Treatment Of Thromboembolic Disorders Research Articles

Unlocking the polymorphic odyssey of Rivaroxaban: a journey of pharmaceutical innovation

The field of pharmaceutical science is a dynamic landscape driven by the pursuit of enhanced drug formulations and improved therapeutic outcomes. One remarkable journey within this domain is the polymorphism odyssey of rivaroxaban, a distinguished anticoagulant inhibitor targeting blood coagulation factor Xa. Rivaroxaban plays a key role in the prevention and treatment of thromboembolic disorders, including myocardial infarction, stroke, and deep venous thromboses. Its intricate crystal structures and diverse solid-state forms, each imparting unique properties affecting solubility, bioavailability, and stability, illuminate the challenges, breakthroughs, and relentless pursuit of superior drug performance in the pharmaceutical development of rivaroxaban. This work not only reflects the scientific commitment to enhancing therapeutic efficacy but also underscores the dynamic interplay between chemical innovation and clinical application in drug development.

Design and Preclinical Characterization Program toward Asundexian (BAY 2433334), an Oral Factor XIa Inhibitor for the Prevention and Treatment of Thromboembolic Disorders.

Activated coagulation factor XI (FXIa) is a highly attractive antithrombotic target as it contributes to the development and progression of thrombosis but is thought to play only a minor role in hemostasis so that its inhibition may allow for decoupling of antithrombotic efficacy and bleeding time prolongation. Herein, we report our major efforts to identify an orally bioavailable, reversible FXIa inhibitor. Using a protein structure-based de novo design approach, we identified a novel micromolar hit with attractive physicochemical properties. During lead modification, a critical problem was balancing potency and absorption by focusing on the most important interactions of the lead series with FXIa while simultaneously seeking to improve metabolic stability and the cytochrome P450 interaction profile. In clinical trials, the resulting compound from our extensive research program, asundexian (BAY 2433334), proved to possess the desired DMPK properties for once-daily oral dosing, and even more importantly, the initial pharmacological hypothesis was confirmed.

Comparative pharmacokinetics of two formulations of 2.5-mg rivaroxaban in healthy Korean subjects.

Rivaroxaban is a direct factor Xa inhibitor used for the prevention and treatment of thromboembolic disorders. The objective of this study was to compare the pharmacokinetic profiles of two rivaroxaban formulations after a single dose of rivaroxaban (2.5-mg tablet) in healthy Korean subjects. This study was a randomized, open-label, single-dose, two-period, crossover study that included 34 healthy adult subjects under fasting conditions. The test drug (Yuhan rivaroxaban tablet) or reference drug (Xarelto tablet) was administered in each period. Serial blood samples were collected up to 36 hours post-dose. Plasma concentrations were measured by LC-MS/MS. Pharmacokinetic parameters, including maximum plasma concentration (Cmax) and area under the plasma concentration-time curve from time zero to the last measurable concentration (AUCt), were determined by non-compartmental analysis. The 90% confidence intervals (CIs) for the ratio of the geometric means of Cmax and AUCt for the test drug/reference drug were calculated to evaluate pharmacokinetic equivalence. A total of 28 subjects were included in the pharmacokinetic analysis. The geometric mean ratios (90% CI) of the test drug/reference drug for rivaroxaban were 1.0140 (0.9794-1.0499) for AUCt and 0.9350 (0.8797-0.9939) for Cmax. All adverse events (AEs) were mild, and there was no significant difference in the incidence of AEs between the formulations. The pharmacokinetic parameters of rivaroxaban were compared between the test and reference drug, and both formulations were bioequivalent. The newly developed rivaroxaban tablet is safe and well tolerated as the reference drug (ClinicalTrials.gov identifiers: NCT05418803).

Factor XI inhibitors — a breakthrough in prevention and treatment of thromboembolic disorders

Available pharmacological antithrombotic prophylaxis is associated with a low but noticeable risk of bleeding. Hence, there is an ongoing search for effective antithrombotic medications which do not significantly increase bleeding risk. Factor XI inhibitors seem to fulfil these requirements. Results of presently available randomized clinical trials on the use of factor XI inhibitors in venous thromboembolism prophylaxis and prevention of arterial thrombosis are presented.

Assessment of knowledge and counseling practice of warfarin among pharmacists in UAE: A cross-sectional study.

Warfarin has long been regarded as the cornerstone anticoagulant for patients requiring long-term prevention or treatment of thromboembolic disorders. With adequate knowledge and counseling skills, hospital and community pharmacists can play a major role in enhancing warfarin therapy. to evaluate the knowledge and counseling practices toward warfarin among community and hospital pharmacists in UAE. A cross-sectional study involving community and hospital pharmacies was conducted with online questionnaire which was submitted to pharmacists focusing on their pharmacotherapeutic knowledge and patient education toward warfarin in UAE. Data were collected within 3 months (July, August and September 2021). SPSS Version 26 was used to analyze the data. The survey questions were sent to expert researchers in pharmacy practice for comments on their relevancy, clarity, and essentiality. Among the target population sample size of 400 pharmacists were approached. Majority of the pharmacists in UAE (157/400, 39.3%) had 1-5 years of experience. Most of the participants (52%) have fair knowledge about warfarin and (62.1%) of them have fair counseling practices about warfarin. Hospital pharmacists have more knowledge than community pharmacists (Mean rank, Independent pharmacy: 166.30, Chain pharmacy: 138.01, Hospital pharmacy: 252.27, p<0.05) and they have better counseling practice than community pharmacists (Mean rank, Independent pharmacy: 188.83, Chain pharmacy: 170.18, Hospital pharmacy: 222.90, p<0.05). The study's participants had moderate knowledge and counseling practices of warfarin. As a result, specialized training in warfarin therapy management for pharmacists is needed to improve therapeutic outcomes and avoid complications. Moreover, conferences or online courses should be held to train pharmacists on how to provide professional counseling to patients.

Opposite Response to Vitamin K Antagonists: A Report of Two Cases and Systematic Review of Literature.

Vitamin K antagonists (VKAs) are used in the prophylaxis and treatment of thromboembolic disorders. Despite a high efficacy, their narrow therapeutic window and high response variability hamper their management. Several patients experience fluctuations in dose–response and are at increased risk of over- or under-anticoagulation. Therefore, it is essential to monitor the prothrombin time/international normalized ratio to determine the so-called stable dose and to adjust the dosage accordingly. Three polymorphisms, CYP2C9∗2, CYP2C9∗3 and VKORC1-1639G>A, are associated with increased sensitivity to VKAs. Other polymorphisms are associated with a request for a higher dose and VKA resistance. We described the clinical cases of two patients who were referred to the Clinical Pharmacology and Pharmacogenetics Unit of the University Hospital of Salerno for pharmacological counseling. One of them showed hypersensitivity and the other one was resistant to VKAs. A systematic review was performed to identify randomized clinical trials investigating the impact of pharmacogenetic testing on increased sensitivity and resistance to VKAs. Although international guidelines are available and information on the genotype-guided dosing approach has been included in VKA drug labels, VKA pharmacogenetic testing is not commonly required. The clinical cases and the results of the systematically reviewed RCTs demonstrate that the pharmacogenetic-based VKA dosing model represents a valuable resource for reducing VKA-associated adverse events.

Discovery of Milvexian, a High-Affinity, Orally Bioavailable Inhibitor of Factor XIa in Clinical Studies for Antithrombotic Therapy.

Factor XIa (FXIa) is an enzyme in the coagulation cascade thought to amplify thrombin generation but has a limited role in hemostasis. From preclinical models and human genetics, an inhibitor of FXIa has the potential to be an antithrombotic agent with superior efficacy and safety. Reversible and irreversible inhibitors of FXIa have demonstrated excellent antithrombotic efficacy without increased bleeding time in animal models (Weitz, J. I., Chan, N. C. Arterioscler. Thromb. Vasc. Biol. 2019, 39 (1), 7-12). Herein, we report the discovery of a novel series of macrocyclic FXIa inhibitors containing a pyrazole P2' moiety. Optimization of the series for (pharmacokinetic) PK properties, free fraction, and solubility resulted in the identification of milvexian (BMS-986177/JNJ-70033093, 17, FXIa Ki = 0.11 nM) as a clinical candidate for the prevention and treatment of thromboembolic disorders, suitable for oral administration.

Calculatin the Effective Intravenous Heparin Dose: Comparison between Lean and Actual Body Weight-Based Dosing in Obese Patients

Abstract Background: Despite the availability of newer anticoag-ulants, unfractionated heparin remains a mainstay anticoagulant for atrial fibrillation, acute coronary syndrome with or without percutaneous intervention, treatment and prevention of deep vein thrombosis (DVT), pulmonary embolism (PE), and other thromboembolic disorders. Aim of Study: The aim of this study is to investigate if intravenous heparin dosing based on lean body weight (LBW) of obese patients would be safe and effective in achieving activated partial thromboplastin time (APTT) within 24 hours compared to the usual practice. Patients and Methods: This is a case-control study con-ducted in Cardiology Department Sammanaud General Hos-pital from May 2017 to May 2018 to investigate if intravenous heparin dosing based on LBW of obese patients would be safe and effective in achieving target APTT within 24 hours compared to the usual practice. The study included 50 obese patients with a diagnosis of atrial fibrillation, suspected or confirmed deep venous thrombosis or pulmonary embolism, unstable angina or Non ST elelvation myocardial infarction with hemodynamic stability, or peripheral vascular disease. Patients aged >18 years randomized into two groups (1) and (2). Results: Studies found that unfractionated heparin dosage adjustments based on the patient's LBW provided therapeutic anticoagulation more rapidly and safely, but protocols based on total body weight increase the risk of a supra-therapeutic PTT. Conclusion: Unfractionated heparin remains a mainstay anticoagulant for atrial fibrillation, acute coronary syndrome with or without percutaneous intervention, treatment and prevention of deep vein thrombosis (DVT), pulmonary embo-lism (PE), and other thromboembolic disorders. As lean body weight contributes to approximately 99% of a drug's clearance, it is useful for guiding dosing in obesity. These findings may enhance the utility of LBW as body descriptor instead of TBW in calculating the effective doses of UFH in treatment of thromboembolic disorders.

Pharmacists' warfarin therapy knowledge and counseling practices in the Eastern Province, Saudi Arabia: A cross-sectional study.

ABSTRACTContext:For several decades, warfarin has been considered the mainstay anticoagulant for patients who require long-term prevention or treatment of thromboembolic disorders in outpatient settings. Hospital and community pharmacists––with adequate knowledge level and counseling skills––can play a significant role in improving warfarin therapy.Aims:The aim of this study was to assess the hospital and community pharmacists’ warfarin therapy knowledge and counseling practices in the eastern province of Saudi Arabia.Materials and Methods:A cross-sectional study was conducted for 2 months. A self-administered questionnaire was designed focusing on warfarin mechanism of action, indications, safety profile, management of toxicity, monitoring, drug/food interactions, and patient education. The questionnaire was distributed among a random sample of hospital and community pharmacists in the Eastern Province of Saudi Arabia.Results:One hundred and fifty-three pharmacists participated in the study; ninety-seven of them were hospital-based (63.4%), whereas the remaining were community pharmacists. Participant’s mean years’ of experience was 5.67. In terms of the percentage of right answers, hospital pharmacists showed significantly better warfarin therapy knowledge than community pharmacists (P = 0.026). The percentages of right answers were 31.3% for drug/food interactions, 49.9% for safety profile/management of toxicity, 53.3% for patient education, and 58.2% for monitoring warfarin safety/efficacy. Neither the participants’ educational level nor their duration of experience had a significant correlation with the percentage of right answers (P = 0.22 and 0.61)Conclusion:Inadequate knowledge and inappropriate practices were encountered among study participants, especially community pharmacists. Therefore, specialized training of pharmacists about warfarin therapy management is essential to optimize therapeutic outcomes and prevent complications.

The Genetics of Warfarin Dose-Response Variability in Africans: An Expert Perspective on Past, Present, and Future.

Coumarins such as warfarin are prescribed for prevention and treatment of thromboembolic disorders. Warfarin remains the most widely prescribed and an anticoagulant of choice in Africa. Warfarin use is, however, limited by interindividual variability in pharmacokinetics and a narrow therapeutic index. The difference in patients' pharmacodynamic responses to warfarin has been attributed to genetic variation in warfarin metabolism and molecular targets (e.g., CYP2C9 and VKORC1) and host-environment interactions. This expert review offers a synthesis of human genetics studies in Africans with respect to pharmacogenetics-informed warfarin dosing. We identify areas that need future research attention or could benefit from harnessing existing pharmacogenetics knowledge toward rational and optimal therapeutics with warfarin in African patients. A literature search was conducted until January 2019. A total of 343 articles were retrieved from nine African countries: Botswana, Ethiopia, Egypt, Ghana, Kenya, South Africa, Sudan, Tanzania, and Mozambique. We found 19 studies on genetics of warfarin treatment specifically among Africans. Genes examined included CYP2C9, VKORC1, CYP4F2, APOE, CALU, GGCX, and EPHX1. CYP2C9*2 and *3 alleles were highly frequent among Egyptians, while rare in other African populations. CYP2C9*5, *8, *9, and *11, and VKORC1 Asp36Tyr genetic variants explained warfarin variability in Africans better, compared to CYP2C9*2 and *3. In Africa, there is limited pharmacogenetics data on warfarin. Therefore, future research and funding commitments should be prioritized to ensure safe and effective use of warfarin in Africa. Lessons learned in Africa from the science of pharmacogenetics would inform rational therapeutics in hematology, cardiology, and surgical specialties worldwide.

Evaluation of oral anticoagulants with vitamin K epoxide reductase in its native milieu

AbstractWarfarin, acenocoumarol, phenprocoumon, and fluindione are commonly prescribed oral anticoagulants for the prevention and treatment of thromboembolic disorders. These anticoagulants function by impairing the biosynthesis of active vitamin K–dependent coagulation factors through the inhibition of vitamin K epoxide reductase (VKOR). Genetic variations in VKOR have been closely associated with the resistant phenotype of oral anticoagulation therapy. However, the relative efficacy of these anticoagulants, their mechanisms of action, and their resistance variations among naturally occurring VKOR mutations remain elusive. Here, we explored these questions using our recently established cell-based VKOR activity assay with the endogenous VKOR function ablated. Our results show that the efficacy of these anticoagulants on VKOR inactivation, from most to least, is: acenocoumarol > phenprocoumon > warfarin > fluindione. This is consistent with their effective clinical dosages for stable anticoagulation control. Cell-based functional studies of how each of the 27 naturally occurring VKOR mutations responds to these 4 oral anticoagulants indicate that phenprocoumon has the largest resistance variation (up to 199-fold), whereas the resistance of acenocoumarol varies the least (<14-fold). Cell-based kinetics studies show that fluindione appears to be a competitive inhibitor of VKOR, whereas warfarin is likely to be a mixed-type inhibitor of VKOR. The anticoagulation effect of these oral anticoagulants can be reversed by the administration of a high dose of vitamin K, apparently due to the existence of a different enzyme that can directly reduce vitamin K. These findings provide new insights into the selection of oral anticoagulants, their effective dosage management, and their mechanisms of anticoagulation.

Effects of the oral, direct factor Xa inhibitor edoxaban on routine coagulation assays, lupus anticoagulant and anti-Xa assays

Edoxaban is an oral direct factor Xa inhibitor for prophylaxis and treatment of thromboembolic disorders. The effects on common coagulation assays are clinically valuable information and in certain clinical situations a quick assessment of the anticoagulant is wanted. Our aim was to investigate the effect of edoxaban on routine coagulation methods and evaluate anti-Xa assays, commonly used for other direct factor Xa inhibitors, for estimation of the drug concentration. Edoxaban was spiked to plasma samples from healthy subjects in the concentration range 0–742 µg/L and analyzed using different reagents for activated partial thromboplastin time (APTT) and prothrombin time (PT). Assays for antithrombin, activated protein C resistance, lupus anticoagulant (LA) and chromogenic anti-Xa assays were also included. Edoxaban displayed similar effects in vitro to other oral direct Xa inhibitors. The concentration needed to double the coagulation time varied between assays and reagents; 539–758 µg/L for the APTT and between 329 and 2505 µg/L for the PT. Edoxaban gave false high antithrombin activities in assays based on Xa-inhibition. Two integrated assays for LA, both based on activation with dilute Russell’s viper venom, displayed different results. Chromogenic anti-Xa assays displayed linear dose-response curves with edoxaban up to approximately 500 µg/L. In conclusion, therapeutic concentrations of edoxaban variably affect different coagulation assays, and even different reagents within an assay group. In comparison with other oral Xa-inhibitors, the in vitro effects of edoxaban were more similar to rivaroxaban than apixaban. For measurement of edoxaban concentration in plasma, it is possible to use the chromogenic anti-Xa assays.

Comparison of reversal activity and mechanism of action of UHRA, andexanet, and PER977 on heparin and oral FXa inhibitors

AbstractAnticoagulants such as unfractionated heparin (UFH), low-molecular-weight heparins (LMWHs), fondaparinux, and direct oral anticoagulants (DOACs) targeting thrombin (IIa) or factor Xa (FXa) are widely used in prevention and treatment of thromboembolic disorders. However, anticoagulant-associated bleeding is a concern that demands monitoring and neutralization. Protamine, the UFH antidote, has limitations, while there is no antidote available for certain direct FXa inhibitors. Improved antidotes in development include UHRA (Universal Heparin Reversal Agent) for all heparin anticoagulants; andexanet alfa (andexanet), a recombinant antidote for both direct FXa inhibitors and LMWHs; and ciraparantag (PER977), a small-molecule antidote for UFH, LMWHs, and certain DOACs. The binding affinities of these antidotes for their presumed anticoagulant targets have not been compared. Here, isothermal titration calorimetry (ITC) was used to determine the affinity of each antidote for its putative targets. Clotting and chromogenic FXa assays were used to characterize neutralization activity, and electron microscopy was used to visualize the effect of each antidote on clot morphology in the absence or presence of anticoagulant. ITC confirmed binding of UHRA to all heparins, and binding of andexanet to edoxaban and rivaroxaban, and to the antithrombin–enoxaparin complex. PER977 was found to bind heparins weakly, but not the direct FXa inhibitors studied. For UHRA and andexanet, an affinity at or below the micromolar level was found to correlate with neutralization activity, while no reversal activity was observed for the PER977/anticoagulant systems. Standard metrics of clot structure were found to correlate weakly with PER977's activity. This is the first study comparing 3 antidotes in development, with each exerting activity through a distinct mechanism.

The Rivaroxaban Program and the Management of Unmet Needs in Thromboembolic Disease.

Rivaroxaban is a non-vitamin K antagonist oral anticoagulant that acts as a direct factor Xa inhibitor, and is widely used for the prevention and treatment of thromboembolic disorders. As further knowledge gaps are identified in thrombosis management, the rivaroxaban research program has expanded in an attempt to elucidate the wider benefits of rivaroxaban. An increased understanding of the interactions taking place within the coagulation cascade may support a broader role for rivaroxaban (2.5 mg twice daily [bid] or 5 mg bid) in the setting of vascular protection, either alone or in combination with an antiplatelet agent. The aim of this article is to describe the potential role of rivaroxaban in the context of vascular protection and provide an overview of recently completed and ongoing randomized controlled trials of rivaroxaban in the areas of stroke prevention, venous protection and vascular protection.

Improvement of time in therapeutic range with warfarin by pharmaceutical intervention.

Background Anticoagulation therapy with warfarin is highly effective for prevention and treatment of thromboembolic disorders. Nevertheless, its management is challenging especially in developing countries, where the medical access is difficult and patient education is poor. Objective To determine the effect of pharmaceutical intervention (PI) on the time in therapeutic range (TTR) of a group of anticoagulated patients from our referral center. Method A group of consecutive outpatients previously treated by usual medical care underwent PI. The intervention consisted in the identification and avoidance of food and drug interactions, the confection of medication schedule charts and education to patients regarding side effects and drug monitoring. Mean TTR before and after PI was compared through the Wilcoxon test for repeated measures. Regression analyses were performed to assess the relationship between a TTR level under 65% and potential explanatory variables. Results Mean TTR before PI was 37.4±23.5% and after PI it raised to 67.0±24.9%. Mean change in TTR was +29.5% (95% CI 14.5-44.6; p<0.001). Before PI only 4 patients had TTR values above 65%, in contrast to 18 patients after PI. Conclusion Our findings support the efficacy of PI to improve TTR values in patients treated with warfarin.

Ringhalexin from Hemachatus haemachatus: A novel inhibitor of extrinsic tenase complex.

Anticoagulant therapy is used for the prevention and treatment of thromboembolic disorders. Blood coagulation is initiated by the interaction of factor VIIa (FVIIa) with membrane-bound tissue factor (TF) to form the extrinsic tenase complex which activates FX to FXa. Thus, it is an important target for the development of novel anticoagulants. Here, we report the isolation and characterization of a novel anticoagulant ringhalexin from the venom of Hemachatus haemachatus (African Ringhals Cobra). Amino acid sequence of the protein indicates that it belongs to the three-finger toxin family and exhibits 94% identity to an uncharacterized Neurotoxin-like protein NTL2 from Naja atra. Ringhalexin inhibited FX activation by extrinsic tenase complex with an IC50 of 123.8 ± 9.54 nM. It is a mixed-type inhibitor with the kinetic constants, Ki and Ki’ of 84.25 ± 3.53 nM and 152.5 ± 11.32 nM, respectively. Ringhalexin also exhibits a weak, irreversible neurotoxicity on chick biventer cervicis muscle preparations. Subsequently, the three-dimensional structure of ringhalexin was determined at 2.95 Å resolution. This study for the first time reports the structure of an anticoagulant three-finger toxin. Thus, ringhalexin is a potent inhibitor of the FX activation by extrinsic tenase complex and a weak, irreversible neurotoxin.

Les calcifications vasculaires sous anti-vitamines K : un effet indésirable méconnu

Despite the availability of new oral anticoagulants, vitamin K antagonists (VKA, such as fluindione, acenocoumarol or warfarin) remain currently the goal standard medicines for oral prevention or treatment of thromboembolic disorders. They inhibit the cycle of the vitamin K and its participation in the enzymatic gamma-carboxylation of many proteins. The VKA prevent the activation of the vitamin K-dependent blood clotting factors limiting thus the initiation of the coagulation cascade. But other proteins are vitamin K-dependent and also remain inactive in the presence of VKA. This is the case of matrix Gla-protein (MGP), a protein that plays a major inhibitory role in the development of vascular calcifications. Several experimental and epidemiological results suggest that the use of the VKA could promote the development of vascular calcifications increasing thus the cardiovascular risk. This risk seems to be higher in patients with chronic kidney disease or mellitus diabetes who are more likely to develop vascular calcifications, and may be due to a decrease of the MGP activity. This review aims at summarizing the data currently available making vascular calcifications the probably underestimated side effects of VKA.

VKORC1 polymorphisms and warfarin maintenance dose in population of Sakha (Yakuts).

Vitamin K antagonists are effective in the prevention and treatment of thromboembolic disorders. Warfarin is one of the most widely prescribed vitamin K antagonists in the world [1, 2]. It has a narrow therapeutic range and a given dose may result in a large inter-individual variation of response. Insufficient dose may fail to prevent thromboembolism, while an overdose increases the risk of bleeding. Patient-specific factors (e.g., age, body size, race, concurrent diseases, and medications) explain some of the variability in warfarin dosage, but genetic factors influencing warfarin response explain a significantly higher proportion of this variability [3]. Molecular analysis of the gene that encodes the target enzyme vitamin K epoxide reductase complex 1 (VKORC1) strongly suggests that its genetic variations greatly affect the individual response to oral anticoagulants [4-7]. To evaluate effects of VKORC1 polymorphisms on warfarin dose excess anticoagulation (INR >4.0) in the population of Sakha (S) patients. 53 patients (29-women, 24-men) with atrial fibrillation (68%), congestive heart failure (60%), hypertension (49%) and cardiac valve replacement (26%) were recruited. The age range was 26-80 years, with a mean age of 62.87 ± 12.57 years.International normalized ratio and plasma warfarin concentrations were determined. Genotyping was carried out by RT-PCR (real-time PCR). The three genetic polymorphisms of the gene VKORC1 G3673A (rs9923231) were studied: normal (GG), heterozygous (GA) and homozygous (AA). Fisher exact probability test and chi-square test (with Yates correction) were applied to compare data among the AA and GG + GA groups; also Mann-Whitney test was used. The median maintenance daily dose of warfarin among AA carriers was 3.0 mg/day [1.25-7.5 mg], while in GG and GA patients it was 3.13 mg/day [1.88-7.92 mg]. The mean daily warfarin dosage was higher in GG and GA genotype carriers 4.05 mg/day (SD ± 1.7) than in patients with AA genotype 3.13 (SD ± 1.5). Differences are of borderline significance (p = 0.054). Of the 41 patients who required warfarin doses of less than 5 mg, 28 (63%) were found to be AA carriers and 14 (37%) were GG, GA carriers. Differences were not quite significant (p = 0.072). Among 31 homozygous polymorphism carriers 2 (4%) patients developed overanticoagulation (INR >4.0), while among 22 normal and heterozygous polymorphisms carriers only 3 (6%) patients developed overanticoagulation (INR >4.0). Differences were not statistically significant (p = 0.36). No significant association between VKORC1 polymorphisms and the frequency of excess anticoagulation (INR >4.0) was found. This may be explained by the number of cases included. AA polymorphisms compared to other polymorphisms shows borderline difference in the warfarin dose. The results can be used for the development of a pharmacogenetic-guided warfarin dosing algorithm.

A Universal Heparin Antidote with Negligible Effect on Fibrin(ogen) and Plasma Coagulation

Background and ObjectiveAnticoagulants play a pivotal role in the treatment of thromboembolic disorders. Haemorrhage in surgical patients receiving anticoagulants is a major concern. Antidotes are administered to counteract anticoagulation and to restore normal hemostasis. To date, protamine sulphate (PS), a cationic polypeptide is the only clinically approved antidote for unfractionated heparin. PS has toxic side effects and limitations. Inability of PS to completely reverse low molecular weight heparins and fondaparinux is due to its low binding affinity to these drugs. However, PS interacts with coagulation proteins such as fibrinogen to form aggregates which leads to cardiovascular adverse effects. Recently, we developed a synthetic universal heparin reversal agent (UHRA) with high binding affinity to heparins. In vivo studies revealed that UHRA completely reverse the activity of all clinical available parenteral anticoagulants and is nontoxic. This study aims to demonstrate the nontoxic nature of UHRA by assessing its influence on fibrinogen, fibrin clot architecture, plasma clotting and clot lysis.MethodsUHRA was developed by incorporating tertiary amine based heparin binding groups on a dendritic hyperbranched polyglycerol scaffold and capping it with methoxy polyethylene glycol chains. Recalcification and tissue factor (TF) initiated turbidimetric plasma clotting assays was performed to understand the impact of UHRA on coagulation system. The interaction of UHRA on fibrinogen was investigated by fibrinogen aggregation assay, fibrin polymerization assay and by spectroscopic analysis (fluorescence and circular dichroism (CD)). The influence of UHRA on fibrin clot architecture was evaluated by scanning electron microscopy (SEM).The anticoagulant neutralization (heparins) by UHRA was studied by fluorogenic thrombin generation assay (TGA) in human platelet-rich plasma (PRP). The lysis of TF-induced plasma clot containing UHRA or PS exposed to exogenous tissue plasminogen activator (t-PA) was studied by turbidimetric assay.Results and discussionResults from the plasma clotting assays showed that UHRA did not alter the clotting parameters compared to PS (TF initiated lag time and maximum absorbance, control vs UHRA 200 mcg/mL, p=0.21 and 0.16, respectively; lag time and maximum absorbance in recalcification, control vs UHRA 200mcg/mL, p=0.08 and 0.13, respectively) suggesting that UHRA has no effect on coagulation system at the concentration studied (Figure 1). Unlike protamine, the fibrinogen aggregation and fibrin polymerization assay was not influenced by UHRA over a broad range of concentrations from 0.05mg/mL to 1mg/mL. Together with tryptophan fluorescence quenching measurements (Figure 2) and fibrinogen secondary structure measurements corroborates that UHRA is not interacting with fibrinogen. The results are quite different from PS and other synthetic cationic polymers which interact with fibrinogen eliciting aggregation and conformational changes. Fibrin clots generated in presence of UHRA (even at 0.5 mg/mL) showed similar structure and fiber size remains same as normal fibrin clot (control vs UHRA 0.5 mg/mL clot, p= 0.12) (Figure 3). On the other hand, fibrin clots formed in the presence of 0.05mg/mL PS (clinical dose) increased the fiber size and changed the clot structure dramatically (control vs PS 0.05mg/mL clot, p< 0.0001). Our plasma clot lysis studies in the presence of exogenous t-PA demonstrate that UHRA did not enhance clot degradation unlike protamine. UHRA restored thrombin levels in anticoagulated PRP (heparinized) demonstrating the efficacy.Conclusion and significanceOur studies demonstrate that universal heparin antidote, UHRA, has negligible impact on fibrinogen, fibrin polymerization, clot structure, clot degradation and the coagulation system revealing their excellent hemocompatibility compared to protamine. Our results support the fact that UHRA could be an ideal antidote to restore hemostasis following invasive surgical procedures and to address bleeding complications by heparin based anticoagulants. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Rare Complication of Low-Molecular-Weight Heparin Therapy: Bladder Hematoma

Introduction: Low-molecular-weight heparin is one of the most preferred agents used in the prevention and treatment of thromboembolic disorders. Case Report: An 85-year-old female patient was admitted for 3 days with ongoing and growing vaginal bleeding. Bladder hematoma and postrenal acute renal failure, rare complications of low-molecular-weight heparins, are presented in this case. Conclusion: Patients who have complaints about vaginal bleeding or hematuria should be questioned about a detailed history and the use of low-molecular-weight heparin. Low-molecular-weight heparin in rare cases may lead to bleeding, and postrenal acute renal failure should be kept in mind.