Characterization of the binding of skeletal muscle myosin to fibrin in trauma

Abstract

BACKGROUND:

The procoagulant phenotype of skeletal muscle myosin (SkM) includes the promotion of thrombin generation to form fibrin, and subsequent increased resistance of fibrin clots to fibrinolysis. The release of SkM into circulation following localized tissue injury is hypothesized to promote systemic hypercoagulation. Yet, in trauma patients and in animal models of tissue injury, an unexplained decrease in SkM levels has been observed.

METHODS:

Plasma samples were collected from orthopedic trauma patients at admission and 6 weeks later and from pigs subjected to experimentally extensive muscle injury. Plasma SkM and myoglobin levels were measured. The binding of SkM to fibrin was quantified in vitro using fluorescence imaging assays. Fibrinolysis was measured using turbidimetric assays and thromboelastography.

RESULTS:

SkM plasma levels in trauma patients were lower at admission compared to 6 weeks later, while plasma myoglobin levels were higher at admission than at 6 weeks. In the pig injury model, plasma SkM levels at 24 hours after injury were decreased, while myoglobin levels were increased. In vitro, SkM bound to fibrin, but not fibrinogen, with an apparent K_d of 0.18 μM and was associated with a decrease in the level of SkM in solution. Fluorescence imaging of the fibrin clots confirmed the presence of SkM within fibrin clots, which increased the resistance of the fibrin clot to fibrinolysis.

CONCLUSIONS:

Direct binding of SkM to fibrin may explain the reduced circulating SkM levels observed after trauma and may help identify potential roles for SkM in trauma-associated coagulopathy.

Trauma is a leading cause of mortality in individuals under 50, causing 4.4 million deaths globally each year.¹ Trauma-induced coagulopathy (TIC) contributes to preventable mortality following trauma, through bleeding, thrombotic, and mixed phenotypes.^2–4 While shock is associated with a hypocoagulable phenotype, tissue injury is associated with a hypercoagulable state characterized by the release of tissue factor, histones, and damage-associated molecular patterns, which may act in concert to promote coagulation while suppressing fibrinolysis.^5–7 Shock and tissue injury frequently coexist, creating a complex hemostatic environment.

Myosins are motor proteins that hydrolyze ATP, bind actin, and generate force. They include skeletal, cardiac, and smooth muscle isoforms, each adapted to its tissue. Skeletal muscle myosin (SkM), the principal contractile component of sarcomeres in skeletal muscle, appears throughout the body and in plasma.^8,9 The procoagulant role of SkM was first suggested by a genetic variant in the MYH gene cluster at chr17p13.1, which was linked to unprovoked venous thromboembolism.^10–12 Subsequent studies demonstrated that SkM promotes thrombin generation through interaction with coagulation factors Xa, Va, and XI.^13–15 Cardiac muscle myosin is similarly procoagulant and exacerbates myocardial damage caused by ischemia/reperfusion in mice.¹⁶ These findings raise the possibility that SkM released from damaged muscle during trauma may regulate the kinetics of the coagulation cascade. A recent study observed an association between SkM deficiency and coagulopathy in trauma patients.¹⁷ To investigate this link, we assessed SkM release after trauma in patients and animals. We found that while plasma myoglobin levels increased after injury, plasma SkM levels decreased. Given the strong binding affinity of SkM for fibrin,¹⁸ we hypothesized that SkM may be incorporated into fibrin clots following injury, explaining the observed decrease in plasma SkM.

METHODS

Patient With Orthopedic Trauma

Serum samples from 10 patients were collected as part of an ongoing double-blinded randomized controlled trial at a level one academic trauma center (institutional review board approval number #00022441). This single-center, pilot, double-blind randomized controlled trial was designed to evaluate the feasibility of intravenous iron therapy following orthopedic trauma.¹⁹ Patients aged 18 to 89 who sustained a lower extremity or pelvis fracture and demonstrated hemoglobin between 7 and 11 g/dL during their inpatient stay were included. Exclusion criteria included active hemorrhage requiring ongoing blood product resuscitation in excess of two units of packed red cells, multiple planned procedures, pre-existing hematologic disorders or chronic inflammatory states, and vulnerable populations. We used biobanked samples from this ongoing study for analysis of SkM levels. Demographic and injury characteristics, including age, sex, body mass index, and Injury Severity Score, were recorded.

Pig Model of Extensive Muscle Injury

The ARRIVE guideline, Supplemental Digital Content 4, http://links.lww.com/TA/F204, was used to ensure proper reporting of methods, results, and discussion of animal studies. Twelve female Yorkshire-Landrace crossbred pigs weighing 40 to 50 kg were fasted for 18 hours prior to surgery. Water was available ad libitum. On the day of the experiment, animals were sedated, intubated, and mechanically ventilated with maintenance isoflurane at 1% to 3% throughout surgery. Surgery involved placing arterial and venous catheters and a small midline laparotomy to place a catheter in the bladder for urine sampling. Following the placement of the catheters, baseline arterial blood samples were collected. Animals received ketamine via continuous infusion for sedation at a minimum dose of 5 mg/kg/h. A captive bolt gun (Schermer Stunner Model MKL) was used to administer a traumatic crush injury via a 21-gauge charge. The bolt gun was placed on the rear hindquarters of the animal, targeting the muscle mass midline to the femur. Two blasts were applied to each hindquarter. Swine received maintenance IV fluid with 5% dextrose-lactated Ringer’s solution at a rate of 10 mL/kg/h for the first 3 hours after intervention, 5 mL/kg/h for the following 3 hours, and 2.5 mL/kg/h for the remainder of the experiment. Blood was collected again 24 hours after the injury.

Coagulation Profiles

Human and pig plasma were prepared via centrifugation of citrated blood (in 0.32% wt/vol sodium citrate; BD) at 2,000g for 10 minutes twice. To measure prothrombin time (PT), 50 μL plasma was incubated with 100 μL Innovin (Thermo Fisher), and the time to clot formation was determined using a KC4 Coagulation Analyzer (Trinity Biotech). For activated partial thromboplastin time (aPTT), 50 μL plasma was incubated with 50 μL aPTT reagent (Thermo Fisher) for 3 minutes. Clotting was initiated with CaCl₂ (50 μL, 8.3 mM final), and the time to clot formation was measured. For recalcified clotting time, 50 μL plasma was incubated with 50 μL of HBS buffer (25 mM HEPES, 150 mM NaCl, pH 7.4). Clot formation was initiated with CaCl₂ (50 μL, 8.3 mM final), and the time to clot formation was recorded.

Myoglobin Measurement

Human and pig plasma myoglobin levels were measured using Human Myoglobin ELISA kits (Abcam) and Pig Myoglobin MYO-9 ELISA kits (Life Diagnostics) per the manufacturer’s instructions.

SkM Measurement

Immunoblotting was performed to evaluate SkM levels.^5,9,17 Plasma samples were size fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions. Proteins were then transferred to polyvinylidene fluoride membranes, and the blots were probed with either anti-MYH antibody (for human plasma; #sc-53095, Santa Cruz Biotechnology) or an in-house anti-SkM chain antibody (for pig plasma; clone 22D4) followed by an HRP-conjugated secondary antibody. Blots were imaged on a ProteinSimple FluorChem and Jess Imaging system. The SkM band at 250 kD corresponds to an intact SkM heavy chain, while the 160 kD band represents the heavy meromyosin fragment of SkM heavy chain. SkM band intensities were quantified using ImageJ and reported as fold change, with a value of 1 defined at admission (for human plasma) or pre-trauma (for pig plasma).

SkM Preparation

SkM from rabbit muscle (Sigma-Aldrich) was dialyzed against a buffer containing 600 mM NaCl and 50 mM Tris (pH 7.4), followed by centrifugation at 21,000g for 1 minute.¹⁴

Coomassie Blue Staining and Western Blot

An aliquot containing 6 μM fibrinogen and 100 nM SkM was incubated for 10 minutes prior to the addition of 10 U/mL thrombin (Sigma-Aldrich) and 6 mM CaCl₂. After a 30-minute incubation to allow fibrin clot formation, the fibrin clot was removed, and the remaining supernatant was collected. Samples were either separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions and stained with Coomassie blue or transferred to polyvinylidene fluoride membrane for immunoblotting with an anti-MYH antibody (#sc-376157, Santa Cruz) followed by an HRP-conjugated secondary antibody. In parallel, 100 nM human serum albumin (Thermo Fisher) was used as a control. For plasma-based experiments, citrated pooled normal plasma was incubated with 100 nM SkM, and coagulation was initiated either by 10 U/mL thrombin or tissue factor (1 nM Innovin) in the presence of 6 mM CaCl₂. After a 30-minute incubation, the fibrin clot was removed, and the supernatant was collected for immunoblotting analysis.

Characterization of Fibrin and Fibrinogen Binding to SkM

SkM was prepared at 10 μg/mL by diluting the concentrated SkM stock (4.4 mg/mL) into 0.1 M Na₂CO₃ buffer (pH 9.6). For plate coating, 50 μL of this SkM solution was mixed with 50 μL of 0.1 M Na₂CO₃ buffer (pH 9.6) and added to each well of a 96-well microtiter plate, resulting in a final concentration of 5 μg/mL SkM per well. Plates were incubated overnight at 4 °C, then blocked with 1% bovine serum albumin (BSA) for 1 hour at 37 °C, washed three times with buffer, and incubated with increasing concentrations of either fibrin-FITC (0–1 μM; Innovative Research) or fibrinogen-FITC (0–10 μM; Innovative Research). Incubations were performed in HEPES-NaHCO₃ buffer (150 mM NaCl, 10 mM NaHCO₃, 10 mM HEPES, 5.5 mM dextrose, 0.1% BSA, pH 7.1) supplemented with 2 mM CaCl₂ for 1 hour at 37 ° C. Following three washes with HEPES buffer, bound fluorescence was measured at 485 nm excitation (bandwidth 9 nm) and 520 nm emission (bandwidth 20 nm). Apparent dissociation constants ($K_d^{\text{app}}$) were determined by fitting specific binding data to a one-site binding isotherm. All fluorescence signals were corrected for nonspecific binding by subtracting the signal from BSA-coated control wells. The specific bound fluorescence, B, was then modeled as a function of total ligand concentration, C, using the following nonlinear least-squares regression model:

$$B\left(C\right)=\frac{B_{\text{max}}\cdot C}{K_{\text{d}}^{\text{app}}+C}.$$

This analysis assumes that the total ligand concentration approximates the free ligand concentration. The reported $K_{\text{d}}^{\text{app}}$ values represent the best-fit parameter, expressed with a ± 95% CI. For experiments where saturation was not observed within the tested concentration range, an apparent $K_{\text{d}}^{\text{app}}$ could not be determined; in these cases, the affinity was reported as > C_max, where C_max is the highest ligand concentration tested.

Fluorescence Imaging for Fibrin Clot

SkM was fluorescently labeled using an Atto 647N Protein Labeling Kit (Sigma-Aldrich). Fibrinogen (6 μM), containing 1% of FITC-labeled fibrinogen (Invitrogen), was incubated with varying concentrations of Atto 647-labeled SkM for 10 min. Coagulation was then initiated by the addition of 10 U/mL thrombin and 6 mM CaCl₂ for 30 min. The fibrin clot was then collected and imaged using a BZ-X810 fluorescence microscope (Keyence) at ×4 magnification.

Fibrin Generation

Citrated pooled normal plasma (50 μL) was incubated with varying concentrations of SkM, followed by incubation with 50 μL of HBS for 3 minutes. CaCl₂ (50 μL, 8.3 mM final) was added to each plasma mixture. Fibrin formation was monitored by measuring turbidity at an absorbance of 405 nm for 30 minutes using an Infinite M200 spectrophotometer (Tecan). The lag time required to initiate fibrin formation and the time to reach half-maximum turbidity (T_1/2 max) were obtained.

Fibrinolysis Assay

In the purified system, a fibrin clot was generated in a 96-well plate using a 100 μL reaction mixture containing 3 μM fibrinogen, 0.25 U/mL thrombin, varying concentrations of SkM, with the addition of 11 nM plasmin. Turbidity was monitored over time at 340 nm to assess clot formation and subsequent fibrinolysis. For plasma, clot lysis was assessed in which tissue plasminogen activator (tPA)-mediated fibrinolysis of a thrombin-induced clot through turbidity change, as described.¹⁶ Briefly, 100 μL of a reaction mixture containing thrombin (0.5 U/mL final), CaCl₂ (2.3 mM final), and tPA (100 ng/mL final) in HBS buffer was added to 100 μL of citrated pooled normal plasma pretreated with varying concentrations of SkM. Following mixing, 100 μL of the mixture was transferred to a 96-well plate, and turbidity was measured over time at 405 nm. Complete clot lysis time (T fibrinolysis) was defined as the interval from maximum turbidity to complete clot dissolution, and half clot lysis time (T_1/2 fibrinolysis) was defined as the time from the maximum turbidity to the midpoint of the decrease in turbidity.

Thromboelastography

Viscoelastic properties of the fibrin clot were evaluated using citrated whole blood obtained from healthy volunteers. Blood samples were treated with tPA 150 ng/mL in the presence or absence of SkM (100 nM) and analyzed using a TEG 5000 hemostasis analyzer system (Haemonetics). The initiation phase of clot formation was measured as R-time, defined as the time from the test until clot formation begins. The degree of fibrinolysis was quantified as LY30 (%), defined as the percentage reduction in clot amplitude 30 minutes after reaching maximum clot strength.

Statistical Analysis

Statistical analyses were performed using GraphPad Prism 10.0 (GraphPad Software Inc.). Data are presented as mean ± SD. Comparisons between two different time points within the same individuals were performed using the Wilcoxon signed-rank test. Comparisons between two groups were conducted using the Mann-Whitney U test as appropriate. For comparisons involving three or more groups, one-way ANOVA was applied. A p value < 0.05 was considered statistically significant. Based on the observed data in plasma SkM levels, post hoc power calculations indicated that detecting the observed differences with 90% power at α of 0.05 would require ~29 patients per group and 7 animals per group.

RESULTS

Reduced Plasma SkM Levels are Observed Following Injuries Compared to Recovery in Orthopedic Trauma Patients

Serum from 10 patients who sustained a lower extremity or pelvis fracture was analyzed to evaluate coagulation profiles and muscle injury markers after tissue trauma. The cohort had a mean age of 61 ± 14.4 years, was 30% male, with a mean body mass index of 29.9 ± 6.9, and an Injury Severity Score of 10.9 ± 5.4 (Supplemental Table 1, Supplemental Digital Content 2, http://links.lww.com/TA/F202). Blood samples were collected during the primary inpatient admission and again at the 6-week follow-up. Coagulation parameters, including PT, aPTT, and recalcified clotting time, did not differ significantly between admission and 6-week follow-up (Fig. 1A). Plasma myoglobin levels were significantly elevated at admission compared to the 6-week follow-up (18.3 ± 14.3 ng/mL vs. 5.5 ± 5.1ng/mL), reflecting muscle injury associated with musculoskeletal trauma (Fig. 1B). In contrast, plasma SkM levels were lower at admission than at the 6-week follow-up (Fig. 1C). We detected a 2.2-fold increase in the 250 and 160 kD SkM bands between admission and follow-up.

Figure 1.

Reduced SkM levels following injuries compared to recovery in orthopedic trauma patients. Coagulation profiles of orthopedic trauma patients at admission and at 6-week follow-up, including (A) prothrombin time (PT), activated partial thromboplastin time (aPTT), and recalcified clotting time. (B, C) Plasma markers of muscle injury, myoglobin and skeletal muscle myosin (SkM), at admission and 6-week follow-up. Data are mean ± SD (n = 10). Significance (*P < .05) was determined by Wilcoxon signed-rank test.

Reduced SkM Levels After Extensive Muscle Trauma Compared to Baseline in a Swine Model

Based on our observations in patients with bone fractures, we next used a pig model of extensive muscle injury to investigate changes in coagulation phenotype and muscle markers during muscle injury. Twelve pigs were anesthetized and subjected to bilateral hindlimb muscle contusion using a bolt gun. Blood samples were collected at baseline and 24 hours postinjury. The aPTT and recalcified clotting time did not differ at 24 hours compared with baseline, but the PT was significantly prolonged (14.4 ± 0.6 vs. 17.5 ± 0.6 s), indicating coagulopathy after extensive muscle injury (Fig. 2A). Plasma myoglobin levels were elevated at 24 hours compared with baseline (0.09 ± 0.04 vs. 3.8 ± 4.2 μg/mL, Fig. 2B). In line with a previous study,⁵ plasma SkM levels were lower 24 hours posttrauma compared with baseline (Fig. 2C). We detected over an 80% reduction in the 250 and 160 kD SkM bands, respectively, prior to and following injury.

Figure 2.

Reduced SkM levels after extensive muscle trauma compared to baseline in a swine model. Coagulation profiles of pigs before and 24 hours after muscle injury, including (A) prothrombin time (PT), activated partial thromboplastin time (aPTT), and recalcified clotting time. (B, C) Plasma levels of myoglobin and skeletal muscle myosin (SkM) were obtained before and 24 hours after muscle injury. Data are mean ± SD (n = 12). Significance (*P < .05) was determined by Wilcoxon signed-rank test.

Consumption of SkM During Fibrin Clot Formation

We next examined the dynamics of SkM levels during fibrin formation in vitro. We hypothesized that SkM binding to fibrin depletes circulating SkM levels. In a purified system, SkM was incubated with excess molar fibrinogen, with both proteins being detectable by Coomassie blue staining or Western blotting with an anti-SkM antibody following electrophoresis. Thrombin and CaCl₂ were added to initiate coagulation and convert soluble fibrinogen into an insoluble fibrin clot. We first confirmed that the generation of an insoluble fibrin clot resulted in the clearance of soluble fibrinogen. Under these conditions, SkM was also cleared from the solution, as observed by Coomassie blue staining (Fig. 3A) or by Western blotting using an anti-SkM antibody (Fig. 3B). The addition of hirudin to prevent fibrin generation preserved fibrinogen and SkM levels. We confirmed that SkM levels were unaffected by the enzymatic activity of thrombin alone (Figs. 3A, B). In contrast, the concentration of albumin remained constant over time (Fig. 3C), excluding nonspecific binding or physical entrapment of proteins during fibrin formation as a mechanism of nonspecific clearance. Finally, we translated our studies from a purified system to a plasma-based system. In plasma, the concentration of SkM significantly decreased following fibrin formation induced by either thrombin or tissue factor (Fig. 3D). The loss of SkM was reversed by the addition of the thrombin-specific inhibitor, hirudin.

Figure 3.

Consumption of SkM during fibrin clot formation. Skeletal muscle myosin (SkM) was incubated with fibrinogen, and coagulation was initiated by adding thrombin and CaCl₂. After 30 minutes, the fibrin clot was removed, and the supernatant was analyzed by (A) Coomassie blue staining and (B) Western blotting using anti-SkM antibodies. (C) Human albumin was used in parallel as a control and analyzed by Coomassie blue staining. (D) Plasma was incubated with SkM, and coagulation was initiated with either thrombin or tissue factor. In select studies, the thrombin-specific inhibitor hirudin was added. Following clot formation and removal, the supernatant was blotted with anti-SkM antibodies. FG, fibrinogen; TF, tissue factor.

Binding of Fibrin to SkM

Saturation binding assays were performed to quantify the affinity of SkM for fibrin(ogen). SkM was placed on 96-well plates, and the binding of fibrin-FITC or fibrinogen-FITC was evaluated. Total fluorescence increased in a concentration-dependent manner for both ligands (Fig. 4). Fibrin-FITC displayed a rapid increase in fluorescence at submicromolar concentrations, reaching a plateau at ~1 μM (Fig. 4A). Nonlinear regression analysis of the specific binding component indicated a $K_{\text{d}}^{\text{app}}$ of 0.18 μM and a maximal binding (B_max) of 20.8 RFU (n = 3), reflective of a strong interaction between fibrin and SkM. In contrast, fibrinogen-FITC exhibited a nearly linear increase in fluorescence up to 10 μM without reaching saturation, indicating substantially weaker binding (Fig. 4B). Because no plateau was observed, a reliable $K_{\text{d}}^{\text{app}}$ could not be determined. As a negative control, BSA immobilized on the plates showed only weak interactions with both fibrin-FITC and fibrinogen-FITC, confirming the specificity of the observed binding (data not shown).

Figure 4.

Binding of SkM to fibrin and fibrinogen. Specific binding of (A) fibrin-FITC and (B) fibrinogen-FITC to skeletal muscle myosin (SkM) immobilized on 96-well plates is shown. Plates were incubated for 1 hour at 37 °C in 20 mM HEPES-NaHCO₃ buffer, pH 7.1, with 2 mM CaCl₂; 1% bovine serum albumin was used as a control for nonspecific binding. (A) Fibrin-FITC binding reached saturation at submicromolar concentrations. The curve represents nonlinear least-squares regression to a one-site binding isotherm. The apparent dissociation constant ($K_{\text{d}}^{\text{app}}$) was 0.18 μM, and maximal binding (B_max) was 20.8 RFU. (B) Fibrinogen-FITC binding increased nearly linearly over the tested concentration range (0–10 μM) without reaching saturation, indicating substantially weaker interaction with SkM. Because no plateau was observed, an accurate $K_{\text{d}}^{\text{app}}$ could not be determined. Error bars represent the SD from three independent replicates.

Incorporation of SkM Into Fibrin Clot

We investigated whether SkM was incorporated into fibrin clots. Clots were formed by adding thrombin and CaCl₂ to a fibrinogen solution or plasma containing 1% of FITC-labeled fibrinogen and SkM labeled with Atto 647N. In both systems, we observed colocalization of fluorescently labeled SkM with fibrin within the fibrin network (Figs. 5A, B).

Figure 5.

SkM is incorporated into the fibrin clot. (A) Fibrinogen containing 1% of FITC-labeled fibrinogen was incubated with varying concentrations of Atto 647-labeled skeletal muscle myosin (SkM) for 10 minutes. Coagulation was then initiated by adding thrombin and CaCl₂ for 30 minutes before the fibrin clot was imaged. (B) Plasma containing 1% of FITC-labeled fibrinogen was incubated with 100 nM Atto 647-labeled SkM for 10 minutes, and coagulation was initiated with thrombin and CaCl₂, and the fibrin clot was imaged after 30 minutes. FG, fibrinogen; Veh, vehicle.

Incorporation of SkM Enhances Clot Resistance to Fibrinolysis

We next examined whether SkM affected clot formation and stability. Consistent with previous reports,¹³ SkM exhibited procoagulant activity in a fibrin generation assay, as evidenced by the shortened lag time (6.0 ± 0.7 min in control vs. 4.3 ± 0.2 min and 4.1 ± 0.5 min with 30 nM and 100 nM SkM) and reduced T_1/2 max (15.7 ± 0.8 min vs. 9.2 ± 0.2 min and 8.0 ± 0.4 min; Fig. 6A; Supplemental Fig. 1A, Supplemental Digital Content 3, http://links.lww.com/TA/F203) in the presence of SkM. Turbidity assays were used to study the effect of SkM on fibrinolysis. While SkM did not significantly alter the complete lysis time or T_1/2 fibrinolysis in the purified system (Fig. 6B; Supplemental Fig. 1B, Supplemental Digital Content 3, http://links.lww.com/TA/F203), it markedly prolonged both parameters in plasma. The complete lysis time increased from 83.3 ± 5.0 minutes to 119.7 ± 10.9 minutes and 119.2 ± 5.4 minutes, while the T_1/2 fibrinolysis increased from 36.4 ± 1.8 minutes to 58.5 ± 14.4 minutes and 65.5 ± 6.4 minutes in the presence of 30 and 100 nM SkM, respectively (Fig. 6C; Supplemental Fig. 1C, Supplemental Digital Content 3, http://links.lww.com/TA/F203). In whole blood, SkM shortened the R-time as measured by thromboelastography in the presence of tPA (Fig. 6D; Supplemental Fig. 1D, Supplemental Digital Content 3, http://links.lww.com/TA/F203).²⁰ However, SkM did not affect LY30 (51.5% vs. 47.0%, p = 0.7; Fig. 6D), the angle, or the maximal amplitude for lysis (data not shown).

Figure 6.

The effects of SkM on fibrin generation and fibrinolysis. (A) Fibrin generation assay in recalcified plasma pretreated with varying concentrations of skeletal muscle myosin (SkM) for 10 minutes, and coagulation was initiated by adding 25 mM CaCl₂. Lag time and time to reach half-maximum (T_1/2 max) were quantified. (B) Fibrinolysis assay using a 100-μL aliquot containing fibrinogen, thrombin, CaCl₂, plasmin, and different concentrations of SkM. Turbidity at 340 nm was monitored at 37 °C for 20 minutes during which a clot was rapidly formed (turbidity increase) and subsequently resolved (turbidity decrease). Complete and half clot lysis times were measured. (C) Fibrinolysis assay using a 100-μL aliquot containing plasma, thrombin, CaCl₂, tissue plasminogen activator (tPA), and different concentrations of SkM. Turbidity at 405 nm was monitored at 37 °C for 120 minutes. Complete and half clot lysis times were measured. (D) Thromboelastography in the presence of tPA with or without SkM. The initiation phase of clot formation was assessed by measuring R-time, and fibrinolysis was assessed by measuring LY30 (%). Data are mean ± SD (n = 3). Significance (*P < .05) was determined by One-way ANOVA and Mann-Whitney U test.

DISCUSSION

Elucidating the mechanisms by which tissue injury alters coagulation and fibrin biology could inform strategies to mitigate hematologic complications following trauma. We report decreased plasma SkM after muscle injury and propose that incorporation of SkM into fibrin clots may explain this phenomenon. Orthopedic trauma patients and experimentally injured pigs exhibited increased plasma myoglobin levels after trauma compared to recovery or baseline. Conversely, plasma SkM levels decreased after injury. SkM was previously shown to promote thrombin activation,^11,13 while diminished plasma SkM levels are associated with coagulopathy in trauma patients.¹⁷ We hypothesized that SkM binds fibrinogen and is incorporated into fibrin clots after injury, explaining the decrease in plasma SkM. Our in vitro results showed that, during clot formation, SkM disappeared from the supernatant and colocalized with the fibrin network.

Our fluorescence binding assays demonstrated distinct interaction profiles for fibrin-FITC and fibrinogen-FITC with SkM. Fibrin-FITC exhibited a strong and saturable interaction with SkM, with an apparent dissociation constant $K_{\text{d}}^{\text{app}}$ of 0.18 μM, consistent with previous surface plasmon resonance measurements reporting a K_d of 0.9 μM.¹⁸ Despite these methodological differences, both approaches indicate submicromolar affinity, confirming a robust interaction between fibrin and SkM. In contrast, fibrinogen-FITC displayed a nearly linear increase in fluorescence over the concentrations tested, without reaching a clear saturation, indicating weaker binding to SkM. Although fibrinogen produced higher absolute fluorescence signals, this does not necessarily reflect a greater number of binding sites, but may be due to differences in fluorophore properties, extent of labeling fibrinogen versus fibrin, or ligand behavior.^21–23 The lack of a plateau suggests a higher K_d for fibrinogen relative to fibrin-FITC and a less specific interaction with SkM. These findings emphasize that fibrin and fibrinogen engage SkM through distinct binding behaviors. The potential mechanisms for the binding of SkM to fibrin remain under debate. While one study suggested noncovalent binding,¹⁸ others proposed non-disulfide covalent cross-linking regulated by transglutaminase activity.²⁴ In the present study, experiments in a purified system demonstrated that SkM can interact with fibrin independently of transglutaminase activity.

Previous studies demonstrated the consumption of other plasma proteins in fibrin clots. Clots in human plasma can passively trap hemoglobin, immunoglobulins, and β-lipoproteins.²⁵ The cytoskeletal protein actin is incorporated in a manner dependent on actin filament length, affecting the viscoelastic properties of fibrin clots and inhibiting fibrinolysis.²⁶ Our results showed similar inhibition of fibrinolysis by SkM, suggesting an avenue for future research characterizing the mechanical properties of SkM-fibrin clots. While our in vitro results demonstrated that fibrin clots can consume SkM, other pathways, such as concomitant release of myosin-binding proteins, enzymatic degradation, or clearance by organs, may decrease SkM levels in vivo.²⁷ Moreover, trauma patients often receive fluids or blood transfusions, raising the possibility that a dilution effect could decrease SkM levels. However, in this case, we would expect a similar reduction in myoglobin levels. Instead, we observed an increase in myoglobin levels, suggesting that dilution alone is unlikely to explain the decrease in SkM. Of note, a previous study reported that while lower plasma SkM levels were associated with coagulopathy, overall plasma SkM levels in the trauma cohort were not statistically different from those of control subjects.¹⁷ In the cited study, blood samples were collected immediately upon arrival to the emergency department, reflecting the very early phase of injury, whereas in the present study, samples were obtained at a later time point (~2 d after injury), capturing a delayed interval during which SkM redistribution or consumption may occur.

Trauma is associated with a broad spectrum of hemostatic responses that reflect the combined effects of tissue injury, inflammation, activation of coagulation, and consumption of coagulation factors. While severe trauma with hemorrhagic shock can produce profound systemic disturbances in coagulation, tissue injury disrupts the endothelium, leading to tissue factor exposure and inflammatory responses, including release of histones and damage-associated molecular patterns.⁵ SkM released from damaged muscle may contribute to tissue injury–mediated coagulopathy. A prior study reported that cardiac muscle myosin aggravates myocardial injury in a murine ischemia/reperfusion injury model.¹⁶ SkM drives thrombin generation by providing a surface for the prothrombinase complex of factor Xa and factor Va¹³ and stabilizes fibrin clots against degradation by plasmin.¹⁸ Our results demonstrated accelerated clotting and resistance to fibrinolysis in the presence of SkM, consistent with previous findings that tissue injury promotes hypercoagulability and impairs fibrinolytic activity.^5,6 Given these findings, consumption of SkM in tissue injury–induced coagulation may reflect the extent of TIC. Indeed, unlike tissue injury, rhabdomyolysis alone is rarely associated with thromboembolic complications, except in cases where disseminated intravascular coagulation develops due to dysregulated coagulation activation.^28,29 This implies that the procoagulant and antifibrinolytic effects of SkM depend on concurrent coagulation activation. By itself, SkM does not initiate coagulation; rather, it acts as a modulator that amplifies the process once it begins.

Further research is needed to connect our in vitro results to clinical outcomes, including blood loss, thromboembolic events, and mortality. This study was limited by small sample sizes and a few time points in the clinical study and animal model. A larger study would be required to compare the range and time-dependent variability of SkM in the plasma in a healthy patient population. Measuring SkM in an animal model immediately after tissue injury would provide insight into the initial release of SkM from damaged muscle. The patient cohort represented relatively low-severity, nonshock trauma, which may not fully capture the spectrum of TIC. Furthermore, since we did not evaluate fibrinolysis activity in plasma samples from patients and animals, we are unable to relate this activity to SkM levels in individuals. Future studies incorporating muscle histology in the pig injury model are needed to determine whether microvascular coagulation occurs at sites of muscle injury. In addition, future studies are needed to characterize the stoichiometric relationship between SkM and fibrin and to assess how varying SkM-to-fibrinogen ratios affect clot formation and stability. Research on the textural properties of meat products has explored the rheological properties of myosin-fibrin gels,²⁴ and similar investigations could characterize the effect of SkM on the rheological properties of human fibrin clots. In summary, our current findings document the decrease in SkM following traumatic injury and suggest an explanation for this phenomenon by demonstrating that SkM disappears from solution as it becomes incorporated into fibrin clots.

Supplementary Material

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