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. 1999 Dec;104(12):1731-7.
doi: 10.1172/JCI8399.

Defective platelet aggregation and increased resistance to thrombosis in purinergic P2Y(1) receptor-null mice

C Léon  1 , B Hechler, M Freund, A Eckly, C Vial, P Ohlmann, A Dierich, M LeMeur, J P Cazenave, C Gachet
Affiliations

Defective platelet aggregation and increased resistance to thrombosis in purinergic P2Y(1) receptor-null mice

C Léon et al. J Clin Invest. 1999 Dec.

Abstract

ADP is a key agonist in hemostasis and thrombosis. ADP-induced platelet activation involves the purinergic P2Y(1) receptor, which is responsible for shape change through intracellular calcium mobilization. This process also depends on an unidentified P2 receptor (P2cyc) that leads to adenylyl cyclase inhibition and promotes the completion and amplification of the platelet response. P2Y(1)-null mice were generated to define the role of the P2Y(1) receptor and to determine whether the unidentified P2cyc receptor is distinct from P2Y(1). These mice are viable with no apparent abnormalities affecting their development, survival, reproduction, or the morphology of their platelets, and the platelet count in these animals is identical to that of wild-type mice. However, platelets from P2Y(1)-deficient mice are unable to aggregate in response to usual concentrations of ADP and display impaired aggregation to other agonists, while high concentrations of ADP induce platelet aggregation without shape change. In addition, ADP-induced inhibition of adenylyl cyclase still occurs, demonstrating the existence of an ADP receptor distinct from P2Y(1). P2Y(1)-null mice have no spontaneous bleeding tendency but are resistant to thromboembolism induced by intravenous injection of ADP or collagen and adrenaline. Hence, the P2Y(1) receptor plays an essential role in thrombotic states and represents a potential target for antithrombotic drugs.

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Figures

Figure 1
Figure 1
Disruption of the P2Y1 receptor gene in mice. (a) Gene targeting strategy: structure and partial restriction map of the P2Y1 gene, targeting construct, and targeted allele. The filled box represents the coding sequence. Restriction endonuclease sites: B, BamHI; arrowheads, PCR primers. (b) Southern blot analyses of ES clones identified as positive by PCR and of tail DNA from P2Y1–/– and P2Y1+/+ mice. The 12-kb wild-type and 9.5-kb targeted BamHI fragments were identified using a probe located outside the 3′ limit of the construct (shown in a). Clones 187 and 26 (bold) were selected to be injected into blastocysts.
Figure 2
Figure 2
In vitro aggregation responses of P2Y1–/– washed mice platelets as compared with wild-type. P2Y1–/– mice platelet aggregation is lost in response to ADP and strongly impaired in response to 1.25 μg/mL collagen, whereas the lag phase, measured from the addition of the agonist to starting of aggregation, is prolonged in response to 5 μg/mL collagen (25 seconds in P2Y1–/– versus 15 seconds in wild-type). In contrast, responses to thrombin and to U46619 were modified only at threshold concentrations. Bar: 1 minute.
Figure 3
Figure 3
Conserved potentiating effect of ADP through activation of the P2cyc receptor. (a) Serotonin induces shape change only in both P2Y1+/+ and P2Y1–/– platelets. Simultaneous addition of serotonin and ADP restores aggregation in P2Y1–/– platelets, an effect inhibited by the selective P2cyc antagonist AR-C99631MX. (b) Intracellular calcium mobilization in response to ADP is lost in P2Y1 knock-out mice. (c) ADP still inhibits cAMP formation in P2Y1–/– mice. Cyclic AMP levels were measured in intact washed platelets exposed to: vehicle (open bars), 1 μM PGE1 (gray bars), PGE1 plus 5μM ADP (black bars), PGE1 plus ADP in the presence of 2 μM AR-C69931MX (striped bars). These data represent 1 experiment performed in duplicate, representative of 3 separate experiments giving similar results.
Figure 4
Figure 4
P2cyc receptor–mediated platelet aggregation with high concentrations of ADP. (a) Addition of 100 μM ADP to P2Y1–/– mice platelets induced a gradual increase of light transmission, without optically detectable shape change, up to 20% above the baseline at 5 minutes (upper-right tracing), which is inhibited by 2 μM AR-C69931MX (lower-right tracing). (b) Transmission electron microscopy of resting (left) or 100 μM ADP-induced small aggregates of 5–10 P2Y1–/– mice platelets (right). Bars: 0.5 μm.
Figure 5
Figure 5
Hemostasis and thrombosis. (a) Bleeding time of P2Y1+/+ and P2Y1–/– mice. Each point represents 1 individual (P2Y1+/+, n = 43; P2Y1–/–, n = 80). (b) P2Y1–/– mice platelet counts are not modified after bolus injections of ADP whereas P2Y1+/+ mice platelet count is decreased by more then 50%. Each bar is the mean of 4 mice. (c) Mortality by thromboembolism in response to a bolus injection of a mixture of collagen (0.5 mg/kg body weight) and adrenaline (60 μg/kg body weight) is reduced by 50% in P2Y1–/– mice. Results are expressed as the percentage of the total number of animals tested.
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