Thrombin Activity Research Articles

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 (<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.

Total Phenolic, Flavonoid and Carotinoid Content and Anticoagulation Activities of Sapparin Tablet

Aims: Sapparin tablet, a medicine used for the treatment of blood diseases specially curing blood thickening or impure blood, liver disease in Mongolian Traditional Medicine. The objectives of the study were to determine total biological active substances and analyze the anticoagulation activity of the Sapparin.
 Study Design: Experimental study.
 Place and Duration of Study: Department of Chemistry and Technology and Department of Pharmacology, Institute of Traditional Medicine and Technology of Mongolia.
 Methodology: Quantitative determination of the total active constituents (phenolic, flavonoid, and carotinoids) of the methanol extracts of Sapparin was performed by using Folin-Ciocalteu reagent, aluminium chloride reagent by spectrophotometry.
 A totally of forty weighing between 220-250 gm were used. Effect of Sapparin was assessed on coagulation parameters following 7, 14, 21 and 28 days administration of 37 mg/kg, 56 mg/kg, 113 mg/kg to healthy rats. The blood coagulation parameters such as prothrombin time (PT) and the activated partial thromboplastin time (aPTT) were measured by means of Quick’s one-stage assay and modified aPTT assay respectively in the rats. Additionally, thrombin activity test was estimated in rats with PT assay using a hemagglutination analyzer. The levels of serum X and von Willebrand factor were measured in Sapparin and control groups by ELISA.
 Results: The total content of the phenols measured as 5.33±0.0005%, flavonoids as 12.95± 2.21% and carotinoids as 4.31±0.96%.
 There was significant increase in all assays except fibrinogen, prothrombin time, thrombin, aPTT. Sapparin treatment significantly reduced levels of serum X factor and von Willebrand factor was significantly decreased in rats.
 Conclusion: Results of this study suggest that Sapparin shows considerable anti-anticoagulant activity in animals and has potential to reduce cardiovascular morbidity and mortality.

A Conformational Change of Complement C5 Is Required for Thrombin-Mediated Cleavage, Revealed by a Novel Ex Vivo Human Whole Blood Model Preserving Full Thrombin Activity.

Thrombin activation of C5 connects thrombosis to inflammation. Complement research in whole blood ex vivo necessitates anticoagulation, which potentially interferes with the inflammatory modulation by thrombin. We challenged the concept of thrombin as an activator of native C5 by analyzing complement activation and C5 cleavage in human whole blood anticoagulated with Gly-Pro-Arg-Pro (GPRP), a peptide targeting fibrin polymerization downstream of thrombin, allowing complete endogenous thrombin generation. GPRP dose-dependently inhibited coagulation but allowed for platelet activation in accordance with thrombin generation. Spontaneous and bacterial-induced complement activation by Escherichia coli and Staphylococcus aureus, analyzed at the level of C3 and C5, were similar in blood anticoagulated with GPRP and the thrombin inhibitor lepirudin. In the GPRP model, endogenous thrombin, even at supra-physiologic concentrations, did not cleave native C5, despite efficiently cleaving commercially sourced purified C5 protein, both in buffer and when added to C5-deficient serum. In normal serum, only exogenously added, commercially sourced C5 was cleaved, whereas the native plasma C5 remained intact. Crucially, affinity-purified C5, eluted under mild conditions using an MgCl2 solution, was not cleaved by thrombin. Acidification of plasma to pH ≤ 6.8 by hydrochloric or lactic acid induced a C5 antigenic change, nonreversible by pH neutralization, that permitted cleavage by thrombin. Circular dichroism on purified C5 confirmed the structural change during acidification. Thus, we propose that pH-induced conformational change allows thrombin-mediated cleavage of C5 and that, contrary to previous reports, thrombin does not cleave plasma C5 in its native form, suggesting that thrombin cleavage of C5 may be restricted to certain pathophysiological conditions.

Synergistic activation of thrombin and angiotensin II receptors revealed by bioluminescence resonance energy transfer.

We recently reported a physical interaction between the angiotensin II (AngII) receptor (AT1R) and thrombin receptor (PAR1) in HEK293 cells using bioluminescence resonance energy transfer (BRET) technology. This was characterized by thrombin trans-activating AT1R and the synergistic responses of the AT1R-PAR1 complex. Here, we investigated the other face of the coin by examining the effect of AT1R on PAR1 activity using BRET. AngII/AT1R did not promote PAR1 activation in the absence of thrombin. However, the combination of thrombin and AngII resulted in their synergistic/allosteric action. Moreover, AngII/AT1R potentiated the maximal thrombin responses, suggesting specific conformational changes within the AT1R-PAR1 complex. Overall, our data confirm the functional AT1R-PAR1 interplay and further support the implication of both AT1R and PAR1 protomers in their synergistic interaction as previously reported.

Abstract MP03: Histone-mediated Platelet Activation And Microvesicle-induced Thrombin Generation In COVID-19

Background: Severe COVID-19 leads to inflammation and coagulopathy with progression to multiple organ failure. Several prothrombotic mechanisms have been proposed, including NETosis with release of citrullinated histones, platelet hyperactivation, and generation of procoagulant microvesicles. Aims: We hypothesized that histones derived from NETosis promote platelet activation and release of microvesicles that mechanistically contribute to thrombin generation and thrombosis in COVID-19. Methods: Platelet poor plasma (PPP) was prepared from 136 COVID-19 patients enrolled in a multicenter randomized clinical trial comparing standard prophylactic dose to intermediate dose enoxaparin in hospitalized patients with COVID-19 (NCT04360824). We also prepared PPP and washed platelets from 53 healthy subjects. We measured citrullinated histone H3 (H3Cit) by ELISA, microvesicles by flow cytometry, and thrombin generation using the Calibrated Automated Thrombogram. Ex vivo platelet adhesion and thrombus growth on collagen was measured in a microfluidic chamber. Results: Compared to healthy subjects, COVID-19 patients had elevated plasma levels of H3Cit and platelet-derived microvesicles. When PPP was triggered with exogenous tissue factor and phospholipids, higher endogenous thrombin potential, peak thrombin, and velocity index were observed in COVID-19 patients compared to healthy subjects. Increased thrombin generation with COVID-19 PPP was evident even when triggered without added phospholipids, suggesting the presence of procoagulant microvesicles in COVID-19 plasma. Enhanced thrombin generation also was observed after transfer of microvesicles isolated from COVID-19 patient plasma to control PPP. Incubation of control platelets with PPP from COVID-19 patients but not healthy subjects caused increased platelet-dependent thrombin generation and larger platelet-thrombi under venous shear stress. These effects of COVID-19 plasma on platelet-dependent thrombin generation and platelet-thrombi formation were inhibited by a histone aptamer. Conclusions: We conclude that histone-mediated platelet activation promotes thrombosis in COVID-19 in part via increased thrombin generation driven by microvesicles.

Features of thrombocyte-vascular hemostasis in women with abnormal uterine bleeding and submucosal uterine fibroids

Objective – to investigate the features of platelet-vascular hemostasis in women with abnormal uterine bleeding on the background of submucosal uterine fibroids.Material and methods. The study included 30 women with abnormal uterine bleeding with submucosal uterine fibroids. Features of thrombocyte-vascular hemostasis were studiedResults. There are new data in relation to the features of certain links of the adjusting system of the aggregate state of blood on a background development of post hemorrhage anaemia. It is clearly shown that changes in the system of adjusting of the aggregate state of blood in women with a severe degree of anaemisation are the display of subclinical inopexi.Conclusions. Changes in the fibrinolytic potential of blood in women with chronic posthemorrhagic anemia on the background of abnormal uterine bleeding and submucosal uterine fibroids are secondary, due to the activation of thrombin and fibrinogenesis by external mechanisms.

Effect of Vibrio-Derived Extracellular Protease vEP-45 on the Blood Complement System.

Simple SummaryAn extracellular Vibrio protease called vEP-45 can activate the complement system through an alternative pathway by cleaving key complement precursors, including C3 and C5, to their active forms, which can induce innate immunity.Vibrio vulnificus is a pathogenic bacterium that can causes wound infections and fetal septicemia. We have reported that V. vulnificus ATCC29307 produces an extracellular zinc-metalloprotease (named vEP-45). Our previous results showed that vEP-45 can convert prothrombin to active thrombin and also activate the plasma kallikrein/kinin system. In this study, the effect of vEP-45 on the activation of the complement system was examined. We found that vEP-45 could proteolytically convert the key complement precursor molecules, including C3, C4, and C5, to their corresponding active forms (e.g., C3a, C3b, C4a, C4b, and C5a) in vitro cleavage assays. C5b production from C5 cleavage mediated by vEP-45 was not observed, whereas the level of C5a was increased in a dose-dependent manner compared to that of the non-treated control. The cleavage of the complement proteins in human plasma by vEP-45 was also confirmed via Western blotting. Furthermore, vEP-45 could convert C3 and C5 to active C3a and C5a as a proinflammatory mediator, while no cleavage of C4 was observed. These results suggest that vEP-45 can activate the complement system involved in innate immunity through an alternative pathway.

Distinct brain regional proteome changes in the rTg-DI rat model of cerebral amyloid angiopathy.

Cerebral amyloid angiopathy(CAA), a prevalent cerebral small vessel disease in the elderly and a common comorbidity of Alzheimer's disease, is characterized by cerebral vascular amyloid accumulation, cerebral infarction, microbleeds, and intracerebral hemorrhages and is a prominent contributor to vascular cognitive impairment and dementia. Here, we investigate proteome changes associated with specific pathological features in several brain regions of rTg-DI rats, a preclinical model of CAA. Whereas varying degrees of microvascular amyloid and associated neuroinflammation are found in several brain regions, the presence of microbleeds and occluded small vessels is largely restricted to the thalamic region of rTg-DI rats, indicating different levels of CAA and associated pathologies occur in distinct brain regions in this model. Here, using SWATHLC-MS/MS, we report specific proteomic analysis of isolated brain regions and employ pathway analysis to correlate regionally specific proteomic changes with uniquely implicated molecular pathways. Pathway analysis suggested common activation of tumor necrosis factor α (TNFα), abnormal nervous system morphology, and neutrophil degranulation in all three regions. Activation of transforming growth factor-β1 (TGF-β1) was common to the hippocampus and thalamus, which share high CAA loads, while the thalamus, which uniquely exhibits thrombotic events, additionally displayed activation of thrombin and aggregation of blood cells. Thus, we present significant and new insight into the cerebral proteome changes found in distinct brain regions with differential CAA-related pathologies of rTg-DI rats and provide new information on potential pathogenic mechanisms associated with these regional disease processes.

FM-CATH, A Novel Cathelicidin From Fejervarya Multistriata, Shows Therapeutic Potential for Treatment of CLP-Induced Sepsis.

Sepsis is an exacerbated inflammatory reaction induced by severe infection. As important defensive molecules in innate immunity, several AMPs are reported to prevent septic shock. In this study, we characterized a novel cathelicidin, FM-CATH, from the frog skin of F. multistriata. FM-CATH was found to adopt an amphipathic α-helix structural in membrane-mimetic environments and possess favorable antimicrobial effects against bacteria and fungus. In addition, it triggered the agglutination of bacteria. It could also strongly bind to LPS and LTA. Additionally, FM-CATH affected the enzymatic activities of thrombin, plasmin, β-tryptase, and tPA, leading to coagulation inhibition in vitro and in vivo. Finally, we observed that FM-CATH improved survival rate and inhibited pathological alteration, bacterial count, serum biochemistry, and pro-inflammatory cytokine expression in the cecal ligation and puncture-induced sepsis mice. Taken together, these findings suggest that FM-CATH might be served as a promising agent for the treatment of sepsis.

Redox manipulation of enzyme activity through physiologically active molecule

SummaryThe effective utility of physiologically active molecules is crucial in numerous biological processes. However, the regulation of enzyme functions through active substances remains challenging at present. Here, glutathione (GSH), produced in cells, was used to modulate the catalytic activity of thrombin without external stimulus. It was found that high concentrations of GSH was more conducive to initiate the cleavage of compound AzoDiTAB in the range of concentration used to mimic the difference between cancer and normal cells, which has practical implications for targeting cancel cells since GSH is overexpressed in cancer cells. Importantly, GSH treatment caused the deformation of G4 structure by cleaving AzoDiTAB and thus triggered the transition of thrombin from being free to be inhibited in complex biological systems. This work would open up a new route for the specific manipulation of enzyme-catalyzed systems in cancer cells.

Thrombin Inhibition Prevents Endothelial Dysfunction and Reverses 20-HETE Overproduction without Affecting Blood Pressure in Angiotensin II-Induced Hypertension in Mice.

Angiotensin II (Ang II) induces hypertension and endothelial dysfunction, but the involvement of thrombin in these responses is not clear. Here, we assessed the effects of the inhibition of thrombin activity by dabigatran on Ang II-induced hypertension and endothelial dysfunction in mice with a particular focus on NO- and 20-HETE-dependent pathways. As expected, dabigatran administration significantly delayed thrombin generation (CAT assay) in Ang II-treated hypertensive mice, and interestingly, it prevented endothelial dysfunction development, but it did not affect elevated blood pressure nor excessive aortic wall thickening. Dabigatran’s effects on endothelial function in Ang II-treated mice were evidenced by improved NO-dependent relaxation in the aorta in response to acetylcholine in vivo (MRI measurements) and increased systemic NO bioavailability (NO2− quantification) with a concomitant increased ex vivo production of endothelium-derived NO (EPR analysis). Dabigatran treatment also contributed to the reduction in the endothelial expression of pro-inflammatory vWF and ICAM-1. Interestingly, the fall in systemic NO bioavailability in Ang II-treated mice was associated with increased 20-HETE concentration in plasma (UPLC-MS/MS analysis), which was normalised by dabigatran treatment. Taking together, the inhibition of thrombin activity in Ang II-induced hypertension in mice improves the NO-dependent function of vascular endothelium and normalises the 20-HETE-depedent pathway without affecting the blood pressure and vascular remodelling.

Biologically active peptides derived from the Antarctic hydrobionts

The development of novel, more efficient antioxidants is one of the most perspective approaches for the treatment of diseases related to oxidative stress. Due to their numerous beneficial health effects, antioxidant peptides draw significant research attention. The aim of this study was to investigate the antioxidant potential of the peptides derived from the Antarctic hydrobionts and the effect of the peptides on key factors of blood clotting. The peptide fractions were isolated from the tissue of the Antarctic hydrobionts by sequential precipitation with perchloric acid and ethanol. All hydrobionts have peptides with antioxidant activity, but their effectiveness is different for different species. The peptides of Antarctic krill are effective at scavenging 1,1-diphenyl-2-picrylhydrazyl radicals, superoxide anion radicals, and nitric oxide radicals, while the peptides of Antarctic jellyfish exhibit metal-chelating ability and hydrogen peroxide scavenging activity. The peptides of Antarctic limpet possess hydroxyl radical scavenging activity and provide a slight membrane-protective activity. In addition, the peptides derived from the Antarctic hydrobionts decreased the amidolytic activity of thrombin and increased the ability of thrombin to polymerize the fibrinogen. Obtained results showed that natural peptides of the Antarctic hydrobionts possess various types of biological activities and can be used as antioxidants or molecules affecting blood coagulation.

Tailoring the effect of antithrombin-targeting therapy in haemophilia A using in silico thrombin generation

Factor (F) VIII deficiency causes bleeding in haemophilia A patients because of the reduced formation of procoagulant enzyme thrombin, which is needed to make the blood clot. We measured the dynamics of coagulation in haemophilia A patients by measuring thrombin generation (TG). Additionally, we quantified the procoagulant process of prothrombin conversion and anticoagulant process of thrombin inhibitor complex formation. In haemophilia A, prothrombin conversion is severely reduced, causing TG to be low. Nevertheless, the thrombin inactivation capacity of these patients is comparable to that in healthy subjects, leading to a severe imbalance between procoagulant and anticoagulant processes and a subsequent increased bleeding risk. A novel therapy in haemophilia A is the targeting of anticoagulant pathway, e.g. thrombin inhibitor antithrombin (AT), to restore the haemostatic balance. We simulated the effect of AT reduction on TG in silico. Lowering AT levels restored TG dose-dependently and an AT reduction of 90–95% led to almost normal TG in most patients . However, the variation in response to AT reduction was large between patients, indicating that this approach should be tailored to each individual patients. Ideally, TG and thrombin dynamics simulation could in the future contribute to the management of patients undergoing AT targeting therapy.

Applying Neural Networking Identifies Antiphospholipid Syndrome Patients with an Accuracy of 98.7%

Background: The antiphospholipid syndrome (APS) is characterized by the presence of antiphospholipid antibodies predominantly directed against β2-glycoprotein I. The syndrome is associated with an increased risk of thrombosis and pregnancy morbidity. In APS patients an altered thrombin generation (TG) profile has recently been described; however, it was not possible to differentiate between patients and healthy controls based on the value of a single TG parameter.Therefore, we used a novel machine learning approach called ‘neural networking’ to look for hidden patterns in patient and healthy subject data. A neural network is a computing system inspired by the human brain and consists of a collection of artificial neurons. These systems can learn to classify samples (healthy or APS patient) using examples from a database containing many input variables (i.e. training). Once the network has been trained and tested it can be used to classify new samples as healthy or APS patient.Aim: To develop a neural network to predict APS in subjects based on TG results.Materials and methods: 31antiphospholipid syndrome patients and 44 age- and sex-matched healthy subjects were enrolled in the study. Patients on anticoagulant therapy were excluded from the study. Thrombin generation was measured at 1 pM tissue factor (TF) and thrombin dynamics analysis was performed to study prothrombin conversion and thrombin inactivation. The sensitivity of the activated protein C system was tested by measuring TG in the presence and absence of 20 nM thrombomodulin (TM).A neural network was trained and tested to predict whether a subject is a patient or a healthy subject using Matlab software. The input of the neural network consisted of TG data (lag time, time-to-peak, peak height, ETP and TM effect) and thrombin dynamics data (total and maximal prothrombin conversion and thrombin inactivation).Results: Thrombin generation was measured in platelet poor plasma at 1 pM TF and prothrombin conversion parameters were determined computationally (Figure 1). The lag time was significantly prolonged in APS patients and the peak height was elevated. Time-to-peak and ETP were comparable between healthy subjects and APS patients, but the inhibitory effect of TM was significantly less in APS patients than healthy controls (p<0.001). The total amount of prothrombin converted during thrombin generation (PCtot) did not differ between healthy subjects and patients, but the maximum rate of prothrombin conversion (PCmax) was significantly elevated in APS patients.The neural networking tool was used to develop a classification network. The network consists of an input layer (thrombin generation and thrombin dynamics data, 1 layer of hidden processing neurons and two outputs (either APS patient or healthy subject) (Figure 2). After a training and testing phase, we obtained a network that accurately predicts a subject's class (APS patient or healthy) based on the given input values with an accuracy of 98.7%.Discussion:APS is a complex condition associated with thrombosis, pregnancy morbidity and alterations of diagnostic variables, for example in the thrombin generation profile. Single TG parameters are unable to predict whether or not a subject has APS, but a neural network based on TG and thrombin dynamics data correctly classifies APS patients in 98.7% of the cases. We have found a novel approach that is helpful in correctly categorizing APS patients and therefore might be included to improve treatment strategies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Genetic Fusion to Factor XIII B-Subunit Improves the Pharmacokinetic Properties of Factor IX

Background: Hemophilia B is a X-linked recessive bleeding disorder characterized by coagulation factor IX (FIX) deficiency. FIX concentrates are used in the prevention and treatment of bleeding episodes in patients with hemophilia B. Prophylaxis is currently considered as the optimal care for severe hemophilia. For patients and their families one of the major problems with prophylaxis is the need for frequent venepunctures. The half-life of standard FIX concentrates is approximately 18 h, which requires 2 or 3 intravenous infusions per week to achieve bleeding prevention. Prolonging the half-life of FIX can therefore reduce the frequency of infusions. Recently, extended half-life FIX concentrates have been developed using two major strategies: i) (glyco) PEGylation(1), ii) genetic fusion to immunoglobulin Fc fragments(2), or to albumin(3). These technologies may have some potential disadvantages such as reduced specific activity, unknown long-term adverse effects of PEG exposure and immunological reactivity which are currently under investigation.The aim of the present study was to develop a new strategy to prolong half-life of recombinant human FIX molecule (rFIX) using an innovative approach of fusion.Factor XIII (FXIII) is a protransglutaminase that circulates in plasma as a heterotetramer composed of two catalytic A subunits and two B subunits (FXIIIB). FXIIIB subunits act as a carrier protein that stabilizes the structure of the A subunits and protects FXIII from proteolysis. It has been reported that a primary deficiency of FXIIIB resulted in the secondary rapid loss of A subunit from the plasma (4). Thus, FXIII-B is responsible for the long half-life of the FXIII molecule (10 to 12 days).We designed a new rFIX molecule fused to coagulation FXIII subunit B (FXIIIB) and separated by a short linker sequence containing an activated Factor X-cleavable site (rFIX-LXa-FXIIIB). Thus, we expected that the carrier protein will be removed as soon as traces of (activated factor X) FXa will be generated, leaving rFIX free to perform its enzymatic role in the tenase complex.Methods: Recombinant FIX-LXa-FXIIIB molecules were expressed in human hepatic Huh-7 cell line and purified via a two-step protocol prior to in vitro and in vivo biochemical and pharmacokinetic characterizations.Results: rFIX-LXa-FXIIIB was correctly expressed by Huh-7 cells at the expected molecular weight of 150 kDa and presented similar procoagulant activity compared to rFIX-WT (106.5 UI/dL ; p> 0.05). We showed that the LXa linker was cleavable by FXa, releasing free rFIX molecule before its activation. After cleavage, rFIX was fully activable by activated FXI. Both rFIX-WT and rFIX-LXa-FXIIIB showed similar thrombin generation capacity both in vitro and in vivo after injection in FIX-deficient mice (Area under the thrombin generation curve =300nM.min ; p>0.05).The half-life (T1/2) of rFIX-LXa-FXIIIB molecule was determined in wild-type mice and rats. In mice, as commercially available extended half-life FIX products, the improvement of the half-life was very limited and in rats, it was 2.5 fold longer compared to rFIX-WT (T1/2 10h vs 4h ; p<0.05).A potential advantage of this new molecule is its capacity to bind to fibrinogen via FXIIIB. It has been recently reported that excess plasma FXIIIB2 is not free, but rather circulates bound to fibrinogen (5). We can hypothesize that the rFIX-LXa-FXIIIB fusion protein can circulate bound to fibrinogen and might therefore enhance/accelerate the transformation of fibrinogen into fibrin. In vivo experiments using mice saphenous vein bleeding model are currently conducted in our laboratory to test this hypothesis.Conclusion: We report here a new fusion protein strategy using FXIIIB. The fusion protein has normal specific activity and normal thrombin generating capacity, associated with improved pharmacokinetics in small animal models. These encouraging results suggest that genetic fusion of rFIX to FXIIIB might be a valuable concept to improve the half-life of FIX molecules. The binding capacity of this novel fusion protein to fibrinogen might be an additional advantage that may improve haemostatic capacity of the molecule.1. Negrier C et al. Blood. 2011;118:2695-701.2. Peters RT et al. Blood. 2010;115:2057-64.3. Metzner HJ et al. Thromb Haemost. 2009;102:634-44.4. Miloszewski K et al. Br J Haematol. 1970;19:685-90.5. Byrnes JR et al. Blood. 2016 Oct 13;128:1969-1978 DisclosuresLe Quellec:LFB: Honoraria; Novo Nordisk: Honoraria; Shire: Honoraria. Negrier:Baxter: Research Funding; Alnylam: Research Funding; Bayer: Honoraria, Research Funding, Speakers Bureau; Biogen/SOBI: Honoraria, Research Funding, Speakers Bureau; CSL Behring: Honoraria, Research Funding, Speakers Bureau; Inspiration: Research Funding; Novo Nordisk: Honoraria, Research Funding, Speakers Bureau; Octapharma: Honoraria, Research Funding, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau; Baxalta: Honoraria, Speakers Bureau; LFB: Honoraria, Speakers Bureau. Dargaud:BMS: Honoraria, Research Funding; Baxalta/Shire: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; LFB: Honoraria, Research Funding; CSL Behring: Honoraria, Research Funding; Bayer: Honoraria, Research Funding; Stago: Honoraria; Octapharma: Honoraria, Research Funding; Alnylam: Research Funding.

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.

Preferential Inhibition of Fibrin Mediated Thrombosis in Rodent Models By JNJ-9375, a Novel exosite1 Thrombin Inhibitor

We have previously reported an improved therapeutic index (TI) for JNJ-9375 vs apixaban in rat and cynomolgus monkey models of venous thrombosis and bleeding. We have also reported that JNJ-9375 preferentially inhibited the fibrin component of the thrombus without significant impact on the platelet component under low and high shear conditions in a human translational model of thrombosis (Badimon chamber model). We hypothesized that the novel mode of action of JNJ-9375 which by selectively inhibiting exosite 1 substrates from binding to thrombin (such as fibrinogen and PAR-1) may allow for other thrombin interactions at the active site and exosite 2 including platelet activation by PAR-4 and GP1b.In the present studies, we have compared the antithrombotic effects of JNJ-9375, the active site thrombin inhibitor dabigatran and the factor Xa inhibitor apixaban in well characterized and widely used rat AV-shunt and carotid ferric chloride models of venous and arterial thrombosis, respectively. JNJ-9375, dabigatran and apixaban all maximally inhibited thrombus formation in a dose dependent manner in the rat AV-shunt model of venous thrombosis. Significant inhibition of thrombus weights of 50%, 44% and 57% were observed at 0.3 mg/kg, i.v., 0.1 mg/kg, i.v. and 1 mg/kg, i.v. with JNJ-9375, dabigatran and apixaban respectively. Dose dependent prolongation in coagulation parameters were also observed ex-vivo with JNJ-9375 (TT, ECT, aPTT and PT), dabigatran (TT, ECT, aPTT) and apixaban (aPTT and PT). In addition, there were dose dependent increases in drug exposure.In the rat ferric chloride model of arterial thrombosis, JNJ-9375, dabigatran and apixaban administration also resulted in dose dependent inhibition of thrombus formation as assessed by the time to occlusion (TTO) of the carotid artery and area under the curve (AUC) for carotid blood flow. However, the dose required to inhibit arterial thrombosis Vs venous thrombosis varied substantially for the different agents. With JNJ-9375 we only observed a significant inhibition of arterial thrombosis (TTO and AUC) at a dose of 10 mg/kg, i.v. (Vs 0.3 mg/kg in the AV-shunt model) which represents a 33-fold shift in the dose required to inhibit arterial thrombosis (Vs venous thrombosis). In contrast, we observed a significant inhibition of arterial thrombosis with dabigatran (AUC) and apixaban (TTO and AUC) at doses of 0.3 mg/kg, i.v. and 3 mg/kg, i.v. (Vs 0.1 mg/kg and 1 mg/kg in the AV-shunt model) respectively. These doses of dabigatran and apixaban represent a 3-fold shift in the dose required to inhibit arterial thrombosis (Vs venous thrombosis). We have also documented dose dependent prolongations in coagulation parameters and drug exposure with all three agents in the these studies. In addition, we performed ex-vivo platelet aggregation in platelet rich plasma with various platelet agonists (ADP, collagen, AA, rat and human thrombin) in those animals dosed with JNJ-9375. As anticipated and consistent with its mechanism of action, JNJ-9375 only inhibited platelet aggregation to thrombin stimulation but not with other platelet agonists confirming the specificity of JNJ-9375. We observed about 50%, >95% and >95% inhibition of thrombin-induced platelet aggregation at 1, 3 and 10 mg/kg, i.v. with JNJ-9375 respectively. However, we only observed inhibition of arterial thrombosis at 10 mg/kg.In summary, we have demonstrated dose dependent inhibition of thrombosis with JNJ-9375, dabigatran and apixaban in venous and arterial model of thrombosis in rats. However, we have observed a larger difference in the dose required for inhibition of arterial Vs venous thrombosis with JNJ-9375 (33-fold) compared to a more modest 3-fold shift observed for dabigatran and apixaban. These findings with JNJ-9375 demonstrate an anticoagulant profile that preferentially inhibits fibrin mediated clot formation with a more modest impact on platelet driven thrombosis in the present models. This differential profile may account in part for the improved TI over apixaban we had reported earlier in animal models. This profile suggests JNJ-9375 which is currently in early clinical development may also be more amenable to dosing in combination with antiplatelet therapy for indications such as acute coronary syndromes and needs to be tested experimentally. DisclosuresChintala:Janssen Research & Development: Employment. Li:Janssen R&D: Employment. Huang Devine:Janssen Research & Development: Employment. Bunce:Janssen R&D: Employment.

A CRISPR-Cas9 Generated Etv6 mutant Mouse Exhibits Decreased Platelet Counts and Functional Platelet Defects

We and others recently described families with autosomal dominant thrombocytopenia, variable platelet function defects, and a predisposition to leukemia caused by heterozygous germline mutations in ETV6 . One of these mutations in ETV6, encoding an amino acid change at position 214 (P214L), was described in five independent families. The P214L mutation, located in the central domain of ETV6, was shown to disrupt nuclear localization of the ETV6 protein and to block megakaryocyte maturation of human cells overexpressing the mutant protein in vitro . Additionally, RNA seq of platelets from patients with the ETV6-P214L mutation showed downregulation of cytoskeletal transcripts and transcripts involved in platelet production and function. To further study the effect of the P214L mutation on ETV6 and on platelet production and function, we have generated a CRISPR-Cas9 knock-in mouse with the equivalent mutation to the human P214L in the mouse Etv6 ortholog (in the corresponding amino acid P216 in mice). CRISPR-Cas9 knock-in mice were generated by designing short guide RNAs specific to the site of Etv6 encoding the proline to be mutated. Additionally, a ssDNA oligo was designed encoding the mutation (cca>cta, P>L) and flanking homologous sequence to provide a template for recombination. Heterozygous and homozygous mice were viable. Heterozygous by heterozygous crosses yielded ratios not significantly different from expected Mendelian ratios: wild type (WT) 18.0%, heterozygous 63.0%, and homozygous 19.1% (p=0.18, Chi-squared test). Complete blood counts were obtained by retro-orbital bleeds and showed a mild but significant decrease in platelet counts in homozygous mice (WT 967.3 x 103/uL, heterozygous 840 x 103/uL, homozygous 807.5 x 103/uL, p=0.03). Western blot indicated normal expression of Etv6 protein in mouse bone marrow. Immunofluorescence and confocal imaging of megakaryocytes from these mice showed an increased percentage of small, immature megakaryocytes in homozygous mice compared to WT mice. Flow cytometry analysis suggested normal distribution of the hematopoietic stem cell compartment. These data are consistent with the patient phenotype and in vitro data, which showed that the P214L mutation resulted in variable thrombocytopenia, immature megakaryocytes, and normal ETV6 protein expression.Considering the downregulation of transcripts related to platelet function in patients with the P214L mutation and the variable platelet function defects reported in patients with germline ETV6 mutations, we characterized the platelet function of Etv6 mutant mice. Platelets from homozygous mice showed decreased platelet activation upon high (10 ug/ml) and low (2 ug/ml) collagen stimulation as measured by light transmission aggregometry (p=0.041 and 0.019, respectively). Additionally, homozygous mice had decreased markers of platelet activation (thrombin and convulxin) including granule secretion (P-selectin exposure) and aIIbb3 activation (JONA binding).In conclusion, we have generated a CRISPR-Cas9 knock-in mouse with the orthologous mutation to the human P214L recurrent germline mutation. Mice homozygous for this mutation recapitulate the human phenotype of decreased platelet counts and impaired megakaryocyte maturation. Additionally, mice homozygous for this mutation show a significant defect in platelet activation, further supporting that Etv6 is important for platelet function. Although complete knock-outs of Etv6 are embryonic lethal and Etv6 is expressed in the hematopoietic stem cell, we did not find a significant hematopoietic stem cell defect in our mutant mice. This finding suggests that the most important function of Etv6 is further downstream in hematopoiesis. The Etv6 P214L mouse represents a reliable model for studying the role of Etv6 in megakaryocyte biology, platelet function, and potentially future studies in leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

Measuring the Anticoagulation Effects of Exosite I Thrombin Inhibitors: Utility and Limitations of Clotting Assays, Thromboelastography, and Calibrated Automated Thrombography

The ability to anticoagulate patients at elevated risk for thrombosis without inducing a bleeding diathesis remains an unmet need of patient care both in a hospital setting and at home. Traditional anticoagulants including warfarin and heparin effectively inhibit clotting, but incur a bleeding risk and require continued monitoring after a stable profile has been established. Current generation direct oral anticoagulants (DOACs) including dabigatran, rivaroxaban, and apixaban overcome many of the liabilities of warfarin and heparin (e.g. lack of target specificity and need for monitoring), but are still susceptible to bleeding diathesis. Thus there remains a need for novel anticoagulants that offer an improved therapeutic index. The recombinant human antibody JNJ-64179375 (JNJ-9375) is a novel thrombin inhibitor that binds to the exosite I region with high affinity and selectivity. In contrast to DOACs that inhibit the protease active site, JNJ-9375 inhibits the interaction of thrombin with its substrates including fibrinogen and PAR-1 while retaining active site and exosite II mediated interactions. This targeted and distinct mechanism of action which modulates the actions of thrombin offers the potential for efficacious anticoagulation with the potential for reduced risk of bleeding compared to active site-specific DOACs.To assess the ability to measure exosite I-specific thrombin inhibitors effects in blood, we compared JNJ- 9375 to the active site ligand dabigatran in clotting assays, thromboelastography (TEG), and calibrated automated thrombography (CAT) assays. We focused on clinically relevant concentrations of these inhibitors based on the efficacious doses reported in the RE-LY study for dabigatran (NCT00262600) and a Phase 1 study for JNJ-9375 (NCT02949206). In conventional clotting assays (prothrombin time (PT), activated partial thromboplastin time (aPTT), and thrombin time (TT)) dabigatran and JNJ-9375 each prolonged clotting times in a dose-dependent manner. TT assays were the most sensitive to dabigatran and JNJ-9375, with 80 nM dabigatran prolonging the clotting time by 5-fold and 100 nM JNJ-9375 prolonging the clot time 4-fold over vehicle control. PT and aPTT assays were less sensitive than the TT assay, requiring ≥ 300 nM dabigatran and ≥ 1 µM JNJ- 9375 to double the PT or aPTT clotting times. In TEG assays both JNJ-9375 and dabigatran prolonged the reaction time (R), with 200 nM JNJ-9375 prolonging R by 1.3- to 1.5-fold and 100 nM dabigatran prolonging R from 1.6- to 1.8-fold over buffer control. In CAT assays initiated with 5 pM tissue factor (PPP Reagent, Stago), dabigatran inhibited peak thrombin and Endogenous thrombin potential (ETP) with an approximate IC50 of 1 µM. In contrast, titration of JNJ-9375 up to 1 µM increased both the Peak thrombin and ETP ≥ 20% above buffer control. This increase in peak thrombin and ETP with JNJ-9375 was also observed with other exosite I-specific ligands such as hirugen, suggesting a general effect of exosite I binding on the primary endpoints of the CAT assay. This insensitivity of the CAT assay to exosite I ligands likely reflects the inability of these molecules to inhibit proteolysis of the peptidyl substrate z-Gly-Gly-Arg-AMC, which directly binds to the active site and is used as the readout of thrombin activity.In summary, of the assays tested the TT clotting assay offers the greatest sensitivity to detect multiple classes of direct thrombin inhibitors in plasma, while TEG can detect direct thrombin inhibitors in whole blood. In contrast, the CAT assay is insensitive to exosite I ligands such as JNJ-9375. These results highlight the importance of the relationship between the endpoint of a given coagulation assay and the mechanism of action of the anticoagulant being tested. DisclosuresBunce:Janssen Research & Development: Employment. Huang Devine:Janssen Research & Development: Employment. Chintala:Janssen Research & Development: Employment.

No changes of parameters nor coagulation activation in healthy subjects vaccinated for SARS-Cov-2

BackgroundRecent reports of thrombotic events after SARS-Cov-2 vaccination raised concern. However, modifications of the most common coagulation parameters after vaccination are unknown. AimsWe measured parameters of coagulation including (i) basic coagulation tests, (ii) procedures aimed to assess the ex-vivo potential capacity to generate thrombin and (iii) in vivo thrombin activity. We also assessed anti-platelet factor 4 (aPF4) with two methods. DesignLaboratory measurements were performed for a cohort of subjects (n = 30) before (baseline) and after (7 and 21days after first dose, and 14days after second dose) SARS-Cov-2 vaccination. ResultsAll subjects received the Pfizer-BioNTech vaccine, and none developed symptomatic thrombotic events during the study period. None of the parameters showed clinically relevant variations at different time-points before and after vaccination. Only platelet count showed a slight increase, and F1.2 and the thrombin generation parameters ETP and ETP-TM ratio, showed a small decline, at the last time-point after vaccination when compared to baseline. aPF4 was negative in all the subjects, except two, who were positive (one with the chemiluminescent and the other with the ELISA assay). ConclusionsThe study shows no modifications of the coagulation parameters nor the presence of biochemical signs of coagulation activation following the administration of the Pfizer-BioNTech vaccine.