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Platelet HIF-2α induces a prothrombotic state
firmed by incubating platelets with Mn2+, which crosses the surface membrane similarly to Ca2+ and quenches Fura-2 fluorescence.23 When MnCl2 (2 mM) was added to Fura-2-labeled platelets pretreated with hypoxia-mimet- ics, there was an abrupt decrease in fluorescence (by 54.15% and 68.56% in DMOG- and DFO-treated cells, respectively) (Figure 4F), validating the entry of divalent cations into platelets stimulated with hypoxia-mimetics. The rise in intracellular calcium and release of EV induced in the presence of hypoxia-mimetics underscored a signif- icant shift of platelet biology to a prothrombotic pheno- type.
Platelets from patients with chronic obstructive pul- monary disease have higher expression of HIF-2a and PAI-1 than those from healthy counterparts
The a subunit of HIF is stabilized under low oxygen conditions,2 as well as upon exposure of cells to non- hypoxic stimuli such as thrombin.7 During the process of hemostasis platelets are stimulated by agonists leading to the formation of tightly packed aggregates, which restrict access of molecular oxygen to individual cells. We, there- fore, examined hypoxic adaptation of platelets and their reaction to hemostatic stimuli. HIF-2a was found to increase significantly and progressively with time when platelets were exposed to a low oxygen environment (1% O2, 5% CO2 and 94% N2) (Figure 1A, C). Strikingly, phys- iological agonists, such as thrombin, ADP and collagen, profoundly enhanced HIF-2a expression in platelets in a normoxic environment (Figure 1B, D), which reflects the presence of oxygen-independent regulation of HIF-2a, too, in these cells.
As the turnover of HIF protein is known to be high, we studied its dynamic regulation in enucleate platelets, which have restricted protein-synthesizing ability. Pretreatment of platelets with puromycin partially pre- vented the rise in HIF-2a elicited in the presence of hypoxia or platelet agonists (Figure 1E, F, H, I), which was consistent with active synthesis of HIF-2a in platelets. p38 MAPK has earlier been implicated in upregulation of HIF- 1a in vascular smooth muscle cells.7 As human platelets are known to express p38 MAPK,28 we determined its role in regulating the synthesis of HIF-2a in thrombin-activat- ed platelets. Pretreatment of platelets with 20 μM and 40 μM SB202190 (an inhibitor of p38 MAPK) led to signifi- cant decreases in the levels of HIF-2a (by 16.43% and 28.77%, respectively) (Figure 1G, J), thus implicating p38 MAPK in HIF-2a generation in platelets. It cannot be ruled out that other factors are involved in the regulation of HIF- 2a synthesis in platelets.
The proteasome is known to degrade HIF in a pVHL- dependent manner upon hydroxylation of its proline residues by prolyl hydroxylases in the presence of oxy- gen. Lysosomes, too, have recently been implicated in cleavage of HIF either by macroautophagy9 or by chaper- one-mediated autophagy.10 It has been demonstrated that platelets possess a functionally active proteasome system.29 In order to understand the role of proteasome peptidase activity in determining HIF stability in platelets, we examined changes in HIF-2a expression in the pres- ence of proteasome inhibitors, PSI and MG 132. Attenuation of proteasome activity was associated with significant rises in the levels of HIF-2a in platelets (Figure 2A, D), suggestive of constitutive proteasomal degrada- tion of HIF-2a. Next, we examined the role of lysosomes and macroautophagy in HIF-2a proteolysis by pretreat- ing cells with bafilomycin A1 (which blocks the activity of vacuolar-ATPase proton pumps), chloroquine (which neutralizes the acidic environment within the lysosome compartment) or 3-methyladenine (a selective inhibitor of macroautophagy). Inhibition of lysosomal activity as well as macroautophagy resulted in significant enhance- ment of HIF-2a levels in platelets (Figure 2B, C, E, F). Taken together, these results imply that the proteolysis of HIF-2a by proteasomes and lysosomes is involved in the regulation of the steady-state level of the factor in platelets.
PAI-1 is a serine protease responsible for the stabiliza-
tion of thrombi. Platelets are known to synthesize func-
COPD is a serious global public health problem charac- terized by progressive, persistent airflow limitation.36 Patients with COPD are reported to have ‘hyperactive’ platelets in the circulation and an increased risk of
12,14,15,37
atherothrombotic events. As expression of HIF-2a
and PAI-1 was significantly higher in platelets exposed to hypoxic stress, we investigated whether platelets from patients with COPD, who are exposed to low arterial PaO2, have greater expression of these proteins. We per- formed western blot analysis on platelets isolated from ten patients with COPD (PaO2 <60 mmHg) and an equal number of age-matched healthy controls (PaO2 >90 mmHg). Platelets from patients with COPD were found to have significantly higher expression of HIF-2a as well as PAI-1 (by 82.50% and 80%, respectively) when compared to platelets from healthy controls (Figure 5A-D).
Platelets from high altitude residents have higher expression of HIF-2a and PAI-1 than those
from lowlander counterparts
As people in oxygen-compromised environments such as those living at a high altitude are reported to have a higher incidence of thrombosis,11 we next studied the expression of HIF-2a and PAI-1 in platelets from individu- als living 2200 m above sea level. Coherently with data from patients with COPD, high altitude residents, too, were found to have significantly higher expression of platelet-specific HIF-2a and PAI-1 (increased by 164.28% and 164.26%, respectively) compared to their lowlander counterparts (Figure 5E-H).
Discussion
ia-inducible factor, HIF-1a is known to be ubiquitously
Of the three isoforms of the catalytic subunit of hypox-
expressed while the presence of the HIF-2a and HIF-3a is
cell-specific.4,5 For example, HIF-2a is expressed in human
3a has been reported to be expressed in lung epithelial 5
embryonic kidney 293 cells26 and neutrophils27 while HIF-
cells. In this study, we demonstrated, for the first time, that there is significant expression of HIF-2a in human platelets (Figure 1). Enucleate platelets are known to carry functional mRNA transcripts of specific genes with limit- ed protein synthesizing ability.38 We found significant expression of both HIF-1a and -2a mRNA in platelets (Online Supplementary Figure S1) although HIF-1a, unlike HIF-2a, was hardly detectable on western blot analysis (data not shown).
30
is a target gene for HIF-2a in renal carcinoma cells. We
21
tionally active PAI-1 from pre-existing mRNA. PAI-1, too,
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