Role of GPIbαlpha Clustering and N-linked Carbohydrates in the Clearance of Refrigerated Platelets

Abstract: The thesis focuses on understanding the mechanisms by which: 1) the macrophage alphaM-subunit recognizes betaN-acetylglucosamine (betaGlcNAc) residues on the von Willebrand factor receptor complex ((GPIbalpha,beta/IX)2V or vWfR) on refrigerated platelets and 2) refrigeration changes vWfR to elicit recognition through alphaMbeta2. 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 a previously unsuspected platelet clearance mechanism. We found that (1) macrophages recognize betaGlcNAc residues of N-linked glycans on clustered GPIbalpha subunits following short-term refrigeration (2 h) of platelets in the absence of plasma and (2) phagocytosis and clearance are mediated by the alphaMbeta2 integrin receptor of macrophages. Galactosylation of GPIbalpha blocks ingestion by the macrophage alphaMbeta2 and allows short-term refrigerated murine platelets to circulate but does not prevent the removal of platelets stored long-term in plasma. Work detailed in this thesis demonstrates that the ingestion of short-term refrigerated platelets is dependent on the alphaM lectin-domain, not the I-domain which is involved in the recognition of most alphaMbeta2 ligands. To address this question, CHO cells were directed to express different alphaM/alphaX receptor subunit chimeras and the relative contribution of alphaM-subdomains to platelet ingestion evaluated in these cells. Critically, the recognition and ingestion of refrigerated platelets by CHO cells occurs only when the alphaM-subunits contain the alphaM lectin-subdomain. The I- or cation binding subdomains of the alphaM-subunit are not required. Soluble recombinant alphaM lectin-domain, but not a soluble alphaM I-domain, also inhibited the phagocytosis of refrigerated platelets by differentiated macrophages and Sf9 cells expressing solely recombinant alphaM lectin-domain constructs bound refrigerated platelets. We conclude, therefore, that refrigeration exposes N-glycan betaGlcNAc residues on vWfR which are recognized by the lectin-domain of alphaMbeta2 to initiate platelet clearance. Next, the relationship between vWfR clustering/conformational changes and refrigeration was investigated. Clustering of vWfR is detectable by fluorescent resonance energy transfer (FRET) measured by flow cytometry. Refrigeration of platelets for 24 h markedly increases the FRET efficiency between GPIbalpha and GPV subunits, whereas the FRET between GPIbalpha and alphaIIb is unaltered. We conclude that vWfR aggregation begins immediately following refrigeration but becomes maximal only after extended refrigeration. A panel of monoclonal antibodies (mAbs) that recognize different vWfR subunits was employed to further probe for structural changes. We found that certain epitopes on GPIbalpha become cryptic as platelets are refrigerated, possibly due to clustering of the vWfR complex, and that the rate of epitope sequestration due to clustering is slowed in the presence of plasma. Changes in binding efficacy of the mAbs are not caused by the loss of GPIbalpha from the platelet surface as determined by immunoblotting of total GPIbalpha. Some vWf binding in cold plasma was detected that may influence the binding of mAbs which bind to GPIbalpha near its vWf binding site. These further changes in vWfR in platelets refrigerated long-term in plasma may be related to the additional phagocytic mechanisms involved in their removal.