The S100A10 Pathway Mediates an Occult Hyperfibrinolytic Subtype in Trauma Patients

Abstract

Objective: 

To determine the characteristics of trauma patients with low levels of fibrinolysis as detected by viscoelastic hemostatic assay (VHA) and explore the underlying mechanisms of this subtype.

Background: 

Hyperfibrinolysis is a central component of acute traumatic coagulopathy but a group of patients present with low levels of VHA-detected fibrinolysis. There is concern that these patients may be at risk of thrombosis if empirically administered an antifibrinolytic agent.

Methods: 

A prospective multicenter observational cohort study was conducted at 5 European major trauma centers. Blood was drawn on arrival, within 2 hours of injury, for VHA (rotation thromboelastometry [ROTEM]) and fibrinolysis plasma protein analysis including the fibrinolytic mediator S100A10. An outcomes-based threshold for ROTEM hypofibrinolysis was determined and patients grouped by this and by D-dimer (DD) levels.

Results: 

Nine hundred fourteen patients were included in the study. The VHA maximum lysis (ML) lower threshold was determined to be <5%. Heterogeneity existed among patients with low ML, with survivors sharing similar clinical and injury characteristics to patients with normal ML values (5–15%). Those who died were critically injured with a preponderance of traumatic brain injury and had a 7-fold higher DD level (died vs. survived: 103,170 vs. 13,672 ng/mL, P < 0.001). Patients with low ML and high DD demonstrated a hyperfibrinolytic biomarker profile, low tissue plasminogen activator levels but high plasma levels of S100A10. S100A10 was negatively correlated with %ML (r = −0.26, P < 0.001) and caused a significant reduction in %ML when added to whole blood ex-vivo.

Conclusions: 

Patients presenting with low ML and low DD levels have low injury severity and normal outcomes. Conversely, patients with low ML but high DD levels are severely injured, functionally coagulopathic and have poor clinical outcomes. These patients have low tissue plasminogen activator levels and are not detectable by ROTEM. S100A10 is a cell surface plasminogen receptor which may drive the hyperfibrinolysis in these patients and which when shed artificially lowers %ML ex-vivo.

Trauma patients rapidly develop an acute traumatic coagulopathy (ATC) which is associated with poor outcomes including transfusion requirements, critical care utilization, and death.^1–3 Hyperfibrinolysis is a central characteristic of ATC^4,5 and early empiric antifibrinolytic administration has been shown to improve overall survival.^6–8 There is controversy as to whether antifibrinolytics should be administered empirically or given only to patients with diagnostic evidence of hyperfibrinolysis.^9,10 This controversy centers on the potential risks of inducing a thrombotic event in patients without active fibrinolysis and the role of viscoelastic hemostasis assays (VHAs) in the diagnosis of hyperfibrinolysis.¹¹ This confusion has led to a wide variation in antifibrinolytic practice worldwide.

We have previously shown that a large number of patients with biomarker evidence of hyperfibrinolysis are not detected by rotation thromboelastometry (ROTEM) viscoelastic devices.¹² Around 60% of trauma patients had an “occult” hyperfibrinolysis with elevated plasmin–antiplasmin (PAP) and D-dimer (DD) levels, increased mortality and blood product utilization, but no evidence of hyperfibrinolysis on ROTEM viscoelastic assay. Several studies have subsequently identified that high mortality is associated with a hypofibrinolytic pattern on thromboelastography and that this represents a group of patients in whom fibrinolysis has been switched off.^13,14 This fibrinolysis “shutdown” pattern appears to be the most common fibrinolytic phenotype and the implication is that administration of antifibrinolytics to these patients could induce arterial or venous thrombotic events and death. It remains unclear though whether all low VHA detected lysis is abnormal; whether this is a heterogeneous group not differentiated by VHA; what are the underlying mechanisms leading to these different patterns of fibrinolysis; and which are associated with poor outcomes.

We hypothesized that not all low levels of fibrinolysis as detected by VHA were pathological. Our first aim was to determine whether low VHA-fibrinolysis represented a homogeneous or heterogeneous group of patients. If heterogeneous, we aimed to determine which patients had poor outcomes and to understand how they might be identified within the whole low VHA-fibrinolysis cohort. We further aimed to characterize the injury characteristics and outcomes of these patients, to shed light on the mechanism of this form of trauma-induced coagulopathy. We conducted an analysis of clinical data and samples from trauma patients prospectively enrolled into our platform study of trauma-induced coagulopathy.

METHODS

The study was first approved by East London and The City Research Ethics Committee (07/Q0603/29) and then subsequently by each local ethics committee and conducted according to the Declaration of Helsinki. Deferred written informed consent was obtained from each patient or their next of kin. Blood samples from healthy volunteers taking no regular medications were obtained after written informed consent (07/Q0702/24).

Study Design

A prospective multicenter observational cohort study, the Activation of Coagulation and Inflammation in Trauma (ACIT) study (UK CRN ID 5637), was conducted at 5 European major trauma centers in London, Oslo, Copenhagen, Amsterdam, and Cologne. All centers are members of the International Trauma Research Network. Patients were excluded from ACIT if they arrived >2 hours postinjury; were transferred from another hospital; received more than 2000 mL crystalloid prehospital; or had sustained burns of over 5% of their body surface area. Patients were retrospectively excluded if they declined to give consent to the use of their research samples, had severe liver disease, a known pre-existing bleeding diathesis, or were taking anticoagulant medication (excluding aspirin) preinjury. Of patients in the ACIT study recruited between January 2008 and July 2014, all adults (>15 yrs) who were either severely injured (ISS ≥ 15), shocked (lactate ≥ 2 mEq/L or base deficit ≥ 4 mEq/L), coagulopathic (international normalized ratio [INR] > 1.2 or ROTEM EXTEM CA5 ≤ 35 mm) or transfused ≥ 4 packed red blood cell (PRBC) units within the first 12 hours and did not receive tranexamic acid (TXA) had multiple biomarker assays performed as part of the Targetted Action for Curing Trauma Induced Coagulopathy program of work and are analyzed in this study.¹⁵

Data Collection

Research personnel at each center screened and enrolled patients. Data were collected prospectively and included patient demographics, time of injury, mechanism of injury (blunt or penetrating), Injury Severity Score (ISS), vital signs on-scene and on arrival in the emergency department (ED), total number of blood products and volume of intravenous fluids administered within the first 12 hours from injury. Patients were observed for 28 days from injury for the occurrence of venous thromboembolic events (deep vein thrombosis or pulmonary embolism), and overall mortality.

Blood Sampling

The baseline research blood sample was drawn within 20 minutes of the patient's arrival in the ED along with standard trauma laboratory tests. A conventional coagulation screen was performed for the determination of prothrombin time (PT) and INR and a point-of-care arterial blood gas analysis performed simultaneously for base deficit calculation.

Blood for ROTEM analysis was collected in a 2.7 mL citrated vacutainer (0.109 Molar/3.2% sodium citrate; Becton, Dickinson and Company, Plymouth, UK). Blood for coagulation and fibrinolysis protein assays was collected in a 4.5 mL glass citrated vacutainer (0.109 Molar/3.2% sodium citrate; Becton, Dickinson and Company, Plymouth, UK). The filled 4.5 mL vacutainer was centrifuged within 1 hour of collection and double-spun plasma subsequently stored at −80°C.

Functional Coagulation Analysis

Functional coagulation analysis was performed within 1 hour of blood drawn at 37°C on a ROTEM delta instrument (Tem International GmbH, Munich, Germany) using the automated electronic pipette according to the manufacturer's instructions. The methodology and parameters of ROTEM have been described previously.¹⁶ The EXTEM assay, measuring tissue-factor initiated clotting, was run for 60 minutes and the maximum lysis (ML) determined. For spiking experiments, recombinant S100A10 (Abbexa, Cambridge, UK) or vehicle was added to the ROTEM cup prior to the start of the test.

Coagulation and Fibrinolysis Plasma Protein Assays

Plasma stored at −80°C was thawed to 37°C immediately before all analyses. Prothrombin fragment 1 + 2 (prothrombin time Frag 1 + 2; Enzygnost F 1 + 2 (monoclonal); Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany), tissue plasminogen activator (tPA; Asserachrom tPA, Diagnostica Stago, Asnières sur Seine, France), plasminogen activator inhibitor-1 (PAI-1; Asserchrom PAI-1; Diagnostica Stago), plasmin-α2-antiplasmin complex (PAP; PAP micro enzyme-linked immunosorbent assay; DRG Instruments GmbH, Marburg, Germany), urokinase (uPA, Abcam, UK) and S100A10 (Abbexa, Cambridge, UK) were measured using sandwich enzyme-linked immunosorbent assays. Fibrinogen levels (Siemens Thrombin reagent, Sysmex UK) and α2-antiplasmin (Siemens Berichrom α2-antiplasmin; Sysmex UK) were determined in the hospital laboratories with a Sysmex CS2100i automated analyzer (Sysmex, UK) according to standard protocols. Latex immunoassays were used to quantify the levels of D-dimer (Siemens Innovance D-dimer; Sysmex, UK) also with the Sysmex CS2100i automated analyzer.

Definitions

Major hemorrhage was defined as administration of at least 4 units of PRBCs within 12 hours of admission, and massive hemorrhage as at least 10 PRBC units in 12 hours. Severe traumatic brain injury was defined as a brain abbreviated injury score of 3 or higher. Coagulopathy was defined as an INR > 1.2. We defined VHA hyperfibrinolysis as an ML >15%.¹²

Statistical Analysis

Statistical analysis was performed using GraphPad Prism version 5 (GraphPad Software Inc, San Diego, CA) and Microsoft Excel 2013 (Microsoft Inc, Redmond, USA). Normal quantile plots were used to test for normal distribution. Nonparametric data are expressed as median (interquartile range) and analyzed using Mann–Whitney U test or Kruskal–Wallis test with Dunn multiple comparison test. Percentages were analyzed using Chi-squared or Fisher exact tests. Correlation between continuous variables was assessed with Spearman correlation coefficient. Trends in survival were compared with Log-rank (Mantel-Cox) test. A P value of < 0.05 was considered statistically significant.

RESULTS

A total of 2465 subjects were enrolled in the ACIT study during the recruitment period across the 5 study sites. Of these, 1318 patients met the inclusion criteria and were eligible for enrolment into this study. Twenty-five patients coenrolled in the CRASH-3 trial were excluded, along with 169 who received TXA, 196 who did not have a baseline ROTEM performed and 14 who did not have a DD measured, leaving 914 patients for analysis.

Determination of ROTEM Maximum Lysis Thresholds for hypofibrinolysis

We first determined our study threshold for low VHA fibrinolysis as this has not been previously defined for ROTEM (Fig. 1). A histogram of mortality for each % ML suggested a lower inflection point at less than 5% and below (Fig. 1A). As confirmation a receiver operating characteristic curve for mortality was generated using patients with a ML value of ≤15%. The point of ML <5.5% had the optimal performance by the Youden index with a sensitivity of 61.6% and specificity of 58.4%. Mortality was doubled in patients with ML <5% (15 vs. 7.1% for ML 5–15%, P<0.001). We therefore defined ML_LOW as ML <5%; ML_NORMAL as ML 5–15%; and ML_HIGH as ML>15%.¹² These 3 groups of patients showed a U-shaped mortality curve, similar to previous studies of hypofibrinolysis in trauma^13,14 (Fig. 1B).

FIGURE 1: Patients exhibiting VHA hypofibrinolysis below ML 5% have an increased overall mortality, incidence of coagulopathy, and red cell transfusion requirement compared with patients with normal fibrinolysis. High D-dimer levels differentiate those patients with the worst clinical outcomes irrespective of ML value. A, Histogram of 28-day mortality for each % maximum lysis (ML) on admission showing a lower inflection point at ML < 5% (dashed line). B, Overall 28-day mortality showed a U-shaped distribution when patients were grouped according to admission ML value. ML_LOW < 5%; ML_NORMAL 5 to 15% and ML_HIGH >15%. ∗∗∗P < 0.001 versus ML_NORMAL. C, D-dimer levels were significantly elevated in those patients who died compared with survivors, irrespective of ML group. ∗∗∗P < 0.001 comparing survived versus died within each ML group. D, Histogram of arrival coagulopathy (INR > 1.2) incidence for each % ML on admission showing a lower inflection point at ML < 5% (dashed line). E, The incidence of coagulopathy demonstrated a U-shaped distribution similar to mortality. ∗∗∗P < 0.001 versus ML_NORMAL. F, Patients with coagulopathy on admission had significantly higher D-dimer levels compared with patients without coagulopathy, irrespective of admission ML. ∗∗∗P < 0.001 comparing coagulopathy versus no coagulopathy within each ML group. G, Histogram of major hemorrhage (requirement of ≥ 4 red blood cell units in first 12 h) for each % ML on admission suggesting a similar lower inflection point at ML < 5% (dashed line). H, Presence of major hemorrhage demonstrated a similar U-shaped distribution. ∗∗P < 0.01, ∗∗∗P < 0.001 versus ML_NORMAL. I, Patients treated for major hemorrhage had significantly higher D-dimer levels irrespective of admission ML value. ∗∗∗P < 0.001 comparing major hemorrhage versus no major hemorrhage within each ML group. Box-whisker plots represent median, interquartile range with 5th and 95th percentiles.

Characterization of Patterns Associated With Mortality in the ML_LOW Cohort

To explore the heterogeneity of the ML_LOW group, we began by examining the differences between ML_LOW patients who lived and those who died. The survivors in this group had very similar admission characteristics, injury patterns, and outcomes to ML_NORMAL patients. The ML_LOW survivors were statistically older, more shocked and more severely injured, but these differences were small (Table 1). Their outcomes in terms of VTE, massive transfusion rates, critical care, and hospital lengths of stay were the same as for ML_NORMAL patients. In contrast, the ML_LOW patients who died were much older, critically injured (median ISS 29), had lower GCS than any other cohort (median 7) and 85% of them had a traumatic brain injury (Table 1).

TABLE 1: Characteristics of the Study Cohort

ML_LOW patients who died were nearly 3 times as likely to receive a massive transfusion and were 4 times as likely to be coagulopathic on admission (63% INR >1.2) than ML_LOW patients who survived (Table 1). Despite the low ML, DD levels in patients who died were extremely high (median 103,170 ng/mL), compared with 13,672 ng/mL in survivors (P < 0.001, Table 1, Fig. 1C). These DD differences were replicated when analyzing coagulopathic versus noncoagulopathic patients and red cell transfusion requirements (Fig. 1D–I). For further analysis we therefore set a DD threshold at 30,000 ng/mL (approximately the upper quartile in ML_NORMAL patients) and used this as a biological marker to explore the heterogeneity within the ML_LOW patient cohort.

More than half (58%) of patients with ML_LOW on admission had low DD levels. Patients with ML_LOW and DD_LOW were essentially identical to patients who were ML_NORMAL in terms of injuries, admission physiology, and outcomes (Table 2). The only significant difference between the 2 groups was that ML_LOW + DD_LOW patients were more likely to have received prebaseline crystalloid (51 vs. 37%, P = 0.002).

TABLE 2: Characteristics and Outcomes of Patients Grouped by Fibrinolytic Profile

Characteristics of Patients Presenting With Low ML But High DD Levels

Mortality was 30% in the ML_LOW + DD_HIGH cohort compared with only 3% in the ML_LOW + DD_LOW group (P < 0.001, Table 2). Deaths occurred later in the ML_LOW + DD_HIGH cohort compared with ML_HIGH + DD_HIGH patients, who tended to die on the day of admission (Fig. 2A, Table 2). ML_LOW + DD_HIGH patients were more severely injured overall (median ISS 29) and were more than twice as likely to have a severe head or torso injury as the ML_LOW + DD_LOW cohort. ML_LOW + DD_HIGH patients were also more shocked and coagulopathic (by INR) on admission and had greater fluid and blood product requirements in the first 24 hours than those with ML_LOW + DD_LOW. Overall ML_LOW + DD_HIGH patients appeared to be severely injured and bleeding and were more like the ML_HIGH + DD_HIGH group. The ML_HIGH + DD_HIGH patients had a greater severity of shock and much higher transfusion requirements, but were less likely to have sustained a severe traumatic brain injury (Table 2).

FIGURE 2: Patients with ML_LOW + DD_HIGH demonstrate increased mortality and have a hyperfibrinolytic biomarker profile despite low levels of the plasminogen activators tPA and uPA. A, Kaplan–Meier curves for survival in patients grouped by fibrinolytic profile. B, Prothrombin fragment (PF 1 + 2) levels across all maximum lysis (ML) and D-dimer (DD) groups with the highest levels seen in the ML_LOW + DD_HIGH patients. C, Plasmin-α2-antiplasmin complex (PAP) across all ML and DD groups indicating high levels of fibrinolytic activity in patients with DD_HIGH irrespective of ML. D, Tissue plasminogen activator (tPA) across all ML and DD groups. Despite an overall hyperfibrinolytic biomarker profile, patients with ML_LOW + DD_HIGH did not exhibit the rise in tPA seen in those patients with ML_HIGH + DD_HIGH. E, Urokinase-type plasminogen activator (uPA) levels were similar across all ML and DD groups. F, Plasminogen activator inhibitor (PAI-1) levels were similar across all ML and DD groups. ^P < 0.05, ML_LOW + DD_LOW vs ML_NORMAL; ^∗ P < 0.05, ^∗∗ P < 0.001, ML_LOW + DD_LOW versus ML_LOW + DD_HIGH; ⁺ P < 0.05, ⁺⁺ P < 0.001, ML_LOW + DD_HIGH versus ML_HIGH + DD_HIGH. Box-whisker plots represent median, interquartile range with 5th and 95th percentiles.

Coagulation Profiles of Patients With Low ML But High DD Levels

We examined the fibrinolytic mechanisms in these patient cohorts (Fig. 2B–F). Across all low D-dimer cohorts, prothrombin fragment (PF1 + 2), fibrinogen, plasmin-antiplasmin complex (PAP), tPA, urokinase-type plasminogen activator (uPA), and PAI-1 levels were the same.

Patients with ML_LOW + DD_HIGH had the highest levels of prothrombin fragments of all cohorts, associated with a reduction in fibrinogen levels (Fig. 2B, Supplementary Table 2, https://links.lww.com/SLA/B393). As expected, they also had high PAP levels indicative of fibrinolytic activity (Fig. 2C). However, surprisingly, tPA levels in patients with ML_LOW + DD_HIGH were low and comparable to the ML_NORMAL and ML_LOW + DD_LOW cohorts (Fig. 2D). Plasma levels of uPA were the same across all groups, as were PAI-1 levels (Fig. 2E, F). Antiplasmin levels were low in the ML_LOW + DD_HIGH group (72 vs. ML_NORMAL 102 u/dL, P < 0.001) and even lower in ML_HIGH + DD_HIGH patients (median 42.0 u/dL, P < 0.001 vs. ML_NORMAL – Supplementary Table 2, https://links.lww.com/SLA/B393).

Mechanism of Occult Hyperfibrinolysis in Trauma Patients

ML_LOW + DD_HIGH patients therefore had high thrombin generation and high fibrinolytic activity but no elevations in the expected plasminogen activators nor increased levels of plasminogen/plasmin inhibitors. We therefore proceeded to measure levels of S100A10, a principally membrane-bound mediator of fibrinolysis in a subgroup of 179 patients. Clinical characteristics and coagulation profiles were similar to the main cohort (supplemental Tables 1 and 2, https://links.lww.com/SLA/B393). Plasma S100A10 levels were low in the DD_LOW cohorts but significantly elevated in both the ML_LOW + DD_HIGH and ML_HIGH + DD_HIGH patients (Fig. 3A). Plasma S100A10 levels were strongly correlated with both PAP (r = 0.58, P < 0.001) and DD (r = 0.56, P < 0.001) levels (Fig. 3B, C). S100A10 levels were strongly associated with injury severity (Fig. 3D). In particular, patients with TBI (abbreviated injury score Head 3 + ) had much higher plasma levels of S100A10 than those without (S100A10: 437 vs. 55 pg/mL, P < 0.001). TBI patients with higher plasma S100A10 (above the median 120 pg/mL) had a 58% mortality, compared with only 6% for those with low S100A10 levels (P < 0.001), as well as a higher incidence of coagulopathy (31 vs. 0%, P = 0.009) and PRBC transfusion (38 vs. 6%, P = 0.01).

FIGURE 3: S100A10 is associated with occult hyperfibrinolysis but reduces clot breakdown on viscoelastic testing. A, Levels of S100A10 in trauma patients grouped by fibrinolytic profile. ^∗∗∗ P < 0.001 versus ML_NORMAL, Mann–Whitney U test. B and C, Relationship between S100A10 and plasmin-antiplasmin (PAP) complex levels (B) and D-dimer levels (C). S100A10 in quartiles; ^∗ P < 0.05 ^∗∗ P < 0.01 ^∗∗∗ P < 0.001 versus Q1, Kruskal–Wallis test with Dunn multiple comparisons test D: S100A10 levels in patients with mild (ISS 1–4), moderate (ISS 5–15), severe (ISS 16–25), and critical (ISS>25) injuries. ^∗∗ P < 0.01 ^∗∗∗ P < 0.001 versus ISS 1–4, Kruskal–Wallis test with Dunn multiple comparisons test. E, Effect of S100A10 on maximum clot lysis (ML) measured by viscoelastic testing. Rotational thromboelastometry (ROTEM) was performed in tissue factor-stimulated blood samples from healthy volunteers (n = 6) in the presence of recombinant S100A10 (10 ng/mL) or vehicle. Lines indicate mean. ^∗∗∗ P < 0.001, paired t test. F, Clot lysis on ROTEM in trauma patients from ML_LOW and ML_NORMAL groups stratified by S100A10 quartiles. ^∗ P < 0.05 ^∗∗ P < 0.01 ^∗∗∗ P < 0.001 versus Q1, Kruskal–Wallis test with Dunn multiple comparisons test (B, C, and F). Box-whisker plots represent median, interquartile range with 5th and 95th percentiles.

As S100A10 is known to bind tPA, we further hypothesized that S100A10 would bind tPA in the ROTEM cup, thus reducing ex-vivo plasmin generation and therefore the VHA-measured fibrinolysis. We added 10 ng/mL S100A10 to the whole blood of 6 healthy volunteers and saw a significant reduction in %ML (Fig. 3E). In the ML_LOW and ML_NORMAL combined cohort, plasma S100A10 levels negatively correlated with %ML (r = −0.26, P < 0.001, Fig. 3F) but were still positively correlated with PAP (r = 0.56, P < 0.001) and DD levels (r = 0.28, P < 0.001). There was no relationship between plasma levels of S100A10 and plasma tPA levels.

DISCUSSION

In this multicenter study of over 900 patients, we have shown that low levels of fibrinolysis as measured by VHAs are physiological in over half of all cases and have a mortality rate and transfusion requirement which is comparable to patients with normal VHA levels of fibrinolysis. Conversely, a subgroup of patients with low VHA detected fibrinolysis are severely injured, functionally coagulopathic, have high transfusion requirements and a 30% mortality. These patients have high levels of thrombin, plasmin, and DD generation but have low levels of plasminogen activators including tPA. We have identified S100A10 as a possible explanation for localized activation of plasmin on cell surfaces in these patients which when shed into the plasma artificially lowers the observed VHA maximum lysis parameter ex-vivo. Together these results support the existence of an occult hyperfibrinolysis in severely injured patients, not detectable by existing diagnostic tools and which is associated with very poor outcomes.

S100A10 is a membrane-bound plasminogen receptor which complexes with Annexin 2 in a hetero-tetramer formation to bind tPA and plasminogen on the cell surface in close proximity to the uPA receptor.^17–19 S100A10 is widely expressed in body tissues and particularly in the brain^20,21 and the complex is upregulated at times of hypoxic stress.²² S100A10 levels are detectable in the blood but the majority of cellular plasmin is formed on the endothelium with S100A10 catalyzing the tPA mediated activation of plasminogen.^23,24 The classical presentation of Acute Promyelocytic Leukaemia is a hemorrhagic and fibrinolytic phenotype characterized by normal levels of tPA but high surface expression of S100A10 on Acute Promyelocytic Leukaemia cells.²⁵ Our findings suggest that S100A10 is exposed by tissue injury leading to plasmin generation and hyperfibrinolysis without the need for high levels of circulating tPA.²⁶ These findings suggest a mechanism that may contribute to the catastrophic coagulopathy often seen with severe traumatic brain injury, the severity of which is not apparent on existing diagnostic tests.

Plasmin has a very short half-life and S100A10 is known to increase plasmin autoproteolysis.²⁴ Ex-vivo detection of fibrinolysis by VHA devices relies on the presence of plasmin to lyse clot forming in the measuring cup. We have shown that the addition of S100A10 to blood reduces VHA-detected lysis. In patients with low tPA levels there is no other available activator of plasminogen ex-vivo and no fibrinolysis will be detected. When tPA levels are high, the effect of S100A10 will be overcome and fibrinolysis will be revealed. These findings are entirely consistent with all observed studies of hyperfibrinolysis in trauma patients.

There has been controversy in the literature as to whether ATC patients with low maximum lysis but high D-dimer levels (ML_LOW + DD_HIGH) have ongoing hyperfibrinolysis or have had their fibrinolysis shutdown at some time immediately before blood sampling. These patients are severely injured and have large on-going blood product requirements. They have a high incidence of coagulopathy with an underlying biochemistry of high PAP and DD levels. Taken together these features suggest active hyperfibrinolysis. It is difficult to support a fibrinolysis shutdown model that would require explanation of how tPA levels could have suddenly become low given that the active pathological processes of injury, shock and bleeding have not yet been controlled and PAI-1 levels are not elevated. Localized hyperfibrinolysis via cell-bound activation of plasminogen is a more plausible mechanism and matches the observed on-going bleeding and transfusion requirements in these patients. This occult hyperfibrinolysis in ATC does not preclude the likely existence of a later fibrinolytic shutdown in trauma patients due to upregulation of PAI-1 and other control mechanisms.

Tranexamic acid's effective reduction of mortality in trauma patients appears to extend beyond reducing blood loss and death from bleeding to affect all-cause mortality.^6,27 Our results suggest that severe tissue injury can induce hyperfibrinolysis through S100A10 and tranexamic acid may therefore reduce local as well as systemic hemorrhage. In traumatic brain injury this might have a profound effect on outcome and we found S100A10 levels correlated closely with coagulopathy, blood use and mortality in severe TBI patients. S100A10 may provide the mechanistic rationale for the use of tranexamic acid in TBI and the results of on-going clinical trials are anticipated soon.^28,29 Overall, our results confirm an occult hyperfibrinolysis which is prevalent in trauma patients and supports the empiric use of tranexamic acid until new diagnostics are available. Restricting treatment only to patients with VHA detected lysis may exclude 80% of trauma patients with high DD levels who might benefit from tranexamic acid.

The inclusion criteria for this study prevent us from determining the absolute incidence of each of these subtypes of TIC. As patients with minor injuries and without shock were excluded, overall rates of hyperfibrinolysis are likely to be overestimates compared with a general trauma population. As the ML_LOW + DD_HIGH S100A10-driven subtype is essentially exclusive to blunt trauma, its incidence will also vary depending on rates of blunt trauma seen in specific institutions. Our exclusion of patients who had already received tranexamic acid is also likely to have led to an underestimate of the numbers in the ML_HIGH subtypes and contributed to the relatively low incidence of massive transfusion in this cohort. There are several aspects of this work that require further experimental research. This clinical study cannot definitively state that the ML_LOW + DD_HIGH patients still have active fibrinolysis after the point of sampling. Our finding of high circulating levels of the primarily tissue-bound fibrinolytic activator S100A10 is circumstantial, but coupled with grossly elevated PAP & DD and no obvious inhibitors, is highly suggestive of active lysis, as are subsequent high blood product requirements. It will require both experimental evidence of active lysis and clinical trial evidence of the effect of TXA in this subgroup to confirm this. The mechanisms of exposure and release of S100A10 also need exploring, especially in the context of traumatic brain injury. Again, both clinical and experimental studies will be required to understand the role of S100A10 in trauma-induced coagulopathy.

We have shown that VHA-detected lysis (ML) is a poor indicator of both the fibrinolytic state and clinical outcomes of patients. Absolute DD levels were more strongly associated with outcomes. Patients with low DD levels had good outcomes regardless of ML. In contrast a specific phenotype of patients exists with elevated DD but low ML who have poor outcomes. These patients are critically injured and have a high incidence of severe traumatic brain injury. We have identified the tissue-bound S100A10 fibrinolytic mediator as a candidate mechanism for hyperfibrinolysis in these patients that also results in an artificially low ML. S100A10 exposure after critical injury represents a new precision marker and therapeutic opportunity in trauma-induced coagulopathy.