Pharmacological profile of asundexian, a novel, orally bioavailable inhibitor of factor XIa.

BACKGROUND: Activated coagulation factor XI (FXIa) contributes to the development and propagation of thrombosis but plays only a minor role in hemostasis; therefore, it is an attractive antithrombotic target. OBJECTIVES: To evaluate the pharmacology of asundexian (BAY 2433334), a small molecule inhibitor targeting FXIa, in vitro and in various rabbit models. METHODS: The effects of asundexian on FXIa activity, selectivity versus other proteases, plasma thrombin generation, and clotting assays were evaluated. Antithrombotic effects were determined in FeCl2 - and arterio-venous (AV) shunt models. Asundexian was administered intravenously or orally, before or during thrombus formation, and with or without antiplatelet drugs (aspirin and ticagrelor). Potential effects of asundexian on bleeding were evaluated in ear-, gum-, and liver injury models. RESULTS: Asundexian inhibited human FXIa with high potency and selectivity. It reduced FXIa activity, thrombin generation triggered by contact activation or low concentrations of tissue factor, and prolonged activated partial thromboplastin time in human, rabbit, and various other species, but not in rodents. In the FeCl2 -injury models, asundexian reduced thrombus weight versus control, and in the arterial model when added to aspirin and ticagrelor. In the AV shunt model, asundexian reduced thrombus weight when administered before or during thrombus formation. Asundexian alone or in combination with antiplatelet drugs did not increase bleeding times or blood loss in any of the models studied. CONCLUSIONS: Asundexian is a potent oral FXIa inhibitor with antithrombotic efficacy in arterial and venous thrombosis models in prevention and intervention settings, without increasing bleeding.

Lecanoric acid mediates anti-proliferative effects by an M phase arrest in colon cancer cells.

Lichen extracts containing, among other compounds, depsides such as evernic acid, atranorin, and lecanoric acid possess anti-proliferative effects. We aimed to identify lichen metabolites that are responsible for the observed anti-proliferative effects. We performed cytotoxicity, cell colony, cell cycle and apoptosis assays in various cell lines or primary immune cells. We analyzed several cell cycle proteins and apoptosis-related proteins to gain insights into the underlying mechanism. All depsides reduced the viability of the tested cell lines (HCT-116, HEK293T, HeLa, NIH3T3, RAW246.7) in a cell line-dependent manner with lecanoric acid being the most effective. Atranorin did not influence the cell cycle or colony formation in HCT-116 cells, but induced apoptosis in HCT-116 cells. Evernic acid showed no anti-proliferative effects. Lecanoric acid inhibited cell colony formation already at 0.03 µg/ml in HCT-116 cells and induced a G2 cell cycle block in several cell lines. Moreover, lecanoric acid arrested the cell cycle, presumably in the M phase, since expression of cyclin B1 and phosphorylated histone H3 was upregulated, whereas the inactive cyclin-dependent kinase 1 (CDK1) was reduced in HCT-116 cells. Most importantly, cell death induced by lecanoric acid was more prominent in cancer cells than in primary human immune and endothelial cells. In conclusion, lecanoric acid seems to mediate its anti-proliferative effects via arrest of cells in the M phase. Our data suggest lecanoric acid may be a potential new candidate for anti-cancer therapy, because it has anti-proliferative effects on cancer cell lines, and does not affect primary immune cells.

Antioxidants as Therapeutic Agents in Acute Respiratory Distress Syndrome (ARDS) Treatment-From Mice to Men.

Acute respiratory distress syndrome (ARDS) is a major cause of patient mortality in intensive care units (ICUs) worldwide. Considering that no causative treatment but only symptomatic care is available, it is obvious that there is a high unmet medical need for a new therapeutic concept. One reason for a missing etiologic therapy strategy is the multifactorial origin of ARDS, which leads to a large heterogeneity of patients. This review summarizes the various kinds of ARDS onset with a special focus on the role of reactive oxygen species (ROS), which are generally linked to ARDS development and progression. Taking a closer look at the data which already have been established in mouse models, this review finally proposes the translation of these results on successful antioxidant use in a personalized approach to the ICU patient as a potential adjuvant to standard ARDS treatment.

Characterization of ACE Inhibitors and AT1R Antagonists with Regard to Their Effect on ACE2 Expression and Infection with SARS-CoV-2 Using a Caco-2 Cell Model.

Blood-pressure-lowering drugs are proposed to foster SARS-CoV-2 infection by pharmacological upregulation of angiotensin-converting enzyme 2 (ACE2), the binding partner of the virus spike (S) protein, located on the surface of the host cells. Conversely, it is postulated that angiotensin-renin system antagonists may prevent lung damage caused by SARS-CoV-2 infection, by reducing angiotensin II levels, which can induce permeability of lung endothelial barrier via its interaction with the AT1 receptor (AT1R). METHODS: We have investigated the influence of the ACE inhibitors (lisinopril, captopril) and the AT1 antagonists (telmisartan, olmesartan) on the level of ACE2 mRNA and protein expression as well as their influence on the cytopathic effect of SARS-CoV-2 and on the cell barrier integrity in a Caco-2 cell model. RESULTS: The drugs revealed no effect on ACE2 mRNA and protein expression. ACE inhibitors and AT1R antagonist olmesartan did not influence the infection rate of SARS-CoV-2 and were unable to prevent the SARS-CoV-2-induced cell barrier disturbance. A concentration of 25 µg/mL telmisartan significantly reduced the virus replication rate. CONCLUSION: ACE inhibitors and AT1R antagonist showed neither beneficial nor detrimental effects on SARS-CoV-2-infection and cell barrier integrity in vitro at pharmacologically relevant concentrations.

Protein expression profiling suggests relevance of noncanonical pathways in isolated pulmonary embolism.

Patients with isolated pulmonary embolism (PE) have a distinct clinical profile from those with deep vein thrombosis (DVT)-associated PE, with more pulmonary conditions and atherosclerosis. These findings suggest a distinct molecular pathophysiology and the potential involvement of alternative pathways in isolated PE. To test this hypothesis, data from 532 individuals from the Genotyping and Molecular Phenotyping of Venous ThromboEmbolism Project, a multicenter prospective cohort study with extensive biobanking, were analyzed. Targeted, high-throughput proteomics, machine learning, and bioinformatic methods were applied to contrast the acute-phase plasma proteomes of isolated PE patients (n = 96) against those of patients with DVT-associated PE (n = 276) or isolated DVT (n = 160). This resulted in the identification of shared molecular processes between PE phenotypes, as well as an isolated PE-specific protein signature. Shared processes included upregulation of inflammation, response to oxidative stress, and the loss of pulmonary surfactant. The isolated PE-specific signature consisted of 5 proteins: interferon-γ, glial cell line-derived neurotrophic growth factor, polypeptide N-acetylgalactosaminyltransferase 3, peptidyl arginine deiminase type-2, and interleukin-15 receptor subunit α. These proteins were orthogonally validated using cis protein quantitative trait loci. External replication in an independent population-based cohort (n = 5778) further validated the proteomic results and showed that they were prognostic for incident primary isolated PE in individuals without history of VTE (median time to event: 2.9 years; interquartile range: 1.6-4.2 years), supporting their possible involvement in the early pathogenesis. This study has identified molecular overlaps and differences between VTE phenotypes. In particular, the results implicate noncanonical pathways more commonly associated with respiratory and atherosclerotic disease in the acute pathophysiology of isolated PE.

Nrf2-A Molecular Target for Sepsis Patients in Critical Care.

The transcription factor NF-E2 p45-related factor 2 (Nrf2) is an established master regulator of the anti-oxidative and detoxifying cellular response. Thus, a role in inflammatory diseases associated with the generation of large amounts of reactive oxygen species (ROS) seems obvious. In line with this, data obtained in cell culture experiments and preclinical settings have shown that Nrf2 is important in regulating target genes that are necessary to ensure cellular redox balance. Additionally, Nrf2 is involved in the induction of phase II drug metabolizing enzymes, which are important both in degrading and converting drugs into active forms, and into putative carcinogens. Therefore, Nrf2 has also been implicated in tumorigenesis. This must be kept in mind when new therapy approaches are planned for the treatment of sepsis. Therefore, this review highlights the function of Nrf2 in sepsis with a special focus on the translation of rodent-based results into sepsis patients in the intensive care unit (ICU).

Comprehensive platelet phenotyping supports the role of platelets in the pathogenesis of acute venous thromboembolism - results from clinical observation studies.

BACKGROUND: The pathogenesis of arterial and venous thrombosis is in large part interlaced. How much platelet phenotype relates to acute venous thromboembolism (VTE) independent of the underlying cardiovascular profile is presently poorly investigated. METHODS: Platelet count and mean platelet volume (MPV), platelet aggregation in whole blood and platelet rich plasma (PRP), platelet-dependent thrombin generation (TG) and platelet surface activation markers were measured under standardized conditions. Machine learning was applied to identify the most relevant characteristics associated with VTE from a large array (N = 58) of clinical and platelet-related variables. FINDINGS: VTE cases (N = 159) presented with lower platelet count and MPV vs controls (N = 140). Whole blood aggregation showed shorter collagen/Epinephrine closure times in cases, particularly within acetylsalicylic acid (ASA) users. Within ASA users, higher PRP aggregation after adenosine diphosphate (ADP), epinephrine, collagen and arachidonic acid was observed in cases vs controls. Within non-ASA and/or subjects on anticoagulants, cases presented with lower aggregation after ADP and collagen vs controls. Lower platelet-dependent TG, higher CD63 on resting and lower PAC-1 expression after collagen/ADP in-vitro stimulated platelets further characterized VTE cases vs controls, independent of therapy. Lasso regression analysis identified 26 variables associated with VTE of which 69% were platelet-related. INTERPRETATION: Comprehensive phenotyping of platelet function identified a large proportion of low responders to ASA in VTE cases. Lower platelet-dependent TG and lower platelet reactivity after ex-vivo stimulation characterized the "platelet exhausted syndrome" in cases. Finally, from a large array of covariates including clinical risk factors, platelet biomarkers comprised 69% of all selected variables differentiating VTE cases vs controls. FUNDING: German Federal Ministry of Education and Research, CTH-Mainz and Bayer AG.

The dual FXa/thrombin inhibitor SATI prevents fibrin and platelet deposition in hypercoagulant rats.

INTRODUCTION: Systemic hypercoagulation is often a severe complication of infective and inflammatory diseases, which overcome the hemostatic balance and lead to multiple thrombotic occlusions in the microvasculature and organ damage and is related to high mortality rates. SATI is a potent dual inhibitor of FXa and thrombin with antithrombotic efficacy in venous and arterial thrombosis models. In this study, the antithrombotic efficacy of SATI was investigated in a microthrombosis model in rats with an induced hypercoagulant state. MATERIALS AND METHODS: The hypercoagulant state was generated by infusion of TF in sixty rats (12 groups, consisting of 5 rats each). SATI was administered in two different doses by constant infusion and its antithrombotic efficacy was investigated using two different approaches: 1) measuring 125I-fibrin deposition in various organs and 2) continuous whole-body imaging of 111In-platelet biodistribution in anesthetized animals. RESULTS: After start of the TF infusion in rats with radioactively-labeled fibrinogen, the radioactivity was accumulated in liver, spleen, kidney, and mostly in the lung as a consequence of fibrin generation. SATI efficiently reduced the pulmonary deposition of fibrin in a dose- and time-dependent manner. In the SATI groups the splenic and renal radioactivity was enhanced at later time points probably as consequence of the clearance of 125I-fibrin(ogen). Imaging of rats that received 111In-platelets prior to systemic TF administration showed retention of the radioactivity mainly in the lungs in the control group. SATI efficiently blocked the platelet accumulation in the lungs and increased platelet recruitment by the spleen. CONCLUSIONS: SATI is a promising candidate for prevention of microcirculatory disturbances by inhibiting fibrin deposition and platelet accumulation in the lungs and thereby conferring organ protection. Both methods used in this study are suitable for investigating the antithrombotic efficacy of new drugs in microthrombosis. Continuous imaging of 111In-platelets allowed for follow-up of thrombus formation in living animals without the need for tissue harvesting.

Targeted inhibition of activated protein C by a non-active-site inhibitory antibody to treat hemophilia.

Activated protein C (APC) is a plasma serine protease with antithrombotic and cytoprotective functions. Based on the hypothesis that specific inhibition of APC's anticoagulant but not its cytoprotective activity can be beneficial for hemophilia therapy, 2 types of inhibitory monoclonal antibodies (mAbs) are tested: A type I active-site binding mAb and a type II mAb binding to an exosite on APC (required for anticoagulant activity) as shown by X-ray crystallography. Both mAbs increase thrombin generation and promote plasma clotting. Type I blocks all APC activities, whereas type II preserves APC's cytoprotective function. In normal monkeys, type I causes many adverse effects including animal death. In contrast, type II is well-tolerated in normal monkeys and shows both acute and prophylactic dose-dependent efficacy in hemophilic monkeys. Our data show that the type II mAb can specifically inhibit APC's anticoagulant function without compromising its cytoprotective function and offers superior therapeutic opportunities for hemophilia.

ADTRP regulates TFPI expression via transcription factor POU1F1 involved in coronary artery disease.

Genomic variants in both ADTRP and TFPI genes are associated with risk of coronary artery disease (CAD). ADTRP regulates TFPI expression and endothelial cell functions involved in the initiation of atherosclerotic CAD. ADTRP also specifies primitive myelopoiesis and definitive hematopoiesis by upregulating TFPI expression. However, the underlying molecular mechanism is unknown. Here we show that transcription factor POU1F1 is the key by which ADTRP regulates TFPI expression. Luciferase reporter assays, chromatin-immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA) in combination with analysis of large and small deletions of the TFPI promoter/regulatory region were used to identify the molecular mechanism by which ADTRP regulates TFPI expression. Genetic association was assessed using case-control association analysis and phenome-wide association analysis (PhenGWA). ADTRP regulates TFPI expression at the transcription level in a dose-dependent manner. The ADTRP-response element was localized to a 50 bp region between -806 bp and -756 bp upstream of TFPI transcription start site, which contains a binding site for POU1F1. Deletion of POU1F1-binding site or knockdown of POU1F1 expression abolished ADTRP-mediated transcription of TFPI. ChIP and EMSA demonstrated that POU1F1 binds to the ADTRP response element. Genetic analysis identified significant association between POU1F1 variants and risk of CAD. PhenGWA identified other phenotypic traits associated with the ADTRP-POU1F1-TFPI axis such as lymphocyte count (ADTRP), waist circumference (TFPI), and standing height (POU1F1). These data identify POU1F1 as a transcription factor that regulates TFPI transcription in response to ADTRP, and link POU1F1 variants to risk of CAD for the first time.

Plasma Kallikrein Contributes to Coagulation in the Absence of Factor XI by Activating Factor IX.

OBJECTIVES: FXIa (factor XIa) induces clot formation, and human congenital FXI deficiency protects against venous thromboembolism and stroke. In contrast, the role of FXI in hemostasis is rather small, especially compared with FIX deficiency. Little is known about the cause of the difference in phenotypes associated with FIX deficiency and FXI deficiency. We speculated that activation of FIX via the intrinsic coagulation is not solely dependent on FXI(a; activated FXI) and aimed at identifying an FXI-independent FIX activation pathway. Approach and Results: We observed that ellagic acid and long-chain polyphosphates activated the coagulation system in FXI-deficient plasma, as could be demonstrated by measurement of thrombin generation, FIXa-AT (antithrombin), and FXa-AT complex levels, suggesting an FXI bypass route of FIX activation. Addition of a specific PKa (plasma kallikrein) inhibitor to FXI-deficient plasma decreased thrombin generation, prolonged activated partial thromboplastin time, and diminished FIXa-AT and FXa-AT complex formation, indicating that PKa plays a role in the FXI bypass route of FIX activation. In addition, FIXa-AT complex formation was significantly increased in F11-/- mice treated with ellagic acid or long-chain polyphosphates compared with controls and this increase was significantly reduced by inhibition of PKa. CONCLUSIONS: We demonstrated that activation of FXII leads to thrombin generation via FIX activation by PKa in the absence of FXI. These findings may, in part, explain the different phenotypes associated with FIX and FXI deficiencies.

Androgen inhibits key atherosclerotic processes by directly activating ADTRP transcription.

Low androgen levels are associated with an increased risk of coronary artery disease (CAD), thrombosis and myocardial infarction (MI), suggesting that androgen has a protective role. However, little is known about the underlying molecular mechanism. Our genome-wide association study identified the ADTRP gene encoding the androgen-dependent TFPI regulating protein as a susceptibility gene for CAD and MI. The expression level of ADTRP was regulated by androgen, but the molecular mechanism is unknown. In this study, we identified the molecular mechanism by which androgen regulates ADTRP expression and tested the hypothesis that androgen plays a protective role in cardiovascular disease by activating ADTRP expression. Luciferase assays with an ADTRP promoter luciferase reporter revealed that androgen regulated ADTRP transcription in a dose- and time-dependent manner, and the effect was abolished by three different androgen inhibitors, including pyrvinium pamoate, bicalutamide, and cyproterone acetate. Chromatin-immunoprecipitation showed that the androgen receptor bound to a half androgen response element (ARE, TGTTCT) located at +324bp from the ADTRP transcription start site. The ARE is required for concentration-dependent transcriptional activation of ADTRP. HL-60 monocyte adhesion to EAhy926 endothelial cells (ECs) and transmigration across the EC layer, the two processes critical to development of CAD and MI, were inhibited by androgen, but the effect was rescued by ADTRP siRNA and exacerbated by overexpression of ADTRP and its downstream genes PIK3R3 and MIA3. These data suggest that one molecular mechanism by which androgen confers protection against CAD is stimulation of ADTRP expression.

Dual inhibition of thrombin and activated factor X attenuates disseminated intravascular coagulation and protects organ function in a baboon model of severe Gram-negative sepsis.

BACKGROUND: Inhibition of procoagulant pathways may improve outcome in sepsis. We examined whether a dual short-acting thrombin (factor II) and factor X (FX)a inhibitor (SATI) ameliorates sepsis-induced disseminated intravascular coagulation (DIC) and is organ-protective. METHODS: Escherichia coli were infused for 2 h in 22 anesthetized baboons. The control (CO) group (n = 8) received sterile isotonic solution only. In the treatment groups, SATI was administered starting 15 minutes after the end of the bacterial exposure. In the low-dose group (LD-SATI, n = 8), SATI was infused with 75 µg/kg/h for the first hour, followed by 23 µg/kg/h until the end of the study. In the high-dose SATI group (HD-SATI, n = 6), 225 µg/kg/h was administered for the first hour followed by continuous infusion of 69 µg/kg/h until termination of the study. RESULTS: Sepsis-induced DIC was attenuated, as reflected by lower peak thrombin-antithrombin complexes (threefold) and D-dimer levels (twofold) in both SATI groups compared to the CO. This coincided with strongly improved cell/organ protection assessed by decreased levels of lactate dehydrogenase (threefold), creatinine (twofold), aspartate aminotransferase (threefold), and amylase (twofold) compared to the CO group. Anuria, which started at 8 h in the CO group, was prevented in both SATI groups. Peak interleukin-6 release at 12 h was prevented in the treatment groups. In both SATI groups, fewer catecholamines were necessary and no bleeding complications were observed. CONCLUSIONS: Dual inhibition of thrombin and FXa preserved activation of coagulation, protected organ function and ameliorated inflammation in severe Gram-negative sepsis in baboons. SATI could be a novel therapeutic agent against sepsis-induced DIC.

Platelet function in baboons and humans - A comparative study of whole blood using impedance platelet aggregometry (Multiplate®).

BACKGROUND: Platelets play a pivotal role in coagulation, inflammation and wound healing. Suitable animal models that have the potential to mimic human platelet function are limited. The objective of the current study was to compare platelet aggregation response in the whole blood of baboons and humans using impedance aggregometry. METHODS: Blood was drawn from 24 anesthetised male baboons and 25 healthy volunteers. The platelet aggregation response was determined by impedance aggregometry (Multiplate®). Platelets in the hirudinised whole blood samples were stimulated with four different activators: adenosine diphosphate (ADP), collagen (COL), thrombin receptor activating peptide-6 (TR1AP), and activation of PAR-4 thrombin receptor subtype (TR4AP) at standard concentrations. Higher than standard concentrations were tested in a subgroup of the animals. RESULTS: The cell counts showed no differences between baboons and humans. The platelet aggregation response was significantly lower in baboons compared to humans when stimulated with the platelet agonists ADP (p<0.0001), COL (p=0.021) and TR4AP (p<0.0001). TR1AP did not stimulate platelet aggregation in the baboon blood. Doubling the concentration of ADP and of TR4AP significantly increased the AUC compared to the standard concentration. In contrast, increased COL levels did not further increase the AUC. CONCLUSION: The current study revealed that testing the platelet function in baboon blood by impedance aggregometry is feasible with ADP, COL and TR4AP, but not with TR1AP. Compared to humans, the aggregation response is lower in baboons. Considering the limitations in accordance to these results, baboons might represent a potential species for further platelet research.

Expression of pro-inflammatory genes in human endothelial cells: Comparison of rivaroxaban and dabigatran.

INTRODUCTION: In addition to its central role in coagulation, thrombin is involved in non-hemostatic activities such as inflammation. Direct inhibition of thrombin activity (e.g. with dabigatran) or reducing its generation by inhibition of Factor Xa (e.g. with rivaroxaban) may therefore have anti-inflammatory effects. MATERIALS AND METHODS: Microarray experiments were performed to identify transcriptome-wide changes in mRNA expression levels induced by thrombin in the presence and absence of the PAR-1 antagonist vorapaxar in primary human umbilical vein endothelial cells (HUVECs). On this basis, HUVECs were incubated with recalcified plasma, with or without rivaroxaban (0.3-3000nM), dabigatran (0.3-10,000nM), or vorapaxar (0.3-10nM). Expression levels of preselected pro-inflammatory genes were quantified by real-time PCR. RESULTS: Vorapaxar abolished 67 of the 69 transcripts altered by more than twofold on addition of thrombin to HUVECs. ELAM-1, VCAM-1, ICAM-1, MCP-1, IL-8, CXCL1, and CXCL2 were among the genes most strongly induced by thrombin. Inflammatory gene expression after stimulation of thrombin generation was concentration-dependently suppressed by vorapaxar, dabigatran, and rivaroxaban. However, dabigatran at low concentrations (3-300nM) increased significantly the expression levels of CXCL1, CXCL2, IL-8, ELAM-1, MCP-1, and tissue factor. CONCLUSION: In HUVECs, plasma-induced transcriptional changes are mediated by thrombin-induced PAR-1 activation. Rivaroxaban downregulated the expression of pro-inflammatory markers and tissue factor to a similar extent to dabigatran.

Effects of Rivaroxaban on Platelet Activation and Platelet-Coagulation Pathway Interaction: In Vitro and In Vivo Studies.

INTRODUCTION: Activation of coagulation and platelets is closely linked, and arterial thrombosis involves coagulation activation as well as platelet activation and aggregation. In these studies, we investigated the possible synergistic effects of rivaroxaban in combination with antiplatelet agents on thrombin generation and platelet aggregation in vitro and on arterial thrombosis and hemostasis in rat models. MATERIALS AND METHODS: Thrombin generation was measured by the Calibrated Automated Thrombogram method (0.5 pmol/L tissue factor) using human platelet-rich plasma (PRP) spiked with rivaroxaban (15, 30, or 60 ng/mL), ticagrelor (1.0 µg/mL), and acetylsalicylic acid (ASA; 100 µg/mL). Tissue factor-induced platelet aggregation was measured in PRP spiked with rivaroxaban (15 or 30 ng/mL), ticagrelor (1 or 3 µg/mL), or a combination of these. An arteriovenous (AV) shunt model in rats was used to determine the effects of rivaroxaban (0.01, 0.03, or 0.1 mg/kg), clopidogrel (1 mg/kg), ASA (3 mg/kg), and combinations on arterial thrombosis. RESULTS: Rivaroxaban inhibited thrombin generation in a concentration-dependent manner and the effect was enhanced with ticagrelor and ticagrelor plus ASA. Rivaroxaban and ticagrelor also concentration-dependently inhibited tissue factor-induced platelet aggregation, and their combination increased the inhibition synergistically. In the AV shunt model, rivaroxaban dose-dependently reduced thrombus formation. Combining subefficacious or weakly efficacious doses of rivaroxaban with ASA or ASA plus clopidogrel increased the antithrombotic effect. CONCLUSION: These data indicate that the combination of rivaroxaban with single or dual antiplatelet agents works synergistically to reduce platelet activation, which may in turn lead to the delayed/reduced formation of coagulation complexes and vice versa, thereby enhancing antithrombotic potency.

Reversal of rivaroxaban-induced anticoagulation with prothrombin complex concentrate, activated prothrombin complex concentrate and recombinant activated factor VII in vitro.

INTRODUCTION: Anticoagulation therapies carry a risk of bleeding; reversal agents may be beneficial in cases of severe bleeding even for anticoagulants with a relatively short half-life, such as the oral factor Xa inhibitor rivaroxaban. MATERIALS AND METHODS: We investigated the in vitro reversal effect of prothrombin complex concentrate (PCC; 0.2-1.0U/mL), activated PCC (aPCC; 0.2-1.0U/mL) and recombinant activated factor VII (rFVIIa; 5-50µg/mL) on rivaroxaban-induced (200-1000ng/mL) changes in prothrombin time (PT) and thrombin generation (TG) in plasma, and in thromboelastometry (clotting time [CT]) in whole blood from healthy subjects. RESULTS: All three agents were partially effective in reversing rivaroxaban-induced anticoagulation but showed different profiles. rFVIIa and aPCC were more effective than PCC in reversing prolongations of PT, CT and TG lag time; rFVIIa was more effective than aPCC. However, the reversal effect reached a plateau with a maximal effect of approximately 50%. Inhibition of maximum thrombin concentration was slightly reversed by these agents; aPCC was the most effective. In contrast, inhibition of endogenous thrombin potential (ETP) was strongly reversed by aPCC, with significant increases over baseline at low rivaroxaban concentrations. Compared with aPCC, PCC showed a similar but less effective reversal profile. rFVIIa reversed ETP inhibition by approximately 50%. CONCLUSIONS: The extent of reversal by aPCC, PCC and rFVIIa was dependent on the parameter measured in rivaroxaban-anticoagulated plasma or blood. ETP measurements may have predictive power for assessing the reversal potential of PCC or aPCC and may be used to indicate an increased prothrombotic risk.

Thrombin has biphasic effects on the nitric oxide-cGMP pathway in endothelial cells and contributes to experimental pulmonary hypertension.

BACKGROUND: A potential role for coagulation factors in pulmonary arterial hypertension has been recently described, but the mechanism of action is currently not known. Here, we investigated the interactions between thrombin and the nitric oxide-cGMP pathway in pulmonary endothelial cells and experimental pulmonary hypertension. PRINCIPAL FINDINGS: Chronic treatment with the selective thrombin inhibitor melagatran (0.9 mg/kg daily via implanted minipumps) reduced right ventricular hypertrophy in the rat monocrotaline model of experimental pulmonary hypertension. In vitro, thrombin was found to have biphasic effects on key regulators of the nitric oxide-cGMP pathway in endothelial cells (HUVECs). Acute thrombin stimulation led to increased expression of the cGMP-elevating factors endothelial nitric oxide synthase (eNOS) and soluble guanylate cyclase (sGC) subunits, leading to increased cGMP levels. By contrast, prolonged exposition of pulmonary endothelial cells to thrombin revealed a characteristic pattern of differential expression of the key regulators of the nitric oxide-cGMP pathway, in which specifically the factors contributing to cGMP elevation (eNOS and sGC) were reduced and the cGMP-hydrolyzing PDE5 was elevated (qPCR and Western blot). In line with the differential expression of key regulators of the nitric oxide-cGMP pathway, a reduction of cGMP by prolonged thrombin stimulation was found. The effects of prolonged thrombin exposure were confirmed in endothelial cells of pulmonary origin (HPAECs and HPMECs). Similar effects could be induced by activation of protease-activated receptor-1 (PAR-1). CONCLUSION: These findings suggest a link between thrombin generation and cGMP depletion in lung endothelial cells through negative regulation of the nitric oxide-cGMP pathway, possibly mediated via PAR-1, which could be of relevance in pulmonary arterial hypertension.

Reversal of rivaroxaban anticoagulation by haemostatic agents in rats and primates.

Rivaroxaban is an oral, direct factor Xa inhibitor for the management of thromboembolic disorders. Despite its short half-life, the ability to reverse rivaroxaban anticoagulation could be beneficial in life-threatening emergencies. The potential of prothrombin complex concentrate (PCC; Beriplex®), activated PCC (aPCC; FEIBA®) or recombinant activated factor VII (rFVIIa; NovoSeven®) to reverse rivaroxaban in rats and baboons was investigated. Anaesthetised rats pre-treated with intravenous rivaroxaban (2 mg/kg) received intravenous rFVIIa (100/400 µg/kg), PCC (25/50 U/kg) or aPCC (50/100 U/kg) after initiation of bleeding. Clotting times and bleeding times (BTs) were recorded. Rivaroxaban was administered as an intravenous 0.6 mg/kg bolus followed by continuous 0.6 mg/kg/hour infusion in baboons. Animals received intravenous aPCC 50 U/kg (2 U/kg/minute) or rFVIIa 210 µg/kg. BT and clotting parameters were measured. In rats pretreated with high-dose rivaroxaban, PCC 50 U/kg, aPCC 100 U/kg and rFVIIa 400 µg/kg significantly reduced BT vs rivaroxaban alone (5.4 ± 1.4-fold to 1.5 ± 0.4-fold [p<0.05]; 3.0 ± 0.4-fold to 1.4 ± 0.1-fold [p<0.001]; and 3.5 ± 0.7-fold to 1.7 ± 0.2-fold [p<0.01] vs baseline, respectively). In baboons pre-infused with rivaroxaban and then given aPCC, BT increased by 2.0 ± 0.2-fold and aPCC returned BT to baseline for the duration of its infusion. rFVIIa reduced BT from 2.5 ± 0.3-fold over baseline to 1.7 ± 0.3-fold over baseline. Prolongation of prothrombin time was reduced by PCC, aPCC and rFVIIa in both species. Rivaroxaban reduced thrombin-antithrombin levels; application of PCC and aPCC, but not rFVIIa, increased these levels. In conclusion, PCC, aPCC or rFVIIa have the potential to reverse the anticoagulant and anti-haemostatic effects of rivaroxaban.

Similarities in thromboelastometric (ROTEM®) findings between humans and baboons.

INTRODUCTION: Interest in visco-elastic testing in different clinical scenarios has increased but few data are available on thromboelastometric findings in primates. MATERIALS AND METHODS: Blood cell count (hemoglobin, hematocrit, platelet count), coagulation parameters (prothrombin time, International Normalized Ratio, fibrinogen), and ROTEM® (Tem International GmbH, Munich, Germany) variables were analyzed using blood from 25 anesthetized male baboons and 21 non-anesthetized healthy volunteers. The platelet component of the clot was calculated as the difference in maximum clot elasticity (MCE) between the whole blood clot (EXTEM test) and the fibrin-based clot (FIBTEM test). In subgroups of each species, 10 µg abciximab was added to the regular FIBTEM reagent (cytochalasin D) for additional platelet inhibition. RESULTS: Blood cell count was comparable between humans and primates. Both fibrinogen concentration (p<0.0001) and maximum clot firmness (MCF) in FIBTEM assays were significantly lower in baboons (p>0.0001, and p=0.006, respectively). PT, INR, and clotting time in NATEM assays were significantly prolonged in humans compared with baboons. MCF in NATEM, EXTEM and INTEM assays was not different between baboons and humans. Clot lysis in NATEM, EXTEM and INTEM assays was significantly higher in humans (p<0.0001). In contrast FIBTEM clot lysis was significantly higher in baboons (p=0.01). Addition of abciximab into the FIBTEM assay resulted in a significant reduction in MCF and MCE (p<0.001) and, consequently, the platelet component increased similar in both humans and baboons (p<0.001). CONCLUSION: Activated ROTEM® tests revealed broad similarities between humans and baboons. ROTEM® assays developed for use in humans can also be used in baboons.