The goal of these studies is to determine the pathway(s) by which hepatocytes ingest platelets to accelerate their clearance from the circulation. Until recently, the only well-established mechanisms affecting platelet survival were antibody-mediated platelet clearance, consumption of platelets by coagulation reactions and loss due to massive bleeding. An effort to address a practical problem, how to refrigerate platelets for transfusion, led us to define an unsuspected carbohydrate-based platelet clearance mechanism where the alphaM-beta 2 receptor on Kupffer cells recognized clustered betaGlcNAc moieties on GPIba of short-term (< 4h) stored platelets. We have now made the striking observation that mouse platelets lacking sialic acid (refrigerated for 48h or platelets from sialyl-transferase STSGallV null mice are removed in the liver, principally by hepatocytes, not macrophages. Because exposed galactose residues increase on the surface as platelets circulate, and after long-term storage as revealed by lectin binding experiments, we postulate that sialic acid normally covers the galactose residues and permits platelet survival. We further hypothesize that increases in galactose exposure induce recognition by the Asialoglycoprotein receptor (ASGPR or HL- 1/2) on hepatocytes. Platelet modifications that markedly enhance clearance will be used to determine the role of sialic acid in platelet survival: 1) ST3GallV null platelets; 2) enzymatically desialylated platelets; and 3) wild type platelets refrigerated for > 48h.
Aim 1 will document and characterize the uptake of long-term stored platelets, ST3GallV null platelets and neuraminidase-treated platelets by hepatocytes in real time, quantify their clearance rates in mice, determine the contribution of macrophage clearance (Aim 1A), and define the role of the ASGP receptor(s) in initiating ingestion (Aim 1B). We will determine whether a combination of galactosylation and sialylation will improve long-term stored platelet and ST3GallV-/- platelet survival (Aim 1C). We will also investigate if platelets lose sialic acid while circulating, a process that could contribute to platelet aging and clearance (Aim 1D), and whether resialylation can improve survival (Aim 1E). These experiments will, therefore, establish a new clearance mechanism for senile platelets. While the intravital experiments should demonstrate HL-1/2 function in the clearance of chilled murine platelets in vivo, other approaches are necessary to establish that human platelets can suffer the same fate.
In Aim 2, therefore, we propose to utilize the in vitro endocytosis of desialylated platelets by HepG2 cells as a simple readout (relative to transfusion studies) (Aim 2A) to define the nature of the host platelet glycoproteins carrying the galactose moieties recognized by HL-1/2 (hepatocytes) (Aim 2B) or MGL1 (macrophages) receptors (Aim 2D). We will further determine if platelet resialylation prevents the ingestion of desialylated platelets in vitro (Aim 2C). ? ? ?
|Lee-Sundlov, Melissa M; Ashline, David J; Hanneman, Andrew J et al. (2017) Circulating blood and platelets supply glycosyltransferases that enable extrinsic extracellular glycosylation. Glycobiology 27:188-198|
|Li, Renhao; Hoffmeister, Karin M; Falet, Hervé (2016) Glycans and the platelet life cycle. Platelets 27:505-11|
|Hoffmeister, Karin M; Falet, Hervé (2016) Platelet clearance by the hepatic Ashwell-Morrell receptor: mechanisms and biological significance. Thromb Res 141 Suppl 2:S68-72|
|Li, June; van der Wal, Dianne E; Zhu, Guangheng et al. (2015) Desialylation is a mechanism of Fc-independent platelet clearance and a therapeutic target in immune thrombocytopenia. Nat Commun 6:7737|
|Grozovsky, Renata; Giannini, Silvia; Falet, Hervé et al. (2015) Novel mechanisms of platelet clearance and thrombopoietin regulation. Curr Opin Hematol 22:445-51|
|Grozovsky, Renata; Giannini, Silvia; Falet, Hervé et al. (2015) Regulating billions of blood platelets: glycans and beyond. Blood 126:1877-84|
|Spitalnik, Steven L; Triulzi, Darrell; Devine, Dana V et al. (2015) 2015 proceedings of the National Heart, Lung, and Blood Institute's State of the Science in Transfusion Medicine symposium. Transfusion 55:2282-90|
|Bender, Markus; Giannini, Silvia; Grozovsky, Renata et al. (2015) Dynamin 2-dependent endocytosis is required for normal megakaryocyte development in mice. Blood 125:1014-24|
|Grozovsky, Renata; Begonja, Antonija Jurak; Liu, Kaifeng et al. (2015) The Ashwell-Morell receptor regulates hepatic thrombopoietin production via JAK2-STAT3 signaling. Nat Med 21:47-54|
|Begonja, Antonija Jurak; Pluthero, Fred G; Suphamungmee, Worawit et al. (2015) FlnA binding to PACSIN2 F-BAR domain regulates membrane tubulation in megakaryocytes and platelets. Blood 126:80-8|
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