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Murine paired immunoglobulin-like receptors B (PIRB), as the ortholog of human leukocyte immunoglobulin-like receptor B2 (LILRB2), is involved in a variety of biological functions. Here, we found that PIRB and LILRB2 were expressed in mouse and human platelets, respectively. PIRB intracellular domain deletion (PIRB-TM) mice had thrombocythemia and significantly higher proportions of megakaryocytes in bone marrow. Agonist-induced aggregation and spreading on immobilized fibrinogen were facilitated in PIRB-TM platelets. The rate of clot retraction in platelet-rich plasma containing PIRB-TM platelets was also increased. Characterization of signaling confirmed that PIRB associated with phosphatases Shp1/2 in platelets. The phosphorylation of Shp1/2 was significantly downregulated in PIRB-TM platelets stimulated with collagen-related peptide (CRP) or on spreading. The results further revealed that the phosphorylation levels of the linker for activation of T cells, SH2 domain-containing leukocyte protein of 76kDa, and phospholipase C were enhanced in PIRB-TM platelets stimulated with CRP. The phosphorylation levels of FAK Y397 and integrin β3 Y759 were also enhanced in PIRB-TM platelet spread on fibrinogen. The PIRB/LILRB2 ligand angiopoietin-like-protein 2 (ANGPTL2) was expressed and stored in platelet α-granules. ANGPTL2 inhibited agonist-induced platelet aggregation and spreading on fibrinogen. The data presented here reveal that PIRB and its ligand ANGPTL2 possess an antithrombotic function by suppressing collagen receptor glycoprotein VI and integrin αIIbβ3-mediated signaling.

Abstract 3356 The platelet ADP receptor P2Y12 plays a prominent role in amplifying platelet activation, aggregation and thrombus formation, and the P2Y12 inhibitor Clopidogrel is widely used clinically to treat coronary artery, peripheral vascular and cerebrovascular diseases. Recently, several publications analyzed the TRITON-TIMI 38 clinical trial (test the efficacy and safety of Prasugrel, a newly FDA approved thienopyridine P2Y12 inhibitor) revealing an increase in multiple types of solid tumors with Prasugrel use, casting doubt on the safety of anti-platelet therapy targeting P2Y12. In this study, we used animal models to investigate the safety of targeting P2Y12, and to elucidate the probable mechanism for the effects of P2Y12 antagonists on metastasis. Tumor growth and metastasis are basic features of malignant cancer. Pulmonary metastasis in the experimental, and spontaneous mouse metastasis models were used to identify the role of P2Y12, and the effects of its inhibitors in tumor growth and metastasis. In the experimental pulmonary metastasis group, one week before tail vein injection of 2×105 melanoma B16 cells, wild type (WT) and P2y12–/– mice were treated every other day with PBS, Clopidogrel or Prasugrel by oral gavage and continue in whole experiment process. Tumor nodules were counted, and the average nodule sizes measured at 20 days after B16 cell injection. P2y12–/– mice had fewer lung metastatic loci than the lungs of the WT controls. The experimental and control lungs had similar size nodules. But Clopidogrel dose dependently (1.5mg/kg and 30mg/kg) caused an increase of metastatic loci and loci size in both WT and P2y12–/– mice. Prasugrel (0.2mg/kg and 4mg/kg) did not affect the number or size of metastatic loci produced in either strain. These results demonstrated that p2y12 deficiency negatively affected cancer metastasis.In contrast, Clopidogrel, but not Prasugrel, promotes metastasis and cancer growth. Importantly, Clopidogrel induced metastasis promotion and growth was not P2Y12 dependent. A plausible explanation of this difference between the two drugs may reside in the fact that both are pro-drugs of thienopyridine family. Presumably, activation of the pro-drug clopidogrel, but not Prasugrel produces one or more metabolites able to promote cancer growth and metastasis in vivo. This supposition was tested, in part by asking if the effects of Clopidogrel are limited to the B16 model of metastasis. The Lewis lung carcinoma (LLC) spontaneous pulmonary metastasis model was used for this purpose. LLC cells (2.0×106) were implanted intradermally in each mouse. Surgery was done 14 days after cell implantation to completely remove the primary tumors (about 1.0g or 1cm3 in size). One month later, lungs were removed, rinsed with PBS, and pulmonary metastatic nodules counted and measured. P2Y12 deficiency decreased the spontaneous formation of pulmonary metastatic loci, but had no effect on the primary tumor. Also, Clopidogrel, dose dependently promoted lung metastasis. Next, the mechanism of the effect of P2Y12 absence on tumor metastasis was investigated. In vitro aggregation experiments revealed that B16 melanoma cells directly interact with p2y12+/+ platelets and cause platelet-cancer cell aggregation. In contrast, P2Y12 deficiency results in significantly less or no cancer cell-platelet aggregation. Interestingly, LLC cells do not cause platelet aggregation. Immunohistochemistry and immunofluorescence analyses of lung samples from the spontaneous metastasis model indicated an obvious attenuation of recruitment of VEGFR1+ bone marrow derived cell clusters, and extracellular matrix fibronectin deposition in lungs, which presumably are required for metastatic niche formation. Moreover, this effect is transplantable, as wild-type mice reconstituted with p2y12–/– bone marrow have a similar phenotype as P2y12–/– mice. These results imply that the mechanism of P2Y12 function on cancer metastasis is complicated, probably mediated through regulation of both cancer cell-platelet direct interaction and metastatic niche formation. Further work is needed to resolve this issue. In summary, it is clear that P2Y12 is a safe drug target for anti-thrombotic therapy. But, metabolic derivatives of the pro-drug Clopidogrel apparently promote cancer metastasis and growth; whereas the newly approved pro-drug Prasugrel lacks that effect in animal models, and hopefully in humans. Disclosures: No relevant conflicts of interest to declare.

Abstract 3252 PTEN (Phosphatase and tensin homologue deleted in chromosome 10) is a well-known tumor suppressor. PTEN dephosphorylates PtdIns(3,4,5)P3 into PtdIns(4,5)P2 and thereby negatively regulates PIP3-downstream signaling. According to present studies, PTEN is also a protein tyrosine phosphatase. In 2010 (Blood. 2010;116(14):2579–81), we reported that hematopoietic specific PTEN deficiency causes an increased platelet count, a shortened tail vein bleeding time, and enhanced platelet activation in response to stimulation by collagen and other agonists along with augmented collagen-induced phosphorylation of platelet Akt at Ser473. The PI3K specific inhibitor LY294002 almost completely blocked collagen-induced WT platelet aggregation, but had less effect on PTEN-deficient platelets. This result implies that PTEN may regulate collagen-induced platelet activation via both PI3K/Akt signaling and an yet to be identified pathway. In this study, we investigated the mechanism underlying PTEN regulation of collagen-induced platelet activation. It is well-known that the GPVI receptor signaling cascade is similar to that of T- and B-cell immune receptors. This signaling cascade relies on the formation of a signalosome composed of the transmembrane adapter LAT (linker for activation of T cells), the two cytosolic adapters SLP-76 and Gads, and the activation of PLCγ2. Accordingly, we investigated the phosphorylation of LAT, SLP-76 and PLCγ2 in PTEN-deficient and WT platelets in response to low level collagen (2μg/ml), with and without LY294002 pre-incubation. The platelet lysate was immunoprecipitated with 4G10, a specific anti-phosphotyrosine monoclonal antibody, to detect tyrosine phosphorylation. Interestingly, we found that in the presence of LY294002, the phosphorylation levels of LAT, SLP-76 and PLCγ2 were very high in PTEN-deficient platelets while the phosphorylation of these molecules was hardly detectable in WT platelets, all in response to low level collagen. These results suggested that PTEN may also regulate platelet activation through LAT, SLP-76 and PLCγ2. This hypothesis was supported by co-immunopricipitation experiments demonstrating that PTEN interacts with LAT in platelets upon collagen stimulation. Moreover, we also found that PTEN Ser380 phosphorylation was correlated with collagen-induced WT platelet activation without LY294002, and PTEN Ser380 phosphorylation could be inhibited by CK2 inhibitor DMAT and PDK1 inhibitor II, but not by Akt inhibitor SH6 or the GSK3β inhibitor LiCl. In contrast, the PDK1 inhibitor II blocked collagen-induced activation of CK2. Radioactivity assays indicated that collagen-induced enzyme activity activation of CK2 was blocked by the PDK1 inhibitor II. Ser380 phosphorylation is thought to reflect the loss of PTEN phosphatase function. So, because inhibition of PI3K, and PDK1 do not result in phosphorylation of PTEN S380 and the resulting loss of PTEN phosphatase function, it may be true that PDK1 inactivates PTEN. Therefore, we conclude that the mechanism of PTEN involvement in collagen-induced platelet activation is complicated, and PTEN plays a key role in GPVI mediated platelet activation probably through down-regulating both the PI3K/Akt and PI3K-PDK1-LAT pathways. We also investigated PTEN's function in αIIbβ3 mediated platelet activation. Platelet spreading and clot retraction experiments were performed. We found that PTEN deficiency significantly promoted clot retraction, but had no effect on platelet spreading on immobilized fibrinogen. Moreover, U46619 and thrombin induced PTEN ser380 phosphorylation was inhibited in β3 deficient platelet. These results indicate that PTEN is also involved in αIIbβ3 mediated outside-in signaling. Although the molecular mechanism of PTEN modulation of αIIbβ3 mediated outside-in signaling is unclear, our results may partially explain why PTEN deficiency also enhances platelet aggregation and secretion in response to agonists other than collagen. Disclosures: No relevant conflicts of interest to declare.