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S.N. Chaurasia et al.
above sea level, and an equal number of age-matched, lowlander controls. Platelets were isolated from blood and subjected to west- ern blot analysis.
Results
cated in the upregulation of HIF-1a in vascular smooth muscle cells.7 As this kinase is known to be expressed in human platelets and activated by thrombin,28 we studied its influence on HIF-2a expression in platelets. Pre-treat- ment of platelets with SB202190, an inhibitor of p38 MAPK, led to a significant decrease in thrombin-induced HIF-2a expression in a dose-dependent manner (inhibi- tion by 16.43% and 28.77% with 20 and 40 μM of SB202190, respectively) (Figure 1G, J).
Human platelets express HIF-2a
HIF-2a is known to be expressed in a cell-specific man-
26,27
ner unlike HIF-1a, which is ubiquitously expressed.
HIF-2α turnover in human platelets
HIF is known to be degraded by proteasomes aided by
Here, for the first time, we report that normoxic, unstim- ulated human platelets in the circulation express HIF-2a and that the expression of this factor is increased consid- erably upon exposure of the platelets to hypoxic stress (Figure 1A). However, we did not detect HIF-1a in platelets using specific antibodies in any of the above experimental conditions (data not shown).
As enucleate platelets with restricted protein synthesiz- ing ability carry functional mRNA for a limited number of genes, we next examined the expression of HIF transcripts in these cells by quantitative PCR. The quantification cycle (Cq) of GAPDH (the endogenous control) was deter- mined at 21 ± 2 while the Cq for HIF-1a and -2a were determined at 24 ± 2 and 25 ± 2.5, respectively, consistent with the presence of mRNA for both these isoforms in platelets. Non-specific amplification was ruled out by melt peak analysis (Online Supplementary Figure S1). Data from droplet digital PCR also supported the expression of mRNA for both HIF-1a and -2a in platelets (data not shown).
the activities of prolyl hydroxylases and the pVHL-E3 lig- ase complex. Lysosomes, too, have recently been implicat- ed in HIF proteolysis by either macroautophagy9 or chap- erone-mediated autophagy.10 The presence of a functional- ly active ubiquitin-proteasome system in human platelets has already been demonstrated.29 In order to ascertain pro- teasomal degradation of HIF-2a in platelets, we treated normoxic cells with proteasome inhibitors, PSI (50 μM) and MG132 (50 μM), for 30 min. Attenuation of protea- some peptidase activity was associated with a significant rise in HIF-2a level in platelets (Figure 2A, D). Next, in order to determine the role of lysosomes in HIF-2a prote- olysis, we pre-incubated platelets with either bafilomycin A1 (250 nM) (which blocks the activity of vacuolar- ATPase proton pumps) or chloroquine (50 μM) (which neutralizes the acidic environment within the lysosome compartment) for 30 min. Platelets were then exposed to hypoxia (1% O2, 5% CO2, and 94% N2) for 30 min or thrombin (1 U/mL) for 10 min at 37°C. Remarkably, each of the inhibitors significantly increased the levels of HIF- 2a in both hypoxic as well as thrombin-stimulated platelets (Figure 2B, C, E, F). Furthermore, we also deter- mined the contribution of macroautophagy in HIF-2a pro- teolysis. Cells were pretreated with 3-methyladenine (5 mM) (an inhibitor of macroautophagy) for 30 min and then exposed to hypoxia for 30 min. Inhibition of macroautophagy by 3-methyladenine led to significant increases in HIF-2a levels in platelets as compared to lev- els in the vehicle-treated control (Figure 2B, E), thus impli- cating macroautophagy in the degradation of HIF-2a under hypoxic conditions. Taken together, the results shown in Figure 2 suggest that HIF-2a in platelets is degraded through both proteasomal and lysosomal prote- olytic systems.
Hypoxia and hypoxia-mimetics induce prothrombotic states through shedding of extracellular vesicles and synthesis of PAI-1 in human platelets
Platelets are known to remain ‘hyperactive’ in oxygen- compromised states11-13 potentially leading to thrombotic episodes. Thrombus stabilization is facilitated by PAI-1, a member of the serine protease-inhibitor superfamily. Platelets synthesize functionally active PAI-1 from pre- existing mRNA.21 PAI-1 has been shown to be the target gene of HIF-2a in renal carcinoma cells.30 As HIF-2a expression is induced in platelets under hypoxia or upon exposure to hypoxia-mimetics such as DMOG (1 mM) and DFO (1 mM) (which stabiize HIF-2a by inhibition of prolyl hydroxylases), we next asked whether synthesis of PAI-1, too, is induced in platelets under these conditions. Exposure of human platelets to hypoxia (Figure 3A, D), DMOG and DFO (Figure 3B, E) upregulated the expres- sion of PAI-1 by 50.06%, 41.35% and 51.87%, respective-
Expression of HIF-2a in human platelets is augmented upon exposure to either hypoxic stress or physiological agonists
The oxygen-sensing a subunit of HIF is stabilized under oxygen-compromised states,2 as well as upon exposure of cells to non-hypoxic stimuli such as thrombin.7 In order to examine hypoxic adaptation of platelets, we incubated the cells under low oxygen concentration (1% O2, 5% CO2, and 94% N2) for the indicated periods. Expression of HIF- 2a increased significantly and progressively with time under hypoxia (Figure 1A, C). Platelets stored under nor- moxia for similar durations also exhibited minor increases in HIF-2a expression, although significantly less than those under hypoxia (data not shown). Interestingly, expo- sure to physiological agonists (thrombin, 1 U/mL; ADP, 10 μM; or collagen 10 μg/mL) for 10 min evoked significantly higher expression of HIF-2a in platelets in a normoxic environment, compared with unstimulated counterparts (Figure 1B, D). This observation underscored the presence of oxygen-independent HIF regulation, too, in platelets. As a thrombus is composed of stimulated platelets with restricted access to oxygen, these cells would have aug- mented HIF-2a expression.
Regulation of HIF demands a consistent turnover and generation of polypeptides from mRNA transcripts. As enucleate platelets have remarkably limited capacity for protein synthesis due to a restricted pool of transcripts, we next studied HIF-2a mRNA-protein translation by pre- incubating platelets with puromycin (10 mM) before exposure to either hypoxia or thrombin. Puromycin decreased HIF-2a expression in hypoxia-exposed (1% O2, 5% CO2, and 94% N2, 30 min) as well as thrombin-stimu- lated (1 U/mL, 10 min) platelets by 22.72% and 33.34%, respectively (Figure 1E, F, H, I). p38 MAPK has been impli-
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haematologica | 2019; 104(12)