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The α-globin chain of hemoglobin potentiates tissue plasminogen activator induced hyperfibrinolysis in vitro

Morton, Alexander P. MD; Hadley, Jamie B. MD; Ghasabyan, Arsen MPH; Kelher, Marguerite R. MS; Moore, Ernest E. MD; Bevers, Shaun MS; Dzieciatkowska, Monika PhD; Hansen, Kirk C. PhD; Cohen, Mitchell S. MD; Banerjee, Anirban PhD; Silliman, Christopher C. MD, PhD

Author Information
Journal of Trauma and Acute Care Surgery: January 2022 - Volume 92 - Issue 1 - p 159-166
doi: 10.1097/TA.0000000000003410
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Fibrinolysis is a physiologic process that ensures vascular patency, and multiple processes can affect the cellular and plasma fibrinolytic activity.1 Although fibrinolysis may be protective, systemic hyperfibrinolysis (HF) can lead to death from exsanguination.2–6 Approximately 25% of severely injured patients (New Injury Severity Score [NISS], >15) develop trauma-induced coagulopathy (TIC), which was initially attributed to impaired thrombin generation or systemic HF.3–5,7,8 The mechanisms driving TIC have been attributed to a number of mechanisms including altered platelet activity, endothelial dysfunction, circulating extracellular vesicles, altered thrombin generation, hypoperfusion/ischemia leading to the elevation of tissue plasminogen activator (tPA), and a reduction in serine protease inhibitors (serpins) and clotting factors by activated protein C.3–5,7–12

Severe injury may distort physiological clot remodeling toward the two extremes: systemic HF and fibrinolysis shutdown, with both resulting in increased mortality versus similarly injured patients with normal clot remodeling.13–16 Thrombelastography (TEG) provides not only measures of clot strength, the maximal amplitude (MA), but also the kinetics of clot formation (reaction time [R time] and angle) and clot remodeling, which includes total dissolution, systemic HF, and total cessation of clot remodeling (fibrinolytic shutdown).7,15,17–20 The lysis time 30 (Ly30) allows for segregation among patients who present with systemic HF, physiologic fibrinolysis (PF), and fibrinolytic shutdown (SD), and the differences among the groups become greater with the addition of exogenous tPA to the TEG assay. These tPA-challenged TEGs are an excellent predictor of massive transfusion in the severely injured.13,19–24 Elevated tPA levels are associated with HF through plasmin activation, and similarly, patients with congenital plasminogen activator inhibitor 1 (PAI-1) deficiency may develop HF.15,25–27 Conversely, SD has been associated with increases in PAI-1 in patients postoperatively and trauma patients, the source of which is likely from platelets, endothelial cells, or organ parenchyma.28–31 Thus, in the severely injured, the tPA:PAI-1 balance seems to be of importance in tipping the state of fibrinolysis toward the extremes of HF or SD; however, the triggers for HF and SD remain undefined.26–31

In short, severe injury causes the release of intracellular proteins into the circulation many of which may bind or interact with plasma proteins including PAI-1 or tPA.24,32–34 Hemoglobin is the most abundant intracellular protein in RBCs and hemoglobin-based oxygen carriers potentiate HF in vitro in tPA-challenged TEGs from healthy volunteers.35,36 Therefore, we hypothesize that hemoglobin or one of its subunits, namely, the α-globin chain, potentiates HF in vitro by directly affecting plasmin activity and may be one of the causes of severe systemic HF. The HF group exhibits some of the highest mortality in the severely injured.5,12,15,16



Human serum albumin, hemoglobin A (HbA), tPA, human plasminogen (PLG), ColorpHast Indicator Strips pH 6.5 to 10.0, and phenylmethylsulfonyl fluoride (PMSF) were obtained from Sigma-Aldrich (St. Louis, MO). Recombinant human, full-length α-globin chain and β-globin chain were purchased from Abcam (Cambridge, MA) and GeneTex (Irvine, CA), respectively. Enzyme-linked immunosorbent assays (ELISAs) for plasmin–α2-antiplasmin (PAP), thrombin-antithrombin (TAT) complexes, and a plasmin activity assay kit were purchased from BioVision (Milpitas, CA). Plasminogen-depleted plasma was obtained from GE Healthcare Life Sciences (Pittsburgh, PA).


This pilot study consisted of consecutive trauma patients (n = 130) meeting criteria for the highest level of activation at Denver Health Medical Center, a level I trauma center, from April 2014 to April 2016. These patients were assigned to the Trauma Activation Protocol approved by the Colorado Multi-institutional Review Board with a waiver of consent.32 The criteria are patients older than 18 years and traumatic injury with any of the following: (a) blunt trauma with a systolic blood pressure of <90 mm Hg, (b) mechanically unstable pelvic injury, (c) penetrating neck/torso injuries with a systolic blood pressure of <90 mm Hg, or (d) gunshot wounds to the neck/torso or stab wounds to the neck/torso that require endotracheal intubation.32 Patients who had traumatic brain injury, received blood products, being treated with anticoagulants, or transferred from another facility were excluded. Patients (29 of 30) had blood drawn within 60 minutes of emergency department (ED) arrival, except one whose blood was obtained 3 hours and 25 minutes post–ED presentation, and these samples were obtained before transfusion. A number of these patients were reported previously from the PF and systemic HF groups and are reported here with permission of the corresponding author (permission from A.B.).32 Before transfusing any blood products, citrated and heparinized whole blood and platelet-free plasma samples were obtained upon arrival and tPA-challenged TEG assays were completed as described.19,37 Targeted proteomic analysis using stable isotope internal standards and ELISA measurements of selected proteins from the coagulation and fibrinolytic cascades were completed.32 The groups were stratified by the Ly30 obtained from tPA-challenged TEGs before any further analyses: 10 consecutive injured patients with HF (Ly30 ≥ 50%), 10 with fibrinolysis shutdown (SD) (Ly30 < 5%), 10 with PF (5% ≤ Ly30 ≤ 20%), and 10 healthy controls.32


Citrated whole blood was collected from healthy volunteers after obtaining informed consent under a Colorado Multi-institutional Review Board protocol. No study subject had a coagulation disorder, nor were they taking any medications that affect coagulation or fibrinolysis, including aspirin or nonsteroidal anti-inflammatory drugs. Both groups had a median age of 30, contained 15 subjects, and all females were younger than 54 years.

Tissue plasminogen activator–challenged (75 ng/mL) TEG assays were completed using the TEG 5000 Thrombelastography Hemostasis Analyzer (Haemonetics, Niles, IL).19,37 All TEGs were performed on citrated native (CN) samples in the presence of increasing concentrations of human HbA, the α-globin chain, or the β-globin chain, versus normal saline (NS). All TEGs yielded the following variables: R time (time to clot formation), angle (rate of clot propagation), MA, maximal clot strength, and percent clot lysis 30 minutes after reaching MA (Ly30).19,37

Proteomics and ELISA Measurement of Plasma Proteins

Target proteomics was performed using multiple reaction monitoring of stable isotope peptides (QconCAT reagents), and endogenous matching peptides from a tryptic digest of plasma from the injured patients (n = 30) and healthy volunteers (n = 10) were completed as described previously.32,38–40 Enzyme-linked immunosorbent assays for PAP and TAT complexes were completed per the manufacturer’s instructions.

Surface Plasmon Resonance

Hemoglobin A and the α- and β-globin chains were concentrated using Microcon concentrators (Millipore, Burlington, MA) in phosphate-buffered saline (PBS) (PBS, buffer pH 7.35 + 0.005% Tween20, PBS) with centrifugation at 8,000g for 75 minutes, to a final volume of 200 μL and a final concentration of 195 μM, as determined by a bicinchoninic acid protein assay. The α- and β-globin chains were dialyzed against PBS for binding to the ligand PLG.

Stock solutions and concentrated samples were stored at −20°C <24 hours before analysis. Plasminogen (ligand) was coupled to a chip to determine binding characteristics.41 The analytes tPA, HbA, and α- or β-globin chains were then flowed over the PLG ligand, and the surface plasmon resonance (SPR) (protein-protein interaction) of the analytes to PLG were analyzed on a Biacore 3000 instrument (GE Healthcare, Piscataway, NJ) in the Biophysics Core of the University of Colorado, Denver. Standard amine-coupling chemistry was used to immobilize PLG or tPA to CM5 sensor chip surfaces at 25°C in separate experiments, and kinetics experiments were completed PLG-coupled CM5 surface for four different ligands using different concentrations in duplicate.41 Buffer blanks were used to double reference the obtained kinetic data. Raw sensogram data were processed and fit using the Scrubber software package (version 2.0b; BioLogic Software, Campbell, Australia; The analyte was stripped from the ligand by washing with 0.5 M NaCO3 after each binding reaction before running the next reaction.41

Plasmin AMC Generation Assays

A plasmin activity kit was adapted to determine plasmin activity in solution. The fluorescence is generated when the fluorophore AMC (7-amino-4-methylcoumarin) is cleaved by plasmin from a small polypeptide. The assay was performed in a white flat bottom 96-well microtiter plates and included assay buffer, plasma, α-globin chain, or PMSF, initiated by tPA. Phosphate-buffered saline served as the negative control, and PMSF was used to inhibit tPA activity. The reaction mixture was pH 7.35, verified by using ColorpHast Indicator Strips, pH 6.5–10.0. Plasmin activity measurements were taken following the addition of all reagents to the well and every 3 minutes (excitation, 360 nm; emission, 450 nm) for 18 minutes, and the calculated slopes were compared and expressed as relative light units (RLU) per minute.

Statistical Analysis

Statistical analyses and graphs were completed using GraphPad (Prism Software, San Diego, CA) software. The data were initially analyzed with the Shapiro-Wilk test to determine distribution. The data are presented as mean ± standard error of the mean if normally distributed and as median ± interquartiles (25%–75%) if not. Statistical significance was calculated on normally distributed data using an independent analysis of variance followed by a Bonferroni test for multiple comparisons or if nonparametric, by the Kruskal-Wallis test followed by a Dunn’s test for multiple comparisons.


Patient Demographics and Clinical TEG Analysis

Patients with HF were the most severely injured as quantified by the NISS and Glasgow Coma Scale scores (Table 1). There was not a difference among the injured patient groups in age or body mass, although the HF patients evidenced a lower plasma pH with higher base deficits than the PF or SD patients (Table 1). Importantly, there was no difference in time to blood sample collection after injury because 29 of 30 samples were acquired within 1 hour with the longest time to collection being 3 hours 25 minutes in a female who was stabbed and did not require any crystalloid in the field. Seventy percent of HF patients died, and the mortality was zero in the PF and SD patient groups. Although the citrated rapid TEGs (R-TEGs), with added tissue factor, were used for clinical decisions, because R-TEGs are faster and the samples do not clot, we used the tPA-challenged TEGs, which allow for greater separation in the Ly30 among patient groups (Table 1).20,22,42 The groups satisfied the CN-TEG cutoffs and the R-TEG cutoffs for HF, PF, and SD as previously published (data not shown).

TABLE 1 - Patient Demographics and Clinical Laboratory Assessment
Physiologic Lysis (n = 10) HF (n = 10) Shutdown (n = 10)
Median (Q1–Q3) Median (Q1–Q3) Median (Q1–Q3)
Age, y 36.4 (23.4–59.6) 32.3 (24.6–43.4) 36.4 (30.3–44.9)
Men, % 90 80 80
Blunt mechanism, % 30 60 50
NISS 17.0 (9.0–22.0) 46.5 (38.0–51.8) 17.0 (14.5–27.0)
 Head/neck 0.0 (0.0–0.0) 0.0 (0.0–3.5) 0.0 (0.0–1.8)
 Chest 0.0 (0.0–3.0) 3.5 (3.0–5.0) 0.0 (0.0–2.3)
 Abdomen/pelvis 0.0 (0.0–3.0) 2.0 (0.0–3.5) 1.0 (0.0–3.0)
 Extremities 0.0 (0.0–2.0) 1.0 (0.0–2.3) 0.0 (0.0–2.0)
First measurement (ED)
 SBP, mm Hg 128.0 (99.5–132.0) 86.0 (82.0–130.0) 110. (100.0–138.0)
 GCS 15.0 (9.0–15.0) 3.0 (3.0–3.0) 15.0 (12.5–15.0)
 pH 7.3 (7.3–7.3) 6.9 (6.8–7.2) 7.3 (7.3–7.3)
 Base excess, mEg/L −5.0 (−7.0 to −2.0) −11.0 (−17.0 to −3.0) −8.0 (−8.8 to −5.8)
 Hematocrit, % 40.3 (38.9–44.0) 31.4 (27.6–37.4) 40.4 (38.2–41.1)
 INR 1.1 (1.1–1.2) 2.0 (1.8–3.4) 1.1 (1.1–1.2)
 PTT 27.5 (25.3–28.2) 66.1 (20.6–98.9) 25.8 (23.9–28.0)
 Fibrinogen No data 68.0 (49.6–107.0) 143.0 (122.0–164.0)
 Death, % 0 70 0
 R 7.8 (6.9–8.4) 8.3 (6.6–12.9) 6.4 (4.5–7.7)
 Angle 60.7 (58.2–61.9) 48.2 (28.0–50.1) 65.9 (62.2–71.1)
 MA 60.0 (57.8–62.5) 20.3 (14.0–31.6) 62.0 (55.6–64.3)
  Ly30 7.6 (6.9–8.3) 80.0 (73.0–88.9) 1.1 (0.5–1.5)
AIS, Abbreviated Injury Scale; GCS, Glasgow Coma Scale; INR, international normalized ratio; PTT, partial thromboplastin time; SBP, systolic blood pressure.

Proteomics and Selected Analysis of the Coagulation and Fibrinolytic Cascades of Injured Patients

Proteomic analysis of the plasma from injured patients demonstrated that all severely injured patients had increased concentrations of both α- and β-globin chains versus healthy controls (p < 0.01), and the HF patients were also different from SD and PF patients (p < 0.01) without a significant decrease in haptoglobin (Table 2). Hyperfibrinolysis patients also had decreased levels of antithrombin and PLG versus healthy controls, and the SD and PF groups (p < 0.01) (Table 2). Importantly, the ELISA data showed that HF patients had increased TAT complexes, for example, increased thrombin activation, and PAP complexes, for example, increased plasmin activation, as compared with healthy controls and PF and SD patient groups (Table 2). The total tPA and tPA activation, increased tPA:PAI-1 complexes, were elevated in all trauma groups versus the healthy controls, as well as being significantly increased in the HF group versus all other groups (Table 2). Lastly, both total PAI-1 and PAI-1 activity were significantly elevated in the SD groups versus the PF and HF groups and the healthy controls (p < 0.05) with all three injured groups showing increased tPA activation, for example, higher circulating levels of tPA:PAI-1 complexes, versus the healthy controls (Table 2).

TABLE 2 - Selected Proteomics and Enzymes from the Coagulation and Fibrinolytic Cascades
QConCat Analysis, Median (Q1–Q3)
Protein Healthy Controls HF Shutdown Physiologic
Hemoglobin, α subunit 0.3 (0.2–0.7) 4.5 (1.3–6.6)* 1.1 (0.48–3.5)* 0.8 (0.6–1.1)*
Hemoglobin, β subunit 1.5 (1.2–4.2) 9.1 (2.8–20.4)* 4.7 (2.4–13.4) 4.5 (2.7–7.9)
Haptoglobin 28.3 (16.2–36.5) 22.8 (9.4–30.5) 26.1 (23.0–27.9) 29.6 (16.3–42.4)
ELISA Analysis
α2-Antiplasmin, μg/mL 1,122.4 ± 196.7 890.9 ± 217.1 1,367.6 ± 251.8 921.2 ± 151.5
Antithrombin III, μg/mL 526.5 ± 44.0 253.2 ± 44.7** 464.8 ± 35.3 382.4 ± 47.3
Thrombin, ng/mL 144.4 ± 12.1 133.5 ± 13.8 153.5 ± 27.3 134.2 ± 15.3
PLG, μg/mL 1,127.8 ± 188.2 529.6 ± 120.8** 995.2 ± 228.1 1,187.2 ± 116.7
Plasmin antiplasmin, ng/mL 0.5 ± 0.05 415.7 ± 199.0** 4.8 ± 3.8 2.8 ± 1.0
Thrombin antithrombin, ng/mL 4.4 ± 0.6 9.0 ± 1.5** 6.4 ± 0.7 8.5 ± 1.4**
 Activity, U/mL 0.002 ± 0.001 0.89 ± 0.2** 0.001 ± 0.001 0.03 ± 0.01**
 Total, ng/mL 3.6 ± 0.8 10.9 ± 2.3** 14.9 ± 3.1** 10.3 ± 2.1**
 Activity, U/mL 4.0 ± 1.6 1.9 ± 0.1 79.3 ± 26.6** 5.0 ± 2.0
 Total, ng/mL 7.6 ± 2.6 8.41 ± 2.3 115. ± 35.2** 13.0 ± 3.8
tPA:PAI-1, ng/mL 5.8 ± 1.3 15.4 ± 3.7** 24.0 ± 4.9** 16.5 ± 3.4**
*p < 0.01 from healthy controls.
**p < 0.05 from healthy controls.


In whole blood from healthy donors, HbA and neither the α- nor the β-globin chains affected any aspect of the CN-TEGs, including the R time, angle, MA, or Ly30 (data not shown). To mimic the injured phenotype tPA (75 ng/mL)–challenged TEGs were completed with the addition of NS (control) or serial dilutions of HbA, α-globin chain, and β-globin chain. Hemoglobin A had no effect on the R time, angle, and MA (data not shown) as well as the tPA-provoked fibrinolysis: median Ly30s being 25% (control), 17% (41 nM), 17% (123 nM), and 18% (204 nM) (p = 0.48) (Fig. 1). Addition of the α-globin chain decreased the R time at 550 nM and 920 nM versus NS (Fig. 1A) (p < 0.05) but did not affect the angle (Fig. 1B) or the MA (Fig. 1C), and enhanced tPA-provoked fibrinolysis: Ly30s of 49.7% (550 nM) and 48.5% (920 nM) versus NS (31.3%) (p < 0.05) (Fig. 1D). Addition of the β-globin chain significantly decreased the R time at 920 nM alone (Fig. 1A) and did not affect any other parameters of the tPA-challenged TEGs including the angle, MA, or the Ly30 (Fig. 1). Tranexamic acid (1 mM) significantly inhibited the tPA-induced lysis by 95 ± 4% (n = 7) as expected.

Figure 1
Figure 1:
The α- and β-globin chains affect R time and Ly30 of tPA-challenged TEGs. Whole blood was drawn from healthy controls, and tPA (75 ng/mL)–challenged TEGs were completed. Four TEG parameters were measured. (A) R time: the α-globin chain at the two highest concentrations (3 μg/mL and 5 μg/mL) significantly shortened the R time as did the highest concentration of the β-globin chain (5 μg/mL). (B) Angle: neither the α-globin chain nor the β-globin chain affected the angle in tPA-challenged TEGs at any of the concentrations employed. (C) The MA was unaffected by either the α-globin chain or the β-globin chain. (D) The β-globin chain significantly increased the Ly30 in a concentration-dependent manner, whereas the β-globin chain did not affect Ly30 in the tPA-challenged TEGs (n = 7, *p < 0.05 vs. WB and NS). WB, whole blood.

Surface Plasmon Resonance

Surface plasmon resonance is considered a criterion standard for measuring the affinity of a biomolecular interaction between two proteins and succeeded the yeast two hybrid assay for identifying the affinity of two proteins.43 As a control, PLG was coupled to the chip, and free tPA (75 ng/mL) was passed over it to measure the coupling efficiency and dissociation constant (KD) of the known enzyme: substrate pair.18 The tPA bound with a KD of 43 nM, and with repetitions, the calculated mean ± SD KD was 48 ± 12 nM (Table 3). To ensure that there was no nonspecific protein-protein interaction, the analyte and ligand were reversed, which yielded identical results (data not shown). Serial dilutions of α-globin chain were then passed over chips coupled to PLG yielding a KD of 93 ± 14 nM (Table 3). Hemoglobin A and the β-globin subunit demonstrated very weak molecular interactions with PLG:HbA:PLG (3.6 ± 0.3 μM) and β-globin subunit:PLG (4.0 μM), respectively. The protein-protein interactions were repeated with similar lack of any physical binding reaction between HbA or the β-globin chain, and the PLG ligand bound to the chip (data not shown) (Table 3).

TABLE 3 - Coupling Efficiency of Ligands to PLG
Flow Over Chip Bound to the Chip K D, nM
tPA PLG 48.0 ± 12
α-Globin chain PLG 93.0 ± 14
β-Globin chain PLG 4,000*
HbA PLG 3,600 ± 300*
*Two replicates exhibited no physical association.

Plasmin Activity Assays

The addition of tPA did not generate plasmin from PLG-free plasma (negative control, results not shown). When normal plasma was used, NS served as a negative control and induced a baseline level of plasmin generation of 1,992 ± 1,186 RLU/min (Fig. 2). Tissue plasminogen activator (75 ng/mL) caused a significant (3.2 ± 0.5-fold) increase in plasmin activity (6,374 ± 997 RLU/min) versus NS (n = 7, p < 0.05) (Fig. 2). The addition of α-globin chain (5 μM) significantly augmented tPA-induced plasmin activity by 1.85 ± 0.6-fold (11,474 ± 1,989 RLU/min; n = 7, p < 0.05) (Fig. 2). Furthermore, preincubation with the protease inhibitor PMSF (50 μM) significantly inhibited the tPA-induced increase in plasmin activity by 92 ± 4% (n = 7, p < 0.05) (Fig. 2).

Figure 2
Figure 2:
The α-globin chain augments plasmin activity. Plasmin activity is shown as a function of treatment group. The plasma alone demonstrates a baseline amount of plasmin activity. The addition of 75 ng/mL of tPA induced a significant increase over the plasma alone. The addition of 5 μg of the α-globin chain significantly increased the plasmin activity in response to tPA. In contrast, the addition of the protease inhibitor PMSF (50 nM) significantly inhibited tPA-induced plasmin activity (n = 7, *p < 0.05 from control, † p < 0.05 from tPA).


Following severe injury and before blood component resuscitation, α-globin chains accumulate in the plasma from all injured patients, regardless of TEG-defined groups, when compared with healthy controls. The patients who exhibit HF had significantly greater amounts of the α-globin chain as compared with the SD and PF patients, whereas the β-globin chain only significantly increased in the HF patients with normal haptoglobin levels present in all groups. Similar to previous reports, the HF patients have increased thrombin-antithrombin and plasmin–α2-antiplasmin complexes, decreased PLG, and increased tPA activity versus all other groups indicating increased systemic activation of plasmin most likely by tPA.32 Both total tPA and tPA:PAI-1 complexes were increased in the injured groups versus the healthy controls; thus, the patients investigated in this report seem to be representative of injured patients who have undergone significant ischemia leading to tPA activation; however, as far as the patients with HF are concerned, the stringent criteria of using a tPA-challenged TEG selected the most severely injured patients who had significant HF, hence the increased morbidity and mortality in this subset of patients.13,15,21,24,44,45 Moreover, trauma-related hemolysis was reported decades ago; the HF group, which is the most severely injured, is likely evidencing such hemolysis; and all of the blood samples were drawn within 60 minutes of ED arrival and may have precluded the drop in haptoglobin in these severely injured patients.46

To evaluate traumatic injury in healthy samples, tPA-challenged TEGs were completed and the injured patients were stratified by their state of fibrinolysis: HF, SD, or PF. Importantly, tPA-challenged TEGs have shortened R times because tPA induces activation of factor V and augments the initiation of plasma-based coagulation.47 The addition of the α-globin chain significantly increased the Ly30 and decreased the R time in tPA-challenged TEGs, while addition of the β-globin subunit only decreased the R time at the highest concentration used, which could be due to its ability to provide a nonspecific scaffold to allow for more rapid activation of the coagulation cascade in the TEG cup. The intact hemoglobin molecule, HbA, did not affect any tPA-challenged TEG parameters. To examine possible protein-protein interactions, SPR experiments were completed and showed that the α-globin chain binds to PLG with a similar KD of tPA binding to PLG, a known enzyme-substrate pair.35 This binding of α-globin chain to PLG significantly augments the fibrinolytic activity of plasmin in vitro, providing a possible mechanism for HF in severely injured patients by increasing and/or stabilizing the enzymatic activity of plasmin through direct binding of the α-globin chain to plasmin (Fig. 3). Importantly, plasmin activation is downstream for tPA, and tPA activity was increased by the ischemia related to significant injury to the highest concentration in the HF group without increased amounts of PAI-1 seen in the SD group. In addition, it is vital to remember that the proteomic analysis does not preserve the tetramers or the a-b dimers of circulating free hemoglobin; therefore, the data are limited to the individual chains. One would expect the presence of α-globin chains and β-globin chains in very similar concentrations if hemolysis alone was the impetus for their release into the circulation as seen in autoimmune hemolytic anemia and hemolytic crises in patients with sickle cell anemia, thalassemia, glucose-6-phosphate dehydrogenase deficiency, or hereditary spherocytosis, and the monomeric α-globin chain is cleared directly by haptoglobin, whereas the β-globin chain is not.48,49 The relative increase in the α-globin chain as denoted by the 1 to 2 ratio of the α-globin chain to the β-globin chain in the HF group is curious and may be due to its release from the vascular wall in which it is present in the vascular endothelium at the myoendothelial junctions in which it functions to control vascular tone.50,51

Figure 3
Figure 3:
Proposed schema of the α-globin augmentation of plasmin activity. Plasminogen circulates in a reversible complex with HRG, a competitive inhibitor, which can encounter the α-globin chain of hemoglobin (α-globin). In response to fibrin endothelial cells release tPA, which then activated PLG, which is bound to the α-globin chain, causes increased plasmin activity. HRG, histidine-rich glycoprotein.

Severe injury induces the release and accumulation of intracellular proteins from damaged tissue or hemolysis into the circulation, which may alter hemostasis and may be grouped by the state of fibrinolysis: SD, HF, and physiologic.15,23,32,34,46,52 Mortality has been reported to be greatest in the HF group, elevated in the SD group, and least in patients with PF.15 Clot stability (Ly30) seems to be crucial in TIC with systemic HF accounting for approximately 25% of injured patients (NISS, >15) with mortality as high as 70%.4,5,8,15 Thus, HF is a dangerous condition that requires better molecular description to ultimately decrease mortality in the severely injured. Importantly, these severely injured HF patients exhibit increased thrombin activity, TAT complexes, plasmin activity, and increased PAP complexes, and such increased plasmin activity is initiated by tPA with possible augmentation by a cofactor, the α-globin chain, which directly binds to PLG and increases plasmin activity.32 The conversion of PLG to plasmin involves a well-defined group of coagulation factors and serine proteases that may be augmented by “moonlighting” protein mediators in severely injured patients, which can be intracellular proteins released by injury and ischemia due to concomitant hemorrhagic shock.35 The α-globin chain may represent one such protein with the ability to tightly bind PLG and change the activity of plasmin following activation. Patients with significant HF as diagnosed by TEG or rotational thrombelastography should receive prompt treatment with tranexamic acid and activation of a massive transfusion protocol, if warranted, with further resuscitation directed by either TEG or rotational thrombelastography.

The intact HbA protein did not affect any tPA-challenged TEG parameters and the α- and β-globin chains did not alter any of the parameters in CN-TEGs. Investigations of the PLG interactome using affinity chromatography revealed that the α-globin chain is a candidate interactor with PLG and the reported in vitro data identify a possible role for α-globin chains by binding to PLG and augmenting plasmin activity leading to HF in the severely injured.23,35 The ability of the α-globin chain to augment plasmin activity may be restricted to the severely injured in whom ischemia increases the release of tPA, which in turn may induce systemic, indiscriminate activation of plasmin resulting in weaker clots and HF.32 Importantly, in these severely injured patients, the plasma haptoglobin concentrations were in the normal range; however, these haptoglobin measurements were upon arrival at the ED within minutes to hours of injury. Previous data have shown that severe injury does cause hemolysis and haptoglobin levels were decreased on the day of injury with normalization by day 3, and our most severely injured patients in the HF groups had α- and β-globin chains in their plasma.46 In this study, the exact timing of the blood draws with regard to injury or ED arrival was not documented such that the normal haptoglobin levels seen here may preclude the described drop in haptoglobin, reported by others, and even in the face of massive hemolysis it may take hours to deplete the haptoglobin reserves.46 Moreover, it is unlikely that the observed increases in α-globin chain and β-globin chain were due to phlebotomy-related hemolysis because all groups were drawn under a stringently controlled protocol with identical equipment and procedures, and yet, only the HF and SD groups had increased α-globin and β-globin chains.

This pilot study has several limitations, including small sample size, the study patients being from a single level I trauma center at moderate altitude, and the samples collected being from only the initial time point. Even moderate altitude may affect wound healing, platelet biology, and coagulation so that these studies should be repeated at lower elevations.53–56 However, it is the initial TEG that is used for therapeutic decisions, which may include the administration of tranexamic acid and activation of trauma transfusion protocols, among others. Surprisingly, none of the SD patients died, and one may expect some mortality in this group especially compared with those with PF.13,15,21,24,44,45 In addition, the HF patients had higher NISS and increased bases excesses compared with the other two groups of trauma patients. Despite these limitations, the interaction of the α-globin chain with plasmin and the resulting role in fibrinolysis need to be verified in larger studies of the severely injured.

The importance of the α-globin chain in the hemoglobin molecule has been well documented; however, it has novel functions in other tissues.35,50,51 The α-globin chain is present in the vessel wall in the myoendothelial junctions and is a mediator of vascular tone in the resistance arteries.50,51 It is also found in the central nervous system and in other cells so that its presence in the plasma may also be due to injury rather than hemolysis alone, although patients with traumatic brain injury were excluded.57 In these severely injured patients, the serum haptoglobin concentrations were in the normal range, which may argue against widespread hemolysis being the source of the high plasma levels of the α-globin chain; however, the reported data come from the initial plasma draw from these injured patients and may precede a decrease in haptoglobin, which has been reported in the severely injured.46 Given the altered physiology and circulation of the severely injured, more work is required to delineate the source of the α- and β-globin chains and to characterize the function of the α-globin chain as part of the PLG interactome and its role in the severely injured, especially with respect to TIC.


A.P.M. performed experiments and wrote the manuscript. J.B.H. performed experiments and helped to write the manuscript. A.G. helped to accrue clinical data and store these data and was instrumental in making the figures. M.R.K. helped with these experiments, analyzed the data, finalized all figures, constructed the tables, and helped to craft the manuscript. E.E.M. helped to design the project, analyzed the data, and helped to write the manuscript. S.B. performed all of the SPR experiments. M.D. completed all mass spectrometry, and K.C.H. helped to obtain mass spectrometry data, analyzed these data and the SPR data, and verified that the data were accurate. A.B. helped to generate the original hypothesis, analyzed the data, and helped to write the manuscript. M.S.C. helped to analyze the data to write the manuscript. C.C.S. devised most of the assays, ensured that the data were complete with proper controls, finalized the manuscript, and presented the data correctly. All authors have read the manuscript and approve it in its present form.


The authors declare no conflicts of interest. This work was supported by Vitalant Research Institute, Denver, CO; grants P50-GM049222, 1RM1GM131968-02, and T32-GM008315 from National Institute of General Medical Sciences and National Institutes of Health; and grant W81XWH-12-2008 from the Department of Defense.


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α-Globin chain; injury; plasmin; plasminogen; surface plasmon resonance

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