Loss of P2Y1 receptor desensitization does not impact hemostasis or thrombosis despite increased platelet reactivity in vitro

Abstract

Background: The hemostatic plug formation at sites of vascular injury is strongly dependent on rapid platelet activation and integrin-mediated adhesion and aggregation. However, to prevent thrombotic complications, platelet aggregate formation must be a self-limiting process. The second-wave mediator adenosine diphosphate (ADP) activates platelets via Gq-coupled P2Y₁ and Gi-coupled P2Y₁₂ receptors. After ADP exposure, the P2Y₁ receptor undergoes rapid phosphorylation-induced desensitization, a negative feedback mechanism believed to be critical for limiting thrombus growth.

Objective: The objective of this study was to examine the role of rapid P2Y₁ receptor desensitization on platelet function and thrombus formation in vivo.

Methods: We analyzed a novel knock-in mouse strain expressing a P2Y₁ receptor variant that cannot be phosphorylated beyond residue 340 (P2Y₁^340-0P), thereby preventing the desensitization of the receptor.

Results: P2Y₁^340-0P mice followed a Mendelian inheritance pattern, and peripheral platelet counts were comparable between P2Y₁^{340-0P/340-0P} and control mice. In vitro, P2Y₁^{340-0P/340-0P} platelets were hyperreactive to ADP, showed a robust activation response to the P2Y₁ receptor-selective agonist, MRS2365, and did not desensitize in response to repeated ADP challenge. We observed increased calcium mobilization, protein kinase C substrate phosphorylation, alpha granule release, activation of the small GTPase Rap1, and integrin inside-out activation/aggregation. This hyperreactivity, however, did not lead to increased platelet adhesion or excessive plug formation under physiological shear conditions.

Conclusion: Our studies demonstrate that receptor phosphorylation at the C-terminus is critical for P2Y₁ receptor desensitization in platelets and that impaired desensitization leads to increased P2Y₁ receptor signaling in vitro. Surprisingly, desensitization of the P2Y₁ receptor is not required for limiting platelet adhesion/aggregation at sites of vascular injury, likely because ADP is degraded quickly or washed away in the bloodstream.

Keywords: P2Y1 receptor; hemostasis; platelets; signaling; thrombosis.

Figure 1.. Desensitization of the P2Y₁ receptor in mouse and human platelets.

A) Washed platelets from either mouse (open circles) or human (closed circles) blood were pretreated with 3 μM MRS2365 for the indicated times prior to stimulation of 10 μM ADP. The data are presented as the mean ± SD of the percentage of the rate of aggregation (measured by impedance) at 0 seconds of pretreatment. Dotted lines indicate 50% decrease in relative response; t_1/2 (mouse) = 21.25 s; t_1/2 (human) = 18.72 s; n = 12 for human; n = 9 for mouse. B) Human whole blood was pretreated with either 3 μM MRS2365 (closed squares; P2Y₁-specific agonist) or 10 μM serotonin (open squares; 5-HT_2A-specific agonist) for the indicated times prior to the addition of 10 μM ADP or 5 μM epinephrine, respectively, and platelet aggregation was measured by impedance. The data are presented as the mean ± SD of the percentage of the maximum aggregation at 0 seconds of pretreatment. *p<0.05; **p<0.01; ****p<0.0001. n= 6. C) Desensitization of the P2Y₁ receptor was quantified in whole blood from wild-type (black bars) or P2Y₁^−/− mice (open bars). Blood samples were pretreated with vehicle or 3 μM MRS2365 for 90s prior to addition of 10 μM ADP alone or simultaneous addition of 10 μM ADP and 10 μM serotonin. Data are presented as the mean ± SD of the aggregation AUC as a percentage of wild-type, vehicle stimulated with ADP (measured by impedance). ****p<0.0001. n = 6 for WT; n = 7 for P2Y₁^−/−.

Figure 2.. Platelets from P2Y₁^{340-0P/340-0P} mice are hyper-responsive to ADP and protected from desensitization.

A,B) Platelets in PRP from P2Y₁^+/+ (black lines; black bars) and P2Y₁^{340-0P/340-0P} (red lines; red bars) mice were stimulated with the indicated doses of ADP and analyzed by standard aggregometry. A) Representative aggregometry traces; B) Area under the curve (AUC) quantification measured from t=0 to t=6 min (n=3 per group). C,D) Integrin activation and α-granule secretion in diluted whole blood from P2Y₁^+/+ (black bars) and P2Y₁^{340-0P/340-0P} (red bars) mice stimulated with the indicated doses of ADP and analyzed by flow cytometry. C) MFI for JON/A-PE staining (activated αIIbβ3, n=5/group); D) MFI for anti P-selectin-647 (α-granule marker, n=4/group). E,F) P2Y₁ receptor desensitization following repeated stimulation with ADP. E) Representative aggregation traces of PRP prepared from P2Y₁^+/+ (black lines) and P2Y₁^{340-0P/340-0P} (red lines) mice pretreated with 2 U/mL of apyrase. Arrows indicate repeated stimulation of PRP with 200 μM of ADP. F) Quantification of peak aggregation (P2Y₁^+/+ - black bars; P2Y₁^{340-0P/340-0P} - red bars) (n=3 per group). Statistical significance was determined by 2-way ANOVA (***p<0.001, ****p<0.0001). All data are expressed as the mean ± SEM.

Figure 3.. Platelets from P2Y₁^{340-0P/340-0P} mice exhibit prolonged ADP-promoted Ca²⁺ mobilization, PKC activation and Rap1-GTP loading.

A) Washed platelets from P2Y₁^+/+ (black line; black bars) or P2Y₁^{340-0P/340-0P} (red line; red bars) mice were loaded with Fluo-4 and Fura-red, stimulated with ADP at indicated concentrations and analyzed by flow cytometry. i) cytosolic Ca²⁺ mobilization (representative traces of 3 independent experiments). ii) quantification of peak Ca²⁺ levels and iii) AUC of cytosolic Ca²⁺ measured from t=0 to t=5 min. B,C) Washed platelets from P2Y₁^+/+ and P2Y₁^{340-0P/340-0P} mice were stimulated with either 1 μM ADP or 500 nM Phorbol-12-myristate-13-acetate (PMA; direct activator of PKC) for the indicated times before lysis and immunoblotting for PKC substrate phosphorylation. B) Representative blot; C) Quantification (n=3 per group). D,E) Washed platelets from P2Y₁^+/+ and P2Y₁^{340-0P/340-0P} mice were stimulated with 1 μM ADP for indicated time prior to lysis and pulldown of activated Rap1-GTP with RAL-GDS beads and immunoblotting for Rap1. D) Representative blot; E) Quantification of Rap1-GTP/Total Rap1 (n=3 per group). Statistical significance was determined by 2-way ANOVA (*p<0.05, **p<0.01). All data are expressed as the mean ± SEM.

Figure 4.. Enhanced activation response of P2Y₁^{340-0P/340-0P} platelets to the P2Y₁-selective agonist, MRS2365.

A,B) Aggregometry analysis of PRP from P2Y₁^+/+ (black lines; black bars) and P2Y₁^{340-0P/340-0P} (red lines; red bars) mice stimulated with indicated doses of MRS2365. A) Representative traces; B) AUC quantification measured from t=0 to t=6 min (n=3-4 per group). C,D) Integrin activation and α-granule secretion in diluted whole blood from P2Y₁^+/+ (black bars) or P2Y₁^{340-0P/340-0P} (red bars) mice stimulated with the indicated doses of MRS2365 and analyzed by flow cytometry. MFI for JON/A-PE staining (activated αIIbβ3, n=5/group); D) MFI for anti P-selectin-647 (α-granule marker, n=5/group). E,F) Washed platelets from P2Y₁^+/+ and P2Y₁^{340-0P/340-0P} mice were stimulated with MRS2365 for indicated time prior to lysis and pulldown of activated Rap1-GTP with RAL-GDS beads and immunoblotting for Rap1. D) Representative blot; E) Quantification of the ratio of Rap1-GTP to total Rap1 (n=3 per group). Statistical significance was determined by 2-way ANOVA (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). All data are expressed as the mean ± SEM.

Figure 5.. P2Y₁^{340-0P/340-0P} platelets are hyper-responsive to collagen but not Par4p.

A,B) Aggregometry analysis of PRP from P2Y₁^+/+ (black lines; black bars) and P2Y₁^{340-0P/340-0P} (red lines; red bars) mice stimulated with indicated doses of PAR4p. A) Representative traces; B) AUC quantification measured from t=0 to t=6 min (n=3-4 per group). C,D) Aggregometry analysis of PRP from P2Y₁^+/+ (black lines; black bars) and P2Y₁^{340-0P/340-0P} (red lines; red bars) mice stimulated with indicated doses of collagen. C) Representative traces; D) AUC quantification measured from t=0 to t=6 min (n=3-4 per group). Statistical significance was determined by 2-way ANOVA (*p<0.05, ****p<0.0001). All data are expressed as the mean ± SEM.

Figure 6.. Normal adhesive function of P2Y₁^{340-0P/340-0P} platelets under flow conditions in vitro and in vivo.

A) Microfluidics chamber studies. Whole blood from P2Y₁^+/+ (black lines) and P2Y₁^{340-0P/340-0P} (red lines) mice was perfused over immobilized collagen at venous (400s⁻¹) or arterial (1,600s⁻¹) shear rates and platelet adhesion was quantified (n=4-5 per group). Representative micrographs at t = 10 minutes are shown, platelets in red. Scale bar = 25 μm B) Arterial thrombosis. P2Y₁^+/+ (black bars) and P2Y₁^{340-0P/340-0P} (red bars) mice were subjected to the FeCl₃-induced carotid artery injury model (2.5% w/v for 5 min) while monitoring blood flow using an ultrasound probe to determine the time to vessel occlusion (n=8/group). Dotted line indicates the end of observation period. C) Venous thrombosis. Thrombus weights from P2Y₁^+/+ (black bars) and P2Y₁^{340-0P/340-0P} (red bars) mice subjected to IVC stenosis model (n=4-6/group). D,E) Hemostasis. D) P2Y₁^+/+ (black bars) and P2Y₁^{340-0P/340-0P} (red bars) mice were subjected to the saphenous vein laser injury model of hemostasis to quantify bleeding times following injury (5-7 injuries per mouse, 3 mice/group). E) Quantification of platelet accumulation at the site of injury for 10 minutes after laser ablation of endothelium (P2Y₁^+/+, black lines; P2Y₁^{340-0P/340-0P} red lines) (5-7 injuries per mouse, 3 mice/group). Statistical significance was determined by unpaired Students’ T-test for B-D. All data are expressed as the mean ± SEM.