scholarly journals Factor Xa Inhibitory Profile of Apixaban, Betrixaban, Edoxaban and Rivaroxaban Does Not Fully Reflect Their Biologic Spectrum

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2520-2520
Author(s):  
Fakiha Siddiqui ◽  
Siddharth Mehrotra ◽  
Vishnu Venkitasubramony ◽  
Rithik Raina ◽  
Lorenzo Storino Ramacciotti ◽  
...  
Keyword(s):  
Whole Blood ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Anticoagulant Activity ◽  
Kinetic Method ◽  
Factor Xa ◽  
Biochemical Properties ◽  
Therapeutic Effects ◽  
Distinct Individual

Abstract Introduction: There are four oral anti-Xa drugs currently available for clinical use in various indications. These drugs are claimed to mediate their therapeutic effects by solely targeting factor Xa. While these agents are structurally similar, their biochemical properties and their effects on blood coagulation differ. Such differences may impact their safety and efficacy profile. The purpose of this study was to demonstrate the differences among factor Xa inhibitors in terms of their in vitro anticoagulant activity and other biochemical effects. Materials and Methods: Commercially obtained powdered forms of Apixaban, Betrixaban, Edoxaban and Rivaroxaban were profiled in this study. Stock solutions of each drug were prepared at 1mg/ml. To investigate the effect on the whole blood clotting profile, thromboelastographic studies were carried out over a concentration range of 0.5 - 2.5 ug/ml and whole blood activated clotting time (ACT) was measured at 1.0 and 2.5 ug/ml. The anticoagulant profile in citrated human pool plasma was measured at concentrations of 0.062-1.0 ug/ml using such tests as prothrombin time (PT) and activated partial thromboplastin time (aPTT). The anti-Xa effects of each agent were measured using a kinetic amydolytic method. The inhibitory potency was calculated in terms of IC-50. Thrombin generation inhibition studies on each drug were carried out in human pool plasma in a concentration range of 0.0-1.0 ug/ml using calibrated aotomated thrombogram (CAT) assay (Diagnostica Stago, Paris, France). Fibrinokinetics studies were carried out using an optical kinetic method, where thrombin was used to trigger clot formation. All results were compiled in terms of mean + 1 SD of 3-5 replicates. Results: All of the anti-Xa agents produced concentration and assay-dependent effects in these studies. The summary of each agent's effects at selected fixed concentrations and the IC-50 of the anti-Xa activity is given in the Table. In the whole blood ACT at 2.5ug/ml, Edoxaban showed the strongest anti-coagulant effects followed by Rivaroxaban > Betrixaban, whereas Apixaban showed minimal effects. In the TEG analysis at 1ug/ml, Edoxaban exhibited stronger anti-coagulant effects as measured by various TEG parameters, including R-time, K-time, alpha, and MA. Edoxaban and Rivaroxaban showed comparable effects followed by Betrixaban, whereas Apixaban exhibited weaker effects. In the PT assay at 1ug/ml, Edoxaban showed stronger effects, whereas Apixaban, Betrixaban and Rivaroxaban were comparable. aPTT at 1ug/ml revealed that Edoxaban was the strongest anti-Xa inhibitor followed by Betrixaban, whereas Apixaban and Rivaroxaban were comparable. In the anti-Xa assay Edoxaban was stronger (IC-50 = 340ng/ml, 0.62uM) than Apixaban (IC-50 =400ng, 0.87uM), Rivaroxaban (IC-50 = 840ng, 1.9uM) and Betrixaban (IC = >1000ng, >2.22 uM). In the thrombin generation assays at 1ug/ml, Apixaban showed the strongest inhibitory activity (IC-50 = 50ng/ml, 108nm) followed by Edoxaban (IC-50 = 58ng/ml, 108nm), Betrixaban (IC-50 = 60ngml, 133nm) while Rivaroxaban showed relatively weaker activity (IC-50 = 100ng/ml, 299nm). In the fibrinokinetics study at 1ug/ml, the anti-Xa agents produced varying degrees of inhibition with Rivaroxaban (67%), Edoxaban (42%), Apixaban (32%) and Betrixaban (12%). Summary and Conclusion: These results demonstrate that the measured anti-Xa activity alone does not fully reflect the overall biologic spectrum of these agents. Assay dependent variations are exhibited by each of these drugs, revealing distinct individual profiles. Edoxaban was the only anti-Xa agent which consistently exhibited relatively stronger inhibitory profile which was proportional to its anti-Xa activity. These studies indicate that the oral anti-Xa drugs may modulate the hemostatic system through additional mechanisms independent of the inhibition of factor Xa. Table. Table. Disclosures No relevant conflicts of interest to declare.

Generic and Branded Versions of Argatroban Exhibit Differential Anticoagulant Effects In Whole Blood, Plasma Based Assays and Thrombin Generation Assays

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 5129-5129
Author(s):  
Jawed Fareed ◽  
Debra Hoppensteadt ◽  
Omer Iqbal ◽  
Jeanine M. Walenga ◽  
Bruce E Lewis
Keyword(s):  
Protein Interactions ◽  
Whole Blood ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Anticoagulant Effect ◽  
Thrombin Time ◽  
Clotting Time ◽  
Generic Products

Abstract Abstract 5129 Several generic versions of argatroban) (Mitsubishi; Tokyo, Japan) have been introduced in Japan (Argaron, Gartban, Slovastan). In addition, other generic versions of argatroban are being considered by the European and North American regulatory bodies. While the generic versions of argatroban exhibit similar antithrombin potency (Ki values), because of the differential compositional variations their anticoagulant effects in whole blood systems may differ due to their cellular and plasmatic protein interactions. Branded and generic versions of argatroban may exhibit differential anticoagulant actions in the whole blood and plasma based assays due to their differential interactions with blood cells, platelets and plasma proteins. Three generic versions of argatroban that are commercially available in Japan namely Argaron, Gartban and Slovastan and a powdered version of generic argatroban (Lundbeck) were compared with the branded argatroban. Native whole blood thrombelastographic (TEG) analysis was carried out at 0.1 ug/mL, the Activated Clotting Time (ACT) assay was carried out in a concentration range of 0–10 ug/mL, and such coagulation tests as the PT/INR, aPTT, Heptest, and calcium thrombin time were performed. Plasma retrieved from the supplemented whole blood was also assayed. Ratios of the clotting time test values from whole blood and plasma were calculated. Retrieved plasma samples were also assayed in the thrombin generation assays (TGA). All of the different versions of argatroban produced a concentration dependent anticoagulant effect in the native whole blood TEG and ACT. In the TEG, while argatroban and Slovastan showed a similar effect, Gartban, Argaron and a powdered generic showed weaker effects. Argatroban was also different in the ACT assay. At a concentration of 5 ug/ml the ACTs were, Arg 340+15.2 secs, S 297+10.5 secs, G 292.0+19.1 secs and A 285.2+21.7 secs. In the citrated whole blood systems, all agents produced a concentration dependent anticoagulant effect; however, the generic versions produced a stronger anticoagulant effect in comparison to branded argatroban (p<0.001). In the PT assay at 5 ug/mL, argatroban showed 32 ± 3 sec vs 40–50 sec for the generic products. Similarly in the aPTT, Heptest and thrombin time tests argatroban was weaker than the generic products. Differences among generic versions were also evident. Similar results were obtained in the retrieved plasma, however the ratio of whole blood over plasma varied from product to product. The IC50 of the generic and branded argatrobans in the TGA were also different. These results show that while in the thrombin inhibition assays generic and branded argatroban may show similar effects, these agents exhibit assay dependent differences in the whole blood and plasma based assays. Such differences may be more evident in the in vivo studirs where endothelial cells and other interactions may contribute to product individuality. Therefore, based on the in vitro antiprotease assays, generic argatrobans may not be considered equivalent and require a multi-parametric study. Currently available generic argatrobans may not be equivalent in the in vivo anticoagulant effects. Therefore, clinical validation of the clinical equivalence for these drugs is warranted. Disclosures: No relevant conflicts of interest to declare.

Effect of Increased Tissue Factor Pathway Inhibitor (TFPI) Levels On Factor Xa Inhibition and Global Hemostasis in the Presence of TFPI-Antagonistic Aptamer BAX 499.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2207-2207 ◽  
Author(s):  
Michael Dockal ◽  
Rudolf Hartmann ◽  
Sabine Knappe ◽  
Michael Palige ◽  
Willibald Kammlander ◽  
...  
Keyword(s):  
Inhibitory Activity ◽  
Whole Blood ◽  
Plasma Levels ◽  
Thrombin Generation ◽  
Anticoagulant Activity ◽  
Reaction System ◽  
Factor Xa ◽  
Model Systems ◽  
Bleeding Tendency ◽  
Fold Excess

Abstract Abstract 2207 In a recent hemophilia clinical trial, patients' TFPI plasma levels substantially increased after intravenous or subcutaneous administration of TFPI-inhibitory aptamer BAX 499. To better understand the consequences of elevated plasma full-length (fl)-TFPI and the potency of BAX 499, the TFPI-inhibitory activity of a wide concentration range of BAX 499 was tested at levels up to 10 nM fl-TFPI in two model systems and in two global hemostatic coagulation assays. Factor Xa (FXa) inhibition assay measured the influence of BAX 499 on the interaction between TFPI and FXa. BAX 499 at 1000 nM efficiently inhibited fl-TFPI within the physiological concentration (< 0.5 nM). However, with increasing TFPI, inhibition of TFPI by BAX499 became considerably less efficient, reflected by increasing EC50 and decreasing maximum inhibition. At substantially elevated TFPI concentrations (10 nM) EC50 increased ∼20-fold, with TFPI retaining ∼70% of its FXa inhibitory activity. Similar results were observed in a more complex reaction system (TF-FVIIa-catalyzed FX activation). These results suggest that BAX 499 is a partial inhibitor of TFPI, efficiently neutralizing TFPI at low concentrations and less efficiently inhibiting TFPI at high TFPI levels. In thrombin generation assays with FVIII-inhibited plasma, a substantial excess of BAX 499 was required to compensate the anticoagulant activity of increasing fl-TFPI. Based on peak thrombin values, fl-TFPI levels that could be neutralized by a given BAX 499 concentration were determined. For example, a ∼50-fold excess of BAX 499 neutralized 0.2 nM TFPI added to FVIII-inhibited plasma, whereas a 140-fold excess was required for neutralization of 7.3 nM fl-TFPI added. An equivalent ROTEM experiment performed in FVIII-inhibited whole blood confirmed these findings. In summary, the data showed that even a high concentration of BAX 499 (1 μM) was not able to counteract the strong inhibitory effect of 10 nM fl-TFPI on FXa, on TF-FVIIa-catalyzed FX activation, on thrombin generation in plasma and clot formation in whole blood, due to the partially inhibitory properties of BAX 499. The current study offers an explanation for increased bleeding tendency in hemophilia patients associated with the ≥25-fold elevated fl-TFPI plasma levels after BAX 499 administration. BAX 499 does not appear to be able to compensate the anticoagulant activity of increased TFPI levels caused by partial inhibition in patients treated with BAX 499. Disclosures: No relevant conflicts of interest to declare.

In Vitro Characterization of a Homogeneous Low-Molecular Weight Heparin with Reversible Anticoagulant Activity

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4263-4263
Author(s):  
Matthew F Whelihan ◽  
Yongmei Xu ◽  
Jian Liu ◽  
Nigel S. Key
Keyword(s):  
Molecular Weight ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Anticoagulant Activity ◽  
Low Molecular Weight ◽  
Thrombin Generation Assay ◽  
In Vitro Characterization ◽  
Fixed Concentration ◽  
Contact Pathway

Abstract Introduction. Due to their increased half-life over unfractionated heparin (UFH) and marked decrease in the incidence of heparin induced thrombocytopenia (HIT), low molecular weight heparins (LMWH) are the most widely prescribed heparin in the US. However owing to their incomplete reversibility with protamine, LMWHs (such as Enoxaparin) carry the risk of bleeding. The synthetic pentasaccharide, Fondaparinux, also lacks a specific antidote. We recently published (Xu et al. Nat. Chem. Biol. 2014) on a new class of synthetic LMWH that is not renal-excreted and offers the benefit of reversal by protamine. The new compound, dubbed “Super 12-mer”, is a 3,483 Da dodecasaccharide consisting of an antithrombin (AT) binding moiety with repeating units of IdoA2S-GlcNS6S (S is sulfate) and two 3-O-sulfate groups which afford the ability to bind protamine. We sought to characterize this new compound in a series of biochemical and global coagulation assays to better characterize its efficacy as a new reversible anticoagulant. Methods. Factor (F) Xa-AT inhibition assays were performed in both purified and plasma-based systems. The Super 12-mer was further tested in a purified prothrombinase system, as well as by tissue factor-initiated thrombin generation assays in contact pathway inhibited citrated plasma. Results. In vitro FXa inhibition studies indicated the IC50 to be 2-fold higher (49 ng/mL, 24 nM) than was previously reported. Nevertheless, the Super 12-mer anti FXa activity was approximately 2-fold greater than Enoxaparin at identical concentrations. However, the anti FXa activities of the Super 12-mer and Enoxaparin in plasma-based systems were roughly equivalent. Prothrombinase experiments indicated that both the Super 12-mer and Enoxaparin were equivalent in their ability to inhibit FXa in complex with FVa. When the two heparinoids were compared in a plasma-based thrombin generation assay (TGA), their effects on thrombin generation were nearly identical with a 50% reduction in peak thrombin generation occurring at approximately 325 nM heparinoid. When protamine is titrated against a fixed concentration of Super 12-mer (625 nM), the Super 12-mer displays a complete reconstitution of thrombin generation. Conclusions. In plasma and purified systems, the Super 12-mer displayed virtually identical efficacy in FXa inhibition compared to Enoxaparin. In buffered systems, the Super 12-mer was approximately 2-fold more active than Enoxaparin against FXa suggesting the Super 12-mer may have other binding partners in plasma. Interestingly, FXa inhibition in prothrombinase was essentially identical between the two heparinoids. Unlike Enoxaparin however, the Super 12-mer displayed near complete reversibility with protamine in TGAs. A significant lag in thrombin generation was observed when protamine was added, consistent with a previous report (Ni Ainle et al. Blood 2009) that protamine itself can act as an anticoagulant by interfering with FV activation. These data show that the Super 12-mer has almost identical efficacy to Enoxaparin in terms of FXa inhibition, while displaying significant reversibility with protamine. Taken together with the fact that this compound can be safely used in renal-impaired patients, the Super 12-mer is a promising new heparanoid anticoagulant with a potentially enhanced safety profile. Disclosures No relevant conflicts of interest to declare.

scholarly journals Bovine Heparin Compared to Porcine Heparin to Demonstrate Bioequivalence

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1253-1253
Author(s):  
Marco Khiella ◽  
Walter Jeske ◽  
Jeanine Walenga ◽  
Omer Iqbal ◽  
Rajan Laddu ◽  
...  
Keyword(s):  
Whole Blood ◽  
Thrombin Generation ◽  
Anticoagulant Activity ◽  
Maximum Amplitude ◽  
Clot Formation ◽  
K Value ◽  
Significant Difference ◽  
Heparin Monitoring ◽  
The U.S

Abstract Introduction: Heparin prevents blood clots from forming in patients undergoing heart surgeries, dialysis, multiple other procedures, and for medical treatment of thrombosis such as associated with cancer. Currently, all heparin products in the U.S. are derived from the intestinal mucosa of pigs. Seventy-five percent of the crude porcine heparin used to make the active pharmaceutical ingredient (API) comes from outside the U.S., with a majority originating from China. Reintroduction of bovine heparin into the U.S. market would expand sources for this critical drug, thus addressing concerns about potential shortages and product adulteration. This study was undertaken to determine the bioequivalence of bovine and porcine heparins using assays relevant to the clinical setting. The assays used in this study were chosen because they overcome limitations of the conventional APTT for heparin monitoring. Selected TEG and ACT assays use whole blood which better simulates physiologic conditions and assesses the full in vitro anticoagulation potential of heparin; these assays are also routinely used clinically for heparin monitoring. The thrombin generation assay is state-of-the-art for hemostatic clinical lab testing, and it provides a more sensitive endpoint of the final generation of thrombin once coagulation is activated. Heparins were studied at concentrations used clinically and tested by assays sensitive to these concentrations. Methods: Bovine heparin API (BH; 7 lots) and US clinical grade porcine heparin (PH: 3 lots) were tested in parallel using recalcified whole blood thromboelastography (TEG; Haemonetics), celite activated clotting time (ACT; Hemochron), and thrombin generation (TGA; Diapharma). Fresh blood was obtained from healthy volunteers under an IRB approved protocol (n=6 per group). Previous work from our lab (Jeske W, Thrombosis & Hemostasis Societies of North America, P57, 2018) identified a weaker potency of BH than PH when compared on an equigravimetric basis in pharmacopeial assays; however, equivalent potency could be obtained when BH was standardized against PH on a unitage basis. In this study, heparins were studied on both a gravimetric and a unitage basis for a comprehensive evaluation. The potency of the BH was determined by pharmacopeial compliant anti-Xa and anti-IIa chromogenic assays cross-referenced to the porcine USP Heparin Reference Standard. Results: All results are depicted in Table 1. For the TEG, the R value, time of latency from start to initial fibrin formation, and the K value, time to achieve a defined clot strength due to thrombin generation and platelet activation, bovine heparin and porcine heparin did not demonstrate a significant difference in anticoagulant activity. The TEG angle, a measure of the speed of fibrin build-up and cross-linking (clot strengthening or rate of clot formation), and the maximum amplitude (MA) value, a function of the maximum of fibrin and platelet binding representing the strongest point of fibrin clot formation, also revealed no significant difference between bovine and porcine heparin anticoagulant activity. For the ACT, at concentrations of 10 µg/mL and 25 µg/mL, there were no statistically significant differences between BH and PH. For the TGA, measuring the time delay until the initiation of thrombin generation following tissue factor (TF) activation, the highest amount of thrombin generated after TF activation, and the total amount of thrombin generated after TF activation (AUC), showed a trend that PH was more potent than BH, but wide variation in the results did not allow for statistical differences to be identified. Conclusion: The results of this investigation demonstrate that bovine heparin produces an equivalent anticoagulant activity as porcine heparin in the TEG, ACT, and TGA assay systems. The equivalent ACT results, in particular, were unexpected since gravimetric amounts of heparins were evaluated. As for all heparins, standardization of bovine heparin in accordance with the process used for porcine heparin will assure equivalent anticoagulant activity among bovine and porcine heparins in whole blood, plasma-based, and pharmacopeial assays. This study further demonstrates that the current assays used to monitor porcine heparin can be similarly used to monitor bovine heparin. Disclosures Walenga: Eurofarma: Research Funding.

scholarly journals Thrombin Generation Capacity of Prothrombin Complex Concentrate in an in Vitro Dilutional Model

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4380-4380
Author(s):  
Henri M.H. Spronk ◽  
Rolf Rossaint ◽  
Henskens M.C. Yvonne ◽  
Rene van Oerle ◽  
Hugo Ten Cate ◽  
...  
Keyword(s):  
Whole Blood ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Coagulation Factors ◽  
Thrombin Generation Assay ◽  
Trauma Induced Coagulopathy ◽  
Healthy Donors ◽  
Ringer’S Lactate ◽  
Generation Capacity

Abstract Abstract 4380 Background: There is a growing use of prothrombin complex concentrates (PCCs) for the treatment of trauma-induced coagulopathy, which is addressed to their propensity to increase thrombin generation. Despite considerable differences in composition of commercially available PCCs, there is lack of data investigating the procoagulant capacity of different PCCs. Methods: The vitamin K-dependent coagulation factors, heparin, and antithrombin were assessed in five commercially available PCCs. The procoagulant potential of the PCCs was assessed in plasma and whole blood from 4 healthy donors by means of classical coagulation assays, thrombin generation assay and thromboelastometry. In order to reflect coagulopathy, whole blood was diluted with 20, 40, 60, and 80% Ringer's lactate solution. Results: The five different PCCs were characterised by comparable levels of factors II, VII, IX and X (all around 20–30 IU/mL), whereas the heparin (0 to 17.6 IU/mL) and antithrombin (0.06 to 1.29 IU/mL) levels were remarkably different between manufactures. In vitro dilution of blood induced a prolongation of the PT and aPTT, and attenuation of thrombin generation and ExTem induced thromboelastometry. Overall, non- or low-heparin containing PCCs restored the in vitro dilutional coagulopathy, whereas PCCs containing heparin has an anticoagulant effect. The thrombin generation assay showed to be the most sensitive method for assessment of PCC effects. Conclusions: This study shows that most available PCCs are not balanced regarding their pro-and anticoagulants. The effect of measured differences in thrombin generation among different PCCs require further investigations to elaborate the clinical meaning in the treatment of trauma induced coagulopathy. Disclosures: No relevant conflicts of interest to declare.

Use of Thrombin Generation Assay As Tool for the Evaluation of the Antithrombotic Samenesse of Enoxaparine Copies

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5092-5092
Author(s):  
Grigorios T. Gerotziafas ◽  
Patrick Van Dreden ◽  
Aurélie Rousseau ◽  
Amir Khaterchi ◽  
Jeanine M. Walenga ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cancer Cells ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Factor Xa ◽  
Inhibitory Potency ◽  
Thrombin Generation Assay ◽  
Pancreatic Cancer Cells ◽  
Branded Product

Abstract Background . The protection rights of LMWHs expired or are expiring, so the extent and nature of the studies required to obtain a market authorization for LMWH copies is an issue of debate. The actual situation, which is characterized by the increasing number of enoxaparin copies available worldwide, makes the definition of criteria for the biological similarity between LMWHs copies and the original product a real need. Aim . The present study, conducted in vitro, offers an analytical approach for the comparison of branded enoxaparin and its copies using a standard pharmacological assay (the measurement of the specific anti-Xa activity) and the calibrated automated thrombogram performed in human platelet rich or platelet poor plasma (PRP and PPP) as well as in an experimental model of hypercoagulability induced by cancer cells. The study aims to provide an integrated rational for the determination of the criteria for the similarity and sameness of branded and copies of enoxaparin. Methods . The studied copies of enoxaparin were Cutenox®, Dilutol®, Enoxa®, Fibrinox®, Loparin®, Lupenox®, Novex®, Noxprin®, Versa®. Samples of PPP and PRP from 15 volunteers were spiked with 2-20 µg/ml of branded (Lovenox®) or copies of enoxaparin. The specific anti-Xa activity in PPP was measured with the Rotachrom® assay. Thrombin generation in PPP and PRP in the presence of tissue factor or pancreatic cancer cells BXPC3 was assessed with the Calibrated Automated Thrombogram. Among thrombogram parametersn the Mean Rate Index (MRI) of TG propagation phase of thrombin generation was analysed. The concentrations inhibiting 50% (IC50) the MRI were calculated. Results . The specific inhibitory potency of enoxaparin copies against factor Xa ranged from 0.072 to 0.088 anti-Xa IU/μg, being lower as compared to that of the branded enoxaparin (0.095 anti-Xa IU/μg). the branded enoxaparin induced a concentration dependent inhibition of thrombin generation. The IC50 values revealed significant differences among the studied copies of enoxaparin and between each one of them and the branded enoxaparin. The potency of each copy of enoxaparin to inhibit thrombin generation as compared to the branded product varied in the three experimental systems. the presence of platelets or pancreatic cancer cells in human plasma induces significant modifications of the inhibitory potency of enoxaparin copies on thrombin generation, which distinguish them not only from the branded enoxaparin but also among them. Conclusion . the copies of enoxaparin which respond to the criteria of sameness to the branded enoxaparin according to the pharmacological criteria endorsed by health authorities demonstrate a significant variability regarding their inhibitory potency on thrombin generation. The presence of platelets and cancer cells significantly influence the variability of the antithrombotic efficiency of the enoxaparin copies as compared to the branded product. The findings of the present study underline the need for the elaboration of functional criteria which evaluate the global antithrombotic capacity of enoxaparin copies in order to evaluate their potential sameness with the branded drug. Disclosures No relevant conflicts of interest to declare.

STUDIES ON THE MECHANISM OF THE ANTITHROMBOTIC ACTION OF A CHEMICALLY SYNTHESIZED HEPARIN PENTASACCHARIDE

1987 ◽  
Author(s):  
L Bara ◽  
J M Walen-ga ◽  
M Petitou ◽  
M Samama ◽  
J Fareed ◽  
...  
Keyword(s):  
Whole Blood ◽  
Thrombin Generation ◽  
Ex Vivo ◽  
Dose Range ◽  
Factor Xa ◽  
Blood Platelet ◽  
Venous Stasis ◽  
Synthetic Substrate ◽  
Inhibition Of Thrombin

A chemically synthesized heparin pentasaccharide (PS) (Instir tut Choay, Paris, France) has been reported to exhibit an antithrombotic action in a rabbit stasis induced thrombosis model, in an IV dose range of 25-200 ug/kg (0.5 to 5 ug/ml circulating concentrations) . Ex vivo plasma analysis from treated animals revealed expected angi-factor Xa activity byboth amidolytic and coagulant (Heptest®) methods. Nodirect inhibitory effect against factor IIa by amidolytic or coagulant methods was observed. Global anticoagulant activities were not found by PT and APTT methods; however, a hypocoagulable thrombelastographic pattern was demonstrated for native whole blood. Platelet activation remained unaffected at the antithrombotic dosages of PS. An attempt was made to more specifically elucidate the anti-factor Xa mediated antithrombotic mechanism of action of PS. The effect of this agent was studied in several thrombin generation assays in human and rabbit plasmas supplemented in vitro with a 0-5 ug/ml concentrations of PS. The following systems were used: 1) activated prothrombin complex (FEIBA®) , thromboplastin-Ca+2/synthetic substrate; 2) prothrombin complex concentrate (Konyne), thrombo- plastin-Ca+2/synthetic substrate; 3) cephalin-ellagic acid-Ca+2/ synthetic substrate (modified Fischer method); 4)cephalin-Ca / synthetic substrate (modified Ofosu method); 5) FEIBA /FPA re- dioimmunoassay; 6)whole blood prothrombin consumption/clot; and 7) cephalin-Ca+2/clot (Hicks-Pitney method). All assays produced a concentration-dependent effect to a 35-50% inhibition of generated thrombin with 4-5 ug/ml PS concentrations. Assay #4 also revealed that although PS inhibited the generation of thrombin, the thrombin that was generated was inhibitedby natural plasma proteins at normal kinetic rates. In ex vivo studies similar concentration-dependent inhibition of thrombin generation was observed using assays #3 and #4. These results indicate that the inhibition of induced venous stasis thrombosis is associated with the inhibition of thrombin generation byPS without a direct inhibition of pre-formed thrombin. Furthermore, these results indicate that thrombotic events may be controlled at pre-thrombin coagulation stages by agents with sole anti-factor Xa activity.

Ancylostoma caninum Anticoagulant Peptide Blocks Metastasis In Vivo and Inhibits Factor Xa Binding to Melanoma Cells In Vitro

1998 ◽  
Vol 79 (05) ◽  
pp. 1041-1047 ◽  
Author(s):  
Kathleen M. Donnelly ◽  
Michael E. Bromberg ◽  
Aaron Milstone ◽  
Jennifer Madison McNiff ◽  
Gordon Terwilliger ◽  
...  
Keyword(s):  
Active Site ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Melanoma Cells ◽  
Factor V ◽  
Ancylostoma Caninum ◽  
Metastatic Activity

SummaryWe evaluated the in vivo anti-metastatic activity of recombinant Ancylostoma caninum Anticoagulant Peptide (rAcAP), a potent (Ki = 265 pM) and specific active site inhibitor of human coagulation factor Xa originally isolated from bloodfeeding hookworms. Subcutaneous injection of SCID mice with rAcAP (0.01-0.2 mg/mouse) prior to tail vein injection of LOX human melanoma cells resulted in a dose dependent reduction in pulmonary metastases. In order to elucidate potential mechanisms of rAcAP’s anti-metastatic activity, experiments were carried out to identify specific interactions between factor Xa and LOX. Binding of biotinylated factor Xa to LOX monolayers was both specific and saturable (Kd = 15 nM). Competition experiments using antibodies to previously identified factor Xa binding proteins, including factor V/Va, effector cell protease receptor-1, and tissue factor pathway inhibitor failed to implicate any of these molecules as significant binding sites for Factor Xa. Functional prothrombinase activity was also supported by LOX, with a half maximal rate of thrombin generation detected at a factor Xa concentration of 2.4 nM. Additional competition experiments using an excess of either rAcAP or active site blocked factor Xa (EGR-Xa) revealed that most of the total factor Xa binding to LOX is mediated via interaction with the enzyme’s active site, predicting that the vast majority of cell-associated factor Xa does not participate directly in thrombin generation. In addition to establishing two distinct mechanisms of factor Xa binding to melanoma, these data raise the possibility that rAcAP’s antimetastatic effect in vivo might involve novel non-coagulant pathways, perhaps via inhibition of active-site mediated interactions between factor Xa and tumor cells.

Assays for Phospholipid-Dependent Formation of Thrombin and Xa: A Potential Method for Quantifying Lupus Anticoagulant Activity

1991 ◽  
Vol 66 (04) ◽  
pp. 453-458 ◽  
Author(s):  
John T Brandt
Keyword(s):  
Specific Activity ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Clinical Importance ◽  
Potential Method ◽  
Quantitative Expression ◽  
Increased Risk ◽  
Apparent Association

SummaryLupus anticoagulants (LAs) are antibodies which interfere with phospholipid-dependent procoagulant reactions. Their clinical importance is due to their apparent association with an increased risk of thrombo-embolic disease. To date there have been few assays for quantifying the specific activity of these antibodies in vitro and this has hampered attempts to purify and characterize these antibodies. Methods for determining phospholipid-dependent generation of thrombin and factor Xa are described. Isolated IgG fractions from 7 of 9 patients with LAs were found to reproducibly inhibit enzyme generation in these assay systems, permitting quantitative expression of inhibitor activity. Different patterns of inhibitory activity, based on the relative inhibition of thrombin and factor Xa generation, were found, further substantiating the known heterogeneity of these antibodies. These systems may prove helpful in further purification and characterization of LAs.

The Anticoagulant Properties of a Modified Form of Protein S

1988 ◽  
Vol 60 (02) ◽  
pp. 298-304 ◽  
Author(s):  
C A Mitchell ◽  
S M Kelemen ◽  
H H Salem
Keyword(s):  
Monoclonal Antibody ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor Xa ◽  
Protein S ◽  
Human Neutrophil Elastase ◽  
Factor X ◽  
Sds Page

SummaryProtein S (PS) is a vitamin K-dependent anticoagulant that acts as a cofactor to activated protein C (APC). To date PS has not been shown to possess anticoagulant activity in the absence of APC.In this study, we have developed monoclonal antibody to protein S and used to purify the protein to homogeneity from plasma. Affinity purified protein S (PSM), although identical to the conventionally purified protein as judged by SDS-PAGE, had significant anticoagulant activity in the absence of APC when measured in a factor Xa recalcification time. Using SDS-PAGE we have demonstrated that prothrombin cleavage by factor X awas inhibited in the presence of PSM. Kinetic analysis of the reaction revealed that PSM competitively inhibited factor X amediated cleavage of prothrombin. PS preincubated with the monoclonal antibody, acquired similar anticoagulant properties. These results suggest that the interaction of the monoclonal antibody with PS results in an alteration in the protein exposing sites that mediate the observed anticoagulant effect. Support that the protein was altered was derived from the observation that PSM was eight fold more sensitive to cleavage by thrombin and human neutrophil elastase than conventionally purified protein S.These observations suggest that PS can be modified in vitro to a protein with APC-independent anticoagulant activity and raise the possibility that a similar alteration could occur in vivo through the binding protein S to a cellular or plasma protein.

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