A dry immersion model of microgravity modulates platelet phenotype, miRNA signature, and circulating plasma protein biomarker profile

Ground based research modalities of microgravity have been proposed as innovative methods to investigate the aetiology of chronic age-related conditions such as cardiovascular disease. Dry Immersion (DI), has been effectively used to interrogate the sequelae of physical inactivity (PI) and microgravity on multiple physiological systems. Herein we look at the causa et effectus of 3-day DI on platelet phenotype, and correlate with both miRomic and circulating biomarker expression. The miRomic profile of platelets is reflective of phenotype, which itself is sensitive and malleable to the exposome, undergoing responsive transitions in order to fulfil platelets role in thrombosis and haemostasis. Heterogeneous platelet subpopulations circulate at any given time, with varying degrees of sensitivity to activation. Employing a DI model, we investigate the effect of acute PI on platelet function in 12 healthy males. 3-day DI resulted in a significant increase in platelet count, plateletcrit, platelet adhesion, aggregation, and a modest elevation of platelet reactivity index (PRI). We identified 15 protein biomarkers and 22 miRNA whose expression levels were altered after DI. A 3-day DI model of microgravity/physical inactivity induced a prothrombotic platelet phenotype with an unique platelet miRNA signature, increased platelet count and plateletcrit. This correlated with a unique circulating protein biomarker signature. Taken together, these findings highlight platelets as sensitive adaptive sentinels and functional biomarkers of epigenetic drift within the cardiovascular compartment.


Dry Immersion Recovery
Day -x -3 Pre -   Figure S2: Gating strategy for the analysis of platelet VASP phosphorylation. Platelets were gated by side and forward scatter (A) and then by expression of CD61 (B). VASP-P phosphorylation was quantified by median and geometric mean fluorescence intensities (C). All data was analysed with Flowjo software. Effect of dry immersion on overall platelet VASP phosphorylation. Graph represents the mean ± SEM of each parameter at each time point. PRI = Platelet reactivity index. (D). The platelet population was identified by its forward and side scatter distribution and by expression of platelet specific antibody (CD61-PE) and 10,000 platelet events were gated. The value of the "corrected" MFI (MFIc) for each tube was obtained. MFIc was obtained after subtraction of the negative control tube (T3) from the value obtained for VASP-P (tubes one and two). The results from this test are labelled by the manufacturer as the "platelet reactivity index" (PRI) expressed as a percentage change in VASP fluorescence intensity between resting (+PGE1) and activated (+ADP stimulated) platelets. The PRI % was calculated from the median MFI using the calculation - For comparisons the PRI was also calculated from the mean fluorescence intensities. The working range of this assay was between 0-100%.  (50)) and analysis (background subtraction, minimum track length) setting were adjusted, allowing optimal particle identification for platelet poor plasma. The number and duration of captures was set to 15 x 60 second captures, providing 15 replicates of the same sample. After the 15 videos for each sample were taken, the NTA software tracked the Brownian motion of individual vesicles by automatically locating and following the centre of each and every particle, measuring the average distance it moved per frame. NTA analysed the raw data, and calculated size and concentration and displayed different particle parameters (size versus relative intensity versus number) against each other. Settings were optimised and maintained between samples. For each 60 second video, the concentration and size of the particles (from 0-1000nm) were recorded. An experiment summary file was automatically generated, displaying the concentration of the sample at each vesicle size. The sum of the concentrations at each size were calculated and the average taken.   Reorganisation of the actin cytoskeleton is a key event in platelet activation and adhesion, and therefore this pathway was of interest with regard to platelet function. Genes circled in red are predicted targets for multiple miRNAs, and genes circled in yellow are predicted targets of single miRNA. Permission was kindly granted by the Kanehisa Laboratory for the use of KEGG software. Figure S8: KEGG map of the ECM -receptor interaction pathway. Platelets express 5 different integrin's, which facilitate platelet adhesion to ECM proteins including collagen, laminin and fibronectin, amongst others, during platelet adhesion. This pathway was therefore chosen as it was relevant to platelet adhesion functions. Genes circled in red are predicted targets for multiple miRNAs, and genes circled in yellow are predicted targets of single miRNA. Permission was kindly granted by the Kanehisa Laboratory for the use of KEGG software.

Preparation of platelets
After blood draw, vacutainers/syringes were gently mixed by inversion. Blood was centrifuged w/o brake at 150xg for 10 minutes at RT. PRP was removed with a transfer pipette and placed into a fresh 50ml tube. Platelets were isolated from PRP by centrifugation, involving pelleting platelets and subsequent resuspension in a suitable buffer. The pH of PRP was brought to 6.5 using ACD. Prostaglandin E1 (PGE1), a platelet aggregation inhibitor, was added (1µl per 1 ml of PRP). PRP was centrifuged at 2000xg for 12 minutes with the brake on to pellet the platelets. The supernatant (PFP) was carefully removed and discarded. The platelet pellet was carefully resuspended in 1ml of buffer (either PBS/PFE/JNL) by gently pipetting up. A further 1ml of buffer was added with 2µl of PGE1 and the washing step was repeated twice, adding PGE1 before each wash. The platelet pellet was resuspended in buffer to the required concentration and platelets were allowed to sit at room temperature for 45 minutes to allow the PGE1 to dissipate. Resuspended platelets were transferred to a fresh tube for further analysis.

Gel filtered platelets
For a very pure platelet population, gel filtered platelets were prepared. This is a gentler method of isolating platelets whereby they are passed through a gel filtration column of sepharose 2B [43].

Leukocyte (CD45) depletion of platelets
Washing platelets removes plasma contaminants, effectively diluting out plasma proteins and therefore leaving a pure platelet sample. Where a highly pure platelet population is required, further purification was necessary. For RNA analysis, the lesser RNA content of platelets may be contaminated by a relatively small number of white blood cells (WBCs). To prepare CD45-depleted platelets for RNA analysis, EasySep TM magnetic technology was used. Cells are cross linked to EasySep TM magnetic particles using the Tetrameric Antibody Complex (TAC) technology and easily separated from unwanted message rich leukocytes (and CD45+ cellsa leukocyte specific marker) using the magnet. PFE Buffer (PBS + FBS + EDTA) was fresh each day RNA was prepared. For 19.5 ml of PFE buffer, 0.4 ml of FBS, 40 µl of 0.5 M EDTA and 19.5 ml of PBS were prepared in a 50 ml tube. This assay included the preparation of PRP as previously described. Platelets were suspended in 1 ml of PFE buffer after the second centrifugation step in a FACS tube. 1 ml of PFE buffer was added and mixed by gentle pipetting and platelets were counted. The EasySep TM whole blood depletion cocktail was then added to washed platelets. This sample was mixed gently and incubated for 15 minutes at RT. The EasySep TM magnetic nanoparticles were mixed vigorously to ensure they were in a uniform suspension and 200µl was added to the sample mixture. The sample was incubated at room temperature for 10 minutes. PFE buffer was added to a total volume of 5mls and the sample was mixed gently. The tube was placed into the magnet and incubated without the cap for 10 minutes at RT. The magnet was inverted, pouring off the supernatant into a 15ml RNAse-free tube. Platelets in the fresh tube were CD45-depleted platelets. 5 µl PGE1 was added to the CD45-depleted platelets and mixed by inversion. The tube was centrifuged at 2000xg for 10 minutes at RT with the brake on to pellet the platelets. The plasma supernatant was removed, and the pellet was ready for RNA extraction.

Microvesicle quantification and analysis
The NanoSight NS300 and Syringe Pump were used to quantify microvesicles (exosomes and microparticles) in PFP samples. Nanoparticle tracking analysis technology (NTA) used in this device combines the properties of light scattering and Brownian motion to attain measurements including concentration and size distribution of particles in a liquid suspension.
Particles in a beam path scatter light and are visualised by 20x magnification microscope fitted with a video camera. The camera functions at 30 frames per second and captures a video file of the particles moving under Brownian motion. The software tracks particles individually and uses the Stokes-Einstein equation to calculate their hydrodynamic diameters and particle size-Where Kb is Boltzmann's constant, T is temperature, n is solvent viscosity, and Dt is particle diffusion coefficient (hence sphere equivalent). The syringe was rinsed with 1ml of 10 % EtoH followed with 1ml of PBS. This procedure was performed between each sample. A 1:2500 dilution of PFP and high pure PBS was used after serial dilution optimisation (dilution 1=4µl of sample and 996µl of PBS, dilution 2=100µl of dilution 1 and 900µl of high pure PBS). Samples were kept on ice until analysis.