Clinical Applications, Pitfalls, and Uncertainties of Thrombin Generation in the Presence of Platelets

Abstract

Platelet-dependent thrombin generation is a helpful tool to assess ex vivo the interaction between platelets and plasma coagulation factors in the initiation, amplification, and inhibition of thrombin generation (TG). This review article discusses the most relevant available data on the clinical applications of fluorogenic TG, the most widely used TG assay, performed in the presence of platelets, i.e., in platelet-rich plasma. With respect to prothrombotic states, arterial hypertension and obesity were the most prominent cardiovascular conditions linked to increased platelet-dependent TG. In addition, platelet-associated hypercoagulability, assessed by the TG assay, has been shown in individuals with active cancer. In terms of bleeding, platelet-dependent TG has been applied to assess bleeding risk in individuals with hemophilia, von Willebrand disease, and Glanzmann thrombasthenia as well as in subjects with other congenital or acquired coagulation factor deficiencies. In addition to risk prediction, a role of the TG assay has been suggested in monitoring antiplatelet therapy in prothrombotic conditions and replacement therapy in bleeding diathesis. Finally, for the routine clinical use and as a biomarker of disease development and progression, better standardization and clinical validation of platelet-dependent TG are still needed.readings from a fluorometer are recorded and calculated by means of the Thrombinoscope software (Thrombinoscope BV). In principle, the measurements in PRP are assessed in fresh material, whereas TG in PFP is assessed in frozen, stored material. For TG measurements in PRP, coagulation is triggered by adding 20 µL exogenous tissue factor (TF) to 80 µL PRP (with adjusted platelet concentration of 150,000 platelets/µL), whereas for TG measurements in PFP, 20 µL exogenous TF and 4 µM phospholipids are added to 80 µL plasma. After prewarming for 10 min at 37 • C in the fluorimeter, the reaction is started by adding 20 µL of a low-affinity fluorogenic substrate for thrombin (Z-Gly-Gly-Arg-AMC) and calcium chloride mixture (FluCa). TG is measured as a function of a calibration signal obtained in a sample from the same plasma (PRP or PFP) after addition of a fixed amount of thrombin-α2-macroglobulin complex (20 µL of thrombin calibrator) and 20 µL of FluCa. The TG method is extensively reviewed in [5][6][7]. Parameters derived from a TG curve and most frequently reported in the studies are lag time, time to minimum thrombin formed expressed in minutes (min); peak height, maximum concentration of thrombin formed expressed in nM thrombin; and endogenous thrombin potential (ETP) or area under the curve expressed as nM of thrombin formed per minute.The aim of this article was to review the available evidence for the potential clinical application of platelet-dependent TG in both prothrombotic and bleeding conditions, as depicted in Figure 1. Aspects preventing its broader application and routine clinical practice will also be discussed as uncertainties and limitations of thrombin generation in the...

Thrombin generation abnormalities in Quebec platelet disorder

“…We studied TG without thrombomodulin, as recommended for bleeding disorder investigations. 1,2 Given the F I G U R E 5 The effect of added tranexamic acid on thrombin generation by Quebec platelet disorder (QPD) and control platelet-rich plasma and platelet-poor plasma. The table inset compares TG endpoints for control and QPD PRP, tested with and without added drug (TXA).…”

“…The table inset compares TG endpoints for control and QPD PRP, tested with and without added drug (TXA). The panels compare the effects of TXA on PRP vs PPP as % differences in TG endpoints compared to baseline (P values indicated) for control and QPD samples complex abnormalities in QPD PRP, it would be challenging to address if QPD alters activated FV downregulation by activated protein C/ protein S. 1,2,5 It would be interesting to use other types of samples and CAT to test if platelet FV is important for maximal TG when plasma FV and TFPI levels are normal. Potentially, this could be done using severe FV-deficient platelets re-suspended in normal plasma and/or PRP from persons with gray platelet syndrome.…”

“…It remains possible that other pathological changes to QPD platelets contribute to their TG abnormalities, including the deficiency of MMRN1. We studied TG without thrombomodulin, as recommended for bleeding disorder investigations 1,2 . Given the complex abnormalities in QPD PRP, it would be challenging to address if QPD alters activated FV downregulation by activated protein C/protein S 1,2,5 …”

“…Assessments of thrombin generation (TG) by calibrated automated thrombograms (CAT) have provided insights on how bleeding disorders alter blood coagulation 1,2 . An important advantage is the ability to test TG with both platelet‐poor (PPP) and platelet‐rich plasma (PRP) 1,2 . Recently, tissue factor pathway inhibitor (TFPI) levels have emerged to be an important determinant of TG and bleeding 3‐5 .…”

Comparative Analysis of Thrombin Calibration Algorithms and Correction for Thrombin-α2macroglobulin Activity

“…The clinical application of the thrombin generation (TG) assay has been suggested by multiple research studies, and it is often used for bionanalytical assessment in clinical trials [ 1 , 2 ]. The TG assay, while uniquely sensitive to the mechanism of action of numerous drugs developed for treatment of bleeding and thrombotic diseases [ 3 , 4 , 5 , 6 , 7 ], remains hard to standardize and validate for routine use outside of the expert central laboratories [ 8 , 9 , 10 , 11 , 12 , 13 ]. Modern automated TG assays are based on fluorogenic peptide substrates which release fluorophore in a relatively slow reaction with thrombin [ 11 , 14 , 15 , 16 , 17 , 18 ].…”