During the previous 5 years of this project, we have defined one receptor-counter receptor pair that? removes washed platelets, chilled for 2-4h, from the circulation. We proposed that chilling causes the? GPIb/V/IX receptor complex (vWfR) to cluster on the surface of chilled platelets bringing exposed beta-N-acetyl? glucosamine (beta-GlcNAc) residues on N-linked glycans of the GP1b-alpha subunit together, which? leads to their recognition by the lectin domain of alphaM-beta2 receptors on phagocytes in the liver. Coverage? of exposed beta-GlcNAc on the GP1b-alpha chain by galactose (galactosylation) rescues the loss of? circulation in mice of platelets chilled under these conditions. However, storage of platelets in plasma? in the cold for 48 h induces further changes that lead to a loss of circulation not rescued by? galactosylation. We now postulate that long-term chilling (equal to or greater than 48h) of platelets in plasma leads to a? """"""""hyperclustering"""""""" of the vWFR. This increases the density of galactose residues in clusters on? galactosylated platelets such that they reach a critical density that now results in clearance by? hepatocytes and/or macrophages using their Asialoglycoprotein receptor (ASGPR), which recognizes? exposed galactose. GP1b-alpha is linked to the underlying actin skeleton by filamin A (FLNa). We? previously showed that chilling induces platelets to remodel their cytoskeleton and assemble actin.? Aim 1 will determine if GP1b-alpha is involved in the removal of platelets stored in plasma for 48h and if the? long-term changes are due to the binding of plasma components to platelets. If GP1b-alpha is central to the? changes, we will investigate the role of FLNa and gelsolin as well as the underlying actin-connection in? vWfR clustering. A pure population of FLNa-null platelets using Ore driven by the hematopoetic? specific promoter GATA1 in mice using our conditional loxP allele of FLNa has been established. We? will determine if platelets lacking FLNa or gelsolin circulate in wild type (WT) mice. We postulate that? FLNa null platelets will be cleared. These results will therefore get at the mechanism, and importance,? of clustering in platelet survival and in vivo function (in collaboration with Project 1- Dr. Wagner). If? platelets lacking FLNa circulate, we will determine if they fail to cluster the vWf receptor in the cold? following short-term storage in buffer or long-term storage in plasma. We will also investigate if? prolonged cold platelet storage dissociates the GP1b-alpha-FLNa/b complex.
Aim 2 will determine: (1) if? sialylation facilitates the survival of refrigerated (> 48 hrs) galactosylated platelets; (2) identify platelet? acceptor proteins for UDP-galactose and CMP-sialic acid; (3) determine which organ/cells clear? galactosylated and/or sialylated platelets (in collaboration with Project 3 - Dr. von Andrian); (4)? evaluate in vivo function of modified and refrigerated platelets (in collaboration with Project 1 - Dr.? Wagner); and (5) establish a humanized adaptive immune system in mice that will allow us to assess? the effects of platelet modification on refrigerated human platelet survival and function in vivo (in? collaboration with Projects 1 and 3 - Drs. Wagner and von Andrian). Studies in this area have been? limited by a lack of good animal models that evaluate both human platelet circulation and function? following transfusion.
Aim 3, if necessary, will identify GP1b-alpha independent changes in murine platelets? refrigerated (> 48 hrs) in plasma under blood bank conditions that target them for removal and identify? the phagocytic receptor that mediates the removal. The overall goal of this work is to identify new? targets that can be modulated to prevent cold-induced platelet clearance and develop methodology to? block them.
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