Euglobulin Clot Lysis Time Reveals a High Frequency of Fibrinolytic Activation in Trauma

Abstract

Activation of the fibrinolytic system plays a central role in the host response to trauma. There is significant heterogeneity in the degree of fibrinolysis activation at baseline that is usually assessed by whole blood thromboelastography (TEG). Few studies have focused on plasma markers of fibrinolysis that could add novel insights into the frequency and mechanisms of fibrinolytic activation in trauma. Global fibrinolysis in plasma was assessed using a modified euglobulin clot lysis time (ECLT) assay in 171 major trauma patients and compared to commonly assessed analytes of fibrinolysis. The median ECLT in trauma patients was significantly shorter at 8.5 hours (IQR, 1.3–19.5) compared to 19.9 hours (9.8–22.6) in healthy controls (p<0.01). ECLT values ≤2.5^th percentile of the reference range were present in 83 (48.5%) of trauma patients, suggesting increased fibrinolytic activation. Shortened ECLT values were associated with elevated plasmin-antiplasmin (PAP) complexes and free tissue plasminogen activator (tPA) levels in plasma. Sixteen (9.2%) individuals met the primary outcome for massive transfusion, here defined as the critical administration threshold (CAT) of 3 units of packed red cells in any 60-minute period within the first 24 hours. In a univariate screen, plasma biomarkers associated with CAT included D-dimer (p<0.001), PAP (p<0.05), free tPA (p<0.05) and ECLT (p<0.05). We conclude that fibrinolytic activation, measured by ECLT, is present in a high proportion of trauma patients at presentation. The shortened ECLT is partially driven by high tPA levels and is associated with high levels of circulating PAP complexes. Further studies are needed to determine whether ECLT is an independent predictor of trauma outcomes.

Introduction

Trauma-induced coagulopathy (TIC) occurs as an early response to severe trauma, and is associated with a poor prognosis [1]. TIC is a clinical entity with a complex pathophysiology that includes a spectrum of imbalances between the procoagulant, anticoagulant and fibrinolytic systems [2]. Antifibrinolytic agents have been shown to be effective in reducing blood loss and improving mortality when administered in the early phase of management of patients with severe trauma [3]. There is significant heterogeneity in the reported incidence and degree of activation of the fibrinolytic system in response to trauma [4–7]. This has led to wide variation in practice patterns in both the definition of the state of fibrinolysis and the subsequent use of antifibrinolytic agents [4–8]. Towards establishing consensus, the International Society on Thrombosis and Haemostasis (ISTH) has recently highlighted a pressing need for additional biomarker studies to improve the mechanistic understanding of fibrinolysis in trauma and to streamline laboratory-based definitions [2].

Thromboelastography (TEG) and rotational thromboelastometry (ROTEM) are viscoelastic whole blood assays that can function as a rapid point of care test to detect spontaneous clot lysis in severe acute trauma. TEG, at currently investigational clinical cutoffs, has been used to define three separate phenotypes of fibrinolysis in whole blood; a) hyperfibrinolysis, b) physiologic fibrinolysis, and c) fibrinolysis shutdown. Each phenotype has postulated mechanisms, differing transfusion requirements and prognostic value [6]. However, the performance of TEG to identify trauma patients with hyperfibrinolysis has been questioned by some investigators, and a subgroup of patients with “occult hyperfibrinolysis” (not detectable by TEG) has been proposed [4,8]. To better identify the cohort of patients with occult hyperfibrinolysis, there is increasing interest in the incorporation of plasma biomarkers of fibrinolysis, such as plasmin-antiplasmin (PAP) complexes or D-dimer into the laboratory definition of fibrinolytic activation [7,8]. Most studies have focused on analytes with rapid turnaround time; however, few studies have attempted to correlate these biomarkers with global plasma-based assays of fibrinolysis such as the euglobulin clot lysis time (ECLT) which is still sometimes considered to be the ‘gold standard’ measure of fibrinolysis [9,10].

Using available samples from a prospective observational study at a single Level 1 Trauma Center in the United States, we describe the distribution of a panel of plasma-based biomarkers of fibrinolysis including ECLT in trauma patients at the time of presentation, and demonstrate the presence of fibrinolytic activation in a higher proportion of patients than suspected using viscoelastic assays.

Methods

Study Population

All trauma patients transported to the Mayo Clinic Emergency Department (ED) by ambulance or air transport, as a Level 1 or 2 trauma activation, from February 2011 to June 2014 were assessed for suitability for enrollment into a parent longitudinal cohort study assessing thrombin generation kinetics as predictors of symptomatic venous thromboembolism after trauma [11]. All patients with available plasma samples remaining after completion of the primary study were included in this sub-cohort analysis. Patients who received tranexamic acid before or after sample collection were not analyzed in this study. The study was approved by the Mayo Clinic Institutional Review Board and patient consent was a pre-requiste for enrollment into the parent study.

Clincal Variables

In accordance with the protocol-driven enrolment into the parent study, recorded data included baseline demographics, baseline and time-dependent clinical characteristics, including systolic and diastolic blood pressure (SBP and DBP, respectively) in the Emergency Department, injury severity score (ISS), type of injury (blunt or penetrating), traumatic brain injury (TBI) diagnosis and severity, patient age and sex, body mass index (BMI) and all-cause mortality. Transfusion therapy was based on Mayo Clinic Trauma Center transfusion guidelines as directed by the medical provider.

Bleeding Definitions

To reduce the potential for both survivor and immortal time bias that is present in some prior publications that utilized total transfusion within 24hrs of injury, we used the critical administration threshold (CAT) definition as defined by Savage et al., i.e. receipt of 3 or more packed red blood cell units in any 60-minute interval in the initial 24 hour period [12]. The patients were dichotomized into either attainment of CAT or not.

Sample Collection

Blood samples from the 171 patients included in this study were collected into 3.2% sodium citrate tubes on admission to the Mayo Clinic Trauma Center. All samples were obtained within a 2-hour window after trauma, and before administration of any antifibrinolytic or transfusion therapy. For 129 of these patients, the results from point of care TEG performed on non-anticoagulated whole blood were also available. In addition, control samples from 114 healthy individuals were collected at Mayo Clinic or at the University of North Carolina. For all samples, platelet free plasma (PFP) was prepared by double centrifugation at 3,000g for 15 minutes at room temperature within 1 hour of collection. PFP aliquots were then stored at −80°C, until analysis.

Reagents

Human plasminogen-free fibrinogen was purchased from Aniara Diagnostica (West Chester, OH, USA). Ovalbumin (98% purity, grade V) was purchased from Millipore-Sigma (St. Louis, MO, USA). Re-lipidated tissue factor (TF) (Dade Innovin, B4212–40 Siemens Healthcare, Erlangen, Germany), acetic acid (glacial, 100%, reagent grade), mineral oil (light, C₁₆H₁₀N₂Na₂O₇S₂, reagent grade), and 96-well microplates (polystyrene, flat bottom, non-tissue culture treated plate, Falcon) were purchased from Fisher Scientific (Hampton, NH, USA). Human alpha thrombin was purchased from Haematologic Technologies (Essex Junction, VT, USA). Recombinant tissue-type plasminogen activator (tPA) was from Innovative Research (Novi, MI, USA). Distillated water (diH₂O) was prepared using a Direct Q3 system (Millipore-Sigma, St. Louis, MO, USA). Tris Buffered Saline (10x, 500 mM Tris, 1500 nM NaCl) was prepared in house and the pH was adjusted to 7.4. Synthetic phospholipid vesicles (PS:PE:PC ratios of 15:41:44) were a gift from Dr. Dougald Monroe, University of North Carolina. For kinetic optical density measurements, a Synergy H1 spectrophotometer (BioTek, Winooski, VT, USA) was used.

Global assays of fibrinolysis.

For all samples, two separate assays were used to evaluate global fibrinolysis, namely the euglobulin clot lysis time (ECLT) and the tPA challenged clot lysis time (tPA-CLT). The ECLT is a sensitive tool that detects endogenous hyperfibrinolysis, whereas the tPA-CLT measures clot lysis initiated by the addition of tPA to clotted plasma. We have previously described the test characteristics as they are performed in our laboratory_, as well as the advantages and disadvantages of the two approaches [13,14].

[A]. Euglobulin clot lysis time.

In short, to obtain the euglobulin fraction of plasma, PFP was diluted 1:10 in dH₂O and acidified to pH 5.9, followed by incubation for 30 min on ice. After centrifugation (1,000xg, 6 minutes), the supernatant was removed, and the residue was dissolved to ½ of the original plasma volume in tris-buffered saline. The euglobulin fraction of plasma was then mixed with plasminogen-free fibrinogen (250 µg/ml), ovalbumin (4%) and clotted with thrombin (17.6 nM). To prevent desiccation of the sample, mineral oil was added to the top of each well. Optical density changes were then read in a microplate reader (λ=405 nm, 37°C) for 24 hours. The euglobulin clot lysis time (ECLT – representing the time from half maximal clotting to half maximal lysis) was the primary end point.

B] The tPA-challenged clot lysis assay (tPA-CLT)

was performed in 96-well microplates, and turbidity was read in a spectrophotometer (λ 405 nm, 37^◦C), as previously described[13,14]. In brief, coagulation and fibrinolysis were simultaneously initiated in PFP by re-calcification, addition of tissue factor (TF, 1 pM), PS:PC:PE phospholipid vesicles (20µM) and tPA (0.625 nM). The low concentration of tPA was chosen for its increased sensitivity to hypofibrinolysis [14], but requires a prolonged (24 hour) reading time. To prevent the clot from drying out, mineral oil was added on the top of each well. The tPA-CLT (the time from half maximal clotting to half maximal lysis) was recorded as the primary endpoint.

For both the ECLT and tPA-CLT assays, data were obtained using Gen5 software and processed using Shiny apps developed by Dr. Colin Longstaff [15].

In addition, for 129 of these patients, results of a point of care TEG using kaolin as a trigger in non-anticoagulated whole blood were available. The TEG 5000 model was utilized according the standard operating procedure per the manufacturer (Haemonetics®). Patients were stratified into TEG-defined fibrinolysis phenotypes using previously defined cutoffs [6], as follows: a) hyperfibrinolytic - spontaneous clot lysis at 30 minutes (Ly30) >3%, b) physiologic fibrinolysis, defined by Ly30 0.9–3.0%, and c) fibrinolytic shutdown, defined by Ly30 values <0.9%.

C]. ELISAs.

Plasma free tPA and plasmin-α₂antiplasmin (PAP) complexes were detected using commercially available kits from Molecular Innovations (Novi, MI, USA). S100A10 kits were from Abbexa, Cambridge, UK. Plasma D-dimer levels were measured using the Abcam ELISA kits (Cambridge, UK).

Statistical Analysis

All continuous variables are described as medians with interquartile ranges for non-parametric data or means with standard deviation for normally distributed data. Categorical variables are expressed as percentages. Comparison between median ECLT or tPA-CLT in the trauma cohort versus normal controls was done using the Mann Whitney U test. Comparison between baseline clinical characteristics and achievement of the CAT stratified by ECLT-defined hyperfibrinolysis (≤ 4.6 hours, equivalent to the 2.5^th centile of healthy controls) was performed using the Mann Whitney U test for continuous variables, and the Fisher’s exact test for categorical variables. A univariate screen using logistic regression was used to calculate the odds ratio for CAT with 95% confidence interval for each of the candidate plasma-based fibrinolysis biomarkers, as well as TEG Ly30 and ISS. All analyses were conducted in IBM SPSS Statistics version 26.0.0.0 (IBM Inc, Armonk, NY), GraphPad Prism version 7.05 (GraphPad Software Inc, San Diego, CA).

Results

Clinical Characteristics

Plasma samples were available for 171 individuals. The median age was 52 years, with an ISS of 14 at presentation. The baseline characteristics and plasma biomarkers of the trauma patients are demonstrated in Table 1. The majority of patients were admitted due to blunt trauma (n=159), while 12 patients had penetrative injuries. TBI was diagnosed in 68 (39.8%) patients, 53 of whom fully recovered without disability. In total, 16 (9.2%) of these subjects met the critical administration threshold (CAT) of blood product usage. There was one death during follow up.

Table 1:

Baseline Characteristics of Trauma Patients

Variables	Trauma patients	Missing
	(n=171)	(n)
Sex Male
(n,%)	125 (73.1%)	0
ISS
Median (IQR)	14 (9–22)	4
Age (years)
Median (IQR)	52 (30–63)	0
SBP (mmHg)
Mean±SD	118 ± 26.3	1
DPB (mmHg)
Mean±SD	71 ± 16.3	5
HR (bpm)
Mean±SD	96 ± 22.4	0
PAP (μg/ml)
Median (IQR)	4.06 (2.44–8.20)	3
D-Dimer (μg/ml)
Median (IQR)	6.45 (1.69–16.85)	3
S100A10 (ng/ml)
Median (IQR)	1.88 (0.36–4.36)	3
free tPA (IU/ml)
Median (IQR)	0.35 (0.24–0.54)	3
ECLT (hours)
Median (IQR)	8.50 (1.2–19.45)	0
tPA-CLT (hours)
Median (IQR)	1.73 (1.48–2.71)	2
TEG LY30 (%)
Median (IQR)	1.0 (0.10–4.40)	40
CAT
(n,%)	16 (9.4%)	0

Abbreviations: CAT = critical administration threshold, ISS = injury severity score, SBP = systolic blood pressure, DBP = diastolic blood pressure, IQR = interquartile range, TEG = thromboelastography, tPA = tissue type plasminogen activator, PAP = plasmin-antiplasmin complex, ECLT = euglobulin clot lysis time, tPA-CLT = tPA-challenged clot lysis time

Fibrinolysis Biomarkers

The global assays of fibrinolysis in plasma i.e. ECLT and tPA-CLT, were both significantly different in trauma patients compared to healthy controls (Figure 1). As previously described [13,14], the range of ECLT values in healthy controls was very wide with a suggestion of a bimodal distribution (Figure 1). The median ECLT in trauma patients was 8.5 hours compared to 19.9 hours in controls (p<0.001). 83 of 171 trauma patients (48.5%) had ECLT values that were < 2.5^th percentile of the reference range in healthy controls, indicating a high frequency of fibrinolytic activation on presentation. The tPA-CLT assay revealed two sub-populations of patients, with clustering of patient values at both extremes. The median tPA-CLT in trauma patients was 1.73 hours, compared to 2.44 hours in controls (P<0.001). 62 of 169 (36.6%) trauma patients had clot lysis times <2.5^th percentile of controls. Only 4 (2.4%) trauma subjects had tPA-CLT values that were >97.5^th percentile of the reference population, suggesting similar proportions of the trauma and control populations that were ‘tPA-resistant’ according to this assay.

Figure 1.

Global assays of fibrinolysis in trauma subjects compared to healthy controls (NCs). The left-hand portion of the figure compares the euglobulin clot lysis time (ECLT) for normal controls and trauma patients. The right-hand portion demonstrates the plasma clot lysis times with exogenously added tissue-type plasminogen activator (tPA), again comparing trauma patients to controls. In the latter assay, the 2 missing data points are due to the plasma from 2 patients being opaque and non-evaluable. Abbreviations: CLT = clot lysis time, ****p<0.0001

The 2.5^th percentile of ECLT in normal controls corresponds to 4.6 hours. We used this arbitrary threshold to define trauma patients with evidence of hyperfibrinolysis. Compared to subjects with an ECLT ≥4.6 hours, individuals with ECLT <4.6 hours were of similar age, slightly more likely to be male (77% vs 70%, p=0.39), have a similar ISS, but no difference in lowest measured systolic or diastolic blood pressure or maximum heart rate on presentation (Table 2). However, notable differences in the levels of several plasma biomarkers of fibrinolysis were observed between the ECLT-defined ‘hyperfibrinolysis’ or ‘no hyperfibrinolysis’ cohorts (Table 2). Figure 2 demonstrates the distribution of these individual biomarkers stratified by ECLT-defined hyperfibrinolysis. Notably, subjects with ECLT <4.6 hours had more elevated free tPA levels (<0.01), higher PAP (p<0.01), shorter tPA-CLT (p<0.0001) and higher D-dimers (p=0.046).

Table 2:

Baseline clinical characteristics, biomarker analyses and outcomes stratified by ECLT

	ECLT
Variables	<4.6 hours (N=83)	≥4.6 hours (N=88)	p value
Sex Male			0.39
(n, %)	64 (77%)	61 (70%)
ISS			0.38
Median (IQR)	14.0 (9.0–22.0)	14 (6–22)
Age (years)			0.31
Median (IQR)	53 (30–63)	50 (28–62)
SBP (mmHg)			0.91
Mean±SD	117.6±29.27	118.1±23.18
DBP (mmHg)			0.87
Mean±SD	70.1±16.6	70.0±16.20
Heart rate (bpm)			0.07
Mean±SD	93.2±23.1	98.6±21.6
PAP (µg/ml)			<0.01
Median (IQR)	5.6 (2.6–10.3)	3.7 (2.2–5.4)
D-Dimer (µg/ml)			0.046
Median (IQR)	10.0 (2.4–20.5)	4.3 (1.1–13.2)
S100A10 (ng/ml)			0.59
Median (IQR)	1.9 (0.5–4.4)	1.6 (0.3–4.5)
free tPA (IU/ml)			0.01
Median (IQR)	0.36 (0.25–0.72)	0.31 (0.24–0.41)
tPA-CLT (hours)			<0.0001
Median (IQR)	1.6 (1.3–1.9)	2.5 (1.6–4.2)
TEG LY30 (%)			0.155
Median (IQR)	1.3 (0.2–5.4)	0.9 (0.0–3.4)
CAT
n (%)	13 (15.7%)	3 (3.4%)	0.01

Abbreviations: CAT = critical administration threshold, ISS = injury severity score, SBP = systolic blood pressure, DBP = diastolic blood pressure, TEG = thromboelastography, tPA = tissue type plasminogen activator, PAP = plasmin-antiplasmin complex, ECLT = euglobulin clot lysis time, tPA-CLT = tPA-challenged clot lysis time

Figure 2.

Association of individual fibrinolysis biomarkers with global hyperfibrinolysis defined by ECLT in trauma patients. The plasma levels of PAP, D-dimer, tPA-CLT and free tPA are plotted for trauma patients with or without ECLT-defined hyperfibrinolyis (<4.6 hours or >4.6 hours, respectively). Abbreviations: ECLT = euglobulin clot lysis time, PAP = plasmin-antiplasmin complexes, tPA-CLT = tissue type plasminogen activator challenged clot lysis time, tPA = tissue type plasminogen activator. Data are shown as median and IQR with whiskers showing 2.5–97.5th percentile range, *p<0.05; **p<0.01, ****p<0.0001

TEG data were available on 129 (77%) subjects. According to accepted criteria for TEG-defined fibrinolytic phenotypes [6,8], 58 (45%) presented with a ‘hyperfibrinolytic’ phenotype, 34 (26%) demonstrated ‘physiologic fibrinolysis’ and 37 (29%) had ‘fibrinolysis shutdown’(Figure 3). These proportions are similar to previously reported cohorts (6). Of interest however, subjects with TEG-defined hyperfibrinolysis only partially overlapped with ECLT-defined hyperfibrinolysis (Figure 3).

Figure 3.

Distribution of ECLT-defined hyperfibrinolysis subgroup according to TEG-defined fibrinolysis phenotypes. TEG fibrinolysis phenotypes were assigned to each of the 129 trauma patients according to the Ly30 value (x-axis). Within each of these 3 categories, proportion of patients with ECLT-defined status is superimposed. Patients with ECLT-defined hyperfibrinolysis are shown in black-stripes bars; those without are represented by grey bars. “n” represents the number of patients in each TEG defined group. <4.6 hours = ECLT less than 4.6 hours. Abbreviations: TEG - thromboelastography

We did not observe any difference in global assays of fibrinolysis between patients with blunt versus penetrative injury, although plasma levels of D-Dimer were dramatically higher in the blunt injury group compared to those with penetrative injury (15.52±22.79 vs. 9.57±30.23 ug/ml ug/ml, mean±SD, p<0.001) (Figure 4). No association between ECLT and trauma severity by ISS was found. However, patients who were diagnosed with severe TBI with resultant disability demonstrated more hyperfibrinolysis by ECLT compared to patients with no TBI (5.57±6.92 hours vs. 11.22±9.39 hours, mean±SD, p<0.05).

Figure 4.

Type of trauma (A-C) and traumatic brain injury (D-F) influence on euglobulin clot lysis time (ECLT), free tPA and D-Dimer. Box plots show median ± IQR; whiskers 2.5–97.5th percentile range. TBI – traumatic brain injury. ns – nonsignificant *p<0.05; **p<0.01

Finally, we evaluated whether the difference in ECLT between trauma patients and controls remained after adjustment for the time of sample draw, since diurnal variation is known to influence fibrinolytic activity [16,17]. Normal control samples were drawn during the workday, predominantly in the morning hours (8–11am). When ECLT values from normal controls were compared to trauma patients grouped according to time of blood draw, the difference remained highly significant in the 6am to midnight ranges (6am-noon p<0.001, noon-6pm p<0.0001, 6pm-midnight p<0.001) (Figure 5). However, no significant difference was observed between normal controls and patients from whom blood was drawn between midnight and 6am.

Figure 5.

Dependence of euglobulin clot lysis time (ECLT) on time of blood draw. Box plots show median ± IQR; whiskers 2.5–97.5th percentile range. NC- normal controls. **-p<0.01, ***- p<0.001, **** - p<0.0001.

Association with CAT

As an exploratory analysis, we performed a univariate screen for biomarker association with the CAT outcome. We found that several clinical and laboratory biomarkers were strongly associated with CAT including ISS (p<0.0001), systolic and diastolic blood pressure (p<0.001 and p<0.05, respectively), D-dimer (p<0.001), PAP (p<0.05), free tPA (p<0.05) and ECLT (p<0.05) (Supplementary Table 1). Of the 11 subjects with available TEG data who also met the CAT transfusion threshold, 4 had TEG-defined hyperfibrinolysis, 2 demonstrated physiologic fibrinolysis, and 5 were in the fibrinolysis shutdown category. When compared to those with fibrinolysis shutdown, there was no difference in attainment of CAT in those with TEG-defined physiologic fibrinolysis (OR 0.67 (95% CI, 0.09–4.35), p=0.64) or hyperfibrinolysis (OR 1.33 (95% CI, 0.31–5.39), p=0.68).

Discussion

This study addressed the hypothesis that plasma-based assays may help to define the frequency and mechanisms of fibrinolytic activation in trauma. Examining a panel of fibrinolytic biomarkers in plasma samples obtained on presentation, we demonstrated that a global assay of fibrinolysis (the ECLT) reveals a high prevalence of fibrinolytic activation. Furthermore, ECLT-defined hyperfibrinolysis in trauma patients was associated with elevated plasma levels of free tPA and PAP complexes. Finally, by the criteria we used, almost half of the patient cohort would be considered to have ECLT-defined hyperfibrinolysis, but in poor agreement with TEG-defined hyperfibrinolysis.

To our knowledge, this is the largest cohort of trauma patients for whom the degree of activation of fibrinolysis was assessed using the ECLT. We were surprised to find a poor correlation between the hyperfibrinolysis phenotype defined by TEG and that defined by the ECLT. Specifically, ECLT-defined hyperfibrinolytic subjects were detected in all three TEG-defined fibrinolysis categories. Furthermore, in our exploratory analyses, TEG had poor discrimination for identifying individuals reaching the CAT threshold. However, larger scale studies are needed to define the relative strength of association of the ECLT and viscoelastic whole blood assays with massive transfusion outcomes.

Viscoelastic whole blood assays have been widely adopted in the clinical management of trauma [18]. The portability and rapid sample turnaround, combined with a point of care utility are major assets that have contributed to their widespread adoption. In addition, the holistic nature of viscoelastic assays as a tool to detect the contribution of all the elements of both the cellular and non-cellular blood components to clot formation and subsequent lysis further add to their appeal. Several groups have defined the range of fibrinolysis parameters on TEG or ROTEM that have been used to define thresholds for the administration of antifibrinolytic agents and/or transfusion in acutely injured patients [18,19]. However, it is generally acknowledged that standardization of viscoelastic instruments is a major challenge [20–22]. Others have questioned whether TEG is sensitive or specific enough to define the pathophysiologic aspects of endogenous fibrinolysis, and/or to direct the need for anti-fibrinolytic therapy [7,22,23]. As a first step to reconciling these controversies, we undertook this exploratory study to compare traditional plasma-based assays of global fibrinolysis to TEG-reported parameters in a cohort of trauma patients.

This study has several important limitations. First, not all consecutive samples from the primary study were available for analysis. We utilized available samples from the biorepository at Mayo Clinic which were collected to address a different hypothesis [11]. As we do not have complete information on the patients for whom samples were not available, the possibility of selection bias remains. Second, the number of patients who required massive transfusion – the clinical endpoint of interest – was limited. We are thus mindful that our analyses exploring the associations with CAT are exploratory, due to lack of power and hence the risk for a type 2 error. Third, we excluded patients who received TXA to avoid its influence on the plasma assays. The use of TXA is likely correlated with more severe trauma; thus, the generalizability of our findings to patients receiving TXA prior to their ED assessment is limited. Fourth, in contrast to previously published work [4,24,25], we used a different outcome to define massive transfusion. Fifth, due to the limited volume of available plasma samples, we were not able to measure other fibrinolytic analytes, including PAI1. Sixth, blood samples were processed at room temperature, which has been shown to be non ideal for measuring endogenous fibrinolytic activity [26]. Finally, a potential limitation of the study is the 2-hour window between the traumatic event and sample collection, which compares to TEG assessment within 15 minutes of injury in some studies [27]. Since it has been observed that the TEG-defined fibrinolytic phenotype is dynamic and rapidly evolving in many patients [27], it is possible that we may have underestimated the number of patients with an initial ‘hyperfibrinolytic’ phenotype.

The use of global assays of fibrinolysis such as the ECLT has fallen out of favor due to the impracticalities of obtaining rapid clinically actionable information, a requirement that is particularly pertinent in trauma. Unlike traditional plasma coagulation assays that can be completed in seconds, plasma assays for fibrinolysis are slow and usually need to be performed over the course of hours. We also acknowledge other limitations for each of the global fibrinolysis assays. In the ECLT, plasminogen and plasminogen activators are enriched relative to natural inhibitors. This modification therefore renders the assay less sensitive to hypofibrinolysis. On the other hand, the tPA-CLT assay does not exclude the dominant fibrinolytic inhibitors, but essentially tests the sensitivity of a plasma clot to exogenously added tPA. We speculate that these methodologic distinctions could therefore explain the lack of an observed association between the tPA-CLT and CAT with transfusion outcome, despite the skewing of lysis times towards more rapid clot lysis in the trauma cohort compared to controls. However, further studies are required to test this hypothesis.

Based on previously defined percentiles for the ECLT in healthy subjects [13,14], almost 50% of the current subjects with trauma had ECLTs that were below the 2.5^th percentile of the normal range, indicating that detectable fibrinolytic activation is likely highly prevalent in trauma. However, the clinical relevance of this finding is as yet unclear. While these plasma-based assays in their current format are clearly unsuitable for point of care adaptation, they do provide unique and otherwise unattainable information on the fibrinolytic process following trauma, and do have the advantage that they can be standardized and performed on batched, frozen plasma aliquots. As such, larger studies are needed to explore this approach in the expectation that novel pharmacologic targets can be identified and approaches developed to mitigate severe hemorrhage in trauma.

Highlights.

1. The euglobulin clot lysis time (ECLT) is a global assay of fibrinolysis that detects a high frequency (49%) of hyperfibrinolysis in trauma patients at the time of presentation.

2. An abnormally shortened ECLT in trauma is associated with elevated free tPA, plasmin- antiplasmin complexes and D-dimer levels.