Longitudinal assessment of coagulation potential before, during, and following an in vitro fertilization cycle.

INTRODUCTION: The association between estrogen and hypercoagulability is well-established but little is known about coagulation dynamics during IVF. Our goal was to measure coagulation potential prior to, during, and following an IVF cycle and to investigate differences by conception outcome. MATERIALS AND METHODS: Patients undergoing IVF with fresh embryo transfer at a single academic center using oral contraceptive pills for cycle batching underwent evaluation of thrombin generation using the calibrated automated thrombogram at multiple points during the IVF cycle. Multiple thrombin generation parameters were compared across timepoints and by IVF cycle outcome using ANOVA repeated measures analysis. RESULTS: Of the 17 patients included, 11 conceived. There was a significant increase in peak and total thrombin generation in the entire cohort between the pre-treatment natural follicular phase and following a short course of oral contraceptive pills used for cycle batching. Further increase in these parameters was seen at the time of oocyte retrieval. In the pre-treatment natural follicular phase, patients who conceived had lower peak thrombin generation. There were changes throughout the cycle for factors II, V, VIII, X, XI, XII, antithrombin, and tissue factor pathway inhibitor. Only Factor XI was distinguishable by conception status; values were lower at all visits in patients who conceived. CONCLUSION: Increases in coagulation potential are seen in patients undergoing IVF following a short course of oral contraceptive pills for cycle batching and continue during controlled ovarian hyperstimulation. Those who conceived were seen to have lower peak thrombin generation in the pre-treatment natural follicular phase.

Thrombin Generation Assay in Antiphospholipid Antibodies Positive Subjects as a Personalized Thrombotic Risk Assessment: State of the Art and Perspectives.

PURPOSE OF THE REVIEW: Thrombotic risk assessment in antiphospholipid positive (aPL +) subjects is a major challenge, and the study of in vitro thrombin generation (thrombin generation assays (TGA)) could provide useful information. Activated protein C (APC) sensitivity is involved in thrombotic events in antiphospholipid syndrome patients. We summarized methods used to assess APC sensitivity with TGA and evaluated the prognostic role of APC resistance through literature search. RECENT FINDINGS: APC resistance induced by aPL is a complex pathway. Several cross-sectional studies assessed APC sensitivity to understand thrombotic event mechanisms in aPL + subjects. Only one prospective cohort had investigated the prognostic impact of APC resistance in aPL + subjects, with a positive and significant correlation between APC sensitivity and the risk of thrombosis during the follow up (hazard ratio, 6.07 [95% CI, 1.69-21.87]). APC resistance assessed with TGA could be associated with thrombotic events in aPL + subjects.

Patients with moderate hemophilia A and B with a severe bleeding phenotype have an increased burden of disease.

BACKGROUND: Patients with moderate hemophilia express varying bleeding phenotypes. OBJECTIVES: To assess the burden of disease in patients with moderate hemophilia and a mild or severe phenotype incorporating the thrombin generation profile. METHODS: This sub-study of the 6th Hemophilia in the Netherlands study, analyzed data of adults with moderate hemophilia A or B. Patient characteristics and information on bleeding tendency, joint status, and quality of life were obtained from electronic patient files and self-reported questionnaires. A severe bleeding phenotype was defined as an annual bleeding rate ≥5, an annual joint bleeding rate ≥3, and/or the use of secondary/tertiary prophylaxis, and a mild phenotype vice versa. TG was measured with the Nijmegen Hemostasis Assay. RESULTS: This study included 116 patients: 21% had a severe phenotype of whom 46% used prophylaxis. Patients with a severe phenotype treated on demand reported a higher median annual bleeding rate (7), annual joint bleeding rate (3), and more frequently an impaired joint (77%) than patients with a severe phenotype on prophylaxis (2; 0; 70%) or patients with a mild phenotype (0; 0; 47%). Furthermore, patients with a severe phenotype treated on demand experienced a more decreased quality of life. Despite similar factor activity levels, patients with a severe phenotype had a lower thrombin peak height and thrombin potential (0.7%; 0.06%) than patients with a mild phenotype (21.3%; 46.8%). CONCLUSION: Patients with moderate hemophilia and a severe phenotype treated on demand displayed a high burden of disease as well as a low thrombin generation profile advocating them toward more intensive prophylactic treatment.

Assessment of DOAC in GEriatrics (Adage Study): Rivaroxaban/Apixaban Concentrations and Thrombin Generation Profiles in NVAF Very Elderly Patients.

BACKGROUND: Although a growing number of very elderly patients with atrial fibrillation (AF), multiple conditions, and polypharmacy receive direct oral anticoagulants (DOACs), few studies specifically investigated both apixaban/rivaroxaban pharmacokinetics and pharmacodynamics in such patients. AIMS: To investigate: (1) DOAC concentration-time profiles; (2) thrombin generation (TG); and (3) clinical outcomes 6 months after inclusion in very elderly AF in-patients receiving rivaroxaban or apixaban. METHODS: Adage-NCT02464488 was an academic prospective exploratory multicenter study, enrolling AF in-patients aged ≥80 years, receiving DOAC for at least 4 days. Each patient had one to five blood samples at different time points over 20 days. DOAC concentrations were determined using chromogenic assays. TG was investigated using ST-Genesia (STG-ThromboScreen, STG-DrugScreen). RESULTS: We included 215 patients (women 71.1%, mean age: 87 ± 4 years), 104 rivaroxaban and 111 apixaban, and 79.5% receiving reduced-dose regimen. We observed important inter-individual variabilities (coefficient of variation) whatever the regimen, at C max [49-46%] and C min [75-61%] in 15 mg rivaroxaban and 2.5 mg apixaban patients, respectively. The dose regimen was associated with C max and C min plasma concentrations in apixaban (p = 0.0058 and p = 0.0222, respectively), but not in rivaroxaban samples (multivariate analysis). Moreover, substantial variability of thrombin peak height (STG-ThromboScreen) was noticed at a given plasma concentration for both xabans, suggesting an impact of the underlying coagulation status on TG in elderly in-patients. After 6-month follow-up, major bleeding/thromboembolic event/death rates were 6.7%/1.0%/17.3% in rivaroxaban and 5.4%/3.6%/18.9% in apixaban patients, respectively. CONCLUSION: Our study provides original data in very elderly patients receiving DOAC in a real-life setting, showing great inter-individual variability in plasma concentrations and TG parameters. Further research is needed to understand the potential clinical impact of these findings.

A convenient and economical strategy for multiple-target electrochemiluminescence detection using peroxydisulfate solution.

As various aptasensors are adopted in clinical diagnosis, the development of convenient multiple-target determination is a field of ever-increasing interests. Herein, a label-free and amplified electrochemiluminescence (ECL) sensing platform was constructed to detect multiple targets of hemin, glucose and thrombin (TB) using peroxydisulfate (S2O82-) solution, which was one of the most convenient and economical ECL systems. It was worth mentioning that the target-induced bi-enzyme cascade catalysis reaction was developed to increase the ECL response strongly of S2O82- solution due to the production of (1O2)2* from the inter-reaction between reactive oxygen species (ROS) and sulfate radical (SO4•-). Specifically, with the layer-by-layer assembly of multi-walled carbon nanotubes (MWCNTs), glucose oxidase (GOx) and gold nanoparticles (AuNPs) as the interface, the guanine-rich (G-rich) thrombin aptamer (TBA) was anchored for hemin (target 1) detection, due to the electrocatalysis of hemin/G-quadruplex as a horseradish peroxidase mimicking DNAzyme (HRP-DNAzyme) towards dissolved oxygen for ROS generation. Second, in the presence of glucose (target 2), the ECL intensity was improved because glucose was the substrate of the bi-enzyme cascade catalysis reaction. Third, when TB (target 3) was sequentially incubated based on the above-mentioned aptasensor, the bi-enzyme catalysis was inhibited to decrease the ECL signal, due to the steric hindrance effect of the TB protein. As a result, the aptasensor achieved the nanomolar detection for hemin (3.33 nM), the micromolar detection for glucose (0.33 µM) and the femtomolar detection for TB (3.33 fM), respectively.

Nursing students' decision-making regarding postpartum hemorrhage: An exploration using the recognition-primed decision model.

AIM: To explore the knowledge content and structure of nursing students' decision-making in a high-stake clinical situation of postpartum hemorrhage using the Recognition-Primed Decision Model. BACKGROUND: According to research on clinical judgment, a nurse's expectations for a patient situation are central to the clinical decision-making process. However, little research has addressed the expectation concept and its relationship with the nurse's knowledge. Grounded in the naturalistic decision-making paradigm, the Recognition-Primed Decision Model provides a potential framework to describe the content and structure of nurses' knowledge and expectations as they unfold in high-stake clinical situations, such as postpartum hemorrhage. As it is typically used in studies of expert decision-making, it is crucial to test the adequacy of the Model with a student population and refine the research methods for using this framework. DESIGN: Descriptive design where qualitative data were analyzed using qualitative and quantitative methods. METHODS: A convenience sample of 53 students enrolled in a maternal and child health course in the Fall of 2021 was formed. As part of an online exercise to prepare for a simulation, they read a vignette presenting the story of a woman experiencing postpartum hemorrhage and recorded their answers to questions designed to probe their decision-making. Recordings were transcribed and subjected to content analysis based on the four components of recognition according to the Recognition-Primed Decision Model (i.e., cues, expectations, goals and actions). FINDINGS: All participants recognized the postpartum hemorrhage. Their knowledge was organized into clusters representing the potential causes (i.e., tone, trauma, tissue and thrombin) and consequences (i.e., hemodynamic instability) of postpartum hemorrhage, as well as other potential issues (e.g., pain and comfort, baby and partner, infection). Although students could identify relevant cues and actions, they had difficulties articulating their longer-term goals and expectations for the mother and care outcomes. CONCLUSIONS: This study showed the potential of the Recognition-Primed Decision Model to organize the content and structure of the knowledge that supported nursing students' decision-making in a high-stake situation. The findings suggest that their knowledge disproportionately focuses on the cause-and-effect relations between cues and actions. They invite further consideration of longer-term goals and expectations in nursing education to prepare students to anticipate events and assess patient responses appropriately.

Assessment of coagulation markers, serum zinc and copper in children with beta-thalassemia major in Egypt.

BACKGROUND: Thalassemia is one of the commonest single gene disorders usually associated with many complications. Coagulation changes as well as trace elements levels alterations have been described in children with ß thalassemia. Activation of coagulation can be assessed by measuring thrombin-antithrombin (TAT) complex, plasmin-antiplasmin (PAP) complex and ß-thromboglobulin (ß-TG). METHODS: A total of 200 children and adolescents were enrolled in the study; 100 were from the Al-Azhar University hospital's pediatric hematology clinic diagnosed as thalassemia major, while the other 100 were apparently healthy volunteers who acted as the control group. Complete blood count, liver function test, kidney function tests, TAT complex, PAP complex, ß-TG as indicators of coagulation changes, serum zinc and copper were performed on all participants. RESULTS: Significantly higher levels of TAT complex, PAP complex and ß-TG in thalassemia children than the controls. Decreased serum zinc and increased serum copper levels in thalassemia children compared to the controls. A negative correlation was observed between the serum level of TAT and hemoglobin level, besides the negative correlation of TAT complex and ß-TG with the serum zinc. CONCLUSION: Thalassemia major was associated with increased serum level of coagulation activation markers, increased serum copper while decreased serum zinc.

Assessment of Rotational Thromboelastometry and Thrombin Generation Assay to Identify Risk of High Blood Loss and Re-Operation After Cardiac Surgery.

Introduction: We aimed to investigate parameters for prediction of post-operative blood loss and re-operation in patients who underwent cardiopulmonary bypass. Methods: Thrombin generation assay, activated partial thromboplastin time, activated clotting time and rotational thromboelastometry (ROTEM) tests were performed at 4 time points in 65 patients: before skin incision (T1), after heparin injection (T2), after protamine reversal (T3) and before skin closure (T4). Results: Pre-operative endogenous thrombin potential (ETP) and peak thrombin levels were significantly lower in patients with high post-operative blood loss (≥ 800 mL) within 24 h than in those with low blood loss (< 800 mL). Clotting time (CT), maximal clotting firmness, clotting firmness time and alpha angle values of ROTEM measured at T2, T3 or T4 were significant predictors for high post-operative blood loss. An increase in CT-EXTEM over 4 time points was significant in patients who had a re-operation within 48 h compared to their counterparts. Conclusions: This study indicates that pre-operative ETP could predict high post-operative blood loss and that intra-operative ROTEM also helps to stratify risks of high post-operative blood loss and re-operation.

Dabigatran and Wet AMD, Results From Retinal Pigment Epithelial Cell Monolayers, the Mouse Model of Choroidal Neovascularization, and Patients From the Medicare Data Base.

Background: Age-related macular degeneration (AMD), the leading cause of irreversible blindness in elderly Caucasian populations, includes destruction of the blood-retina barrier (BRB) generated by the retinal pigment epithelium-Bruch's membrane complex (RPE/BrM), and complement activation. Thrombin is likely to get access to those structures upon BRB integrity loss. Here we investigate the potential role of thrombin in AMD by analyzing effects of the thrombin inhibitor dabigatran. Material and Methods: MarketScan data for patients aged ≥65 years on Medicare was used to identify association between AMD and dabigatran use. ARPE-19 cells grown as mature monolayers were analyzed for thrombin effects on barrier function (transepithelial resistance; TER) and downstream signaling (complement activation, expression of connective tissue growth factor (CTGF), and secretion of vascular endothelial growth factor (VEGF)). Laser-induced choroidal neovascularization (CNV) in mouse is used to test the identified downstream signaling. Results: Risk of new wet AMD diagnosis was reduced in dabigatran users. In RPE monolayers, thrombin reduced TER, generated unique complement C3 and C5 cleavage products, led to C3d/MAC deposition on cell surfaces, and increased CTGF expression via PAR1-receptor activation and VEGF secretion. CNV lesion repair was accelerated by dabigatran, and molecular readouts suggest that downstream effects of thrombin include CTGF and VEGF, but not the complement system. Conclusions: This study provides evidence of association between dabigatran use and reduced exudative AMD diagnosis. Based on the cell- and animal-based studies, we suggest that thrombin modulates wound healing and CTGF and VEGF expression, making dabigatran a potential novel treatment option in AMD.

Simple method for assessment of activities of thrombin inhibitors through their molecular structure parameters.

Thrombi (blood clots) form in blood vessels in thromboembolic disorders, which are among the main reasons for death in the world. A novel approach is presented to predict thrombin inhibitory activities ((log(103/Ki) (nM)) of some classes of non-peptidic thrombin inhibitors. The largest reported data set of log(103/Ki) for 260 thrombin inhibitors are used to derive and test the new model where it can be easily applied through a computer code. The new model is derived and tested based on 201 and 59 experimental data, respectively, where its reliability is established by external and internal validations. The reliability of the novel correlation is compared with the complex 3D-QSAR method CoMSIA based on donor hydrogen bond, electrostatic interactions, steric occupancy, local hydrophobicity, and acceptor hydrogen bond fields. The values of correlation coefficient (R2), and root mean squared error (RMSE) for 138/34 data of training/test sets, where the predicted results of complex CoMSIA calculations were available, are 0.9173/0.6010 (R2), and 0.2503/0.4911 (RMSE) as well as 0.8753/0.3888 (R2), and 0.3287/0.6358 (RMSE) for the new and CoMSIA models, respectively. Further statistical parameters also confirm high reliability, precision, accuracy, and the goodness-of-fit of the simple model.

Correlation between Thrombin Generation, Standard Coagulation Assays, and Viscoelastic Assays for Hemostatic Assessment in Critically Ill Children.

BACKGROUND: Accurate assessment of hemostatic function is essential to guide care in critically ill children with acute and acquired coagulopathies. Thrombin generation (TG) provides a global assessment of procoagulant and anticoagulant factors and is commonly used in hemostasis research laboratories. Our objective was to determine the correlation of clinically available hemostasis assays with TG in critically ill children. METHODS: Children (<18 years old, >3 kg in weight) in the intensive care unit were enrolled from March 2016 to December 2019 in a prospective 2-center study. Coagulation tests were prothrombin time, activated thromboplastin time, anti-Xa assay, viscoelastic assays (thromboelastography [TEG], rotational thromboelastometry [ROTEM]), and TG (induced by 20 pM tissue factor in platelet poor plasma and reported as endogenous thrombin potential [ETP; nM*min]). Data are reported as median (interquartile range) or Spearman coefficient (ρ). RESULTS: Patients (n = 106, age 10.2 years [3.8-15.3]) were divided into 3 groups: (a) no anticoagulation (n = 46), (b) anticoagulation (unfractionated heparin) without extracorporeal life support (n = 34), or (c) with extracorporeal life support (n = 26). ETP was decreased in anticoagulated compared to non-anticoagulated patients (group 1: 902.4 [560.8-1234], group 2: 315.6 [0.0-962.2], group 3: 258.5 [0.0-716.6]; P < 0.0001). Across all patients, ETP correlated best with TEG kinetic time (TEG-K), in min (ρ = -0.639), followed by TEG reaction time, in min (ρ = -0.596). By group, ETP correlated best with international normalized ratio for group 1 (ρ = -0.469), TEG-K time for group 2 (ρ = -0.640), and anti-Xa for group 3 (ρ = -0.793). CONCLUSIONS: Standard and viscoelastic assays have varying correlation with TG in critically ill children. TEG-K time had the most consistent moderate correlation with ETP across all groups.

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

BACKGROUND: 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. OBJECTIVES: (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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Assessment of plasminogen activator inhibitor-1(PAI1) and thrombin activitable fibrinolysis inhibitor (TAFI) in Egyptian children with hemophilia A.

Patients with hemophilia A display varied bleeding phenotypes not correlated with degree of deficiency of factor VIII level. We investigated Plasminogen Activator Inhibitor 1(PAI1) level and Thrombin Activatable Fibrinolysis Inhibitor (TAFI) also known as Carboxypeptidase B2 (CPB2) level in Patients with hemophilia A and their possible correlation with bleeding tendency. Twenty-six patients attending in hematology unit of pediatric department were included in this study. In addition, fourteen apparently healthy subjects matched ages and genders were included as control group. The International Society of Thrombosis Bleeding Assessment Tool (ISTH/BAT) was used to assess bleeding score in patients. Plasma levels of Plasminogen Activator Fibrinolysis Inhibitor (PAI1) and Thrombin Activatable Fibrinolysis Inhibitor (TAFI) zymogen were measured by enzyme-linked immunosorbent assay (ELIZA). As compared to controls, hemophilic patients had significantly high bleeding score, low PAI 1 level and high TAFI level. There was no significant correlation between bleeding score by ISTH/BAT and patient severity. PAI 1 and TAFI level have no significant correlation with patient severity. PAI 1 level was statistically significant different between intense and non-intense hemorrhagic groups, while TAFI level has no significant correlation with bleeding phenotype. PAI 1 and TAFI levels had significantly correlation between patients and controls. PAI-1 level had statistically significant correlation with bleeding phenotype, while TAFI level failed to show any correlation between intense and non-intense hemorrhagic groups. So, PAI-1 levels may have predictive value of bleeding tendency in hemophiliacs.

The modification of the thrombin generation assay for the clinical assessment of hypercoagulability in patients receiving heparin therapy.

BACKGROUND: Heparin diminishes thrombin generation (TG) because it decreases the survival time of thrombin in plasma. Under heparin therapy, the TG curve therefore does not reflect the true hemostatic status of the patient. AIM: We investigated how far the in vitro addition of a heparin antagonist can restore the underlying TG capacity. MATERIALS & METHODS: Five different heparin antagonists were tested: polybrene, protamine sulfate, heparinase type 1, heparinase HEP-TEM, and (Z-GGR)2 -rhodamine (P2Rho). RESULTS AND CONCLUSION: Polybrene, P2Rho, and heparinase HEP-TEM effectively neutralized heparin at prophylactic and therapeutical doses of both low molecular weight and unfractionated heparin. The advantages and limits of each molecule and the most favorable combinations of TG-trigger and antagonist are discussed.

Preclinical assessment of thrombin-preconditioned human Wharton's jelly-derived mesenchymal stem cells for neonatal hypoxic-ischaemic brain injury.

Hypoxic-ischaemic encephalopathy (HIE) is a type of brain injury affecting approximately 1 million newborn babies per year worldwide, the only treatment for which is therapeutic hypothermia. Thrombin-preconditioned mesenchymal stem cells (MSCs) exert neuroprotective effects by enriching cargo contents and boosting exosome biogenesis, thus showing promise as a new therapeutic strategy for HIE. This study was conducted to evaluate the tissue distribution and potential toxicity of thrombin-preconditioned human Wharton's jelly-derived mesenchymal stem cells (th-hWJMSCs) in animal models before the initiation of clinical trials. We investigated the biodistribution, tumorigenicity and general toxicity of th-hWJMSCs. MSCs were administered the maximum feasible dose (1 × 105 cells/10 µL/head) once, or at lower doses into the cerebral ventricle. To support the clinical use of th-hWJMSCs for treating brain injury, preclinical safety studies were conducted in newborn Sprague-Dawley rats and BALB/c nude mice. In addition, growth parameters were evaluated to assess the impact of th-hWJMSCs on the growth of newborn babies. Our results suggest that th-hWJMSCs are non-toxic and non-tumorigenic in rodent models, survive for up to 7 days in the brain and hold potential for HIE therapy.

Preliminary hemocompatibility assessment of an innovative material for blood contacting surfaces.

Over the years, several devices have been created (and the development of many others is currently in progress) to be in permanent contact with blood: mechanical circulatory supports represent an example thereof. The hemocompatibility of these devices largely depends on the chemical composition of blood-contacting components. In the present work, an innovative material (hybrid membrane) is proposed to fabricate the inner surfaces of a pulsatile ventricular chamber: it has been obtained by coupling a synthetic polymer (e.g., commercial polycarbonate urethane) with decellularized porcine pericardium. The hemocompatibility of the innovative material has been preliminarily assessed by measuring its capacity to promote thrombin generation and induce platelet activation. Our results demonstrated the blood compatibility of the proposed hybrid membrane.

Development of an Image-Based HCS-Compatible Method for Endothelial Barrier Function Assessment.

The recent renascence of phenotypic drug discovery (PDD) is catalyzed by its ability to identify first-in-class drugs and deliver results when the exact molecular mechanism is partially obscure. Acute respiratory distress syndrome (ARDS) is a severe, life-threatening condition with a high mortality rate that has increased in frequency due to the COVID-19 pandemic. Despite decades of laboratory and clinical study, no efficient pharmacological therapy for ARDS has been found. An increase in endothelial permeability is the primary event in ARDS onset, causing the development of pulmonary edema that leads to respiratory failure. Currently, the detailed molecular mechanisms regulating endothelial permeability are poorly understood. Therefore, the use of the PDD approach in the search for efficient ARDS treatment can be more productive than classic target-based drug discovery (TDD), but its use requires a new cell-based assay compatible with high-throughput (HTS) and high-content (HCS) screening. Here we report the development of a new plate-based image cytometry method to measure endothelial barrier function. The incorporation of image cytometry in combination with digital image analysis substantially decreases assay variability and increases the signal window. This new method simultaneously allows for rapid measurement of cell monolayer permeability and cytological analysis. The time-course of permeability increase in human pulmonary artery endothelial cells (HPAECs) in response to the thrombin and tumor necrosis factor α treatment correlates with previously published data obtained by transendothelial resistance (TER) measurements. Furthermore, the proposed image cytometry method can be easily adapted for HTS/HCS applications.

In vitro assessment and phase I randomized clinical trial of anfibatide a snake venom derived anti-thrombotic agent targeting human platelet GPIbα.

The interaction of platelet GPIbα with von Willebrand factor (VWF) is essential to initiate platelet adhesion and thrombosis, particularly under high shear stress conditions. However, no drug targeting GPIbα has been developed for clinical practice. Here we characterized anfibatide, a GPIbα antagonist purified from snake (Deinagkistrodon acutus) venom, and evaluated its interaction with GPIbα by surface plasmon resonance and in silico modeling. We demonstrated that anfibatide interferds with both VWF and thrombin binding, inhibited ristocetin/botrocetin- and low-dose thrombin-induced human platelet aggregation, and decreased thrombus volume and stability in blood flowing over collagen. In a single-center, randomized, and open-label phase I clinical trial, anfibatide was administered intravenously to 94 healthy volunteers either as a single dose bolus, or a bolus followed by a constant rate infusion of anfibatide for 24 h. Anfibatide inhibited VWF-mediated platelet aggregation without significantly altering bleeding time or coagulation. The inhibitory effects disappeared within 8 h after drug withdrawal. No thrombocytopenia or anti-anfibatide antibodies were detected, and no serious adverse events or allergic reactions were observed during the studies. Therefore, anfibatide was well-tolerated among healthy subjects. Interestingly, anfibatide exhibited pharmacologic effects in vivo at concentrations thousand-fold lower than in vitro, a phenomenon which deserves further investigation.Trial registration: Clinicaltrials.gov NCT01588132.

Use of population PK/PD approach to model the thrombin generation assay: assessment in haemophilia A plasma samples spiked by a TFPI antibody.

The thrombin generation (TG) assay is a well-established tool to capture the clotting potential of any healthy or haemophiliac subject. It measures ex vivo the kinetics of thrombin activation throughout the coagulation. Clinical studies allowed to create two databases gathering the coagulation factor levels and the thrombin generation profile of 40 healthy and 40 haemophiliac A (HA) subjects. Besides, portions of all HA samples were spiked with increasing levels of a TFPI antibody (considered as a possible therapeutic target) and corresponding TG profiles were determined. The non-linear mixed-effect (NLME) modelling aims at describing and explaining the experimentally observed important variability of the TG curves between subjects and the individual effects of spiking with a TFPI antibody. The models consist of an empirical description of the TG kinetics, accounting for an additive residual error and between-subject variability on its parameters. Factor VIII and TFPI were found to significantly explain and reduce the variability of the TG of haemophilia A samples. Besides, the model is shown to correctly reproduce the variability in the response to the ex vivo spiking with the TFPI antibody, by combining the empirical description of TG to a simple Hill equation that accounts for the binding between TFPI and different doses of its antibody. Such models can be useful for clinical practice, with the analysis and comparison of the distributions of TG profiles in healthy and haemophilia populations; and also for research, with the analysis of the effect of TFPI and its neutralization on individual TG profiles.

High-Dose Epinephrine Enhances Platelet Aggregation at the Expense of Procoagulant Activity.

Platelet activation is characterized by shape change, granule secretion, activation of fibrinogen receptor (glycoprotein IIb/IIIa) sustaining platelet aggregation, and externalization of negatively charged aminophospholipids contributing to platelet procoagulant activity. Epinephrine (EPI) alone is a weak platelet activator. However, it is able to potentiate platelet activation initiated by other agonists. In this work, we investigated the role of EPI in the generation of procoagulant platelets. Human platelets were activated with convulxin (CVX), thrombin (THR) or protease-activated receptor (PAR) agonists, EPI, and combination thereof. Platelet aggregation was assessed by light transmission aggregometry or with PAC-1 binding by flow cytometry. Procoagulant collagen-and-THR (COAT) platelets, induced by combined activation with CVX-and-THR, were visualized by flow cytometry as Annexin-V-positive and PAC-1-negative platelets. Cytosolic calcium fluxes were monitored by flow cytometry using Fluo-3 indicator. EPI increased platelet aggregation induced by all agonist combinations tested. On the other hand, EPI dose-dependently reduced the formation of procoagulant COAT platelets generated by combined CVX-and-THR activation. We observed a decreased Annexin-V-positivity and increased binding of PAC-1 with the triple activation (CVX + THR + EPI) compared with CVX + THR. Calcium mobilization with triple activation was decreased with the higher EPI dose (1,000 µM) compared with CVX + THR calcium kinetics. In conclusion, when platelets are activated with CVX-and-THR, the addition of increasing concentrations of EPI (triple stimulation) modulates platelet response reducing cytosolic calcium mobilization, decreasing procoagulant activity, and enhancing platelet aggregation.