Anti-factor IIa Research Articles

Establishment and Validation of an Automated System for the Antifactor IIa Assay: A Case Study of Potency Assessment of a Pharmaceutical Gel Formulation.

Antithrombotic agents and anticoagulant drugs, such as those from the heparin family, are employed in clinical settings for the prevention and treatment of clotting, thromboembolism, and wound healing. The potency assessment of antithrombotic agents is typically conducted using antifactor IIa assay with manual systems which are time-consuming and often lack repeatability. Here, we present a novel automated system that significantly enhances assay repeatability, attaining an outstandingly low relative standard deviation (RSD) % of only 0.6% for repeatability. This system has been applied to a pharmaceutical gel formulation for wound healing developed by Abdi Ibrahim Pharmaceuticals R&D Center as a case study for validation. The automated system demonstrated substantial improvements over manual systems in linearity (R2 = 0.9927), precision, accuracy, specificity, and robustness. The system aligns with the European Pharmacopoeia specifications, promising to enhance quality control across pharmaceutical formulations and conduct absorbance-based end-point assays within the pharmaceutical industry while offering increased throughput and cost-effectiveness.

Chromogenic and Clot-Based Bivalirudin Assays for Monitoring Anticoagulation.

Direct thrombin inhibitors (DTIs) are usually monitored with the activated partial thromboplastin time (aPTT) or activated clotting time (ACT). Both are complex assays with multiple enzymatic steps, and performance may be influenced by physiologic and pathologic factors unrelated to the DTI. Simpler systems, such as clot-based dilute thrombin time (dTT) and chromogenic anti-factor IIa assays, have been developed for monitoring DTIs, but there is limited data on their performance in clinical settings. Medical records of patients who received bivalirudin between March 2020 and April 2022 at a single institution were reviewed for demographic data and adverse outcomes. Plasma samples drawn for aPTT testing were analyzed with chromogenic anti-IIa and dTT bivalirudin assays. Results were compared to bivalirudin dosing. Results of aPTT assays from 32 patients were compared with the chromogenic (n = 136) and dTT (n = 120) bivalirudin assays. Correlations between the aPTT and the chromogenic and dTT assays were poor (Spearman coefficients 0.55 and 0.62, respectively). There was a stronger correlation when results of the chromogenic and dTT assays were compared to each other (Spearman coefficient 0.92). When assay results were compared to bivalirudin dose, there were stronger correlations with the chromogenic and dTT assays than with the aPTT (Spearman coefficients 0.51, 0.63 and 0.22, respectively). There was considerable variation between results of specific bivalirudin assays and the aPTT. While bivalirudin assay results correlated better with administered drug dose, suggesting improving reliability, more studies are needed to determine if there is correlation between testing and clinical outcomes.

Potency evaluation of unfractionated heparins commercialized in Brazil through anti-factor Xa and anti-factor IIa chromogenic tests and coagulation assay

Potency evaluation of unfractionated heparins commercialized in Brazil through anti-factor Xa and anti-factor IIa chromogenic tests and coagulation assay

Population pharmacokinetic analysis for dabigatran etexilate in Chinese patients with non-valvular atrial fibrillation.

We aimed to develop a pharmacokinetic (PK) and pharmacodynamic (PD) model from healthy Chinese subjects and real-world non-valvular atrial fibrillation (NVAF) patients. We also investigated meaningful intrinsic and extrinsic factors and related biomarkers for bleeding events. We characterized the integrated PK/PD models based on rich PK/PD data [dabigatran concentration, activated partial thromboplastin time (APTT), prothrombin time (PT), and anti-factor IIa (anti-FIIa) activity] from 118 healthy volunteers and sparse PD data [APTT, PT, and anti-FIIa] from 167 patients with NVAF after verifying the model extrapolation performance. We also documented the correlations between PD biomarkers and clinically relevant bleeding events over one year. Next, we used the final integrated PK/PD model (a two-compartment, linear model with first-order absorption) to evaluate the influence of dosage and individual covariates on PD parameters. The age, high-density liptein cholesterol (HDL-C), and creatinine clearance (CrCL) improved the PK model fit. The linear direct-effects PD model described the correlation between APTT, PT, and anti-FIIa and plasma concentration. CrCL improved the PD model fit. Anti-FIIa was more sensitive to the increase in dabigatran exposure than APTT and PT in the PD model. Therefore, fixed dabigatran doses could be prescribed for patients with NVAF without adjusting for age and HDL-C. We observed an elevated bleeding tendency with higher peak and trough values of APTT, PT, and anti-FIIa. Randomized studies should be performed to evaluate the efficacy and safety of low-dose dabigatran in Chinese patients with NVAF.

Deaminative-cleaved S. monotuberculatus fucosylated glycosaminoglycan: Structural elucidation and anticoagulant activity

Stichopus monotuberculatus is a tropical sea cucumber species and used as a folk medicine and tonic food. In this study, a fucosylated glycosaminoglycan (SmFG), the depolymerized SmFG (dSmFG) and its oligosaccharide fractions were prepared. The SmFG and its depolymerized products were comprised of a chondroitin-sulfate-E backbone, and various sulfated fucose side chains, including an unusual disaccharide side chain connected to the C-3 position of D-glucuronic acid (GlcA) or GlcA-ol. A peeling reaction occurred during the deaminative depolymerization process. The dSmFG and its fractions showed strong anticoagulant activity by selectively inhibiting intrinsic tenase complex, and had no anti-factor IIa, Xa and VIIa activity. The anticoagulant activity reduced with the decrease of molecular weight, and the unusual branch and novel reducing end may enhance the anticoagulant activity. These findings can provide significant information for development and utilization of depolymerized products from SmFG in food and pharmaceutical industries.

Enzymatic synthesis of low molecular weight heparins from N-sulfo heparosan depolymerized by heparanase or heparin lyase

Low-molecular-weight heparin (LMWH) is prepared from the controlled chemical or enzymatic depolymerization of animal sourced heparins. It has been widely used as an anticoagulant. Concerns about the shortcomings of animal-derived heparin and the contamination of supply chain demand biochemical approaches for synthesizing LMWH. In the present study, two LMWHs were enzymatically synthesized from low molecular weight N-sulfated heparosan (LMW-NSH) cleaved by recombinant hydrolase, endo-β-glucuronidase, (HepBp) or heparin lyase III (HepIII), followed by subsequent sulfotransferase modifications. Structural characterization shows that LMWH chains prepared using HepBp had a saturated uronic acid residue at their reducing ends, while chains of LMWH prepared using HepIII had an unsaturated uronic acid residue at their non-reducing end. Both LMWHs had anti-factor Xa and anti-factor IIa activities comparable to enoxaparin. This approach demonstrates that the hydrolase, HepBp, can be used to prepare a new type of LMWH that has no unsaturated uronic acid at its non-reducing end.

Analysis of Inter-Observer Agreement of Adjudication of Colors of Pad Colors of Doac Dipstick to Determine Presence or Absence of Direct Oral Anticoagulants in Outpatients' Urine Samples

Background: The efficacy and safety of direct oral anticoagulants (DOACs) in patients with thromboembolic disease is closely related to patient's adherence to therapy. Objective documentation of drug intake is a useful tool for patients' education and improvement of adherence to treatment but may be improved by accurate point-of-care (POCT) testing. Results of DOAC Dipstick may be one of these methods but may depend on interpretation variability.Aim: We aim to analyze the inter-observer agreement of DOAC Dipstick near-patient device in outpatients on stable anticoagulation with rivaroxaban (R), apixaban (A) and dabigatran (D).Methods: A prospective observational cohort study was performed including patients on active treatment with R, A, and D for secondary VTE prevention. All participants were routinely assessed for DOACs' plasma concentration using STA ® Liquid anti-Xa and STA ® Liquid anti-IIa chromogenic assays , and creatinine clearance (Cockroft -Gault equation). DOAC Dipstick test was performed in patients' urine samples were performed by trained staff according the instructions for use. The assessment was based on pads'colors which are specific for indicating the presence and absence of factor -Xa (FXA) and thrombin inhibitors (THI). In order to assess inter-observer agreement, the study nurse performing the test and the medication-prescribing physician were in charge of evaluating independently each test strip. THI pad and FXA pad served as negative control for patients treated with R and A, and D, respectively. Inter-observer agreement was calculated by Cohen's kappa coefficient (kappa index).Results: A new interim analysis shortly before study termination was performed after enrolment of 79 patients (female/ male 44/35, age 56 ± 18 years, mean and standard deviation). Of those, 20% (n=17) were treated for deep vein thrombosis, 13% (n=10) for pulmonary embolism (PE), 40% (n=32) for recurrent thromboembolic disease, 18.3% (n=13), for cancer-associated thrombosis, 6 for antiphospholipid syndrome (n= 5), 2 % (n=2) for AF. 60 % (n=48 ) were treated with R, 37% (n=29) with A and 3% (n=2) with D. All patients had normal renal function. Anti-factor Xa levels were determined with a median value of 156.6±129.2 ng/ml and anti-factor IIa levels with 191.66±110.34 ng/mL. The inter- observer agreement of colors of FXA and THI pads of urine samples was 0.99 for positivity for R, A, and D. Pads that served as negative controls were assessed correctly as negative by both observers in all cases (kappa index 1.0).Conclusion: Given the encouraging results, the ongoing study should allow the device's validation as an accurate, easy-to-use assessment tool for determination of the presence or absence of DOACs in patients' urine samples also based on a very low inter-observer variability. The aforementioned data confirm the results of the first interim analysis presented in the ISTH 2021 congress. DisclosuresHarenberg: DOACSENCE: Other: Founder and managing director.

Use of Bivalirudin-Specific Monitoring Assays in Ventricular Assist Device Patients

Increasing numbers of pediatric ventricular assist device (VAD) patients are being anticoagulated with the parenteral direct thrombin inhibitor bivalirudin because it is reportedly associated with fewer bleeding and thrombotic events. With expanded use, management is shifting from a handful of experts to a wider pool of clinicians and trainees, increasing the importance of identifying broadly acceptable, standardized monitoring assays. The pharmacokinetics of bivalirudin have not been well-studied in the pediatric population and drug monitoring in all ages has been problematic for critically ill patients who require intermediate or longer-term therapeutic anticoagulation. The dilute thrombin time (dTT), available in many clinical laboratories, has been suggested as a potentially superior alternative to the activated partial thromboplastin time (aPTT), but results have been inconsistent. As clinical use of the dTT (c-dTT) for monitoring bivalirudin increased at our institution, we sought to evaluate the performance of commercially available, “research only” functional bivalirudin assays with calibrators and controls to measure bivalirudin's anticoagulation effect, utilizing residual plasmas and clinical data from VAD patients treated with bivalirudin. Residual citrated, platelet poor plasma samples from clinically ordered laboratory tests in VAD patients were collected and stored frozen at -70 oC from February 8, 2018, to January 4, 2021. With IRB approval, the samples were analyzed in conjunction with medical record review. Two experimental assay kits were utilized, ex-dTT: a dilute thrombin time assay (Hemoclot, Hyphen-Biomed, FR) and ex-anti FIIa: a chromogenic anti-factor IIa assay (BiophenDTI, Hyphen-Biomed, FR). Bivalirudin calibrators (Biophen, Hyphen-Biomed, FR) were used to develop a standard curve for the assays. Controls of low and high (1.5 and 4 microgram/mL) bivalirudin (Biophen, Hyphen-Biomed, FR) were used for quality control of the assays. Results from the two experimental assays were compared with available standard laboratory monitoring when results were available. In total, 115 residual plasma samples from 11 different patients (up to 16 samples per patient) were analyzed. Subjects included 1 adult (37 yr.) and 10 pediatric patients (0-18 yr.). There was excellent correlation between the two experimental assays (Fig. 1A). Correlation was good between the c-dTT and each of the experimental assays; however, with a clinical laboratory platform change (instrument, reagents) midway through sample collection, the c-dTT results shifted substantially in their corresponding estimate of bivalirudin concentration (Figs 1B and C). Activated partial thromboplastin time (aPTT) had poor correlation with the c-dTT and with each of the experimental assays (Fig. 1D). Here we report results from two commercially available kits that estimate bivalirudin concentration using dTT or chromogenic anti-factor IIa assays, in comparison with clinically generated results from VAD patients treated with bivalirudin. Our findings agree with previous observations that the aPTT shows poor correlation with dTT assays (clinically used and experimental) over days and weeks of anticoagulation. The two experimental assays had excellent correlation with each other and good correlation with the c-dTT; however, the fact that the c-dTT results shifted dramatically with a clinical laboratory platform change is illustrative of the need for a bivalirudin-specific monitoring assay as an essential tool for improving outcomes at our center and across centers. More research is needed to understand which type of monitoring assay (dTT or anti-FIIa) may be better suited to particular clinical circumstances. [Display omitted] DisclosuresLorts: Abbott: Consultancy; Medtronic: Consultancy; Berlin Heart: Consultancy; Syncardia: Consultancy; Abiomed: Consultancy.

Reversal Activity and Toxicity of Heparin-Binding Copolymer after Subcutaneous Administration of Enoxaparin in Mice.

Uncontrolled bleeding after enoxaparin (ENX) is rare but may be life-threatening. The only registered antidote for ENX, protamine sulfate (PS), has 60% efficacy and can cause severe adverse side effects. We developed a diblock copolymer, heparin-binding copolymer (HBC), that reverses intravenously administered heparins. Here, we focused on the HBC inhibitory activity against subcutaneously administered ENX in healthy mice. BALB/c mice were subcutaneously injected with ENX at the dose of 5 mg/kg. After 110 min, vehicle, HBC (6.25 and 12.5 mg/kg), or PS (5 and 10 mg/kg) were administered into the tail vein. The blood was collected after 3, 10, 60, 120, 360, and 600 min after vehicle, HBC, or PS administration. The activities of antifactors Xa and IIa and biochemical parameters were measured. The main organs were collected for histological analysis. HBC at the lower dose reversed the effect of ENX on antifactor Xa activity for 10 min after antidote administration, whereas at the higher dose, HBC reversed the effect on antifactor Xa activity throughout the course of the experiment. Both doses of HBC completely reversed the effect of ENX on antifactor IIa activity. PS did not reverse antifactor Xa activity and partially reversed antifactor IIa activity. HBC modulated biochemical parameters. Histopathological analysis showed changes in the liver, lungs, and spleen of mice treated with HBC and in the lungs and heart of mice treated with PS. HBC administered in an appropriate dose might be an efficient substitute for PS to reverse significantly increased anticoagulant activity that may be connected with major bleeding in patients receiving ENX subcutaneously.

Anti-thrombotic activity of phenolic acids obtained from Salvia miltiorrhiza f. alba in TNF-α-stimulated endothelial cells via the NF-κB/JNK/p38 MAPK signaling pathway.

Over the past 100years, Salvia miltiorrhiza f. alba (Lamiaceae) (RSMA) roots have been used to cure thromboangiitis obliterans (TAO) in local clinics. This study aimed to confirm the anti-thrombotic efficacy of 12 phenolic acids obtained from RSMA and to clarify the possible underlying mechanisms. The results of quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) experiments demonstrated that most of the phenolic acids markedly inhibited PAI-1 protein and mRNA levels but increased t-PA protein and mRNA levels in TNF-α-induced EA.hy926 cells (P < 0.05 or 0.001), with lithospermic acid displaying the strongest effect. In vitro anticoagulation and antiplatelet aggregation assays showed that lithospermic acid and salvianolic acid B significantly prolonged prothrombin time (PT), activated partial thromboplastin time (APTT), decreased fibrinogen concentration (FIB), and inhibited platelet aggregation induced by adenosine diphosphate (ADP) in rat blood. Both lithospermic acid and salvianolic acid B markedly down-regulated the expression of factor Xa and factor IIa on the external surface of EA.hy926 cells and demonstrated significant anti-factor IIa and anti-factor Xa activity using chromogenic substrates in vitro. Western blot results revealed that both lithospermic acid and salvianolic acid B also significantly inhibited the expression of TF, p-p65, p-p38, and pJNK proteins induced by TNF-α. These results indicated that all of the phenolic acids appeared to have some anti-thrombotic activity, with salvianolic acid B and lithospermic acid markedly decreasing the chance of thrombosis by regulating the NF-κB/JNK/p38 MAPK signaling pathway in response to TNF-α.

Preparation of Low Molecular Weight Heparin from a Remodeled Bovine Intestinal Heparin.

Bovine intestinal heparins are structurally distinct from porcine intestinal heparins and exhibit lower specific anticoagulant activity (units/mg). The reduced content of N-sulfo, 3-O-sulfo glucosamine, the central and critical residue in heparin's antithrombin III binding site, is responsible for bovine intestinal heparin's reduced activity. Previous studies demonstrate that treatment of bovine intestinal heparin with 3-O-sulfotransferase in the presence of 3'-phosphoadenosine-5'-phosphosulfate afforded remodeled bovine heparin with an enhanced activity reaching the United States Pharmacopeia's requirements. Starting from this remodeled bovine intestinal heparin, we report the preparation of a bovine intestinal low molecular weight heparin having the same structural properties and anti-factor IIa and anti-factor Xa activities of Enoxaparin. Moreover, this bovine intestinal heparin-derived "Enoxaparin" showed comparable platelet factor-4 binding affinity, suggesting that it should exhibit similarly low levels of heparin induced thrombocytopeneia, HIT.

Sulfated non-anticoagulant low molecular weight heparin in the prevention of cancer and non-cancer associated thrombosis without compromising hemostasis

IntroductionCancer-associated thrombosis (CAT) accounts for about 20% of all cases of Venous Thromboembolism (VTE). Tissue factor (TF) is documented to be highly expressed on cancer cells and pathological angiogenic endothelial cells. Here, we used a novel oxidized sulfated ultra-LMWH, S-NACH, which is devoid of anti-factor Xa and IIa activities with limited to no systemic anticoagulant effects. This sulfated form has enhanced binding to vascular endothelial cells (EC) and releases and potentiates the action of tissue factor pathway inhibitor (TFPI). S-NACH binds with high affinity to EC, releases and binds to EC TFPI, and promotes vascular antithrombotic effect with limited to no risk of bleeding complications. Materials and methodsWe investigated the effects of S-NACH on clot kinetics in vitro and in vivo. Also, we investigated the effects of S-NACH on CAT mediated by human acute leukemia cells (K562) and human pancreatic cancer cells (SUIT2). ResultsS-NACH was associated with ~3-fold increase of TFPI 2 levels within 3 h. Also, S-NACH reversed the hypercoagulability state that is associated with cancer cells in vitro. In vivo, S-NACH at 20 mg/kg subcutaneously (SC) had no effect on bleeding time compared to both tinzaparin and enoxaparin at 5 mg/kg SC. S-NACH did not show any anti-IIa or anti-Xa activities in comparison to tinzaparin and enoxaparin (p < 0.001). ConclusionData suggest the importance of S-NACH through its EC binding, EC TFPI release and its interaction with TFPI in enhancing its activity in the prevention of cancer and non-cancer associated thrombosis with limited to no bleeding complications.

Different Coagulation Indicators in Predicting Clinical Outcomes for Patients With Direct Oral Anticoagulants: A Systematic Review and Meta-analysis

PurposeThere are many anticoagulant test indexes available for direct oral anticoagulants (DOACs), but how to select the appropriate index and the index cutoff values are still controversial. This is the first study, to our knowledge, to assess the association of different coagulation indicators with clinical outcomes among DOACs using a meta-analysis of observational studies. MethodsA medical literature search was conducted using PubMed, Web of Science, EMBASE, ClinicalTrials.gov, and the Cochrane Library from inception to February 2020. Studies that reported relationships between coagulation indexes and clinical outcomes or the diagnostic value of coagulation assays were included in the analysis. FindingsA total of 17 articles (7 meta-analyses and 10 systematic reviews) from 8904 citations were included in the analysis. In the analysis of bleeding events with coagulation indexes for DOACs, for peak prothrombin time level (cutoff value of 19–25 s), the pooled results found a sensitivity of 0.61 (95% CI, 0.44–0.75) and a specificity of 0.71 (95% CI, 0.49–0.86). For rivaroxaban, the trough anti–factor Xa concentration (AXA-C) (cutoff value of 400–500 ng/mL) had a sensitivity of 0.53 (95% CI, 0.16–0.87) and a specificity of 0.87 (95% CI, 0.71–0.94), with a diagnostic odds ratio of 7 (95% CI, 2–32). For apixaban, trough AXA-C had a sensitivity of 0.85 (95% CI, 0.60–0.96) and a specificity of 0.83 (95% CI, 0.52–0.95). The AUC of the AXA-C peak was higher than that of the trough AXA-C for apixaban, with a higher sensitivity and specificity. Compared with trough concentration of anti–factor IIa for dabigatran, the peak concentration had a higher specificity (98%) at the cutoff value of 484 ng/mL. In the analysis of thromboembolic events with coagulation indexes for DOACs, peak and trough prothrombin time values were not typically correlated with subsequent symptomatic venous thromboembolism, without a sensitivity or specificity higher than 90%. Trough AXA-C had a sensitivity of 100% and but a low specificity (<50%) for rivaroxaban-apixaban. Trough AXA-C had a sensitivity of 100% and a specificity of 32% with a cutoff value of 108 ng/mL for dabigatran. ImplicationPeak prothrombin time (19–25 s) and AXA-C had a better predictive value on bleeding outcomes for rivaroxaban and apixaban, whereas peak concentration of anti–factor IIa activity can be an indicator for dabigatran. Coagulation indexes might not be a good indicator of thromboembolic events of DOACs. Because the limited studies focused on association of coagulation indicators and clinical outcomes, more studies are needed to verify this in the future.

Comparative Studies on the Anticoagulant Profile of Branded Enoxaparin and a New Biosimilar Version.

Low molecular weight heparins (LMWH) represent depolymerized heparin prepared by various methods that exhibit differential, biochemical and pharmacological profiles. Enoxaparin is prepared by benzylation followed by alkaline depolymerization of porcine heparin. Upon the expiration of its patent, several biosimilar versions of enoxaparin have become available. Heparinox (Sodic enoxaparine; Cristália Produtos Químicos Farmacêuticos LTDA, Sao Paulo, Brazil) is a new biosimilar form of enoxaparin. We assessed the molecular weight and the biochemical profile of Heparinox and compared its properties to the original branded enoxaparin (Lovenox; Sanofi, Paris, France). Clotting profiles compared included activated clotting time, activated partial thromboplastin time (aPTT), and thrombin time (TT). Anti-protease assays included anti-factor Xa and anti-factor IIa activities. Thrombin generation was measured using a calibrated automated thrombogram and thrombokinetic profile included peak thrombin, lag time and area under the curve. USP potency was determined using commercially available assay kits. Molecular weight profiling was determined using high performance liquid chromatography. We determined that Heparinox and Lovenox were comparable in their molecular weight profile. Th anticoagulant profile of the branded and biosimilar version were also similar in the clot based aPTT and TT. Similarly, the anti-Xa and anti-IIa activities were comparable in the products. No differences were noted in the thrombin generation inhibitory profile of the branded and biosimilar versions of enoxaparin. Our studies suggest that Heparinox is bioequivalent to the original branded enoxaparin based upon in vitro tests however will require further in vivo studies in animal models and humans to determine their clinical bioequivalence.

Influence of sulfated polysaccharides from Ulva lactuca L. upon Xa and IIa coagulation factors and on venous blood clot formation

The interest in bioactive compounds from natural sources, such as marine organisms, has increased considerably in recent years. Among these compounds, sulfated polysaccharides from seaweed exhibit a broad spectrum of biological activities. Sulfated polysaccharides from green algae are still poorly investigated. For this reason, in this study, using unusual methodologies, such as extraction conditions, FACE, and RAMAN, we investigated the structural features of F50UL and F70UL sulfated polysaccharides from Ulva lactuca L. and their distinct in vitro anticoagulant and in vivo antithrombotic activities. Sulfated polysaccharides of U. lactuca were obtained by enzymatic proteolysis with ALCALASE® and fractionated by acetone precipitation. F50Ul and F70Ul sulfated polysaccharides with higher yield were partially chemically characterized by Fluorophore-assisted carbohydrate electrophoresis (FACE) and RAMAN spectroscopy analysis and submitted to an in vitro screening by APTT, PT, TT, and anti-factor Xa and IIa tests. The venae cavae ligature experimental model for the analysis of in vivo antithrombotic activity of F50Ul and F70Ul sulfated polysaccharides were also performed. The U. lactuca L. sulfated polysaccharides characterization by FACE and RAMAN showed a typical ulvans structure that contains as principal component rhamnose, but other monosaccharides (uronic acid, glucose, and galactose) are present. F50Ul (0.1–1.0 μg/μl) showed anticoagulant activity in vitro. However, F70Ul that has a similar composition did not present these effects. Also, only F50Ul sulfated polysaccharides (≥5 μg/g) showed a great in vivo antithrombotic concentration-dependent and time-dependent activity. In summary, we demonstrate the use of unusual extraction and characterization analysis procedures for U. lactuca L. sulfated polysaccharides and the ability of F50Ul to reduce the weight of thrombus in rats probably by the association with factors Xa and IIa inhibition. These results provide strong evidence of the anticoagulant potential of these sulfated polysaccharides isolated from Ulva lactuca L.

Anticoagulant Heparin Mimetics via RAFT Polymerization.

Heparin, a sulfated polysaccharide derived from animal sources, is the most commonly used parenteral anticoagulant drug, but it suffers from significant safety and supply issues. Herein, we describe the preparation of heparin mimetic homo- and copolymers via the reversible addition-fragmentation chain transfer (RAFT) polymerization in water of commercially available (non-carbohydrate) sulfonated and carboxylated monomers. The anticoagulant activities of the polymers were assessed by activated partial thromboplastin time (APTT), thrombin clotting time (TCT), and for the more promising polymers, thrombin generation, antifactor Xa, and antifactor IIa assays. Sulfonated homopolymers studied herein displayed low cytotoxicity and significant anticoagulant activity in APTT, TCT, and thrombin generation assays. In addition, copolymers of sodium styrenesulfonate and acrylic acid [poly(SSS-co-AA)] displayed unprecedented antifactor IIa activity. This study demonstrates the potential of RAFT polymers as alternative anticoagulants for biomedical applications.

Effect of Sulfated NON-Anticoagulant Low Molecular Weight Heparin in Cancer and NON-Cancer Associated Thrombosis

Introduction: The process of thrombosis depends on a complex interaction between different factors including vascular endothelial activation, platelet activation and adhesion. Blood coagulation process initiated by plasma protease factor VIIa in complex with the membrane protein tissue factor. Cancer-associated thrombosis (CAT) accounts for about 20% of all cases of Venous Thromboembolism (VTE). Tissue factor (TF) is documented to be highly expressed on the cancer cells and pathological angiogenic endothelial cells. Subsequent theories proposed that the cancer cells activate the coagulation cascade, as the oncogenes cause tumor transformation, which in turn causes the cancer tissues to be able to express different pro-coagulant proteins. VTE risk factors can be divided into cancer- and therapy-associated factors. Low molecular weight heparin (LMWH) is used for prophylaxis and treatment of deep vein thrombosis and CAT as it significantly shortened both duration of thromboembolic crisis and hospitalization. Safety concerns associated with the bleeding risks are the major barrier to dose optimization of treatments. Here, we used the novel sulfated non-anticoagulant LMWH, referred to as S-NACH, which has a similar activity of LMWH but devoid of systemic anti-factor Xa and IIa activity. The sulfated form has enhanced S-NACH binding to the vascular endothelial cells, release TFPI and potentiate the action of tissue factor pathway inhibitors (TFPI). We investigated the effects of S-NACH on clot kinetics in comparison with LMWH using thrombelstography (TEG) and examined its in vitro and in vivo effects on vascular endothelial release capacity of TFPI. Also we investigate the effects of S-NACH on cancer-associated thrombosis-mediated by human pancreatic cancer cells (SUIT2). Methods: We conducted both in vitro and in vivo experiments using our compound (S-NACH) to test its efficacy on coagulation properties in comparison to LMWH (enoxaparin). We compared the effects on the coagulation time, time to clot initiation and thrombus strength level in vitro, with and without HUVEC endothelial cell line coated into cups used for the TEG analysis. TFPI levels were also estimated in both in vitro and in vivo models at different doses of S-NACH. In addition, the effect of S-NACH on factors Xa and IIa was assessed in mice and rabbits. Also, weused pancreatic cancer cell line (SUIT2) to test the efficiency of blood coagulation in the presence of S-NACH, and Enoxaparin at 1-10 μg. Results: TEG assay have shown that, increasing concentrations of S-NACH (0.1 - 10 ug) did not affect the clotting time (R) and maximum amplitude of the clot formation (MA), while LMWH (enoxaparin) was associated with marked increased clotting time (R) and decreased maximum amplitude of the clot formation (MA) in comparison to control. Using HUVEC cell line coated cups; both S-NACH and LMWH were associated with increased clotting time (R) and decreased maximum amplitude of the clot formation (MA). This is due to the ability of HUVEC cell to release of TFPI. In the in vivo study, administration of enoxaparin inhibited both factors Xa and IIa, while administration of S-NACH did not affect the activities of these coagulation factors. S-NACH caused 3 folds greater tissue factor pathway inhibitor (TFPI) release from human endothelial cells and in mice versus enoxaparin. On the other hand, TEG assay shows significantly decreased clotting time (R-value) and increased clot strength in blood from healthy donors when adding pancreatic SUIT2 cells. Data from the TEG assay demonstrated lack of effects of S-NACH or TFPI alone on clot kinetics in human. In contrast, S-NACH (1-10 μg) in the presence of 0.01-0.1 ng TFPI resulted in a dose-dependent suppression of clot strength and prolongation of clotting time (Figure 1). Conclusion: S-NACH is devoid of anti-thrombin binding and inhibition of systemic anti-thrombin-dependent coagulation factors such as factor Xa and factor IIa but has an optimal releasing capacity of vascular endothelial TFPI that is associated with antithrombotic activities without any effect on hemostasis. Also, S-NACH prevents CAT without any effects on hemostasis. Disclosures No relevant conflicts of interest to declare.

Novel reversal agents and laboratory evaluation for direct-acting oral anticoagulants (DOAC): An update.

Novel oral anticoagulants (NOACs) are no longer “novel” but their reversal agents definitely are. Although NOACs enjoy high clinical efficacy, monitoring and reversal of their effect is a challenge which this review attempts to surmount. Ideally, for NOAC activity measurement, specific anti-Factor IIa levels and anti -Factor Xa levels should be monitored (chromogenic assays), but such tests are not readily available. Modifications of the existing coagulation tests catering to this unmet need for quantification of DOAC activity have been reviewed. The available United States Food and Drug Administration (FDA) approved reversal agents, idarucizumab for dabigatrin and andexanet alfa for anti-Xa direct acting oral anticoagulants have given promising results but are prohibitively priced. Medline, Embase, and Scopus databases were thoroughly searched for clinical trials on laboratory investigations and specific as well as non-specific reversal-agents for DOACs.

Structural and activity variability of fractions with different charge density and chain length from pharmaceutical heparins.

Heparin is a structurally complex polysaccharide used as a clinical anticoagulant. It is comprised of a heterogeneous mixture of polysaccharide chains having a variety of sequences and lengths. The production methods and regulatory controls of pharmaceutical heparins have changed over the years. This study assesses the structural and activity uniformity of the polysaccharide chains comprising two contemporary heparin products. The heparin fractions with different sizes and charges were separated with size exclusion and ion exchange chromatography. The fractions were analyzed for their molecular weight properties, di- and tetrasaccharide compositions, and anti-factor IIa and anti-factor-Xa activities. The distribution of these properties through chains of different lengths and ones with different charge density were compared. The results demonstrate that with the increase in heparin purity, activity and molecular weight required by the current pharmacopeia, the uniformity of pharmaceutical heparin products have increased.

Detection of lupus anticoagulant in the era of direct oral anticoagulants.

Lupus anticoagulant (LAC) is an in vitro phenomenon determining a phospholipid-dependent elongation of clotting times. The presence of LAC associated with anticardiolipin (aCL) and anti-β2 glycoprotein I (anti-β2GPI) antibodies is strongly associated with thrombosis and pregnancy morbidity. Direct oral anticoagulants (DOACs) targeting thrombin and factor Xa are currently widely use to prevent and treat venous and arterial thromboembolism. Some concern has, however, been expressed about the possibility of false laboratory results during LAC assessment in patients taking these drugs. Several in vitro studies, spiking DOACs into normal plasma as well as ex vivo at peak levels in treated patients, led in false-positive LAC. The dilute Russell Viper Venom time is the assay that is most influenced by rivaroxaban, edoxaban, dabigatran and less by apixaban. Both screening and confirmatory tests have resulted equally prolonged for activated partial thromboplastin time and have not led to false-positive results, but this may depend on the type of reagent used for the test. Taipan/Ecarin snake venoms ratios, has been recommend by some investigators as they do not seem to be affected by rivaroxaban or edoxaban, but these tests are neither standardized nor generally available in clinical practice. In conclusion, for the time being it does not seem advisable to carry out LAC testing during anti-factor Xa and anti-factor IIa treatment because of the risk of false-positive results. Whenever needed in deciding the suspension of DOACs or in case of recurrent thrombosis, LAC determination should be carried out at trough better if DOAC concentration is known.