Hypoxia Modulates Platelet Purinergic Signalling Pathways

Abstract

Background: Hypoxia resulting from ascent to high-altitude or pathological states at sea level is known to increase platelet reactivity. Previous work from our group has suggested that this may be adenosine diphosphate (ADP)-specific. Given the clinical importance of drugs targeting ADP pathways, research into the impact of hypoxia on platelet ADP pathways is highly important.

Methods: Optimul aggregometry was performed on plasma from 29 lowland residents ascending to 4,700 m, allowing systematic assessment of platelet reactivity in response to several platelet agonists. Aggregometry was also performed in response to ADP in the presence of inhibitors of the two main ADP receptors, P2Y₁ and P2Y₁₂ (MRS2500 and cangrelor, respectively). Phosphorylation of vasodilator-stimulated phosphoprotein (VASP), a key determinant of platelet aggregation, was analysed using the VASPFix assay.

Results: Hypobaric hypoxia significantly reduced the ability of a fixed concentration of cangrelor to inhibit ADP-induced aggregation and increased basal VASP phosphorylation. However, in the absence of P2Y receptor inhibitors, we did not find evidence of increased platelet sensitivity to any of the agonists tested and found reduced sensitivity to thrombin receptor-activating peptide-6 amide.

Conclusion: Our results provide evidence of increased P2Y₁ receptor activity at high altitude and suggest down-regulation of the P2Y₁₂ pathway through increased VASP phosphorylation. These changes in ADP pathway activity are of potential therapeutic significance to high-altitude sojourners and hypoxic sea level patients prescribed platelet inhibitors and warrant further investigation.

Fig. 1

Ascent profile. Baseline testing was performed in April 2017, 2 months prior to the expedition. Subjects landed in La Paz (3,700 m) where they spent four nights before ascending to Huayna Potosi Base Camp (4,700 m) by bus on day 5 where they stayed for the remainder of the study. Optimul aggregometry, VASPFix and full blood count samples were collected at baseline and on day 11. On day 6, only optimul aggregometry was performed.

Fig. 2

Platelet counts and oxygen saturations. ( A ) Oxygen saturation was measured at baseline and on day 6 and day 11 of the expedition. Data points are represented as semi-translucent circles, with summary box plots superimposed. Data were analysed by a one-way repeated-measure analysis of variance (ANOVA) followed by a Tukey's HSD test. ( B ) Platelet count was measured at baseline and on day 11 of the expedition. Data points are represented as semi-translucent circles, with summary box plots superimposed. Data were analysed by paired Student's t -test. *** p  < 0.001, * p  < 0.05.

Fig. 3

The effect of hypoxia on platelet activation pathways. Dose–response curves of platelet aggregation in response to ( A ) arachidonic acid (AA), ( B ) adenosine diphosphate (ADP), ( C ) collagen, ( D ) epinephrine, ( E ) thrombin receptor-activating peptide (TRAP)-6 amide and ( F ) U46619. Data are mean percentage aggregation ± standard error of the mean (SEM), and best fit curves optimised to these mean values. EC ₅₀ s were compared by one-way analysis of variance (ANOVA) followed by Tukey's HSD post hoc tests where appropriate. p -Values reported were adjusted to account for family-wise error rate using Bonferroni corrections. ** p  < 0.01 vs. baseline.

Fig. 4

The effect of hypoxia on adenosine diphosphate (ADP)-induced platelet aggregation in the presence of fixed doses of inhibitors. Platelet-rich plasma (PRP) was incubated for 30 minutes with ( A ) 1 µM MRS2500 (P2Y ₁ inhibitor) or ( B ) 100 nM cangrelor (P2Y ₁₂ inhibitor). Data are mean percentage aggregation ± standard error of the mean (SEM), and best fit curves optimised to these mean values. EC ₅₀ and R _max values were compared by one-way analysis of variance (ANOVA) followed by Tukey's HSD post hoc tests where appropriate. p -Values reported were adjusted to account for family-wise error rate using Bonferroni corrections. ** p  < 0.01 vs. baseline.

Fig. 5

The effect of hypoxia on platelet vasodilator-stimulated phosphoprotein (VASP) phosphorylation. Platelet-rich plasma (PRP) was incubated for 6 minutes with phosphate-buffered saline (PBS), iloprost or adenosine diphosphate (ADP) + Iloprost before addition of VASPFix. Flow cytometry was used to identify the fluorescein isothiocyanate (FITC) median fluorescence (MF), reflecting the degree of VASP phosphorylation. ( A ) Raw FITC MF for each condition at each time point. Data are mean FITC MF ± standard error of the mean (SEM). Data were compared by paired t -tests with p -values adjusted by Bonferroni correction. ( B ) Percentage increase in FITC MF induced by addition of iloprost. ( C ) Percentage decrease induced by ADP addition. ( B , C ) Individual data points are represented by semi-translucent circles with box plots superimposed. Data were compared by paired t -tests. ** p  < 0.01, *** p  < 0.001.

Fig. 6

Proposed model of hypoxia-induced changes to purinergic signalling. Our findings are in red, while those of other studies examining the impact of hypoxia on platelet signalling are in blue. Our data suggest that hypoxia up-regulates the basal level of vasodilator-stimulated phosphoprotein (VASP) phosphorylation, a key determinant of platelet aggregation downstream of the P2Y ₁₂ receptor. Other work demonstrating increased nitric oxide (NO) levels at altitude provides a possible mechanism for this finding. Phosphorylated-VASP (VASP-P) inhibits the expression of active α _IIb β ₃ expression, which links to Kiouptsi et al's finding that hypoxic platelets have lower expression of active α _IIb β ₃ in response to adenosine diphosphate (ADP). Increased P2Y ₁ pathway activity would be consistent with Tyagi et al's finding that calpains are up-regulated by hypoxia. Since we found no change in overall sensitivity to ADP, it may be that the changes produced by hypoxia in these two pathways counteract one another. AC, adenylate cyclase; ADP, adenosine diphosphate; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanine monophosphate; GC, guanylate cyclase; PI3K, phosphoinositide 3-kinase.