Temporal phenotyping of circulating microparticles after trauma: a prospective cohort study

The dynamic process of coagulation after traumatic injuries may deteriorate towards a trauma-induced coagulopathy (TIC) in which the early platelet dysfunction has a pivotal role [1]. Besides platelets, small cell-derived subcellular vesicles are suggested mediators of coagulation playing a crucial role at the conjunction of the cellular and plasmatic coagulation, circulating in both, healthy and diseased individuals [2, 3]. Although platelet-derived MP (PDMP) are the most abundant of all circulating MP, endothelial- and monocytes-derived MP (EDMP, MDMP) also participate in cellular signalling, coagulation and haemostasis by inducing platelet aggregation or developing a platelet- and fibrin-rich thrombus [3‐6]. In detail, the procoagulative effects of MPs are mediated inter alia by externalized phospholipids such as phosphatidylserine (PS, while not all MP are PS-positive), tissue factor or von-Willebrand factor (vWF) [6, 7]. The role of MP as major coagulation stimulant became evident as ex-vivo generated PDMP were estimated having 50- to 100-fold higher procoagulant activity as activated platelets [8].

With respect to traumatic injuries, the comparability of studies on MP measurements remains challenging due to heterogeneity of trauma severity and injury pattern, the patient’s coagulation status, and the occurrence of TIC. For this reason, high level of both, procoagulant PS-positive PDMP and red-cell derived MP (RDMP) were reported by Curry et al., while Windelov and colleagues determined lower level of PS-PDMP in trauma patients who were characterized by impaired coagulation [9, 10]. Likewise, Matijevic and colleagues reported that the traumatic injury and the occurrence of TIC differentially affect MP quantity. Patients with TIC have significantly lower counts of MP than patients without. This observation was accompanied by a shift in the PDMP:EDMP ratio from healthy individuals (100:1) and trauma patients (6:1) and thus, increased EDMP, RDMP and leukocyte-derived MP (LDMP) as well as PDMP level tending to decrease after trauma when compared to healthy controls [11]. However, Balvers et al. measured lower PDMP and comparable EDMP and RDMP level after trauma compared to healthy individuals [12].

In this study, we determine MP quantity after polytrauma capturing a size range between 0.3 to 0.9 μm by a highly sensitive flow cytometry assay. The present study aimed to determine how (i) MP levels differ in polytraumatic patients from those in minor injured and healthy individuals, (ii) MP distribution changes over time (up to 72 h post-injury) and (iii) MP levels correlate with the coagulation systems displayed by functional coagulation assays.

This observational study could demonstrate that

MPs, which are released from various cell types, have been described in both, diseased and healthy individuals. The MP majority (> 80%) derive from platelets, while smaller quantities derive from endothelial cells and leukocytes [2]. In the analysed healthy individuals, this relation of PDMP to EDMP could be reproduced. Variations in MP’s concentration or cellular origin have been reported for various medical conditions [11, 15]. However, recent literature is split over the role of MP following major trauma. As well increased unspecified MP and PDMP levels were reported as others determined decreased values following trauma [9‐12]. More consistently was reported, that patients with impaired coagulation or TIC show lower levels of PS-PDMP [10, 11]. Interestingly, while Matijevic et al. observed an increase of EDMP levels independently of the presence of TIC, Park et al. found EDMP only in one of 59 injured patients [11, 16]. In comparison to the presented data, previous studies included patients with a wide range and in mean lower injury severity [9, 10, 12]. Focusing on most severely injured patients (median ISS 26.5) and comparing them to minor injuries and healthy individuals, we could demonstrate that PDMP and EDMP levels increase depending on and correlating to the injury severity. Particularly, positive correlations between the ISS and endothelial MP determined by CD144 and CD62e could be shown by the present study indicating that these markers might serve as promising biomarker for injuries of the epithelium and injury severity. Similarly, CD144⁺ vesicles had been suggested as potential marker for endothelial injuries in other clinical settings [17].

In healthy individuals only low levels of circulating EDMP were detectable. Following polytrauma, we observed significant 8.5 and 13.1-fold EDMP increased compared to monotrauma patients and healthy individuals, which is in line with the PROMMTT cohort in which 20-fold higher EDMP numbers were measured [11]. This endothelial reaction to trauma appears double-edged on their influence on the clot formation. On the one hand, as shown the present study, EDMP correlated negatively with the INTEM clotting time and PS⁺-EDMP even correlated negatively with aPTT and INR (Table 2). In addition to TF⁺- and PS⁺-bearing PDMP, which are known to have a higher procoagulant activity than platelets [18], our results show the EDMP’s procoagulant potential. Previously, it has been described that an increasing concentration of EDMP shortens the plasma clotting time in a presumable TF-/FVII-dependant manner [6, 19]. Jy and colleagues could demonstrate that a subset of EDMP carry unusually large vWf multimers, promoting platelet aggregation and increasing the stability of the aggregates formed [5]. On the other hand, PS-negative EDMP correlated negatively with FIBTEM A10 and FIBTEM MCF, displaying a certain anticoagulant effect. Lacroix et al. described that in pathological settings EDMP bear tissue plasminogen activator and therefore postulated EDMP as “a circulating source for fibrinolysis” [20]. In addition, subsets of EDMP have membrane-bound endothelial Protein C receptors [21]. Consequently, activated membrane-bound Protein C retains its anticoagulant activity [21], which is in line with findings by Matijevic and colleagues, as higher EDMP levels correlated negatively with thrombin generation [11]. However, we could not observe this association in the presented study as an impaired thrombin generation could be expected to affect EXTEM values. Nevertheless, MP and especially EDMP express both, activators and inhibitors of coagulation, and therefore seem to stabilise the haemostatic balance [22].

As recommended in current guidelines, preventing the development and treating TIC is of outmost importance for patients’ outcome following severe trauma [23]. Since increased MP levels seem to improve haemostasis and might represent an endogenous compensation mechanism, the additional administration of pro-haemostatic agents might lead to spontaneous clotting resulting in thromboembolic events. Aside from trauma, elevated levels of circulating MP are associated with thrombotic events in cancer patients [24, 25]. However, in the present cohort we observed only one thromboembolic event 14 days after trauma. As patients were treated according to current guidelines and guided by viscoelastic testing, the risk of adverse events due to increased MP levels seems manageable. In contrast, MP levels might be helpful determining the point when thromboprophylaxis is indicated. Furthermore, the administration of artificial MP might become an additional tool in haemostatic therapy. Nevertheless, this remains fully speculative and further studies have to clarify MPs’ signalling and detailed function.

However, several PDMP populations correlated positively with 28 days mortality, which is in contrast to previous studies [9‐11]. In a cohort analysed by Curry et al., mortality correlated negatively with PS-positive PDMP values [9]. Going into detail of the fatal cases in the presented study, two patients died early after trauma showing low MP values, while one patient, having the highest overall MP values, died 14 days after trauma by pulmonary embolism. Therefore, it appears obvious, that the presented cohort might be too small to draw any further conclusions regarding patient outcome. Interestingly, besides the mentioned studies suggesting a protective effect of MP due to effects on the coagulation system [9‐11], a recent study could demonstrate reduced MP levels in patients with ARDS (Acute Respiratory Distress Syndrome) [26].

As could be expected, PDMP and EDMP levels were increased at hospital admission of trauma patients. Comparable to a previous study, we could not observe any statistically significant trend of MP levels over 72 h after trauma [27]. Although our trauma cohort was more concise regarding injury severity compared to the patients analysed by Park et al., still differences in injury pattern and in the early in-hospital treatment might contribute to this MP development [27].

Several limitations have to be admitted interpreting the presented study. The results derive from a small cohort, but in comparison to previous studies focusing on the relevance of MP following trauma, we were able to include comparably serious injured patients. Certainly, a larger cohort might have led to more significant results regarding the PDMPs procoagulatoric potential. Furthermore, the presented conclusions are based on correlations, which do not provide causal evidence. Although general trends in the posttraumatic increase of MPs are comparable to previous studies, absolute numbers differ in part distinctively [7, 9‐12, 27]. This underlies the importance to strive for a standardisation in pre-analytical variables and flow-cytometric protocols according to the recommendations by the International Society on Thrombosis and Haemostasis [13].

In conclusion, this study could demonstrate that PDMP and EDMP levels increase significantly following polytrauma correlating with injury severity. Although severe coagulopathy was not observed, EDMP levels were associated with improved coagulation parameters suggesting their essential for regulating blood coagulation after trauma. However, further studies analysing the procoagulative and fibrinolytic potential are needed.