Platelets, non-nucleated blood components first described over 130 years ago, are recognized as the primary cell regulating hemostasis and thrombosis. The vascular importance of platelets has been attributed to their essential role in thrombosis, mediating myocardial infarction, stroke, and venous thromboembolism. Increasing knowledge on the platelets' role in the vasculature has led to many advances in understanding not only how platelets interact with the vessel wall but also how they convey changes in the environment to other circulating cells. In addition to their well-described hemostatic function, platelets are active participants in the immune response to microbial organisms and foreign substances. Although incompletely understood, the immune role of platelets is a delicate balance between its pathogenic response and its regulation of thrombotic and hemostatic functions. Platelets mediate complex vascular homeostasis via specific receptors and granule release, RNA transfer, and mitochondrial secretion that subsequently regulates hemostasis and thrombosis, infection, and innate and adaptive immunity.

Influenza infection increases the incidence of myocardial infarction but the reason is unknown. Platelets mediate vascular occlusion through thrombotic functions but are also recognized to have immunomodulatory activity. To determine if platelet processes are activated during influenza infection, we collected blood from 18 patients with acute influenza infection. Microscopy reveals activated platelets, many containing viral particles and extracellular-DNA associated with platelets. To understand the mechanism, we isolate human platelets and treat them with influenza A virus. Viral-engulfment leads to C3 release from platelets as a function of TLR7 and C3 leads to neutrophil-DNA release and aggregation. TLR7 specificity is confirmed in murine models lacking the receptor, and platelet depletion models support platelet-mediated C3 and neutrophil-DNA release post-influenza infection. These findings demonstrate that the initial intrinsic defense against influenza is mediated by platelet–neutrophil cross-communication that tightly regulates host immune and complement responses but can also lead to thrombotic vascular occlusion.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a cytokine with potential therapeutic value against cancers because of its selective cytotoxicity to many transformed, but not normal, cells. The ''decoy receptors'' TRAIL-R3 (TR3) and TRAIL-R4 (TR4) were believed to negatively regulate TRAIL-induced cytotoxicity by competing for ligand binding with TRAIL-R1 (TR1) and TRAIL-R2 (TR2). Here, we show that inhibition of TRAILinduced apoptosis by TR4 critically depends on its association with TR2 via the NH 2-terminal preligand assembly domain overlapping the first partial cysteine-rich domain of both receptors. By contrast, ligand binding by TR4 is dispensable for its apoptosis inhibitory function, thereby excluding the possibility that TR4 was a ''decoy'' to inhibit apoptosis by binding up TRAIL. In primary CD8 ؉ T cells, which express only TR2 and TR4 and are resistant to TRAIL-induced apoptosis, stimulation with phorbol myristate acetate abrogated the ligand-independent interaction between TR2 and TR4 and enhanced their sensitivity to TRAIL-induced apoptosis. Hence, whereas most TNF receptors normally form only homotrimeric complexes, the preligand assembly domains in TR2 and TR4 permit mixed complex formation as a means to regulate apoptosis induction. We propose that TR4 is a ''regulatory'' rather than ''decoy'' receptor that inhibits apoptosis signaling by TRAIL through this previously uncharacterized ligand-independent mechanism. decoy receptors T he regulation of cell death by members of the TNF family plays a critical role in immune function and homeostasis (1). TRAIL is a TNF-like cytokine that selectively induces apoptosis in many tumor cells, but not in normal cells. Administration of recombinant TRAIL or antibodies against TR2 in several experimental tumor models exhibited potent antitumor activity with minimal hepatic toxicity (2-6). Moreover, recombinant TRAIL or agonist TRAIL receptor antibody often synergizes with chemotherapy or radiation to induce tumor-cell apoptosis (7-10). This unique property of TRAIL has prompted many to vaunt it as a potential therapeutic agent against malignant diseases. Despite its potency against tumor cells, the physiological function of TRAIL is largely unknown, although some reports have implicated TRAIL to be involved in tumor surveillance (11), target cell killing by various immune effector cells (12,13), and the regulation of innate immune responses (14).TRAIL binds to five distinct TNF receptor (TNFR)-like receptors, TR1 (TRAIL-R1͞DR4), TR2 (TRAIL-R2͞DR5͞ Killer͞Trick), TR3 (TRAIL-R3͞DcR1͞LIT͞TRID), TR4 (TRAIL-R4͞DcR2͞TRUNDD), and the soluble receptor osteoprotegerin (OPG). OPG is a soluble receptor that also binds another TNF-like cytokine called TRANCE͞RANK-L and may have a more prominent role in bone and myeloid cell development than in regulating TRAIL-induced apoptosis. The four membrane-anchored TRAIL receptors contain two complete cysteine-rich domains (CRDs) for ligand binding that are preceded at the NH 2 termini by a highly conserved...

The role of platelets in regulating vascular homeostasis has expanded beyond mediation of haemostasis and thrombosis. The discovery of platelet RNA and the presence of subpopulations of platelets containing varying amounts of RNA suggest a role for platelet transcripts in vascular function. As the RNA in anucleated platelets is biologically functional and may transfer to other vascular cells, we hypothesised that platelet RNA diminishes over the lifespan of the platelet with diminishing platelet size due to horizontal cellular transfer. The purpose of this study is to determine if platelet RNA variance is the result of horizontal cellular transfer between platelets and other vascular cells. Utilising platelet sorting and RNA sequencing, we found that smaller platelets contained a more diverse set of transcripts than larger platelets. Further investigation using fluorescence imaging, gene expression analyses and in vitro and in vivo modelling revealed that platelets take up RNA from other vascular cells in a complex manner, revealing a dynamic role for platelets in modulating vascular homeostasis through bidirectional RNA transfer. The resultant RNA profile heterogeneity suggests unique functional roles for platelets dependent on size and complexity. This study expands our basic understanding of platelet function and heterogeneity and is the first to evaluate endogenous vascular RNA uptake and its relation to platelet processes. Our findings describe a novel endogenous phenomenon that can help elucidate the platelet's role in these non-thrombotic and haemostatic fields, as well as present potential for diagnostic and therapeutic development.

Platelets, anucleated cells with a central role in hemostasis and inflammation, contain messenger RNAs and microRNAs of unknown functionality and clinical relevance. Historically, platelet RNA was viewed as merely a remnant of platelet biogenesis; however, several studies now refute this assumption. Studies have shown that platelets can actively translate RNA to protein and that specific RNA profiles correlate with select human clinical phenotypes. These studies support a more fluid role for platelet RNA in platelet function and disease development. Our lab and others have recently studied the platelet's unique ability to transfer RNA to recipient cells and the effect this transfer has on the recipient cells' functions. This transfer may represent a previously unknown form of vascular cell communication and modulation. Unlike the well-characterized thrombotic properties of platelets, the nature and purpose of platelet RNA transfer has not been determined, partly due to limitations in techniques used to manipulate platelet RNA profiles. Defining the mechanism of RNA transfer and its role in the vascular system will allow for the better understanding of how platelets function in both their traditional thrombotic role and non-traditional functions, potentially having widespread implications in several fields.

Summary Our understanding of platelets, anucleate cells with a traditional role in hemostasis and inflammation, has developed greatly over the last decade. Platelets' role in the systemic response of the body to vascular injury, inflammation, and infection has expanded as has our understanding of their importance to the body's regulation of these processes. One recently explored mechanism by which platelets regulate the body's inflammatory and immune response is through its endogenous RNA. Platelets' messenger RNA (mRNAs) and microRNA (miRNAs) profiles have been shown to reflect disease and disease risk factors and have been correlated with select human clinical phenotypes. Developing an understanding of platelet transcripts in the circulation elucidates how platelets function in both their traditional thrombotic role and non‐traditional functions and may have widespread implications in several fields including thrombosis, infection, cancer, and systemic inflammation.

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Previously we reported (O. Ab; EORTC, 2010) that an antibody-maytansinoid conjugate (AMC) composed of an anti-FOLR1 antibody conjugated to the cytotoxic maytansinoid, DM4, via the disulfide-containing linker, SPDB, was potent in killing FOLR1-expressing cancer cells in vitro and in vivo. In light of the favorable results noted, we assessed the optimal antibody, linker, and maytansinoid agent for an AMC targeting FOLR1, as reported here. Antibody selection. Anti-FOLR1 antibodies were generated by immunizing mice with human FOLR1-expressing cells, and a panel of FOLR1-specific antibodies was identified by flow cytometry binding assay. Several FOLR1-antibodies with high binding affinity to both human and monkey FOLR1 were chosen for further evaluation and were humanized using ImmunoGen's resurfacing technology. Antibodies were conjugated to DM1 via the non-cleavable SMCC linker and the conjugates tested for activity against FOLR1-positive KB cells in vitro and in vivo. All conjugates had comparable cytotoxic potencies in vitro. However, the in vivo anti-tumor activity of one conjugate, M9346A-SMCC-DM1, was significantly better than that of SMCC-DM1 conjugates of other FOLR1 antibodies. Based on this finding, the M9346A antibody was chosen for further development. Linker/maytansinoid selection. The M9346A antibody was linked to DM1 or DM4 via the disulfide-containing cleavable linkers SPP, SPDB or sulfo-SPDB, or via the non-cleavable SMCC linker. We compared the in vitro cytotoxic activities of these conjugates on KB, Igrov-1 and Jeg-3 cell lines. The conjugates with cleavable linkers displayed markedly greater in vitro activities than the SMCC conjugate. We then examined the in vivo activities of the conjugates in FOLR1-positive KB- and Ovcar 3-tumor models. Again, we found that the conjugates with cleavable linkers were more active in vivo than the noncleavable conjugate. Among the conjugates with cleavable linkers, the sulfo-SPDB-DM4 conjugate was the most active conjugate against the Ovcar-3 model, it had activity comparable to that of the SPDB-DM4 conjugate against KB tumors, and both were more active than the SPP-DM1 conjugate in the two xenograft models. Taking into consideration that sulfo-SPDB-DM4 was the most efficacious design in vivo and the potential of the hydrophilic sulfo-SPDB-linker to enable better activity against PgP-expressing cells (previously reported data), M9346A-sulfo-SPDB-DM4 was selected to be the candidate for development and designated IMGN853. IMGN853 is a promising candidate for the treatment of FOLR1-expressing tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4576. doi:10.1158/1538-7445.AM2011-4576