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.

CYK-1/Formin activation in cortical RhoA signaling centers promotes organismal left-right symmetry breaking.

Proper left-right symmetry breaking is essential for animal development, and in many cases, this process is actomyosin-dependent. In Caenorhabditis elegans embryos active torque generation in the actomyosin layer promotes left-right symmetry breaking by driving chiral counterrotating cortical flows. While both Formins and Myosins have been implicated in left-right symmetry breaking and both can rotate actin filaments in vitro, it remains unclear whether active torques in the actomyosin cortex are generated by Formins, Myosins, or both. We combined the strength of C. elegans genetics with quantitative imaging and thin film, chiral active fluid theory to show that, while Non-Muscle Myosin II activity drives cortical actomyosin flows, it is permissive for chiral counterrotation and dispensable for chiral symmetry breaking of cortical flows. Instead, we find that CYK-1/Formin activation in RhoA foci is instructive for chiral counterrotation and promotes in-plane, active torque generation in the actomyosin cortex. Notably, we observe that artificially generated large active RhoA patches undergo rotations with consistent handedness in a CYK-1/Formin-dependent manner. Altogether, we conclude that CYK-1/Formin-dependent active torque generation facilitates chiral symmetry breaking of actomyosin flows and drives organismal left-right symmetry breaking in the nematode worm.

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.

Pineapple Lectin AcmJRL Binds SARS-CoV-2 Spike Protein in a Carbohydrate-Dependent Fashion.

The highly glycosylated spike protein of SARS-CoV-2 is essential for infection and constitutes a prime target for antiviral agents and vaccines. The pineapple-derived jacalin-related lectin AcmJRL is present in the medication bromelain in significant quantities and has previously been described to bind mannosides. Here, we performed a large ligand screening of AcmJRL by glycan array analysis, quantified the interaction with carbohydrates and validated high-mannose glycans as preferred ligands. Because the SARS-CoV-2 spike protein was previously reported to carry a high proportion of high-mannose N-glycans, we tested the binding of AcmJRL to the recombinantly produced extraviral domain of spike protein. We could demonstrate that AcmJRL binds the spike protein with a low-micromolar KD in a carbohydrate-dependent fashion.

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.

Aspirin or Rivaroxaban for VTE Prophylaxis after Hip or Knee Arthroplasty.

BACKGROUND: Clinical trials and meta-analyses have suggested that aspirin may be effective for the prevention of venous thromboembolism (proximal deep-vein thrombosis or pulmonary embolism) after total hip or total knee arthroplasty, but comparisons with direct oral anticoagulants are lacking for prophylaxis beyond hospital discharge. METHODS: We performed a multicenter, double-blind, randomized, controlled trial involving patients who were undergoing total hip or knee arthroplasty. All the patients received once-daily oral rivaroxaban (10 mg) until postoperative day 5 and then were randomly assigned to continue rivaroxaban or switch to aspirin (81 mg daily) for an additional 9 days after total knee arthroplasty or for 30 days after total hip arthroplasty. Patients were followed for 90 days for symptomatic venous thromboembolism (the primary effectiveness outcome) and bleeding complications, including major or clinically relevant nonmajor bleeding (the primary safety outcome). RESULTS: A total of 3424 patients (1804 undergoing total hip arthroplasty and 1620 undergoing total knee arthroplasty) were enrolled in the trial. Venous thromboembolism occurred in 11 of 1707 patients (0.64%) in the aspirin group and in 12 of 1717 patients (0.70%) in the rivaroxaban group (difference, 0.06 percentage points; 95% confidence interval [CI], -0.55 to 0.66; P<0.001 for noninferiority and P=0.84 for superiority). Major bleeding complications occurred in 8 patients (0.47%) in the aspirin group and in 5 (0.29%) in the rivaroxaban group (difference, 0.18 percentage points; 95% CI, -0.65 to 0.29; P=0.42). Clinically important bleeding occurred in 22 patients (1.29%) in the aspirin group and in 17 (0.99%) in the rivaroxaban group (difference, 0.30 percentage points; 95% CI, -1.07 to 0.47; P=0.43). CONCLUSIONS: Among patients who received 5 days of rivaroxaban prophylaxis after total hip or total knee arthroplasty, extended prophylaxis with aspirin was not significantly different from rivaroxaban in the prevention of symptomatic venous thromboembolism. (Funded by the Canadian Institutes of Health Research; ClinicalTrials.gov number, NCT01720108 .).

Biuret-A Crucial Reaction Intermediate for Understanding Urea Pyrolysis To Form Carbon Nitrides: Crystal-Structure Elucidation and In Situ Diffractometric, Vibrational and Thermal Characterisation.

The crystal structure of biuret was elucidated by means of XRD analysis of single crystals grown through slow evaporation from a solution in ethanol. It crystallises in its own structure type in space group C2/c (a=15.4135(8) Å, b=6.6042(3) Å, c=9.3055(4) Å, Z=8). Biuret decomposition was studied in situ by means of temperature-programmed powder XRD and FTIR spectroscopy, to identify a co-crystalline biuret-cyanuric acid phase as a previously unrecognised reaction intermediate. Extensive thermogravimetric studies of varying crucible geometry, heating rate and initial sample mass reveal that the concentration of reactive gases at the interface to the condensed sample residues is a crucial parameter for the prevailing decomposition pathway. Taking these findings into consideration, a study on the optimisation of carbon nitride synthesis from urea on the gram scale, with standard solid-state laboratory techniques, is presented. Finally, a serendipitously encountered self-coating of the crucible inner walls by graphite during repeated synthetic cycles, which prove to be highly beneficial for the obtained yields, is reported.

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.

Con: Tolvaptan for autosomal dominant polycystic kidney disease-do we know all the answers?

According to recent literature, tolvaptan ameliorates the natural decline of renal function in autosomal dominant polycystic kidney disease. Tolvaptan is an orally available vasopressin V2 receptor antagonist. We describe herein the remaining questions and problems: it is unclear from the published work what influence tolvaptan has on total kidney volume. The consequences of hepatotoxicity for the subsequent dosing of tolvaptan have not been reported. A vasopressin V2 antagonist will cause polyuria and polydipsia and tolvaptan may influence quality of life (QOL), however, there are no QOL data. The cost-effectiveness of tolvaptan is borderline. It is unknown at which stage of renal failure tolvaptan therapy may have to be stopped. There are no established criteria to determine the ineffectiveness of tolvaptan. It is presently undecided whether a steady high water intake is able to imitate the renal effects of tolvaptan. Finally, the cause of worsening glomerular filtration rate after the start of tolvaptan is unknown.

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.

Synthesis and Characterization of the First Borosulfates of Magnesium, Manganese, Cobalt, Nickel, and Zinc.

The first magnesium, manganese, cobalt, nickel, and zinc borosulfates were synthesized employing solvothermal conditions starting from the superacid H[B(HSO4)4] and the respective metal powders (Mg, Ni, Zn) or oxides (MnO2, CoO). α- M4[B2O(SO4)6] ( M = Mg, Mn, Co, Ni, Zn) crystallize isotypically in a new structure type in P3̅ (No. 147) with Z = 1, a = 793.59(4)-810.86(9) pm, and c = 743.98(4)-775.09(9) pm. The oligomeric anion comprises unprecedented dimeric open-branched quadruple tetrahedra { oB, 4 t}[B2O(SO4)6]8-, which are connected via M2O9 dimers to give a three-dimensional network. Upon mild heating, we observed a phase change from α-Mg4[B2O(SO4)6] to ß-Mg4[B2O(SO4)6], yielding a further new structure type in P3̅ (No. 147) with Z = 3, a = 1391.96(6) pm, and c = 748.54(3) pm. The reaction of MgB2 with SO3 yields Mg[B2(SO4)4] crystallizing in C2/ c with Z = 4, a = 1744.28(10) pm, b = 531.45(3) pm, c = 1429.06(8) pm, and ß = 126.323(2)° showing phyllosilicate topology. UV/vis spectroscopy on α- TM4[B2O(SO4)6] ( TM = Co, Ni) confirms the valence state of the TM and reveals that borosulfates are weakly coordinating host structures. Structure relationships between the presented crystal structures and similar borophosphates are shown. The results of vibrational spectroscopy as well as magnetic and thermal measurement investigations are discussed.

Switching between Exonucleolysis and Replication by T7 DNA Polymerase Ensures High Fidelity.

DNA polymerase catalyzes the accurate transfer of genetic information from one generation to the next, and thus it is vitally important for replication to be faithful. DNA polymerase fulfills the strict requirements for fidelity by a combination of mechanisms: 1) high selectivity for correct nucleotide incorporation, 2) a slowing down of the replication rate after misincorporation, and 3) proofreading by excision of misincorporated bases. To elucidate the kinetic interplay between replication and proofreading, we used high-resolution optical tweezers to probe how DNA-duplex stability affects replication by bacteriophage T7 DNA polymerase. Our data show highly irregular replication dynamics, with frequent pauses and direction reversals as the polymerase cycles through the states that govern the mechanochemistry behind high-fidelity T7 DNA replication. We constructed a kinetic model that incorporates both existing biochemical data and the, to our knowledge, novel states we observed. We fit the model directly to the acquired pause-time and run-time distributions. Our findings indicate that the main pathway for error correction is DNA polymerase dissociation-mediated DNA transfer, followed by biased binding into the exonuclease active site. The number of bases removed by this proofreading mechanism is much larger than the number of erroneous bases that would be expected to be incorporated, ensuring a high-fidelity replication of the bacteriophage T7 genome.