An automated analysis method enabling the screening of covalent thrombin and factor XIIa inhibitors via liquid chromatography-mass spectrometry.

An automated sample preparation and separation method for the analysis of various enzyme-inhibitor combinations using liquid chromatography (LC) coupled to mass spectrometry (MS) is presented. As conventional anticoagulants have several drawbacks, the most severe being the elevated risk of internal bleedings, it is necessary to develop new-generation anticoagulants with reduced side effects. Therefore, the screening of potential inhibitors against anticoagulation targets like thrombin and FXIIa is important to design a potent and selective inhibitor. To facilitate the analysis of numerous enzyme-inhibitor covalent complexes, automation of the analysis using an LC system with a user-defined injection sequence is helpful. The developed method ensures comparable reaction conditions like reaction time and temperature for all enzyme-inhibitor complexes. Furthermore, it prevents time-consuming manual sample preparation and potential manual errors. To achieve good reproducibility with relative standard deviation of approximately 3% for three-fold determination, multiple cleaning steps were added to the automated sample preparation. Subsequently, this method was applied to screen a variety of 15 aminopyrazole- and aminotriazole-based inhibitors with a covalent mechanism of action against thrombin and to test two covalent inhibitors for FXIIa. Successful complex formation and acylation of the catalytic center of the enzymes was monitored using deconvoluted mass spectra and the matching mass shifts of the acyl moiety of the analyzed inhibitors. The inhibitors' structure directly influenced reaction yields. Sterically demanding aminotriazoles and acyl moieties both affected the product formation negatively. However, the screening yielded several promising candidates for new covalent thrombin inhibitors, which might find their application as prospective anticoagulants.

Comparison of 90-Day Adverse Events Associated With Aspirin and Potent Anticoagulation Use for Venous Thromboembolism Prophylaxis: A Cohort Study of 72,288 Total Knee and 35,142 Total Hip Arthroplasty Patients.

BACKGROUND: While aspirin is acceptable for venous thromboembolism (VTE) prophylaxis following total joint arthroplasty in most patients, more potent agents are used in patients considered higher risk for VTE. We evaluated the efficacy and safety of aspirin versus potent anticoagulation agents following total knee arthroplasty (TKA) and total hip arthroplasty (THA). METHODS: A cohort study of 72,288 TKA and 35,142 THA from the Kaiser Permanente Total Joint Replacement Registry was performed (2009 to 2019). Identified medications were aspirin, factor Xa inhibitors, low-molecular-weight heparin (LMWH), and warfarin. A validated VTE risk score was assigned to each patient. Propensity score-weighted logistic regressions were used to evaluate 90-day VTEs. Noninferiority testing was performed with a margin of 1.25 using the upper bound (UB) of the 1-sided 95% CI. RESULTS: For TKA, aspirin was not inferior to LMWH (odds ratio [OR] = 0.77, UB = 1.09) and warfarin (OR = 0.64, UB = 0.90); there was no evidence to support noninferiority of aspirin compared to factor Xa inhibitors. Findings were consistent for THA (LMWH: OR = 0.59, UB = 0.75; warfarin: OR = 0.69, UB = 0.89). TKA was considered higher risk for VTE, whereas aspirin use demonstrated noninferiority compared to warfarin (OR = 0.54, UB = 0.81), we lacked evidence of noninferiority when compared to LMWH and factor Xa inhibitors. We lacked evidence of noninferiority of aspirin versus any potent anticoagulation in higher-risk THA. CONCLUSION: Aspirin was found to be effective and safe for VTE prevention in primary total joint arthroplasty, including in patients considered higher risk for VTE. LEVEL OF EVIDENCE: III.