PAR4 Inhibition Reduces Coronary Artery Atherosclerosis and Myocardial Fibrosis in SR-B1/LDLR Double Knockout Mice.

BACKGROUND: SR-B1 (scavenger receptor class B type 1)/LDLR (low-density lipoprotein receptor) double knockout mice fed a high-fat, high-cholesterol diet containing cholate exhibit coronary artery disease characterized by occlusive coronary artery atherosclerosis, platelet accumulation in coronary arteries, and myocardial fibrosis. Platelets are involved in atherosclerosis development, and PAR (protease-activated receptor) 4 has a prominent role in platelet function in mice. However, the role of PAR4 on coronary artery disease in mice has not been tested. METHODS: We tested the effects of a PAR4 inhibitory pepducin (RAG8) on diet-induced aortic sinus and coronary artery atherosclerosis, platelet accumulation in atherosclerotic coronary arteries, and myocardial fibrosis in SR-B1/LDLR double knockout mice. SR-B1/LDLR double knockout mice were fed a high-fat, high-cholesterol diet containing cholate and injected daily with 20 mg/kg of either the RAG8 pepducin or a control reverse-sequence pepducin (SRQ8) for 20 days. RESULTS: Platelets from the RAG8-treated mice exhibited reduced thrombin and PAR4 agonist peptide-mediated activation compared with those from control SRQ8-treated mice when tested ex vivo. Although aortic sinus atherosclerosis levels did not differ, RAG8-treated mice exhibited reduced coronary artery atherosclerosis, reduced platelet accumulation in atherosclerotic coronary arteries, and reduced myocardial fibrosis. These protective effects were not accompanied by changes in circulating lipids, inflammatory cytokines, or immune cells. However, RAG8-treated mice exhibited reduced VCAM-1 (vascular cell adhesion molecule 1) protein levels in nonatherosclerotic coronary artery cross sections and reduced leukocyte accumulation in atherosclerotic coronary artery cross sections compared with those from SRQ8-treated mice. CONCLUSIONS: The PAR4 inhibitory RAG8 pepducin reduced coronary artery atherosclerosis and myocardial fibrosis in SR-B1/LDLR double knockout mice fed a high-fat, high-cholesterol diet containing cholate. Furthermore, RAG8 reduced VCAM-1 in nonatherosclerotic coronary arteries and reduced leukocyte and platelet accumulation in atherosclerotic coronary arteries. These findings identify PAR4 as an attractive target in reducing coronary artery disease development, and the use of RAG8 may potentially be beneficial in cardiovascular disease.

Previous Ischemic Stroke Significantly Alters Stroke Risk in Newly Diagnosed Cancer Patients.

BACKGROUND: Previous ischemic stroke (IS) is a risk factor for subsequent IS in the general population; it is unclear if this relationship remains true in patients with cancer. Our objective was to examine the association between previous IS and risk for future IS in individuals newly diagnosed with cancer. METHODS: We conducted a retrospective population-based matched cohort study of newly diagnosed adult cancer patients (excluding nonmelanoma skin cancers and primary central nervous system tumors) in Ontario, Canada from 2010 to 2020; those with prior IS were matched (1:4) by age, sex, year of cancer diagnosis, cancer stage, and cancer site to those without a history of stroke. Cumulative incidence function curves were created to estimate the incidence of IS. Subdistribution adjusted hazard ratios (aHRs) and 95% CIs were calculated, where death was treated as a competing event. Multivariable analysis was adjusted for imbalanced baseline characteristics. RESULTS: We examined 65 525 individuals with cancer, including 13 070 with a history of IS. The median follow-up duration was 743 days (interquartile range, 177-1729 days). The incidence of IS following cancer diagnosis was 261.3/10 000 person-years in the cohort with prior IS and 75.3/10 000 person-years in those without prior IS. Individuals with prior IS had an increased risk for IS after cancer diagnosis compared with those without a history (aHR, 2.68 [95% CI, 2.41-2.98]); they also had more prevalent cardiovascular risk factors. The highest risk for stroke compared with those without a history of IS was observed in the gynecologic cancer (aHR, 3.84 [95% CI, 2.15-6.85]) and lung cancer (aHR, 3.18 [95% CI, 2.52-4.02]) subgroups. The risk of IS was inversely correlated with lag time of previous stroke; those with IS 1 year before their cancer diagnosis had the highest risk (aHR, 3.68 [95% CI, 3.22-4.22]). CONCLUSIONS: Among individuals with newly diagnosed cancer, those with IS history were almost 3× more likely to experience a stroke after cancer diagnosis, especially if the prediagnosis stroke occurred within 1 year preceding cancer diagnosis.

What can ex vivo experiments contribute to clinical haemostasis management?

A recent ex vivo study found that post-cardiopulmonary bypass platelet defects can be restored with supplemental fibrinogen, but the clinical significance of this finding will require further study. We propose that the best management strategy for achieving haemostasis in bleeding surgical patients is to identify individualised coagulation defects and then use a targeted therapeutic approach that addresses each identified defect systematically.

Apixaban to Prevent Venous Thromboembolism in Patients with Cancer.

BACKGROUND: Patients with active cancer have an increased risk of venous thromboembolism, which results in substantial morbidity, mortality, and health care expenditures. The Khorana score (range, 0 to 6, with higher scores indicating a higher risk of venous thromboembolism) has been validated to identify patients with cancer at elevated risk for this complication and may help select those who could benefit from thromboprophylaxis. METHODS: We conducted a randomized, placebo-controlled, double-blind clinical trial assessing the efficacy and safety of apixaban (2.5 mg twice daily) for thromboprophylaxis in ambulatory patients with cancer who were at intermediate-to-high risk for venous thromboembolism (Khorana score, ≥2) and were initiating chemotherapy. The primary efficacy outcome was objectively documented venous thromboembolism over a follow-up period of 180 days. The main safety outcome was a major bleeding episode. RESULTS: Of the 574 patients who underwent randomization, 563 were included in the modified intention-to-treat analysis. Venous thromboembolism occurred in 12 of 288 patients (4.2%) in the apixaban group and in 28 of 275 patients (10.2%) in the placebo group (hazard ratio, 0.41; 95% confidence interval [CI], 0.26 to 0.65; P<0.001). In the modified intention-to-treat analysis, major bleeding occurred in 10 patients (3.5%) in the apixaban group and in 5 patients (1.8%) in the placebo group (hazard ratio, 2.00; 95% CI, 1.01 to 3.95; P = 0.046). During the treatment period, major bleeding occurred in 6 patients (2.1%) in the apixaban group and in 3 patients (1.1%) in the placebo group (hazard ratio, 1.89; 95% CI, 0.39 to 9.24). CONCLUSIONS: Apixaban therapy resulted in a significantly lower rate of venous thromboembolism than did placebo among intermediate-to-high-risk ambulatory patients with cancer who were starting chemotherapy. The rate of major bleeding episodes was higher with apixaban than with placebo. (Funded by the Canadian Institutes of Health Research and Bristol-Myers Squibb-Pfizer Alliance; AVERT ClinicalTrials.gov number, NCT02048865.).

Adherence to apixaban for extended thromboprophylaxis after major abdominal or pelvic surgery for cancer: A prospective cohort study.

BACKGROUND: Due to lack of data, direct oral anticoagulants are not considered by guidelines for venous thromboembolism (VTE) prophylaxis after cancer surgery. Adherence to low-molecular-weight heparin injections in this setting is sometimes poor. AIM: Analysis of adherence to oral apixaban for extended thromboprophylaxis. METHODS: Consecutive patients discharged after major surgery for abdominal/pelvic cancer and considered eligible for extended prophylaxis were offered apixaban 2.5 mg twice daily. Primary outcomes were adherence metrics-proportion of prescriptions filled, persistence (not prematurely discontinued), proportion of days covered (PDC) based on apixaban pill counts, and modified Morisky medication adherence scale at Days 28-30. Secondary outcomes were bleeding, VTE, and serious adverse events until Day 90. RESULTS: We included 53 patients, 51 were analyzed. Of 45 patients with prescriptions all had it filled (95% confidence interval [CI], 92%-100%). Persistence was 98% (95% CI, 90%-100%). PDC was ≥80% for 48 patients (94%; 95% CI, 84%-99%). We found good adherence (0/6 answers "yes") in 75% and moderate (1/6 answers "yes") in 25%. No major bleed or VTE occurred while on apixaban. CONCLUSION: Our results support good adherence with apixaban for VTE prophylaxis up to 28 days after major abdominal or pelvic cancer surgery.

The association of thrombin generation with bleeding outcomes in cardiac surgery: a prospective observational study.

BACKGROUND: Cardiac surgery with cardiopulmonary bypass (CPB) is associated with coagulopathic bleeding. Impaired thrombin generation may be an important cause of coagulopathic bleeding but is poorly measured by existing hemostatic assays. We examined thrombin generation during cardiac surgery, using calibrated automated thrombography, and its association with bleeding outcomes. METHODS: We conducted a prospective observational study in 100 patients undergoing cardiac surgery with CPB. Calibrated automated thrombography parameters were expressed as a ratio of post-CPB values divided by pre-CPB values. The association of thrombin generation parameters for bleeding outcomes was compared with conventional tests of hemostasis, and the outcomes of patients with the most severe post-CPB impairment in thrombin generation (≥ 80% drop from baseline) were compared with the rest of the cohort. RESULTS: All 100 patients were included in the final analysis, with a mean age of 63 (12) yr, 31 (31%) female, and 94 (94%) undergoing bypass and/or valve surgery. Post-CPB, peak thrombin decreased by a median of 73% (interquartile range [IQR], 49-91%) (P < 0.001) and total thrombin generation, expressed as the endogenous thrombin potential (ETP), decreased 56% [IQR, 30-83%] (P < 0.001). In patients with ≥ 80% decrease in ETP, 21% required re-exploration for bleeding compared with 7% in the rest of the cohort (P = 0.04), and 48% required medical or surgical treatment for hemostasis compared with 27% in the rest of the cohort (P = 0.04). CONCLUSIONS: Thrombin generation is significantly impaired by CPB and associated with higher bleeding severity. Clinical studies aimed at the identification and treatment of patients with impaired thrombin generation are warranted.

RéSUMé: CONTEXTE: La chirurgie cardiaque avec circulation extracorporelle (CEC) est associée à des saignements sur coagulopathie. L'altération de la génération de thrombine peut constituer une cause importante de saignement sur coagulopathie, mais elle est mal mesurée par les tests d'hémostase existants. Nous avons examiné la génération de thrombine pendant la chirurgie cardiaque à l'aide d'une thrombographie automatisée calibrée ainsi que son association avec les issues hémorragiques. MéTHODE: Nous avons réalisé une étude observationnelle prospective portant sur 100 patients bénéficiant d'une chirurgie cardiaque sous CEC. Les paramètres de thrombographie automatisée calibrée ont été exprimés sous forme du rapport entre les valeurs post-CEC divisées par les valeurs pré-CEC. L'association des paramètres de génération de thrombine pour les issues hémorragiques a été comparée aux tests conventionnels de l'hémostase, et les issues des patients présentant l'altération post-CEC la plus prononcée dans la génération de thrombine (baisse ≥ 80 % par rapport aux valeurs de base) ont été comparées au reste de la cohorte. RéSULTATS: Les 100 patients ont tous été inclus dans l'analyse finale, avec un âge moyen de 63 (12) ans, 31 (31 %) femmes et 94 (94 %) subissant une chirurgie de pontage et / ou une chirurgie valvulaire. Après la CEC, le pic de thrombine a diminué d'une médiane de 73 % (écart interquartile [ÉIQ], 49 à 91 %) (P < 0,001) et la génération de thrombine totale, exprimée en potentiel de thrombine endogène (PTE), a diminué de 56 % [ÉIQ, 30­83 %] (P < 0,001). Chez les patients présentant une diminution ≥ 80 % du PTE, 21 % ont nécessité une nouvelle exploration pour dépister les saignements, comparativement à 7 % dans le reste de la cohorte (P = 0,04), et 48 % ont nécessité un traitement médical ou chirurgical pour l'hémostase, comparativement à 27 % dans le reste de la cohorte (P = 0,04). CONCLUSION: La génération de thrombine est significativement altérée par la CEC et associée à des saignements plus graves. Des études cliniques visant à identifier et à traiter les patients présentant une altération de la génération de thrombine sont recommandées.

Anti-Thrombotic Therapy to Ameliorate Complications of COVID-19 (ATTACC): Study design and methodology for an international, adaptive Bayesian randomized controlled trial.

BACKGROUND: Mortality from COVID-19 is high among hospitalized patients and effective therapeutics are lacking. Hypercoagulability, thrombosis and hyperinflammation occur in COVID-19 and may contribute to severe complications. Therapeutic anticoagulation may improve clinical outcomes through anti-thrombotic, anti-inflammatory and anti-viral mechanisms. Our primary objective is to evaluate whether therapeutic-dose anticoagulation with low-molecular-weight heparin or unfractionated heparin prevents mechanical ventilation and/or death in patients hospitalized with COVID-19 compared to usual care. METHODS: An international, open-label, adaptive randomized controlled trial. Using a Bayesian framework, the trial will declare results as soon as pre-specified posterior probabilities for superiority, futility, or harm are reached. The trial uses response-adaptive randomization to maximize the probability that patients will receive the more beneficial treatment approach, as treatment effect information accumulates within the trial. By leveraging a common data safety monitoring board and pooling data with a second similar international Bayesian adaptive trial (REMAP-COVID anticoagulation domain), treatment efficacy and safety will be evaluated as efficiently as possible. The primary outcome is an ordinal endpoint with three possible outcomes based on the worst status of each patient through day 30: no requirement for invasive mechanical ventilation, invasive mechanical ventilation or death. CONCLUSION: Using an adaptive trial design, the Anti-Thrombotic Therapy To Ameliorate Complications of COVID-19 trial will establish whether therapeutic anticoagulation can reduce mortality and/or avoid the need for mechanical ventilation in patients hospitalized with COVID-19. Leveraging existing networks to recruit sites will increase enrollment and mitigate enrollment risk in sites with declining COVID-19 cases.

Thrombin generation and bleeding in cardiac surgery: a clinical narrative review.

This narrative review discusses the role of thrombin generation in coagulation and bleeding in cardiac surgery, the laboratory methods for clinical detection of impaired thrombin generation, and the available hemostatic interventions that can be used to improve thrombin generation. Coagulopathy after cardiopulmonary bypass (CPB) is associated with excessive blood loss and adverse patient outcomes. Thrombin plays a crucial role in primary hemostasis, and impaired thrombin generation can be an important cause of post-CPB coagulopathy. Existing coagulation assays have significant limitations in assessing thrombin generation, but whole-blood assays designed to measure thrombin generation at the bed-side are under development. Until then, clinicians may need to institute therapy empirically for non-surgical bleeding in the setting of normal coagulation measures. Available therapies for impaired thrombin generation include administration of plasma, prothrombin complex concentrate, and bypassing agents (recombinant activated factor VII and factor eight inhibitor bypassing activity). In vitro experiments have explored the relative potency of these therapies, but clinical studies are lacking. The potential incorporation of thrombin generation assays into clinical practice and treatment algorithms for impaired thrombin generation must await further clinical development.

RéSUMé: Ce compte rendu narratif discute du rôle de la génération de thrombine dans la coagulation et le saignement en chirurgie cardiaque, des méthodes de laboratoire pour le dépistage clinique d'une génération de thrombine altérée et des interventions hémostatiques disponibles qui peuvent être utilisées pour améliorer la génération de thrombine. Une coagulopathie après la circulation extracorporelle (CEC) est associée à des pertes de sang excessives et à des complications pour les patients. La thrombine joue un rôle essentiel d'hémostase primaire, et une génération de thrombine altérée peut constituer une cause importante de coagulopathie post-CEC. Les analyses de coagulation existantes comportent d'importantes limites en ce qui touche à l'évaluation de la génération de thrombine, mais des analyses de sang complet conçues pour mesurer la génération de thrombine au chevet sont en cours d'élaboration. En attendant, les cliniciens pourraient devoir amorcer un traitement de manière empirique pour prendre en charge les saignements non chirurgicaux dans un contexte de valeurs de coagulation mesurées normales. Les traitements disponibles pour une génération de thrombine altérée comprennent l'administration de plasma, de concentrés de complexe prothrombinique, et d'agents de contournement (bypass) (facteur VII recombinant activé et activité de contournement de l'inhibiteur du facteur VIII). Des expériences in vitro ont exploré l'activité thérapeutique relative de ces traitements, mais les études cliniques manquent. L'intégration potentielle d'analyses de génération de thrombine dans la pratique clinique et d'algorithmes de traitement pour une génération de thrombine altérée doit attendre des développements cliniques plus poussés.

Emerging anticoagulant strategies.

Despite the introduction of direct oral anticoagulants (DOACs), the search for more effective and safer antithrombotic strategies continues. Better understanding of the pathogenesis of thrombosis has fostered 2 new approaches to achieving this goal. First, evidence that thrombin may be as important as platelets to thrombosis at sites of arterial injury and that platelets contribute to venous thrombosis has prompted trials comparing anticoagulants with aspirin for secondary prevention in arterial thrombosis and aspirin with anticoagulants for primary and secondary prevention of venous thrombosis. These studies will help identify novel treatment strategies. Second, emerging data that naturally occurring polyphosphates activate the contact system and that this system is critical for thrombus stabilization and growth have identified factor XII (FXII) and FXI as targets for new anticoagulants that may be even safer than the DOACs. Studies are needed to determine whether FXI or FXII is the better target and to compare the efficacy and safety of these new strategies with current standards of care for the prevention or treatment of thrombosis. Focusing on these advances, this article outlines how treatment strategies for thrombosis are evolving and describes the rationale and approaches to targeting FXII and FXI. These emerging anticoagulant strategies should address unmet needs and reduce the systemic underuse of anticoagulation because of the fear of bleeding.

Effect of Different Doses of Acetylsalicylic Acid on the Antithrombotic Activity of Clopidogrel in a Mouse Arterial Thrombosis Model.

Objective- Dual-antiplatelet therapy with acetylsalicylic acid and a P2Y12 antagonist, such as clopidogrel, is the standard of care for acute coronary syndromes. However, the drugs have divergent effects on the formation of cAMP, an inhibitory second messenger. Thus, by inhibiting the synthesis of prostacyclin, acetylsalicylic acid reduces cAMP formation, whereas clopidogrel potentiates it. Therefore, with higher doses of acetylsalicylic acid, the potentiation of cAMP production by clopidogrel may be attenuated, which could limit the antithrombotic potential of the drug combination. The purpose of this study was to examine this possibility in vivo. Approach and Results- Mice were given oral acetylsalicylic acid at varying doses, oral clopidogrel (5 mg/kg body weight), or both. At doses of 0.15 and 0.6 mg/kg, acetylsalicylic acid inhibited arachidonic acid-induced platelet aggregation, but only 0.6 mg/kg acetylsalicylic acid, or higher, decreased the plasma levels of 6-keto-prostaglandin-F1α, the stable metabolite of prostacyclin. When given with clopidogrel, laser injury-induced arterial thrombi were significantly larger with the 0.6 mg/kg dose of acetylsalicylic acid than with the 0.15 mg/kg dose. Thrombi in mice treated with clopidogrel and the 0.15 mg/kg dose of acetylsalicylic acid were smaller than in mice treated with clopidogrel alone, suggesting that acetylsalicylic acid can add to the antithrombotic effect of clopidogrel but that higher doses of acetylsalicylic acid blunt the antithrombotic effect of clopidogrel. Conclusions- These findings support the use of lower, prostacyclin-preserving, doses of acetylsalicylic acid in conjunction with clopidogrel.

Rosuvastatin Reduces Aortic Sinus and Coronary Artery Atherosclerosis in SR-B1 (Scavenger Receptor Class B Type 1)/ApoE (Apolipoprotein E) Double Knockout Mice Independently of Plasma Cholesterol Lowering.

OBJECTIVE: Rosuvastatin has been widely used in the primary and secondary prevention of coronary heart disease. However, its antiatherosclerotic properties have not been tested in a mouse model that could mimic human coronary heart disease. The present study was designed to test the effects of rosuvastatin on coronary artery atherosclerosis and myocardial fibrosis in SR-B1 (scavenger receptor class B type 1) and apoE (apolipoprotein E) double knockout mice. APPROACH AND RESULTS: Three-week-old SR-B1-/-/apoE-/- mice were injected daily with 10 mg/kg of rosuvastatin for 2 weeks. Compared with saline-treated mice, rosuvastatin-treated mice showed increased levels of hepatic PCSK9 (proprotein convertase subtilisin/kexin type-9) and LDLR (low-density lipoprotein receptor) message, increased plasma PCSK9 protein but decreased levels of hepatic LDLR protein and increased plasma total cholesterol associated with apoB (apolipoprotein B) 48-containing lipoproteins. In spite of this, rosuvastatin treatment was associated with decreased atherosclerosis in both the aortic sinus and coronary arteries and reduced platelet accumulation in atherosclerotic coronary arteries. Cardiac fibrosis and cardiomegaly were also attenuated in rosuvastatin-treated SR-B1-/-/apoE-/- mice. Two-week treatment with rosuvastatin resulted in significant decreases in markers of oxidized phospholipids in atherosclerotic plaques. In vitro analysis showed that incubation of bone marrow-derived macrophages with rosuvastatin substantially downregulated cluster of differentiation (CD)36 and inhibited oxidized LDL-induced foam cell formation. CONCLUSIONS: Rosuvastatin protected SR-B1-/-/apoE-/- mice against atherosclerosis and platelet accumulation in coronary arteries and attenuated myocardial fibrosis and cardiomegaly, despite increased plasma total cholesterol. The ability of rosuvastatin to reduce oxidized phospholipids in atherosclerotic plaques and inhibit macrophage foam cell formation may have contributed to this protection.

Animal Models of Thrombosis From Zebrafish to Nonhuman Primates: Use in the Elucidation of New Pathologic Pathways and the Development of Antithrombotic Drugs.

Thrombosis is a leading cause of morbidity and mortality worldwide. Animal models are used to understand the pathological pathways involved in thrombosis and to test the efficacy and safety of new antithrombotic drugs. In this review, we will first describe the central role a variety of animal models of thrombosis and hemostasis has played in the development of new antiplatelet and anticoagulant drugs. These include the widely used P2Y12 antagonists and the recently developed orally available anticoagulants that directly target factor Xa or thrombin. Next, we will describe the new players, such as polyphosphate, neutrophil extracellular traps, and microparticles, which have been shown to contribute to thrombosis in mouse models, particularly venous thrombosis models. Other mouse studies have demonstrated roles for the factor XIIa and factor XIa in thrombosis. This has spurred the development of strategies to reduce their levels or activities as a new approach for preventing thrombosis. Finally, we will discuss the emergence of zebrafish as a model to study thrombosis and its potential use in the discovery of novel factors involved in thrombosis and hemostasis. Animal models of thrombosis from zebrafish to nonhuman primates are vital in identifying pathological pathways of thrombosis that can be safely targeted with a minimal effect on hemostasis. Future studies should focus on understanding the different triggers of thrombosis and the best drugs to prevent each type of thrombotic event.

Arterial thrombosis is accelerated in mice deficient in histidine-rich glycoprotein.

Factor (F) XII, a key component of the contact system, triggers clotting via the intrinsic pathway, and is implicated in propagating thrombosis. Although nucleic acids are potent activators, it is unclear how the contact system is regulated to prevent uncontrolled clotting. Previously, we showed that histidine-rich glycoprotein (HRG) binds FXIIa and attenuates its capacity to trigger coagulation. To investigate the role of HRG as a regulator of the intrinsic pathway, we compared RNA- and DNA-induced thrombin generation in plasma from HRG-deficient and wild-type mice. Thrombin generation was enhanced in plasma from HRG-deficient mice, and accelerated clotting was restored to normal with HRG reconstitution. Although blood loss after tail tip amputation was similar in HRG-deficient and wild-type mice, carotid artery occlusion after FeCl3 injury was accelerated in HRG-deficient mice, and HRG administration abrogated this effect. To confirm that HRG modulates the contact system, we used DNase, RNase, and antisense oligonucleotides to characterize the FeCl3 model. Whereas DNase or FVII knockdown had no effect, carotid occlusion was abrogated with RNase or FXII knockdown, confirming that FeCl3-induced thrombosis is triggered by RNA in a FXII-dependent fashion. Therefore, in a nucleic acid-driven model, HRG inhibits thrombosis by modulating the intrinsic pathway of coagulation.

Evolving use of new oral anticoagulants for treatment of venous thromboembolism.

The new oral anticoagulants (NOACs), which include dabigatran, rivaroxaban, apixaban, and edoxaban, are poised to replace warfarin for treatment of the majority of patients with venous thromboembolism (VTE). With a rapid onset of action and the capacity to be administered in fixed doses without routine coagulation monitoring, NOACs streamline VTE treatment. In phase 3 trials in patients with acute symptomatic VTE, NOACs have been shown to be noninferior to conventional anticoagulant therapy for prevention of recurrence and are associated with less bleeding. Rivaroxaban and dabigatran are already licensed for VTE treatment in the United States, and apixaban and edoxaban are under regulatory consideration for this indication. As the number of approved drugs increases, clinicians will need to choose the right anticoagulant for the right VTE patient. To help with this decision, this review (1) compares the pharmacologic profiles of the NOACs, (2) outlines the unique design features of the phase 3 trials that evaluated the NOACs for VTE treatment, (3) reviews the results of these trials highlighting similarities and differences in the findings, (4) provides perspective about which VTE patients should receive conventional treatment or are candidates for NOACs, and (5) offers suggestions about how to choose among the NOACs.

Abnormal megakaryocyte development and platelet function in Nbeal2(-/-) mice.

Gray platelet syndrome (GPS) is an inherited bleeding disorder associated with macrothrombocytopenia and α-granule-deficient platelets. GPS has been linked to loss of function mutations in NEABL2 (neurobeachin-like 2), and we describe here a murine GPS model, the Nbeal2(-/-) mouse. As in GPS, Nbeal2(-/-) mice exhibit splenomegaly, macrothrombocytopenia, and a deficiency of platelet α-granules and their cargo, including von Willebrand factor (VWF), thrombospondin-1, and platelet factor 4. The platelet α-granule membrane protein P-selectin is expressed at 48% of wild-type levels and externalized upon platelet activation. The presence of P-selectin and normal levels of VPS33B and VPS16B in Nbeal2(-/-) platelets suggests that NBEAL2 acts independently of VPS33B/VPS16B at a later stage of α-granule biogenesis. Impaired Nbeal2(-/-) platelet function was shown by flow cytometry, platelet aggregometry, bleeding assays, and intravital imaging of laser-induced arterial thrombus formation. Microscopic analysis detected marked abnormalities in Nbeal2(-/-) bone marrow megakaryocytes, which when cultured showed delayed maturation, decreased survival, decreased ploidy, and developmental abnormalities, including abnormal extracellular distribution of VWF. Our results confirm that α-granule secretion plays a significant role in platelet function, and they also indicate that abnormal α-granule formation in Nbeal2(-/-) mice has deleterious effects on megakaryocyte survival, development, and platelet production.

A specific fluorescence resonance energy quenching-based biosensor for measuring thrombin activity in whole blood.

BACKGROUND: At sites of vessel injury, thrombin acts as the central mediator of coagulation by catalyzing fibrin clot formation and platelet activation. Thrombin generation is most frequently measured in plasma samples using small-molecule substrates; however, these have low specificity for thrombin and limited utility in whole blood. Plasma assays are limited because they ignore the hemostatic contributions of blood cells and require anticoagulation and the addition of supraphysiological concentrations of calcium. OBJECTIVES: To overcome these limitations, we designed and characterized a fluorescence resonance energy quenching-based thrombin sensor (FTS) protein. METHODS: The fluorescence resonance energy quenching pair of mAmetrine and tTomato, separated by a thrombin recognition sequence, was developed. The protein was expressed using Escherichia coli, and purity was assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The cleavage of FTS was monitored by fluorescence using excitation at 406 nm and emission at 526 nm and 581 nm. RESULTS: Compared with small-molecule substrates, the FTS demonstrated high specificity for thrombin; it is not cleaved by thrombin or inhibited by α2-macroglobulin and interacts with thrombin's anion-binding exosite I. The FTS can effectively measure thrombin generation in plasma and in finger-prick whole blood, which allows it to be developed into a point-of-care test of thrombin generation. The FTS does not inhibit standard thrombin-generation assays. Lastly, FTS-based thrombin generation in nonanticoagulated finger-prick blood was delayed but enhanced compared with that in citrated plasma. CONCLUSION: The FTS will broaden our understanding of thrombin generation in ways that are not attainable with current methods.