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

Abstract

Thrombin generation assay (TGA) is a sensitive method for the assessment of the global clotting potential of plasma. This kinetic assay can detect both hypocoagulable and hypercoagulable conditions: delayed or reduced thrombin generation leading to a prolonged clotting time, or induced thrombin activity, shifting the coagulation cascade toward thrombosis. The purpose of this study is to qualify the TGA in nonhuman primates (NHP) and rats for its use during nonclinical in vivo and in vitro studies. Blood was drawn from nonanesthetized animals, and platelet-poor plasma was obtained after double centrifugation; coefficients of variation were <10% for all derived parameters of thrombin generation assessed with 5 pM of tissue factor. Thrombin generation was evaluated in vitro in rat and NHP plasmas with ascending doses of unfractionated heparin (UFH), recombinant tissue factor, and anticoagulant compounds. Thrombin generation was decreased with UFH and anticoagulant compounds, but was increased in the presence of tissue factor, in a dose-dependent manner. In a rat model of inflammation, animals were administered a low dose of lipopolysaccharides. Thrombin generation measurements were decreased 3 hours post-LPS administration with a nadir at 24 hours, while thrombin–antithrombin complexes reached a peak at 8 hours, supporting an earlier production of thrombin. In conclusion, these data demonstrated that TGA can be performed in vitro for screening of compounds expected to have effects on coagulation cascade, and thrombin generation can be measured at interim time points during nonclinical in vivo studies in rats and NHP.

The thrombin generation capability of the Chacma baboon (Papio ursinus): implications for haemostatic disease models

“…Regardless of the differences outlined above, our Chacma baboon ETPs are still more similar to human ETPs than pig, rabbit 9 , Sprague–Dawley rat, and Cynomolgus monkey ( Macaca fascicularis ) 10 , ETPs are. Siller-Matula et al 9 , after performing TGAs on six members of each species using a similar method to ours, report median ETPs of 2043 nM.minute on pigs and 6295 nM.minute on rabbits, which represents differences of − 52% and + 49%, respectively, from the corresponding human median of 4235 nM.minute.…”

“…Poitout-Belissent et al 10 investigated compound-related effects on coagulability in Sprague–Dawley rats, and Cynomolgus and Rhesus monkeys, using a TGA method similar to ours, and report baseline mean values for the rats and rhesus monkeys used in their experiments, which can be used for indirect comparison. The mean ETP of 664.3 nM.minute reported for rats is 84% lower than the median ETP reported for humans by Siller-Matula et al 9 , who reported a much higher median ETP for the same rat species (3075 nM.minute, which represents a difference of only − 27% from the human median), highlighting the large interindividual variation possibly present in rats, which may also complicate the use of this species in coagulation experiments.…”

“…The mean ETP of 664.3 nM.minute reported for rats is 84% lower than the median ETP reported for humans by Siller-Matula et al 9 , who reported a much higher median ETP for the same rat species (3075 nM.minute, which represents a difference of only − 27% from the human median), highlighting the large interindividual variation possibly present in rats, which may also complicate the use of this species in coagulation experiments. Although mean baseline values for Cynomolgus monkeys are not reported, Poitout-Belissent et al 10 do provide reference ranges for various TGA parameters for this species, which they calculated to investigate intra-and interassay variation. Cynomolgus monkey ETPs ranged from 1072.5 to 1827.5 nM.minute (n = 13).…”

“…Whether Chacma baboon thrombin generation is similar to that of other non-human primate species requires investigation and cannot simply be assumed, since interspecies variation has not only been described in both the endogenous thrombin potentials (of humans, rats, pigs and rabbits) and the thrombin generation lag phase times (of humans, rats, pigs, sheep and rabbits), as measured by the thrombin generation assay (TGA) 9 , but also in the Endogenous Thrombin Potentials (ETPs) of different non-human primates, such as Cynomolgus and Rhesus monkeys 10 .…”

Comparative Thrombin Generation in Animal Plasma: Sensitivity to Human Factor XIa and Tissue Factor

“…Moreover, there is a limited amount of studies that compare TG between various species [ 8 , 10 , 15 , 16 ]. When interspecies differences were evaluated [ 9 , 17 , 18 , 19 ], the authors suggested that while some animals might have TG similar to humans, others might not. This type of data can bring useful evidence forward for coagulation investigations that are species specific.…”