INCREASED PLATELET MITOCHONDRIAL FUNCTION CORRELATES WITH CLOT STRENGTH IN A RODENT FRACTURE MODEL

Abstract

Background:

Thromboelastographic measures of clot strength increase early after injury, portending higher risks for thromboembolic complications during recovery. Understanding the specific role of platelets is challenging due to a lack of clinically relevant measures of platelet function. Platelet mitochondrial respirometry may provide insight to global platelet function, but has not yet been correlated with functional coagulation studies.

Methods:

Wistar rats underwent anesthesia and either immediate sacrifice for baseline values [n=6] or (1) bilateral hindlimb orthopedic injury [n=12], versus (2) sham anesthesia [n=12] with terminal phlebotomy/hepatectomy after 24 hours. High resolution respirometry was used to measure basal respiration, mitochondrial leak, maximal oxidative phosphorylation, and Complex IV activity in intact platelets; Complex-I and Complex-II driven respiration was measured in isolated liver mitochondria. Results were normalized to platelet number and protein mass, respectively. Citrated native thromboelastography (TEG) was performed in triplicate.

Results:

Citrated native TEG maximal amplitude (MA) was significantly higher (81.0 ± 3.0 vs. 73.3 ± 3.5 mm, p<0.001) in trauma compared to sham rats 24 hours after injury. Intact platelets from injured rats had higher basal oxygen consumption (17.7 ± 2.5 vs. 15.1 ± 3.2 pmol/s*10⁸ cells, p=0.045), with similar trends in mitochondrial leak rate (p=0.19) when compared to sham animals. Overall, platelet basal respiration significantly correlated with TEG-MA (r=0.44, p=0.034). As a control for sex-dependent systemic mitochondrial differences, females displayed higher liver mitochondria Complex-I driven respiration (895.6 ± 123.7 vs. 622.1 ± 48.7 mmol e⁻/min/mg protein, p=0.02); as a control for systemic mitochondrial effects of injury, no liver mitochondrial respiration differences were seen.

Conclusions:

Platelet mitochondrial basal respiration is increased after injury and correlates with clot strength in this rodent hindlimb fracture model. Several mitochondrial-targeted therapeutics exist in common use that are underexplored but hold promise as potential antithrombotic adjuncts that can be sensitively evaluated in this preclinical model.

Background

Previous studies utilizing both static coagulation assays and dynamic thromboelastography (TEG) have reported the presence of early hypercoagulability in 85% of injured patients on admission, and early TEG-based hypercoagulability predicts later thromboembolic complications during recovery.^1–3 Of particular interest, Coleman et al. demonstrated persistent TEG maximal amplitude-based (TEG-MA) hypercoagulability present at 48 hours after injury, which was associated with greater risk for thrombotic complications.⁴ Similarly, both small and large animal preclinical trauma models have demonstrated emergence of early TEG-MA based hypercoagulability at 4–6 hours after injury that persists for at least 3 days and perhaps longer.^5,6 Despite current thromboprophylaxis strategies addressing enzymatic coagulation with heparinoids, TEG MA and TEG G-value have both shown to be independent predictors of thrombotic complications after orthopedic injury and surgical procedures,^7–9 and previous work by Kornblith et al. demonstrated that platelets are the major contributor to TEG-MA values early after injury.¹⁰ Biomarkers of platelet activation are known to significantly increase early and persistently in severely injured patients, suggesting higher activation and function of circulating platelets;¹¹ however, multiple aggregation-focused modalities have paradoxically demonstrated that circulating platelets have impaired aggregation in patients sustaining trauma, with increased dysfunction correlating with injury severity.^11,12 These conflicting findings highlight the limitations of existing assays used to evaluate clinically meaningful post-injury platelet biology.

Measurement of mitochondrial respiration in circulating platelets may be a useful assay focusing on agonist-independent platelet activation potential. Indeed, mitochondrial dysfunction has been implicated in multiple pathologic systemic processes, such as acute respiratory distress disorder and sepsis,^13,14 and has also been used to specifically evaluate the functional properties of donated platelets under various storage conditions.^15,16 Analysis of mitochondrial bioenergetics with high resolution respirometry (HRR) allows for direct assessment of aerobic respiration, with specific substrate and inhibitor protocols allowing for sensitive analysis of each electron transport chain (ETC) subunit. A thorough understanding of platelet mitochondrial bioenergetics after injury may provide important clinical prognostic information, more sensitively identify effects of antiplatelet agents, and suggest new targets for platelet-focused antithrombotic therapies.

In order to address this knowledge gap in post-injury platelet dysfunction, we aimed to evaluate the effects of injury on platelet mitochondrial respiration and TEG-based platelet function. While the vast majority of patients do not present with active hemorrhage, >45% sustain some form of orthopedic injury; therefore, we elected to use a previously validated and clinically relevant rodent model of hypercoagulability after isolated orthopedic injury⁶ to compare mitochondrial bioenergetics of circulating platelets to that of isolated liver mitochondria to evaluate for platelet-specific as well as potential systemic mitochondrial effects after injury. Compared to uninjured animals, we hypothesized that platelet mitochondrial respiration would be increased and less efficient after orthopedic trauma, while remote solid organ function would not be affected.

Methods

Animal Protocol

Thirty Wistar rats (16 male and 14 female) between 12 to 20 weeks of age were provided from a colony maintained at our institution. All experimental protocols were approved by the Institutional Animal Care and Use Committee and carried out in accordance with both the “Guide for the Care and Use of Laboratory Animals” from the National Institutes of Health and the guidelines of the Animal Welfare Act. Animals were housed 2 to 3 per cage at 22°C in a12-hour light/dark cycle and allowed free access to Teklad 8604 chow (Inotiv, Inc.; Chicago, IL) and water. The ARRIVE 2.0 guidelines for animal research were used for reporting of study design, conductance and results. (Supplement 1)

To obtain baseline values, an initial cohort of 3 male rats and 3 female rats (n=6) underwent inhalational anesthesia with 3% to 5% isoflurane, laparotomy and terminal blood draw via aortic puncture. Blood was collected in 3.2% sodium citrate and K₂EDTA (BD Vacutainer^®;Becton, Dickinson, and Company; Franklin Lakes, NJ) for TEG and high resolution respirometry (HRR), respectively. Complete hepatectomy was performed, and liver tissue placed in cold MSM buffer (220mM mannitol, 70mM sucrose, 5mM MOPS at pH 7.4).

A second cohort of both male and female (n=24) rats were anesthetized and subjected to either sham anesthesia (n=12; 6 male and 6 female) or bilateral hindlimb orthopedic injury (n=12; 7 male and 5 female), which involves (1) controlled soft tissue and muscular injury to muscle beds behind both femurs by 30-second crush with a clamp, (2) fibular fracture using a 14-gauge needle, and (3) injection of 1.5 mL of 0.5 g/mL bone homogenate to the injury site, followed by intraperitoneal injection of carprofen.⁶ Terminal blood and liver samples were obtained 24 hours after the initial procedure as described above.

Thromboelastography

After gently rocking for 30 minutes at room temperature, citrated “native” whole blood samples were recalcified and analyzed using TEG 5000 Hemostasis Analyzers (Haemonetics Corp.,Boston, MA) according to manufacturer’s instructions. Due to operator and enzymatic coagulation variability, samples were run in triplicate and averages used in data analysis. Parameters used for analysis include R time, K time, α-angle, maximal amplitude (MA), G value, and lysis percentage 30 minutes after MA (LY-30).

Platelet Isolation

EDTA tubes containing fresh whole blood were centrifuged at 300 × g for 15 minutes at 21°C. The resultant supernatant, containing a platelet rich plasma (PRP) was then pipetted off and centrifuged for 5 minutes at 4600 × g at 21°C, yielding a platelet pellet and nearly cell-free platelet poor plasma (PPP). The platelet pellet was then resuspended in 560μL of PPP to produce an “ultra-rich” platelet plasma (URPP). 60μL of this URPP was used to obtain the final platelet concentration using an automated Hemavet 950 Hematology System (Drew Scientific, Inc.; Miami Lakes, FL).

Intact Platelet High Resolution Respirometry

Oxygen consumption by isolated platelets was measured using an Oroboros FluoRespirometer (Oroboros Instruments, GmbH; Innsbruck, Austria). All experiments were performed at 37°C in 2mL glass chambers with the stirrer speeds set to 750 rpm. Data was recorded with DatLab 7 software (Oroboros) at two second intervals. Intact, non-permeabilized platelets (URPP) were suspended in a mitochondrial respiration medium (MiR05) containing EGTA (0.5mM), MgCl₂ (3mM), lactobionic acid (60mM), taurine (20mM), KH₂PO4₂ (10mM), HEPES (20mM), and D-sucrose (110mM) (Oroboros), with an average of 7.3 × 10⁷ platelets/chamber. After stabilization of basal O₂ consumption, supported by endogenous substrates and endogenous ADP, resting routine cellular respiration was recorded (Basal). Oligomycin (0.25μM, inhibiting flow of protons through ATP-synthase) was added to obtain a LEAK rate, or oxygen flux independent of ADP-phosphorylation. A maximal oxidative capacity (MaxOXPHOS) was measured after titration of an uncoupler (0.25μM FCCP per addition or 0.09μM valinomycin and 0.03μM nigericin per addition), until no further increase in respiratory rate was seen. We then added 0.5μM rotenone (Complex-I inhibitor) and 2.5μM antimycin-A (Complex-III inhibitor) to measure residual non-mitochondrial cellular respiration after electron transport chain shutdown (Non-Mito). Finally, Complex-IV capacity (CIV) was measured after the addition of 3mM ascorbate and 0.3mM N,N,N’,N’-tetramethyl-p-phenylenediamine (TMPD; see representative curve in Figure 1). All respiratory rates were normalized to 1 × 10⁸ platelets and reported as pmol O2/(s × 1×10⁸cell). To correct for background respiration, non-mitochondrial oxygen flux rates were subtracted from Basal, LEAK, MaxOXPHOS and CIV rates prior to analysis.

Figure 1.

Representative intact platelet respirometry tracing

Using an Oroboros O2k-FluoRespirometer, oxygen flux was directly measured to determine intact platelet mitochondrial respiration rates under various conditions. First, routine respiration (‘Basal’) was recorded after stabilization in the chamber. Oligomycin (0.25μM) was added to obtain a ‘LEAK’ rate, or cellular respiration independent of ADP-phosphorylation. Maximal oxidative capacity (‘MaxOXPHOS’) was measured after titration of an uncoupler (0.25μM FCCP per addition or 0.09μM valinomycin and 0.03μM nigericin per addition). We then added 0.5μM rotenone (Complex-I inhibitor) and 2.5μM antimycin-A (Complex-III inhibitor) to measure residual non-mitochondrial cellular respiration (‘Non-Mito’). Finally, Complex-IV capacity (‘CIV’) was measured after the addition of 3mM ascorbate and 0.3mM N,N,N’,N’-tetramethyl-p-phenylenediamine, or TMPD.

Liver Mitochondria Isolation

Liver mitochondria were isolated following previously established protocols with minor modifications.^17,18 In brief, liver tissue was washed in ice cold MSM buffer (220 mM mannitol, 70 mM sucrose, and 5 mM Mops at pH 7.4) then minced with a razor blade. The minced tissue was added to 20 ml ice-cold isolation buffer (MSM buffer supplemented with 2 mM EDTA and 0.2% fatty acid-free BSA) and homogenized in a 30 mL glass homogenizer and a Teflon pestle (pestle was shaved down by 0.1 mm in diameter) for 5 strokes. The homogenate was centrifuged at 300×g for 10 min at 4 °C. The supernatant was then centrifuged at 3000×g for 10 min at 4 °C, and the mitochondrial pellet was washed three times in ice-cold MSM. After final resuspension in ice-cold MSM, mitochondrial protein concentration was determined using the DC Protein assay (Bio-Rad Laboratories: Hercules, CA).

Isolated Liver Mitochondria High Resolution Respirometry

Liver mitochondria were analyzed with an Oroboros O2k-FluoRespirometer (O2k, Oroboros Instruments). Mitochondrial respiration was measured at 37°C in FAO buffer (10 mM KH₂PO₄, 5 mM MgCl₂, 30 mM KCl, 1 mM EDTA, 75 mM Tris, pH 7.5) by adding either 20 mM glutamate plus 5 mM malate or 20 mM succinate followed by 2 mM ADP (see representative curve in Figure 2A). The respiratory control ratio (RCR) was calculated as the ratio between ADP-stimulated (state 3) and resting (state 2) respiration rates. As a surrogate for reactive oxygen species (ROS) generation, mitochondrial hydrogen peroxide generation was measured simultaneously with oxygen consumption by using the Amplex red assay which includes horse radish peroxidase (HRP, 1 U/mL), superoxide dismutase (SOD, 5 U/mL), and Amplex Red (10 μM;^18,19 see representative curve in Figure 2B)

Figure 2.

Representative isolated liver mitochondria respirometry tracing

Using an Oroboros O2k-FluoRespirometer, oxygen flux was directly measured to determine isolated liver mitochondrial respiration rates of under various conditions. (A) After isolation, mitochondrial respiration was measured using an Oroboros O2k-FluoRespirometer at 37°C in FAO buffer (10 mM KH₂PO₄, 5 mM MgCl₂, 30 mM KCl, 1 mM EDTA, 75 mM Tris, pH 7.5) by adding either 20 mM glutamate plus 5 mM malate or 20 mM succinate (‘State 2) followed by 2 mM ADP (‘State 3’), depicted in the top image. (B) Mitochondrial hydrogen peroxide generation was measured simultaneously with oxygen consumption by using the Amplex red assay which includes horse radish peroxidase (HRP, 1 U/mL), superoxide dismutase (SOD, 5 U/mL), and Amplex Red (10 μM).

Statistical Analysis

All data was normally distributed, and therefore presented as mean ± SD. Comparisons between groups were performed using Student’s t test or two-way analysis of variance (ANOVA) with post-hoc Tukey’s test where appropriate. To assess for relationships between TEG and various HRR rates, Pearson’s correlation with t testing was performed. An α of 0.05 was considered significant. All data analysis was performed by the authors using Prism 9.0 (GraphPad Software, Boston, MA) and Stata 17.0 (StataCorp., College Station, TX)

Results

Rats used for baseline characteristics (n=6) were 12.9 ± 0.1 weeks old with an average weight of 274.4 ± 77.4g. Male rats were heavier (344 ± 16.7 vs. 204.8 ±13.8g, p<0.001), but there were no significant differences in age or glucose (both p>0.4). Given known sex dimorphism in both coagulation as well as mitochondrial function, we first evaluated for potential effects of sex as a biological variable. We found no differences in baseline thromboelastography measures of clot strength or stability (Figure 3A), with aggregate TEG MA and G-values measured at 71.8 ± 2.83mm and 12.9 ± 1.90 kd/sec, respectively. However, when evaluating isolated liver mitochondrial function, females were noted to have significantly higher Complex-I driven respiration (895.6 ± 123.7 vs. 622.1 ± 48.7 mmol e⁻/min/mg protein, p=0.02) with a similar non-significant trend in Complex-II driven respiration (1449 ± 336.2 vs. 1110 ± 393.8 mmol e⁻/min/mg protein, p=0.32); ROS production was similar (p=0.099 and p=0.40, respectively; Figure 3B). The clinical relevance of this enzyme-specific difference in liver mitochondrial function between sexes warrants further investigation outside the scope of our current study. No sex differences in baseline intact platelet mitochondrial respiratory rates between male and female rats were identified (Figure 3C; all p>0.43).

Figure 3.

Effects of sex as a biological variable on whole blood thromboelastography, isolated liver mitochondrial respirometry, and intact platelet mitochondrial respirometry

Whole blood, intact platelets, and isolated liver mitochondria were collected from anesthetized rats at baseline. (A) Citrated native thromboelastography (TEG) was used to assess maximal amplitude (MA) of clot formation and clot stability (G-value). (B) Isolated liver mitochondria were assessed for Complex-I and Complex-II driven respiration, as well as reactive oxygen species (ROS) generation. (C) Intact platelets were assessed for basal, leak, and maximal respiratory rates.

To evaluate for the effects of orthopedic injury, rats 16.7 ± 3.0 weeks old with an average weight of 301.3 ± 75.0g were used (n=23). There were no differences in age (17.3 ± 2.9 vs. 16.0 ± 3.0 weeks, p=0.31) or weight (305.5 ± 72.9 vs. 297.4 ±79.9g, p=0.8) between trauma and sham animals, but injured rats were found to have significantly lower glucose levels 24 hours after the initial procedure (113.9 ±14.8 vs. 140.8 ± 23.8 mg/dL, p=0.006). Thromboelastographic and respirometry findings are summarized in Table 1. Specifically, rats subjected to the orthopedic trauma model displayed significantly elevated MA (81.0 ± 3.0 vs. 73.3 ± 3.5mm, p<0.001) and G-value (22.2 ± 5.1 vs. 14.1 ± 2.6 kd/sec, p<0.001) compared to sham at 24 hours (Figure 4A); there were no differences in citrated native TEG R-time, K-time, alpha angle, or LY-30 (data not shown). To evaluate for systemic mitochondrial effects not specific to platelets, we performed HRR on isolated liver mitochondria in sham- and trauma-treated rats. There were no significant differences in isolated liver mitochondria Complex-I driven respiration (679.5 ±162.9 vs. 634.6 ± 270.2 mmol e⁻/min/mg protein, p=0.61), Complex-II driven respiration (1118.8 ± 233.4 vs. 958.2 ± 469.7 mmol e⁻/min/mg protein, p=0.27) or ROS production between trauma and sham (Figure 4B). Intact platelets from injured rats had higher basal oxygen consumption (17.7 ± 2.5 vs. 15.1 ± 3.2 pmol/s*10⁸ cells, p=0.045), with similar trends in mitochondrial LEAK rates (p=0.19) when compared to sham animals, indicating some degree of hyperactivity and possible membrane dysfunction. Maximal oxidative phosphorylation did not differ between the two groups (p=0.71); however, interestingly, MaxOXPHOS rates were not higher than basal respiration, suggesting limited substrate availability (Figure 4C). When assessing sex dependent effects of trauma, no significant differences were noted between males and females. Overall, platelet basal respiration significantly correlated with native TEG MA (Pearson’s r=0.41, p=0.05; Figure 5A), with weak correlations to G-value (r=0.36, p=0.09; Figure 5B).

Table 1.

Citrated native thromboelastography parameters and mitochondrial respiratory

	Treatment
Parameter	Sham (n=12)	Trauma (n=12)	p-value
Citrated Native TEG
R time (min)	7.97 ± 1.36	8.22 ±1.98	0.73
K time (min)	2.39 ± 0.69	2.36 ± 0.53	0.90
α-angle (degrees)	61.8 ± 5.5	64.3 ± 5.1	0.26
Maximal amplitude (mm)	73.3 ± 3.5 *	81.0 ± 3.0 *	<0.001
G value (kd/sec)	14.1 ± 2.6 *	22.2 ± 5.1 *	<0.001
LY-30 (%)	0.04 ± 0.05	0.13 ± 0.16	0.054
Intact Platelet Respiration (pmol/s*108 cells)
Basal	15.1 ± 3.25*	17.7 ± 2.50*	0.045
LEAK	1.77 ± 1.13	3.01 ± 2.95	0.19
MaxOXPHOS	15.7 ± 4.73	15.0 ± 3.62	0.71
Complex IV	1356 ± 663	1248 ± 612	0.69
Liver Mitochondrial Respiration (mmol e − /min/mg prot)
Complex-I driven	634.6 ± 270.2	679.5 ± 162.9	0.61
Complex II-driven	958.2 ± 469.7	1118.8 ± 233.4	0.27

Rates: Thromboelastography (TEG) parameters, intact platelet mitochondrial respiratory rates, and isolated liver mitochondrial respiratory rates of animals 24 hours after undergoing either sham anesthesia or bilateral hindlimb orthopedic injury. Data is displayed as mean ± SD.

Figure 4.

Effects of orthopedic injury on whole blood thromboelastography, isolated liver mitochondrial respirometry, and intact platelet mitochondrial respirometry

Whole blood, intact platelets, and isolated liver mitochondria were collected from rats treated with either sham procedure or orthopedic injury 24h after treatment. (A) Citrated native thromboelastography (TEG) was used to assess maximal amplitude (MA) of clot formation and clot stability (G-value). (B) Isolated liver mitochondria were assessed for Complex-I and Complex-II driven respiration, as well as reactive oxygen species (ROS) generation. (C) Intact platelets were assessed for basal, leak, and maximal respiratory rates.

Figure 5.

Correlations between intact platelet basal respiration and thromboelastography-based clot strength and stability

Whole blood and intact platelets were collected from rats treated with either sham procedure or orthopedic injury 24h after treatment, and correlations between respirometry and thromboelastography assessed in aggregate. In aggregate, platelet mitochondrial basal respiration significantly correlated to (A) citrated native thromboelastography maximal amplitude (MA) with similar, yet insignificant correlations to G-value (B). Scatter plots of individual values, line of best fit (solid line) and 95% confidence intervals are provided.

Discussion

Mitochondrial oxygen consumption serves as a marker of overall cellular stimulation, and alterations in platelet mitochondrial electron transport chain capacity have been implicated in a number of pathologic processes. In a previous prospective study of patients with severe sepsis, both basal and maximal platelet mitochondrial respiratory rates were higher in non-survivors, with maximal respiratory rates having a significant association with the degree of organ failure.²⁰ However, the relationships between platelet mediated hypercoagulability after injury and mitochondrial function remain underexplored.

In our current pilot study, we sought to evaluate the relationships between platelet mitochondrial function and TEG parameters in a previously-validated rodent model of hypercoagulability after orthopedic injury. Here, we identified a significant correlation between clot strength (TEG MA) and platelet mitochondrial basal respiratory rates, which are increased after injury, suggesting a post-injury hypermetabolic and relative hypercoagulable state. Although insignificant, platelet mitochondrial leak rates were also increased in trauma animals, suggesting some degree of mitochondrial dysfunction. Furthermore, using liver mitochondria as a surrogate marker of systemic mitochondrial effects, these mitochondrial functional changes appeared specific to platelets. Our findings parallel a previous human ex vivo study on human donated platelets by Villarroel et al., who demonstrated positive associations between basal mitochondrial respiration and platelet activation marker expression following agonism with thrombin receptor activating peptide, and described decrements in both based on storage duration.¹⁵

The hypermetabolic platelet cellular state and resultant increased clot strength seen in this study suggest potential novel therapeutic targets to mitigate post-injury hypercoagulability in injured patients. Studies of circulating platelet bioenergetics in rats with streptozotocin-induced diabetes show a similar profile of HRR-assessed elevated basal respiratory rate as well as elevation of flow cytometry-assessed platelet activation markers in diabetic compared to non-diabetic rats, consistent with the hypermetabolic/hypercoagulable phenotype observed after orthopedic injury.²¹ In a follow-up study, treatment of streptozotocin-diabetic rats with metformin normalized basal respiration, resting P-selectin expression, and thrombin-induced P-selectin expression to levels similar to non-diabetic rats, suggesting that metformin mitigates the hypermetabolic/hypercoagulable phenotype of diabetic platelets.²² In mouse models, metformin was further shown to reduce platelet activation, aggregation, and adhesion, and to reduce thrombus burden in induced venous and arterial thrombosis models while preserving clinical bleeding time compared to aspirin.²³ Beyond metformin, several additional antioxidant compounds have been shown to maintain platelet function^24,25 and modulate platelet activation in response to various agonists through reactive oxygen species scavenging.^26,27 Overall, these data suggest that HRR may provide key insight into preclinical development of novel therapeutic agents that hold promise to mitigate hypercoagulability, with potentially superior bleeding side effect profiles compared to current thromboembolic prophylaxis standards.

Several key limitations must be considered when interpreting our limited data and lack of power analysis in this initial characterization. First, our correlations between platelet mitochondrial basal respiration and TEG-based measures of clot strength/stability represent general associations between the two biologic parameters and require more specific validation using additional platelet functional testing modalities (such as platelet activation, aggregometry, and adhesion assays); however, higher basal respiratory rates after trauma are associated with TEG MA measures of clot strength, suggesting a clinically significant relationship. Additionally, classic TEG MA relays broad information related to clot strength that is not specific to platelets alone. Additional assays, such as platelet mapping TEG, will provide more optimal information by evaluating the specific contributions of fibrin polymerization to overall MA.

Secondly, as seen in other studies using respirometry to evaluate platelet mitochondrial bioenergetics, there is tremendous sensitivity in oxygen consumption at such low respiratory rates when compared to other tissues. Although leukocyte counts remained below threshold values during automated cell counting, formal negative selection for leukocytes was not performed, introducing potential low-level leukocyte contamination that may be incorrectly interpreted as platelet activity. Future work will require specific leukocyte depletion to rule this out.

Given the rapid development of platelet associated hypercoagulability in other animal models,^5,6 platelet mitochondrial assessment at additional earlier and later time points is needed to identify the temporal peak, as well as the longitudinal duration, of platelet bioenergetic alteration present after injury, both of which have critical implications for therapeutic initiation and duration that remain uncharacterized. Finally, the use of resting circulating platelets to evaluate the pathophysiology of post-traumatic thrombotic complications poses a unique set of limitations. Although we suggest global platelet hyperactivity, activated platelets actively involved in thrombus formation are not assessed in the peripheral circulating platelet population accessed here. Taken together, our findings suggest that assessment of platelet mitochondrial function provides valuable information in the study of post-injury hypercoagulability, and may provide a vital tool in developing optimized thromboprophylactic strategies in trauma patients.