P2Y12 regulates platelet adhesion/activation, thrombus growth, and thrombus stability in injured arteries

Abstract

The critical role for ADP in arterial thrombogenesis was established by the clinical success of P2Y12 antagonists, currently used at doses that block 40-50% of the P2Y12 on platelets. This study was designed to determine the role of P2Y12 in platelet thrombosis and how its complete absence affects the thrombotic process. P2Y12-null mice were generated by a gene-targeting strategy. Using an in vivo mesenteric artery injury model and real-time continuous analysis of the thrombotic process, we observed that the time for appearance of first thrombus was delayed and that only small, unstable thrombi formed in P2Y12-/- mice without reaching occlusive size, in the absence of aspirin. Platelet adhesion to vWF was impaired in P2Y12-/- platelets. While adhesion to fibrinogen and collagen appeared normal, the platelets in thrombi from P2Y12-/- mice on collagen were less dense and less activated than their WT counterparts. P2Y12-/- platelet activation was also reduced in response to ADP or a PAR-4-activating peptide. Thus, P2Y12 is involved in several key steps of thrombosis: platelet adhesion/activation, thrombus growth, and stability. The data suggest that more aggressive strategies of P2Y12 antagonism will be antithrombotic without the requirement of aspirin cotherapy and may provide benefits even to the aspirin-nonresponder population.

Figure 1

Targeting the P2Y₁₂ locus. (a) The neomycin resistance gene in pLNL is flanked by a 4-kb (5′) and 6-kb (3′) fragment from the P2Y₁₂ genomic locus. HindIII, XbaI, XhoI, and NotI sites were introduced by PCR. The WT P2Y₁₂ coding exon is denoted as ORF. ORF, open reading frame.(b) Southern hybridizations of XbaI-digested mouse genomic DNA from progeny of breeding pairs heterozygous at the mutant allele. The XbaI fragment detected by the indicated probe (gray oval in Figure 1a) is reduced from 9.1 to 8.3 kb due to introduction of a new XbaI site in the targeted locus. Intro, introduction.

Figure 2

Aggregation measurements in PRP obtained from WT (+/+), P2Y₁₂^–/– (–/–), or P2Y₁₂^+/– (+/–) mice in response to (a) 0.5 μM ADP, (b) 10 μM ADP, (c) 1 μg/ml collagen, (d) 20 μg/ml collagen, (e) 1 mM mTRAP, and (f) 2.5 mM mTRAP. Traces are representative of data obtained from two independent experiments (blood from four mice pooled for each experiment). (g) Tail bleeding times for WT (n = 14), P2Y₁₂^+/– (n = 18), and P2Y₁₂^–/– mice (n = 15) littermates. Values are expressed as mean ± SEM. *P < 0.01 versus WT and P2Y₁₂^+/– mice (using Dunnett test).

Figure 3

Inhibition of cAMP by ADP and epinephrine in WT, P2Y₁₂^+/–, and P2Y₁₂^–/– mice. Data are expressed as the mean ± SD of triplicate measurements normalized to cAMP levels in the presence of 50 μM forskolin (100%) and are representative of two independent experiments. HET, heterozygous.

Figure 4

(a) In vivo arterial thrombotic profile of WT, P2Y₁₂^+/–, and P2Y₁₂^–/– mice. (b) Modulation of the thrombotic profile upon epinephrine treatment. B, bleaching; Epi, epinephrine. Vertical arrows correspond to the time when filter paper was removed from the artery.

Figure 5

Characteristics of the in vivo thrombotic process. (a) A comparison of the time for appearance of first thrombus greater than 20 μm for all three P2Y₁₂ genotypes, WT, P2Y₁₂^+/–, and P2Y₁₂^–/–, with and without addition of either ASA, epinephrine (1, 10, or 50 μM), or epinephrine plus CT51464 (+CT). (b) P2Y₁₂^–/– thrombotic process was characterized by constant embolization of thrombi greater than 25 μm and less than 50 μm (dotted bars) between 8 and 15 minutes after injury, whereas only a few large thrombi (greater than 50 μm, black bars) embolized in both WT and P2Y₁₂^+/– mice before occlusion of the blood vessel. Intravenous injection of epinephrine accelerated the thrombotic process but did not fully restore stability. When more than 50 thrombi embolized during the 7-minute period, a value of 50 was fixed. This explains the lack of error bars for the P2Y₁₂^–/– groups treated with 1 and 10 μM epinephrine. (c) Time for occlusion of mesenteric arteries. P2Y₁₂ deficiency significantly increased (P < 0.0001) the time for occlusion. P2Y₁₂^+/– mice presented an increased time for occlusion (P < 0.01 versus WT). ASA treatment of WT mice prolonged the time for occlusion (P < 0.05). Epinephrine (50 μM) restored occlusion in P2Y₁₂^–/– mice. GP IIb-IIIa antagonism in epinephrine-treated mice prevented occlusion, but was accompanied by severe bleeding at the site of vascular surgery (n = 5–12 animals).

Figure 6

Perfusion chamber experiments. (a) Washed platelets resuspended in presence of botrocetin were perfused through a human vWF-coated capillary at 871/s for 4 minutes. (b) Non-anticoagulated whole blood was perfused over fibrinogen at 871/s for 2.5 minutes. No differences were observed between WT and P2Y₁₂^–/– blood. (c) Twenty-five perfusion chambers were used to establish the mean gray level thrombotic profile in the 345-μm diameter capillary, including WT, P2Y₁₂^–/–, and P2Y₁₂^–/– mice treated with ASA. (d) ASA-treated or untreated non-anticoagulated blood of WT or P2Y₁₂^–/– mice was perfused over collagen at 871/s. Epinephrine infusion (50 μM) corrects the inhibition observed with the combination P2Y₁₂ deficiency/ASA uptake (10 mg/kg). Values were expressed as the mean ± SEM of at least six animals per group.*P < 0.001.

Figure 7

Level of platelet activation at the apex of arterial thrombi. Thrombi formed after a 2.5-minute perfusion period of WT (a, c, and e) and P2Y₁₂^–/– (b, d, and f) blood at 840/s over collagen. Fixed (but not permeabilized) thrombi were stained for P-selectin (a and b), Gas6 (c and d), and with Alexa 488-fibrinogen (e and f).

Figure 8

Flow-cytometric analysis of Alexa 488-fibrinogen binding to resting platelets, 0.6 mM mTRAP-treated platelets, and 5 μM ADP-treated platelets, in the presence or absence of 10 μM epinephrine-activated (Epi) and ADP plus epinephrine–activated platelets. Thick line represents P2Y₁₂^–/– platelets; thin line WT platelets. Epinephrine (1 and 10 μM) did not induce binding of Alexa 488-fibrinogen in absence of 1 mM CaCl₂ (not shown). The presence of epinephrine in ADP- and mTRAP-activated P2Y₁₂^–/– platelets gave a pattern similar to that of WT platelets treated with the same agonist in the absence of epinephrine. Data shown are representative of at least three experiments performed in duplicate.

Figure 9

P2Y₁₂ at the crossroads of the arterial thrombotic process. Upon vascular injury, various thrombogenic components (i.e., collagen/vWF) are exposed to arterial blood flow. Platelet membrane GP Ibα recognizes the activated conformation of vWF, inducing various signals inside the platelets, and leads to GP IIb-IIIa activation. ADP released from platelet-dense granules participates in the firm adhesion/activation step by interaction with P2Y₁₂, leading to activation of GP IIb-IIIa. Simultaneously, collagen-induced platelet activation through GP VI (42) leads to GP IIb-IIIa activation, resulting in adhesion and aggregation. ADP-P2Y₁₂ interactions (downstream of GP Ibα signaling and subsequent stimulation of the thrombin receptor) (43) also mediate the recruitment of circulating platelets to a growing thrombus. P2Y₁₂ might also affect thrombus stability as a result of low surface expression of known secondary platelet agonists (P-selectin, Gas6, CD40L [see ref. 44] not shown on the scheme). PAR, protease-activated receptor.