Zhongren Ding

ADP receptors--targets for developing antithrombotic agents.

Platelet P2 receptors--P2Y1, P2Y12, and P2X1--constitute the means by which adenine nucleotides can activate platelets. Coactivation of the Galphaq-coupled P2Y1 and Galphai2-coupled P2Y12 receptors is necessary for ADP-mediated platelet activation, which forms the basis of using P2 antagonists as antithrombotic drugs. P2Y1 receptor antagonists inhibit platelet activation, while P2Y1 knockout mice show longer bleeding times than normal mice but few other problems; however, its ubiquitous expression in other tissues renders P2Y1 questionable as an antithrombotic target. The P2Y12 receptor is expressed nearly exclusively in platelets and brain, making it an attractive antithrombotic target. Antagonists for the P2Y12 receptor have been developed that either require metabolic activation to covalently inhibit P2Y12 and are irreversible, or simply are competitive in nature and thus reversible. Ticlopidine and clopidogrel are irreversible P2Y12 antagonists and have been repeatedly proven as clinical antithrombotic agents. In addition, a recently reported P2Y12 antagonist, CS-747, shows promise as a future antithrombotic drug. The AR-C series of compounds represent reversible P2Y12 antagonists and have been used extensively to characterize the function of P2Y12 in platelets. Clinical studies show that AR-C69931MX is as effective as clopidogrel; furthermore, the combination of AR-C69931MX (cangrelor) and clopidogrel confers greater antagonism of P2Y12 than either antagonist alone. The P2X1 receptor is a calcium channel that functions to potentiate agonist-induced platelet shape change, and its inhibition or loss has little if any effect on hemostasis. A combination of P2Y1 and P2Y12 antagonists may represent an additional course of antithrombotic treatment.

Identification of a Potent Inverse Agonist at a Constitutively Active Mutant of Human P2Y12 Receptor

Human platelets express two P2Y receptors: Gq-coupled P2Y1, and Gi-coupled P2Y12. Both P2Y1 and P2Y12 are ADP receptors on human platelets and are essential for ADP-induced platelet aggregation that plays pivotal roles in thrombosis and hemostasis. Numerous constitutively active G protein-coupled receptors have been described in natural or recombinant systems, but in the P2Y receptors, to date, no constitutive activity has been reported. In our effort to identify G protein coupling domains of the human platelet ADP receptor, we constructed a chimeric hemagglutinin-tagged human P2Y12 receptor with its C terminus replaced by the corresponding part of human P2Y1 receptor and stably expressed it in Chinese hamster ovary-K1 cells. It is interesting that the chimeric P2Y12 mutant exhibited a high level of constitutive activity, as evidenced by decreased cAMP levels in the absence of agonists. The constitutive activation of the chimeric P2Y12 mutant was dramatically inhibited by pertussis toxin, a Gi inhibitor. The constitutively active P2Y12 mutant retained normal responses to 2-methylthio-ADP, with an EC50 of 0.15 ± 0.04 nM. The constitutively active P2Y12 mutant caused Akt phosphorylation that was abolished by the addition of pertussis toxin. Pharmacological evaluation of several P2Y12 antagonists revealed (E)-N-[1-[7-(hexylamino)-5-(propylthio)-3H-1,2,3-triazolo-[4,5-d]-pyrimidin-3-yl]-1,5,6-trideoxy-β-d-ribo-hept-5-enofuranuronoyl]-l-aspartic acid (AR-C78511) as a potent P2Y12 inverse agonist and 5′-adenylic acid, N-[2-(methylthio)ethyl]-2-[(3,3,3-trifluoropropyl)thio]-, monoanhydride with (dichloromethylene)bis[phosphonic acid] (AR-C69931MX) as a neutral antagonist. In conclusion, this is the first report of a cell line stably expressing a constitutively active mutant of human platelet P2Y12 receptor and the identification of potent inverse agonist.

Nucleotide-Binding Oligomerization Domain 2 Receptor Is Expressed in Platelets and Enhances Platelet Activation and Thrombosis

Background— Pattern recognition receptor nucleotide-binding oligomerization domain 2 (NOD2) is well investigated in immunity, but its expression and function in platelets has never been explored. Method and Results— Using reverse transcription polymerase chain reaction and Western blot, we show that both human and mouse platelets express NOD2, and its agonist muramyl dipeptide induced NOD2 activation as evidenced by receptor dimerization. NOD2 activation potentiates platelet aggregation and secretion induced by low concentrations of thrombin or collagen, and clot retraction, as well. These potentiating effects of muramyl dipeptide were not seen in platelets from NOD2-deficient mice. Plasma from septic patients also potentiates platelet aggregation induced by thrombin or collagen NOD2 dependently. Using intravital microscopy, we found that muramyl dipeptide administration accelerated in vivo thrombosis in a FeCl3-injured mesenteric arteriole thrombosis mouse model. Platelet depletion and transfusion experiments confirmed that NOD2 from platelets contributes to the in vivo thrombosis in mice. NOD2 activation also accelerates platelet-dependent hemostasis. We further found that platelets express receptor-interacting protein 2, and provided evidence suggesting that mitogen activated-protein kinase and nitric oxide/soluble guanylyl cyclase/cGMP/protein kinase G pathways downstream of receptor-interacting protein mediate the role of NOD2 in platelets. Finally, muramyl dipeptide stimulates proinflammatory cytokine interleukin-1&bgr; maturation and accumulation in human and mouse platelets NOD2 dependently. Conclusions— NOD2 is expressed in platelets and functions in platelet activation and arterial thrombosis, possibly during infection. To our knowledge, this is the first study on NOD-like receptors in platelets that link thrombotic events to inflammation.Background Pattern recognition receptor NOD2 (nucleotide binding oligomerization domain 2) is well investigated in immunity, its expression and function in platelets has never been explored.

Integrin αIIb-Mediated PI3K/Akt Activation in Platelets

Integrin αIIbβ3 mediated bidirectional signaling plays a critical role in thrombosis and haemostasis. Signaling mediated by the β3 subunit has been extensively studied, but αIIb mediated signaling has not been characterized. Previously, we reported that platelet granule secretion and TxA2 production induced by αIIb mediated outside-in signaling is negatively regulated by the β3 cytoplasmic domain residues R724KEFAKFEEER734. In this study, we identified part of the signaling pathway utilized by αIIb mediated outside-in signaling. Platelets from humans and gene deficient mice, and genetically modified CHO cells as well as a variety of kinase inhibitors were used for this work. We found that aggregation of TxA2 production and granule secretion by β3Δ724 human platelets initiated by αIIb mediated outside-in signaling was inhibited by the Src family kinase inhibitor PP2 and the PI3K inhibitor wortmannin, respectively, but not by the MAPK inhibitor U0126. Also, PP2 and wortmannin, and the palmitoylated β3 peptide R724KEFAKFEEER734, each inhibited the phosphorylation of Akt residue Ser473 and prevented TxA2 production and storage granule secretion. Similarly, Akt phosphorylation in mouse platelets stimulated by the PAR4 agonist peptide AYPGKF was αIIbβ3-dependent, and blocked by PP2, wortmannin and the palmitoylated peptide p-RKEFAKFEEER. Akt was also phosphorylated in response to mAb D3 plus Fg treatment of CHO cells in suspension expressing αIIbβ3-Δ724 or αIIbβ3E724AERKFERKFE734, but not in cells expressing wild type αIIbβ3. In summary, SFK(s) and PI3K/Akt signaling is utilized by αIIb-mediated outside-in signaling to activate platelets even in the absence of all but 8 membrane proximal residues of the β3 cytoplasmic domain. Our results provide new insight into the signaling pathway used by αIIb-mediated outside-in signaling in platelets.

BF061, a novel antiplatelet and antithrombotic agent targeting P2Y12 receptor and phosphodiesterase

The addition of phosphodiesterase (PDE) inhibitors has been reported to potentiate the antithrombotic effects of P2Y₁₂ antagonists without increasing bleeding risk. In this study, we report that a potent antiplatelet agent, 2-ethylthio-6-phenethylaminoadenosine (BF061), inhibits platelet activation and thrombosis via P2Y₁₂ antagonism and PDE inhibition. We explored the antiplatelet mechanism of BF061 by measuring cAMP, cGMP levels, PDE activity, and the interaction between ADP and P2Y₁₂ using atomic force microscopy. The antithrombotic effect of BF061 was evaluated in mice using intravital microscopy in FeCl₃₋induced mesenteric and laser-induced cremasteric arterial thrombosis models. BF061 robustly inhibited platelet aggregation and ATP release induced by multiple platelet agonists via P2Y₁₂ antagonism and PDE inhibition. Interestingly, despite being structurally similar to BF061, P2Y₁₂ receptor antagonist AR-C69931MX had no effect on human platelet PDE. In FeCl3-induced mesenteric arterial thrombosis model, BF061 effectively prevented thrombus formation similarly to clopidogrel; it also reduced thrombus volume in laser-injured cremaster arteriole model. In contrast, BF061 induced dramatically less bleeding at an antithrombotic dose compared to clopidogrel. In summary, we developed a novel antiplatelet and antithrombotic agent targeting both P2Y₁₂ and PDE. Given the prevalence of combined antiplatelet therapy in clinical practice, an antiplatelet agent bearing dual activities may have therapeutic advantage as a potential antithrombotic drug.

Salvianolic acid B inhibits platelets as a P2Y12 antagonist and PDE inhibitor: Evidence from clinic to laboratory

Salviae miltiorrhiza (Danshen) has been used for thousands of years in China and some other Asian countries to treat atherothrombotic diseases. Salvianolate which consists of three water-soluble ingredients purified from Salviae miltiorrhiza, has been approved by Chinese SFDA to treat coronary artery disease. So far, there is no evidence clearly showing the clinical efficiency of salvianolate and the underlying mechanism. This study is to evaluate the effects of salvianolate on platelets in patients with acute coronary syndrome and explore the underlying mechanism. We evaluated the effects of salvianolate on platelets in patients with acute coronary syndrome by measuring ADP-induced PAC-1 binding and P-selectin expression on platelets. Salvianolate significantly potentiated the antiplatelet effects of standard dual antiplatelet therapy. We also investigated the antiplatelet effects of salvianolatic acid B (Sal-B), the major component which composes 85% of salvianolate. Sal-B inhibits human platelet activation induced by multiple agonists in vitro by inhibiting phosphodiesterase (PDE) and antagonizing P2Y12 receptor. For the first time, we show the antiplatelet efficiency of salvianolate in ACS patients undergoing treatment with clopidogrel plus aspirin, and demonstrate that Sal-B, the major component of salvianolate inhibits human platelet activation via PDE inhibition and P2Y12 antagonism which may account for the clinical antiplatelet effects of salvianolate. Our results suggest that Sal-B may substitute salvianolate for clinical use.

BF0801, a novel adenine derivative, inhibits platelet activation via phosphodiesterase inhibition and P2Y12 antagonism

Though antiplatelet drugs are proven beneficial to patients with coronary heart disease and stroke, more effective and safer antiplatelet drugs are still needed. In this study we report the antiplatelet effects and mechanism of BF0801, a novel adenine derivative. BF0801 dramatically inhibited platelet aggregation and ATP release induced by ADP, 2MeSADP, AYPGKF, SFLLRN or convulxin without affecting shape change in vitro . It also potentiated the inhibitory effects of adenosine-based P2Y12 antagonist AR-C69931MX or phosphodiesterase (PDE) inhibitor IBMX on platelet aggregation. The cAMP levels in both resting and forskolin-stimulated platelets were increased by BF0801 suggesting its PDE inhibitor activity, which is further confirmed by the concentration-dependent suppression of BF0801 on the native and recombinant PDE. Similar to AR-C69931MX, BF0801 drastically inhibited 2MeSADP- induced adenylyl cyclase inhibition in platelets indicating its P2Y12 antagonism activity, which is substantiated by the inhibition of BF0801 on the interaction between ADP and P2Y12 receptor expressed in CHO-K1 cells measured by atomic force microscopy. Moreover, we confirmed the antiplatelet effects of BF0801 using platelets from rats intravenously given BF0801. In summary, for the first time we developed a novel adenine derivative bearing dual activities of PDE inhibition and P2Y12 antagonism, which may have therapeutic advantage as a potential antithrombotic drug.

Tumor vascular disrupting agent 5,6‐dimethylxanthenone‐4‐acetic acid inhibits platelet activation and thrombosis via inhibition of thromboxane A2 signaling and phosphodiesterase

5,6‐Dimethylxanthenone‐4‐acetic acid (DMXAA) is a tumor vascular disrupting agent under clinical trials as an adjacent antitumor agent. DMXAA is structurally similar to flavone‐8‐acetic acid (FAA), an old tumor vascular disrupting agent with antiplatelet and antithrombotic effects. In contrast to FAA, which causes bleeding in tumor patients, no bleeding has been reported in patients receiving DMXAA. Whether DMXAA also affects platelet function is not clear.

Increased platelet activation and thrombosis in transgenic mice expressing constitutively active P2Y12

Summary.  Background:  In our previous in vitro study, we reported a constitutively active chimeric P2Y12 (cP2Y12) and found that AR‐C78511 is a potent inverse agonist at this receptor. The role of cP2Y12 in platelet activation and thrombosis is not clear.

P2Y12 protects platelets from apoptosis via PI3k-dependent Bak/Bax inactivation.

Summary.  Background: Platelet ADP receptor P2Y12 is well studied and recognized as a key player in platelet activation, hemostasis and thrombosis. However, the role of P2Y12 in platelet apoptosis remains unknown. Objectives: To evaluate the role of the P2Y12 receptor in platelet apoptosis. Methods: We used flow cytometry and Western blotting to assess apoptotic events in platelets treated with ABT‐737 or ABT‐263, and stored at 37 °C, combined with P2Y12 receptor antagonists or P2Y12‐deficient mice. Results: P2Y12 activation attenuated apoptosis induced by ABT‐737 in human and mouse platelets in vitro, evidenced by reduced phosphatidylserine (PS) exposure, diminished depolarization of mitochondrial inner transmembrane potential (ΔΨm) and decreased caspase‐3 activation. Through increasing the phosphorylation level of Akt and Bad, and changing the interaction between different Bcl‐2 family proteins, P2Y12 activation inactivated Bak/Bax. This antiapoptotic effect could be abolished by P2Y12 antagonism or PI3K inhibition. We also observed the antiapoptotic effect of P2Y12 activation in platelets stored at 37 °C. P2Y12 activation improved the impaired activation responses of apoptotic platelets stressed by ABT‐737. In platelets from mice dosed with ABT‐263 in vivo, clopidogrel or deficiency of P2Y12 receptor enhanced apoptosis along with increased Bak/Bax activation. Conclusions: This study demonstrates that P2Y12 activation protects platelets from apoptosis via PI3k‐dependent Bak/Bax inactivation, which may be physiologically important to counter the proapoptotic challenge. Our findings that P2Y12 blockade exaggerates platelet apoptosis induced by ABT‐263 (Navitoclax) also imply a novel drug interaction of ABT‐263 and P2Y12 antagonists.