Preanalytic processing of rat plasma influences thrombin generation and fibrinolysis assays

Abstract

Preanalytic processing and sex have significant effects on many functional measures of hemostasis in rats. A standardized double centrifugation protocol to prepare PPP is recommended for future studies.

Monitoring Compound-Related Effects on Coagulability in Rats and Cynomolgus and Rhesus Monkeys by Thrombin Generation Kinetic Measurement

“…Indeed, platelets and microparticles provide phospholipid membranes that support the assembly of coagulation complexes that catalyze thrombin generation. 4 Application of this standardized procedure resulted in acceptable validation results with CVs below 10% for all derived thrombin generation parameters. Important intra-and interassay variability of TGA Figure 3.…”

“…Skilled technicians collected the blood by clean draws on quiet nonanesthetized animals with only minimal restraint, limiting the activation of the coagulation pathway by the release of tissue factor during venous puncture. 4,9 Platelet-poor plasma was obtained from blood, rapidly after collection by double centrifugation in order to avoid any contamination by residual platelets debris or microparticles that may influence thrombin generation. Indeed, platelets and microparticles provide phospholipid membranes that support the assembly of coagulation complexes that catalyze thrombin generation.…”

“…The prolongation of these coagulation times indicates a deficiency, dysfunction, or inhibition of one or more coagulation factors, but their capability to detect subtle changes remains limited because the clot formation occurs when only a small portion of the thrombin potentially present in plasma is produced. 4 These assays are also not designed to detect increases in coagulation factor activities or compound-induced hypercoagulability in nonclinical studies, which remains a major cause of compound withdrawal. 5 Thrombin generation assay (TGA) detects delayed thrombin generation, leading to a prolonged clotting time, and also increased thrombin activity, leading to an increased risk of thrombosis.…”

Interspecies comparison of simultaneous thrombin and plasmin generation

“…Interestingly, guinea pigs (the species with the lowest measured TG parameters) show compatibility with human TF 38 . Finally, plasminogen from several species also seems to be compatible with human tPA 5,41,42 . Current data suggests that interspecies incompatibility in TF and tPA can contribute to small changes in hemostasis assays, nonetheless the overall comparison of species studied here and compared with humans, reflects similarities and differences in species hemostasis based on our STPGA assay.…”

“…Human PH values were larger than in rat, swine and rabbit, while guinea pig PH values were the smallest. Rat and rabbit are commonly used species to study TG [3][4][5]9,[29][30][31][32] . Among these studies, several used TF concentrations consistent with our study, and subsequently PH values were also similar to those presented in our study 3,4,9,29,30 .…”