D121 Located within the DRY Motif of P2Y12 Is Essential for P2Y12-Mediated Platelet Function

Figures

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  Figure 1. 2-MeSADP-mediated aggregation is interrupted in platelets from PDS25. (A) Representative tracings showing aggregation of platelets from PDS25 and healthy control donors that were stimulated with 100 nM 2-MeSADP following pretreatment of 100 nM AR-C69931MX or not and monitored for 3 min to observe aggregation. (B) Western blot showing Akt phosphorylation in platelets from PDS25 or a healthy control following stimulation with 2-MeSADP. Total Akt was used as a loading control. (C) Representative tracings showing aggregation and secretion of platelets from a healthy control donor and PDS25 stimulated with the indicated concentration of AYPGKF. (D) Quantitation of aggregation of platelets from healthy control donors and PDS25 stimulated with the indicated concentration of AYPGKF. (E) Quantitation of ATP secretion from platelets stimulated with the indicated concentration of AYPGKF. * p < 0.05 vs. healthy control, n = 5.

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  Figure 2. VASP phosphorylation is enhanced, while Rap1b-GTP formation is reduced in platelets from PDS25 stimulated with 2-MeSADP. (A) Representative Western blot showing VASP phosphorylation in response to either 100 nM 2-MeSADP or 10 mM epinephrine for 3 min with or without PGE1 pretreatment. Total VASP was used to assess loading. (B) Quantitation of VASP phosphorylation as described in A. (C) Representative Western blot showing Rap1b-GTP in platelets from PDS25 or normal control donors stimulated with 100 nM 2-MeSADP alone or in combination with 10 μM epinephrine. Rap1b was used to assess loading. (D) Quantitation of GTP-Rap1b expressed as a ratio of total Rap1b as seen in C. H = Healthy control, P = PDS25. * p < 0.05 vs. control, n = 3.

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  Figure 3. Gz-mediated aggregation is intact in platelets from PDS25, while thrombus formation on collagen is inhibited in whole blood from PDS25. (A) Representative aggregation tracings of platelets isolated from PDS25 or healthy controls stimulated with 100 nM 2-MeSADP, 10 μM epinephrine, or both. (B) Quantitation of aggregation as represented in panel A. * p < 0.05 vs. healthy control stimulated with 100 nM 2-MeSADP, n = 4. (C) Representative images of thrombus formation following whole blood (from PDS25 or healthy controls) flow over a collagen-coated surface. (D) Quantitation of thrombus area resulting from whole blood flow over collagen. * p < 0.05 vs. healthy control, n = 5.

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  Figure 4. 2-MeSADP-mediated platelet aggregation is interrupted in platelets from P2Y12 D127N knock-in mice. (A) Representative agarose gel showing the banding pattern of WT, heterozygous (Het), and homozygous D127N knock-in (KI) mice. (B) Representative tracings showing WT, Het, and KI platelet aggregation in response to 10 nM 2-MeSADP. (C) Quantitation of platelet aggregation in response to 10 nM 2-MeSADP as described in (B). (D) Representative tracings showing WT, Het, and KI D127N platelet aggregation in response to 100 nM 2-MeSADP. Quantitation of platelet aggregation in response to 10 nM 2-MeSADP as described in (E). * p < 0.05 vs. WT, n = 5.

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  Figure 5. D127N KI mouse platelets have a significantly inhibited response to ADP. (A) Representative tracings showing aggregation in response to either 5 or 10 μM ADP of platelets isolated from WT, Het, or KI mice. (B,C) Quantitiation of platelet aggregation in response to 5 or 10 μM ADP. (D) Representative aggregation and secretion tracings of platelets isolated from WT, Het, and KI mice stimulated with 200 or 500 μM AYPGKF. (E,F) Quantitation of aggregation (E) and secretion (F) from WT, Het, and KI platelets stimulated with 200 μM AYPGKF. (G,H) Quantitation of aggregation (G) and secretion (H) from WT, Het, and KI platelets stimulated with 500 μM AYPGKF.

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  Figure 6. Hemostasis and thrombosis are interrupted in P2Y12 D127N knock-in mice. (A) Time for bleeding to stop following excision of the distal 3 mm of the tail. (B–D) Representative images showing blood flow in the carotid arteries of WT, Het, and KI mice following administration of 7.5% FeCl3. (E) Quantitation of time to vessel occlusion following FeCl3 injury to the carotid artery. * p < 0.05 vs. WT using Wilcoxon test, n = 9–11.