Detection of endogenous tissue factor levels in plasma using the calibrated automated thrombogram assay

BackgroundThe calibrated automated thrombogram (CAT) assay measures thrombin generation in plasma.ObjectiveUse the CAT assay to detect endogenous tissue factor (TF) in recalcified platelet-rich plasma (PRP) and platelet-free plasma (PFP).MethodsBlood from healthy volunteers was collected into citrate and incubated at 37 °C with or without lipopolysaccharide (LPS) for 5 hours. PRP and PFP were prepared and clotting was initiated by recalcification. Thrombin generation was measured using the CAT assay.ResultsThe lag time (LT) was significantly shortened in PRP prepared from LPS-treated blood compared with untreated blood (10 ± 3 min versus 20 ± 6 min), and this change was reversed by the addition of inactivated human factor VIIa. LPS stimulation did not change the peak thrombin. Similar results were observed in PFP (21 ± 4 min versus 35 ± 5 min). LPS stimulation also significantly reduced the LT of PRP and PFP derived from blood containing citrate and a factor XIIa inhibitor. Finally, a low concentration of exogenous TF shortened the LT of PFP prepared from unstimulated, citrated blood without affecting the peak thrombin.ConclusionChanges in LT in the CAT assay can be used to monitor levels of endogenous TF in citrated plasma.