Anticoagulant Effect Of Heparin Research Articles

HEMA-Lysine-Based Cryogels for Highly Selective Heparin Neutralization.

Unfractionated heparin (UFH) and its low-molecular-weight fragments (LMWH) are widely used as anticoagulants for surgical procedures and extracorporeal blood purification therapies such as cardiovascular surgery and dialysis. The anticoagulant effect of heparin is essential for the optimal execution of extracorporeal blood circulation. However, at the end of these procedures, to avoid the risk of bleeding, it is necessary to neutralize it. Currently, the only antidote for heparin neutralization is protamine sulphate, a highly basic protein which constitutes a further source of serious side events and is ineffective in neutralizing LMWH. Furthermore, dialysis patients, due to the routine administration of heparin, often experience serious adverse effects, among which HIT (heparin-induced thrombocytopenia) is one of the most severe. For this reason, the finding of new heparin antagonists or alternative methods for heparin removal from blood is of great interest. Here, we describe the synthesis and characterization of a set of biocompatible macroporous cryogels based on poly(2-hydroxyethyl methacrylate) (pHEMA) and L-lysine with strong filtering capability and remarkable neutralization performance with regard to UFH and LMWH. These properties could enable the design and creation of a filtering device to rapidly reverse heparin, protecting patients from the harmful consequences of the anticoagulant.

Abstract 11378: Reference Intervals of Activated Clotting Time in Pediatric Patients Undergoing Cardiac Surgery

Introduction: Monitoring the anticoagulant effect of heparin is critical to prevent thrombosis and/or bleeding during cardiac surgery. This evaluation is usually performed by assessing the activated clotting time (ACT) with a POC device. Despite the relevance and widespread use of ACT, there are currently limited reference values to use as a target. Furthermore, compared to the adult population, infants have been described with a longer ACT, reinforcing the need to establish a separate reference interval for this segment of the population. Aim: The present work aims to propose reference intervals of ACT in pediatric patients. Methods: We collected data on 155 consecutive patients, ranging in age from 1 day to 18 years, undergoing cardiac surgery from 01-2019 to 01-2021 at Sainte-Justine hospital. Prior to surgery, patients were given UFH as a bolus of 3 mg/kg, and Hemochron Signature Elite ACT+ (Accriva Diagnostics, Inc.) was used to measure ACT before and promptly after heparin injection. Data were subsequently analyzed by using a parametric method. Lower and upper reference limits are the values at the 2.5 and 97.5 percentile, respectively. Results: The ACT before heparin injection followed a normal distribution with a lower reference limit at 85 seconds and an upper limit at 147 seconds compared to the range of 70-120 seconds proposed for the general population. Interestingly, the ACT after heparin bolus exhibited a non-gaussian distribution. Since neonates have been previously described with a greater heparin resistance compared to older infants; patients were separated into two age groups: patients < 1-month of age (Group A; n=31) and patients > 1-month (group B; n=124). Afterward, we performed a log transformation of the ACT values after heparin injection to approximate a Gaussian distribution, yielding mean ACT value of 392 seconds (limits 291-529 seconds) for Group A and 473 seconds (limits 314-996 seconds) for Group B (Welch test p value = 4.4 10 -6 ). No significant difference in ACT values prior to heparin bolus was found between the two groups. Conclusion: Overall, our results provide reference values of ACT measured using an Hemochron device, in a pediatric context, paving the way for improved patient care during cardiac surgery.

Running after Activated Clotting Time Values in Patients Receiving Direct Oral Anticoagulants: A Potentially Dangerous Race. Results from a Prospective Study in Atrial Fibrillation Catheter Ablation Procedures

Background: Activated Clotting Time (ACT) guided heparinization is the gold standard for titrating unfractionated heparin (UFH) administration during atrial fibrillation (AF) ablation procedures. The current ACT target (300 s) is based on studies in patients receiving a vitamin K antagonist (VKA). Several studies have shown that in patients receiving Direct Oral Anticoagulants (DOACs), the correlation between ACT values and UFH delivered dose is weak. Objective: To assess the relationship between ACT and real heparin anticoagulant effect measured by anti-Xa activity in patients receiving different anticoagulant treatments. Methods: Patients referred for AF catheter ablation in our centre were prospectively included depending on their anticoagulant type. Results: 113 patients were included, receiving rivaroxaban (n = 30), apixaban (n = 30), dabigatran (n = 30), and VKA (n = 23). To meet target ACT, a higher UFH dose was required in DOAC than VKA patients (14,077.8 IU vs. 9565.2 IU, p < 0.001), leading to a longer time to achieve target ACT (46.5 min vs. 27.3 min, p = 0.001). The correlation of ACT and anti-Xa activity was tighter in the VKA group (Spearman correlation ρ = 0.53), compared to the DOAC group (ρ = 0.19). Despite lower ACT values in the DOAC group, this group demonstrated a higher mean anti-Xa activity compared to the VKA group (1.56 ± 0.39 vs. 1.14 ± 0.36; p = 0.002). Conclusion: Use of a conventional ACT threshold at 300 s during AF ablation procedures leads to a significant increase in UFH administration in patients treated with DOACs. This increase corresponds more likely to an overdosing than a real increase in UFH requirement.

Laboratory Assays to Address the Problem of Heparin-Protamine Balance Following Neonatal Open Heart Surgery

Introduction: Neonates are especially susceptible to bleeding during and after cardiopulmonary bypass (CPB) surgery seemingly by virtue of a proportionately larger foreign surface exposure area for blood, significant hemodilution, and a coagulation system that differs from older patients. During CPB, heparin is administered to prevent thrombin formation. After CPB, it is reversed with protamine sulfate. Although administered to reverse anticoagulation, high levels of protamine sulfate paradoxically inhibit blood coagulation. This study evaluated the effect of heparin anticoagulation and protamine sulfate reversal on the accuracy of diagnostic coagulation tests and bleeding outcomes in a neonatal population undergoing CPB for congenital heart defects in the first 30 days of life.Methods: Blood samples were obtained at baseline (Draw 1), on CPB (Draw 2), following protamine reversal (Draw 3) and upon admission to the cardiac intensive care unit (Draw 4). Thrombin time (±protamine), FXa activity, activated partial thromboplastin time (APTT), Rotational Thromboelastometry (ROTEM) and Calibrated Automated Thrombography (CAT) were performed. Excessive bleeding was defined by meeting one or more of the following criteria: ≥ 84 mL/kg total chest tube output (CTO) in the first 24 hours, ≥ 7 mL/kg/hr CTO for ≥2 consecutive hours in the first 12 hours, or re-exploration in the first 24 hours for bleeding or tamponade.Results: This prospective observational study enrolled 44 neonates. Of these, 16 (36%) had excessive bleeding. The thrombin time assay was sensitive to heparin but was only minimally impacted by protamine sulfate, unless heparin was in the sample. Therefore, it was selected as the “gold-standard” assay for measurement of heparin. Relative heparin levels, determined by rate of fibrin formation, inversely correlated with thrombin generation measured using CAT, particularly in comparison to the thrombin generation rate in Draw 4 samples (exponential fit R2= 0.333). Higher plasma heparin concentration was present in 85% of the patients at Draw 4 than at Draw 3, indicating postoperative heparin rebound. This was important because, although the plasma heparin concentration in bleeders varied greatly, those patients with the two highest heparin concentrations at Draw 4 had excessive bleeding. The FXa activity assay was altered by protamine and by heparin. Therefore, samples with elevated FXa activity but normal thrombin time likely had high protamine. There were 4 such samples and 2 were bleeders. Additional coagulation assays were compared to the thrombin time assay to assess their performance for detection of heparin. ROTEM and APTT assays also correlated weakly (R2= 0.0531 with P value 0.1527 and R2 = 0.1549 with P value 0.0099, respectively) with the thrombin time assay at Draw 4. However, no correlation was present at Draw 3.Conclusions: Bleeding remains a common complication in neonates undergoing CPB for repair of heart defects. While there are many possible causes for the bleeding, excess heparin or protamine may contribute to its severity in a subset of cases. The simple to perform and inexpensive thrombin time test readily and accurately detects excess heparin. Increased use of this test may be beneficial, particularly for detection of heparin rebound that is observed in approximately 85% of patients. Excess protamine may also contribute to bleeding in these patients, and can be assessed using a combination of thrombin time and factor Xa activity assays. DisclosuresMast:Novo Nordisk: Research Funding.

Host-Guest Complex for Heparin Binding and Sensing

Heparin is a naturally occurring highly charged polyanion and a well-known anticoagulant widely used in surgical practice.[1] More specifically, it is a negatively charged sulfated polysaccharide belonging to the group of glycosaminoglycans. The anticoagulant effect of heparin is based on its ability to activate antithrombin-III, which, subsequently, inactivates vital coagulation cascade substance such as thrombin and factor Xa consequently preventing fibrin and clot formation.[2] To balance and neutralize heparin dosing, a heparin inhibitor is needed, and commonly, arginine-rich protein protamine sulfate (PS) is used for this purpose.[3] However, in recent years there has been an interest to investigate alternatives for protamine due to its adverse effects like hypotension and serious allergic reactions.[4]In this study, a host-guest complex capable of selective heparin binding and sensing is presented.[5] Heparin binding affinity was studied in solution with a variety of polycationic macrocyclic hosts, a pillar[5]arene and multiple resorcin[4]arenes, by dynamic light scattering, dye displacement assay, isothermal titration calorimetry, and anti-Xa assay. The measurements reveal the significant importance of multivalency in the electrostatic host-heparin binding in competitive, application-relevant media. Additionally, to monitor heparin concentration, a host-guest indicator displacement assay was performed by following the free and bound state of methyl orange dye in UV-Vis spectroscopic experiments. Furthermore, this colorimetric sensing based on the tertiary host-guest-heparin supramolecular assembly was utilized in the construction of a calibration curve in a range of blood plasma concentrations. Figure 1

Heparin resistance in severe thermal injury: A prospective cohort study.

BackgroundLow molecular-weight heparin (LMWH) is routinely administered to burn patients for thromboprophylaxis. Some studies have reported heparin resistance, yet the mechanism(s) and prevalence have not been systematically studied. We hypothesized that nucleosomes, composed of histone structures with associated DNA released from injured tissue and activated immune cells in the form of neutrophil extracellular traps (NETs or NETosis), neutralize LMWH resulting in suboptimal anticoagulation, assessed by reduction in anti-factor Xa activity.MethodsBlood was sampled from >15% total body surface area (TBSA) burn patients receiving LMWH on days 5, 10 and 14. Peak anti-factor Xa (AFXa) activity, anti-thrombin (ATIII) activity, cell-free DNA (cfDNA) levels and nucleosome levels were measured. Mixed effects regression was adjusted for multiple confounders, including injury severity and ATIII activity, and was used to test the association between nucleosomes and AFXa.ResultsA total of 30 patients with severe burns were included. Mean TBSA 43% (SD 17). Twenty-three (77%) patients were affected by heparin resistance (defined by AFXa activity <0.2 IU/mL). Mean peak AFXa activity across samples was 0.18 IU/mL (SD 0.11). Mean ATIII was 81.9% activity (SD 20.4). Samples taken at higher LWMH doses were found to have significantly increased AFXa activity, though the effect was not observed at all doses, at 8000 IU no samples were heparin resistant. Nucleosome levels were negatively correlated with AFXa (r = −0.29, p = 0.050) consistent with the hypothesis. The final model, with peak AFXa as the response variable, was adjusted for nucleosome levels (p = 0.0453), ATIII activity (p = 0.0053), LMWH dose pre-sample (p = 0.0049), drug given (enoxaparin or tinzaparin) (p = 0.03), and other confounders including severity of injury, age, gender, time point of sample.ConclusionsHeparin resistance is a prevalent issue in severe burns. Nucleosome levels were increased post-burn, and showed an inverse association with AFXa consistent with the hypothesis that they may interfere with the anticoagulant effect of heparin in vivo and contribute to heparin resistance. Accurate monitoring of AFXa activity with appropriate therapy escalation plans are recommended with dose adjustment following severe burn injury.

Host-Guest Complex for Heparin Binding and Sensing

Heparin is a naturally occurring highly charged polyanion and a well-known anticoagulant widely used in surgical practice.[1] More specifically, it is a negatively charged sulfated polysaccharide belonging to the group of glycosaminoglycans (Scheme 1). The anticoagulant effect of heparin is based on its ability to activate antithrombin-III, which, subsequently, inactivates vital coagulation cascade substance such as thrombin and factor Xa consequently preventing fibrin and clot formation.[2] To balance and neutralize heparin dosing, a heparin inhibitor is needed, and commonly, arginine-rich protein protamine sulfate (PS) is used for this purpose.[3] However, in recent years there has been an interest to investigate alternatives for protamine due to its adverse effects like hypotension and serious allergic reactions.[4]In this study, a host-guest complex capable in selective heparin binding and sensing is presented.[5] Heparin binding affinity was studied in solution with a variety of polycationic macrocyclic hosts, a pillar[5]arene and multiple resorcin[4]arenes, by dynamic light scattering, dye displacement assay, isothermal titration calorimetry, and anti-Xa assay. The measurements reveal the significant importance of multivalency in the electrostatic host-heparin binding in competitive, application-relevant media. Additionally, to monitor heparin concentration, a host-guest indicator displacement assay was performed by following the free and bound state of methyl orange dye in UV-Vis spectroscopic experiments. Furthermore, this colorimetric sensing based on the tertiary host-guest-heparin supramolecular assembly was utilized in the construction of a calibration curve in a range of blood plasma concentrations.

Heparin Forms Polymers with Cell-free DNA Which Elongate Under Shear in Flowing Blood

Heparin is a widely used anticoagulant which inhibits factor Xa and thrombin through potentiation of antithrombin. We recently identified that the nucleic acid stain SYTOX reacts with platelet polyphosphate due to molecular similarities, some of which are shared by heparin. We attempted to study heparin in flowing blood by live-cell fluorescence microscopy, using SYTOX for heparin visualisation. Immunostaining was performed with monoclonal antibodies directed against various heparin-binding proteins. In addition, we studied modulation of heparin activity in coagulation assays, as well its effects on fibrin formation under flow in recalcified whole blood. We found that SYTOX-positive polymers appear in heparinised blood under flow. These polymers typically associate with platelet aggregates and their length (reversibly) increases with shear rate. Immunostaining revealed that of the heparin-binding proteins assessed, they only contain histones. In coagulation assays and flow studies on fibrin formation, we found that addition of exogenous histones reverses the anticoagulant effects of heparin. Furthermore, the polymers do not appear in the presence of DNase I, heparinase I/III, or the heparin antidote protamine. These findings suggest that heparin forms polymeric complexes with cell-free DNA in whole blood through a currently unidentified mechanism.

Relationship between antithrombin III genetic polymorphism and individual variation in anticoagulant effect of heparin in patients of different nationalities

Objective To evaluate the relationship between antithrombin Ⅲ(ATⅢ)genetic polymorphism and individual variation in anticoagulant effect of heparin in the patients of different nationalities. Methods Sixty patients of Uighur nationality and 60 patients of Han nationality, aged 18-64 yr, with body mass index of 21-25 kg/m2, of American Society of Anesthesiologists physical statusⅠ-Ⅲ, undergoing elective cardiac surgery under cardiopulmonary bypass, were divided into Uighur group and Han group, respectively.ATⅢ gene polymorphism was detected by polymerase chain reaction(A/G was heterozygous, A/A and G/G were homozygous). Heparin sodium was intravenously injected at 5 min before the start of cardiopulmonary bypass with an amount of ACT ≥ 480 s. The amount of heparin and protamine, intraoperative blood transfusion and postoperative 24 h drainage(pericardium, mediastinum/thoracic cavity)were recorded.Activated partial thromboplastin time and prothrombin time were measured at 10 min before operation and 24 h after operation. Results Compared with Han group, the amount of heparin, ratio of protamine to heparin for heparin neutralization and requirement for intraoperative blood transfusion were significantly decreased, the postoperative drainage volume was decreased, activated partial thromboplastin time was shortened at 24 h after operation, the frequency of A/A genotype was increased and the frequency of G/G genotype was decreased at ATⅢ gene single-nucleotide polymorphism sites rs5877 and rs5878, and the minimum allele(A>G)frequency was increased in Uighur group(P<0.05). Conclusion ATⅢ gene polymorphism may be one of the mechanisms underlying individual variation in anticoagulant effect of heparin between the patients of Uighur nationality and Han nationality. Key words: Antithrombin Ⅲ; Polymorphism, single nucleotide; Heparin; Ethnic groups

Rivaroxaban for the treatment of venous thromboembolism in patients with nephrotic syndrome and low AT-III: A pilot study.

The anticoagulation effect of heparin requires adequate serum antithrombin (AT)-III levels. Rivaroxaban, however, exhibits its anticoagulation effects independent of AT-III. The aim of the present study was to evaluate the efficacy and safety of rivaroxaban as a treatment for venous thromboembolism in patients with AT-III deficiency due to nephrotic syndrome. Patients with nephrotic syndrome and low serum concentration and functional activity of AT-III and venous thromboembolism were randomly assigned to the rivaroxaban group (n=8) and low weight molecular heparin group (n=8). The patients were treated for 4 weeks and evaluated at weeks 2 and 4. The primary endpoint was thrombus dissolution or a >90% decrease in thrombus volume in 4 weeks. Secondary endpoints included an increase in the volume of the pre-existing thrombosis and safety assessments. In each of the two groups, 7/8 patients achieved a primary endpoint. At week 2, 5 patients in the rivaroxaban group and 4 in the low weight molecular heparin group had achieved the primary endpoint. Notably, at week 2 the patients whose AT-III levels and functional activity remained low in the low weight molecular heparin group did not achieve the primary endpoint. The adverse effects were similar in both groups, with no severe hemorrhage observed. In conclusion, the results of this pilot study demonstrate that rivaroxaban may be an effective, safe, single-agent approach for treating vein thromboembolism in patients with nephrotic syndrome and low AT-III levels. The potential benefits of rivaroxaban over low weight molecular heparin treatment require further investigation with a larger sample size in order to validate the findings of the present study.

Micro-electromechanical film bulk acoustic sensor for plasma and whole blood coagulation monitoring

Monitoring blood coagulation is an important issue in the surgeries and the treatment of cardiovascular diseases. In this work, we reported a novel strategy for the blood coagulation monitoring based on a micro-electromechanical film bulk acoustic resonator. The resonator was excited by a lateral electric field and operated under the shear mode with a frequency of 1.9GHz. According to the apparent step-ladder curves of the frequency response to the change of blood viscoelasticity, the coagulation time (prothrombin time) and the coagulation kinetics were measured with the sample consumption of only 1μl. The procoagulant activity of thromboplastin and the anticoagulant effect of heparin on the blood coagulation process were illustrated exemplarily. The measured prothrombin times showed a good linear correlation with R2=0.99969 and a consistency with the coefficient of variation less than 5% compared with the commercial coagulometer. The proposed film bulk acoustic sensor, which has the advantages of small size, light weight, low cost, simple operation and little sample consumption, is a promising device for miniaturized, online and automated analytical system for routine diagnostics of hemostatic status and personal health monitoring.

Clinical use and the Italian demand for antithrombin.

Antithrombin (AT) is a single-stranded glycoprotein synthesised in the liver with a plasma concentration of approximately 150 μg/mL and a molecular weight of approximately 59,000 Da1. It is a complex molecule with multiple biologically important properties. It is a serine protease inhibitor (serpin) that inactivates many enzymes in the coagulation cascade2,3. Indeed, it is the key inhibitor of the coagulation system and is estimated to provide 80% of the inhibitory activity against thrombin but it also inhibits activated factors X, IX, VII, XI, and XII. AT has a great affinity for thrombin and is also known as heparin cofactor as it is responsible for the anticoagulant effect of heparin. Heparins markedly accelerate the rate of complex formation between AT and the serine proteases thus increasing AT inhibitory activity 5,000–40,000-fold4. AT also has remarkable anti-inflammatory properties, several of which result from its actions in the coagulation cascade. Other anti-inflammatory properties of AT involve direct interactions with endothelial cells, leading to the release of prostacyclin, which also mediates its anti-platelet effect3. In addition, recent evidence of interactions of the endothelial cell growth factors bFGF (basic fibroblast growth factor) and VEGF (vascular endothelial cell growth factor) with a heparin-like molecule in matrix provide the rationale for further investigation into the possible role of AT as a potent anti-angiogenic factor5. Normal values of AT activity in the plasma range from 80% to 120%. In normal conditions, its biological half-life is 1.5–2.5 days. In conditions of acquired deficiency and in the presence of heparin, the half-life of AT can be notably shorter, being reduced to only a few hours6.

Heparin supplement counteracts the prohemostatic effect of prothrombin complex concentrate and factor IX concentrate: An in vitro evaluation

IntroductionCoagulation factor concentrates like factor IX (FIX) and prothrombin complex concentrate (PCC) can contain anticoagulant substances that may hamper their procoagulant effectiveness in the treatment of hemophilia B or reversal of oral anticoagulation, as well as the laboratory assessment thereof. The aim of the present study was to evaluate the influence of anticoagulant heparin supplement on the prohemostatic potential of different PCCs and FIX concentrates. Materials and methodsProhemostatic potential was evaluated in vitro employing PT/aPTT, thrombography (TGA) and thromboelastography (TEG) with FIX deficient plasma, vitamin K antagonist (VKA)-anticoagulated plasma and plasma anticoagulated with rivaroxaban. ResultsMost PCCs contained heparin, while heparin was detected in 1 out of 4 examined FIX concentrates. All heparin-containing clotting factor concentrates showed severely hampered prohemostatic effects when therapeutic doses were added to anticoagulated plasmas. Upon heparin removal, comparable prohemostatic effects were observed. Of importance is the notion that the anticoagulant effect of heparin was enhanced by rivaroxaban, resulting in a 7 fold increased PT sensitivity towards heparin in the presence of 500μg/L rivaroxaban. ConclusionsCompositional differences between clotting factor concentrates should be taken into account. Therapeutic levels of heparin may be co-infused when treating emergency bleeds with high prohemostatic drug doses, particularly those recommended in the reversal of non-VKA anticoagulants such as rivaroxaban by PCC. Given the relative short half-life of heparin compared to vitamin K-dependent clotting factors, an anticoagulant heparin effect shortly after concentrate infusion should be considered clinically and while interpreting laboratory coagulation parameters.

Sudden Thrombosis in Coronary Artery Bypass Grafting Surgery

Sudden Thrombosis in Coronary Artery Bypass Grafting Surgery

Novel HIT antibody detection method using Sonoclot® coagulation analyzer

IntroductionSince heparin-induced thrombocytopenia (HIT), caused by the generation of antibodies against platelet factor 4 (PF4)/heparin complexes (HIT antibodies), may induce serious complications due to thrombosis, a prompt diagnosis is desirable. Functional tests with platelet activation to detect HIT antibodies are useful for diagnosis of HIT, in particular 14C-selotonin release assay (SRA). However, they are complicated and so can be performed only in limited laboratories. We tested if a blood coagulation test using Sonoclot® analyzer can serve for the detection of HIT antibodies. Materials and MethodsA murine monoclonal antibody (HIT-MoAb) against PF4/heparin complexes was used as an alternative to human HIT antibodies. To the mixture of HIT-MoAb and heparin (0.5 U/mL, final), whole blood obtained from a healthy volunteer was added, and then the activated clotting time (ACT), clot rate (CR), and area under the curve (AUC) were measured with Sonoclot® analyzer for 30minutes. ResultsThe HIT-MoAb (30 to 100μg/mL, final) concentration dependently suppressed the anticoagulation activity (prolongation of ACT and decrease of CR and AUC) of heparin. ConclusionsThe suppression of anticoagulation effect of heparin by HIT-MoAb was demonstrated by measurements using Sonoclot® analyzer. This method may provide a new tool for screening of HIT antibodies.

Neutralizing the anticoagulant activity of ultra-low-molecular-weight heparins using N-acetylglucosamine 6-sulfatase.

Heparin has been the most commonly used anticoagulant drug for nearly a century. The drug heparin is generally categorized into three forms according to its molecular weight: unfractionated (UF, average molecular weight 13000), low molecular weight (average molecular weight 5000) and ultra-low-molecular-weight heparin (ULMWH, average molecular weight 2000). An overdose of heparin may lead to very dangerous bleeding in patients. Protamine sulfate may be administered as an antidote to reverse heparin's anticoagulant effect. However, there is no effective antidote for ULMWH. In the current study, we examine the use of human N-acetylglucosamine 6-sulfatase (NG6S), expressed in Chinese hamster ovary cells, as a reversal agent for ULMWH. NG6S removes a single 6-O-sulfo group at the non-reducing end of the ULMWH Arixtra(®) (fondaparinux), effectively removing its ability to bind to antithrombin and preventing its inhibition of coagulation factor Xa. These results pave the way to developing human NG6S as an antidote for neutralizing the anticoagulant activity of ULMWHs.

Discovery of Anticoagulant Drugs: A Historical Perspective

The history of the traditional anticoagulants is marked by both perseverance and serendipity. The anticoagulant effect of heparin was discovered by McLean in 1915, while he was searching for a procoagulant in dog liver. Link identified dicumarol from spoiled sweet clover hay in 1939 as the causal agent of the sweet clover disease, a hemorrhagic disorder in cattle. Hirudin extracts from the medicinal leech were first used for parenteral anticoagulation in the clinic in 1909, but their use was limited due to adverse effects and difficulties in achieving highly purified extracts. Heparins and coumarins (i.e.: warfarin, phenprocoumon, acenocoumarol) have been the mainstay of anticoagulant therapy for more than 60 years. Over the past decades, the drug discovery paradigm has shifted toward rational design following a target-based approach, in which specific proteins, or "targets", are chosen on current understandings of pathophysiology, small molecules that inhibit the target's activity may be identified by high-throughput screening and, in selected cases, these new molecules can be developed further as drugs. Despite the application of rational design, serendipity has still played a significant role in some of the new discoveries. This review will focus on the discovery of the main anticoagulant drugs in current clinical use, like unfractionated heparin, low-molecular-weight heparins, fondaparinux, coumarins (i.e.: warfarin, acenocoumarol, phenprocoumon), parenteral direct thrombin inhibitors (DTIs) (i.e.: argatroban, recombinant hirudins, bivalirudin), oral DTIs (i.e.: dabigatran) and oral direct factor Xa inhibitors (i.e.: rivaroxaban, apixaban).

Inhibition of Red Blood Cell-Prothrombinase by Heparin and a Covalent Antithrombin-Heparin Complex

Abstract 1179 Introduction:Various hematological conditions, including sickle cell anemia, beta-thalassemia and preeclampsia, have been linked with the presence of procoagulant red blood cells (RBCs). Overt exposure of phosphatidylserine (PS) on surfaces of RBCs has been a suspect for increased thrombotic complications in these patients. Asymmetrical distribution of PS at the membrane surface may provide docking sites for the formation of the procoagulant prothrombinase complex, which further aids in amplification of the coagulation cascade leading to subsequent thrombosis. In acute thrombotic complications, heparin is used as the first line of treatment in these patients. However, little is known regarding heparin's efficiency to pacify the procoagulant RBC-prothrombinase system. Therefore, we sought to determine the rate of RBC-prothrombinase inhibition by antithrombin and unfractionated heparin (AT+UFH) versus a novel covalent complex of AT and heparin (ATH). Methods:Thrombin generation was used to assess functionality of the phosphatidic acid (PA)-induced RBC-prothrombinase system, using a thrombin-specific substrate S-2238™. Discontinuous second order rate constant assays were carried out to obtain k2 values for inhibition of free or prothrombinase-bound Xa by AT+UFH or ATH. Freshly isolated RBCs were activated using PA and calcium for 15 min at room temperature. Activated RBCs were incubated with factors Xa and Va, Ca2+ and pefabloc®-TH (thrombin inhibitor) in different wells of a 96-well plate for 3 min at 37 °C. Prothrombin was then simultaneously added to all wells and allowed to react for 3 min at 37 °C, followed by addition of AT+UFH or ATH inhibitors to each well at specific time intervals. Reactions were neutralized by simultaneous addition of polybrene, Na2EDTA and Xa-specific substrate S-2222™ in buffer. The remaining Xa enzyme activity was obtained and final k2 values calculated. To investigate the roles of individual components of the prothrombinase complex on the anticoagulant effects of heparin and ATH, additional experiments were performed where components of the complex (prothrombin, activated RBCs or Va) were removed prior to reaction with the inhibitors. Results:PA-induced RBC-prothrombinase yielded a 4-fold greater thrombin generation compared to non-activated RBCs (p<0.05). The k2 values (x 108 M−1min−1) for inhibition of Xa alone by AT+UFH was 3.14 ± 0.11, whereas inhibition of RBC-prothrombinase significantly decreased this value to 2.12 ± 0.22, p<0.02. In comparison, ATH inhibited both free Xa and prothrombinase-complexed Xa at significantly faster rates compared to AT+UFH reactions (5.12 ± 0.27 and 4.85 ± 0.24 (p<0.0001), respectively). No significant differences were observed for free Xa or RBC-prothrombinase inhibition by ATH (p<0.46). When components of the RBC-prothrombinase were removed from reactions, the greatest impact on Xa inhibition was observed from factor Va omission. A significant decrease in inhibition rate was shown for both AT+UFH and ATH (4-fold and 2-fold, respectively) due to factor Va absence. Conclusion:In this study, we report inhibition of the prothrombinase complex on the surface of procoagulant RBCs. Consistent with vesicle and activated platelet systems, RBC-prothrombinase seems to protect Xa from inhibition by AT+UFH, which suggests that heparin efficiency is also compromised when prothrombinase forms on the surface of procoagulant RBCs. Relative to AT+UFH reactions, RBC-prothrombinase was significantly enhanced and no protection of Xa was observed with ATH. From our findings, factor Va may be the major component moderating the protective effect. Overall, these data indicate a clinical advantage for ATH over heparin in treatment of blood-based coagulation in vivo. This study also provides some understanding of how heparin or ATH treatment may interact with procoagulant complexes on the surface of RBCs in patients with red cell disorders that have thrombotic complications. Disclosures:No relevant conflicts of interest to declare.

Optimization of heparin anticoagulation for hemodialysis

Unfractionated heparin remains the most commonly used anticoagulant for extracorporeal therapies worldwide due to cost, years of clinical experience showing effectiveness and safety for outpatient hemodialysis. Most centers administer unfractionated heparin as an initial bolus followed by a constant infusion, which is then stopped prior to the end of the dialysis session. Although the anticoagulant effect of heparin can be monitored at the bedside, most centers take a pragmatic stance for routine outpatient hemodialysis, and adjust bolus doses and infusion rates based on visual inspection of the dialyzer header and venous air detector chamber for clots, and stop the heparin infusion based on the time taken for needle puncture sites to stop bleeding at the end of the hemodialysis session. Heparin is negatively charged and can bind to plasma proteins, leukocytes, and plastic. As such, it is important to achieve adequate mixing of heparin with blood to optimize anticoagulation within the extracorporeal circuit, by administering an intravenous bolus a few minutes prior to connecting the patient to the circuit and ensuring thorough mixing of the heparin infusion.

Peroperative effects of fresh frozen plasma and antithrombin III on heparin sensitivity and coagulation during nitroglycerine infusion in coronary artery bypass surgery

Nitroglycerin (NTG) reduces the anticoagulant effects of heparin and may lead to heparin resistance. Fresh frozen plasma (FFP) and antithrombin III (ATIII) may be used for the treatment of heparin resistance. We aimed to compare the effects of FFP and ATIII on heparin requirement, coagulation parameters, and bleeding in patients undergoing coronary artery bypass graft surgery (CABGS) with moderate dose of intraoperative NTG infusion. Forty-eight patients undergoing CABGS with NTG infusion were randomly allocated to three groups. Group C served as control, whereas the patients in group P received FFP and those in group A received ATIII after anesthesia induction. ATIII activity and coagulation parameters were measured at five different times intraoperatively. Total heparin requirement, heparin consumption, and heparin sensitivity were calculated. ATIII activity and ACT were significantly higher and activated partial thromboplastin time and fibrinogen level were significantly lower during cardiopulmonary bypass in group A than in groups P and C. Heparin sensitivity was significantly higher and total heparin requirement and consumption were significantly lower in ATIII group than in other groups. ATIII administration increases heparin sensitivity and decreases heparin requirements compared with FFP in patients undergoing CABGS with peroperative NTG infusion. ATIII may be preferred to FFP in patients with heparin resistance due to NTG infusion undergoing CABGS.