Fig. 1.

αKG mediated augmentation of prolyl-hydroxylase activity of PHD2 inactivate pAkt in platelets. (a) All 3 isoforms of PHD exist in human platelet. Platelet-rich plasma (PRP) isolated from 3 different healthy individuals, after adjusting to equal number of platelets, was incubated with or without collagen (5 μg/ml) for 5 min and processed for western blotting (WB) of PHD1, PHD2 and PHD3. Densitometry data are mentioned in Fig. S14a. (b-g) PRP was incubated with collagen in a time- and concentration-dependent manner, and (b) expression of pAkt(Ser473), Akt, pAkt1(Thr 308), Akt1, HIF-1α and HIF-2α was measured in platelet pellet using WB. (c-d) Densitometry analysis shows elevated expression of pAkt and pAkt1 after collagen stimulation, other densitometry data are described in Fig. S14b-c. (e) Platelets were incubated with collagen in presence of αKG (0.25 and 0.5 mM) or DKG (10 and 100 μM) and the expression of above signalling molecules and PHD2 was measured using WB. (f-g) Densitometry data show suppression of collagen-induced elevation of pAkt and pAkt1 by αKG, but an elevation of these molecules in presence of DKG. Other densitometry data are described in Fig. S14d-f. (h-i) (h) Immunoprecipitation (IP) of PHD2 from lysate of washed-platelets from above experiment was performed and processed for WB of pAkt; further, IP of pAkt from same lysate and WB for hydroxy proline shows the interaction between the molecules. (i) Densitometry data of pAkt. Other densitometry data are described in Fig. S14g-h. (j-k) PRP from above experiment of Fig. 1c was processed for measuring surface (j) P-Selectin, (k) PS (Annexin-V binding) and (l) GP2b3a activation (PAC-1 binding) using flow cytometry. (m-n) (m) Platelet aggregation was performed using PRP pre-treated with αKG or DKG in response to collagen. (n) Percentage platelet aggregation was measured. Data from similar experiment in response to agonist ADP are described in Fig. S4. (o-p) (o) PRP from healthy individuals was incubated with αKG or DKG and perfused on immobilized collagen surface under arterial flow share condition 25 dyne/cm² and platelet thrombus formation was measured. Scale bar 50 µm. (p) Thrombus area was measured. (q) Secretion of Sphingosine-1-phosphate (S1P) was quantified from supernatant of αKG- and collagen-treated washed platelets using ELISA. Data in above figure are mean ± SEM from 3 independent experiments (one-way ANOVA, *P<0.05. **P<0.01, ***P<0.001, ****P<0.0001 and ns=non-significant).