Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2020 Oct 1.
Published in final edited form as: J Thromb Haemost. 2019 Jul 28;17(10):1733–1745. doi: 10.1111/jth.14563

Platelet-derived extracellular vesicles released after trauma promote hemostasis and contribute to DVT in mice

Mitchell R Dyer 1, Wyeth Alexander 2, Adnan Hassoune 1, Qiwei Chen 1, Jurgis Alvikas 1, Yingjie Liu 1, Shannon Haldeman 1, Will Plautz 2, Patricia Loughran 1,3, Hui Li 4,5, Brian Boone 1, Yoel Sadovsky 4, Prithu Sunnd 6, Brian S Zuckerbraun 1, Matthew D Neal 1
PMCID: PMC6773503  NIHMSID: NIHMS1040959  PMID: 31294514

Abstract

Background:

Traumatic injury can lead to dysregulation of the normal clotting system resulting in hemorrhagic and thrombotic complications. Platelet activation is robust following traumatic injury and one process of platelet activation is release of extracellular vesicles (PEV) that carry heterogenous cargo loads and surface ligands.

Objectives:

We sought to investigate and characterize the release and function of PEVs generated following traumatic injury.

Methods:

PEV content and quantity in circulation following trauma in humans and mice was measured using flow cytometry, size exclusion chromatography, and nanoparticle tracking analysis. PEVs were isolated from circulation and the effects on thrombin generation, bleeding time, hemorrhage control and thrombus formation were determined. Finally, the effect of hydroxychloroquine (HCQ) on PEV release and thrombosis were examined

Results:

Human and murine trauma results in a significant release of PEVs into circulation compared to healthy controls. These PEVs result in abundant thrombin generation, increased platelet aggregation, decreased bleeding times, and decreased hemorrhage in uncontrolled bleeding. Conversely, PEVs contributed to enhanced venous thrombus formation and were recruited to the developing thrombus site. Interestingly, HCQ treatment resulted in decreased platelet aggregation, decreased PEV release, and reduced DVT burden in mice.

Conclusions:

These data demonstrate that trauma results in significant release of PEVs which are both pro-hemostatic and pro-thrombotic. The effects of PEVs can be mitigated by treatment with HCQ, suggesting the potential use as a form of DVT prophylaxis.

Introduction

Traumatic injury is a leading cause of morbidity and mortality worldwide.1 Early mortality is due to hemorrhage; however, patients that survive the initial insult are at risk for microvascular complications leading to organ injury and macrovascular complications such as development of deep venous thrombosis (DVT) or pulmonary embolism (PE).2–4 The mechanisms driving these thrombotic complications are incompletely understood but have been hypothesized to involve activation of the innate immune system with subsequent effects on coagulation.5–10 Platelets have increasingly been recognized as critical mediators of immunothrombosis and are rapidly activated following injury.11,12 Further, there is increasing recognition of the active role played by platelets in the pathogenesis of DVT including active release of critical innate immune ligands and promotion of neutrophil extracellular trap formation.13–16

Platelet activation results in the release of extracellular vesicles (PEVs). PEVs are believed to have diverse roles including cell-cell communication, cargo delivery, as well as both anticoagulant and pro-thrombotic properties.17–23 While the release of extracellular vesicles following trauma have been described24–28, the function, cell of origin, and clinical relevance of this process are not well understood. The release of PEVs may be an initial response to serve in hemorrhage control; however, excessive or persistent release of PEVs may lead to a later pro-thrombotic state and contribute to common thrombotic complications in trauma patients, such as DVT. Anti-platelet therapy in trauma patients is complex as many will have direct clinical contraindications to the use of traditional anti-platelet therapy such as aspirin or clopidogrel. Hydoxychloroquine (HCQ) is known to have anti-platelet effects, has previously been utilized for DVT prophylaxis in post-operative patients, and is known to have a good clinical safety profile and no known associated bleeding risks.29–31 However, whether HCQ can modulate the release of PEVs has not been investigated.

We hypothesized that trauma would lead to release of PEVs that would contribute to thrombotic complications, specifically DVT. We now show that PEVs are released following both human and murine trauma and that these PEVs result in an increase in thrombin generation. Adoptive transfer studies of PEVs from mice following trauma were both pro-hemostatic but also markedly prothrombotic. Finally, we demonstrate the striking effect of HCQ to decrease the formation of PEV following injury and to reduce thrombus burden in a murine model of DVT due, at least in part, to a reduction of PEV production.

Methods

Animals

C57BL/6J (age 8-12 weeks, male) were purchased from Jackson Laboratories. Mice were housed in accordance with University of Pittsburgh (Pittsburgh, PA, USA) and National Institutes of Health (NIH; Bethesda, MD, USA) animal care guidelines. All animal experiments were approved and conducted in accordance with the guidelines set forth by the Animal Research and Care Committee at the University of Pittsburgh.

Murine Polytrauma Model

C57BL/6J male mice were subjected to a validated model of polytrauma consisting of pseudofracture and liver crush as previously described.8,32 Based on prior data, platelet activation peaks 30 minutes following injury in this model, therefore at 30 minutes mice were sacrificed.8 Blood was centrifuged for platelet and PEV analysis via flow cytometry or PEV isolation as described below. Where noted mice were given hydroxychloroquine sulfate (HCQ) (Thermo Fisher), 50mg/kg, via intraperitoneal injection 30 minutes prior to polytrauma or IVC ligation.

PEV Isolation and Quantification

Human blood samples were obtained from an observation cohort of severely injured trauma patients (IRB: PRO08010232) and healthy controls. Venous samples were collected in acid-citrate-dextrose buffer and centrifuged at 200g for 10 minutes to obtain platelet-rich plasma (PRP). Murine blood samples were collected via cardiac puncture and centrifuged for PRP. PEVs were isolated from PRP by serial centrifugation.33 Briefly, PRP was centrifuged at 3000rpm for 5 minutes without brake and the supernatant was collected and the platelet pellet was discarded. Next, this supernatant was centrifuged at 12,000g x 30 minutes to pellet cell debris and residual platelet contamination and a PEV rich supernatant.

To determine the concentration of PEV, PRP samples, either human or murine, were diluted to a standard platelet concentration of 1×105ml. Samples were then incubated with APC-conjugated anti-CD42b monoclonal antibody (mouse IgG1κ; 17-0429-42, eBioscience) and FITC-conjugated anti-CD62P monoclonal antibody (rat LEW IgG1λ; 553744, BD Biosciences) or respective isotype control antibodies. Flow cytometry was carried out with a FACSCanto flow cytometer using DIVA software (BD Biosciences). Platelets were gated based on their characteristic scatter properties and expression of CD42b and CD62P. PEV analysis was performed as described previously.33 The flow cytometer was calibrated using standard size latex beads. PEV were gated for size less than 1µm based on bead size and co-expression of CD42b and CD62P. Finally, PEVs were quantified based on the platelet count and are described as the CD42b+CD62P PEV/CD42b+CD62P platelet.33

Ex-Vivo Generation of PEVs

Murine whole blood was collected in acid-citrate-dextrose buffer via cardiac puncture. PRP was generated as described and platelet concentrations were adjusted to 1×105/ml. Diluted PRP was then treated with thrombin (0.5U/ml) for 15 minutes. Hirudin was added for thrombin neutralization and the stimulated PRP was then centrifuged as described.

Isolation of small PEV (containing exosomes) for Adoptive Transfer Studies

Whole blood from human or mice was collected in acid-citrate-dextrose buffer and centrifuged to obtain platelet poor plasma (PPP). PPP underwent an additional centrifugation at 3000g for 15 minutes. Small PEVs were isolated using the ExoQuick™ Precipitation Solution (System Biosciences) according to the manufacturer’s protocol. In separate experiments, Size Exclusion Chromatography (iZON Science) was performed according to the manufacture’s protocol to isolate small PEV and nanoparticle tracking analysis was performed (Nanosight, Malvern Instruments) to confirm purity. To further isolate platelet-specific small EVs, biotinylated antihuman CD42b (Biorbyt, orb114027) was added to the suspended EVs for 90 minutes at room temperature. Streptavidin Plus UltraLink resin was added for 30 minutes. The incubation sample was centrifuged at 400g for 5 minutes and the supernatant containing platelet specific small EVs was removed. 0.05mM acetic acid was then added to release the immune complexes followed by a final centrifugation at 4000g for 10 minutes and removal of the supernatant which contained the platelet-derived exosomes. Small PEV were analyzed by western blot for CD9 (BioWorld Cat. No. BS3022).

Clotting Assays

Whole blood, human and murine, was incubated with trauma-derived PEVs as indicated for 30 minutes at room temperature and plasma was obtained by centrifugation. PT and aPTT were measured using a hemostasis analyzer (Diagnostica Stago Start4).

Thrombin Generation Assay

Thrombin generation assays (TGAs) in platelet free plasma (PFP) were performed according to previously outlined methods.34 Briefly, 50μL of PFP was incubated with 10μL of TF-phospholipid reagent (Technothrombin® RB, 2pM TF : 4.0μM phospholipid). Human trauma-derived PEV or PEV generated from healthy volunteers were put in dilution buffer (150mM NaCl, 20mM HEPES, 0.1% PEG-8000, pH 7.4) and titrated by concentration into separate wells of the microplate. Following incubation, 50μL of 1mM Z-Gly-Gly-Arg-AMC in 15mM CaCl2 was immediately added to initiate the reaction. Fluorescence was measured on a Synergy™ 2 Multi-Detection Microplate Reader (Biotek). A thrombin calibration curve was produced by using a Technothrombin® thrombin generation assay calibrator.

Platelet Aggregation and Activation

Whole blood impedance aggregometry was performed using a ChronoLog aggregometer (Model 700) according to the manufacturer’s instructions with collagen (2 µg/ml) induction. Where noted, whole blood samples were treated with 1×108 PEVs for 30 minutes prior to assessing aggregation. In separate experiments, mice were injected with PBS or HCQ (50 mg/kg). After 60 minutes whole blood was collected, centrifuged for platelet isolation and diluted to a standard concentration of 1×108/mL. Platelets were treated with thrombin (0.5U/mL) for 15 minutes and flow cytometry was performed as described above to detect platelet activation.

Tail Vein Bleeding Assay and Liver Laceration Model

Mice were anesthetized and the tail tip (3-mm) was removed with a scalpel. Tails were submerged in PBS and bleeding was monitored as previously described.8,35 Bleeding time was quantified as the time from initiation of bleeding to the cessation of blood flow (seconds). Where noted mice were given 1-4×108 trauma-derived PEVs or equal volume PBS via penile vein injection 30 minutes prior to starting the tail bleed assay. IVC ligation was performed in mice following recovery (24 hours) after tail bleed to model DVT after injury. To mimic traumatic hemorrhage, we utilized a validated model of uncontrolled hemorrhage in mice.36 Briefly, mice underwent a midline laparotomy and standard resection of the left middle lobe. Blood loss was quantified (grams). Where noted mice were given trauma-derived PEVs or equal volume PBS via tail vein injection prior to liver laceration.

Deep venous thrombosis (DVT) Model and PEV Adoptive Transfusion

DVT was induced by inferior vena cava (IVC) ligation as described previously.37 Briefly, mice were anesthetized, underwent a midline laparotomy, the IVC was isolated and ligated completely at the level of the renal veins, and all visible side branches are ligated. Posterior branches were left open. Mice recovered and were sacrificed 24 hours following IVC ligation. Thrombi, which included the thrombus and vessel wall, were excised and weighed immediately. Blood was collected and analyzed for PEV quantification as described. For adoptive transfer experiments, age and sex-matched mice were subjected to polytrauma as described. PEVs were isolated and quantified as described. ~1×108 trauma-derived PEVs were then administered via tail vein injection 30 minutes prior to IVC ligation (Supplemental Figure 1). This number of PEVs was significantly lower than the concentration noted following murine polytrauma (~4-5×108/mL) or human trauma (~1×1013/mL) so as to avoid overestimating a pro-thrombotic response by administering supra-physiologic numbers of PEVs. Where noted, HCQ (50mg/kg) was administered either alone or sequentially with trauma-derived PEVs 30 minutes prior to ligation.

Labeled Trauma PEV Adoptive Transfer Experiments

Trauma-derived PEVs were isolated from mice subjected to polytrauma. Isolated PEVs were incubated with CellMask™ deep red plasma membrane stain (Thermo Fisher, C10046) and CellTracker™ deep red dye (Thermo Fisher, C34565) at a concentration of 1:1000 for 30 minutes at 37°C. The solution was centrifuged at 500g for 5 minutes. The supernatant was removed, the labeled PEVs were diluted in PBS and incubated at 37°C for 5 minutes. The solution was then gently centrifuged at 500g for 5 minutes again. The supernatant was again removed the labeled PEVs were then washed x2 in PBS. The final solution containing labeled PEVs was then injected via tail vein into mice 30 minutes prior to IVC ligation. Clots were harvested (24hrs) and processed for immunofluorescence imaging.

Statistical Analysis

All data are presented as mean ± SD for n ≥ 3 unless stated otherwise in the figure legends. Statistical significance was determined with the 2-tailed Student’s t test or 1-way ANOVA with Bonferroni post-hoc testing using Graph Pad Prism software (GraphPad). A p value of less than 0.05 was considered significant.

Results

Trauma results in release of platelet-derived extracellular vesicles (PEVs) in humans and mice

To quantify the specific release of PEVs, samples from human trauma patients were analyzed at two timepoints following injury (0 hour(hospital presentation) and 24 hours later). Patient demographics are presented in Table 1. Compared to healthy controls, trauma patients have a significant increase in circulating PEVs (#CD42b+CD62P PEV/CD42b+CD62P platelet, 6.5±3.9 healthy control vs 18.0±10.6 0hr vs 38.9±17 24hr, p < 0.0001) (Figure 1A). Furthermore, there is an increase in circulating PEVs within trauma patients over time (CD42b+CD62P PEV/CD42b+CD62P platelet, 10.6 0hr vs 38.9±17 24hr, p < 0.001) (Figure 1A). We next investigated the release of PEVs in mice in a model of trauma. Prior work identified maximum platelet activation following injury in our model to be at 30 minutes.38 In a similar fashion, we found traumatic injury in mice results in a significant release of PEVs (CD42b+CD62P PEV/CD42b+CD62P platelet, 0.97±0.79 vs 42.3±9.1, p<0.0001) (Figure 1B).

Table 1.

Patient Demographics

Characteristic
Age (mean ± SD) 46 ± 19
Gender, Male (%) 69
Race, White (%) 77
Mechanism, Blunt (%) 100
ISS (median, IQR) 17 (13, 25.75)
Length of Stay, Days (mean ± SD) 14 ± 13.6
Hypotension, SBP <90 mmHg (%) 70
Emergent Operation (%) 80
Blood Product Requirement 24hr, Y/N (%) 30
In-hospital Mortality (%) 9.5
Admission lactate (mean ± SD) 4.8 ±3.4
Admission INR (mean ± SD) 1.2 ± 0.2
Open in a new tab

Figure 1. Trauma results in release of platelet-derived extracellular vesicles that promote thrombin generation and platelet aggregation.

Figure 1.

Open in a new tab

Quantification of flow cytometry for the proportion of CD42/CD62 positive PEVs per platelet from healthy (control) and trauma patients on presentation and 24 hours following injury (A, n=14 healthy, n=24 0hr, n=16 24hr) and for out of box (control) and polytrauma (trauma) murine samples (B, n=6/experiment). Isolated human trauma-derived PEVs led to a significant increase in thrombin generation (C) which was time and dose dependent (D, n=3 with 3 replications). PEVs from patients who experienced trauma led to significant increase in peak thrombin generation compared to PEV from pooled healthy plasma that was dose and time dependent (E-F, n=3 with 3 replications). For (B) and (C) the doses used refer to the number of microliters (μL) utilized in each thrombin generation run at a PEV concentration of ~1×1013/mL. Note, the reagent (Reagent B) utilized in the thrombin generation assay results in a peak thrombin of 400–600 and therefore serves as baseline control for PEV comparisons. PEVs were generated and isolated by ex vivo stimulation of platelets and added to whole blood and platelet aggregation was assessed (G, n=3 with 3 replications). Finally, trauma-derived PEVs were isolated from mice subjected to polytrauma and added to whole blood and platelet aggregation was determined (H, n=3 with 3 replications). *p<0.0001 **p<0.001 ***p<0.005 ****p<0.0005 ******p<0.01

PEVs released after trauma are prothrombotic

We next sought to evaluate the physiologic effect of trauma PEVs on coagulation and thrombosis. The effect of circulating human trauma-derived PEVs on thrombin generation was assessed. Thrombin generation was significantly increased in a dose-dependent manner (Figure 1C). As well, higher concentrations of PEVs led to more rapid and robust thrombin generation (Figure 1D). Compared to PEVs from healthy controls generated ex vivo with thrombin, PEVs isolated from trauma patients led a significant increase in peak thrombin generation (Figure 1E–F). Tissue factor levels were measured in the human PEVs samples and were found to be negligible, suggesting that thrombin generation was unlikely to be related to the delivery of tissue factor by residual plasma or PEVs (Supplemental Figure 2A). Trauma-derived PEVs had no effect on standard coagulation tests (Supplemental Figure 2B). To investigate the effects of PEV on platelet function, we assessed whole blood platelet aggregation in the presence of PEVs. Aggregation was significantly increased in whole blood incubated with ex-vivo generated PEVs (AUC, 162±28 vs 83±7, p<0.001) (Figure 1G). Finally, platelet aggregation was significantly increased in whole blood with isolated PEVs added from mice subjected to polytrauma (AUC, 67±7 vs 97±5, p<0.01) (Figure 1H).

Trauma PEVs reduce bleeding time but increase subsequent thrombus burden

We hypothesized that PEVs released following trauma may promote hemostasis. Adoptive transfer of murine trauma-derived PEVs was performed and tail bleeding time was measured. Mice transfused with trauma-PEVs had a decreased bleeding time compared to vehicle control (seconds, 147.5±50.5 PBS vs. 127.5±40.7 1×108 PEVs vs 86.4±29 4×108 PEVs, p=0.005) (Figure 2A). The effect of PEV on uncontrolled hemorrhage was assessed in a model of liver laceration and found to significantly decrease blood loss compared to control (g blood loss, 0.64±0.04 PBS vs. 0.39±0.07 4×108 PEVs, p=0.02) (Figure 2B). Trauma patients with significant hemorrhage are at risk of later development of venous thrombotic complications. To directly assess whether this initial pro-hemostatic effect of PEVs would influence venous thrombosis, mice were subjected to tail bleed, recovered and subsequently subjected to IVC ligation 24 hours later. Strikingly, mice transfused with PEVs during the initial tail bleeding experiments had a significantly increased thrombus burden (mg, 13.5±5.1 PBS vs 20.7±3.5 1×108 PEVs vs 21.0±5.9 4×108 PEVs, p=0.02) (Figure 2C).

Figure 2. Trauma-derived platelet extracellular vesicles decrease bleeding time and enhance thrombosis.

Figure 2.

Open in a new tab

To assess in vivo effects of PEVs released following traumatic injury, PEVs were isolated from mice subjected to polytrauma and transfused into mice undergoing tail bleed assay. Trauma-derived PEVs enhance hemostasis and decreased bleeding time (A, n=10–12/group). To reflect bleeding following trauma, we performed a liver laceration model that results in uncontrolled hemorrhage. Transfusion of 4×10^8 PEV resulted in a significant decrease in blood loss (B, n=6/group). Finally, to determine if these PEVs would also have an effect on thrombus formation, a known later complication following trauma, mice subjected to tail bleeding recovered for 24 hours and were then subjected to IVC ligation. Strikingly, mice transfused with PEVs prior to their tail vein bleed had significantly higher thrombus burden compared to control at 24 hours following IVC ligation (C, n=6–8/group). Experimental diagram of liver laceration model that results in uncontrolled intraperitoneal hemorrhage (D). *p<0.01 **p<0.05

Trauma-derived PEVs and small PEVs containing exosomes enhance thrombus burden in murine DVT

To further characterize the effects of PEV on thrombus development outside the setting of bleeding, we performed IVC ligation in mice transfused with 1×108 PEVs. At 24 hours, PEV from stimulated platelets resulted in significantly higher thrombus burden (mg, 22.1±8 PEV vs 10.6±2.1 PBS, p=0.03) (Figure 3A). To extend our investigations into the role of PEVs generated in physiologic settings, PEVs were isolated from mice subjected to polytrauma. Transfusion of trauma-derived PEVs resulted in a significantly higher thrombus weight (mg, 21.2±8.9 vs 10.7±4, p=0.01) (Figure 3B).

Figure 3. Platelet extracellular vesicles and small PEV containing exosomes released after traumatic injury increase thrombus burden and localize to the thrombus site in murine DVT.

Figure 3.

Open in a new tab

PEVs generated and isolated from platelets stimulated with thrombin and then transfused into mice undergoing IVC ligation led to a significant increase in thrombus burden at 24 hours (A, n=4/group). To assess the effect of in vivo released PEVs, isolated PEVs from mice subjected to a model of polytrauma were used in an adoptive transfer to mice undergoing IVC ligation. Trauma-derived PEVs led to a significant increase in thrombus weight at 24 hours (B, n=8–10/group). To further assess the PEV effect, platelet-derived small EVs, which contain exosomes, from mice subjected to traumatic injury were isolated and used in adoptive transfer experiments as in (B). Similar to the prior findings, platelet released small PEVs containing exosomes from trauma resulted in a significant increase in DVT burden as assessed by thrombus weight (C, n=10/group). Finally, to demonstrate the transfused PEVs were localizing to the developing thrombus isolated PEVs were labeled with CellTracker and CellMask. As demonstrated in (D), labeled PEV (red) localize to the thrombus site and interact with platelets (green) suggesting PEVs have direct role in regulating thrombus development.

EV cell populations are heterogeneous and consist of subpopulations. Nomenclature of these subpopulations is non-standard; however, according the International Society for Extracellular Vesicles, small EVs consist of cell populations <100 or <200nM in size and are often referred to as exosomes.39 Small PEV are known to carry numerous thrombotic mediators.40–42 To investigate if there were differential effects of the EV subpopulations released following trauma, small PEVs were isolated from mice subjected to polytrauma and used in adoptive transfer into mice for DVT analysis. To confirm we were working with small PEVs we characterized the isolated subpopulation by size exclusion chromatography, nanoparticle tracking analysis, and western blot analysis for the surface marker CD9 (Supplemental Figure 3). Strikingly, mice transfused with trauma-derived small PEVs had a significant increase in thrombus burden (mg, 14.5±5.3 vs 7.2±4.4, p<0.005) (Figure 3C). Finally, to confirm the transfused PEVs were localizing and participating in thrombus development, trauma-derived PEVs were incubated with dual cell labeling fluorophores and injected via tail vein prior to IVC ligation. As demonstrated in Figure 3D, labeled PEVs are bound throughout the developing thrombus at 24 hours.

Hydroxychloroquine sulfate reduces platelet aggregation and release of platelet extracellular vesicles

Chloroquine and its derivative hydroxychloroquine (HCQ) are synthetic 4-aminoquinolines. Interestingly, HCQ has anti-platelet effects; however, the platelet inhibitory mechanism remains unknown.43–45 To confirm anti-platelet effects of HCQ, mice were treated with HCQ and platelet aggregometry was assessed. HCQ treatment led to a significant decrease in platelet aggregation (AUC, 80±11.2 vs 48.2±13.6, p=0.01) (Figure 4A). We next hypothesized that HCQ would exert anti-platelet actions in part through modulating the release of PEVs. HCQ treatment had no effect on baseline circulating levels of PEVs (#PEV/platelet, 10.5±0.9 vs 10.9 ± 1.1) (Figure 4B). In contrast, PEV release from stimulated platelets was significantly reduced with HCQ (#PEV/platelet, 32.5±4.1 vs 20.6±4.1, p=0.02) (Figure 4B). Finally, the effect of HCQ on platelet activation was investigated. Stimulated platelets from mice treated with HCQ did not appear to have significantly different levels of overall platelet activation as assessed by dual CD41 and CD62 positivity by flow cytometry (% CD41/CD62 dual positivity, 10,000 events, PBS 57.5 ± 16.5 vs. HCQ 46.4 ± 4.4, p=0.4, Supplemental Figure 4). To confirm our ex vivo findings mice were subjected to polytrauma with HCQ and platelet aggregation and PEV release were determined. HCQ led to a significant decrease in platelet aggregation (AUC, 292.5±86.3 vs 55 ± 28.9, p<0.005) and PEV release following injury (#PEV/platelet, 50.1±12 vs 26.2±3.9, p=0.01) (Figure 4C–D).

Figure 4. Hydroxychloroquine sulfate reduces platelet aggregation, platelet extracellular vesicle release, and thrombus burden in murine DVT.

Figure 4.

Open in a new tab

Mice treated with HCQ had a significant decrease in whole blood platelet aggregation (A). To assess the effect of HCQ on release of PEVs, flow cytometry was performed after platelets were stimulated with thrombin. HCQ treatment led to a significant reduction in release of PEVs (B). Moving to assessment of in vivo effects of HCQ, mice were treated with HCQ and subjected to a model of polytrauma. Whole blood platelet aggregation was significantly reduced in mice treated with HCQ (C). Additionally, the release of PEVs following traumatic injury was reduced in mice treated with HCQ (D). IVC ligation results in a significant release of PEV into circulation at 24 hours (E, OOB=out of box control). To further investigate the physiologic effects HCQ treatment, mice were treated with HCQ or control and subjected to IVC ligation. At 24 hours, mice treated with HCQ had a significantly lower thrombus burden assessed by clot weight (F). To determine if the HCQ effect was in part due to inhibition of release of PEVs we next performed IVC ligation in mice treated with both HCQ and isolated trauma-derived PEVs and found that co-treatment with trauma PEVs reversed the beneficial antithrombotic effects of HCQ (G), suggesting HCQ’s antithrombotic mechanism is in part through reduction in release of PEVs. *p<0.05 **p<0.005 ***p<0.01

Hydroxychloroquine sulfate reduces thrombus burden in murine DVT which is reversed by trauma-derived platelet extracellular vesicles

Limited historical use of HCQ as a means of DVT prophylaxis have been reported, although the mechanism is unknown.29,31 First, we confirmed IVC ligation resulted in a significant release of PEVs (Figure 4E). Therefore, we hypothesized HCQ therapy would reduce thrombus burden in murine DVT, in part by modulating PEV release. HCQ treatment had a significant reduction in thrombus burden as assessed by thrombus weight (mg, 12.9±4.7 vs 7.4±3.7, p=0.02) (Figure 4F). If HCQ was reducing thrombus burden through the reduction of PEV release we hypothesized adding back trauma-derived PEVs in mice co-treated with HCQ would overcome the HCQ effect. The addition of trauma-derived PEVs reversed the anti-thrombotic effect of HCQ (mg, 17.6±8.5 vs 7.9±6.1, p=0.02), with no significant difference between PEV+HCQ or PEV alone (Figure 4G). Together, these data suggest that HCQ exerts anti-platelet and anti-thrombotic properties in part through the inhibition of release of PEVs.

Discussion

Traumatic injury is a leading cause of morbidity and mortality worldwide in part due to hemorrhagic and thrombotic complications. The underlying mechanisms that drive these complications are diverse and incompletely understood. We sought to determine the role of PEV release following human murine polytrauma in hemostasis and the subsequent development of DVT. Here, we present evidence that in both human and murine trauma, platelet activation leads to a robust release of platelet-derived extracellular vesicles (PEVs) which both promote hemostasis and drive thrombosis after injury. The release and the effects of these trauma-derived PEVs are attenuated by treatment with hydroxychloroquine sulfate (HCQ).

Platelet activation following traumatic injury leads to release of danger-associated molecular pattern (DAMP) molecules and activation of the innate immune system followed by microvascular and macrovascular thrombotic complications, a process described as immunothrombosis.24 Park et. al demonstrated that trauma results in increased circulating levels of pro-coagulant extracellular vesicles; however, the cell-origin of these extracellular vesicles was not determined.25 Furthermore, later work by the same group did not show correlation between extracellular vesicle levels and coagulopathy.26 Interestingly, Curry et. al demonstrated levels of procoagulant EVs correlated with mortality in trauma patients.28 Our findings significantly expand on not only the release of EVs and specifically PEVs following trauma but demonstrate their important physiologic functions such as hemostasis and contributions to venous thrombosis. It is possible that release of PEVs following injury has a beneficial response in promoting hemostasis, as evidenced by the reduction of tail vein bleed time and intraperitoneal hemorrhage following liver laceration in mice. The pro-hemostatic role of PEVs may represent an evolutionary response to injury where hemorrhage control is an immediate priority, which fits with prior findings that patients with lower level of PEVs following trauma had increased mortality.28 However, this response may be exaggerated in the severely injured patient and/or modern medical care inserts a survivor bias from injuries, whereby an exaggerated PEV release (beneficial for hemostasis) ultimately contributes to later thrombotic complications.

In the present study, we demonstrate that PEVs contribute the thrombosis. It remains unexplored how the response of activated platelets and PEVs alters the dynamics of blood flow following injury. The rheology of blood flow following injury and hemorrhage has been previously investigated46, and preservation of the cell-free layer has been shown to be critical for glycocalyx and endothelial integrity.47 Alterations of flow, both due to hypoperfusion and changes in the cellular composition of blood following injury likely have important implications for the development of thrombosis. Whether this allows enhanced contact with endothelial and/or subendothelium, provoking platelet activation and EV release, remains unexplored. Nonetheless, beyond the simple exposure to increased pro-thrombotic microparticles, the changes in flow dynamics after trauma likely provoke changes in coagulation that are incompletely understood.

Although the role of tissue factor bearing tumor microparticles has been previously implicated in cancer associated VTE, the potential contribution of PEVs to thrombosis following injury was previously unexplored.48–50 We now show that trauma-derived PEVs enhance platelet aggregation and drive thrombin generation with a marked increase in thrombus burden in a murine model of DVT. These findings appear to be independent of tissue factor given low levels in PEV trauma samples, which is consistent with prior reports that PEVs have low tissue factor activity.51 PEVs could be found within the developing thrombus and interacting with platelets. Interestingly, it has been proposed that most of circulating PEVs are cleared rapidly from circulation; however, our data shows that PEVs that remain in circulation can participate in thrombus formation and have persistent effects in the local milieu of the developing thrombus.52,53 Additionally, elevated circulating levels of extracellular vesicles from multiple cell lineages have been found in VTE patients and implicated the pathogenesis of VTE.54–56 As such, PEVs may have the potential to modulate cell interactions and localize to thrombus locations leading to increased thrombus formation.

We also present evidence for the use of HCQ as an anti-platelet, anti-thrombotic prophylaxis in murine DVT. Patients with systemic lupus erythematosus treated with HCQ experienced fewer cardiac events for composite incident coronary artery disease, stroke, or transient ischemic attack.57 Additionally, prior to initiation of VTE prophylaxis with heparins, HCQ given postoperatively reduced DVT incidence in surgical patients compared to no therapy.31 Importantly, bleeding complications have not been reported in the few trials examining the effectiveness of HCQ in DVT prevention.29,31 Moreover, in a study examining the potential antithrombotic benefit of HCQ in patients with anti-phospholipid syndrome, patients treated with HCQ in addition to standard oral anticoagulant therapy had no increased bleeding events.58,59 The trauma literature demonstrates persistently elevated VTE rates despite the use of gold-standard therapy via low-molecular weight heparins, suggesting that perhaps targeting the coagulation cascade alone is insufficient in this high-risk patient population.3,4,60–63 The addition of platelet inhibition for DVT prophylaxis in trauma has been advocated by some but remains controversial.63–65 Whether HCQ represents a potential VTE thromboprophylaxis strategy in addition to current standard of care warrants further clinical investigation. Importantly, however, HCQ is known to impact numerous cell types and cellular pathways in addition to platelets. 10,66 Although HCQ markedly decreases PEV release from platelets, the mechanism of this effect is unknown. Interestingly, work on human platelets has shown HCQ decreases release of arachidonic acid and thromboxane generation in activated platelets.67 In contrast, we did not find a significant difference in platelet activation in response to thrombin stimulation between mice treated with HCQ compared to control; however, our numbers were small and we did not look specifically at release of platelet contents as a marker of activation. This warrants further work in the future to further elucidate the anti-platelet mechanism of HCQ. HCQ is a well-known as an anti-autophagy agent and is also known to regulate NET formation, both of which have been implicated in DVT formation and could contribute to the observed effects in our model.66,68

The present work has a number of important limitations including the methodology for isolation and functional testing of PEVs. First, we isolated PEVs from whole blood samples by centrifugation techniques and characterized them via flow cytometry. This method has been characterized and validated previously33; however, it is possible that extracellular vesicles from other cell types contaminate the isolation. In an attempt to mitigate this, isolated PEVs underwent positive selection utilizing a CD42b resin to purify platelet PEVs and exclude CD42 negative extracellular vesicles, however the presence of retained MVs of different origin and the presence of soluble mediators in the PEV fraction could contribute to the findings. Second, the release of PEVs from activated platelets in trauma likely results in a heterogeneous PEV population in terms of cargo load and cell-surface ligand. As multiple pro-thrombotic ligands have been discovered in or on platelet extracellular vesicles, it is likely there is no individual ligand responsible but rather the collective effect of various agonists concentrated in PEVs.17,22,33,69 The model of induction of DVT, while accepted in the literature, is an artificial mode of thrombus development. Our murine studies were conducted in young, male C57 mice and need to be confirmed in both older and female mice. Finally, while our human data is exciting and highlights the need for prospective assessment of PEV in trauma patients the association with development of DVT, it is preliminary in nature and limited in the comparison group to healthy controls that were not matched to the trauma patients.

In summary, we present evidence that traumatic injury results in substantial release of PEVs. Additionally, we have demonstrated these PEVs have diverse roles including thrombin generation and promotion of hemostasis while subsequently contributing to later DVT development. Furthermore, HCQ treatment led to a decrease in the pathophysiologic thrombotic effects of these PEVs released following trauma. Taken together, we propose that PEVs after trauma may be an important and previously inadequately characterized contributor to thrombosis after injury and that attenuating their production may serve as a potential area of investigation to mitigate the burden of DVT after injury.

Supplementary Material

Supp figS1-4

Key Points:

  1. Traumatic injury results in an abundant release of platelet extracellular vesicles

  2. Platelet extracellular vesicles released following injury promote hemostasis but also contribute to formation of DVT.

Acknowledgments

This work is supported by U.S. National Institutes of Health grants 1 R35 GM119526-01 and UM1HL120877-01, support from the Vascular Medicine Institute at the University of Pittsburgh, the Hemophilia Center of Western Pennsylvania, and the Institute for Transfusion Medicine, as well as the American Association for the Surgery for Trauma Research award (all to MDN). This work was additionally supported by the training grant 5T32GM008516 (to MRD) from the National Institute of Health. The authors would like to thank Ms. Lauryn Kohut for her animal expertise.

Footnotes

Disclosure of Conflicts of Interest

MDN has the following financial relationships to disclose: Consultant, External Scientific Advisor for Anticoagulation Science for Janssen Pharmaceuticals (Johnson & Johnson), Trauma Advisory Board, CSL Behring, and US Patent 9,072,760 TLR4 inhibitors for the treatment of human infectious and inflammatory disorders (issued to Neal, Wipf, Hackam, Sodhi).

References

  • 1.Sauaia AM, Moore FAM, Moore EEM, et al. Epidemiology of Trauma Deaths: A Reassessment. J. Trauma-Injury Infect. Crit. Care 1995;38:185–93. [DOI] [PubMed] [Google Scholar]
  • 2.Heit JA. Epidemiology of venous thromboembolism. Nat. Rev. Cardiol 2015;12(8):464–74. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Pierce C a, Haut ER, Kardooni S, et al. Surveillance bias and deep vein thrombosis in the national trauma data bank: the more we look, the more we find. J. Trauma 2008;64(4):932–6; discussion 936–7. [DOI] [PubMed] [Google Scholar]
  • 4.Park MS, Perkins SE, Spears GM, et al. Risk factors for venous thromboembolism after acute trauma: A population-based case-cohort study. Thromb. Res 2016;144:40–5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Engelmann B, Massberg S. Thrombosis as an intravascular effector of innate immunity. Nat. Rev. Immunol 2012;13(1):34–45. [DOI] [PubMed] [Google Scholar]
  • 6.Saghazadeh A, Hafizi S, Rezaei N. Inflammation in venous thromboembolism: Cause or consequence? Int. Immunopharmacol 2015;28(1):655–665. [DOI] [PubMed] [Google Scholar]
  • 7.Saghazadeh A, Rezaei N. Inflammation as a cause of venous thromboembolism. Crit. Rev. Oncol. Hematol 2016;99:272–285. [DOI] [PubMed] [Google Scholar]
  • 8.Vogel S, Bodenstein R, Chen Q, et al. Platelet-derived HMGB1 is a critical mediator of thrombosis. J. Clin. Invest 2015;125(12):4638–4654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Stark K, Philippi V, Stockhausen S, et al. Disulfide HMGB1 derived from platelets coordinates venous thrombosis in mice. Blood. 2016;128(20):2435–2450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Dyer MR, Chen Q, Haldeman S, et al. Deep vein thrombosis in mice is regulated by platelet HMGB1 through release of neutrophil-extracellular traps and DNA. Sci. Rep 2018;8(1):2068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Koupenova M, Clancy L, Corkrey HA, Freedman JE. Circulating Platelets as Mediators of Immunity, Inflammation, and Thrombosis. Circ. Res 2018;122(2):337–351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Kim S-J, Davis R, Jenne C. Platelets as Modulators of Inflammation. Semin. Thromb. Hemost 2018;44(02):091–101. [DOI] [PubMed] [Google Scholar]
  • 13.Brill A, Fuchs TA, Chauhan AK, et al. von Willebrand factor-mediated platelet adhesion is critical for deep vein thrombosis in mouse models. Blood. 2011;117(4):1400–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Mackman N New insights into the mechanisms of venous thrombosis. J. Clin. Invest 2012;122(7):2331–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Carestia A, Kaufman T, Schattner M. Platelets: New Bricks in the Building of Neutrophil Extracellular Traps. Front. Immunol 2016;7:271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.von Brühl M-L, Stark K, Steinhart A, et al. Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo. J. Exp. Med 2012;209(4):819–35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Burnouf T, Goubran HA, Chou M-L, Devos D, Radosevic M. Platelet microparticles: Detection and assessment of their paradoxical functional roles in disease and regenerative medicine. YBLRE. 2014;28:155–166. [DOI] [PubMed] [Google Scholar]
  • 18.Lacroix R, Dubois C, Leroyer AS, Sabatier F, Dignat-George F. Revisited role of microparticles in arterial and venous thrombosis. J. Thromb. Haemost 2013;11(s1):24–35. [DOI] [PubMed] [Google Scholar]
  • 19.SHANTSILA E, KAMPHUISEN PW, LIP GYH. Circulating microparticles in cardiovascular disease: implications for atherogenesis and atherothrombosis. J. Thromb. Haemost 2010;8(11):2358–2368. [DOI] [PubMed] [Google Scholar]
  • 20.Barry OP, Praticò D, Savani RC, FitzGerald GA. Modulation of monocyte-endothelial cell interactions by platelet microparticles. J. Clin. Invest 1998;102(1):136–44. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Berckmans RJ, Nieuwland R, Böing AN, et al. Cell-derived microparticles circulate in healthy humans and support low grade thrombin generation. Thromb. Haemost 2001;85(4):639–646. [PubMed] [Google Scholar]
  • 22.Forlow SB, Mcever RP, Nollert MU. Leukocyte-leukocyte interactions mediated by platelet microparticles under flow. 2000;95:1317–1323. [PubMed] [Google Scholar]
  • 23.WANG J-G, MANLY D, KIRCHHOFER D, PAWLINSKI R, MACKMAN N. Levels of microparticle tissue factor activity correlate with coagulation activation in endotoxemic mice. J. Thromb. Haemost 2009;7(7):1092–1098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Ogura H, Kawasaki T, Tanaka H, et al. Activated platelets enhance microparticle formation and platelet-leukocyte interaction in severe trauma and sepsis. J. Trauma 2001;50(5):801–9. [DOI] [PubMed] [Google Scholar]
  • 25.Park MS, Owen BAL, Ballinger BA, et al. Quantification of hypercoagulable state after blunt trauma: microparticle and thrombin generation are increased relative to injury severity, while standard markers are not. Surgery. 2012;151(6):831–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Park MS, Xue A, Spears GM, et al. Thrombin generation and procoagulant microparticle profiles after acute trauma: A prospective cohort study. J. Trauma Acute Care Surg 2015;79(5):726–31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Tian Y, Salsbery B, Wang M, et al. Brain-derived microparticles induce systemic coagulation in a murine model of traumatic brain injury. Blood. 2015;125(13):2151–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Curry N, Raja A, Beavis J, Stanworth S, Harrison P. Levels of procoagulant microvesicles are elevated after traumatic injury and platelet microvesicles are negatively correlated with mortality. J. Extracell. vesicles 2014;3(1):25625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Carter AE, Eban R. Prevention of postoperative deep venous thrombosis in legs by orally administered hydroxychloroquine sulphate. Br. Med. J 1974;3(5923):94–95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Achuthan S, Ahluwalia J, Shafiq N, et al. Hydroxychloroquine’s Efficacy as an Antiplatelet Agent Study in Healthy Volunteers. J. Cardiovasc. Pharmacol. Ther 2015;20(2):174–180. [DOI] [PubMed] [Google Scholar]
  • 31.Wu TK, Tsapogas MJ, Jordan FR . Prophylaxis of deep venous thrombosis by hydroxychloroquine sulfate and heparin. Surg. Gynecol. Obstet 1977;145(5):714–8. [PubMed] [Google Scholar]
  • 32.Darwiche SS, Kobbe P, Pfeifer R, et al. Pseudofracture: an acute peripheral tissue trauma model. J. Vis. Exp 2011;50:1–5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Hottz ED, Lopes JF, Freitas C, et al. Platelets mediate increased endothelium permeability in dengue through NLRP3-inflammasome activation. Blood. 2013;122(20):3405–3414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Hemker HC, Giesen P, Al Dieri R, et al. Calibrated Automated Thrombin Generation Measurement in Clotting Plasma. Pathophysiol. Haemost. Thromb 2003;33(1):4–15. [DOI] [PubMed] [Google Scholar]
  • 35.Shukla M, Sekhon UDS, Betapudi V, Li W, Hickman DA, Pawlowski CL, Dyer MR, Neal MD, McCrae KR, Sen Gupta A. In vitro characterization of SynthoPlateTM (synthetic platelet) technology and its in vivo evaluation in severely thrombocytopenic mice. J. Thromb. Haemost 2017;15(2):375–387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Dyer M, Haldeman S, Gutierrez A, et al. Uncontrolled Hemorrhagic Shock Modeled via Liver Laceration in Mice with Real Time Hemodynamic Monitoring. J. Vis. Exp 2017;123:55554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Diaz JA, Obi AT, Myers DD, et al. Critical review of mouse models of venous thrombosis. Arterioscler. Thromb. Vasc. Biol 2012;32(3):556–62. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Vogel S, Bodenstein R, Chen Q, et al. Platelet-derived HMGB1 is a critical mediator of thrombosis. J. Clin. Invest 2015;125(12):4638–54. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Théry C, Witwer KW, Aikawa E, et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J. Extracell. Vesicles 2018;7(1):1535750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Goetzl EJ, Goetzl L, Karliner JS, Tang N, Pulliam L. Human plasma platelet-derived exosomes: effects of aspirin. FASEB J. 2016;30(5):2058–63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Pienimaeki-Roemer A, Kuhlmann K, Böttcher A, et al. Lipidomic and proteomic characterization of platelet extracellular vesicle subfractions from senescent platelets. Transfusion. 2015;55(3):507–521. [DOI] [PubMed] [Google Scholar]
  • 42.Aatonen M, Grönholm M, Siljander P. Platelet-Derived Microvesicles: Multitalented Participants in Intercellular Communication. Semin. Thromb. Hemost 2012;38(01):102–113. [DOI] [PubMed] [Google Scholar]
  • 43.Edwards MH, Pierangeli S, Liu X, et al. Hydroxychloroquine reverses thrombogenic properties of antiphospholipid antibodies in mice. Circulation. 1997;96(12):4380–4. [DOI] [PubMed] [Google Scholar]
  • 44.Achuthan S, Ahluwalia J, Shafiq N, et al. Hydroxychloroquine’s Efficacy as an Antiplatelet Agent Study in Healthy Volunteers. J. Cardiovasc. Pharmacol. Ther 2015;20(2):174–180. [DOI] [PubMed] [Google Scholar]
  • 45.Hu C, Lu L, Wan J-P, Wen C. The Pharmacological Mechanisms and Therapeutic Activities of Hydroxychloroquine in Rheumatic and Related Diseases. Curr. Med. Chem 2017;24(20):2241–9. [DOI] [PubMed] [Google Scholar]
  • 46.Bragin DE, Kameneva MV, Bragina OA, et al. Rheological effects of drag-reducing polymers improve cerebral blood flow and oxygenation after traumatic brain injury in rats. J. Cereb. Blood Flow Metab 2017;37(3):762–775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Johansson PI, Henriksen HH, Stensballe J, et al. Traumatic Endotheliopathy. Ann. Surg 2017;265(3):597–603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Thomas GM, Panicot-Dubois L, Lacroix R, et al. Cancer cell-derived microparticles bearing P-selectin glycoprotein ligand 1 accelerate thrombus formation in vivo. J. Exp. Med 2009;206(9):1913–27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Thomas GM, Brill A, Mezouar S, et al. Tissue factor expressed by circulating cancer cell-derived microparticles drastically increases the incidence of deep vein thrombosis in mice. J. Thromb. Haemost 2015;13(7):1310–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.DAVILA M, AMIRKHOSRAVI A, COLL E, et al. Tissue factor-bearing microparticles derived from tumor cells: impact on coagulation activation. J. Thromb. Haemost 2008;6(9):1517–1524. [DOI] [PubMed] [Google Scholar]
  • 51.Lacroix R, Dubois C, Leroyer AS, Sabatier F, Dignat-George F. Revisited role of microparticles in arterial and venous thrombosis. J. Thromb. Haemost 2013;11:24–35. [DOI] [PubMed] [Google Scholar]
  • 52.RAND ML, WANG H, BANG KWA, PACKHAM MA, FREEDMAN J. Rapid clearance of procoagulant platelet-derived microparticles from the circulation of rabbits. J. Thromb. Haemost 2006;4(7):1621–1623. [DOI] [PubMed] [Google Scholar]
  • 53.Rank A, Nieuwland R, Crispin A, et al. Clearance of platelet microparticles in vivo. Platelets. 2011;22(2):111–116. [DOI] [PubMed] [Google Scholar]
  • 54.Ay C, Freyssinet J-M, Sailer T, Vormittag R, Pabinger I. Circulating procoagulant microparticles in patients with venous thromboembolism. Thromb. Res 2009;123(5):724–6. [DOI] [PubMed] [Google Scholar]
  • 55.Campello E, Spiezia L, Radu CM, et al. Endothelial, platelet, and tissue factor-bearing microparticles in cancer patients with and without venous thromboembolism. Thromb. Res 2011;127(5):473–7. [DOI] [PubMed] [Google Scholar]
  • 56.Chirinos JA, Heresi GA, Velasquez H, et al. Elevation of endothelial microparticles, platelets, and leukocyte activation in patients with venous thromboembolism. J. Am. Coll. Cardiol 2005;45(9):1467–71. [DOI] [PubMed] [Google Scholar]
  • 57.Sharma TS, Wasko MCM, Tang X, et al. Hydroxychloroquine Use Is Associated With Decreased Incident Cardiovascular Events in Rheumatoid Arthritis Patients. J. Am. Heart Assoc 2016;5(1):e002867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.Schmidt-Tanguy A, Voswinkel J, Henrion D, et al. Antithrombotic effects of hydroxychloroquine in primary antiphospholipid syndrome patients. J. Thromb. Haemost 2013;11(10):1927–9. [DOI] [PubMed] [Google Scholar]
  • 59.Belizna C Hydroxychloroquine as an anti-thrombotic in antiphospholipid syndrome. Autoimmun. Rev 2015;14(4):358–362. [DOI] [PubMed] [Google Scholar]
  • 60.Geerts WH, Code KI, Jay RM, Chen E, Szalai JP. A Prospective Study of Venous Thromboembolism after Major Trauma. N. Engl. J. Med 1994;331(24):1601–1606. [DOI] [PubMed] [Google Scholar]
  • 61.Barrera LM, Perel P, Ker K, et al. Thromboprophylaxis for trauma patients. Cochrane Database Syst. Rev 2013;(3):CD008303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 62.Skrifvars MB, Bailey M, Presneill J, et al. Venous thromboembolic events in critically ill traumatic brain injury patients. Intensive Care Med. 2017;43(3):419–428. [DOI] [PubMed] [Google Scholar]
  • 63.Karcutskie CA, Dharmaraja A, Patel J, et al. Association of Anti–Factor Xa–Guided Dosing of Enoxaparin With Venous Thromboembolism After Trauma. JAMA Surg. 2017;40(3):CD008303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 64.Brill JB, Calvo RY, Wallace JD, et al. Aspirin as added prophylaxis for deep vein thrombosis in trauma: A retrospective case-control study. J. Trauma Acute Care Surg 2016;80(4):625–30. [DOI] [PubMed] [Google Scholar]
  • 65.Harr JN, Moore EE, Chin TL, et al. Platelets are dominant contributors to hypercoagulability after injury. J. Trauma Acute Care Surg 2013;74(3):756–62; discussion 762–5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 66.Boone BA, Murthy P, Miller-Ocuin J, et al. Chloroquine reduces hypercoagulability in pancreatic cancer through inhibition of neutrophil extracellular traps. BMC Cancer. 2018;18(1):678. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 67.Nosál’ R, Jančinová V, Danihelová E. Chloroquine: A Multipotent Inhibitor of Human Platelets in Vitro. Thromb. Res 2000;98(5):411–421. [DOI] [PubMed] [Google Scholar]
  • 68.Smith CK, Vivekanandan-Giri A, Tang C, et al. Neutrophil extracellular trap-derived enzymes oxidize high-density lipoprotein: an additional proatherogenic mechanism in systemic lupus erythematosus. Arthritis Rheumatol. (Hoboken, N.J.) 2014;66(9):2532–2544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 69.Merten M, Pakala R, Thiagarajan P, Benedict CR. Platelet microparticles promote platelet interaction with subendothelial matrix in a glycoprotein IIb/IIIa-dependent mechanism. Circulation. 1999;99(19):2577–82. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supp figS1-4
NIHMS1040959-supplement-Supp_figS1-4.docx (12.1MB, docx)

RESOURCES