Thrombomodulin phenotype of a distinct monocyte subtype is an independent prognostic marker for disseminated intravascular coagulation

Abstract

Introduction: Thrombomodulin, which is expressed solely on monocytes, along with tissue factor (TF), takes part in coagulation and inflammation. Circulating blood monocytes can be divided into 3 major subtypes on the basis of their receptor phenotype: classical (CD14brightCD16negative, CMs), inflammatory (CD14brightCD16positive; IMs), and dendritic cell-like (CD14dimCD16positive DMs). Monocyte subtype is strongly regulated, and the balance may influence the clinical outcomes of disseminated intravascular coagulation (DIC). Therefore, we investigated the phenotypic difference in thrombomodulin and TF expression between different monocyte subtypes in coagulopathy severity and prognosis in patients suspected of having DIC.

Methods: In total, 98 patients suspected of having DIC were enrolled. The subtypes of circulating monocytes were identified using CD14 and CD16 and the thrombomodulin and TF expression in each subtype, expressed as mean fluorescence intensity, was measured by flow cytometry. Plasma level of tissue factor was measured by ELISA. In cultures of microbead-selected, CD14-positive peripheral monocytes, lipopolysaccharide (LPS)- or interleukin-10-induced expression profiles were analyzed, using flow cytometry.

Results: The proportion of monocyte subtypes did not significantly differ between the overt and non-overt DIC groups. The IM thrombomodulin expression level was prominent in the overt DIC group and was well correlated with other coagulation markers. Of note, IM thrombomodulin expression was found to be an independent prognostic marker in multivariate Cox regression analysis. In addition, in vitro culture of peripheral monocytes showed that LPS stimulation upregulated thrombomodulin expression and TF expression in distinct populations of monocytes.

Conclusions: These findings suggest that the IM thrombomodulin phenotype is a potential independent prognostic marker for DIC, and that thrombomodulin-induced upregulation of monocytes is a vestige of the physiological defense mechanism against hypercoagulopathy.

Figure 1

Thrombomodulin expression level of inflammatory monocytes (CD14^brightCD16^positive). Levels are based on the prothrombin time (PT) (a) and plasma levels of tissue factor (b), antithrombin (c), and protein C (d). The expression level of thrombomodulin was scaled by an arbitrary unit of mean fluorescence intensity. The upper limit of each box represents the median value, and the bar represents the value of the 25th-75th percentile. ^†P < 0.05, ^‡P < 0.001.

Figure 2

Receiver operating characteristic (ROC) curves and the area under the ROC curves (AUC) for antithrombin, protein C, and thrombomodulin levels of CD14^brightCD16^positive inflammatory monocytes (IM). Curves were used for the diagnosis of overt disseminated intravascular coagulation. SE, standard error.

Figure 3

Kaplan-Meier survival analysis according to proportions and expression levels of thrombomodulin and tissue factor. Proportions and expression levels of (a) classical monocytes (CM), (b) inflammatory monocytes (IM), and (c) dendritic monocytes (DM) are shown. The cutoff values were determined as the values at which the prognostic power to predict 28-day mortality were the highest.

Figure 4

Changes in the proportion and expression phenotype of a monocyte subtype cultured in vitro. Purified monocytes from healthy donors (n = 4) were cultured in vitro for 24 hours with vehicle, 100 mg/dL lipopolysaccharide (LPS), or 10 ng/mL interleukin-10 (IL-10). (a) Changes in the proportion and phenotype of (b) tissue factor and (c) thrombomodulin expression among three monocyte subtypes - classical monocytes (CM), inflammatory monocytes (IM), and dendritic monocytes (DM) - are shown over culture time. MFI, mean fluorescence intensity.