Thrombin generation assays for global evaluation of the hemostatic system: perspectives and limitations

Abstract

The existing techniques to evaluate hemostasis in clinical laboratories are not sensitive enough to detect hypercoagulable and mild hypocoagulable states. Under different experimental conditions, the thrombin generation test may meet these requirements. This technique evaluates the overall balance between procoagulant and anticoagulant forces and has provided new insights in our understanding of the coagulation cascade, as well as of the diagnosis of hypocoagulability and hypercoagulability conditions. Thrombin generated in the thrombin generation test can be quantified as platelet-rich or platelet-poor plasma using the calibrated automated thrombogram method, which monitors the cleavage of a fluorogenic substrate that is simultaneously compared to the known thrombin activity in a non-clotting plasma sample. The calibrated automated thrombogram method is an open system, in which different antibodies, proteins, enzymes and peptides can be introduced to answer specific questions regarding hemostatic processes. The thrombin generation test has great clinical potential, such as in monitoring patients taking anticoagulants and antiplatelet drugs, screening for genetic or acquired thrombotic disorders, and evaluating bleeding risk control in patients with hemophilia using bypass agents or replacement therapy. Different to conventional coagulation tests, the thrombin generation test can be used for an overall evaluation of hemostasis, the results of which can then be used to evaluate specific characteristics of hemostasis, such as prothrombin time, activated partial thromboplastin time, and levels of fibrinogen and other coagulation factors. The introduction of this method will contribute to a better understanding and evaluation of overall hemostatic processes; however, this method still requires standardization and clinical validation.

<b>Extraction, physical-chemical characterization and <i>in vitro</i> inhibitory potential of thrombin generation of crude sulfated polysaccharides from Brazilian tropical seaweeds

“…As the addition of calcium chloride to display the APTT had no influence on the evaluation by in vitro testing (Figure 2), limited values of detection by in vitro APTT test in human plasma treated with G. birdiae crude SPs extract could be speculated because this classical test measures only 5% of thrombin formed in plasma, and the production of thrombin and fibrin could be still occurring in vitro (Castoldi & Rosing, 2011;Duarte et al, 2017).…”

“…Although no considering as an automated method (Duarte et al, 2017), our alternative technique have shown able to evaluate TG in vitro (Rodrigues et al, 2016a;Salles et al, 2017).…”

“…Elevated TG is associated with the risk of venous thrombosis (Castoldi & Rosing, 2011;Duarte et al, 2017) and thrombin interplays between coagulation and inflammation (Pomin, 2012). The TG method led us to a more precise analysis of macroalgae anticoagulants than traditional methods (Castoldi & Rosing, 2011;Duarte et al, 2017).…”

“…The TG method led us to a more precise analysis of macroalgae anticoagulants than traditional methods (Castoldi & Rosing, 2011;Duarte et al, 2017). The diverse classes of SPs prevented the human plasma coagulability induced by both cephalin and thromboplastin and TG parameters would have potential to postulate their mechanisms underlying in vitro, due to the lack of structure-function relationship data of these compounds, as a biotechnological perspective (Nishino et al, 1999;Mourão et al, 2001;Glauser et al, 2009;Zhang et al, 2014).…”

“…Both the activated partial thromboplastin time (APTT) and the prothrombin time (PT) tests are conventionally employed, respectively, for intrinsic and extrinsic coagulation pathway examination. However, these screening assays have limited value to predict risk of circulatory disorder (e.g., bleeding and thrombosis) and, in the last years, thrombin generation (TG)-based coagulation methods have constituted as more representatives of the total clot formation process (Castoldi & Rosing, 2011;Duarte, Ferreira, Rios, Reis, & Carvalho, 2017) to give additional data about plasma alternative anticoagulants (Nishino, Fukuda, Nagumo, Fujihara, & Kaji, 1999;Mourão et al, 2001;Glauser et al, 2009;Zhang et al, 2014;Rodrigues et al, 2016a;Mansour et al, 2017;Salles et al, 2017).…”