New Methodological Approach for Coagulation Assays Using Chicken Plasma Intrinsic Properties

Special Article: Thrombin Inhibitors

J Blood Disord. 2023; 10(1): 1074.

New Methodological Approach for Coagulation Assays Using Chicken Plasma Intrinsic Properties

Prezoto BC¹*; Falla MVA²; Lebrun I²

1Laboratory of Pharmacology, Butantan Institute, São Paulo, SP, Brazil

2Laboratory of Biochemistry, Butantan Institute, São Paulo, SP, Brazil

*Corresponding author: Prezoto BC Laboratory of Pharmacology, Butantan Institute, Av Vital Brasil, 1500, CEP 05503-900, São Paulo, SP, Brazil. Tel: +55-11-2627-9753 Email: [email protected]

Received: May 12, 2023 Accepted: June 13, 2023 Published: June 20, 2023


Background: Sensitivity of classical coagulation assays by using mammalian plasmas to pro- and anticoagulant compounds including venom or toxins occurs on a microscale level (micrograms). Although it improves responses to agonists, recalcification triggers a relatively fast thrombin formation process. The Recalcification Time (RT) of factor XII- deficient Chicken Plasma (CP) is comparatively long (=1800 seconds) when compared to human plasma or others. Our objective was to compare its sensitivity with that presented by human plasma samples to Unfractionated Heparin (UH), a prototype anticoagulant compound, under similar conditions through rotational thromboelastometry.

Methods: To find doses of UH sufficient enough to prolong the Clotting Time (CT) parameter of these activated plasmas to values within their normal RT ranges.

Results: In total, 0.0065±0.0009 IU of UH (n=6) was detected in 260μL of CP samples, but only 0.125±0.012 IU of UH was sufficient to induce a similar effect in activated human plasma samples.

Conclusion: The higher sensitivity of CP to anticoagulants could be useful for (a) detection of anticoagulant compounds in substances of unknown origin; (b) purification procedures of anticoagulant toxins from crude animal venoms and (c) determination of relative potencies of agonists and their selective antagonists such as pharmaceutical agents, antivenoms or natural inhibitors of venom toxins with a better result in kinetic clothing parameters.

Keywords: Coagulation process; Chicken; Factor XII deficiency; Animal models; Biomedical research; Rotational thromboelastometry


The coagulation cascade consists of a complex network of interactions resulting in thrombin-mediated cleavage of fibrinogen to fibrin, which is one major component of a thrombus. The coagulation cascade can be initiated either by the “extrinsic pathway”, the release of tissue factor leading to activation of factor VII to the tissue factor/factor VIIa complex, or by the “intrinsic pathway”, so-called contact activation leading via factors XII, XI and IX to the assembly of the tenase complex consisting of activated factors VIII and IX and Ca2+. Both complexes can activate factor X, which induces the formation of the prothrombinase complex consisting of activated factors X, V and Ca2+ on a phospholipid surface. The latter leads to the activation of thrombin, which in turn cleaves fibrinogen to fibrin [1].

Circulating blood cells, coagulation factors and vascular wall components of mammalian species are considered as targets of a variety of coagulant agonists, including venom toxins [2], which have been investigated both as laboratory reagents and as potential therapeutic agents. The study of venom pro- or anticoagulant toxins has been traditionally assessed directly by simple clotting studies on mammalian plasma samples. However, these classical coagulation assays present some limitations. For example, most of in vitro techniques designed for assaying the coagulant activity of snake venoms or toxins on mammalian plasma samples offer a single parameter for one complex enzymatic process [3]. Attempts to overcome this kind of limitation have been enacted with the use of different strategies, such as the monitoring of viscoelastic changes in plasma or Whole Blood (WB) samples with thromboelastography and, more recently, rotational thromboelastometry. These techniques improve the evaluation of the clotting process, since it monitors several parameters, such as the stages of clot initiation, formation, stability, strength and dissolution [4]. This technology has been used in various studies on the pro- or anticoagulant activities of several snake venoms or toxins on citrated human whole blood, as well as plasma samples of rats and dogs [5–10].

Citrated plasma is the substrate for almost all coagulation-specific laboratory tests and is derived from whole blood drawn into a tube containing liquid 3.2% sodium citrate at a ratio of nine parts whole blood and one part citrate. Citrate acts as a Ca2+ chelating agent to prevent coagulation of the sample so that all the clotting factors are preserved and can be evaluated. Chelation of Ca2+ determines another obvious restriction of classical coagulation assays, since non-recalcified plasma samples such as those used in the minimum coagulant dose require relatively large amounts of venom or toxins [11]. Attempt to improve sensitivity to agonists could be achieved by the addition of Ca2+ and phospholipids, since these cofactors are essential in some enzymatic steps of the coagulation cascade [12,13]. However, if higher sensitivity could be achieved, this strategy simultaneously starts and accelerates enzymatic reactions of the coagulation process; as a consequence, the time interval in which agonists can be assayed becomes limited (to around 600 seconds). This limitation associated with recalcified mammalian plasma samples becomes evident when small amounts of the proteins being tested are available; for example, during screening strategies for purification procedures of individual toxins from crude venoms or during assays for detecting the presence of pro- or anticoagulant substances in samples of unknown origins. This restricted time interval does not allow the elaboration of one typical dose–response curve to agonists or antagonists.

Another strategy to improve sensitivity to agonists by using recalcification could be achieved through the use of reptiles and avian blood or plasma samples, which present a prolonged Recalcification Time (RT) [14].

By testing recalcified factor XII-deficient Chicken Plasma (CP) samples through rotational thromboelastometry, we recently published one study in which we described that addition of the cofactors Ca2+ and phospholipids to this plasma elicited a time lapse sufficient (>1800 seconds) for the elaboration of one typical dose–response curve after testing with the in vitro procoagulant venom of the snake Bothrops jararaca (B. jararaca), displacing its sensitivity to a nanoscale range [15]. A similar strategy was later used to test the response of CP samples to the anticoagulant activity of two venom toxins: crotoxin (from Crotalus durrissus terricus snake venom) [16] and Phospholipase A2 (PLA2) from Apis mellifera bee venom [17]. However, the relative magnitude of the sensitivity of this pharmacological preparation to anticoagulant substances is generally unknown.

Classical coagulation assays such as the Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT) and Thrombin Time (TT) are global coagulation screening tests routinely used for assessment of the coagulation status in patients with suspected acquired deficiencies of coagulation factors of the intrinsic and common pathways of the coagulation system, and are also extensively used to monitor anticoagulant agents, including direct thrombin inhibitors [18,19].

In this study, we challenged chicken and human plasma samples under similar conditions through rotational thromboelastometry to compare their sensitivity to the anticoagulant effect of Unfractionated Heparin (UH), a sulfated polysaccharide and an essential drug that has been used as a clinical anticoagulant worldwide for over 90 years [20]. These assays were conducted under controlled conditions, i.e., these plasma samples were simultaneously activated with standardized doses of one classical activator of coagulation (the aPTT clot reagent, containing ellagic acid and synthetic phospholipids), taking their Recalcification Time (RT) as a reference.

The highest sensitivity of recalcified CP to the in vitro heparin anticoagulant activity (almost 19-fold higher than that presented by human plasma) described in this paper reinforces our previous findings using anticoagulant compounds such as crotoxin from Crotalus durrissus terricus venom and PLA2 from Apis mellifera bee venom.

Materials and Methods


Activated Partial Thromboplastin Time Reagent (aPTT clot), containing ellagic acid plus synthetic phospholipids, was from BIOS Diagnostica (SP, Brazil); pooled 4% citrated normal human plasma (maintained at - 80°C) from Sigma-Aldrich, Inc. (USA); unfractioned sodium heparin (Hepamax-S) (5,000IU/mL) from swine intestinal mucosa, from Blau Farmacêutica (Cotia, SP, Brazil). All chemicals were of analytical reagent grade.


Adult female Gallus gallus domesticus chickens (1.0 to 1.7kg) were used. All birds were a donation from commercial breeding (Granja Ino, São Paulo, SP, Brazil). The animals had free access to water and food, and were kept under a 12h light/dark cycle.

The experimental protocol was approved by the Ethic Committee on Animal Use of the Butantan Institute (protocol CEUA number 6259250918).

Obtention of Citrated Chicken Plasma Samples

Blood samples were collected into syringes containing 1:10 (v/v) 3.2% trisodium citrate and then closed with cotton-yarn. Chicken plasma was obtained after centrifugation at 3000xg for 20 min at 4°C and stored at - 80°C.

Thromboelastometric Assays with Chicken or Human Plasma Samples

Standardization of the activator aPTT clot mean coagulant dose: Clotting Time (CT) parameter (in seconds, from start of the reaction to initial clot formation) of the INTEM (intrinsic pathway thromboelastometry) assay was measured using a computerized ROTEM four channel system (Pentapharm, Munich, Germany), during 3h at 37°C. In summary, before recalcification (with 20μL of 0.2M CaCl2), 260μL of human or chicken plasmas were incubated for 1 min with 60μL of 0.9% NaCl solution containing 0.9% NaCl solution [control group, so-called Recalcification Time (RT)] (Group 1) or crescent doses of the activator aPTT clot reagent, for determination of its Mean Coagulant Dose (MCD) (Group 2). The RT of CP samples in control group is relatively prolonged (=1800 seconds), when compared to that presented by human plasma (almost 600 seconds). For the elaboration of a dose–response curve of CP samples to tested doses of the activator aPTT clot reagent, reference values of 90, 900 and 1800 seconds were standardized as maximum (90%), mean (50%) and minimum (10%) coagulant responses, respectively. On the other hand, for the elaboration of a dose– response curve of human plasma samples to tested doses of the activator aPTT clot reagent, reference values of 30, 300 and 600 seconds were standardized as maximum (90%), mean (50%) and minimum (10%) coagulant responses, respectively (Figure 1).

Citation: Prezoto BC, Falla MVA, Lebrun I. New Methodological Approach for Coagulation Assays Using Chicken Plasma Intrinsic Properties. J Blood Disord. 2023; 10(1): 1074.