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 Ximelagatran was withdrawn from the market due to adverse reactions.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.

Compositional Differences in Commercially Available Prothrombin Complex Concentrates

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4391-4391 ◽  
Author(s):  
Nasir Sadeghi ◽  
Daniel Kahn ◽  
Josephine Cunanan ◽  
Arthur Only ◽  
Debra Hoppensteadt ◽  
...  
Keyword(s):  
Protein Content ◽  
Tissue Factor ◽  
Oral Anticoagulant ◽  
Factor Xa ◽  
Factor Ix ◽  
Thrombin Inhibitors ◽  
Prothrombin Complex Concentrates ◽  
Thrombin Activity ◽  
Sds Page ◽  
Anticoagulant Drugs

Abstract Abstract 4391 Prothrombin complex concentrates (PCCs) are used in the management of bleeding complications with conventional oral anticoagulant drugs such as warfarin. These concentrates are also used in supportive therapy for hemostatic disorders. More recently these agents have been investigated for neutralization of the newer oral anticoagulant drugs such as the direct factor Xa and thrombin inhibitors. Since the activation of these complexes results in the generation of factor Xa and IIa, these agents may potentially neutralize both Xa and thrombin inhibitors. However, the potency of these agents is defined in units which represent the level of factor IX, other factors including factor II, VII, and X are also present, and in unspecified amounts. Moreover, other vitamin K-dependent proteins such as protein C, protein S, and protein Z may also be present. Varying amounts of albumin and other agent such as heparin and antithrombin may also be present as an additive. The purpose of this study is to compare the compositions of the currently available PCCs such as Profilnine®, Beriplex®, Cofact®, Octaplex®, Prothromplex®, and the older agents such as Konyne®, Preconetiv®, and Feiba®. Materials and Methods: Commercially available PCCs were obtained from various suppliers. Each of the individual vials of these concentrates was diluted with saline to obtain a 10 U/ml factor IX solution. Protein content was measured using Lowry's method. Sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) electrophoresis was carried out by dilution of each concentrates to 2 U/ml. Western blot analysis was performed to determine presence of prothrombin, prothrombin-1, and thrombin using anti-human recombinant thrombin antibody capable recognizing these proteins. Tissue factor activation profiles of each PCCs was also studied using Innovin®. The protein composition of native and activated prothrombin complexes was also investigated using surface-enhanced laser desorption/ionization (SELDI) mass spectrometry utilizing the gold chip array (BioRad). Tissue factor mediated thrombin generation by each of the prothrombin complex was studied using a fluorometric method (Technoclone, Vienna, Austria). Results: The total protein content of these PCCs ranged from 18–106 mg/100U. Some of the products were found to contain varying amounts of albumin, antithrombin and heparin as evident in both SDS-PAGE and SELDI analysis. The SDS-PAGE profile of these complexes showed multiple protein bands ranging from 15 to 250 kDa. Beriplex and Profilnine showed fewer bands; Profilnine® mainly exhibited 250, 110, 75 kDa bands and Beriplex® mainly 75 and 66 kDa bands. The other products contain additional bands in the range of 15 to 66 kDa representing albumin and other products. In the SELDI analysis multiple peaks consistent with the SDS-PAGE profile were noted. The immunoblotting showed a major band 70–75 kDa (prothrombin) along with a 50 kDa band representing prethrombin-1. Prior to activation, Feiba® exhibited a distinct additional 37 kDa dense band, (thrombin). SELDI analysis also indicated variable amounts of prothrombin in the products. Upon activation all PCC's were capable of generating thrombin as measured by SELDI and immunoblotting. The prothrombin band completely disappears from all PCCs except Preconetiv®, the prevalent products being prethombin-1 and thrombin. The amount of the prethrombin-1 band varied widely among products; and nearly disappears from all as it is converted to thrombin with time of incubation. The amount of thrombin activity generated from each prothrombin complex was concentration dependent and ranged from 30–1044nm/1.25 units/ml. Octaplex and Cofact produced the strongest thrombin activity whereas Beriplex and Prothromplex produced the least thrombin activity. Conclusions: This study shows that despite standardization in factor IX units, at equivalent IX unit potency these agents widely vary in their composition. Beriplex® and Profilnine® represent purer preparations. Upon activation of prothrombin initiated by tissue factor each complex is capable of generating varying amounts of thrombin. Because of these wide variations in protease generation the relative neutralization potential of each of these PCCs may also differ widely. Thus each of these products should thus be considered as a distinct agent and their efficacies individually determined for a given indication. Disclosures: No relevant conflicts of interest to declare.

Inhibition of Thrombin Generation in Plasma by Inhibitors of Factor Xa

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

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

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.

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.

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.

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

1991 ◽  
Vol 66 (05) ◽  
pp. 559-564 ◽  
Author(s):  
Jerome M Teitel
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Factor Viia ◽  
Factor Xa ◽  
Procoagulant Activity ◽  
Tissue Factor Expression ◽  
Prothrombin Complex Concentrates ◽  
Cell Tissue ◽  
Factor Expression ◽  
Necrosis Factor

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

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.

scholarly journals Treatment of bleeding complications in patients on anticoagulant therapy

Blood ◽  
2019 ◽  
Vol 133 (5) ◽  
pp. 425-435 ◽  
Author(s):  
Siavash Piran ◽  
Sam Schulman
Keyword(s):  
Anticoagulant Therapy ◽  
Vitamin K Antagonists ◽  
Laboratory Data ◽  
Factor Xa ◽  
Thrombin Inhibitors ◽  
Bleeding Complications ◽  
Emergency Setting ◽  
Antifibrinolytic Agents ◽  
Different Types ◽  
Life Threatening

Abstract Anticoagulant therapy is often refrained from out of fear of hemorrhagic complications. The most frequent type of major bleeding is gastrointestinal, but intracranial hemorrhage has the worst prognosis. Management of these complications in patients on anticoagulants should follow the same routines as for nonanticoagulated patients, as described here with the previously mentioned bleeds as examples. In addition, for life-threatening or massive hemorrhages, reversal of the anticoagulant effect is also crucial. Adequate reversal requires information on which anticoagulant the patient has taken and when the last dose was ingested. Laboratory data can be of some help, but not for all anticoagulants in the emergency setting. This is reviewed here for the different types of anticoagulants: vitamin K antagonists, heparins, fondaparinux, thrombin inhibitors and factor Xa inhibitors. Specific antidotes for the latter are becoming available, but supportive care and nonspecific support for hemostasis with antifibrinolytic agents or prothrombin complex concentrates, which are widely available, should be kept in mind.

Sign in / Sign up

Export Citation Format

Share Document