Absence of thrombospondin-2 causes age-related dilated cardiomyopathy

BACKGROUND: The progressive shift from a young to an aged heart is characterized by alterations in the cardiac matrix. The present study investigated whether the matricellular protein thrombospondin-2 (TSP-2) may affect cardiac dimensions and function with physiological aging of the heart. METHODS AND RESULTS: TSP-2 knockout (KO) and wild-type mice were followed up to an age of 60 weeks. Survival rate, cardiac function, and morphology did not differ at a young age in TSP-2 KO compared with wild-type mice. However, >55% of the TSP-2 KO mice died between 24 and 60 weeks of age, whereas <10% of the wild-type mice died. In the absence of TSP-2, older mice displayed a severe dilated cardiomyopathy with impaired systolic function, increased cardiac dilatation, and fibrosis. Ultrastructural analysis revealed progressive myocyte stress and death, accompanied by an inflammatory response and replacement fibrosis, in aging TSP-2 KO animals, whereas capillary or coronary morphology or density was not affected. Importantly, adeno-associated virus-9 gene-mediated transfer of TSP-2 in 7-week-old TSP-2 KO mice normalized their survival and prevented dilated cardiomyopathy. In TSP-2 KO animals, age-related cardiomyopathy was accompanied by increased matrix metalloproteinase-2 and decreased tissue transglutaminase-2 activity, together with impaired collagen cross-linking. At the cardiomyocyte level, TSP-2 deficiency in vivo and its knockdown in vitro decreased the activation of the Akt survival pathway in cardiomyocytes. CONCLUSIONS: TSP-2 expression in the heart protects against age-dependent dilated cardiomyopath

Two novel mutations in the prothrombin gene identified in a patient with compound heterozygous type 1/2 prothrombin deficiency

Thrombosis and Hemostasi

Two novel mutations in the prothrombin gene identified in a patient with compound heterozygous type 1/2 prothrombin deficiency

Thrombosis and Hemostasi

Determination of dabigatran and rivaroxaban by ultra-performance liquid chromatography-tandem mass spectrometry and coagulation assays after major orthopaedic surgery

Major orthopaedic surgery is associated with an increased risk of venous thromboembolism. Direct oral anticoagulants (DOACs) are recommended as thromboprophylactic agents after orthopaedic surgery. Although routine monitoring of DOACs in general is not required, measuring DOAC concentration may be necessary in clinical settings. The effects of DOACs on routine coagulation assays in spiked material are studied extensively, however, few data are available on DOAC concentration in patients after major orthopaedic surgery. We measured trough and peak DOAC concentrations with UPLC-MS/MS and routine coagulation tests in a prospective study including 40 patients receiving thromboprophylactic treatment with dabigatran 220 mg od and 40 patients receiving rivaroxaban 10 mg od after major orthopaedic surgery. For rivaroxaban, the median trough concentration with UPLC-MS/MS was 17.1 ng/mL and median peak concentration was 149 ng/mL. The anti-Xa assay displayed a good correlation, but a positive bias in comparison to the reference method. Furthermore, trough levels were mostly below the LOD of the anti-Xa assay. For dabigatran, the median trough concentration with UPLC-MS/MS was 12.1 ng/mL, and median peak level was 80.8 ng/mL. A positive bias was found when results from coagulation assays were compared to UPLC-MS/MS data. However, the addition of glucuronidated metabolites to dabigatran concentration UPLC-MS/MS data generally resolved most of this bias. Age was found to have a significant influence on dabigatran pharmacokinetics, irrespective of kidney function, whereas no effect of age was found during rivaroxaban treatment. In both treatment groups, female subjects displayed faster pharmacokinetics in comparison to male subjects, although not reaching significance. We conclude that UPLC-MS/MS is the method of choice to measure trough concentrations of DOACs in patients after orthopaedic surgery. Current coagulation assays are not suited for this purpose. We found large heterogeneity in both peak and trough concentrations of DOACs, and showed that pharmacokinetics of novel oral anticoagulants may be influenced by age and gender. Whether patients with high or low trough concentrations are at increased risk for bleeding or thromboembolic events respectively remains to be established

Determination of dabigatran, rivaroxaban and apixaban by ultra-performance liquid chromatography - tandem mass spectrometry (UPLC-MS/MS) and coagulation assays for therapy monitoring of novel direct oral anticoagulants

Background: Three novel direct oral anticoagulants (DOACs) have recently been registered by the Food and Drug Administration and European Medicines Agency Commission: dabigatran, rivaroxaban, and apixaban. To quantify DOACs in plasma, various dedicated coagulation assays have been developed. Objective: To develop and validate a reference ultra-performance liquid chromatography - tandem mass spectrometry (UPLC-MS/MS) method and to evaluate the analytical performance of several coagulation assays for quantification of dabigatran, rivaroxaban, and apixaban. Methods: The developed UPLC-MS/MS method was validated by determination of precision, accuracy, specificity, matrix effects, lower limits of detection, carry-over, recovery, stability, and robustness. The following coagulation assays were evaluated for accuracy and precision: laboratory-developed (LD) diluted thrombin time (dTT), Hemoclot dTT, Pefakit PiCT, ECA, Liquid anti-Xa, Biophen Heparin (LRT), and Biophen DiXal anti-Xa. Agreement between the various coagulation assays and UPLC-MS/MS was determined with random samples from patients using dabigatran or rivaroxaban. Results: The UPLC-MS/MS method was shown to be accurate, precise, sensitive, stable, and robust. The dabigatran coagulation assay showing the best precision, accuracy and agreement with the UPLC-MS/MS method was the LD dTT test. For rivaroxaban, the anti-factor Xa assays were superior to the PiCT-Xa assay with regard to precision, accuracy, and agreement with the reference method. For apixaban, the Liquid anti-Xa assay was superior to the PiCT-Xa assay. Conclusions: Statistically significant differences were observed between the various coagulation assays as compared with the UPLC-MS/MS reference method. It is currently unknown whether these differences are clinically relevant. When DOACs are quantified with coagulation assays, comparison with a reference method as part of proficiency testing is therefore pivotal