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

Thrombomodulin (TM) is a transmembrane glycoprotein that blocks the interaction between thrombin and procoagulant protein substrates and acts as a vascular endothelial cell receptor for thrombin to activate protein C. Activated protein C inactivates factors Va and VIIIa and inhibits further thrombin generation and thus plays an important role in the anticoagulant state of the endothelium [1]. Tissue factor (TF) is an essential cofactor for the initiation of the extrinsic coagulation pathway. TF complexes with factors VII and VIIa and activates factors IX and X, and these activated factors contribute to the generation of thrombin on cell surfaces [2].

Disseminated intravascular coagulation (DIC) is characterized by systemic fibrin formation, resulting from increased generation of thrombin, simultaneous suppression of physiological anticoagulants, and impaired fibrinolysis [3]. A marked impairment in the protein C system worsens coagulopathy because the protein C pathway plays a role in the major regulatory loop that limits thrombin generation. This reduction in the protein C system is caused, in part, by the cytokine-induced decrement in TM activity and free protein S levels and impaired protein synthesis [3, 4].

Monocytes play an important role in the coagulation system [5]. Endothelial cells and circulating monocytes express TF and TM within the vasculature [6]. Dysregulation of TF and TM expressions on cell surfaces may affect intravascular coagulation status. For example, inflammatory cytokines induce monocyte TF expression, which would yield procoagulant diathesis [5]. Also, in numerous pathophysiological conditions, monocyte TM expression was shown to be altered [7‐9]. Therefore, one may speculate that the imbalance of the surface molecule expression of monocytes plays a role in the pathophysiology of DIC. In addition, monocytes, as key components of the humoral and cellular immune system, have been studied for subpopulation changes during infection and inflammatory conditions [10, 11]. Whereas some inflammatory cytokines were known to increase TF of monocytes [12], anti-inflammatory cytokines such as IL-10 and IL-4 could suppress TF expression [13]. Because both inflammatory and anti-inflammatory cytokines are usually elevated in DIC, these cytokines may affect the expression of TF and TM in monocytes.

Monocytes subcategorized by the surface molecules CD14 and CD16 have been classified into three groups: CD14^brightCD16^negative classical monocytes (CMs), which constitute the majority of circulating monocytes; CD14^brightCD16^positive inflammatory monocytes (IMs), which produce proinflammatory cytokines; and CD14^dimCD16^positive dendritic cell-like monocytes (DMs), which have features of differentiated monocytes or tissue macrophages, such as increased migration into tissues [14‐16]. Many studies reported increases in the levels of IMs during inflammatory conditions such as in sepsis, rheumatoid arthritis, and hemolytic uremic syndrome [10, 11, 17]; however, changes in the DMs were variable [17‐19].

In experimental models of sepsis, TF and TM mRNA upregulations through thrombin generation have been reported [7]. Monocyte subtype is strongly regulated, and the modulation of TF and TM expressions on monocyte subtype may influence the clinical outcomes of coagulopathy. Because the number of IMs are increased during inflammatory conditions [10], it can be hypothesized that the expression status of TF and TM on IMs may be a reflection of ongoing coagulopathy. Therefore, we investigated the phenotypic difference in TM and TF expressions among different monocyte subtypes associated with coagulopathy severity and prognosis in patients suspected of having DIC. Furthermore, to explore the changing pattern in expression phenotype of each monocyte subtype induced by both inflammatory stimuli and anti-inflammatory stimuli, the surface expression of TF and TM was investigated in monocytes derived from the in vitro culture of peripheral blood monocytes stimulated with lipopolysaccharide (LPS) and IL-10.

Tightly controlled TF and TM expressions maintain normal rheological properties of the blood. However, various stimuli such as infection and inflammation can induce inflammatory cytokines that increase TF expression and suppress anticoagulant protein expression [22‐24]. This imbalance would eventually yield to the procoagulant diathesis of DIC. Therefore, the changed pattern of TF and TM expressions plays an important role in various pathophysiological conditions. Although the vascular endothelium is known to express TF and TM [6], circulating monocytes are also important cellular sources of TF and TM expressions within vessels [5]. The existence of different populations of monocytes (CMs, IMs, and DMs) is well established, and each population has a distinct antigen phenotype and function [11]. To date, there are no data on the expression pattern of TF and TM in any of these monocyte subpopulations. This study was the first to demonstrate the phenotypic changes of TF and TM in each monocyte subpopulation during DIC.

Interestingly, IM TM expression was prominent in the overt DIC group and had good correlation with other coagulation markers. Of note, IM TM expression was found to be an independent prognostic marker for DIC, which has been the focus of this study. Other phenotypic changes of the monocytes also showed differences between the overt and non-overt DIC, such as the lower TF expression of CMs in the overt DIC group. TF expression of CM was significant in multivariate analysis, but the correlations with other coagulation markers were weak and the differences between the survivor/non-survivor groups were minimal, and this needs to be studied further. When the survivors and non-survivors were compared, the percentage of CM was lower and TM expression on CMs and IMs was higher in the non-survivors. The TM expression on CM was significant in the univariate analysis but was not found to be an independent prognostic factor. In addition, the TM and TF expressions of DMs were higher than those of the IMs, but the mean differences of the TM and TF expressions of DMs between survivors and non-survivor were not significant and the phenotype of DMs was not found to be significant in multivariate analysis. These findings support the clinical relevance and importance of TM rather than TF expression in IMs.

Evaluation of the TF and TM expressions on each monocyte subtype showed positive correlation within each subpopulation of the monocytes. TF is a well-known initiator of coagulation and an important modulator of inflammation induced by proinflammatory cytokines [12], but the TM functions as both an anticoagulant and an anti-inflammatory molecule [25], so it is necessary to understand how TM expression is integrated to maintain homeostasis under hypercoagulable and proinflammatory conditions. TM is known to be transcriptionally upregulated by thrombin, vascular endothelial growth factor, histamine, dibutyryl cAMP, retinoic acid, theophylline, and statin, whereas shear stress, hemodynamic forces, hypoxia, and oxidized low-density lipoprotein suppress its expression [25]. In our study, TM expression tended to increase in hypercoagulable conditions. This finding is consistent with that of the previous in vitro experiment, which showed that viral stimulation increased TM expression in monocytes and endothelial cells [8]. This is also in agreement with the study that showed thrombin-induced upregulation of TM mRNA levels [7] and with the study that showed increased amounts of surface TM on monocytes during meningococcal disease [9]. All of these findings support the general notion that infection or inflammation shifts the hemostatic balance to thrombosis.

Although IM expansion was shown in inflammatory conditions [17‐19], it is currently unclear how to change the TM phenotype of IMs. In our study, the IM TM expression level was highly associated with severe coagulopathy and poor prognosis, but those of CMs and DMs were not. This finding suggests that IMs play a role in maintaining the hemostatic balance of the active anticoagulant system by enhancing TM expression. The vivid reaction of IMs can be speculated from that of a previous study, which states that IMs produce proinflammatory cytokines [11]. The surface-bound TM is theoretically considered to be a regulator of the coagulation cascade in monocytes. However, it remains unclear whether IM TM expression exerts functional activity to dampen hypercoagulation. In our study, coagulopathy was severe in patients with high levels of TM, suggesting that the enhanced expression of TM in IMs plays an insufficient role in regulating the inflammatory sequelae. This change might just be the result of a physiological defense mechanism against hypercoagulopathy [26].

In our result, the percentage of monocyte subpopulations did not significantly differ between the overt and the non-overt DIC groups. Most related studies have compared the monocyte subpopulations between control and sepsis patients [17‐19]. However, our study focused on patients suspected of having DIC (some with a recent inflammatory insult, others with overlaying stimuli in chronic conditions, and others in recovery); thus, the result may not show a clear-cut difference between the overt and the non-overt groups. This heterogeneity within each subgroup may have created a less dramatic difference between the expression level of TF or TM on monocytes as well.

To evaluate the diagnostic value of the IM TM phenotype, we analyzed the AUC value and compared it with that of well-known DIC markers. The AUC for the TM phenotype was significant (0.672) but was lower than that of protein C and antithrombin, suggesting that the IM TM phenotype is not a good diagnostic marker of overt DIC. On the other hand, it was useful for estimating prognosis. IM TM expression remained a significant prognostic factor in multivariate Cox analysis, with a hazard ratio of 19.11 after adjustment for the effect of other coagulation markers. Given that most of the DIC markers are dependent on each other, the IM TM phenotype is expected to be a useful potential marker of prognosis. A future prospective study is needed to verify the prognostic value of this marker.

In vitro culture results showed that the IM proportion increased with culture time in both control and stimulated monocytes. Interestingly, IL-10 induced a high proportion of IMs and a correspondingly low proportion of CMs in comparison with LPS or no treatment. Moreover, IL-10 treatment tended to decrease TF and increase TM, although the difference was minimal. Given that IL-10 is an anti-inflammatory cytokine, these actions are thought to be counter-responsive to the inflammatory stimuli. Our suggestion is in good agreement with a previous report in which the alternative activation of monocytes by IL-10 induced a phenotype that promoted tissue repair and suppressed inflammation [14]. On the other hand, TF expression in all monocyte subpopulations increased in the LPS-treated group, as observed in other studies [13, 24, 27]. An elegant study reported that TF mRNA levels in leukocytes increased during DIC [28]. In our clinical results, TF expression was not a significant marker except in CM, in which low TF expression predicted poor prognosis. It is currently unclear why low TF expression represents poor prognosis. In our data, the TF expression between overt and non-overt DIC was not different, although in vitro culture suggested that LPS induced the expression of both TF and TM. In the in vitro experiment, monocytes from healthy individuals were stimulated with an inflammatory stimulus (LPS), reflecting the basic modulation of TF and TM expressions by an inflammatory insult. However, the studied population is a heterogeneous group even in the overt or non-overt DIC group; thus, the result may not show a clear-cut difference between the overt and the non-overt groups. TM expression did not differ significantly between the three monocyte subpopulations, but LPS treatment upregulated TM at 2 hours in CMs and at 12 to 24 hours in IMs. We [29] and another group [30] previously reported that LPS downregulated TM expression in monocytes. However, we could not demonstrate LPS-induced TM downregulation. We speculate that the difference in expression may be a result of different culture conditions. Previous experiments used a culture of PBMCs that included high numbers of lymphocytes [29, 30], and this potentially produces amounts of inflammatory cytokines that can affect the TM level. In this experiment, we used purified monocytes that contained low numbers of lymphocytes. Upregulation of TM may contribute to the regulation of coagulation by promoting activated protein C, thus suggesting a defense mechanism against the development of extensive microvascular fibrin deposition during DIC. However, as shown in our clinical study, insufficient TM function is expected in monocytes.