Inhibition Of Thrombin Generation Research Articles

Differences in coagulation responses to vascular injury between uninterrupted dabigatran and apixaban - a clinical prospective randomized study

BackgroundThe coagulation response during vascular injury with uninterrupted administration of direct oral anticoagulants (DOACs) has not been elucidated. ObjectiveOur aim was to evaluate differences in coagulation responses after vascular injury between uninterrupted direct thrombin inhibitor and direct factor Xa inhibitor recipients. MethodsPatients scheduled for catheter ablation for atrial fibrillation were randomly assigned to receive dabigatran or apixaban in this prospective, randomized, comparative, parallel-group study. Venous blood was collected three times: 180 minutes after taking the anticoagulant on the day before the procedure, before vascular punctures of the ablation procedure, and 10–15 minutes after the start of vascular punctures. ResultsForty-two patients were enrolled. The prothrombin fragment 1+2 (F1+2) level, the primary endpoint, was much larger after vascular puncture in the uninterrupted dabigatran recipients (median: 83 pmol/L; interquartile range: 56–133 pmol/L) than in the uninterrupted apixaban recipients (median: 1 pmol/L; interquartile range: −3–19 pmol/L; P < 0.001). Antithrombin levels decreased after vascular puncture in dabigatran recipients, and both protein C and antithrombin levels decreased after vascular puncture in apixaban recipients. ConclusionsUnlike uninterrupted apixaban, uninterrupted dabigatran does not inhibit thrombin generation in response to vascular injury.

Fondaparinux Results in Similar Pregnancy Outcomes with Lower Adverse Reaction Rates Compared to Low Molecular Weight Heparin in Chinese Recurrent Miscarriage Women.

Fondaparinux inhibits thrombin generation by inactivating factor Xa, which has the potential to treat recurrent miscarriage (RM). However, more clinical evidence is required to support its application in Chinese women with RM. This research aimed to compare the live birth rate, gestational weeks at delivery, birth weight, Apgar score of newborns, and adverse reaction rates between fondaparinux and low molecular weight heparin (LMWH) in Chinese women with RM. Totally, 132 women with RM treated with fondaparinux or LMWH were included in this retrospective study. According to the corresponding treatment, women with RM were divided into the fondaparinux cohort (N = 45) and LMWH cohort (N = 87). The live birth rate was 68.9% in the fondaparinux cohort and 56.3% in the LMWH cohort, which was not different between the two cohorts (P = 0.161). Multivariable logistics regression analysis suggested that only previous miscarriage times (≥ 4 times vs. < 4 times) were independently related to a lower possibility of live birth in women with RM (odds ratio = 0.431, P = 0.036). It was also observed that gestational weeks at delivery (38.1 ± 1.4 vs. 37.7 ± 1.7 weeks) (P = 0.258), birth weight (2,923.7 ± 355.0 vs. 2,807.8 ± 334.0 g) (P = 0.144), and Apgar score of newborns (9.8 ± 0.5 vs. 9.6 ± 0.8) (P = 0.175) were not different between the fondaparinux cohort and LMWH cohort. Inspiringly, the total adverse reaction rate was reduced in the fondaparinux cohort vs. the LMWH cohort (20.0% vs. 37.9%) (P = 0.036). Fondaparinux results in similar pregnancy outcomes with lower adverse reaction rates compared to LMWH in Chinese women with RM.

Pomab a Novel Monoclonal Type-I Antiphospholipid Antibody, Reduces Thrombin Generation By Selectively Binding to the Open Form of Prothrombin

Background. Anti-prothrombin (anti-PT) antibodies are a type of antiphospholipid antibodies frequently found in antiphospholipid syndrome (APS) patients. Previously, we identified two types of anti-PT antibodies, one binding to epitopes in kringle-1 that are hidden in the closed form but available in the open form (Type-I), and another one in which the epitopes are located in fragment-1 but accessible in both forms (Type-II). Our understanding of their functional characteristics remains limited. Goal. To identify Type-I monoclonal antibodies for mechanistic studies. Methods. Type-I anti-prothrombin antibodies were generated by immunizing mice with fragment-1 and producing monoclonal antibodies using hybridoma technology. Binding mechanisms and epitope recognition for one representative Type-I antibody, POmAb, were elucidated using surface plasmon resonance (SPR), single-molecule FRET (smFRET), and cryo-electron microscopy (cryo-EM). Functional evaluations were performed in human plasma and with purified components. In vivo hemostatic activity was assessed in a laser injury model of the cremasteric arteriole followed by intravital microscopy. Results. Type-I antibodies recognizing kringle-1 were obtained from immunization experiments. One of them, POmAb (Prothrombin Open monoclonal Antibody), was selected for in-depth mechanistic and structural studies. SPR studies showed that POmAb binds to the open form of prothrombin with low nanomolar affinity, and binding was impaired by the mutations S91A and Y93A in kringle-1. smFRET provided evidence of conformational selection, where POmAb preferentially selects the open conformation of prothrombin, thereby shifting the equilibrium towards the open form. Cryo-EM studies of the POmAb-prothrombin complex revealed an extended binding interface in kringle-1 which overlaps with the region occupied by the serine protease domain in the closed form, explaining why POmAb binds to the open form. In human plasma, POmAb prolonged the activated partial thromboplastin time and Russel's viper venom time, though the effect was modest. Further investigations using purified proteins and antibody fragments confirmed that POmAb reduces thrombin generation, although it does not completely abolish it, as the inhibition of thrombin generation occurs through a non-competitive mechanism with prothrombinase. In vivo studies corroborated in vitro findings, further proving reduced fibrin accumulation at the site of injury. Conclusions. We discovered POmAb, a Type-I antiphospholipid antibody. We demonstrated that it selectively binds to the open form of prothrombin, resulting in reduced thrombin generation. POmAb represents a novel tool for studying the role of Type-I antibodies in APS and advances the provocative new idea of conformational trapping of prothrombin as a novel class of anticoagulants.

High plasma levels of C1-inhibitor are associated with lower risk of future venous thromboembolism

BackgroundC1-inhibitor (C1INH) is a broad-acting serine protease inhibitor with anticoagulant activity. The impact of C1INH plasma levels within the normal physiological range on risk of venous thromboembolism (VTE) is unknown. We assessed the association of plasma C1INH levels and VTE risk and evaluated the impact of C1INH on thrombin and plasmin generation in ex vivo assays. MethodsA nested case-control study with 405 patients with VTE and 829 age- and sex-matched controls was derived from the Tromsø Study. Odds ratios (ORs) with 95% confidence intervals (95% CI) for VTE were estimated across plasma C1INH quartiles. Genetic regulation of C1INH was explored using quantitative trait loci analysis of whole exome sequencing data. The effect of plasma C1INH levels on coagulation was evaluated ex vivo by calibrated automated thrombography. ResultsIndividuals with C1INH levels in the highest quartile had a lower risk of VTE (OR 0.68, 95% CI: 0.49-0.96) compared with those with C1INH in the lowest quartile. In subgroup analysis, the corresponding ORs were 0.60 (95% CI: 0.39-0.89) for deep vein thrombosis and 0.85 (95% CI: 0.52-1.38) for pulmonary embolism, respectively. No significant genetic determinants of plasma C1INH levels were identified. Addition of exogenous C1INH to normal human plasma reduced thrombin generation triggered by an activator of the intrinsic coagulation pathway, but not when triggered by an activator of the extrinsic coagulation pathway. ConclusionsHigh plasma levels of C1INH were associated with lower risk of VTE, and C1INH inhibited thrombin generation initiated by the intrinsic coagulation pathway ex vivo.

Potency Adjusted Blended Heparin of Bovine, Ovine, and Porcine Heparin Exhibit Comparable Biologic Effects to Referenced Single-Sourced Porcine Heparin.

Introduction: Bovine and ovine mucosa represent alternate anticoagulants to porcine mucosa for production of unfractionated heparin (UFH). Standardized heparins from various sources can be blended and potency adjusted, blended heparins exhibit comparable effects as single-sourced porcine UFH. This study evaluated the pharmacologic profile of blended heparin and compared their activities to that of single sourced porcine, ovine, and bovine heparins. Methods: The anticoagulant effects of gravimetric and potency-adjusted heparins were evaluated with aPTT, TT, anti-Xa, anti-IIa, ACT, and TGA studies. Protamine sulfate studies were used for neutralization potential of each of the individual heparins. Results: The potency-adjusted heparins demonstrated comparable aPTT, TT, anti-Xa, anti-IIa, and ACT values at all concentrations (U/mL). However, in gravimetric studies, bovine heparin consistently showed lower values with the exception of thrombin generation inhibition studies. The protamine sulfate neutralization studies demonstrated complete neutralization at all concentrations for the potency-adjusted heparins. However, at gravimetric concentrations, minor differences were noted in the neutralization profile in each of these heparins. Conclusion: These studies support the hypothesis that blended heparin from bovine, ovine, and porcine tissue, when standardized in unit-equivalent proportions, exhibits a comparable anticoagulant profile to the single species derived heparins.

Inhibition of platelet-surface-bound proteins during coagulation under flow II: Antithrombin and heparin

Blood coagulation is a self-repair process regulated by activated platelet surfaces, clotting factors, and inhibitors. Antithrombin (AT) is one such inhibitor that impedes coagulation by targeting and inactivating several key coagulation enzymes. The effect of AT is greatly enhanced in the presence of heparin, a common anticoagulant drug. When heparin binds to AT, it either bridges with the target enzyme or induces allosteric changes in AT leading to more favorable binding with the target enzyme. AT inhibition of fluid-phase enzymes caused little suppression of thrombin generation in our previous mathematical models of blood coagulation under flow. This is because in that model, flow itself was a greater inhibitor of the fluid-phase enzymes than AT. From clinical observations, it is clear that AT and heparin should have strong inhibitory effects on thrombin generation, and thus we hypothesized that AT could be inhibiting enzymes bound to activated platelet surfaces that are not subject to being washed away by flow. We extended our mathematical model to include the relevant reactions of AT inhibition at the activated platelet surfaces as well as those for unfractionated heparin and a low molecular weight heparin. Our results show that AT alone is only an effective inhibitor at low tissue factor densities, but in the presence of heparin, it can greatly alter, and in some cases shut down, thrombin generation. Additionally, we studied each target enzyme separately and found that inactivation of no single enzyme could substantially suppress thrombin generation.

Orally delivered rutin in lipid-based nano-formulation exerts strong antithrombotic effects by protein disulfide isomerase inhibition

Thrombosis occurs in both macrovasculature and microvasculature, causing various cardio-cerebral vascular diseases. The lack of effective and safe antithrombotic drugs leads to a public health crisis. Mounting evidence suggests that protein disulfide isomerase (PDI) plays a critical role in the initial stage of thrombus formation, motivating the research of the feasibility of PDI inhibitors as novel anti-thrombotics. Rutin, one of the most potent PDI inhibitors, was reported to suppress platelet aggregation and thrombosis in animal models, but further studies and clinical translation were restricted due to its low aqueous solubility and oral bioavailability. In this work, we fabricated rutin-loaded lipid-based nano-formulation (NanoR) and characterized their physical–chemical properties, release profiles, pharmacokinetic process, and pharmacodynamic function against thrombosis in macrovessels and microvessels. NanoR provided increased solubility and dissolution of rutin to achieve earlier T max and higher C max than the sodium salt of rutin (NaR) after oral gavage. Ex vivo studies demonstrated that NanoR significantly inhibited thrombin generation and clot formation in the plasma of mice. Importantly, such effect was reversed by exogenous recombinant PDI, demonstrating the specificity of the NanoR. In direct current-induced arterial thrombosis model and ferric chloride-induced microvascular thrombosis model, NanoR exhibited greatly enhanced antithrombotic activity compared with NaR. NanoR also showed good safety performance according to tail bleeding assay, global coagulation tests, and histological analysis. Overall, our current results indicated that NanoR offers a promising antithrombotic treatment with potential for clinical translation.

Thrombin generation capacity is enhanced by low antithrombin activity and depends on the activity of the related coagulation factors

BackgroundSupplementation with antithrombin (AT) concentrates is now common in the treatment of congenital and acquired AT deficiency. However, there is no established consensus on the target and timing of supplementation. We aimed to elucidate the effects of AT deficiency on the balance between coagulation activation and inhibition using a thrombin generation assay as in vitro global assay.MethodsSamples were prepared by admixing commercially acquired AT-deficient plasma with < 1% AT activity with pooled normal plasma. The AT activity in each sample was adjusted to 100, 90, 70, 50, 40, 30, 10, 5, and < 1%. A thrombin generation assay was performed in each sample. AT concentrate-spiked samples were also prepared by adjusting the AT activities in four types of the concentrates: one recombinant and three plasma-derived AT concentrates. The final targeted AT activities in the samples were adjusted to 100, 50, 30, and 5% by spiking each concentrate into the AT-deficient plasma. We also prepared samples with five levels of prothrombin time (PT) % in coagulation factors with the AT activity fixed at 30% by dilution by mixing AT-deficient plasma and normal plasma with Owren’s veronal buffer to adjust the coagulation factor activities in several proportions. The theoretical target PT% values were 100, 66, 50, 40, and 30%. A thrombin generation assay was performed on all samples.ResultsThe ability to generate thrombin depended on the AT activity, and the amount of thrombin generation was increased as AT was decreased. Additionally, the amount of thrombin generation was changed significantly when AT activity was ≤ 50%, indicating that AT suppressed thrombin generation. In particular, thrombin generation was remarkable when AT activity was < 30%, and it can be assumed that the prognosis is poor due to organ failure from thrombotic tendency.ConclusionsThe results presented in this basic research were found to be consistent with the clinical findings to date. The mechanism by which 30–50% of AT activity is set as the clinical boundary was elucidated by the thrombin generation assay.

Chemical synthesis and pharmacological properties of heparin pentasaccharide analogues

The pentasaccharide fondaparinux is a synthetic anticoagulant based on heparin antithrombin-binding sequence. Fondaparinux improves safety and predictable pharmacodynamics compared with heparins; however, it requires a complicate synthesis process which contain more than 50 steps of synthesis. Herein, we designed and synthesized four fondaparinux analogues (compounds 1, 2, 3, 4) using a [2+3] convergent synthetic method, which greatly simplified the synthetic process, improved the product yield, and curtailed the expenditures. These synthesized compounds showed stronger anticoagulant activities by factor Xa inhibition (IC50 725–1126 nM vs. 1909 nM for fondaparinux) in the AT-dependent manner. After subcutaneous (s.c.) administration to rats, the compounds displayed long-lasting anti-factor Xa activities and inhibition of thrombin generation ex vivo. Compared with fondaparinux, these compounds were slowly eliminated after s.c. administration to rats, the half-lies (t1/2) were more than 2-fold of that of fondaparinux. These results suggested the pentasaccharide analogues may exhibit better pharmacokinetic and predictable pharmacodynamic characteristics.

Transparent and Highly Flexible Hierarchically Structured Polydimethylsiloxane Surfaces Suppress Bacterial Attachment and Thrombosis Under Static and Dynamic Conditions.

The surface fouling of biomedical devices has been an ongoing issue in healthcare. Bacterial and blood adhesion in particular, severely impede the performance of such tools, leading to poor patient outcomes. Various structural and chemical modifications have been shown to reduce fouling, but all existing strategies lack the combination of physical, chemical, and economic traits necessary for widespread use. Herein, a lubricant infused, hierarchically micro- and nanostructured polydimethylsiloxane surface is presented. The surface is easy to produce and exhibits the high flexibility and optical transparency necessary for incorporation into various biomedical tools. Tests involving two clinically relevant, priority pathogens show up to a 98.5% reduction in the biofilm formation of methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa. With blood, the surface reduces staining by 95% and suppresses thrombin generation to background levels. Furthermore, the surface shows applicability within applications such as catheters, extracorporeal circuits, and microfluidic devices, through its effectiveness in dynamic conditions. The perfusion of bacterial media shows up to 96.5% reduction in bacterial adhesion. Similarly, a 95.8% reduction in fibrin networks is observed following whole blood perfusion. This substrate stands to hold high applicability within biomedical systems as a means to prevent fouling, thus improving performance.

Assessment of thrombin generation in horses using a calibrated automated thrombogram.

The amount of thrombin generated reflects the endogenous thrombin potential (ETP), which depends on the balance of pro- and anticoagulant factors. The calibrated automated thrombogram (CAT) allows for the direct measurement of thrombin generation during the clotting process. (1) To describe the results of the CAT assay in horses, (2) to establish intra-assay and intra- and interindividual variation of thrombin generation in healthy horses, and (3) to compare in vitro low-molecular-weight heparin (LMWH) sensitivity between healthy and sick horses. The hypothesis for the last objective is that inhibition of thrombin generation in sick horses requires higher heparin concentrations. The plasma of 10 healthy mixed breed horses was used for the determination of normal thrombin generation parameters (lag time, time to peak, peak thrombin concentration, and ETP). Five of the healthy horses were compared with five horses with systemic inflammatory response syndrome (SIRS). In vitro heparin sensitivity was determined using LMWH. The intra-assay variation was small (<5%) for all parameters. Relatively large intra- and interindividual variation were observed in healthy horses. Four of the five sick horses with SIRS had a thrombogram compatible with a hypercoagulable state. The in vitro heparin sensitivity test suggested decreased sensitivity to LMWH in hypercoagulable states. The CAT assay could detect coagulopathy in horses. In vivo experiments are needed to confirm that it can be used to monitor responses to LMWH therapy.

Bleeding After Gastric Endoscopic Submucosal Dissection Focused on Management of Xa Inhibitors.

PurposeThe use of direct oral Xa inhibitors (DXaIs) to prevent venothrombotic events is increasing. However, gastrointestinal bleeding, including that related to endoscopic resection, is a concern. In this study, we evaluated bleeding and coagulation times during the perioperative period of gastric endoscopic submucosal dissection (ESD).Materials and MethodsPatients who consecutively underwent gastric ESD from August 2016 to December 2018 were analyzed. Bleeding rates were compared among the 3 groups (antiplatelet, DXaIs, and control). DXaI administration was discontinued on the day of the procedure. Prothrombin time (PT), activated partial thromboplastin time, and the ratio of inhibited thrombin generation (RITG), which was based on dilute PT, were determined before and after ESD.ResultsDuring the study period, 265 gastric ESDs were performed in 239 patients, where 23 and 50 patients received DXaIs and antiplatelets, respectively. Delayed bleeding occurred in 17 patients (7.4%) and 21 lesions (7.1%). The bleeding rate in the DXaI group was significantly higher than that in the other groups (30.4%, P<0.01), and the adjusted odds ratio of bleeding was 5.7 (95% confidence interval, 1.4–23.7; P=0.016). In patients using DXaIs, there was a significant (P=0.046) difference in the median RITG between bleeding cases (18.6%) and non-bleeding cases (3.8%).ConclusionsA one-day cessation of DXaIs was related to a high incidence of bleeding after gastric ESD, and monitoring of residual coagulation activity at trough levels might enable the predicted risk of delayed bleeding in patients using DXaIs.

Sulfated agaran with 4,6-pyruvated form from red seaweed Acanthophora muscoides attenuates thrombin formation: in vitro and ex vivo studies

In vitro studies have described the sulfated agaran from Acanthophora muscoides as an intrinsic inhibitor of thrombin generation (TG), but not in ex vivo assay. This investigation partially characterized a pyruvate fraction with in vitro and ex vivo effects on an intrinsic/extrinsic pathway-induced thrombin generation (TG) continuous model using 36 or 60-fold diluted mice or defibrinated, normal human plasma. Fraction separated by DEAE-cellulose chromatography exhibited charge homogeneity and non-sulfated polysaccharides (&lt;100 kDa) by agarose and polyacrylamide gel electrophoresis, respectively, using Stains-all alone. Fourier Transform Infrared and Nuclear Magnetic Resonance studies indicated a 4,6-pyruvated agaran-structure. The fraction and heparin had no effect on prothrombin time, but there was a preponderant intrinsic rather than extrinsic pathway inhibition in TG assay; themselves, acting on both free and fibrin bound thrombin activity without chromogenic substrate interaction. Both fractions, desulfated and native, anticipated and induced thrombin formation in activators-devoid or normal plasma. In addition, mice pretreated with fraction (20 mg kg-1, intraperitoneally) reduced intrinsically plasma TG ex vivo after 2h. Heparin suppressed TG in vitro, but induced it ex vivo. Therefore, agaran from A. muscoides blocks TG on in vitro and ex vivo studies, suggesting to evaluate the blood coagulability status.

Abstract 110: C1 Esterase Inhibitor Functions As An Anticoagulant In Human And Murine Venous Thrombosis

Factor (F) XII and FXI play a central role in venous thrombus formation in preclinical disease models and contributes to venous thromboembolism (VTE) in humans. Agents targeting FXIa have been developed and effectively prevent VTE in humans. C1 esterase inhibitor (C1INH) is a serine protease inhibitor that inhibits several proteases, including FXII and FXI. In this study, we investigated the anticoagulant activity of C1INH in humans and mice. First, plasma C1INH levels were determined in a population-based nested case-control study consisting of 405 VTE patients and 829 age- and sex-matched controls derived from the Tromsø Study. Participants with plasma C1INH levels in the highest quartile had a significantly lower risk of VTE (odds ratio [OR] of 0.68, confidence interval 0.49-0.96, P&lt;0.05), unprovoked VTE (OR 0.59, confidence interval 0.39-0.89) and pulmonary embolism (OR 0.57, confidence interval 0.34-0.92) compared to participants with C1INH levels in the lowest quartile after adjustment for age and sex. Secondly, plasma-based thrombin generation studies were conducted to assess the anticoagulant function of C1INH. Supplementation of normal human pooled plasma with exogenous human purified C1INH (0.2 and 0.4mg/ml) significantly inhibited thrombin generation initiated with silica (P&lt;0.05) and significantly prolonged the activated partial thromboplastin assay clotting time (P&lt;0.001). Thirdly, administration of a clinically approved human purified C1INH product to mice significantly reduced thrombus weight (P&lt;0.05) in the murine inferior vena cava stenosis model of venous thrombosis. Studies with C1 inhibitor deficient mice and substrate selective C1INH variants are currently ongoing. Our results indicate that C1INH serves as an endogenous anticoagulant with higher plasma levels of C1INH associated with a decreased future risk of VTE. Further, exogenous C1INH effectively inhibits thrombin generation in vitro and venous thrombus formation in a mouse model. Our findings suggest that C1INH may prevent thrombosis caused by activation of the intrinsic coagulation pathway.

Structure‐function of anticoagulant TIX‐5, the inhibitor of factor Xa‐mediated FV activation

BackgroundThe prothrombinase complex consists of factors Xa (FXa) and Va (FVa) on an anionic phospholipid surface and converts prothrombin into thrombin. Both coagulation factors require activation before complex assembly. We recently identified TIX‐5, a unique anticoagulant tick protein that specifically inhibits FXa‐mediated activation of FV. Because TIX‐5 inhibited thrombin generation in blood plasma, it was concluded that FV activation by FXa contributes importantly to coagulation. ObjectiveWe aimed to unravel the structure‐function relationships of TIX‐5. MethodWe used a structure model generated based on homology with the allergen Der F7. ResultsTick inhibitor of factor Xa toward FV was predicted to consist of a single rod formed by several beta sheets wrapped around a central C‐terminal alpha helix. By mutagenesis we could show that two hydrophobic loops at one end of the rod mediate the phospholipid binding of TIX‐5. On the other end of the rod an FV interaction region was identified on one side, whereas on the other side an EGK sequence was identified that could potentially form a pseudosubstrate of FXa. All three interaction sites were important for the anticoagulant properties of TIX‐5 in a tissue factor‐initiated thrombin generation assay as well as in the inhibition of FV activation by FXa in a purified system. ConclusionThe structure‐function properties of TIX‐5 are in perfect agreement with a protein that inhibits the FXa‐mediated activation on a phospholipid surface. The present elucidation of the mechanism of action of TIX‐5 will aid in deciphering the processes involved in the initiation phase of blood coagulation.

Aprotinin Inhibits Thrombin Generation by Inhibition of the Intrinsic Pathway, but is not a Direct Thrombin Inhibitor.

Background Aprotinin is a broad-acting serine protease inhibitor that has been clinically used to prevent blood loss during major surgical procedures including cardiac surgery and liver transplantation. The prohemostatic properties of aprotinin likely are related to its antifibrinolytic effects, but other mechanisms including preservation of platelet function have been proposed. Aim Here we assessed effects of aprotinin on various hemostatic pathways in vitro, and compared effects to tranexamic acid(TXA), which is an antifibrinolytic but not a serine protease inhibitor. Methods We used plasma-based clot lysis assays, clotting assays in whole blood, plasma, and using purified proteins, and platelet activation assays to which aprotinin or TXA were added in pharmacological concentrations. Results Aprotinin and TXA dose-dependently inhibited fibrinolysis in plasma. Aprotinin inhibited clot formation and thrombin generation initiated via the intrinsic pathway, but had no effect on reactions initiated by tissue factor. However, in the presence of thrombomodulin, aprotinin enhanced thrombin generation in reactions started by tissue factor. TXA had no effect on coagulation. Aprotinin did not inhibit thrombin, only weakly inhibited the TF-VIIa complex and had no effect on platelet activation and aggregation by various agonists including thrombin. Aprotinin and TXA inhibited plasmin-induced platelet activation. Conclusion Pharmacologically relevant concentrations of aprotinin inhibit coagulation initiated via the intrinsic pathway. The antifibrinolytic activity of aprotinin likely explains the prohemostatic effects of aprotinin during surgical procedures. The anticoagulant properties may be beneficial during surgical procedures in which pathological activation of the intrinsic pathway, for example by extracorporeal circuits, occurs.

An Ex Vivo Analysis of Thrombin Generation and Thromboelastometry in Patients Undergoing Treatment with Rivaroxaban Demonstrates Sustained Anticoagulant Effect and High Inter-Individual Variability

Background : Rivaroxaban, 20 mg orally administered once daily, is widely used for secondary prevention of venous thromboembolism (VTE) without the need for routine laboratory monitoring. It is licensed for the prevention of VTE after major orthopedic surgery, for the treatment of acute symptomatic VTE, the secondary prevention of VTE and for prevention of stroke in patients with non-valvular atrial fibrillation. Rivaroxaban selectively and competitively inhibits free factor Xa (FXa) and prothrombinase/clot-associated FXa through reversible interactions thereby inhibiting thrombin generation and decreasing fibrin clot formation. The need of laboratory evaluation of rivaroxaban is actually questioned.Aim : In patients treated for secondary prevention of VTE, we evaluated ex vivo the anticoagulant potency of rivaroxaban treatment at different times after drug intake.Methods : 97 patients (39 males, 58 females; median age: 59 years) with idiopathic VTE were treated with rivaroxaban (20 mg/day) for a period longer than 6 months and compared to 30 controls age and sex-matched. Rivaroxaban concentrations (STA®-Liquid Anti-Xa), thrombin generation (PPP-reagent 5 pM TF; calibrated automated thrombogram, Stago France) and whole blood thromboelastometry (5 pM tissue factor; ROTEM® Werfen) were assessed. The interval between drug intake and blood sampling was registered.Results : Treatment with rivaroxaban significantly inhibited thrombin generation (TG) and prolonged clotting time (CT): 93% of patients had TG inferior to the lower normal limit and 77% had CT longer than the upper normal limit. This effect in both assays, was not correlated to its plasma concentrations, it was significant from the 1st hour after drug intake and persisted at similar levels during the daily therapeutic cycle even at low concentrations (30 ng/ml). Rivaroxaban had no effect on clot firmness. The inter-individual coefficient of variation of rivaroxaban concentrations and TG/ROTEM alterations after drug intake ranged from 50% to 100%.Conclusion : Once daily administration of rivaroxaban induces sustained daily anticoagulant effect, detected by TG and ROTEM assays, with no significant changes related to drug concentration reduction. This profile could explain the wide therapeutic window and the favorable benefit risk ratio of the treatment with rivaroxaban. Due to the high variability the identification of patients with extreme values of TG could be useful strategy for treatment optimization on individual basis. DisclosuresNo relevant conflicts of interest to declare.

Biochemical and Pharmacological Differentiation of APC and Recombinant Thrombomodulin with Reference to the Effect on the Hemostatic and Fibrinolytic Processes

Introduction:Both thrombomodulin and activated protein C play an important role in the regulation of hemostasis. Recombinant versions of both the thrombomodulin (rTM) and activated protein C (rAPC) have been developed for clinical applications. Marked differences in their biochemical and pharmacological profiles exist. Such differences in their effects may be due to target sites and other interactions and may have therapeutic and safety implications.Aim:The aim of this study is to investigate the biochemical and pharmacological differences between a clinically developed rTM (ART-123, Asahi Kasei Pharma, Tokyo, Japan) and a plasma derived APC (Haematologic Technologies, Burlington, VT, USA).Materials and Methods:The anticoagulant profile of both agents was investigated in native whole blood using the ACT, TEG and global anticoagulant profile using PT and APTT assays at a concentration range of 0-10 ug/ml. The global anticoagulant profile in plasma based systems in terms of PT and APTT was also investigated. The effect of both of these agents was studied in the antiprotease (Xa and IIa) inhibitory assays and thrombin generation inhibition methods. A modified TEG system was used to study the modulation of tissue plasminogen activator mediated fibrinolysis by these agents.Results:In the ACT assays APC produced a much stronger anticoagulant effect in a concentration range of 0-10 ug/ml with a doubling time at 3 ug/ml, whereas rTM produced marginal effects on this parameter. The TEG profile of APC exhibited much stronger anticoagulant effects in comparison to rTM. At 3 ug/mL, APC produces much longer prolongation of the R and K time parameters in comparison to rTM. APC also facilitated tPA-mediated fibrinolysis in contrast to rTM. In the anti-IIa assays, rTM produced concentration dependent inhibition of thrombin and no inhibition of factor Xa. APC did not have any effect on either Factor Xa or IIa. In the thrombin generation assays, APC produced a stronger inhibition of thrombin generation (IC50=0.4 ug/mL) whereas rTM was relatively weaker (IC50 = 1.2 ug/mL). In the APTT assay APC produced a much stronger anticoagulant effect whereas rTM produced relatively weaker effects. Both agents did not produce any modification of agonist induced platelet aggregation. In terms of anticoagulant potency the APC exhibited 70 + 6 USP U/mg whereas the rTM was much weaker < 10 USP U/mg.Conclusions:These studies demonstrate that APC is a much stronger anticoagulant in comparison to rTM. In addition both agents produced different modulation of fibrinolytic and hemostatic processes. These observations suggest that in clinical indications such as DIC, these agents may have differential safety/efficacy implications. DisclosuresTanaka:Asahi Kasei Pharma America Corporation: Employment. Tsuruta:Asahi Kasei Pharma America Corporation: Employment.

Improved Inhibition of Protease-Activated Receptor 4 (PAR4) with Function-Blocking Antibodies Provides Equivalent Antithrombotic Activity in the Face of the Hyper-Reactive Thr120 PAR4 Variant

Background: Thrombin activates platelets via two protease-activated receptors (PARs), PAR1 and PAR4, and both are targets of antithrombotic drugs - a PAR1 inhibitor is approved for clinical use and PAR4 inhibitors are in trial. However, recent studies report a PAR4 variant (rs773902; encoding Thr120 instead of Ala120) is expressed in up to 80% of individuals, depending on the population, and renders the receptor more sensitive to agonists and less sensitive to antagonists. These studies suggest the effectiveness of current strategies to inhibit PAR4 may vary substantially across the population. Here, we report the generation of a novel function-blocking PAR4 antibody that provides equivalent antithrombotic activity against the Ala120 or Thr120 PAR4 variant.Methods: We generated a panel of anti-PAR4 antibodies and examined their inhibitory activity against the Ala120 and Thr120 PAR4 variants in cultured cells, isolated platelets, and in an ex vivo whole blood thrombosis assay.Results: Our lead function-blocking antibody bound human PAR4 with high selectivity (~16-fold over PARs1, 2 or 3) and affinity (KD ~ 5nM). This antibody provided near-complete inhibition of thrombin cleavage of either the Ala120 or Thr120 PAR4 variant when transfected in HEK293T cells. Platelets isolated from individuals expressing the Thr120 PAR4 variant exhibited increased thrombin-induced aggregation and phosphatidylserine exposure versus those expressing the Ala120 PAR4 variant. Yet the anti-PAR4 antibody inhibited these responses with an equivalent IC50 of ~ 5 µg/ml. Furthermore, this function-blocking anti-PAR4 antibody significantly impaired platelet procoagulant activity in an ex vivo whole blood thrombosis assay. Here, equivalent inhibition of thrombin generation (FRET-based thrombin substrate) and fibrin formation (anti-fibrin antibody) was observed in thrombi generated in blood from individuals expressing the Ala120 or Thr120 PAR4 variant.Conclusion: We generated a highly selective and effective function-blocking anti-PAR4 antibody that impairs thrombin-induced PAR4 cleavage, platelet activation, and pro-thrombotic activity equivalently in the presence of the Ala120 or Thr120 PAR4 variant. These findings reveal a novel approach to PAR4 inhibition that overcomes the impaired sensitivity of existing approaches against the commonly expressed Thr120 PAR4 variant, and provide rationale for such an approach for improved antithrombotic therapy. DisclosuresNo relevant conflicts of interest to declare.

Factor V Atlanta: A Novel Mutation in the F5 Gene Reveals Potential New Cis-Acting Elements Involved in Regulating FV-Short and TFPI Levels

Coagulation factor V (FV) is procofactor essential to normal hemostasis. Recently, a point mutation in exon 13 of the F5 gene (FV-East Texas; FV-ET), which encodes the B-domain, has been shown to result in an inherited moderate bleeding diathesis termed East Texas Bleeding Disorder. The mutation (F5 c. A2350G, according to the latest annotated reference sequence for the F5 transcript, NM_000130.4, originally reported as A2440G) greatly enhances alternative splicing at an otherwise weak splice site, which leads to the production of a new FV isoform, FV-short. FV-short, via an exposed acidic region, binds with high affinity to tissue factor pathway inhibitor alpha (TFPIα) and is thought to stabilize the inhibitor in blood thereby increasing its concentration via changes in clearance mechanisms. High levels of TFPIα as well as other regulatory mechanisms contribute to inhibition of thrombin generation leading to a clinical bleeding phenotype. Another family with a different point mutation (FV-Amsterdam, F5 c.C2588G) downstream from FV-ET was recently identified and found to have similar clinical and laboratory characteristics. A 43 year-old male with a history of recurrent intramuscular hematomas underwent laboratory investigation to identify the etiology of his bleeding tendency. Laboratory testing revealed a prolonged PT and aPTT that failed to correct after 50:50 mixing with normal human plasma (NHP) and normal activity levels of factors II, V, VII, VIII, IX, X, XI, XII, and XIII. A lupus anticoagulant was not detected. A thrombin generation assay revealed a prolonged lag time versus control plasma (20 min vs. 6 min). These findings were similar to FV-ET, and further studies revealed that the concentration of total TFPI and TFPIα were elevated in the patient. Surprisingly however, the levels of TFPI were much higher compared to those measured in FV-ET. Relative to NHP, total TFPI and TFPIα were increased 11-and 22-fold, respectively for the patient compared to 4- and 5-fold for FV-ET (Table 1). Remarkably, a point mutation in exon 13 was not identified. Rather, analysis performed on genomic DNA identified a heterozygous 832 base pair (bp) deletion within exon 13 (c.2413-3244del). This deletion is predicted to result in the omission of 277 amino acids preceding a frameshift and translation of 12 non-native amino acids followed by a stop codon.While this alteration should result in a functionally null allele, causing the patient to have 50% FV levels, the clinical and biochemical findings do not align with this genomic data. Western blotting of the patient's plasma using anti-FV heavy or light chain antibodies revealed the presence of full length (FL)-FV (330 kDa) protein as well as a strong band at 250 kDa representing FV-short. Using recombinant FV-short as a control, quantitative western blotting revealed the patient has very high levels of FV-short (50 nM) compared to FV-ET plasma (5-7 nM; Table 1). To reconcile these data with the genetic information, we performed RT-PCR analysis of blood cell RNA. Several different FV transcripts were identified including a putative FL-FV transcript, a transcript containing the 832 bp frameshift deletion, and a FV-short transcript identical to that from FV-ET. The latter finding was unexpected as the 832 bp deletion is located 3' to the FV-ET donor splice site. We hypothesize that encoded within this novel 832 bp deletion is a sequence that functions as a cis-acting element which regulates FV-short production under normal physiologic conditions. FV-short is constituently expressed to a minor degree in unaffected individuals (~0.2-0.4 nM). Removal of this 832 bp sequence through genetic deletion is predicted to facilitate upregulation of FV-short biosynthesis via enhanced splicing at the FV-ET donor/acceptor splice sites. The identification of the FV-Atlanta deletion and the FV-ET and FV-Amsterdam point mutations highlight a complicated mechanism by which FV-short levels are modulated by alternative splicing. Since FV-short has a major influence on TFPIα levels, these collective findings are clinically relevant and may represent an additional level of regulation existing in the initiation and propagation of coagulation. [Display omitted] DisclosuresZimowski:National Hemophilia Foundation/Shire: Other: Clinical Fellowship Award Recipient. Denning:Expression Therapeutics: Employment. Jhita:Expression Therapeutics: Employment. Rivera:Expression Therapeutics: Employment. Escobar:Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees; NovoNordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees. Kempton:Genentech: Membership on an entity's Board of Directors or advisory committees. Camire:Bayer: Consultancy, Research Funding; Pfizer: Consultancy, Patents & Royalties, Research Funding; Novo Nordisk: Research Funding; Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees.