Histone-driven hypercoagulation contributes to the lethal triad of acute trauma-induced coagulopathy

Abstract

Severe tissue injury and hemorrhagic shock can result in trauma-induced coagulopathy (TIC), acidosis, and hypothermia, denoted as lethal triad. This condition exacerbates trauma complications and contributes to organ dysfunction and mortality, prompting the need to better define its complex pathomechanisms in the blood. Therefore, we developed a standardized ex vivo human whole blood (hWB) model to differentiate the impact of various pathophysiological conditions, damage-associated molecular patterns (DAMPs), and key inflammatory mediators on TIC development. Modelling incremental grades of the lethal triad, we assessed the resulting thrombo-coagulopathy by monitoring platelet activation, clot formation, and mediator release. Both acidosis and hypothermia independently triggered platelet activation (with enhanced surface CD62P/CD63 expression) and platelet consumption, leading to ex vivo clot formation. Completing the lethal triad with additional dilutional coagulopathy revealed even more pronounced effects. Further dissecting underlying cross-talk mechanisms, platelet-rich plasma (PRP) or isolated platelets exposed to similar conditions exhibited no relevant impact on platelet function. Notably, histones-unlike HMGB1, pro-inflammatory cytokines or anaphylatoxins-amplified the lethal triad-driven hypercoagulative TIC response in both hWB and PRP. In conclusion, our findings in a modular, clinically relevant whole blood model highlight histones as a central driver of TIC. Furthermore, tracking early TIC progression in this model provides an effective and efficient platform for evaluating novel therapeutic approaches to target drivers of coagulopathy.

Keywords: Coagulopathy; Hemostasis; Platelets; TIC; Trauma.

Fig. 1

Coagulopathic effects of different grades of acidosis on platelets in whole blood and PRP. (A) Protocol for modelling acidosis in whole blood (WB) and platelet rich plasma (PRP). (B) Analyzed read-out parameters for platelet activation ex-vivo. CD62P is marker for alpha granule exocytosis, CD63 denotes dense (δ) granule secretion. Surface CD154 (CD40L) is released as truncated sCD154 upon platelet activation. (C) Acidosis in ex-vivo whole blood leads to a significant platelet activation, as evident by dropping platelet counts and increasing plasma concentrations of sCD154 and von-Willebrandt factor (vWF), whereas acidosis had no such effect in PRP (D). (E) Flow cytometry analyses of CD62P surface expression on platelets after 30 min incubation of acidotic WB showed no relevant pre-activation and sustained reaction capacity to post-incubation stimulation with 5 µM ADP at low- and midgrade acidosis (°I/II), but marked activation und loss of reaction capacity after °III acidosis. CD63 expression, which is not inducible by 5 µM of ADP, was slightly increased after °III acidosis, as was CD154 expression. (F) Platelets from acidotic PRP showed no pre-activation in terms of CD62P/CD63/CD154 expression, but a slightly enhanced, grade-dependent reaction to subsequent ADP (5 µM) stimulation. (G) ROTEM analyses showed no relevant alterations of intrinsic or extrinsic hemostasis at low-/mid-grade acidosis but revealed significant impairment of clot formation and stability under severe acidotic conditions already at baseline and even more so after 30 min of incubation ex vivo. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns: not significant; MPC: 2-methacryloyloxethyl phosphorylcholine; FPX: fondaparinux; MCF: mean clot firmness; MFI: medium fluorescence intensity. For better visibility, statistically non-significant differences were only marked, if of relevance for the experimental hypothesis.

Fig. 2

Pro-coagulative effects of hypothermia, acidosis and dilution on platelets in whole blood. Whole blood (hWB) was incubated at 30° and 34° respectively and coagulopathic effects of hypothermia with and without Acidosis °II and 3:7 dilution (jonosteril) were compared with normothermic controls (CTRL). The middle panel displays absolute platelet counts, the lower panel shows counts normalized to hemoglobin concentration to subtract diluting effects. Hypothermia at 34 °C exhibited hypercoagulative effects comparable to °II acidosis. This effect was abrogated at severe hypothermia (30 °C). At mild hypothermia (34 °C), 3:7 dilution with crystalline solution caused a slight drop in platelet counts beyond the mere diluting effect, with in-tube clot formation in 1 sample. This effect was abolished at 30 °C. Acidosis at severe hypothermia caused a more consistent drop in platelets counts but less clotting-formation in tubes after 30 min, suggesting an impaired hemostatic capacity of hWB at lower temperatures. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns: not significant. For better visibility, statistically non-significant differences were only marked, if of relevance for the experimental hypothesis.

Fig. 3

Hypercoagulative effects of the lethal triad in hWB and PRP. (A) Protocol for the lethal triad (LT) in human whole blood (hWB) and platelet rich plasma (PRP). LT caused time- and grade-dependent hypercoagulation, with incremental clot formation ex vivo, which was also reflected in decreased relative platelet counts and slightly increased vWF plasma concentration (B). As samples with higher grade acidosis and later time-points showed substantial in-tube clotting, only small volumes of plasma could be retrieved. (C) in PRP, i.e. in the absence of cellular immunity, LT conditions had no pro-coagulative effect. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns: not significant; MPC: 2-methacryloyloxethyl phosphorylcholine; FPX: fondaparinux; Hb: hemoglobin. For better visibility, statistically non-significant differences were only marked, if of relevance for the experimental hypothesis.

Fig. 4

Coagulopathic effect of histones on lethal triad-driven TIC. (A) Ex vivo incubation of hWB with 50 µg/ml histones ex vivo caused clot-formation in a fraction of samples. When incubated under LT conditions, histones caused overwhelming platelet activation and clot formation. (B) The hypercoagulative effect was slightly less pronounced when incubated under sole hypothermic or acidotic conditions, respectively. (C) The effect of histones showed no relevant concentration-dependency in hWB. (D) In PRP, the hypercoagulative effect of 50 µg/ml histones was similarly substantial, with (E) some concentration-dependency. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns: not significant; MPC: 2-methacryloyloxethyl phosphorylcholine; FPX: fondaparinux; Hb: hemoglobin. For better visibility, statistically non-significant differences were only marked, if of relevance for the experimental hypothesis.