Secondary Logo

Journal Logo

Clinical Science Aspects

HDAC Inhibitor Attenuated NETs Formation Induced by Activated Platelets In Vitro, Partially Through Downregulating Platelet Secretion

Chen, Zhenxing; Liu, Chang; Jiang, Yu; Liu, Hanchi; Shao, Lina; Zhang, Kaiyuan; Cheng, Daye; Zhou, Ying§; Chong, Wei

Author Information
doi: 10.1097/SHK.0000000000001518

Abstract

INTRODUCTION

In addition to phagocytosis, oxygen burst and degranulation, neutrophils, as the most abundant innate immune cells in humans, also achieve their immunological function through neutrophil extracellular traps (NETs) formation (1, 2). NETs are web-like structures composed of DNA, histones, and granular proteins such as neutrophil elastase and myeloperoxidase (MPO) (3). Despite their physiological function in restricting the spread of pathogens, accumulating studies in recent years have revealed that uncontrolled NETs formation plays a vital role in organ injury mediated by dysregulated inflammation (3). Thus far, there have been no effective therapies targeting uncontrolled NETs formation, leaving a large obstacle to attenuating organ injury in diseases related to NETs.

Once stimulated with coagulant or pathogenic stimuli, platelets become activated and undergo appropriate changes, including aggregation and secretion, to achieve their function in hemostasis and the immune response (4, 5). Previous studies have demonstrated that neutrophils incubated with preactivated platelets had NETs formation in vitro, and treatment with antiplatelet drugs, platelet deletion, or knockout of adhesive molecules on platelet surfaces in mice resulted in reduced NETs formation under pathogenic conditions, indicating that activated platelets are an important inducer of pathogenic NETs formation (6–8). Organ injury in many diseases was found to be related to dysregulated platelet activation and NETs formation (9–13). So in vitro studies using activated platelets as inducer of NETs may have extensive application prospects.

Histone acetyltransferase (HAT) and histone deacetylase (HDAC) are enzymes that mediate posttranslational modification of histones and nonhistone proteins. HAT catalyzes acetylation of lysine, and HDAC catalyzes deacetylation of acetylated lysine (14). With dynamic acetyl modification, proteins cyclically change their structure and function. Until now, 18 HDACs have been found in humans and are divided into four classes according to homology with yeast. Predecessors found that treatment with suberoylanilide hydroxamic acid (SAHA), an antitumor drug with a nonselective inhibitory effect on class I, class II, and class IV HDACs, attenuated organ injury and coagulant dysfunction in septic mice, but the exact mechanism was still unrevealed (15). In our previous study, reduced elevation in serum H3, a marker of NETs, in septic mice treated with SAHA was found suggesting potential regulatory effect of HDACs on NETs formation (16).

It has been demonstrated that human platelets express functional HDAC6, which belongs to class II HDACs, and modulates the movement of microtubules by mediating the deacetylation of α-tubulin (17, 18). Platelets have multiple granules, including α-granules, δ-granules and lysosomes, and these granules store diverse substances (4). It has also been reported that platelets’ storage granules are attached to microtubules, and their trafficking is regulated by these microtubules (19). In previous study, platelet factor 4 (PF4) and von Willebrand factor (vWF) in platelets, which were usually synthesized by megakaryocytes and stored in α-granules of platelets, were found to be important mediators in NETs formation induced by activated platelets (20). Therefore, we speculate that HDACs participate in the release of α-granules from platelets and that the inhibition of HDACs during platelet activation may attenuate NETs formation induced by activated platelets.

First, a bioreactive system was established to induce and monitor NETs formation with freshly isolated platelets and neutrophils in this study (Fig. 1). Then, we compared NETs formation induced by preactivated platelets in the presence of SAHA with that in the absence of SAHA through trend and quantity of DNA release. Occurrence of NETs was showed by live-cell imaging and immunofluorescence and PF4 in supernatant of platelets after incubation was evaluated with ELISA.

F1
Fig. 1:
A workflow diagram of the bioreactive system for evaluating drug's effect on platelets’ capacity in NETs inducing.

PATIENTS AND METHODS

Reagents

Reagents were obtained from the following manufacturers: Anticoagulant ACD-A (bjbalb.com, GL3002), Histopaque-1119 (Sigma, 11191), Percoll (Solarbio, P8370), RPMI 1640 medium (Solarbio, 90022), HEPES (Solarbio, H8090), TRAP-6 (bjbalb.com, M04564), SAHA (Sigma, SML0061), Sytox green (bjbalb.com, KFS148), 8% paraformaldehyde (Solarbio, P1112), 5% BSA (Solarbio, SW3015), anti-neutrophil elastase antibody [EPR7479] (Abcam, ab131260), goat antirabbit IgG H&L (Alexa Fluor 488) (Abcam, ab150077), DAPI (Solarbio, C0065), human CXCL4/PF4 Quantikine ELISA kit (R&D, DPF40), and Wright stain solution (Solarbio, G1040).

Human samples

Healthy volunteers were recruited from all people visiting the hospital via a recruitment advertisement published in the First hospital of China Medical University. Individuals who met the following criteria were enrolled: age 18to 50 years, without a history of hematological diseases such as aplastic anemia and agranulocytosis, without a history of autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis, without a history of contagious diseases such as AIDS and syphilis, without a history of endocrine diseases such as diabetes and Cushing's disease, without a history of cardiovascular diseases such as hypertension and atherosclerosis, without a history of tumors, without a history of immunosuppressive therapy. Individuals who met the following criteria were excluded from this study: age < 18 or > 50 years, a history of disease mentioned above, a history of infectious disease in the past 2 weeks and a history of taking antiplatelet drugs or anticoagulants in the past 1 month. After recruitment, four individuals (Supplemental Digital Content 1, https://links.lww.com/SHK/B32) participated in this study and each individual donated sample for one to five times depending on personal will. Each individual received venous puncture on median cubital vein to collect 6 mL to 18 mL whole blood each time depending on experiment conducted that day by medical staff in Department of Emergency, First Hospital of China Medical University, and the operation of venous puncture was standardized. Whole blood was collected in citrate tube or EDTA tube depending on subsequent use and sample from each individual was not mixed with others. This study was approved by the research ethics committee of the First Hospital of China Medical University (No. 2019-185-2), and informed consent was obtained from all volunteers.

Platelet isolation and activation

Venous blood was collected in a citrate tube (BD, 364305), and ACD-A was added to blood at a ratio of 10:1.5 (blood:anticoagulant, v:v). Platelet-rich plasma (PRP) was collected after centrifuging the blood at 300 × g for 10 min. Then, PRP was centrifuged at 450 ×g for 5 min to obtain platelets, and isolated platelets were resuspended in RPMI 1640 medium containing 10 mM HEPES. The purity and concentration of isolated platelets were evaluated with an automated blood cell counter, and the concentration of platelets was adjusted to 100×106 cell/mL. After that, platelets were incubated with TRAP-6 (50 μM) in the presence or absence of SAHA (10 μM) at 37°C with 5% CO2 for 30 min.

PF4 measurement by ELISA

After incubation, the platelet suspension was placed on ice for 4 min to stop the reaction. The supernatant was collected after centrifuging the platelet suspension at 14,000 ×g and 4°C for 3 min. Then, PF4 in the supernatant was measured following instructions for the human CXCL4/PF4 Quantikine ELISA kit. Each sample was tested in duplicate.

Neutrophil isolation and NETs induction

To isolate neutrophils, density gradient centrifugation was conducted. In brief, 6 mL of anticoagulated blood that was collected in an EDTA tube (BD, 365900) was transferred to a centrifuge tube containing 6 mL Histopaque-1119. Samples were centrifuged at 800 ×g for 20 min, plasma and the cell layer containing PBMCs and platelets were discarded, and the suspension containing neutrophils was collected and washed with PBS. After washing, the pellets were resuspended in 3 mL PBS, and the suspension was transferred to a centrifuge tube containing 3 mL 75% isosmotic Percoll, 3 mL 70% isosmotic Percoll, and 3 mL 65% isosmotic Percoll. The samples were centrifuged at 800 ×g for 20 min, and the cell layer in 70% isosmotic Percoll was collected and washed with PBS. After washing, the pellets were resuspended in RPMI 1640 medium containing 10 mM HEPES, the cell number was counted with an automated cell counter (Bio-Rad, TC20), and the concentration of the suspension was adjusted to 2×106 cell/mL. Wright's staining was then conducted to validate the purity of the isolated neutrophils (>90%).

To induce NETs formation with activated platelets, platelets that were preactivated as described above were mixed with neutrophils at a ratio of 1:25 (neutrophils:platelets, N:N), and the samples were incubated at 37°C for 2 h. To induce NETs formation with PMA, neutrophils were incubated with 100 nM PMA at 37°C for 3 h.

Quantification of NETs formation with FI of Sytox green

A FI-T kinetic curve based on the FI of Sytox green was established to describe the trend of DNA release. In brief, samples were prepared as described above. The cell suspension was supplemented with 1.25 μM Sytox green and then plated into a microplate (ThermoFisher, 165305) with a transparent bottom and black walls at a density of 105 cells/well. After that, the microplate was transferred to a microplate reader (Tecan Infinite, F200), and the cells were incubated at 37°C for 2 h (3 h for neutrophils stimulated with PMA). Each group has six wells of cell suspension each time. During incubation, the FI in the cell suspension was detected using excited light at 485 nm every 3 min. Then the mean FI of each group was shown in ordinate and every detecting time-point was shown in horizontal ordinate. NETs formation after incubation in each well was quantified with DNA release reflected by FI increase between FI(2 or 3 h) and FI(0 h).

Live-cell imaging

To monitor the process of NETs formation, live-cell imaging was conducted. In brief, samples were prepared as described above. The cell suspension was supplemented with 1.25 μM Sytox green and then plated in a confocal dish at a density of 1.2×106 cells/dish. After that, the cells were incubated at 37°C with 5% CO2. During incubation, the samples were observed with confocal microscopy (Leica, SP5) with a laser at 488 nm at the following timepoints: 0 h, 1.5 to 2 h, and 2.5 to 3 h.

Immunofluorescence

Immunofluorescence was conducted to validate the presence of NETs. In brief, samples were prepared as described above. The cell suspension was plated in a confocal dish at a density of 1.2×106/dish. After incubation at 37°C with 5% CO2 for 2 h (3 h for neutrophils induced with PMA), an equal volume of 8% paraformaldehyde was added to the dish for fixation. Then, the samples were washed with cold PBS and blocked with 5% BSA. After blocking, the samples were stained with rabbit anti-neutrophil elastase antibody (1:70), antirabbit secondary antibody conjugated with Alexa Fluor 488 (1:1,000) and DAPI (10 μg/mL). Finally, the samples were observed by confocal microscopy (Leica, SP5) in xyz mode.

Statistical analysis

The FI-T curve was used to describe trend of DNA release from neutrophils. Comparison between groups was conducted using repeated measurement ANOVA in SPSS 23.0 with general linear model for repeated measurements followed with LSD t test for multiple comparison (P value in repeated measurement ANOVA meets statistical significance, number of groups > 2). Quantity of NETs was reflected by FI of Sytox green expressed as the mean (SD) and comparison between groups was conducted using Student t test (number of group=2) or one-way ANOVA followed by the LSD t test (P value in one-way ANOVA meets statistical significance, number of groups > 2). The concentration of PF4 in supernatant was expressed as the mean (SD) and comparison between groups was conducted using one-way ANOVA followed by the LSD t test (P value in one-way ANOVA meets statistical significance). A P < 0.05 was considered statistically significant.

RESULTS

Activity of isolated platelets and neutrophils

Because fresh platelets and neutrophils were isolated through complicated procedures, the activity of the isolated cells was evaluated. PF4 in the supernatant of platelets incubated with TRAP-6 was elevated compared with that of platelets incubated without TRAP-6 (1743.57 (141.45) versus 95.78 (19.48) ng/mL, P < 0.001), indicating that the isolated platelets were potentially activated (Fig. 2). It is worth mentioning that the supernatant of platelets incubated without TRAP-6 also had PF4 present (95.78 (19.48) ng/mL), which suggested slight activation of platelets due to the isolation procedure and incubation in vitro.

F2
Fig. 2:
ELISA results of PF4 levels in supernatant are shown in the bar graph.

To validate the potential of NETs formation in isolated neutrophils, neutrophils from one volunteer were separated into two groups with different treatment: N (neutrophils incubated alone) and N+PMA (neutrophils incubated with 100 nM PMA). Mean FI of each group at each detecting time-point was shown in FI-T curve (Fig. 3A). Compared with neutrophils incubated alone, neutrophils incubated with 100 nM PMA had different trend of DNA release, more extracellular DNA at end time-point and more DNA release after incubation (Fig. 3B). Live-cell imaging of neutrophils incubated with PMA showed web-like structure formation (Fig. 4 , A and C) after 3 h of incubation, and immunofluorescence measured by xyz scanning using confocal microscopy showed colocalization of NE with web-like DNA (Fig. 5, Supplemental Digital Content 2, https://links.lww.com/SHK/B33), indicating that isolated neutrophils could produce NETs.

F3
Fig. 3:
A, The mean FI of each group was shown in ordinate and every detecting time-point was shown in horizontal ordinate.
F4
Fig. 4:
Live-cell images captured by confocal microscopy are shown.
F5
Fig. 4 (Continued):
Live-cell images captured by confocal microscopy are shown.
F6
Fig. 5:
One layer of images captured by xyz scanning using confocal microscopy in each group is shown.

Induction of NETs formation with preactivated platelets

In this experiment, we induced NETs with preactivated platelets and monitored it with FI-T curve. Neutrophils from one volunteer were divided into four groups with different treatment: N (neutrophils incubated alone), N+(PLT) (neutrophils incubated with platelets preincubated alone), N+(PLT+TRAP) (neutrophils incubated with platelets preincubated with 50 μM TRAP-6), N+(TRAP) (neutrophils incubated with TRAP-6). Mean FI of each group at each detecting time-point was shown in FI-T curve. Compared with neutrophils incubated alone, neutrophils incubated with platelets preincubated with TRAP-6 had different trend of DNA release (Fig. 6A), more extracellular DNA at end time-point and more DNA release after incubation (Fig. 6B). Subsequent live-cell imaging showed the formation of web-like structures (Fig. 4 , B and D), and immunofluorescence measured by xyz scanning using confocal microscopy showed colocalization of NE with web-like DNA, indicating the presence of NETs (Fig. 5, Supplemental Digital Content 3, https://links.lww.com/SHK/B34). Platelets without extra stimulation, which were slightly activated in vitro without TRAP-6, also induced NETs formation but was less powerful than preactivated platelets (Figs. 4 B and D, 5 and 6 and Supplemental Digital Content 4, https://links.lww.com/SHK/B35). Moreover, neutrophils incubated with the same dose of TRAP-6 without platelets did not exhibit such changes (Figs. 4 A and C and 6), indicating that NETs formation was induced by activated platelets and not directly by TRAP-6.

F7
Fig. 6:
A, The mean FI of each group was shown in ordinate and every detecting time-point was shown in horizontal ordinate.

Platelets activated in the presence of the HDACi showed attenuated capacity in NETs inducing

In this experiment, neutrophils from one volunteer were divided into three groups with different treatments: N (neutrophils incubated alone), N+(PLT+TRAP) (neutrophils incubated with platelets preactivated with 50 μM TRAP-6), N+(PLT+TRAP+SAHA) (neutrophils incubated with platelets preactivated with 50 μM TRAP-6 in the presence of 10 μM SAHA). Mean FI in each group at each detecting time-point was shown in FI-T curve. Compared with N, N+(PLT+TRAP+SAHA) had different trend of DNA release (Fig. 7A), more extracellular DNA at end time-point, and more DNA release after incubation (Fig. 7B). Subsequent live-cell imaging showed the formation of web-like structures (Fig. 4 , B and D), and immunofluorescence measured by xyz scanning using confocal microscopy showed colocalization of NE with web-like DNA (Fig. 5, Supplemental Digital Content 5, https://links.lww.com/SHK/B36), indicating that NETs formation was not blocked in N+(PLT+TRAP+SAHA). Compared with N+(PLT+TRAP), N+(PLT+TRAP+SAHA) had different trend of DNA release (Fig. 7A), less extracellular DNA at end time-point, and less DNA release after incubation (Fig. 7B) indicating that platelets activated in the presence of HDACi had attenuated capacity in NETs inducing.

F8
Fig. 7:
A, The mean FI of each group was shown in ordinate and every detecting time-point was shown in horizontal ordinate.

The HDACi attenuated platelet secretion

To explore the mechanism by which SAHA attenuated platelets’ capacity in NETs inducing, isolated platelets were divided into three groups with different treatment: PLT (platelets incubated alone), PLT+TRAP (platelets incubated with 50 μM TRAP-6), PLT+TRAP+SAHA (platelets incubated with 50 μM TRAP-6 in the presence of 10 μM SAHA). Compared with PLT, PLT+TRAP, and PLT+TRAP+SAHA both had higher PF4 in supernatant (Fig. 2) after incubation indicating that isolated platelets had PF4 secretion after TRAP-6 stimulation and it was not blocked by SAHA. Compared with PLT+TRAP, PLT+TRAP+SAHA had less PF4 in the supernatant indicating that platelet secretion induced by TRAP-6 was downregulated in the presence of HDACi (Fig. 2).

DISCUSSION

Despite accumulating evidence in recent years revealing that NETs play a vital role in the pathological process of diverse diseases, there have not been any effective treatments targeting dysregulated NETs formation due to two main obstacles: difficulty of measurement and unclear mechanisms of NETs formation. Therefore, we conducted several experiments in this study with two aims: to establish a bioreactive system for monitoring and studying the effect of drugs on NETs formation in vitro and to test if HDACis could affect NETs formation related to platelet activation.

Establishment of an in vitro bioreactive system

To establish such a system, we have overcome three obstacles: cell isolation, induction of NETs, and detection of NETs.

First, in this study, isolation and preactivation of cells could be completed within 2 h of collection from volunteers. The essential instruments for cell isolation, such as a centrifuge, biosafety cabinet and incubator, made the process easy to conduct in general laboratories. Ten times higher PF4 in supernatant of platelets stimulated with TRAP-6 than unstimulated platelets and formation of NETs in neutrophils stimulated with 100 nM PMA suggest that cell isolation in this study could be achieved with general instruments and the isolated cells were suitable for further functional tests.

Second, we induced NETs formation with activated platelets. Neutrophils have been shown to produce NETs after incubation with activated platelets in vitro(6, 20). McDonald et al. found that mice with platelet deletions or knockout of adhesive molecules on platelet surfaces had reduced NETs formation during sepsis, and it was also reported that mice treated with antiplatelet drugs had reduced NETs formation and lung injury during sterile inflammation, indicating that activated platelets are important inducers of NETs formation in vivo(7, 8, 21). Thus, the induction of NETs by activated platelets provided us with a perspective on the complicated relationship between neutrophils and platelets. In this study, platelets were activated by TRAP-6, which acted as an agonist of PAR-1 and mimicked coagulant stimulus. The capacity of TRAP-6 to activate platelets to release α-granules has been proven by previous studies (22, 23). Additionally, it has been demonstrated that platelets preactivated by TRAP-6 could induce NETs formation in vitro(7, 21). Similarly, we showed here both secretion of PF4 and capacity of NETs induction by TRAP-6-activated platelets.

Third, we detected NETs formation by FI-T curve, live-cell imaging, and immunofluorescence in this study. This is the first study to use FI-T curve to monitor NETs formation induced by activated platelets (24). The FI-T curve was obtained by incubating neutrophils with inducers in the presence of Sytox green and continuously monitoring FI in the cell suspension. Compared with other methods for NETs detection, such as MPO-DNA or NE-DNA, which require procedures to collect NETs first, the FI-T curve was much easier to conduct. Moreover, the FI-T curve could not only quantify NETs formation at one single time-point but also provide us with a global view of NETs formation. For example, in this study we see different trend of DNA release between groups by analyzing trend of FI changing with time. It did not affect the results of quantifying but enhanced the accuracy of drug selection especially for those with a time dependent manner. To validate that these changes were caused by NETs rather than large amounts of dead cells, live-cell imaging was conducted during sample incubation, and the formation of web-like structures made up of DNA was detected. Furthermore, large amounts of dead cells were not found. NETs were further validated by immunofluorescence measured by xyz scanning using confocal microscopy. These results suggest that the FI-T curve could be applied to monitor and quantify NETs formation induced by activated platelets. Specifically, only 100 μL of sample was needed for each well, making high-throughput detection or drug selection for NETs modulation possible. The limitation of our system was that live-cell imaging cannot be conducted in the same sample to validate NETs formation, resulting in the possibility of false-positive results. With the development of live-cell detection technology, such as the recently published EVOS M7000 (ThermoFisher) live-cell imaging system, which provides simultaneous imaging, monitoring, and sample incubation in a 96-well microplate, this problem will be addressed in the future.

The HDACi attenuated the secretion of PF4, resulting in reduced NETs formation induced by activated platelets.

In the past decade, many studies on HDACis have been conducted, and their protective effect on organ injury in hemorrhagic shock, trauma, sepsis, and cardiovascular disease was found (15, 25, 26). Although at the level of epigenetic modulation, attenuated activation of some signaling pathways related to inflammation and downregulated transcription of genes related to proinflammatory cytokines were found, the key mechanism by which HDACis exert their protective effect is still unknown (26–28). Recently, it was reported that HDAC6 participates in the production of platelets at the post-translational level by mediating deacetylation of nonhistone proteins (29). Functional HDAC6 in platelets modulates the movement of microtubules by mediating the deacetylation of nonhistone α-tubulin (17, 18). Interestingly, α-granules, which store functional substances such as PF4 and vWF, are attached to microtubules (19). In addition, it has been reported that the movement of microtubules mediates the trafficking of α-granules in platelets during activation. Additionally, platelet secretion was inhibited by blocking microtubules with antibodies (30). Therefore, we speculated that HDACs participate in the release of α-granules.

In this study, the platelets were incubated with TRAP-6 and SAHA, one of the HDACis, whose target includes HDAC6. Although the elevation of PF4 in the supernatant was not blocked, it was still attenuated compared with that of the supernatant of platelets incubated without SAHA, indicating that attenuation of NETs formation was at least partially due to reduced platelet secretion. It could be explained by the following reasons that the secretion of PF4 was not fully blocked. First, modulating microtubule trafficking may not be effective for α-granules near the membrane after stimulation, and this was supported by reports that the secretion of PF4 was time-dependent, with nearly 50% of the PF4 secreted within 5 min after stimulation, which is earlier than the movement of microtubules, which usually occurred later (17, 18, 23). Second, the activity of HDAC6 may not be fully inhibited by SAHA. Although it has been shown in a previous study that HDAC6 in platelets could be inhibited by another nonselective HDAC inhibitor, TSA, which has a similar inhibitory effect as that of SAHA, whether HDAC6 in platelets could be fully inhibited by SAHA in this study was not evaluated (18). Third, because only PF4 was detected, we could not exclude the possibility that secretion of other substances contained in α-granules was more significantly inhibited. It is possible that only some specific subtypes of the α-granules were modulated by microtubules during stimulation. It was previously reported that there are diverse subtypes of α-granules containing different cargoes (31, 32).

Besides, this study still has limitations in three parts: volunteers, study design, and measurement. First, the amount of participants is small, only four volunteers participate in this study to donate samples. Whether results got in this study is a mass event common to all people should be validated in further study with large sample size. Second, this study only provides evidence that HDACs participate in TRAP-6 induced platelets activation. If it applied to platelets activation induced by other stimulators need to be further studied. Neither could the study tell the exact mechanism of reduced platelet activation and NETs formation, because SAHA was a chemical compound with other potential effect. In further study, we will use other HDAC inhibitor with specific effect to see which HDAC participants in this process. Third, we only evaluated limited markers in this study. In further study, diverse markers will be used to perform a globally evaluation of platelet activation and NETs formation. Although the results from this study were not enough to prove the presence of a pathway in which HDAC6 mediates the movement of microtubules to modulate the release of stored granules from platelets, which could affect many downstream events, including NETs formation, we still provide new insight into the function of HDACs, as well as the modulation of NETs formation.

Acknowledgments

The authors express their thanks to the Department of Emergency and the Institute of Endocrinology in First Hospital of China Medical University for generously providing the experimental materials used in this study.

REFERENCES

1. Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, Weinrauch Y, Zychlinsky A. Neutrophil extracellular traps kill bacteria. Science 303 (5663):1532–1535, 2004.
2. Honda M, Kubes P: Neutrophils and neutrophil extracellular traps in the liver and gastrointestinal system JT Nat Rev Gastroenterol Hepatol; 15 (4):206–221, 2018.
3. Papayannopoulos V. Neutrophil extracellular traps in immunity and disease. Nat Rev Immunol 2018; 18 (2):134–147.
4. van der Meijden PEJ, Heemskerk JWM: Platelet biology and functions: new concepts and clinical perspectives JT Nat Rev Cardiol; 16 (3):166–179, 2019.
5. Middleton EA, Weyrich AS, Zimmerman GA. Platelets in pulmonary immune responses and inflammatory lung diseases. Physiol Rev 2016; 96 (4):1211–1259.
6. Clark SR, Ma AC, Tavener SA, McDonald B, Goodarzi Z, Kelly MM, Patel KD, Chakrabarti S, McAvoy E, Sinclair GD, et al. Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood. Nat Med 13 (4):463–469, 2007.
7. Caudrillier A, Kessenbrock K, Gilliss BM, Nguyen JX, Marques MB, Monestier M, Toy P, Werb Z, Looney MR. Platelets induce neutrophil extracellular traps in transfusion-related acute lung injury. J Clin Invest 122 (7):2661–2671, 2012.
8. McDonald B, Urrutia R, Yipp BG, Jenne CN, Kubes P. Intravascular neutrophil extracellular traps capture bacteria from the bloodstream during sepsis. Cell Host Microbe 12 (3):324–333, 2012.
9. Marx C, Novotny J, Salbeck D, Zellner KR, Nicolai L, Pekayvaz K, Kilani B, Stockhausen S, Burgener N, Kupka D, et al. Eosinophil-platelet interactions promote atherosclerosis and stabilize thrombosis by eosinophil extracellular traps. Blood 134:1859–1872, 2019.
10. Silva JC, Rodrigues NC, Thompson-Souza GA, Muniz VS, Neves JS, Figueiredo RT. Mac-1 triggers neutrophil DNA extracellular trap formation to Aspergillus fumigatus independently of PAD4 histone citrullination. J Leukoc Biol 107:69–83, 2019.
11. Le Joncour A, Martos R, Loyau S, Lelay N, Dossier A, Cazes A, Fouret P, Domont F, Papo T, Jandrot-Perrus M, et al. Critical role of neutrophil extracellular traps (NETs) in patients with Behcet's disease. Ann Rheum Dis 78 (9):1274–1282, 2019.
12. Bryk AH, Prior SM, Plens K, Konieczynska M, Hohendorff J, Malecki MT, Butenas S, Undas A. Predictors of neutrophil extracellular traps markers in type 2 diabetes mellitus: associations with a prothrombotic state and hypofibrinolysis. Cardiovasc F Diabetol 18 (1):49, 2019.
13. McDonald B, Davis RP, Kim SJ, Tse M, Esmon CT, Kolaczkowska E, Jenne CN. Platelets and neutrophil extracellular traps collaborate to promote intravascular coagulation during sepsis in mice. Blood 129 (10):1357–1367, 2017.
14. Falkenberg KJ, Johnstone RW. Histone deacetylases and their inhibitors in cancer, neurological diseases and immune disorders. Nat Rev Drug Discov 13 (9):673–691, 2014.
15. Zhao T, Li Y, Liu B, Wu E, Sillesen M, Velmahos GC, Halaweish I, Alam HB. Histone deacetylase inhibitor treatment attenuates coagulation imbalance in a lethal murine model of sepsis. Surgery 156 (2):214–220, 2014.
16. Li Y, Liu B, Fukudome EY, Lu J, Chong W, Jin G, Liu Z, Velmahos GC, deMoya M, King DR. Identification of citrullinated histone H3 as a potential serum protein biomarker in a lethal model of lipopolysaccharide-induced shock. Surgery 150 (3):442–451, 2011.
17. Sadoul K, Wang J, Diagouraga B, Vitte AL, Buchou T, Rossini T, Polack B, Xi X, Matthias P, Khochbin S. HDAC6 controls the kinetics of platelet activation. Blood 120 (20):4215–4218, 2012.
18. Aslan JE, Phillips KG, Healy LD, Itakura A, Pang J, Mccarty OJ. Histone deacetylase 6-mediated deacetylation of α-tubulin coordinates cytoskeletal and signaling events during platelet activation. Am J Physiol Cell Physiol 305 (12):C1230–C1239, 2013.
19. Doris C, Bulmaro C, Ricardo M, Sirenia G, Galván IJ. Actin filaments and microtubule dual-granule transport in human adhered platelets: the role of alpha-dystrobrevins. Br J Haematol 149 (1):124–136, 2010.
20. Carestia A, Kaufman T, Rivadeneyra L, Landoni VI, Pozner RG, Negrotto S, D’Atri LP, Gomez RM, Schattner M. Mediators and molecular pathways involved in the regulation of neutrophil extracellular trap formation mediated by activated platelets. J Leukoc Biol 99 (1):153–162, 2016.
21. Rossaint J, Herter JM, Van Aken H, Napirei M, Döring Y, Weber C, Soehnlein O, Zarbock A. Synchronized integrin engagement and chemokine activation is crucial in neutrophil extracellular trap-mediated sterile inflammation. Blood 123 (16):2573–2584, 2014.
22. Chatterjee M, Huang Z, Zhang W, Jiang L, Hultenby K, Zhu L, Hu H, Nilsson GP, Li N. Distinct platelet packaging, release, and surface expression of proangiogenic and antiangiogenic factors on different platelet stimuli. Blood 117 (14):3907–3911, 2011.
23. Jonnalagadda D, Izu LT, Whiteheart SW. Platelet secretion is kinetically heterogeneous in an agonist-responsive manner. Blood 120 (26):5209–5216, 2012.
24. Sil P, Yoo DG, Floyd M, Gingerich A, Rada B. High throughput measurement of extracellular DNA release and quantitative NET formation in human neutrophils in vitro. J Vis Exp 2016 (112):e52779, 2016.
25. Zhao TC, Cheng G, Zhang LX, Tseng YT, Padbury JF. Inhibition of histone deacetylases triggers pharmacologic preconditioning effects against myocardial ischemic injury. Cardiovasc Res 76 (3):473–481, 2007.
26. Halaweish I, Nikolian V, Georgoff P, Li Y, Alam HB. Creating a “Pro-survival Phenotype” through histone deacetylase inhibition: past, present, and future. Shock 44: (Suppl 1): 6, 2015.
27. Li Y, Liu B, Gu X, Kochanek AR, Fukudome EY, Liu Z, Zhao T, Chong W, Zhao Y, Zhang D, et al. Creating a “pro-survival” phenotype through epigenetic modulation. Surgery 152 (3):455–464, 2012.
28. Li Y, Alam HB. Creating a pro-survival and anti-inflammatory phenotype by modulation of acetylation in models of hemorrhagic and septic shock. Adv Exp Med Biol 710:107–133, 2012.
29. Messaoudi K, Ali A, Ishaq R, Palazzo A, Sliwa D, Bluteau O, Souquère S, Muller D, Diop KM, Rameau P, et al. Critical role of the HDAC6-cortactin axis in human megakaryocyte maturation leading to a proplatelet-formation defect. Nat Commun 8 (1):1786, 2017.
30. Berry S, Dawicki DD, Agarwal KC, Steiner M. The role of microtubules in platelet secretory release. Biochim Biophys Acta 1012 (1):46–56, 1989.
31. Heijnen H, van der Sluijs P. Platelet secretory behaviour: as diverse as the granules … or not? J Thrombosis Haemostasis Jth 13 (12):2141–2151, 2016.
32. Italiano JE, Richardson JL, Patel-Hett S, Battinelli E, Zaslavsky A, Short S, Ryeom S, Folkman J, Klement GL. Angiogenesis is regulated by a novel mechanism: pro- and antiangiogenic proteins are organized into separate platelet alpha granules and differentially released. Blood 111 (3):1227–1233, 2008.
Keywords:

Antiplatelet; coagulopathy; histone deacetylase inhibitor; neutrophil extracellular traps; platelet activation

Supplemental Digital Content

Copyright © 2020 by the Shock Society