Persistant Inhibition of Thrombin Generation After Intravenous Administration of Enoxaparin in Primates.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2095-2095
Author(s):  
Evangelos Litinas ◽  
Angel Gray ◽  
Nasir Sadeghi ◽  
Josephine Cunanan ◽  
Debra Hoppensteadt ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Time Course ◽  
Conflicts Of Interest ◽  
Clinical Effects ◽  
Plasma Samples ◽  
Thrombin Inhibition ◽  
Biologic Effects ◽  
Antithrombotic Effects ◽  
Sustained Inhibition ◽  
Inhibition Of Thrombin

Abstract Abstract 2095 Poster Board II-72 The biologic half life (T12) of low molecular weight heparin (LMWH) is usually measured in terms of the circulating anti-Xa levels. Enoxaparin represents an unique LMWH whose biologic T12 is relatively longer than most LMWHs. Moreover, it is known that the antithrombotic effects of this agent last longer in comparison to the measurable circulating anti-Xa levels. Therefore besides the anti-Xa activity, additional non-measurable biologic effects are contributory to the clinical effects of this agent. Plasma based thrombin generation assays have recently become available to assess the effects of LMWHs such as enoxaparin. In these assays blood plasma samples are activated using different activators and the generated thrombin inhibition is measured. To measure the time course of thrombin generation inhibitory activity after an IV bolus dose of 0.5 mg/kg of enoxaparin into groups of primates (n=6-8), a commercially available thrombin generation method was employed (Technoclone, Vienna, Austria/DiaPharma, West Chester,OH). Blood samples were drawn from each of the primates injected at varying time points for up to 28 hours. A thromboplastin/phospholipids based reagent was used to generate thrombin and the results were recorded in terms of nm of thrombin formed. The baseline values ranged from 500-900 nm (710±60 nm), although a complete inhibition of thrombin generation was noted at 1 hour (24±8 nm), a slow and gradual reduction in the thrombin generation inhibition was noted with a T12 of 9 hours. Even at 28 hours after the administration of enoxaparin, sustained inhibition of thrombin generation was noted (30-50%). Interestingly, the circulating anti-Xa and anti-IIa activity gradually diminished to an almost non-detectable level at 6 hours. These studies suggest that enoxaparin produces antithrombotic actions by multiple mechanisms. Furthermore thrombin generation methods in plasma samples may provide a more sensitive assay for the monitoring of the effect of LMWH. Disclosures: No relevant conflicts of interest to declare.

Comparison of Branded Enoxaparin (Lovenox) and a Biosimilar Version of Enoxaparin (Fibrinox)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4385-4385
Author(s):  
Walter Jeske ◽  
Elizabeth McGeehan ◽  
Omer Iqbal ◽  
Debra Hoppensteadt ◽  
Jeanine M. Walenga ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Average Molecular Weight ◽  
Molecular Profile ◽  
Distribution Analysis ◽  
Thrombin Time ◽  
Plasma Samples ◽  
Branded Product ◽  
Inhibition Of Thrombin

Abstract Abstract 4385 Several biosimilar versions of branded enoxaparin (Lovenox, Sanofi-Aventis, Paris, France) have recently become available throughout the world. These biosimilar enoxaparin preparations are distributed by multiple suppliers in Asia and in North and South America. Enoxaparin represents a complex mixture of oligosaccharides obtained by alkaline depolymerization of porcine mucosal heparin. It is the most widely used low molecular weight heparin which has been validated for clinical use in multiple indications. While the molecular profile and anti-Xa potencies of some of the biosimilar versions of enoxaparin are comparable, product based differences have been reported amongst some of the biosimilar versions of enoxaparin. The purpose of this study was to compare the biochemical and pharmacologic profile of one biosimilar version of enoxaparin, namely Fibrinox (Sandoz SA, Buenos Aires, Argentina) with the branded product Lovenox. The products were compared in equigravimetric amounts, assuming equivalent potency (100 AXa U/mg). Both products exhibited comparable molecular weight profiles in terms of average molecular weight and oligosachharide distribution. Analysis of the antithrombin binding hexasaccharide fractions of Fibrinox and Lovenox indicated the presence of eight distinct hexasaccharides. The relative proportions these hexasaccharides differed between Fibrinox and Lovenox. The anti-Xa and anti-IIa activities were comparable. In the whole blood clot-based assays such as TEG and ACT, both agents produced similar anticoagulant effects. In the plasma based assays such as the APTT, Heptest and thrombin time, both products showed comparable anticoagulant effects in the normal human pooled plasma samples. However, in plasma samples collected from patients with liver disease who were apparently anticoagulant free, the two products showed differences in their anticoagulant effects in the APTT assay (p<0.05). In the TF mediated thrombin generation assay, Fibrinox produced a stronger inhibition of thrombin generation compared to Lovenox (IC50; Fibrinox, 1.6 μ g/ml, Lovenox 2.2 μ g/ml). No differences were observed between the two products in the agonist induced platelet aggregation assays. However in the 14C serotonin release study, Fibrinox produced a stronger HIT serum mediated 14C release (p<0.05). Differences in the fibrinokinetic profile and the inhibition of thrombin activatable fibrinolytic inhibitor activation were observed with these LMWHs. These studies suggest while both the molecular profile and the pharmacopoeial potency of Fibrinox is similar to the branded product, these drugs can be differentiated in some of the other assays and should be evaluated in terms of additional pharmacologic mechanisims to demonstrate bioequivalence. Disclosures: No relevant conflicts of interest to declare.

Differential inhibition of thrombin generation by vitamin K antagonists alone and associated with low-molecular-weight heparin

2009 ◽  
Vol 102 (07) ◽  
pp. 42-48 ◽  
Author(s):  
Grigoris T. Gerotziafas ◽  
Charlotte Dupont ◽  
Alex C. Spyropoulos ◽  
Mohamed Hatmi ◽  
Meyer M. Samama ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Vitamin K ◽  
Low Molecular Weight Heparin ◽  
Thrombin Generation ◽  
Vitamin K Antagonists ◽  
Low Molecular Weight ◽  
Plasma Samples ◽  
Thrombin Generation Assay ◽  
International Normalised Ratio ◽  
Inhibition Of Thrombin

SummaryVitamin K antagonists (VKA) treatment starts with co-administration of low-molecular-weight heparin (LMWH). The anticoagulation induced by the two drugs is still not well determined. In the present study we used thrombin generation assay to evaluate the hypo-coagulation induced by treatment with VKA and by the combination of VKA with LMWH. Tissue factor triggered thrombin generation in platelet-poor plasma was assessed in samples from 15 healthy volunteers, 97 samples from patients treated with VKA and 41 samples from patients receiving enoxaparin and VKA. Patients were classified according to international normalised ratio (INR) level (<2, 2–3 and >3).In plasma samples from patients treated with VKA having INR 2–3 the inhibition of thrombin generation reached 50% compared to controls. In samples with INR>3 this inhibition was 80%. In samples from patients receiving both LMWH and VKA, thrombin generation was significantly decreased compared to the controls and VKA group. In samples with an INR 2–3 obtained from patients treated with LMWH and VKA, the inhibition of thrombin generation was similar to that observed in samples with an INR>3 obtained from VKA treated patients. Thrombin generation assay is sensitive to detect the global the anticoagulant effect produced by the association of LMWH and VKA. For equal INR dual anticoagulant treatment induces significantly more profound inhibition of thrombin generation compared to treatment with VKA alone. The clinical relevance of this observation merits to be studied in prospective studies in patients with defined indications of anticoagulant therapy.

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.

Depolymerized Holothurian Glycosaminoglycan Inhibits Plasma Thrombin Generation Via Interaction with the Factor IXa Heparin-Binding Exosite

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3081-3081
Author(s):  
Buyue Yang ◽  
John P. Sheehan
Keyword(s):  
Factor Viii ◽  
Thrombin Generation ◽  
Fold Increase ◽  
Factor Ix ◽  
Heparin Binding ◽  
Thrombin Inhibition ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Recombinant Factor Ix ◽  
Inhibition Of Thrombin

Abstract Depolymerized holothurian glycosaminoglycan (DHG) is a fucosylated chrondroitin sulfate that possesses antithrombin-independent antithrombotic properties in rodent thrombosis and dog hemodialysis models. DHG demonstrates significantly less bleeding in template or tail transection assays than therapeutically equivalent doses of heparins. Several potential in vitro mechanisms have been described for DHG, including acceleration of thrombin inhibition by heparin cofactor II (HCII), inhibition of factor VIII activation by thrombin, and inhibition of factor X activation by the intrinsic tenase complex (factor IXa-factor VIIIa). The relevant mechanism(s) for inhibition of tissue factor (TF) induced plasma thrombin generation by DHG were examined in HCII or mock-immunodepleted, and factor-deficient human plasmas, using selected recombinant factor IX(a) with mutations in the heparin-binding exosite. Plasma thrombin generation was detected by fluorogenic substrate cleavage in the presence of corn trypin inhibitor to block contact activation, and compared to a standard curve generated with α2-macroglobulin-thrombin complex. The dose-dependent decrease in velocity index, a parameter reflecting the rate of thrombin generation between lag phase and peak thrombin concentration, was used to compare DHG potency. When triggered by 0.2 pM TF, the EC50 for inhibition of thrombin generation by DHG was 0.16 ± 0.01 μM in both HCII-depleted and mock-depleted plasma, suggesting that DHG acts independently of HCII. When triggered by excess (4 pM) TF, plasma thrombin generation was independent of factors VIII and IX. Under these conditions, the EC50 for DHG inhibition of thrombin generation was increased 13-fold in mock-depleted plasma (2.02 ± 0.09 μM) and 28-fold in HCII-depleted plasma (4.31 ± 0.23 μM). These results suggest that components of the intrinsic tenase complex contribute to inhibition of plasma thrombin generation by DHG, and HCII contributes only at high tissue factor concentrations. In the presence of 0.2 pM TF, Western blotting under nonreducing conditions showed preservation of the prothrombin/meizothrombin band and delayed/reduced thrombin generation in the presence of 0.5 μM DHG, confirming that the inhibition involves reduced prothrombin activation rather than accelerated thrombin inhibition. When triggered by 0.2 pM TF in factor VIII-deficient plasma supplemented with 700 pM factor VIII or thrombin-activated factor VIIIa, the EC50 for inhibition by DHG was 0.41 ± 0.02 μM and 0.44 ± 0.05 μM, respectively. Similarly, the EC50 for DHG inhibition of thrombin generation in factor IX deficient plasma supplemented with 0.2 pM TF and 100% plasma-derived factor IX (90 nM), or 100 pM plasma-derived factor IXa alone, was 0.36 ± 0.01 μM and 0.34 ± 0.02 μM, respectively. Thus, activation of factors VIII and IX do not contribute significantly to the inhibition mechanism for DHG. The contribution of intrinsic tenase activity to DHG inhibition of plasma thrombin generation was assessed using recombinant factor IX(a) mutants with moderate (R170A) or marked (R233A) reductions in heparin affinity. Factor IX deficient plasma was supplemented with 0.2 pM TF and 100% recombinant factor IX, or 100 pM factor IXa, with increasing concentrations of DHG. Similar to plasma-derived factor IX(a), DHG demonstrated an EC50 of 0.38 ± 0.01 μM for inhibition of thrombin generation in the presence of factor IX(a) wild type (WT) zymogen or protease. In the presence of factor IX(a) R170A, the EC50 for DHG was 0.86 ± 0.06 μM and 1.02 ± 0.02 μM, respectively, a 2–3 fold increase relative to WT (P ≤ 0.01). For factor IX(a) R233A, the EC50 for DHG was 3.55 ± 0.47 μM for zymogen and 2.98 ± 0.64 μM for protease, an 8–9 fold increase relative to WT (P ≤ 0.01). Thus, mutations in the factor IXa heparin-binding exosite induced resistance to DHG inhibition of thrombin generation as follows: factor IX(a) R233A&gt; R170A&gt; WT. These findings are consistent with the common mechanism for intrinsic tenase inhibition demonstrated for heparin and DHG in purified systems, and establish the factor IXa heparin-binding exosite as the relevant molecular target for inhibition of plasma thrombin generation by DHG. This antithrombin-independent mechanism likely mediates the antithrombotic efficacy of DHG and related glycosaminoglycans, and may represent a novel therapeutic target with lower bleeding risk.

Enoxaparin Produces Sustained Antithrombotic Effects in Comparison to Fondaparinux and Rivaroxaban: Clinical Implications.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3128-3128
Author(s):  
Walter Jeske ◽  
Nicholas Masse ◽  
Joseph Emanuele ◽  
He Zhu ◽  
Elizabeth McGeehan ◽  
...  
Keyword(s):  
Therapeutic Dose ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Therapeutic Drug ◽  
Moderate Increase ◽  
Antithrombotic Activity ◽  
Blood Samples ◽  
Thrombosis Model ◽  
Duration Of Effect ◽  
Antithrombotic Effects

Abstract Abstract 3128 Poster Board III-65 Enoxaparin (Lovenox®), fondaparinux (Arixtra®) and rivaroxaban (Xarelto®) have been shown to be effective in mediating antithrombotic effects in post-surgical indications. Because of marked differences in the pharmacokinetic and pharmacodynamic behavior of these drugs we hypothesized that after the last dosage, the duration of the residual antithrombotic activity of the various drugs also differs. In order to compare the duration of effect of these three drugs, a rabbit stasis-thrombosis model (RSTM) utilizing FEIBA as the thrombogenic challenge and a rabbit ear bleeding model (REBM) were employed. Individual groups of rabbits (n=5) were treated with doses to mimic prophylactic, therapeutic and supratherapeutic (5x therapeutic) drug levels. Enoxaparin and fondaparinux were administered subcutaneously and rivaroxaban was administered by oral gavage. Blood samples were collected at baseline and at 16-18 hours post-administration of the last dose on day 1 or after 4 days of repeated dosing in the RSTM or at 3 hours post-administration in the REBM. At the 16-18 hour time point, circulating anti-Xa levels were not observed in any of the treatment groups. Despite this, enoxaparin treated animals exhibited a strong antithrombotic response following administration of the therapeutic dose (clot score = 1.3 ± 0.6 vs. 2.7 ± 0.6 for saline controls). Dosing once daily for four days did not enhance the antithrombotic activity of enoxaparin. In the plasmatic thrombin generation assays, a reduction in thrombin generation was noted in samples from enoxaparin-treated animals. For enoxaparin, no increase in bleeding was observed at prophylactic or therapeutic doses when compared to saline. An increase in bleeding (∼5 fold vs. saline) was observed at 3 and 6 hours post-administration of the ‘overdose’. A dose-dependent increase in anti-Xa activity was observed in blood samples collected 3 hours post-administration. For fondaparinux, no increase in bleeding was observed with any of the doses tested. There was a moderate increase in anti-Xa activity in the samples from rabbits treated with the ‘overdose’ of fondaparinux. This level was comparable to that observed with the therapeutic dose of enoxaparin. These studies suggest that enoxaparin produces a sustained antithrombotic effect in contrast to rivaroxaban and fondaparinux. These observations underscore that residual/sustained antithrombotic effects of enoxaparin may be partly responsible for the prolonged antithrombotic actions associated with the clinical use of enoxaparin. Disclosures No relevant conflicts of interest to declare.

Non-Pathogenic Anti-Heparin/PF4 Antibodies Generated by Low Molecular Weight Heparins Mediate Procoagulant Effects.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1091-1091
Author(s):  
Jeanine M. Walenga ◽  
Debra Hoppensteadt ◽  
Evangelos Litinas ◽  
Harry L. Messmore ◽  
Bruce E Lewis ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Biologically Active ◽  
Control Group ◽  
Heparin Group ◽  
Thrombin Generation Assay ◽  
Enoxaparin Group ◽  
Pre Treatment ◽  
Inhibition Of Thrombin

Abstract Abstract 1091 Introduction: While the incidence of symptomatic heparin-induced thrombocytopenia (HIT) is relatively low with the use of low molecular weights heparins (LMWHs), these agents do generate anti-heparin/PF4 antibodies in 10–20% of treated patients. Dosage, duration, and the pathologic predisposition of the patient influence the quantitative and qualitative nature of these antibodies. It has been suggested that these non-pathogenic antibodies (NPAs) which do not produce symptomatic HIT may, nevertheless, be biologically active and mediate thrombogenic responses. The overall pathophysiologic role of NPAs is unknown at this time. Hypothesis: NPAs generated by LMWHs cause coagulation activation and compromise the anticoagulant effects of the administered LMWH. Study Design: Blood plasma samples collected at baseline and day 10 from patients enrolled in orthopedic surgery clinical trials of LMWHs for the prophylactic management of deep vein thrombosis (Lovenox enoxaparin, sanofi-aventis, n=352; Clivarin reviparin, Abbott, n=380) were retrospectively screened for the presence of anti-heparin/PF4 antibodies using the GTI ELISA method (Waukesha, WI). Positive samples were tested by the 14C-SRA to determine if the antibodies were capable of functionally activating platelets. Both ELISA positive and negative samples were evaluated in an assay of thrombin generation (Technothrombin TGA kit, diaPharma, West Chester, OH). Result: In the enoxaparin study, the baseline pre-treatment samples only showed one patient in the heparin control group to be positive by ELISA. On day 10, 11 of 175 (6.3%) enoxaparin patients had a positive ELISA response, whereas 22 of 177 (12.4%) heparin patients were ELISA positive. None of the samples were 14C-SRA positive. In the thrombin generation assay, the ELISA positive samples showed a lesser inhibition of thrombin generation for both the enoxaparin and heparin groups (270 ± 27 nM TGA enoxaparin group; 220 ± 21 nM TGA heparin group) compared to the thrombin generation response of the ELISA negative samples (190 ± 18 nM TGA enoxaparin group; 160 ± 20 nM TGA heparin group). In the reviparin study, none of the patients were ELISA positive at baseline. On day 10, in the reviparin group 19 of 200 (9.5%) patients were ELISA positive, whereas 28 of 180 (15.6%) heparin control patients had a positive ELISA titer. None of the samples were 14C-SRA positive. In comparison to the baseline (pre-treatment), both the reviparin and heparin treated patients showed an inhibition of thrombin generation (410 ± 27 nM TGA baseline vs 180–290 nM with treatment). However, consistent with the above study, those samples that were ELISA antibody positive showed a lesser inhibition of thrombin generation (240 ± 21 nM TGA reviparin group; 210 ± 16 nM TGA heparin group) in comparison to the ELISA negative samples (190 ± 12 nM TGA reviparin group; 180 ± 14 nM TGA heparin group). Interestingly, the D-dimer levels were found to be higher in the ELISA positive samples in all groups for both studies (p<0.05). Conclusion: These studies suggest a potential pathologic role of NPAs. The results of the thrombin generation studies strongly suggest that the generation of NPAs may result in a reduction of the antithrombotic potential of both LMWH and heparin in treated patients. While the exact mechanism of this process is not clear, dosage adjustment may be useful in those patients who generate NPAs. Disclosures: No relevant conflicts of interest to declare.

Thrombin Generation Responses to Human Factor XIa in Plasma of Animal Species

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5142-5142
Author(s):  
Yideng Liang ◽  
Evi B Struble ◽  
Li Ma ◽  
Samuel A Woodle ◽  
Naveen Jha ◽  
...  
Keyword(s):  
Animal Models ◽  
Guinea Pig ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Zealand White Rabbit ◽  
Coagulation Factor ◽  
Species Selection ◽  
Plasma Samples ◽  
Factor Xia ◽  
Generation Test

Abstract Abstract 5142 Thromboembolic events (TEE), including deep venous thrombosis and myocardial infarction, have been reported in patients receiving immunoglobulin intravenous (IVIG). Recent research showed that many TEE-induced IVIG samples were contaminated with human coagulation factor XIa (FXIa). At present, there is no systematic investigation of animal models that can be used to assess FXIa dependent thrombogenicity risk in animals and be predictive for human response. To assist in species selection for in vivo animal studies, we compared the magnitude and kinetic parameters of thrombin generation in commercially available human and animal plasma samples in response to human FXIa. A low sample volume, highly reproducible thrombin generation test was developed for this study. In our assay, thrombin generation in plasma (20 microliters) is initiated by physiological activator tissue factor in the presence of procoagulant phospholipids, sample (FXIa), buffer and calcium (combined volume of all additions to plasma is 20 microliters). The thrombin generation response to human FXIa was evident from increased thrombin peak height and/or shortened time to thrombin peak. The minimal effective FXIa concentration was species-dependent and ranged from below 0. 1 pM in monkey plasma to over 100 nM in BalbC mouse. The FXIa sensitivity of commercial plasma samples was aligned in the following order: Rhesus and Cynomolgus Monkeys > Human Donors > New Zealand White Rabbit and Sprague Dawley Rat > Hartley Guinea Pig and CD1 Mouse > Lewis Rat > Wistar and Fisher 344 Rats > C57BL6 and Blab C Mice. However, further experiments on freshly collected guinea pig plasma demonstrated that preanalytical variables, notably, blood collection and plasma freezing and thawing techniques, are critical to achieve optimal sensitivity of the thrombin generation test to FXIa. Therefore, variable quality of commercial plasma may have affected observed interspecies differences in thrombin generation. Collectively, these findings will help to clarify differences in minimal thrombogenic doses of FXIa observed in various animal models and levels of FXIa associated with thrombotic events in humans. This research was supported by the FDA Office of Women's Health grant (2012). Disclosures: No relevant conflicts of interest to declare.

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 Validation of the Bioequivalence of USP Potency Adjusted Porcine, Ovine, and Bovine Heparins

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 6-6
Author(s):  
Nausheen Baig ◽  
Ahmed Kouta ◽  
Walter Jeske ◽  
Debra Hoppensteadt ◽  
Jeanine Walenga ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Area Under The Curve ◽  
Anticoagulant Effect ◽  
Clotting Time ◽  
Kinetic Assay ◽  
Pharmaceutical Ingredients ◽  
Biologic Effects ◽  
Mass Basis

Introduction: Currently, there is a shortage of porcine heparin due to several factors such as limited availability of porcine mucosa, supply chain issues, and increased usage due to COVID-19. This has warranted the development of heparin from alternate sources such as bovine and ovine mucosa which is abundantly available for this purpose. On a mass basis, commercially available porcine heparins exhibit a similar potency (200 units/mg) to their ovine counterpart (190 units/mg) and a higher potency in contrast to their bovine counterpart (130-150 units/mg). Therefore, at gravimetric levels, the porcine heparins exhibit stronger biochemical and pharmacological effects in various laboratory assays in comparison to bovine heparin and similar effects in comparison to ovine heparins. Since heparin is standardized in biologic units and cross referenced against USP or EP Standard, it is hypothesized that potency equated porcine, ovine, and bovine heparin will exhibit similar biologic activities in laboratory assays carried out in the in vitro setting. The purpose of this study is to compare the biologic properties of the porcine, ovine, and bovine heparin at USP potency equated levels in standardized laboratory assays. Materials and Methods: Active pharmaceutical ingredients (API) of porcine mucosal heparin (200 units/mg) of U.S. origin was commercially obtained from Medefil Inc. (Glendale Heights, IL). Ovine heparin was obtained from Ronnsi Pharmaceutical (Jiangsu, China). Bovine heparin (140 units/mg) was obtained from Kin Master Pharmaceuticals (Posso Fundo, Brazil). All heparins were diluted at a concentration of 100 units/mL in saline. The anticoagulant effect of all heparins were evaluated using the whole blood clotting assays such as the ACT and thromboelastographic methods. Heparins were diluted in citrated human plasma yielding a final concentration range of 0-1 unit/mL. Clot based assays such as aPTT, TT, and prothrombinase induced clotting time (PiCT) were measured. Thrombin generation inhibition assay was carried out using a kinetic assay (CAT system, Diagnositca Stago, Paris, France). Protamine and heparinase neutralization profiles of these agents were also investigated in the plasma-based systems. These assays were then repeated at gravimetric dosages at final concentrations of 0-10 ug/mL. The results collected from these trials were then mathematically converted to units and compared to the results collected from the potency adjusted trials. All results were tabulated and compared, and applicable statistical methods were applied. Results: The USP potency adjusted heparin exhibited comparable anticoagulant effects in both the ACT and TEG assays. At equigravimetric levels porcine and ovine heparins produced comparable anticoagulant effects and bovine heparin produced weaker anticoagulant effect in both assays. In the citrated plasma supplementation studies, all drugs produced similar anticoagulant effects at potency adjusted dosages. In the chromogenic anti-Xa and anti-IIa assays, the behaviors of the agents were also comparable. In the thrombin generation assays, in terms of peak thrombin generation, area under the curve, and lag time, the porcine, ovine, and bovine heparins showed comparable effects. The protamine neutralization profiles of the porcine, ovine, and bovine heparin exhibited variable assay dependent results. Potency adjusted bovine heparin required higher amount of protamine for the complete neutralization of the biologic effects in comparison to the porcine heparin. At gravimetric concentrations, bovine heparins exhibited lower potencies than both the porcine and ovine heparins, which produced similar results. Summary and Conclusion: These results show that at potency adjusted concentrations, the porcine, ovine, and bovine heparin exhibit comparable biochemical and anticoagulant responses in the plasma-based systems. Therefore, the hypothesis that potency equated porcine, ovine, and bovine heparins exhibit comparable biochemical and anticoagulant activities is validated. Thus, the proposed approach to standardize heparins against a common standard in a biologic assay such as the USP method is valid. Furthermore, these results warrant regulatory considerations to fast track the review process for the re-introduction of bovine heparin and approval of bovine heparin as a biosimilar anticoagulant to porcine heparin. Disclosures No relevant conflicts of interest to declare.

Red-Cell Microparticles Released From Stored Packed Cells: Possible Contributing Factor to Adverse Responses to Transfusion

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 342-342 ◽  
Author(s):  
Wenche Jy ◽  
Carlos Bidot ◽  
Max E Johansen ◽  
Lawrence Horstman ◽  
Sherry Shariatmadar ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Time Course ◽  
Conflicts Of Interest ◽  
Blood Bank ◽  
Red Cells ◽  
Procoagulant Activity ◽  
Blood Type ◽  
Storage Lesion ◽  
Rate Of Increase ◽  
Proinflammatory Activity

Abstract Abstract 342 Background. Packed red cells (PC) stored in blood bank undergo a series of changes, so-called “storage lesion”, which increase with time. In view of some but not all recent studies, it is widely believed that transfusion with younger blood carries less risk of adverse reactions than older blood. However, there is no agreement on the “safe” age of blood, nor is it clearly understood why older blood may carry increased risks. The purpose of this study was to identify microparticle-related factors in stored PC at different time intervals that might pose risk of adverse effects. We investigated profiles of cell-derived microparticles (MP), particularly RBC-derived MP (RMP), in stored PC and assessed their procoagulant and inflammatory property. Methods. Twelve bags of fresh packed red cells (PC) of known blood types (A+, B+, AB+, O+) were obtained from blood bank (2-4 days since drawing). All were non-leuko depleted and were stored at 4°C. Time of receipt was considered day 0. At intervals of 0, 10, 20, and 30 days, 40 mL was withdrawn and centrifuged at 1000xg for 20 min to remove cells. The supernatants were then assayed for (1) quantity of different species of cell-derived MP by flow cyteometry comprising (a) RMP defined by CD235b; (b) LMP by CD45; (c) PMP by CD41; (d) EMP by CD144; (e) generic MP by Ulex Europaeus (Ulex) or Annexin V (AnV), (2) procoagulant activity by MP-mediated thrombin generation assay (TGA); (3) MP-mediated proinflammatory activity by CD 11b expression in neutrophils following incubation with RMP. Results. (1) MP Profiles. The time-course of generation of the MP subtypes varied considerably. For RMP, there was little increase before day 10, but then rose rapidly with time, to 180% at 20 days, and to 450% at 30 days. Small amounts of MP derived from leukocytes (LMP), platelets (PMP), and endothelia (EMP) were present in all bags at day 0, generally <10% by number compared to RMP. For LMP, there was no significant change in the first 20 days but was increased significantly at day 30, to 160% of day 0. For PMP, counts rose steadily from day 0 and peaked to 220% of baseline at day 20. For EMP, counts were very low (<1% of RMP) and no change was observed over 30 days. For total MP, defined by Ulex counts and total protein concentrations, the time course was similar to RMP. Results with AnV+ MP showed significant increase from day 10 to day 20. We found no influence of blood type on MP generation. (2) Procoagulant Activity. There was little change in MP-mediated thrombin generation in the first 10 days, but it rose significantly from day 10 to day 20, correlating well with counts of AnV+ MP or RMP. (3) Proinflammatory Activity. Leukocyte CD11b expression induced by MP from the PC bags showed a nearly linear rate of increase from day 0 to day 30, correlating closely with PMP. (4) Exceptions. In 2 of the 12 PC bags, we observed exceptionally high levels of both RMP and PMP (4-8 fold higher than average) from day 0 to day 30. CBC assay showed that all bags contained similar counts of RBC and WBC, with exception of high concentrations of platelets (>200,000/μL) in the two exceptional bags vs. the others (20,000 – 45,000/μL). In the two exceptional bags, coagulant activities and inflammatory potential were also highly elevated compared to the values in the other bags from day 0 to day 30. Conclusions. (i) RMP are the predominant MP species in stored RBC, increasing slowly from day 0 to 10 and thereafter rising exponentially to day 30. (ii) PMP were also present in significant amounts. The time course of RMP and PMP release correlated well with procoagulant and proinflammatory indicators in stored RBC. (iii) Two of the 12 bags (17%) exhibited exceptionally high platelet content and RMP, PMP. The significance of this new finding remains to be clarified. These preliminary results indicate that procoagulant, proinflammatory MP levels increased significantly after 10 days of storage, and that contaminating platelets exacerbate RMP generation. The increase in MP in stored PC constitutes one aspect of the storage lesion and may pose prothrombotic and/or proinflammatory risks in blood transfusions. (Supported by NIH Grant 5R01HL098031-02) Disclosures: No relevant conflicts of interest to declare.

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