Elevated anti-human factor Xa activity in rabbit and rodent plasma: Implications for preclinical assessment of human factor X in animal models of hemostasis

Daniël Verhoef,Annabelle V.R Tjalma,Ka Lei Cheung,Pieter H Reitsma,Mettine H.A Bos

doi:10.1016/j.thromres.2020.11.023

Daniël Verhoef, Annabelle V.R Tjalma + Show 3 more

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https://doi.org/10.1016/j.thromres.2020.11.023

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Journal: Thrombosis Research	Publication Date: Nov 28, 2020
Citations: 5	License type: cc-by

Affiliation: Leiden University Medical Center

Abstract

A wide variety of animal models on thrombosis and hemostasis are used in thrombosis and hemostasis research for the preclinical assessment of hemostatic agents. While the vertebrate coagulome is highly conserved, human and animal plasmas differ considerably when evaluated in coagulation assays such as prothrombin time (PT), activated partial thromboplastin time (APTT), and calibrated automated thrombography (CAT). Here, we have aimed to provide a reference framework for the evaluation of coagulation assays and inhibition of activated human FXa (hFXa) in various animal plasmas. To do so, a side-by-side evaluation of the extrinsic and intrinsic pathway of coagulation was performed by means of PT, APTT, and CAT measurements on (diluted) pooled plasmas from goats, pigs, rabbits, rats, mice, and humans. Plasma anti-FXa activity was assessed by determining the rate of recombinant hFXa inhibition through chromogenic activity analyses and immunoblotting. In general, rabbit, rat, and mouse plasmas exhibited robust clotting upon stimulation of both the extrinsic and intrinsic pathway, produced more thrombin during CAT upon plasma dilution, and displayed relatively high hFXa inhibitory activities. By comparison, goat, porcine, and human plasma displayed a similar profile in PT and APTT assays, produced less thrombin during CAT upon plasma dilution, and displayed comparable hFXa inhibitory activities. In conclusion, the observed differences in clotting parameters and anti-hFXa activity point to a higher anticoagulant threshold in plasma from rabbits, rats, and particularly in mice relative to human, goat, and porcine plasma. Finally, rat plasma was found to be more relevant to the preclinical assessment of human FX(a) in comparison to murine plasma.

Highlights

The vertebrate coagulome comprises a wide variety ofenzymes, cofactors, and inhibitors that maintain an intricate balance between pro, and anticoagulant pathways
Studies that serve to evaluate the procoagulant activity of human coagulation proteins build upon animal models and coagulation assays originally intended for human plasma
Rat, rabbit, pig, and goat are frequently used in preclinical hemostasis ad thrombosis research [30,31], we evaluated the coagulant characteristics of pooled plasmas from these species by means of extrinsic and intrinsic clotting analyses and calibrated automated thrombography (CAT) measurements

Summary

Introduction

The vertebrate coagulome comprises a wide variety of (pro)enzymes, cofactors, and inhibitors that maintain an intricate balance between pro, and anticoagulant pathways. Mouse and rabbit plasma have been observed to exhibit reduced rates of thrombin generation upon tissue factor-dependent activation of coagulation [4,5]. While interspecies differences in coagulation factor levels that regulate thrombin formation may be fundamental to these observations, detailed comparisons. Improved knowledge of global coagulation assays for rodent and non-rodent plasmas will facilitate the use and interpretation of animal models for studies in the field of thrombosis and hemostasis [6]. The evaluation of specific coagulation pa rameters in various species will directly impact the selection of animal models for in vivo studies that are essential in the development of pro coagulant therapeutics, such as for instance coagulation factor X (FX) and variants thereof [7,8,9]

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