Amidolytic Assay Research Articles

Factor XII contact activation can be prevented by targeting 2 unique patches in its epidermal growth factor-like 1 domain with a nanobody

BackgroundFactor (F)XII triggers contact activation by binding to foreign surfaces, with the epidermal growth factor-like 1 (EGF-1) domain being the primary binding site. Blocking FXII surface-binding might hold therapeutic value to prevent medical device–induced thrombosis. ObjectivesTo unravel and prevent EGF-1–mediated FXII surface-binding with a variable domain of heavy chain–only antibody (VHH). MethodsFXII variants with glutamine substitutions of 2 positively charged amino acid patches within the EGF-1 domain were created. Their role in FXII contact activation was assessed using kaolin pull-down experiments, amidolytic activity assays, and clotting assays. FXII EGF-1 domain–specific VHHs were raised to inhibit EGF-1–mediated FXII contact activation while preserving quiescence. ResultsTwo unique, positively charged patches in the EGF-1 domain were identified (upstream, 73K74K76K78H81K82H; downstream, 87K113K). Neutralizing the charge of both patches led to a 99% reduction in FXII kaolin binding, subsequent decrease in autoactivation of 94%, and prolongation of clot formation in activated partial thromboplastin time assays from 36 (±2) to 223 (±13) seconds. Three FXII EGF-1–specific VHHs were developed that are capable of inhibiting kaolin binding and subsequent contact system activation in plasma. The most effective VHH “F2” binds the positively charged patches and thereby dose-dependently extends activated partial thromboplastin time clotting times from 29 (±2) to 43 (±3) seconds without disrupting FXII quiescence. ConclusionThe 2 unique, positively charged patches in FXII EGF-1 cooperatively mediate FXII surface-binding, making both patches crucial for contact activation. Targeting these with FXII EGF-1–specific VHHs can exclusively decrease FXII surface-binding and subsequent contact activation, while preserving zymogen quiescence. These patches thus have potential as druggable targets in preventing medical device–induced thrombosis.

Andexanet Alfa Neutralizes the Anticoagulant Effects of Unfractionated Heparin of Bovine, Ovine and Porcine Origin Almost as Protamine Sulfate.

Andexanet alfa (AA) - zhzo, recombinant coagulation factor Xa, is an approved antidote for oral Xa inhibitors (apixaban and rivaroxaban). Unfractionated heparin (UFH) is commonly used for therapeutic, interventional, and surgical indications. Protamine sulfate (PrSO4) is frequently used to neutralize UFH. This study aimed to investigate the comparative neutralization profiles of AA and PrSO4 for heparins of bovine, ovine, and porcine origin. The neutralization effect of PrSO4 at 25 µg/ml and AA at 100 µg/ml was studied on an approximate surgical/interventional concentration of heparin by supplementing whole blood with each of the heparins at 25 µg/ml. For the clotting profile (activated partial thromboplastin time: aPTT), amidolytic (anti-Xa and anti-IIa), and thrombin generation assay each of the heparin were supplemented from -10-0.62 µg/ml. In the whole blood ACT studies, all three heparins produced strong anti-coagulant effects (400-450 seconds) compared to saline (130-150 seconds). Both AA and PrSO4 almost fully neutralized the anti-coagulant effects of heparins (140-160 seconds). Both antidotes completely reversed the anticoagulant effects of all three heparins in the aPTT and thrombin generation assay. However, PrSO4 was more effective in neutralizing the anti-Xa, and anti-IIa effects than AA, which only partially neutralized these effects. Andexanet alfa at 100 µg/ml effectively neutralizes the therapeutic and surgical/interventional concentrations of heparins in in-vitro settings. While differences in the anti-Xa, and anti-IIa effects between heparins were noted, anti-coagulant effect of these agents in the aPTT assay were comparable. A similar neutralization profile was observed in the ACT and thrombin generation assays by both agents.

Specific inhibition on PAI-1 reduces the dose of Alteplase for ischemic stroke treatment

Administration of recombinant tPA (rtPA, or trade name Alteplase®) is an FDA-approved therapy for acute ischemic stroke (AIS), but poses the risk of hemorrhagic complications. Recombinant tPA can be rapidly inactivated by the endogenous inhibitor, plasminogen activator inhibitor 1 (PAI-1). In this work, we study a novel treatment approach that combines a PAI-1 inhibitor, PAItrap4, with a reduced dose of rtPA to address the hemorrhagic concern of rtPA. PAItrap4 is a highly specific and very potent protein-based inhibitor of PAI-1, comprising of a variant of uPA serine protease domain, human serum albumin, and a cyclic RGD peptide. PAItrap4 efficiently targets and inhibits PAI-1 on activated platelets, and also possesses a long half-life in vivo. Our results demonstrate that PAItrap4 effectively counteracts the inhibitory effects of PAI-1 on rtPA, preserving rtPA activity based on amidolytic and clot lysis assays. In an in vivo murine stroke model, PAItrap4, together with low-dose rtPA, enhances the blood perfusion in the stroke-affected areas, reduces infarct size, and promotes neurological recovery in mice. Importantly, such treatment does not increase the amount of cerebral hemorrhage, thus reducing the risk of cerebral hemorrhage. In addition, PAItrap4 does not compromise the normal blood coagulation function in mice, demonstrating its safety as a therapeutic agent. These findings highlight this combination therapy as a promising alternative for the treatment of ischemic stroke, offering improved safety and efficacy.

A novel recombinant human microplasminogen induced complete posterior vitreous detachment without morphological change of retina in juvenile rabbits

Vitreomacular traction syndrome results from persistent vitreoretinal adhesions in the setting of partial posterior vitreous detachment (PVD). Vitrectomy and reattachment of retina is an effective therapeutic approach. The adhesion between vitreous cortex and internal limiting membrane (ILM) of the retina is stronger in youth, which brings difficulties to induce PVD in vitrectomy. Several clinical investigations demonstrated that intravitreous injection of plasmin before vitrectomy could reduce the risk of detachment. In our study, a novel recombinant human microplasminogen (rhμPlg) was expressed by Pichia pastoris. Molecular docking showed that the binding of rhμPlg with tissue plasminogen activator (t-PA) was similar to plasminogen, suggesting rh μPlg could be activated by t-PA to generate microplasmin (μPlm). Moreover, rhμPlg had higher catalytic activity than plasminogen in amidolytic assays. Complete PVD was found at vitreous posterior pole of 125 μg rhμPlg-treated eyes without morphological change of retina in juvenile rabbits via intraocular injection. Our results demonstrate that rhμPlg has a potential value in the treatment of vitreoretinopathy.

Exploring the Role of Antithrombin in Nephrotic Syndrome-Associated Hypercoagulopathy: A Multi-Cohort Study and Meta-Analysis.

Nephrotic syndrome is associated with an acquired hypercoagulopathy that is thought to drive its predisposition for venous thromboembolism. Previous studies have suggested that urinary antithrombin (AT) loss leading to acquired AT deficiency is the primary mechanism underlying this hypercoagulopathy, but this hypothesis has not been directly tested. The objectives of this study were to test the influence of AT levels on hypercoagulopathy in nephrotic syndrome patient samples and perform meta-analyses to evaluate the likelihood of AT deficiency in patients with nephrotic syndrome. Samples from three independent nephrotic syndrome cohorts were analyzed. AT antigen and activity assays were performed using ELISA and amidolytic assays, respectively. Plasma thrombin generation, albumin, and urine protein-to-creatinine ratios were determined using established methods. Meta-analyses were performed by combining these new data with previously published data. AT levels were not consistently related to either plasma albumin or proteinuria. AT was quantitatively related to hypercoagulopathy in adult nephrotic syndrome, whereas AT activity was inconsistently associated with hypercoagulopathy in childhood nephrotic syndrome. Notably, hypercoagulopathy did not differ between patients with normal AT levels and those with levels below the threshold used to define clinical AT deficiency (<70%). Moreover, ex vivo AT supplementation did not significantly alter hypercoagulopathy in AT-deficient plasma samples. The meta-analyses demonstrated that AT deficiency was not a uniform feature of nephrotic syndrome and was more common in children than adults. These data suggest that AT deficiency plays only a limited role in the mechanisms underlying the acquired hypercoagulopathy of nephrotic syndrome. Moreover, AT deficiency was not present in all patients with nephrotic syndrome and was more likely in children than adults despite the higher risk for venous thromboembolism in adults than children.

Enhanced control of bioactivity of tissue plasminogen activator (tPA) through domain-directed enzymatic oxidation of terminal galactose.

Introduction: In targeted enzyme prodrug constructs, it is critical to control the bioactivity of the drug in its prodrug form. The preparation of such constructs often involves conjugation reactions directed to functional groups on amino acid side chains of the protein, which result in random conjugation and incomplete control of bioactivity of a prodrug, which may result in significant nontarget effect. Thus, more specific method of modification is desired. If the drug is a glycoprotein, enzymatic oxidation may offer an alternative approach for therapeutic glycoproteins. Methods: Tissue plasminogen activator (tPA), a model glycoprotein enzyme, was treated with galactose oxidase (GO) and horseradish peroxidase, followed by thiolation reaction and conjugation with low molecular weight heparin (LMWH). The LMWH-tPA conjugate was isolated by ion-exchange chromatography followed by centrifugal filtration. The conjugate was characterized for its fibrinolytic activity and for its plasminogen activation through an indirect amidolytic assay with a plasmin-specific substrate S-2251 when LMWH-tPA conjugate is complexed with protamine-albumin conjugate, followed by triggered activation in the presence of heparin. Results: LMWH-tPA conjugate prepared via enzymatic oxidation retained ~95% of its fibrinolytic activity with respect to native tPA. Upon complexation with protamine-albumin conjugate, the activity of LMWH-tPA was effectively inhibited (~90%) whereas the LMWH-tPA prepared by random thiolation exhibited ~55% inhibition. Addition of heparin fully generated the activities of both conjugates. Conclusion: The tPA was successfully modified via enzymatic oxidation by GO, resulting in enhanced control of its activity in the prodrug construct. This approach can be applied to other therapeutic glycoproteins.

Bio‐equivalence of Potency Adjusted Approved Heparin Solutions Compared to a Newly Developed Heparin Solution

IntroductionPorcine mucosal heparin solutions with a defined potency adjusted concentrations are widely used for the anticoagulation in medical, surgical and interventional indications. Recently, Sintetica has developed a 20.000 IU (48 ml, 416 IU/ml) solution. The active pharmaceutical ingredient used is a porcine mucosal origin with a specific activity of 194 U/mg. The purpose of this investigation is to compare the Sintetica heparin solution with other commercially available potency adjusted heparin solutions such as Fresenius, Roche, Hospira and Medefil heparins to demonstrate their bio‐equivalence in various biological assays.MethodCommercially available heparin solutions were obtained from Sintetica, Roche, Fresenius Kabi, Hospira and Medefil. USP reference standard was obtained from US Pharmacopeia. USP compliant anti‐Xa and anti‐IIa method based on chromogenic substrate assays were obtained from Aniara Diagnostics (Westchester, OH). Thrombin generation studies were carried out using a fluorokinetic method (Diagnostico France). The quantification of glycosaminoglycan (GAG) content was made by using a fluorescence probe (Red Probe, Munster, Germany). The clot based assays and amidolytic assays were carried out on ACL Elite analyzer. HIT mediated platelet aggregation studies were carried out using HIT antibodies from clinically diagnosed patient serum. All solutions were initially diluted to a working concentration of 100 USP U/ml in sterile saline. Working concentrations were prepared at 10 and 1 U/ml. For the potency measurements calibration curves were prepared with the USP standard for cross‐referencing. NHP was supplemented with each of these heparins in 0 to 1 U/ml concentrations. All other tests were carried out according to standard operating procedures.ResultsIn the USP compliant anti‐Xa and anti‐IIa assays all standardized solutions provided comparable results. Similarly in the clot‐based assays such as the aPTT, thrombin time, prothrombinase‐induced clotting time, all solutions produced comparable anticoagulant effects. Similarly in the thrombin generation assays all products produced comparable inhibitory responses of thrombin generation in terms of peak thrombin, area‐under‐the‐curve and the lag time. The HIT antibody mediated platelet aggregation was similar with all solutions. Total GAG content as measured by Red Probe was also comparable with calculated amounts of 0.520 mg/100U for Sintetica, 0.540 mg/100U for Fresenius, 0.510 mg/100U for Hospira, 0.540 mg/100U for Medefil and 0.530 mg/100Us for Roche’s product.ConclusionThese result demonstrate that commercially available potency adjusted heparin solutions and the developed Sintetica heparin solution exhibit comparable anticoagulant and anti‐protease effects. Based on these results the Sintetica product is similar to the commercially available clinically approved potency adjusted heparin solutions. Furthermore these results suggest that these solutions are interchangeable for clinical use.

USP Potency Adjusted Bovine Mucosal Heparins are Comparable to Porcine Mucosal Heparin and May be Interchangeable for Anticoagulation

IntroductionMost of the heparins used clinically are derived from porcine intestinal mucosa. In contrast to the porcine mucosal heparin (PMH), the active pharmaceutical ingredient (API) of bovine mucosal heparin (BMH) exhibit a somewhat weaker USP potency as cross‐referenced against PMH. We hypothesized that at equivalent potencies as adjusted by using the USP reference, the BMH may exhibit comparable effects.Materials &amp; MethodsMolecular weight profile and in vitro anticoagulant and antiprotease assays were used to compare the BMH with PMH. USP potency was measured in the amidolytic assays. Potency adjustments in reference to USP standard were made by weight basis. Potency adjusted BMH and PMH were injected at identical dosages (50 U/kg and 100 U/kg) to groups of human primates (n=4) via intravenous route and blood samples were collected for a 6 hour period of time.ResultsBMH exhibited higher molecular weight profiles compared to PMH as determined by size exclusion chromatography {BMH (Mw) 18.6 ± 0.5 kDa and PMH 15.4 ± 0.4 kDa}. BMH showed a potency of 130 U/mg whereas PMH showed a potency of 190 U/mg. When the BMH was compared at a potency adjusted concentration with PMH, it showed identical calibration curves in the aPTT and anti‐protease assays. Mean t1/2 ranged from 54 ± 11 min &amp; to 71 ± 18 min for PMH &amp; BMH respectively (p=&gt;0.05). Mean AUC values based on anti‐Xa or anti‐IIa activities were comparable for both heparins. Mean Vd (~60 ml/kg) and Cl (~0.75 ml/kg/min) were also comparable for both heparins.ConclusionPotency adjusted based dosing results in comparable anticoagulants and pharmacokinetic profiles for BMH and PMH. Therefore, such dosing may provide uniform levels of anticoagulation for the parenteral indications for heparins. These observations suggest that potency equated BMH and PMH produced comparable anticoagulant effects and may be interchangeable.

Reference values of coagulation assays performed for thrombophilia screening after a first venous thrombosis and their intra-patient associations

IntroductionNo reference values are currently available for coagulation assays performed for thrombophilia screening prescribed according to guidelines, after a first venous thromboembolic (VTE) event, and we have no idea of the intra-patient associations between results. MethodsWe performed a retrospective study of consecutive prescriptions fulfilling guidelines in a French university hospital from 2010 to 2019 (n = 3842) from the Glims® laboratory information system. We collected results of 12 parameters: aPTT, PT, fibrinogen (Fg), one-stage clotting methods for factors VIII, IX, XI and II (FVIII, FIX, FXI, FII), antithrombin (using an amidolytic assay: AT), protein C and S (using clotting assays: PC and PS) and mixing tests of a lupus-anticoagulant sensitive aPTT and of DRVVT. ResultsWe show the results of the 12 parameters from 3603 individual files with less than 6 missing values, then describe these distributions and correlations between results from 2930 files with no missing value. We give the frequency of results described as indicating a risk of first VTE or of VTE recurrence. We propose 2 quantitative scores linking the 12 parameters at the individual level and reflecting their degree of dispersion with respect to their mean, describe the values of these scores and their associations with thrombophilic results. ConclusionsThese normal values should help laboratory workers to validate process results and to assess their degree of originality. Our 2 scores should help to determine the intra-patient plausibility of associations of results. The usefulness of these laboratory scores for predicting clinically-relevant outcomes deserves to be investigated.

Screening of the Promising Direct Thrombin Inhibitors from Haematophagous Organisms. Part I: Recombinant Analogues and Their Antithrombotic Activity In Vitro.

The success in treatment of venous thromboembolism and acute coronary syndromes using direct thrombin inhibitors has stimulated research aimed at finding a new anticoagulant from haematophagous organisms. This study deals with the comparison between hirudin-1 from Hirudomedicinalis(desirudin), being the first-known and most well-studied natural anticoagulant, along with recombinant analogs of haemadin from the leech Haemadipsa sylvestris, variegin from the tick Amblyomma variegatum, and anophelin from Anopheles albimanus. These polypeptides were chosen due to their high specificity and affinity for thrombin, as well as their distinctive inhibitory mechanisms. We have developed a universal scheme for the biotechnological production of these recombinant peptides as pharmaceutical substances. The anticoagulant activities of these peptides were compared using the thrombin amidolytic activity assay and prolongation of coagulation time (thrombin time, prothrombin time, and activated partial thromboplastin time) in mouse and human plasma. The preliminary results obtained suggest haemadin as the closest analog of recombinant hirudin-1, the active substance of the medicinal product Iprivask (Aventis Pharmaceuticals, USA) for the prevention of deep venous thrombosis in patients undergoing elective hip or knee replacement surgery. In contrast, variegin can be regarded as a natural analog of bivalirudin (Angiomax, The Medicines Company), a synthetic hirudin-1 derivative certified for the treatment of patients undergoing percutaneous coronary intervention and of patients with unstable angina pectoris after percutaneous transluminal coronary angioplasty.

USP Standardized Mixtures of Bovine, Ovine and Porcine Heparin Exhibit Comparable Biologic Effects to Referenced Single Sourced Heparins and May be Interchangeable,

Introduction: Unfractionated heparin (UFH) is the first line anticoagulant for the management of medical indications. UFH complexes with antithrombin to produce strong inhibition of thrombin and factor Xa. The UFHs are standardized using USP compliant amidolytic anti-Xa and IIa methods in defined conditions. Clinically used UFH is solely sourced from porcine mucosal tissue. Because of the shortage of porcine tissue and the African Swine Fever, the supply chain of this anticoagulant is compromised. Thus, there is a need for resourcing of this anticoagulant. Bovine and ovine mucosal sources represent alternate material for production of UFH. Previous studies have shown that bovine and ovine UFH exhibit anticoagulant effects which can be standardized by using the USP method. Additionally, the standardized heparins from various sources can be blended and their potency can be adjusted to exhibit comparable effects as the single sourced UFH. The purpose of this study is to evaluate the pharmacologic profile of the blended heparin and compare these activities to that of the single sourced porcine, ovine and bovine heparins.Methods: Two groups of heparins were evaluated in this study, porcine, ovine, bovine, and the blended heparin in gravimetric measurements (ug/ml) and these same four in potency adjusted measurements (U/ml). The pharmacologic profiles of the heparins in this study were investigated via global anticoagulant assays and anti-protease assays performed in plasma. Clot based assays such as the activated partial thromboplastin time (aPTT) and thrombin time (TT) were used to study the anticoagulant effects of the single source and blended heparins. The amidolytic anti-Xa and IIa assays were used to assess the inhibitory effects of these heparins on these proteases. USP compliant anti-Xa and IIa assays were used to determine potencies of the various heparins. Protamine sulfate (PS) neutralization studies were performed to evaluate the reversal of anticoagulant effects in each of the heparins.Results: The aPTT assay showed that at final concentrations of 5 ug/ml and 2.5 ug/ml porcine heparin significantly (p < .01) prolonged the aPTT compared to ovine, bovine, and blended heparins. When studied with potency adjusted heparins, all heparins demonstrated comparable aPTT values at all concentrations (U/ml). The TT assay showed that porcine and ovine heparins prolonged the TT at 1.25 ug/ml compared to bovine and blended heparins. When studied with potency adjusted heparins, all heparins demonstrated comparable TT values at all concentrations (U/ml). The anti-Xa assay showed that at all final concentrations between 10 ug/ml and 0.625 ug/ml porcine, ovine, and blended heparins produced significantly (p <.001) stronger Xa inhibition than bovine heparin. When studied with potency adjusted heparins, all heparins demonstrated comparable anti-Xa inhibition at all concentrations (U/ml). The anti-IIa assay showed that at final concentrations 2.5 ug/ml, 1.25 ug/ml, and 0.625 ug/ml porcine and ovine heparins produced significantly (p < .05) stronger IIa inhibition than bovine heparin. When studied with potency adjusted heparins, all heparins demonstrated comparable anti-IIa inhibition at all concentrations (U/ml). The USP compliant anti-Xa assay with gravimetric heparins showed potencies of 201, 201, 150, and 184 U for porcine, ovine, bovine, and blended heparins respectively. The USP compliant anti-Xa assay with potency adjusted heparins showed comparable potencies for all four heparins. The USP compliant anti-IIa assay with gravimetric heparins showed potencies of 204, 196, 127, and 167 U for porcine, ovine, bovine, and blended heparins respectively. The USP compliant anti-IIa assay with potency adjusted heparins showed comparable potencies for all four heparins. The protamine sulfate neutralization studies demonstrated complete neutralization at all concentrations for all of the potency adjusted heparins in the aPTT, TT, anti-Xa, and anti-IIa assays.Conclusion: These studies support the hypothesis that a blended heparin product from bovine, ovine, and porcine tissue, when standardized in USP unit-equivalent proportions, exhibits a comparable anticoagulant profile to the single species heparins. These findings suggest that there is a potential for development of blended heparin to stabilize supply chain of this important anticoagulant and warrant clinical validation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Impact of LMWH and Specific Factor Xa Inhibitors, Apixaban and Fondaparinux, on Cancer Cell Biology and Procoagulant Properties of Cancer Microenvironment

Background Cancer patients with venous thromboembolism (VTE) or at risk of VTE are treated with antithrombotic agents. Cancer cells express procoagulant properties and induce hypercoagulability in the microenvironment, that could impact the efficiency of the antithrombotic agents.Aims In the present study, we investigated the interaction between antithrombotic agents with pancreatic cancer cells, as well as with their microenvironment. The impact of apixaban, fondaparinux, enoxaparin and tinzaparin on the procoagulant properties of pancreatic cancer cells BXPC3 was examinated. Reciprocally, we also investigated the impact of BXPC3 on the potency of these antithrombotic agents.Methods BXPC3 cells (400 cells/μl) were exposed for 48 hours to apixaban (2 µg/ml), fondaparinux (2 µg/ml), enoxaparin, tinzaparin (2 anti-Xa IU/ml) or NaCL (control). Then, conditioned media (CM) and BXPC3 cells were harvested, separated and put in contact with normal platelet-poor plasma (PPP). Subsequently, thrombin generation (TG) was assessed using Thrombogram-Thrombinoscope® assay (Diagnostica Stago). Cells' viability was also assessed with the MTT assay. Gene expression for Tissue Factor (TF), Vascular Endothelial Growth Factor (VEGF), Thrombospondin 1 (THSB1) was assessed with RT-qPCR at the cells exposed or not to the antithrombotic agents. Expression of TF protein and activity of cancer cells was assessed using ELISA method. Residual anti-Xa activity in CM was measured using specific amidolytic assays for each antithrombotic agent.Results Apixaban, fondaparinux, enoxaparin, and tinzaparin significantly reduced cell viability by 25%, 12%, 14%, and 11% respectively. In the control experiment non treated BXPC3 cells enhanced TG. Pre-treatment of BXPC3 with the antithrombotic agents did not significantly modify their capacity to trigger and enhance TG. Among the studied agents only apixaban resulted in significant decrease of TF mRNA expression. However, protein expression of TF was not significantly modified by any of the antithrombotic agents. VEGF's mRNA expression was significantly decreased by fondaparinux and enoxaparin. THBS1's mRNA expression was significantly increased by apixaban. The concentrations of the anti-Xa activity of fondaparinux, enoxaparin and tinzaparin in the CM obtained at 48h after exposure of cells were reduced by 27%, 48% and 26% respectively as compared to those initially added in the culture medium. In contrast, the concentration of apixaban in the CM did not significantly change. The CM obtained by cells exposed to apixaban, fondaparinux, enoxaparin and tinzaparin inhibited TG by 70%, 30%, 40% and 90% respectively.Conclusion. Antithrombotic agents reduced the viability of BXPC3 cells. Among the studied agents, apixaban had the most pronounced effect on cells' viability. The antithrombotic agents had a potential downregulating effect on the proangiogenetic properties of BXPC3 via the decrease of VEGF gene expression (fondaparinux and enoxaparin) and enhancement of THBS1 gene expression (apixaban). Nevertheless, preincubation of BXPC3 with the antithrombotic agents did not alter the expression of TF protein and their effect on thrombin generation. Moreover, BXPCE exerted a “degradation” effect on LMWH and fondaparinux. Apixaban appeared to escape from this effect of the cancer cells. A significant inhibitory effect on thrombin generation was exerted by the residual concentrations of the antithrombotic agents in the microenvironment of cancer cells. The ensemble of these data highlight for the first time that the presence of antithrombotic agents in cancer cell microenvironment alters the biology of cancer cells and offer a constant antithrombotic effect in the microenvironment. DisclosuresNo relevant conflicts of interest to declare.

Inhibition of Thrombin and Its Generation By Recombinant Thrombomodulin and Activated Protein C in Whole Blood and Plasma Based Systems

Introduction:Recombinant thrombomodulin (rTM) and activated protein C (APC) are important modulators of coagulation and fibrinolysis. Both agents have been developed for various clinical indications. Beside their direct effect on coagulation, these agents also modulate other components of hemostatic processes to mediate their pharmacologic actions such as the direct inhibition of proteases and their generation.Aim:The aim of this study is to compare the differential inhibition of thrombin and its generation by rTM (ART-123, Asahi Kasei, Tokyo, Japan) and a plasma derived APC (Hematologic Technologies, Burlington, VT, USA).Methods:The inhibitory effects of APC and rTM on thrombin were measured in a plasma based and fluorogenic substrate assays. In the whole blood studies, native blood from healthy volunteers (n=10) was supplemented with rTM and APC at concentrations levels of 0-1.0 ug/ml. Thrombin generation was triggered by adding diluted APTT activator (Triniclot) and the reaction was stopped after 3 minutes. Thrombin generation markers such as F1.2 and TAT were measured using commercially available ELISA methods. In the citrated plasma studies both agents were supplemented at 0-1.0 ug/ml. Thrombin generation studies were carried out using a flouremtric assay (TGA, Techniclone, Vienna, Austria). Lag time peak thrombin generation and area under the curve (AUC) were measured. The IC50 values for each of the agents was calculated.Results:APC did not have any direct inhibitory effects on thrombin in both the clotting and amidolytic assays. However, rTM produced a concentration dependent inhibition of thrombin in both the clot based and amidolytic assays. In the whole blood systems both the APC and rTM produced a concentration dependent inhibition of thrombin generation markers such as F1.2 and TAT. APC produced a slightly stronger inhibition of stronger inhibition of TG as measured by F1.2, IC50 = 0.55 ug/ml compared with thrombomodulin, IC50 = 0.70 ug/ml. In the plasma based system the IC50 for peak thrombin inhibition was 0.46 ug/ml for APC and 0.97 ug/ml for rTM. Additional differences in the lag phase and AUC profiles were also noted. Conclusions: Beside the direct anticoagulant effects both the APC and rTM are capable of inhibiting the TF mediated thrombin generated in whole blood and plasma based systems. APC exhibits slightly stronger inhibitory effects in TG assays in both plasma and whole blood. .Conclusions:In contrast to APC, rTM produces direct inhibitory effects on thrombin in both the amidolytic and clot based assays. Both APC and rTM are capable of inhibiting the TF mediated thrombin generation in whole blood and plasma based systems. APC exhibits slightly stronger inhibitory effects in thrombin generation assays in both plasma and whole blood. These studies suggest that modulation of thrombin and other proteases contribute to the clinical effects of both rTM and APC. DisclosuresTanaka:Asahi Kasei Pharma America Corporation: Employment. Tsuruta:Asahi Kasei Pharma America Corporation: Employment.

Myeloperoxidase has no effect on the low procoagulant activity of silica-free DNA

Blood coagulation and innate immunity are closely interrelated. At sites of inflammation, DNA and myeloperoxidase (MPO) are released from polymorphonuclear leukocytes (PMNs) as an integral component of neutrophil extracellular traps (NETs). NETs exert pleiotropic thrombogenic effects, with DNA-mediated contact activation of factor XII (FXII) likely playing a role. We have previously shown that MPO, a highly cationic protein, regulates coagulation through heteromolecular interactions with various negatively charged structures, including membrane phospholipids and low-molecular-weight heparin. The aims of our current study were to confirm that DNA activates coagulation and to investigate whether its procoagulant activity (PCA) is regulated by PMN-derived MPO. To this end, we used thrombin generation and FXIIa amidolytic activity assays to analyze the PCA of cell-free DNA isolated with silica membrane-based (cfDNA) or silica-free procedures (PaxDNA). cfDNA potently activated FXII and promoted thrombin generation in a concentration-dependent manner, but its PCA was largely attributable to contaminating silica particles. In contrast, pure, i.e. silica-free, PaxDNA was markedly less procoagulant. Although PaxDNA amplified thrombin generation in plasma, it was devoid of any direct FXII activating activity. MPO supershifted both cfDNA and PaxDNA in gel electrophoresis, but only silica-associated PCA of cfDNA was neutralized by MPO independently of its catalytic properties. Moreover, pretreatment with DNase I abolished silica-induced thrombin generation. In summary, we show that pure DNA has rather weak PCA, which is not further inhibited by heteromolecular complex formation with exogenous MPO. Our study thus provides novel mechanistic insights into the regulation of coagulation by extracellular DNA under inflammatory conditions.

Thrombin in the Activation of the Fluid Contact Phase in Patients with Hereditary Angioedema Carrying the F12 P.Thr309Lys Variant.

Hereditary angioedema due to pathogenic FXII variants (HAE-FXII) is a rare dominant disease caused by increased activation of the plasma contact system. The most prevalent HAE-FXII variant, c.1032C > A p.Thr309Lys (FXII309Lys), results in a smaller FXII protein with increased sensitivity to fluid-phase activation by poorly understood mechanisms. We aimed to investigate the functionality of the FXII309Lys variant in 33 HAE-FXIIpatients, 25 healthy controls and 46 patients with congenital disorders of glycosylation (CDG). Activation of the plasma contact system was assessed by western blot and amidolytic assay in basal conditions or after treatment with either artificial or physiological activators. Recombinant wild-type and FXII309Lys variants were expressed in S2 insect (Drosophila) cells. Amidolytic and fibrin generation assays were performed in fresh plasma samples. FXII309Lys samples exhibited an increased electrophoretic mobility comparable with N-glycan-deficient FXII from CDG patients and asialo-FXII generated by neuraminidase treatment. They presented increased sensitivity to activation by dextran sulphate and silica which resulted in the generation of an aberrant 37-kDa heavy chain. We did not observe increased susceptibility of FXII309Lys to proteolysis by exogenous or tPA-generated plasmin. However, both exogenous and endogenous thrombin cleaved the FXII309Lys variant, releasing a 37-kDa fragment and resulting in enhanced proteolytic activation on the fluid phase. This model supports a sequential proteolytic activation process involving thrombin priming of FXII309Lys, followed by kallikrein cleavage and generation of active βFXIIa. The present results and the observation that angioedema episodes in HAE-FXII patients occur predominantly during hypercoagulable situations suggest a key role for thrombin.

Extracts from Uncaria tomentosa as antiplatelet agents and thrombin inhibitors – The in vitro and in silico study

Ethnopharmacological relevanceA wide range of traditional medicine applications of Uncaria tomentosa (Willd. ex Schult.) DC., commonly known as ‘vilcacora’ or ‘cat's claw’, includes blood purification, its anticoagulant properties and its use in haemorrhage therapy. Aim of the studyOur work is devoted to the effects of ethanol and aqueous extracts (1–50 μg/ml) from U. tomentosa leaves and bark on the haemostatic system. The study is based on two main questions: Can these extracts influence the coagulation cascade of blood plasma or the activation of blood platelets? Do they feature any anticoagulant properties? Materials and methodsBlood platelet aggregation was measured in human platelet-rich plasma; the anticoagulant tests were based on the thrombin, prothrombin and the activated partial thromboplastin time. For the thrombin (TH)-inhibitory activity evaluation, the chromogenic substrate S-2238 and fibrinogen, i.e. physiological substrate for this enzyme, were used. In silico studies included the interactions of TH and the main components of the extracts. ResultsThe examined extracts demonstrated slight antiplatelet activity. The thrombin time was slightly prolonged. The most efficient TH inhibitor was the ethanolic fraction from leaves (IC50 = 5.86 and 12.48 μg/ml, for the amidolytic and proteolytic assay, respectively). The plant ingredients interacted with TH within and outside the active site, dependently on the compound. The higher binding affinity was found for procyanidins B2 and C1. ConclusionsThe examined extracts demonstrated slight antiplatelet effects; however, they may be promising candidates for the natural inhibitors of TH, which is critical for the formation of fibrin clot.

Compound heterozygous protein C variants undetectable by common laboratory testing causing purpura fulminans after the neonatal period.

Compound heterozygous protein C variants undetectable by common laboratory testing causing purpura fulminans after the neonatal period.

Antithrombin III-mediated blood coagulation inhibitory activity of chitosan sulfate derivatized with different functional groups

Acylated chitosan sulfate (ChS1), a sulfated polysaccharide with high anticoagulant activity, was chemically synthesized and structurally characterized using FT-IR analysis. The beneficial structural properties and high availability of the sulfate group in ChS1 led to greater anticoagulant activity through both the intrinsic and common pathways with antithrombin III (AT III)-mediated inhibition, particularly involving coagulation factors FXa and FIIa. The analysis of the binding affinities using surface plasma resonance found that the equilibrium dissociation constant (KD) of ChS1 for FXa and FIIa in the presence of AT III was 67.4 nM and 112.6 nM, respectively, indicating the stronger interaction of the AT III/ChS1 complex with the ligands and the inhibition of activated FX and FII. The results of amidolytic assays further demonstrated the stronger inhibition of the proteolytic conversion of factor X by the intrinsic FXase complex and of FII by the prothrombinase complex. Molecular docking analysis further validated the protein-ligand interactions of ChS1 with AT III and their binding affinity.

Nephrotic syndrome-associated hypercoagulopathy is alleviated by both pioglitazone and glucocorticoid which target two different nuclear receptors.

BackgroundThrombosis is a potentially life‐threatening nephrotic syndrome (NS) complication. We have previously demonstrated that hypercoagulopathy is proportional to NS severity in rat models and that pioglitazone (Pio) reduces proteinuria both independently and in combination with methylprednisolone (MP), a glucocorticoid (GC). However, the effect of these treatments on NS‐associated hypercoagulopathy remains unknown. We thus sought to determine the ability of Pio and GC to alleviate NS‐associated hypercoagulopathy.MethodsPuromycin aminonucleoside‐induced rat NS was treated with sham, Low‐ or High‐dose MP, Pio, or combination (Pio + Low‐MP) and plasma was collected at day 11. Plasma samples were collected from children with steroid‐sensitive NS (SSNS) and steroid‐resistant NS (SRNS) upon presentation and after 7 weeks of GC therapy. Plasma endogenous thrombin potential (ETP), antithrombin (AT) activity, and albumin (Alb) were measured using thrombin generation, amidolytic, and colorimetric assays, respectively.ResultsIn a rat model of NS, both High‐MP and Pio improved proteinuria and corrected hypoalbuminemia, ETP and AT activity (p < .05). Proteinuria (p = .005) and hypoalbuminemia (p < .001) were correlated with ETP. In childhood NS, while ETP was not different at presentation, GC therapy improved proteinuria, hypoalbuminemia, and ETP in children with SSNS (p < .001) but not SRNS (p = .330).ConclusionsBoth Pio and GC diminish proteinuria and significantly alleviate hypercoagulopathy. Both Pio and MP improved hypercoagulopathy in rats, and successful GC therapy (SSNS) also improved hypercoagulopathy in childhood NS. These data suggest that even a partial reduction in proteinuria may reduce NS‐associated thrombotic risk.

A comparative evaluation of the CN-6000 haemostasis analyser using coagulation, amidolytic, immuno-turbidometric and light transmission aggregometry assays.

BackgroundThe CN‐6000 (Sysmex Corp.) is a new haemostasis analyser with blood coagulation, amidolytic, immuno‐turbidometric and light transmission aggregometry (LTA) capabilities. Transmitted light is monitored at multiple wavelengths (340, 405, 575, 660, 800 nm), from an LED light source.AimsTo evaluate the performance of the CN‐6000 against a predicate device.MethodsThe CN‐6000 was evaluated against the CS‐5100 (Sysmex) for 14 different tests, using 880 samples from normal subjects, anticoagulated patients, critically ill patients, plasmas with high or low fibrinogen content or abnormal levels of interfering substances. Between‐day assay imprecision was assessed using commercial QC materials (n = 10 replicates on each of 5 days).ResultsAcceptable levels of imprecision were obtained for all assays. Agreement between the two analysers was excellent for all assays. Throughput was 35% higher using the CN‐6000 (337 vs 250 tests per hour for PT, aPTT and fibrinogen). The CN‐6000 also demonstrated improved clot detection in plasmas with high levels of interfering substances as demonstrated by a 29% reduction in “vote‐outs” due to low light transmission (24 vs 34).ConclusionsThe CN‐6000 demonstrated excellent comparability with the predicate instrument and acceptable levels of imprecision in all assays. Improvements in throughput and clot detection in the presence of interfering substances were also shown.