Anticoagulant Potency Research Articles

Patient and Perioperative Variables Affecting 30-Day Readmission for Surgical Complications After Hip and Knee Arthroplasties: A Matched Cohort Study

BackgroundChanges in reimbursement for total hip and knee arthroplasties (THA and TKA) have placed increased financial burden of early readmission on hospitals and surgeons. Our purpose was to characterize factors of 30-day readmission for surgical complications after THA and TKA at a single, high-volume orthopedic specialty hospital. MethodsPatients with a diagnosis of osteoarthritis and who were readmitted within 30 days of their unilateral primary THA or TKA procedure between 2010 and 2014. Readmitted patients were matched to nonreadmitted patients 1:2. Patient and perioperative variables were collected for both cohorts. A conditional logistic regression was performed to assess both the patient and perioperative factors and their predictive value toward 30-day readmission. ResultsTwenty-one thousand eight hundred sixty-four arthroplasties (THA = 11,105; TKA = 10,759) were performed between 2010 and 2014 at our institution, in which 60 patients (THA = 37, TKA = 23) were readmitted during this 5-year period. The most common reasons for readmission were fracture (N = 14), infection (N = 14), and dislocation (N = 9). Thirty-day readmission for THA was associated with increased procedure time (P = .05), length of stay (LOS) shorter than 2 days (P = .04), discharge to a skilled nursing facility (P = .05), and anticoagulation use other than aspirin (P = .02). Thirty-day readmission for TKA was associated with increased tourniquet time (P = .02), LOS <3 days (P < .01), and preoperative depression (P = .02). In the combined THA/TKA model, a diagnosis of depression increased 30-day readmission (odds ratio 3.5 [1.4-8.5]; P < .01). ConclusionRisk factors for 30-day readmission for surgical complications included short LOS, discharge destination, increased procedure/tourniquet time, potent anticoagulation use, and preoperative diagnosis of depression. A focus on risk factor modification and improved risk stratification models are necessary to optimize patient care using readmission rates as a quality benchmark.

Impact of recent guideline changes on aspirin prescribing after knee arthroplasty.

BackgroundPrior to 2012, the American Academy of Orthopaedic Surgeons (AAOS) and American College of Chest Physicians (ACCP) differed in their recommendations for postoperative pharmacologic venous thromboembolism prophylaxis (VTEP) after total joint arthroplasty. More specifically, aspirin (ASA) monotherapy was not endorsed by the ACCP as an acceptable prophylaxis. In 2012, the ACCP supported ASA monotherapy compared with no prophylaxis. Our aim was to investigate the impact of the convergence of ACCP and AAOS recommendations on surgeon prescribing patterns after knee arthroplasty (KA).MethodsThis is a retrospective chart review. We collected data to assess preoperative VTE risk and examined VTEP prescriptions on postoperative day 1 (POD1) and at discharge (D/C) from 7/2008 to 12/2011 (pre-period) and 1/2012 to 7/2014 (post-period). Adult patients undergoing primary and revision KA were identified by ICD-9 procedure codes. Patients on preoperative full-dose anticoagulation and with hypercoagulability disorders were excluded.ResultsOf 368 records reviewed, 329 were included in the analysis. There were no differences between the two period groups for age, sex, BMI, estrogen therapy, malignancy, smoking status, prior VTE, bilateral procedures, or surgery within 3 months. On POD1, in the pre-period, 4.6 % were prescribed ASA monotherapy versus 44.4 % in the post-period (p < 0.001). On D/C, in the pre-period, 13.9 % were prescribed ASA versus 55.6 % in the post-period (p < 0.001).ConclusionsOur results indicate a statistically significant change in orthopedist prescribing patterns after guideline convergence. Furthermore, there was no apparent change in VTE risk between the two study groups when excluding patients necessitating full anticoagulation. Prior literature has shown that the divergence in guidelines influenced physicians away from ASA and toward more potent anticoagulants in order to avoid potential litigation. Once its role in VTEP was supported by the ACCP, it appears that ASA monotherapy was readily and rapidly incorporated into clinical practice. ASA may be favored over other VTEP agents for its lower bleeding risk profile and cost. This study highlights the profound impact clinical practice guidelines have on clinician prescribing patterns. Although prospective randomized trials are needed to compare the efficacy of ASA with other VTEP agents, ASA is now a predominant part of the VTEP armamentarium after KA.

Transformation of Lettuce with rol ABC Genes: Extracts Show Enhanced Antioxidant, Analgesic, Anti-Inflammatory, Antidepressant, and Anticoagulant Activities in Rats.

Lettuce is an edible crop that is well known for dietary and antioxidant benefits. The present study was conducted to investigate the effects of rol ABC genes on antioxidant and medicinal potential of lettuce by Agrobacterium-mediated transformation. Transgene integration and expression was confirmed through PCR and real-time RT-PCR, respectively. The transformed plants showed 91-102% increase in total phenolic contents and 53-65% increase in total flavonoid contents compared to untransformed plants. Total antioxidant capacity and total reducing power increased up to 112 and 133% in transformed plants, respectively. Results of DPPH assay showed maximum 51% increase, and lipid peroxidation assay exhibited 20% increase in antioxidant activity of transformed plants compared to controls. Different in vivo assays were carried out in rats. The transgenic plants showed up to 80% inhibition in both hot plate analgesic assay and carrageenan-induced hind paw edema test, while untransformed plants showed only 45% inhibition. Antidepressant and anticoagulant potential of transformed plants was also significantly enhanced compared to untransformed plants. Taken together, the present work highlights the use of rol genes to enhance the secondary metabolite production in lettuce and improve its analgesic, anti-inflammatory, antidepressant, and anticoagulatory properties.

Pulmonary Embolism Rates Following Total Hip Arthroplasty With Prophylactic Anticoagulation: Some Pulmonary Emboli Cannot Be Avoided

BackgroundA symptomatic pulmonary embolism (PE) after total joint arthroplasty has been described as a “never event.” Despite potent anticoagulants and improvements in patient care, PE continues to occur following total hip arthroplasty (THA). This study evaluates symptomatic PE rates over time in THA patients enrolled in multicenter randomized clinical trials (RCTs) assessing the efficacy of venous thromboembolism prophylaxis regimens. MethodsThe MEDLINE and Cochrane Central Register of Controlled Trials were searched to identify clinical trials assessing prophylactic anticoagulation in patients undergoing THA between January 1995 and December 2015. Inclusion criteria consisted of RCTs evaluating prophylactic anticoagulation in patients undergoing THA. A random effect model was used to combine PE rates across studies. ResultsA total of 21 studies (34,764 patients) were included. Patients were administered low molecular weight heparin (13,590 patients), oral factor Xa inhibitors (6609 patients), oral direct thrombin inhibitors (5965 patients), indirect factors Xa/IIa inhibitors (3444 patients), aspirin (2427 patients), and warfarin (489 patients). Mobile compression was used in 199 patients, and placebo was used in 2041 patients. Across all included studies, the estimated PE rate was 0.21% (95% confidence interval: 0.13%, 0.32%). Between 1997 and 2013, the proportion of PEs did not change in regression analysis. ConclusionAlthough the PE rate was low, it was consistent throughout the 17 years spanning these RCTs, which excluded patients with significant morbidity. These results suggest that even healthy THA patients receiving aggressive anticoagulation still have a risk for PE, and the “never event” designation requires reassessment.

Biological and structural analyses of bovine heparin fractions of intermediate and high molecular weight

Low molecular weight heparin, which is generally obtained by chemical and enzymatic depolymerization of unfractionated heparin, has high bioavailability and can be subcutaneously injected. The aim of the present investigation was to fractionate bovine heparin using a physical method (ultrafiltration through a 10kDa cut-off membrane), avoiding structural modifications that can be caused by chemical or enzymatic treatments. Two fractions with different molecular weights were obtained: the first had an intermediate molecular weight (B-IMWH; Mn=9587Da) and the other had a high molecular weight (B-HMWH; 22,396Da). B-IMWH and B-HMWH have anticoagulant activity of 103 and 154IU/mg respectively, which could be inhibited by protamine. Both fractions inhibited α-thrombin and factor Xa in vitro and showed antithrombotic effect in vivo. Moreover, ex vivo aPTT assay demonstrated that B-IMWH is absorbed by subcutaneous route. The results showed that ultrafiltration can be used to obtain two bovine heparin fractions, which differ on their molecular weights, structural components, anticoagulant potency, and administration routes.

G-Quadruplex Aptamers to Human Thrombin Versus Other Direct Thrombin Inhibitors: The Focus on Mechanism of Action and Drug Efficiency as Anticoagulants.

Thrombin is a key enzyme of blood coagulation system which has multiple functions including pro- and anticoagulant, platelet aggregating and inflammatory activities. Unsurprisingly, this enzyme has been a target for anticoagulant drug development for decades. Among the most interesting direct thrombin inhibitors with intravenous administration route are the following ones: 1) hirudins, proteins with bivalent binding mode to the thrombin, 2) bivalirudin, the peptide with bivalent binding mode to the thrombin, 3) argatroban, the chemical that binds to the thrombin active site, and 4) G-quadruplex DNA aptamers, structured oligonucleotides with an affinity to protein-binding site of the thrombin. Efficiency of all these inhibitors has been studied in vivo in preclinical and clinical trials, as well as in vitro with various tests, allowing to compare them thoroughly. In the review three levels of comparison were used to highlight the features of each inhibitor: 1) thrombin inhibition constants as a characteristic of inhibitor potency in simple enzymatic system; 2) inhibition of fibrin fiber formation and thrombin generation in coagulation cascade as a characteristic of anticoagulant potency in human blood plasma; and 3) therapeutic doses used and therapeutic profiles obtained after intravenous administration into animals and humans. The data clearly demonstrate weak and strong aspects of thrombin binding aptamers providing a solid background for further novel anticoagulant development. &lt;/p&gt;&lt;p&gt.

Paper wasp nest-mediated biosynthesis of silver nanoparticles for antimicrobial, catalytic, anticoagulant, and thrombolytic applications.

Biosynthesis of silver nanoparticles (AgNPs) using nest extract of paper wasp (Polistes sp) was investigated in this work. The AgNPs were characterized by UV–Vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), and transmission electron microscopy (TEM), and evaluated for antibacterial, antifungal, dye degradation, blood anticoagulation, and blood clot dissolution (thrombolytic) activities. The crystalline polydispersed AgNPs with size range of 12.5–95.55 nm absorbed maximally at 428 nm and showed anisotropic structures of sphere, triangle, hexagon, rod, and rhombus. The FTIR data showed prominent peaks at 3426 and 1641 cm−1, which indicate the involvement of phenolics compounds and proteins in the synthesis of AgNPs. The prominence of Ag in the EDX spectra showed that indeed, AgNPs were formed. The AgNPs showed potent antibacterial activities (12–35 mm) against three multi-drug strains of Pseudomonas aeruginosa and Klebsiella granulomatis. While the growth of Aspergillus flavus and Aspergillus niger was completely suppressed, the AgNPs produced growth inhibition of 75.61 % against Aspergillus fumigatus at 100 µg/ml. Furthermore, the AgNPs degraded malachite green to the tune of 93.1 %. The AgNPs also prevented coagulation of blood, while it completely dissolved preformed blood clots within 5 min showing the potent anticoagulation and thrombolytic activities. This study, which is the first of its kind to use nest extract of paper wasp for the synthesis of nanoparticles, has shown that the biosynthesized AgNPs could be deployed for biomedical and catalytic applications.

Anticoagulant properties and cytotoxic effect against HCT116 human colon cell line of sulfated glycosaminoglycans isolated from the Norway lobster (Nephrops norvegicus) shell.

Sulfated glycosaminoglycans (SGNL) were extracted for the first time from Norway lobster (Nephrops norvegicus) shell. The monosaccharide composition analysed by GC/MS revealed the presence of galacturonic acid, glucuronic acid, N-acetylgalactosamine and N-acetylglucosamine. The analysis of SGNL with acetate cellulose electrophoresis in Zn-acetate revealed the presence of heparan sulfate (HS) and dermatan sulfate (DS). SGNL were evaluated for their anticoagulant activities using activated partial thromboplastin time (aPTT), thrombin time (TT) and prothrombine time (PT) tests. After 21h incubation, HCT116 cell proliferation was inhibited (p<0.05) between 39.7 and 54.8% at 1.5-7.5mg/mL of SGNL. SGNL don't show hemolytic activity towards bovine erythrocytes and no cytotoxicity against the normal lymphocytes. The antiproliferative efficacy of these lobster glycosaminoglycans were probably related with the higher sulfate content. SGNL demonstrated promising antiproliferative and anticoagulant potential, which may be used as a novel, effective and promising antithrombotic agent.

Individualized Risk Model for Venous Thromboembolism After Total Joint Arthroplasty

BackgroundVenous thromboembolism (VTE) after total joint arthroplasty (TJA) is a potentially fatal complication. Currently, a standard protocol for postoperative VTE prophylaxis is used that makes little distinction between patients at varying risks of VTE. We sought to develop a simple scoring system identifying patients at higher risk for VTE in whom more potent anticoagulation may need to be administered. MethodsUtilizing the National Inpatient Sample data, 1,721,806 patients undergoing TJA were identified, among whom 15,775 (0.9%) developed VTE after index arthroplasty. Among the cohort, all known potential risk factors for VTE were assessed. An initial logistic regression model using potential predictors for VTE was performed. Predictors with little contribution or poor predictive power were pruned from the data, and the model was refit. ResultsAfter pruning of variables that had little to no contribution to VTE risk, using the logistic regression, all independent predictors of VTE after TJA were identified in the data. Relative weights for each factor were determined. Hypercoagulability, metastatic cancer, stroke, sepsis, and chronic obstructive pulmonary disease had some of the highest points. Patients with any of these conditions had risk for postoperative VTE that exceeded the 3% rate. Based on the model, an iOS (iPhone operating system) application was developed (VTEstimator) that could be used to assign patients into low or high risk for VTE after TJA. ConclusionWe believe individualization of VTE prophylaxis after TJA can improve the efficacy of preventing VTE while minimizing untoward risks associated with the administration of anticoagulation.

Bilateral upper extremity severe ecchymosis in a patient on alirocumab, rivaroxaban, and clopidogrel therapy

The PCSK9 inhibitors, alirocumab and evolocumab, are a new class of powerful LDL cholesterol lowering agents which allow previously unattainable lowering of LDL cholesterol (LDLC). Once past the necessarily restrictive confines of placebo-controlled clinical trials, in current medical practice, some patients with concomitant thromboembolism and arterial disease may also be taking potent anticoagulants and/or anti-platelet agents in addition to PCSK9 inhibitors. There are currently no reports on drug interactions and no warnings when alirocumab or evolocumab are used in conjunction with anticoagulants and/or anti-platelet therapy. Here, we present a case of patient with exceptional LDLC lowering (LDLC < 4 mg/dl) on alirocumab 150 mg/ml every 2 weeks and concomitant anti-platelet (clopidogrel 75 mg/day) and anti-coagulation therapy (rivaroxaban 10 mg/day) because of recurrent non-cardiogenic amaurosis fugax, and transient ischemic attacks, and ischemic stroke associated with heterozygosity for the G20210A prothrombin gene mutation. On this regimen, the patient developed gradually spreading intensive skin ecchymosis and bleeding on both arms. This resolved after stopping alirocumab, clopidogrel, and rivaroxaban, and did not reappear when clopidogrel and rivaroxaban were restarted without alirocumab.

The potential of coumatetralyl enhanced by cholecalciferol in the control of anticoagulant-resistant Norway rats (Rattus norvegicus).

We evaluated the potential of cholecalciferol as an enhancer of the first-generation anticoagulant coumatetralyl in the Westphalia anticoagulant-resistant strain of the Norway rat (Rattus norvegicus Berkenhout), characterised by the Tyr139Cys polymorphism on the VKOR enzyme. Because today only the most potent, but also most persistent anticoagulant rodenticides of the second generation remain available to control this strain, new rodenticide solutions are required. Feeding trials in the laboratory confirmed a significant level of efficacy, which was corroborated by field trials in the Münsterland resistance area. After frequency and level of resistance were assessed by blood clotting response tests, field trials were conducted with bait containing coumatetralyl at 375 mg kg-1 and cholecalciferol at 50 mg kg-1 or 100 mg kg-1 . Control success was 94% when a large rat infestation comprising 42% resistant animals was treated. Another field trial applying the combination to a rat population that had survived a preceding treatment with bromadiolone resulted in a 99.5% control success according to the first census day, but with some increase in rat activity during subsequent census days. The combination of coumatetralyl and cholecalciferol is a promising alternative approach to the most potent second-generation anticoagulants in resistance management, particularly in respect of environmental risks, such as secondary poisoning. © 2016 Society of Chemical Industry.

Development and characterization of an innovative heparin coating to stabilize and protect liposomes against adverse immune reactions

Liposomes have been recognized as excellent drug delivery systems, but when they come in direct contact with different blood components they may trigger an immediate activation of the innate immune system. The aim of the present study was to produce long-circulating, blood-compatible liposomes by developing a construct of liposomes covered by a novel unique heparin complex (CHC; 70 heparin molecules per complex) to avoid recognition by the innate immune system. Unilamellar, cationic liposomes were produced by hand extrusion through a 100-nm polycarbonate membrane. Coating of liposomes with the macromolecular CHC was accomplished by electrostatic interactions. Dynamic light scattering as well as QCM-D measurements were used to verify the electrostatic deposition of the negatively charged CHC to cationic liposomes. The CHC-coated liposomes did not aggregate when in contact with lepirudin anti-coagulated plasma. Unlike previous attempts to coat liposomes with heparin, this technique produced freely moveable heparin strands sticking out from the liposome surface, which exposed AT binding sites reflecting the anticoagulant potentials of the liposomes. In experiments using lepirudin-anticoagulated plasma, CHC-coated liposomes, in contrast to non-coated control liposomes, did not activate the complement system, as evidenced by low C3a and sC5b-9 generation and reduced leakage from the liposomes. In conclusion, we show that liposomes can be successfully coated with the biopolymer CHC, resulting in biocompatible and stable liposomes that have significant application potential.

Design of Potent and Controllable Anticoagulants Using DNA Aptamers and Nanostructures.

The regulation of thrombin activity offers an opportunity to regulate blood clotting because of the central role played by this molecule in the coagulation cascade. Thrombin-binding DNA aptamers have been used to inhibit thrombin activity. In the past, to address the low efficacy reported for these aptamers during clinical trials, multiple aptamers have been linked using DNA nanostructures. Here, we modify that strategy by linking multiple copies of various thrombin-binding aptamers using DNA weave tiles. The resulting constructs have very high anticoagulant activity in functional assays owing to their improved cooperative binding affinity to thrombin due to optimized spacing, orientation, and the high local concentration of aptamers. We also report the results of molecular dynamics simulations to gain insight into the solution conformations of the tiles. Moreover, by using DNA strand displacement, we were able to turn the coagulation cascade off and on as desired, thereby enabling significantly better control over blood coagulation.

Pretreatment with rivaroxaban attenuates stroke severity in rats by a dual antithrombotic and anti-inflammatory mechanism.

Stroke outcome is more favourable in patients receiving oral anticoagulants compared with non-anticoagulated patients. The reasons for this "stroke-attenuating" property of oral anticoagulants are largely unknown. This study examined whether prestroke anticoagulation with rivaroxaban, a novel direct factor Xa inhibitor, influences stroke severity, thrombin-mediated intracerebral thrombus formation and pro-inflammatory processes in a rat model of brain ischaemia/reperfusion injury. Male Wistar rats were anticoagulated with rivaroxaban and subjected to 90 minutes of transient middle cerebral artery occlusion. Infarct size, functional outcome and the occurrence of intracranial haemorrhage (ICH) were assessed until day 7. Thrombin generation was determined by measuring the amount of thrombin/antithrombin complex. Intracerebral thrombus formation was evaluated by histology and Western blot. CD68-immunoreactivity and the expression of cytokines and adhesion molecules were investigated to assess postischaemic inflammation. The integrity of the blood-brain barrier was analysed using fluorescein isothiocyanate-dextran. Rats pretreated with rivaroxaban developed significantly smaller strokes and less severe functional deficits compared with controls. Although rivaroxaban strongly reduced thrombin-mediated thrombus formation, this was not accompanied by an increased risk of ICH. In addition, rivaroxaban dampened the inflammatory response in the ischaemic brain by downregulating ICAM-1 expression and the activation of CD68+-immune cells. In contrast, rivaroxaban had no effect on the integrity of the blood-brain barrier after stroke. Here, we identified reduced thrombo-inflammation as a major determinant of the stroke-protective property of rivaroxaban in rats. Further studies are needed to assess the therapeutic potential of novel oral anticoagulants in the acute phase after a stroke.

Resourcing of Heparin and Low Molecular Weight Heparins from Bovine, Ovine, and Porcine Origin. Studies to Demonstrate the Biosimilarities

Introduction:The currently used unfractionated heparin (UFH) and low molecular weight heparins (LMWH) are mostly derived from Porcine mucosal tissue. Since the technology to manufacture heparin has advanced and the quality assurance practices are in place, improved products with high potency and purity are now available. At the same time the demand for heparins has increased requiring alternate sources to obtain heparin and LMWH. Considering these factors the resourcing of heparins utilizing bovine (cow) and ovine (sheep) tissues is discussed at regulatory and pharmaceutical levels. The pharmaceutical industry has already initiated programs to manufacture Bovine, Ovine, and Porcine heparins and depolymerized enoxaparin from these products some of which are currently in various phases of development. The purpose of this study is to compare 5 individual batches of UFH obtained from Bovine, Ovine, and Porcine origin and their depolymerized product obtained by benzylation followed by alkaline hydrolysis representing enoxaparins.Methods:The molecular profile of the heparins and enoxaparins from various sources were determined using the size exclusion method. A narrow range calibration method was used for comparing the molecular weight of heparin, whereas the EP method was used to cross-reference the molecular weight of enoxaparins. The anticoagulant potency was measured use clot based methods such as aPTT and Thrombin Time. Chromogenic substrate based methods were used to determine the USP potency in terms of anti-Xa and anti-IIa activities (Hyphen Biomedical, Ohio, USA). The interaction between AT and heparins and enoxaparin were investigated in a purified biochemical system, using AT supplemented buffered assay. Thrombin Generation inhibition studies were carried out using a flourometric method (Technoclone, Vienna, Austria). The relative interaction of the heparins and enoxaparins with heparin induced thrombocytopenia (HIT) antibody induced aggregation of platelets were investigated using serum pool obtained from clinically confirmed HIT cases using aggregometry.Results:The molecular profile of the Bovine, ovine, and porcine heparins and enoxaparin were almost identical. In the clot based assays, such as PT and PTT, Porcine and Ovine heparin produce consistently comparable anticoagulant effects, which were stronger in comparison to the bovine derived heparins. In contrast, the enoxaparins derived from these three sources showed minimal differences. In the amidolytic anti Xa and IIa assays both ovine and porcine heparins produced similar inhibitory effects, whereas the bovine heparin exhibited lower activity. In the purified system the Porcine and Ovine preparations consistently showed lower IC50 values for both the thrombin and Xa inhibition in contrast to the bovine heparin. Similar trends were observed in the anti IIa assays. The USP potency of the Porcine and Ovine heparins ranged from 180 to 190u/mg, whereas the Bovine was found to be 130-140 u/mg. The anti-Xa - IIa ratio for the heparin were comparable. The ovine and porcine enoxaparin exhibited comparable potencies which ranged 94-110 u/mg whereas bovine enoxaparin was slightly lower 80-87 u/mg. However the antiXa and anti-IIa ratios were comparable. The AT mediated inhibition of factor Xa and anti-IIa was stronger with heparins in comparison to the enoxaparins. Similarly heparins produced stronger inhibition of thrombin generation in comparison to the enoxaparin. In the HIT screening there was no difference between the HIT responses in the heparins from different species. Similar results were obtained with enoxaparins.Conclusions:These studies show that while bovine, ovine and porcine heparins and enoxaparins exhibit comparable molecular profiles however in some of the functional assays bovine heparin and enoxaparin exhibited somewhat lesser potencies especially in the pharmacopeial assays. No differences were noted in the HIT antibody interactions among heparins and enoxaparins from different species. These studies demonstrate that ovine and porcine heparins are biosimilar and can be developed as such for clinical purposes. The bovine derived heparins exhibit slightly weaker potencies in functional assays despite comparable molecular profile. Potency adjustment for in vivo usage may be required to obtain comparable anticoagulant responses for the bovine heparin and enoxaparin. DisclosuresNo relevant conflicts of interest to declare.

Gold nanoparticles modified with self-assembled hybrid monolayer of triblock aptamers as a photoreversible anticoagulant

We demonstrated that thrombin-binding aptamer-conjugated gold nanoparticles (TBA–Au NPs), prepared from a self-assembled hybrid monolayer (SAHM) of triblock aptamers on Au NPs (13nm), can effectively inhibit thrombin activity toward fibrinogen. The first block poly(adenine) at the end of the triblock TBA was used for the self-assembly on Au NP surface. The second block, in the middle of TBA, was composed of oligonucleotides that could hybridize with each other. The third block, containing TBA15 (15-base, binding to the exosite I of thrombin) and TBA29 (29-base, binding to the exosite II of thrombin) provided bivalent interaction with thrombin. The SAHM triblock aptamers have optimal distances between TBA15 and TBA29, aptamer density, and orientation on the Au NP surfaces. These properties strengthen the interactions with thrombin (Kd=1.5×10−11M), resulting in an extremely high anticoagulant potency. The thrombin clotting time mediated by SAHM TBA15/TBA29–Au NPs was >10 times longer than that of four commercially available drugs (heparin, argatroban, hirudin, or warfarin). In addition, the rat-tail bleeding assay time further demonstrated that the SAHM TBA15/TBA29–Au NPs were superior to heparin. The SAHM TBA15/TBA29–Au NPs exhibited excellent stability in the human plasma (half-life >14days) and good biocompatibility (low cytotoxicity and hemolysis). Most interestingly, the inhibition by SAHM TBA15/TBA29–Au NPs was controllable by the irradiation of green laser, via heat transfer-induced TBA release from Au NPs. Therefore, these easily prepared (self-assembled), low cost (non-thiolated aptamer), photo-controllable, multivalent TBA15/TBA29–Au NPs (high density of TBA15/TBA29 on Au NPs) show good potential for the treatment of various diseases related to blood-clotting disorders. Our study opens up the possibility of regulation of molecule binding, protein recognition, and enzyme activity using SAHM aptamer-functionalized nanomaterials.

The role of factors of the hemostasis system and fibrinolysis in the development of gastroesophageal reflux in patients with chronic obstructive pulmonary disease

Gastroesophageal reflux disease (GERD) and chronic obstructive pulmonary disease (COPD) belong to common pathological conditions comorbid in 25–60 % of cases.Objective: to find probable mechanisms of GERD progressing against the ground of COPD by means of learning various links of the blood clotting system, anticoagulative blood activity and fibrinolysis for further improvement of the ways to correct the disorders found.Methods. 124 patients with COPD were examined including 90 with comorbid GERD. The control group included 24 practically healthy individuals of an appropriate age and sex. Clinical, biochemical, instrumental and statistical methods of examination were applied.Results. The patients were divided into the following groups: the 1st group – 34 patients with COPD, the 2nd group – 30 patients with COPD with endoscopically negative GERD, the 3rd group – 30 patients with COPD and endoscopically positive non-erosive GERD, the 4th group – 30 patients with COPD and endoscopically positive erosive GERD. The following results were found: while examining coagulation hemostasis – decrease of prothrombin time, fibrinogen content; while examining anticoagulation blood potential – decrease of thrombin time, activity of antithrombin III; while examining fibrinolytic blood activity – inhibition of the total fibrinolytic activity (at the expense of enzymatic activity decrease) and compensatory increase of non-enzymatic fibrinolytic activity, decrease of Hageman-dependent fibrinolysis activity.Conclusions. Disorders of coagulation hemostasis, anti-coagulation potential and fibrinolytic blood activity are intensified with the increase of severity of bronchial obstructive syndrome and promote the development of endoscopically positive erosive GERD

Targeting Two Coagulation Cascade Proteases with a Bivalent Aptamer Yields a Potent and Antidote-Controllable Anticoagulant.

Potent and rapid-onset anticoagulation is required for several clinical settings, including cardiopulmonary bypass surgery. In addition, because anticoagulation is associated with increased bleeding following surgery, the ability to rapidly reverse such robust anticoagulation is also important. Previously, we observed that no single aptamer was as potent as heparin for anticoagulating blood. However, we discovered that combinations of two aptamers were as potent as heparin. Herein, we sought to combine two individual anticoagulant aptamers into a single bivalent RNA molecule in an effort to generate a single molecule that retained the potent anticoagulant activity of the combination of individual aptamers. We created four bivalent aptamers that can inhibit Factor X/Xa and prothrombin/thrombin and anticoagulate plasma, as well as the combination of individual aptamers. Detailed characterization of the shortest bivalent aptamer indicates that each aptamer retains full binding and functional activity when presented in the bivalent context. Finally, reversal of this bivalent aptamer with a single antidote was explored, and anticoagulant activity could be rapidly turned off in a dose-dependent manner. These studies demonstrate that bivalent anticoagulant aptamers represent a novel and potent approach to actively and reversibly control coagulation.

Stroke in atrial fibrillation and improving the identification of ‘high‐risk’ patients: the crossroads of immunity and thrombosis

Stroke in atrial fibrillation and improving the identification of ‘high‐risk’ patients: the crossroads of immunity and thrombosis

A Systems Pharmacology Model for Predicting Effects of Factor Xa Inhibitors in Healthy Subjects: Assessment of Pharmacokinetics and Binding Kinetics.

Factor Xa (FXa) emerged as a promising target for effective anticoagulation and several FXa inhibitors are now available for the prevention of venous thromboembolism. However, in previously reported pharmacokinetic/pharmacodynamic (PK/PD) models, the complex coagulation processes and detailed information of drug action are usually unclear, which makes it difficult to predict clinical outcome at the drug discovery stage. In this study, a large-scale systems pharmacology model was developed based on several published models and clinical data. It takes into account all pathways of the coagulation network, and captures drug-specific features: plasma pharmacokinetics and drug-target binding kinetics (BKs). We aimed to predict the anticoagulation effects of FXa inhibitors in healthy subjects, and to use this model to compare the effects of compounds with different binding properties. Our model predicts the clotting time and anti-FXa effects and could thus serve as a predictive tool for the anticoagulant potential of a new compound.