Page 169 - 2019_12-Haematologica-web

Platelet HIF-2α induces a prothrombotic state
hydroxylated HIF-a subunits are ubiquitinated by the von Hippel-Lindau tumor suppressor (pVHL) E3 ligase com- plex and HIF is targeted for proteasomal degradation.2,4 Under hypoxia, oxygen-sensing prolyl hydroxylases fail to hydroxylate HIF-a, leading to this latter’s stabilization. HIF can also be stabilized by non-hypoxic stimuli, includ- ing thrombin,7 as well as by hypoxia-mimetics such as dimethyloxalylglycine (DMOG) and deferoxamine (DFO).8 Interestingly, there have also been recent reports of HIF degradation mediated through either autophagy9 or chaperone-mediated lysosomal autophagy.10
Oxygen-compromised environments such as a high alti- tude and sports are associated with a higher incidence of thrombosis.11 Patients with pathological conditions associ- ated with hypoxia, such as chronic obstructive pulmonary disease (COPD) and sleep apnea, have also been reported to have hyperactive platelets in their circulation as well as an increased risk of thrombosis.12-15 A recent study has cor- related platelet hyperactivity under hypoxic stress with enhanced activity of the cysteine protease calpain.16 Hypoxia has been shown to enhance synthesis of throm- bogenic molecules such as tissue factor17 and plasminogen- activator inhibitor-1 (PAI-1)18 in murine lung cells. Little is known about the mechanistic basis of platelet responses to hypoxia and adaptation of these cells to an oxygen- compromised environment prevalent within cell aggre- gates or fibrin-rich thrombi. Platelets are enucleate cells with restricted ability for de novo protein synthesis by translation. The repertoire of proteins known to be syn- thesized by platelets is limited but includes Bcl-3,19 inter- leukin-1β,20 PAI-1,21 and tissue factor among others.22 The present study adds HIF-2a to this growing list of the platelet translatome. HIF-2a translation is induced in platelets by hypoxia, hypoxia-mimetics and physiological agonists such as collagen, thrombin or ADP. Inhibitors of either protein synthesis or mitogen-activated protein kinase (MAPK) markedly depress HIF-2a synthesis. Our results implicate both proteasome-mediated as well as lysosome-mediated pathways in the degradation of HIF- 2a in platelets. Hypoxia and hypoxia-mimetics induce synthesis of PAI-1 in platelets and shedding of EV, both of which contribute to the evolution of a prothrombotic phe- notype. Consistently with this, mice pretreated with hypoxia-mimetics, which would trigger platelet hypoxia signaling by stabilizing HIF-a, exhibited accelerated arte- rial thrombosis. Circulating platelets from patients with COPD as well as a highland population were found to have significantly higher expression of HIF-2a and PAI-1 compared to their control counterparts, which are findings coherent with the platelet hyperactivity reported in these subjects.11,12
Methods
Ethical approval
Animal experiments were approved by the Central Animal Ethical Committee of Banaras Hindu University. All efforts were made to minimize the number of animals used, and their suffer- ing. Venous blood samples were collected from human partici- pants at the University after obtaining written informed consent, strictly as per recommendations and as approved by the Institutional Ethical Committee of the Institute of Medical Sciences, Banaras Hindu University. The study was conducted according to standards set by the Declaration of Helsinki.
Platelet preparation and materials
Platelets were isolated from fresh venous human blood by dif- ferential centrifugation, as described elsewhere.23 The sources of materials and additional methods are detailed in the Online Supplementary Data.
Western analysis
Platelet proteins were separated by sodium dodecylsulfate poly- acrylamide gel electrophoresis (SDS-PAGE) and electrophoretical- ly transferred onto polyvinylidene fluoride membranes. Following blocking, membranes were incubated with primary antibodies (anti-HIF-1a, 1:500; anti-HIF-2a, 1:500; anti-PAI-1, 1:100; anti- actin, 1:5000) and horseradish peroxidase-conjugated secondary antibodies (goat anti-mouse, 1:1500, for HIF-1a and PAI-1; goat anti-rabbit, 1:2000, for HIF-2a, and 1:40000, for actin). Antibody binding was detected using enhanced chemiluminescence.
Total RNA extraction, reverse transcription and quantitative real-time polymerase chain reaction
RNA was extracted from platelets and reverse transcribed to complementary DNA. The quantitative polymerase chain reac- tion (PCR) was initiated at 95°C for 3 min, followed by 40 cycles of denaturation (10 s at 95°C), annealing (10 s, at 56°C for GAPDH, and at 59.2°C for both HIF-1a and -2a) and extension at 72°C.
Hypoxic stimulation of isolated human platelets
Isolated human platelets were exposed to hypoxia (1% O2, 5% CO2, and 94% N2) for the indicated time periods in an automati- cally controlled hypoxia chamber glove box (Plas-Labs) at room temperature. After completion of incubation, cells were lysed inside the hypoxia chamber to avoid their re-oxygenation.
Isolation and analysis of platelet-derived extracellular vesicles
Platelets were sedimented at 800× g for 10 min followed by 1200× g for 2 min at 22°C to obtain platelet-derived extracellular vesicles (PEV) cleared of platelets. PEV in supernatant were ana- lyzed by a Nanoparticle Tracking Analyzer.
Intravital imaging of mesenteric arteriolar thrombi
Ferric chloride-induced mesenteric arteriolar thrombi in mice were imaged as described previously,24 with minor modifications.
Measurement of intracellular free calcium
Intracellular free calcium was measured in Fura 2-ace- toxymethyl ester (Fura-2 AM)-stained platelets as described in the Online Supplementary Data and calibrated according to the deriva- tion of Grynkiewicz et al.25
Analysis of platelets from patients with chronic obstructive pulmonary disease and individuals living at high altitude
Blood was collected from ten patients suffering from an acute exacerbation of COPD (arterial PaO2 <60 mmHg) admitted to Sir Sunderlal Hospital, Banaras Hindu University, and an equal num- ber of age-matched healthy controls (arterial PaO2 >90 mmHg) (Online Supplementary Table S1). Exclusion criteria for both groups were domiciliary oxygen therapy, active smoking, hypertension, diabetes mellitus, malignancies and use of antiplatelet drugs. Arterial blood gas analysis was carried out using a Cobas B 121 Analyzer. Platelets were isolated from these samples and subject- ed to further studies.
Blood was also collected, with written informed consent, from ten healthy residents from Dhankuta, Nepal, which is 2200 m
haematologica | 2019; 104(12)
2483