Pamela B. Conley

Identification of the platelet ADP receptor targeted by antithrombotic drugs

Platelets have a crucial role in the maintenance of normal haemostasis, and perturbations of this system can lead to pathological thrombus formation and vascular occlusion, resulting in stroke, myocardial infarction and unstable angina. ADP released from damaged vessels and red blood cells induces platelet aggregation through activation of the integrin GPIIb–IIIa and subsequent binding of fibrinogen. ADP is also secreted from platelets on activation, providing positive feedback that potentiates the actions of many platelet activators. ADP mediates platelet aggregation through its action on two G-protein-coupled receptor subtypes. The P2Y 1 receptor couples to Gq and mobilizes intracellular calcium ions to mediate platelet shape change and aggregation. The second ADP receptor required for aggregation (variously called P2YADP , P2YAC, P2Ycyc or P2TAC) is coupled to the inhibition of adenylyl cyclase through Gi. The molecular identity of the G i-linked receptor is still elusive, even though it is the target of efficacious antithrombotic agents, such as ticlopidine and clopidogrel and AR-C66096 (ref. 9). Here we describe the cloning of this receptor, designated P2Y12, and provide evidence that a patient with a bleeding disorder has a defect in this gene. Cloning of the P2Y12 receptor should facilitate the development of better antiplatelet agents to treat cardiovascular diseases.

A specific antidote for reversal of anticoagulation by direct and indirect inhibitors of coagulation factor Xa

Inhibitors of coagulation factor Xa (fXa) have emerged as a new class of antithrombotics but lack effective antidotes for patients experiencing serious bleeding. We designed and expressed a modified form of fXa as an antidote for fXa inhibitors. This recombinant protein (r-Antidote, PRT064445) is catalytically inactive and lacks the membrane-binding γ-carboxyglutamic acid domain of native fXa but retains the ability of native fXa to bind direct fXa inhibitors as well as low molecular weight heparin–activated antithrombin III (ATIII). r-Antidote dose-dependently reversed the inhibition of fXa by direct fXa inhibitors and corrected the prolongation of ex vivo clotting times by such inhibitors. In rabbits treated with the direct fXa inhibitor rivaroxaban, r-Antidote restored hemostasis in a liver laceration model. The effect of r-Antidote was mediated by reducing plasma anti-fXa activity and the non–protein bound fraction of the fXa inhibitor in plasma. In rats, r-Antidote administration dose-dependently and completely corrected increases in blood loss resulting from ATIII-dependent anticoagulation by enoxaparin or fondaparinux. r-Antidote has the potential to be used as a universal antidote for a broad range of fXa inhibitors.

Andexanet Alfa for the Reversal of Factor Xa Inhibitor Activity

BACKGROUND Bleeding is a complication of treatment with factor Xa inhibitors, but there are no specific agents for the reversal of the effects of these drugs. Andexanet is designed to reverse the anticoagulant effects of factor Xa inhibitors. METHODS Healthy older volunteers were given 5 mg of apixaban twice daily or 20 mg of rivaroxaban daily. For each factor Xa inhibitor, a two-part randomized placebo-controlled study was conducted to evaluate andexanet administered as a bolus or as a bolus plus a 2-hour infusion. The primary outcome was the mean percent change in anti-factor Xa activity, which is a measure of factor Xa inhibition by the anticoagulant. RESULTS Among the apixaban-treated participants, anti-factor Xa activity was reduced by 94% among those who received an andexanet bolus (24 participants), as compared with 21% among those who received placebo (9 participants) (P<0.001), and unbound apixaban concentration was reduced by 9.3 ng per milliliter versus 1.9 ng per milliliter (P<0.001); thrombin generation was fully restored in 100% versus 11% of the participants (P<0.001) within 2 to 5 minutes. Among the rivaroxaban-treated participants, anti-factor Xa activity was reduced by 92% among those who received an andexanet bolus (27 participants), as compared with 18% among those who received placebo (14 participants) (P<0.001), and unbound rivaroxaban concentration was reduced by 23.4 ng per milliliter versus 4.2 ng per milliliter (P<0.001); thrombin generation was fully restored in 96% versus 7% of the participants (P<0.001). These effects were sustained when andexanet was administered as a bolus plus an infusion. In a subgroup of participants, transient increases in levels of d-dimer and prothrombin fragments 1 and 2 were observed, which resolved within 24 to 72 hours. No serious adverse or thrombotic events were reported. CONCLUSIONS Andexanet reversed the anticoagulant activity of apixaban and rivaroxaban in older healthy participants within minutes after administration and for the duration of infusion, without evidence of clinical toxic effects. (Funded by Portola Pharmaceuticals and others; ANNEXA-A and ANNEXA-R ClinicalTrials.gov numbers, NCT02207725 and NCT02220725.).

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

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.

Andexanet Alfa for Acute Major Bleeding Associated with Factor Xa Inhibitors

BACKGROUND Andexanet alfa (andexanet) is a recombinant modified human factor Xa decoy protein that has been shown to reverse the inhibition of factor Xa in healthy volunteers. METHODS In this multicenter, prospective, open-label, single-group study, we evaluated 67 patients who had acute major bleeding within 18 hours after the administration of a factor Xa inhibitor. The patients all received a bolus of andexanet followed by a 2-hour infusion of the drug. Patients were evaluated for changes in measures of anti-factor Xa activity and were assessed for clinical hemostatic efficacy during a 12-hour period. All the patients were subsequently followed for 30 days. The efficacy population of 47 patients had a baseline value for anti-factor Xa activity of at least 75 ng per milliliter (or ≥0.5 IU per milliliter for those receiving enoxaparin) and had confirmed bleeding severity at adjudication. RESULTS The mean age of the patients was 77 years; most of the patients had substantial cardiovascular disease. Bleeding was predominantly gastrointestinal or intracranial. The mean (±SD) time from emergency department presentation to the administration of the andexanet bolus was 4.8±1.8 hours. After the bolus administration, the median anti-factor Xa activity decreased by 89% (95% confidence interval [CI], 58 to 94) from baseline among patients receiving rivaroxaban and by 93% (95% CI, 87 to 94) among patients receiving apixaban. These levels remained similar during the 2-hour infusion. Four hours after the end of the infusion, there was a relative decrease from baseline of 39% in the measure of anti-factor Xa activity among patients receiving rivaroxaban and of 30% among those receiving apixaban. Twelve hours after the andexanet infusion, clinical hemostasis was adjudicated as excellent or good in 37 of 47 patients in the efficacy analysis (79%; 95% CI, 64 to 89). Thrombotic events occurred in 12 of 67 patients (18%) during the 30-day follow-up. CONCLUSIONS On the basis of a descriptive preliminary analysis, an initial bolus and subsequent 2-hour infusion of andexanet substantially reduced anti-factor Xa activity in patients with acute major bleeding associated with factor Xa inhibitors, with effective hemostasis occurring in 79%. (Funded by Portola Pharmaceuticals; ANNEXA-4 ClinicalTrials.gov number, NCT02329327 .).

Molecular bases of defective signal transduction in the platelet P2Y12 receptor of a patient with congenital bleeding

We have identified structural attributes required for signal transduction through a seven-transmembrane-domain receptor. Platelets from a patient (AC) with a congenital bleeding disorder had normal shape change but reduced and reversible aggregation in response to 4 μM ADP, similar to normal platelets with blocked P2Y12 receptor. The response to 20 μM ADP, albeit still decreased, was more pronounced and was reduced by a P2Y12 antagonist, indicating some residual receptor function. ADP failed to lower the adenylyl cyclase activity stimulated by prostaglandin E1 in the patients platelets, even though the number and affinity of 2-methylthioadenosine 5′-[33P]diphosphate-binding sites was normal. Analysis of the patients P2Y12 gene revealed a G-to-A transition in one allele, changing the codon for Arg-256 in the sixth transmembrane domain to Gln, and a C-to-T transition in the other allele, changing the codon for Arg-265 in the third extracellular loop to Trp. Neither mutation interfered with receptor surface expression but both altered function, since ADP inhibited the forskolin-induced increase of cAMP markedly less in cells transfected with either mutant P2Y12 as compared with wild-type receptor. These studies delineate a region of P2Y12 required for normal function after ADP binding.

Impaired activation of murine platelets lacking Gαi2

The intracellular signaling pathways by which G protein–coupled receptors on the platelet surface initiate aggregation, a critical process for hemostasis and thrombosis, are not well understood. In particular, the contribution of the Gi pathway has not been directly addressed. We have investigated the activation of platelets from mice in which the gene for the predominant platelet Gαi subtype, Gαi2, has been disrupted. In intact platelets from Gαi2-deficient mice, the inhibition of adenylyl cyclase by ADP was found to be partially impaired compared with wild-type platelets. Moreover, both ADP-dependent platelet aggregation and the activation of the integrin αIIbβ3 (GPIIb-IIIa) were strongly reduced in platelets from Gαi2-deficient mice. In addition, Gαi2-deficient platelets displayed impaired activation at low thrombin concentrations. This defect was mimicked by blocking the adenylyl cyclase–coupled platelet ADP receptor (P2Y12) on wild-type platelets with a selective antagonist. These observations suggest that Gαi2 is involved in the inhibition of platelet adenylyl cyclase in vivo and is a critical component of the signaling pathway for integrin activation by ADP, resulting in platelet aggregation. In addition, thrombin-dependent activation of mouse platelets is mediated, at least in part, by secreted ADP acting on the Gαi2–linked ADP receptor.

Distinct Clathrin‐Coated Pits Sort Different G Protein‐Coupled Receptor Cargo

Upon activation, many G protein‐coupled receptors (GPCRs) internalize by clathrin‐mediated endocytosis and are subsequently sorted to undergo recycling or lysosomal degradation. Here we observe that sorting can take place much earlier than previously thought, by entry of different GPCRs into distinct populations of clathrin‐coated pit (CCP). These distinct populations were revealed by analysis of two purinergic GPCRs, P2Y1 and P2Y12, which enter two populations of CCPs in a mutually exclusive manner. The mechanisms underlying early GPCR sorting involve differential kinase‐dependent processes because internalization of P2Y12 is mediated by GPCR kinases (GRKs) and arrestin, whereas P2Y1 internalization is GRK‐ and arrestin‐independent but requires protein kinase C. Importantly, the β2 adrenoceptor which also internalizes in a GRK‐dependent manner also traffics exclusively to P2Y12‐containing CCPs. Our data therefore reveal distinct populations of CCPs that sort GPCR cargo at the plasma membrane using different kinase‐dependent mechanisms.

Anticoagulants (Thrombin Inhibitors) and Aspirin Synergize With P2Y12 Receptor Antagonism in Thrombosis

Background—This study was designed to determine whether (1) P2Y12 antagonism synergizes with other antithrombotics and (2) anticoagulants (thrombin inhibitors) affect the antithrombotic activity elicited by P2Y12 antagonism. Methods and Results—Thrombosis was achieved by perfusion of human and murine blood through type III collagen–coated capillaries at arterial shear rate. CT50547, a direct-acting P2Y12 antagonist, inhibited thrombosis in PPACK- but not heparin-anticoagulated human blood. In contrast, CT50547 inhibited thrombosis in aspirin-treated individuals independently of the anticoagulant. Thrombin and TXA2 also synergized with P2Y12 in the absence of anticoagulation, because combined treatment of aspirin or C921-78 (a factor Xa inhibitor) with CT50547 or 2-MeSAMP (a P2Y12 antagonist) inhibited the thrombotic process, whereas all treatments failed to inhibit thrombosis when used individually. Synergism was also observed ex vivo when P2Y12-deficient (P2Y12−/−) mice were administered aspirin or coagulation inhibitors (C921-78 and bivalirudin). Finally, using intravital microscopy, we found that both C921-78 and bivalirudin abrogated the thrombotic process in P2Y12+/− mice, whereas each showed only partial efficacy in P2Y12+/+ animals. Conclusions—Our study indicates that (1) thrombin inhibitors and aspirin have a demonstrable synergy of antithrombotic activity with P2Y12 antagonism and (2) the in vitro analysis of the antithrombotic activity of P2Y12 antagonists is affected by the anticoagulant used for blood collection. This suggests that the antithrombotic potential of P2Y12 antagonists in vitro may be overestimated in anticoagulated samples of blood and best achieved in vivo by the inclusion of aspirin and/or a thrombin inhibitor.

Distinct roles for protein kinase C isoforms in regulating platelet purinergic receptor function.

ADP is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors (GPCRs), P2Y1 and P2Y12. We have shown previously that the receptors are functionally desensitized, in a homologous manner, by distinct kinase-dependent mechanisms in which P2Y1 is regulated by protein kinase C (PKC) and P2Y12 by G protein-coupled receptor kinases. In this study, we addressed whether different PKC isoforms play different roles in regulating the trafficking and activity of these two GPCRs. Expression of PKCα and PKCδ dominant-negative mutants in 1321N1 cells revealed that both isoforms regulated P2Y1 receptor signaling and trafficking, although only PKCδ was capable of regulating P2Y12, in experiments in which PKC was directly activated by the phorbol ester phorbol 12-myristate 13-acetate (PMA). These results were paralleled in human platelets, in which PMA reduced subsequent ADP-induced P2Y1 and P2Y12 receptor signaling. PKC isoform-selective inhibitors revealed that novel, but not conventional, isoforms of PKC regulate P2Y12 function, whereas both novel and classic isoforms regulate P2Y1 activity. It is also noteworthy that we studied receptor internalization in platelets by a radioligand binding approach showing that both receptors internalize rapidly in these cells. ADP-induced P2Y1 receptor internalization is attenuated by PKC inhibitors, whereas that of the P2Y12 receptor is unaffected. Both P2Y1 and P2Y12 receptors can also undergo PMA-stimulated internalization, and here again, novel but not classic PKCs regulate P2Y12, whereas both novel and classic isoforms regulate P2Y1 internalization. This study therefore is the first to reveal distinct roles for PKC isoforms in the regulation of platelet P2Y receptor function and trafficking.