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Platelet HIF-2α induces a prothrombotic state
rable to that from platelets exposed to hypoxia (Figure 3C). Hypoxia-mimetics also induced accelerated arterial thrombosis in mice (Figure 3F-H). As intracellular calcium
34
plays a critical role in platelet activation, we determined
the effect of hypoxia-mimetics on Ca2+ flux in human
platelets. Both DMOG and DFO elicited significant rises
in cytosolic Ca2+ (by 2.21- and 1.64-fold, respectively)
when calcium was included in the suspension buffer
(Figure 4D, E). Interestingly, chelation of external calcium
with EGTA led to a substantial drop in the DMOG- or
DFO-induced rise in platelet cytosolic calcium (by 64.70%
and 77.66%, respectively) (Figure 4D, E), a finding consis-
2+
tent with Ca entry from the external medium in the pres-
ence of hypoxia-mimetics. Ca2+ influx was further validat- ed by incubating platelets with Mn2+, which led to quenching of Fura-2 fluorescence in DMOG- and DFO- treated platelets (Figure 4F). Both the rise in intracellular calcium and the release of EV evoked by hypoxia-mimet- ics underscored a significant shift to a prothrombotic phe- notype of platelets marked with higher expression and stabilization of HIF-2a.
ing 2200 m above sea level) would have a platelet pheno- type similar to that of platelets isolated from patients with COPD. Notably, these high altitude residents, too, were found to have significantly higher expression of HIF-2a and PAI-1 in platelets as compared to their lowlander coun- terparts (Figure 5E-H). This is the first report on hypoxic changes in circulating platelets from either patients with COPD or from people living at a high altitude.
In conclusion, our study elucidates adaptive signaling in platelets in response to the challenges of a hypoxic envi- ronment and agonist stimulation, which closely correlate with the microenvironment within a thrombus. Under either of these situations platelets synthesize HIF-2a and the thrombogenic polypeptide PAI-1, and also release abundant numbers of EV. Concordantly, circulating platelets from patients with COPD, which is a condition predisposing to atherothrombotic events, as well as high altitude residents also exhibited significantly higher expression of HIF-2a and PAI-1. We further established that the steady-state level of HIF-2a, which is a short- lived polypeptide with a high turnover rate, is determined in platelets by synthesis in a MAPK-dependent manner and by proteolytic degradation through the proteasomal and lysosomal systems. Since hypoxic adaptation in platelets contributes to the prothrombotic state, targeting platelet hypoxia signaling could be an effective, new-gen- eration anti-thrombotic strategy.
Acknowledgments
This research was supported by a J. C. Bose National Fellowship and grants received by D. Dash from the Department of Biotechnology (DBT) (BT/PR20645/BRB/10/1541/2016), Science and Engineering Research Board (SERB), Department of Science and Technology (DST), Government of India, the Indian Council of Medical Research (ICMR) and the Council of Scientific and Industrial Research (CSIR). SNC is a recipient of a Senior Research Fellowship from the Indian Council of Medical Research (ICMR). We thank M/S Bio-Rad for extending the facility for droplet digital PCR.
Patients with COPD have ‘hyperactive’ platelets in their circulation, which contribute to the prothrombotic attrib- utes of this disease12,37 and cardiovascular mortality.14,39 Recently, neutrophils from patients with COPD were
27 demonstrated to have elevated expression of HIF-2a. We
investigated whether exposure to low arterial PaO2 (<60 mmHg) in COPD would induce changes in circulating platelets similar to those seen in isolated cells subjected to hypoxic stress in vitro. Remarkably, platelets from patients with COPD were found to have significantly higher expression of HIF-2a and PAI-1 as compared to the platelets from their healthy counterparts (Figure 5A-D), which provides a molecular underpinning for the throm- bogenic phenotype associated with COPD.
Oxygen-compromised environments such as high alti- tude are also associated with increased platelet activation,11 attributable to upregulation of the cysteine protease, cal- pain.16 We wondered whether a highland population (liv-
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