Whole blood coagulation assays ROTEM and T-TAS to monitor dabigatran treatment.

BACKGROUND: A rapid and reliable assessment of the dabigatran effect is desirable in dabigatran treated patients with uncontrolled bleeding or before acute surgery. OBJECTIVE: To evaluate how the viscoelastic point-of-care test Rotational thromboelastometry (ROTEM) and Total Thrombus-formation system (T-TAS), which studies thrombus formation under flowing conditions, correlate with dabigatran concentrations in patients with atrial fibrillation (AF). METHOD: ROTEM using the reagents In-tem, Ex-tem, Fib-tem or low tissue factor concentration (TF), and T-TAS with the AR-chip (shear rate 600s-1, representing flow in large arteries) were investigated in whole blood samples. Plasma concentrations were determined by mass spectrometry (LC-MS/MS) at trough and post-dose in 30 patients on dabigatran 150mg BID. RESULTS: Median plasma dabigatran concentrations at trough were 86ng/mL (29-150) and post-dose (2.8h after ingestion) 175ng/mL (67-490). The ROTEM clotting time (CT) correlated strongly with dabigatran concentrations when activated with the reagents Ex-tem (r=0.92, p<0.01) and Fib-tem (r=0.93, p<0.01), while with In-tem and low TF the correlation was weaker (r=0.72 and r=0.36, p<0.01). There were significant but weaker correlations also between dabigatran concentrations and T-TAS variables (r-values 0.39-0.41, p<0.01), aPTT (r=0.70, p<0.01) and PT-INR (r=0.43, p<0.01) respectively. CONCLUSIONS: ROTEM Ex-tem and Fib-tem CT shows a strong correlation with dabigatran concentrations in real-life AF-patients, and results are obtained within minutes. This could make ROTEM useful in acute situations. T-TAS detect differences in hemostasis caused by dabigatran, but the relationships to plasma concentrations of dabigatran are weaker than for ROTEM CT with the settings used in this study.

CD40L expression in plasma of volunteers following LPS administration: A comparison between assay of CD40L on platelet microvesicles and soluble CD40L.

CD40 ligand (CD40L) is a transmembrane protein that is mainly expressed on activated T cells and platelets. This protein, however, may also be shed from cells and circulate in the blood in a soluble form. "Soluble CD40L" has attracted interest as a biomarker as it can interact with CD40 and elicit cellular responses involved in the pathophysiology of various thrombotic and inflammatory conditions. As platelets can release microvesicles following activation, we investigated the expression of CD40L on circulating microvesicles as well as CD40L in plasma, in an experimental model of inflammation in healthy volunteers (i.e., intravenous lipopolysaccharide administration). We studied CD40L quantified as CD40L-positive platelet microvesicles by flow cytometry, and as CD40L in plasma ("soluble CD40L") by an ELISA. Results of these studies showed that levels of CD40L exposed on platelet microvesicles were significantly increased after lipopolysaccharide administration. ELISA measurements of CD40L in plasma ("soluble CD40L") did not show any significant increase in plasma levels over time. Separation of soluble and vesicle-bound CD40L by high-speed centrifugation indicated that the ELISA can also detect CD40L on microvesicles, as a trend toward increased concentrations were observed in the pellet of high-speed centrifuged samples (i.e., in samples in which microvesicles are enriched). Together, these findings suggest that platelet microvesicles are a source of CD40L in the circulation and that CD40L exposure on platelet microvesicles increases following experimentally induced inflammation. Our data also suggest that determining levels of CD40L on microvesicles in plasma samples may provide a more sensitive detection of changes in CD40L expression than measurement of "soluble CD40L" in plasma with an ELISA. In addition, information regarding the cellular source of CD40L can be obtained with a flow cytometry-based microvesicle assay in a way not possible with an ordinary ELISA.