The Christmas season is a time for reflecting on the phenomenon of birth. From a medical standpoint, the period of pregnancy is particularly demanding for the human organism. Pregnancy is a hypercoagulable state associated with an increased risk of venous thrombosis, strongly related to enhanced thrombin generation, from its very first month.1 In this context, the introduction of thromboprophylaxis in women at a high thrombotic risk, such as those with obstetric antiphospholipid syndrome, may be warranted.2 Thrombin, a key serine protease that converts soluble fibrinogen into insoluble fibrin, is generated from its precursor, prothrombin—highly abundant in circulating blood, as compared with other coagulation factors (except fibrinogen).3 Thrombin has a short lifespan (about 1 minute); therefore, key markers reflecting thrombin activity in vivo include prothrombin fragments 1+2 (a direct marker of factor Xa activity and an indirect measure of thrombin generation), stable complexes of thrombin with antithrombin, as well as markers related to fibrin formation, such as fibrinopeptide A (indicating thrombin proteolytic activity). Such measures are assessed mostly in citrated, platelet-poor plasma using immunoenzymatic assays for research purposes. Elevated thrombin generation markers are related to venous thrombosis, acute coronary syndrome, disseminated intravascular coagulation, cancer, trauma, surgery, and other clinical states. Of note, no assay has yet been established for active thrombin assessment in plasma, apart from single reports using refined methods.

Growing evidence supports research on and clinical utility of thrombin generation assays. Assessment of thrombin generation seems to be crucial in clinical evaluation of hypercoagulability and bleeding disorders, as well as monitoring of anticoagulation.4 As compared with in vivo markers of thrombin activity, thrombin generation assessed ex vivo exhibits a potential of plasma (citrated; fresh or frozen; platelet-poor or -rich) to generate active thrombin and monitor the kinetics of its formation in real time upon addition of different concentrations (low, normal, or high) of exogenous tissue factor (TF), a key initiator of the extrinsic pathway. Such an approach, in the presence of TF concentration of approximately 15 pM, allows for the assessment of thrombin generation even in patients receiving therapeutic doses of anticoagulants. Thrombin activation tested in plasma is strictly related to fibrin formation, with a natural interaction between fibrin and thrombin.3 Thrombin cleaves a peptide substrate containing a fluorophore (Z-Gly-Gly-Arg-7-amino-4-methylcoumarin), whose fluorescence is measured. Usually, maximum thrombin concentration is generated within a few minutes, and rapidly neutralized by specific (antithrombin) or broad-spectrum protease inhibitors (α2-macroglobulin), which implies enhanced thrombin generation potential in patients with a deficiency of natural anticoagulants.5 Importantly, thrombin generation measures, such as endogenous thrombin potential and peak thrombin, can be obtained within minutes, and the typical curve is a second derivative of the fluorescence signal measured. The signal is adjusted for thrombin consumption during the experiment and inner filter effects or the background signal (related to plasma features per se), based on thrombin calibrator interaction with tested plasma (neither thrombin nor fibrin is generated in this sample). A recent study showed good performance of a thrombin generation assay in predicting bleeding in patients with acquired hemophilia A on emicizumab prophylaxis, mimicking the activity of activated factor VIII.6 In general, nonsubstitutive therapies enhance thrombin generation.

Thrombin generation describes coagulation in a manner that more closely reflects physiological conditions than conventional coagulation assays, although methods of its assessment require standardization and approval before they can be implemented in routine patient management.7 However, a new generation of analyzers (ST Genesia, Diagnostica Stago S.A.S., Asnières-sur-Seine, France) enable standardization and automation of the thrombin generation test, making it widely accessible for in vitro diagnostic use. Future experience will reveal its role in clinical practice.

We hope that the coming year meets new diagnostic and therapeutic challenges, and that Przegląd Lekarski – Jagiellonian Medical Review will assist in this effort by objectively presenting emerging issues and developments.