban. Whereas DX9065a were not neutralized. Table 1 shows the composite results for the neutralization of oral and parenteral anti-Xa agents at 0.5 ug/ml by andexanet alfa at 100 ug/ml. Conclusion: Our results suggest that andexanet alfa is capable of neutralizing the effects of potent parenteral anti-Xa agents such as otamixaban in an assay dependent fashion. The data also points to the varying inhibitory effects of anti-Xa agents which are differentially neutralized by andexanet alfa. These results also underscore that the in-vitro anti-Xa potency of both the oral and parenteral anti-Xa agents does not fully reflect their inhibitory effects on the overall coagulation process. Nevertheless, andexanet alfa may be a useful agent in the neutralization of parenteral anti-Xa agents. Disclosures No relevant conflicts of interest to declare.

Thrombin Generation in Sickle Cell Disease: Insights From Computerized Automated Thrombography.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2587-2587 ◽  
Author(s):  
Suhita Gayen Betal ◽  
Gregory J Kato ◽  
Marlene Peters Lawrence ◽  
Catherine Seamon ◽  
Yamaja Setty ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Rapid Quenching ◽  
Vascular Pathology ◽  
Control Group ◽  
Peak Time ◽  
Sample Collection ◽  
Early Start ◽  
Cat Assay

Abstract Abstract 2587 Poster Board II-563 Background: SCD is considered to be a hypercoagulable state. Because of the great complexity of the hemostatic system it has not been easy to establish an etiological link between hypercoagulability and SCD vascular pathology. In this study we evaluate hemostatic perturbations in adult SCD patients by employing the computerized automated thrombogram (CAT), a novel thrombin generation assay that provides a global measure of coagulation potential and a direct assessment of the coagulation phenotype (Hemker HC et al.,Thromb Haemost. 2006; 96:553–61). We aim to characterize the coagulation phenotype of the SCD patient by a panel of CAT assay parameters. Methods: A total of 23 SCD patients (HbSS and HbSbeta0Thalassemia; 18 –58 years) were evaluated. Control group consisted of 6 age-matched controls (males=3, females=3). SCD plasma samples were obtained at baseline during routine clinic visits. Platelet poor plasma (PPP) ± corn trypsin inhibitor (CTI) - to minimize variability from activation of contact pathway during sample collection- was analyzed by CAT using 2 different triggers for initiation of thrombin generation: i) High trigger –5pM Tissue factor (TF) with 4uM phospholipid (PL) and ii) Low trigger- 1pM TF with 4uM PL. Five CAT assay parameters were studied – Lag time, Endogenous thrombin potential (ETP), Peak thrombin (Peak), Time to peak (ttPeak) and Start Tail. Students t-test was used to compare means of CAT assay parameters between SCD patients and controls. Lactate dehydrogenase (a biomarker of hemolysis-associated nitric oxide resistance and endothelial dysfunction) correlated with CAT assay parameters including lagtime (r= −0.35, p=0.05); ttPeak (r=−0.45, p<0.02) and start tail (r=−0.47, p=0.01). Summary: In this study interesting differences in kinetics of thrombin generation were noted between SCD patients and controls as follows: Thrombin generation with high trigger demonstrated a rapid burst of thrombin generation in SCD plasma (shorter lagtime, lesser ttpeak) vs. controls. Overall lower and less sustained thrombin generation (i.e. decreased ETP and early start tail) followed this rapid burst in SCD. Thrombin generation with low trigger also showed significantly decreased ttpeak of thrombin generation in SCD with similar overall lower thrombin generation and early start tail. ETP, Peak thrombin level, and ttPeak have shown correlations with clinical hypercoagulable states evaluated by the CAT assay. We speculate that this paradoxical upsurge of thrombin generation with swift attenuation suggests the presence of a compensatory upregulation of Tissue Factor Pathway Inhibitor (TFPI) or anticoagulant responses in SCD that results in rapid quenching of thrombin generation. Additionally, we have previously shown that heme, a product of intravascular hemolysis, upregulates endothelial TF (J Thromb Haemost. 2008; 12:2202–9). The observed relationship between increased LDH and evidence of rapid response of thrombin generation is in keeping with this result. These preliminary data support the use of the CAT assay in further detailed analyses of mechanisms of thrombin generation in SCD. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Prospective Assessment of Biomarkers of Hypercoagulability in Oncological Patients and Healthcare Workers Following Vaccination Against Sars-Cov-2 with the mRNA Vaccine. the Roadmap-COVID-19-Vaccin Study

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3207-3207
Author(s):  
Patrick Van Dreden ◽  
Joseph Gligorov ◽  
Evangelos Terpos ◽  
Mathieu Jamelot ◽  
Michele Sabbah ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Tissue Factor ◽  
Platelet Activation ◽  
Research Funding ◽  
Thrombin Generation ◽  
Cell Activation ◽  
Control Group ◽  
Clotting Time ◽  
Endothelial Cell Activation ◽  
Active Cancer

Abstract Background: COVID-19 has been associated with hypercoagulability, endothelial cell injury and frequent thrombotic complications resulting both from direct effects of the virus on the endothelium and from the 'cytokine storm' resulting from the host's immune response. Since the COVID-19 vaccines have been shown to effectively prevent symptomatic infection including hospital admissions and severe disease, the risk of COVID-19-related thrombosis should be expected to (almost) disappear in vaccinated individuals. However, some rare cases of venous thrombosis have been reported in individuals vaccinated with mRNA vaccines. Thus, there is a sharp contrast between the clinical or experimental data reported in the literature on COVID-19 and on the rare thrombotic events observed after the vaccination with these vaccines. This phenomenon raised some scepticism of even some fear about the safety of these vaccines which could compromise the adhesion of the citizens in the vaccination program. Aims: We conducted a prospective observational study, to explore the impact of vaccination with the BNT162b2 (Pfizer/BioNTech) on blood hypercoagulability and endothelial cell activation and to investigate if this is modified by the presence of active cancer. Methods: In total 229 subjects were prospectively included in the study from April to June 2021. Subjects were stratified in three predefined groups: 127 vaccinated patients with active cancer (VOnco group), 72 vaccinated health care workers (VHcw group) and 30 non vaccinated health individuals (Control group). Blood samples were obtained 2 days after the administration of the first dose of BNT162b2 vaccine and collected in Vacutainer® tubes (0.109 mol/L trisodium citrate). Platelet poor plasma (PPP) was prepared by double centrifugation at 2000 g for 20 minutes at room temperature and plasma aliquots were stored at -80°C until assayed. Samples of PPP were assessed for thrombin generation (TG) with PPP-Reagent® (Thrombogram-Thrombinoscope assay with PPP-Reagent®TF 5pM), E-selectin, D-dimers, (D-Di), Tissue Factor (TFa), procoagulant phospholipid-dependent clotting time (Procag-PPL) and von Willebrand factor (vWF), thrombomodulin (TM), tissue factor pathway inhibitor (TFPI), and platelet factor 4 (PF4). All assays were from Diagnostica Stago (France). The upper and lower normal limits (UNL and LNL) for each biomarker were calculated by the mean±2SD for the control group. Results: All vaccinated subjects showed significantly increased levels of PF4 (71% &gt;UNL, p&lt;0.001), D-Dimers (74% &gt;UNL, p&lt;0.01), vWF (60% &gt;UNL, p&lt;0.01), FVIII (62% &gt;UNL, p&lt;0.01) and shorter Procoag-PPL clotting time (96% &lt;LNL, p&lt;0.001), as compared to controls. Thrombin generation showed significantly higher Peak (60% &gt;UNL, p&lt;0.01), ETP (38% &gt;UNL, p&lt;0.01) and MRI (66% &gt;UNL, p&lt;0.01) but no differences in lag-time in vaccinated subjects as compared to the control group. Vaccinated subjects did not show any increase at the levels of TFa, TFPI, TM and E-selectin in comparison with the control group. The studied biomarkers were not significantly different between the VOnco and VHcw groups. Conclusion: The ROADMAP-COVID-19-Vaccine study shows that administration of the first dose of the BNT162b2 vaccine induced significant platelet activation documented by shorter Procoag-PPL associated with increased levels of PF4. Plasma hypercoagulability was less frequent in vaccinated individuals whereas there was no evidence of significant endothelial cells activation after vaccination. Interestingly, the presence of active cancer was not associated with an enhancement of platelet activation, hypercoagulability, or endothelial cell activation after the vaccination. Probably, the generated antibodies against the spike protein or lead to platelet activation in a FcyRIIa dependent manner that results in PF4 release. The implication of the mild inflammatory reaction triggered by the vaccination could be another possible pathway leading to platelet activation. Nevertheless, vaccination does not provoke endothelial activation even in patients with cancer. The findings of the ROADMAP-COVID-19-Vaccine study support the concept administration of mRNA based vaccines does not directly cause a systematic hypercoagulability. Disclosures Gligorov: Roche-Genentech: Research Funding; Novartis: Research Funding; Onxeo: Research Funding; Daichi: Research Funding; MSD: Research Funding; Eisai: Research Funding; Genomic Heatlh: Research Funding; Ipsen: Research Funding; Macrogenics: Research Funding; Pfizer: Research Funding. Terpos: Novartis: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Genesis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; BMS: Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; GSK: Honoraria, Research Funding. Dimopoulos: Amgen: Honoraria; BMS: Honoraria; Janssen: Honoraria; Beigene: Honoraria; Takeda: Honoraria.

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.

Inhibition of thrombin generation by the zymogen factor VII: implications for the treatment of hemophilia A by factor VIIa

Blood ◽  
2000 ◽  
Vol 95 (4) ◽  
pp. 1330-1335 ◽  
Author(s):  
Cornelis van 't Veer ◽  
Neal J. Golden ◽  
Kenneth G. Mann
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Factor Viia ◽  
Plasma Concentrations ◽  
Factor Xa ◽  
Factor Vii ◽  
Lag Phase ◽  
Single Chain

Factor VII circulates as a single chain inactive zymogen (10 nmol/L) and a trace (∼10-100 pmol/L) circulates as the 2-chain form, factor VIIa. Factor VII and factor VIIa were studied in a coagulation model using plasma concentrations of purified coagulation factors with reactions initiated with relipidated tissue factor (TF). Factor VII (10 nmol/L) extended the lag phase of thrombin generation initiated by 100 pmol/L factor VIIa and low TF. With the coagulation inhibitors TFPI and AT-III present, factor VII both extended the lag phase of the reaction and depressed the rate of thrombin generation. The inhibition of factor Xa generation by factor VII is consistent with its competition with factor VIIa for TF. Thrombin generation with TF concentrations &gt;100 pmol/L was not inhibited by factor VII. At low tissue factor concentrations (&lt;25 pmol/L) thrombin generation becomes sensitive to the absence of factor VIII. In the absence of factor VIII, factor VII significantly inhibits TF-initiated thrombin generation by 100 pmol/L factor VIIa. In this hemophilia A model, approximately 2 nmol/L factor VIIa is needed to overcome the inhibition of physiologic (10 nmol/L) factor VII. At 10 nmol/L, factor VIIa provided a thrombin generation response in the hemophilia model (0% factor VIII, 10 nmol/L factor VII) equivalent to that observed with normal plasma, (100% factor VIII, 10 nmol/L factor VII, 100 pmol/L factor VIIa). These results suggest that the therapeutic efficacy of factor VIIa in the medical treatment of hemophiliacs with inhibitors is, in part, based on overcoming the factor VII inhibitory effect.

Factor Xa Inhibitors Can Be Differentiated in Xa, IIa and Microparticle Generation and Other Whole Blood Based Assays.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 931-931 ◽  
Author(s):  
Omer Iqbal ◽  
Cafer Adiguzel ◽  
Debra Hoppensteadt ◽  
Josephine Cunannan ◽  
Jawed Fareed
Keyword(s):  
Tissue Factor ◽  
Whole Blood ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Final Concentration ◽  
Factor Xa Inhibitors ◽  
Saline Control ◽  
Functional Assay ◽  
Control Value ◽  
Anticoagulant Agents

Abstract Currently used oral anticoagulants such as Vitamin K antagonists have drawbacks, which reportedly limit their safety and efficacy. Oral Factor Xa and IIa inhibitors are claimed to overcome these limitations. Factor Xa inhibitors provide more complete suppression of thrombin generation than Factor IIa inhibitors. Four Factor Xa anticoagulant agents, both direct and indirect, namely A,B,C and D with Ki values 6–200 nM, were studied in various assays at equigravimetric final concentration of 10 ug/ml to determine their relative potencies. Apparent differences in their biochemical profiles were noted in thrombin generation, Factor Xa generation and microparticle generation inhibition assays. Furthermore, the anticoagulant potential of these Xa inhibitors was studied in celite activated clotting time (ACT) and modified celite ACT system using different concentrations of tissue factor. Microparticle generation was performed using agent- supplemented whole blood that was incubated for three minutes with varying concentrations of tissue factor. Following three minutes the reaction was stopped using EDTA stop solution. The functional assay for the determination of microparticle procoagulant activity in the plasma was performed using Zymuphen MP-Activity assay kit from Hyphen BioMed (Neuville-sur-Oise, France). While three of the Xa inhibitors studied showed microparticle generation inhibition levels of 31.37 nM, one agent provided a level of 17.87 nM compared to a saline control value of 39.28nM. Supplementation studies were also carried out in whole blood PT, APTT and Heptest assays in the concentration range of 0 to 10 ug/ml. The whole blood prothrombin time assay showed a maximum of 208.8, 61.3, 77.8 and 15.9 seconds at a final concentration of 10 ug/ml for the respective anticoagulant agents when compared to a normal value of 12.3 seconds. Similarly the whole blood APTT assay showed a maximum of 267.9, 161.8, >300 and 71.9 seconds respectively when compared to a control value of 46.8 seconds. The whole blood Heptest assay showed a varying maximum anticoagulant effect from >300, 42.7, >300 and >300 seconds respectively when compared to a control value of 13.8 seconds. In each of these assays Factor Xa inhibitors showed concentration-dependent effects and had varying potencies. In TF-mediated platelet activation assays the different Xa agents produced varying effects which were not proportionate to their Ki values. Differentiation of Factor Xa inhibitors have a clinical impact in dosage selection, dosage adjustment and their monitoring when given in large dosages. Synthetic factor Xa agents exhibit Ki values of 20–200 nM, while pentasaccharide -AT complex has a Ki value of 60 nM. However, these results do not translate into proportionate anticoagulant effects to their Ki values. Large-scale clinical studies are necessary to validate these preliminary results.

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.

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