One pathway that mediates the rapid removal of transfused platelets exposed to cold for short periods (≤ 2 h) is the clustering of the von-Willebrand factor (VWF) receptor complex. Clustered VWF receptors are recognized by the αMβ2 integrin on hepatic macrophages, which, through its lectin domain, binds exposed βN-acetylglucosamine (βGlcNAc) residues on the clustered GPIbα subunits (

Cell
112
:
87
,
2003
). Coverage of the exposed βGlcNAc residues by galactosylation prevents clearance of platelets from the circulation. We have now investigated detailed changes of the VWF complex on platelets during short (≤ 2 h) and prolonged refrigeration (≥ 48 h). The VWF receptor structure in chilled and rewarmed platelets was probed by flow cytometry using a panel of mAbs directed towards the GPIbα N-terminal (N–T) and GPIX (FITC-AN51 (aa36–59), PE-Cy5-HIP1 (aa59–81), FITC-SZ2 (aa268–282), and PE-GPIX mAb). Binding of the anti-GPIbα mAbs HIP1 and SZ2 as well as the anti-GPIX was diminished 40–50 % following 0.5 h of cooling when compared with room temperature (RT) platelets. The anti -GPIbα N-T mAb AN51 bound similarly to cooled and RT platelets. In contrast, the binding of all anti-VWF complex mAbs was decreased by 50–60 % after refrigeration of platelets in plasma for 48 h when compared to fresh RT platelets. Immunoblots demonstrated that loss of binding did not result from GPIbα shedding. VWF and fibrinogen binding were also increased by ~ 5 % or ~ 20 %, respectively, and P-selectin exposure increased by ~ 30 % in 48 h refrigerated platelets compared to the RT platelets stored for 48 h as determined by flow cytometry. These results suggest that the conformation of the VWF receptor is altered by long term refrigeration which leads to VWF binding and partial platelet activation. Loss of antibody binding is not simply due to GPIbα clustering as incubation of platelets with 5 μM latrunculin A for 1 h, which induces profound GPIbα clustering, increased binding by ~ 30 % compared to untreated platelets, but did not alter the diminished mAb binding to platelets induced by chilling. Refrigerated (≤ 14 days) platelets still adhere well to collagen-coated surfaces under flow (1000 seconds−1 for 2 min) demonstrating that VWF receptor function remains intact. We conclude that refrigeration of platelets in plasma for ≥ 48 h induces both conformational changes and clustering of the VWF complex, to prime it for activation by inducing VWF binding.

Topics:
blood platelets, refrigeration, von willebrand factor receptor, monoclonal antibodies, flow cytometry, fluorescein-5-isothiocyanate, acetylglucosamine, antibodies, chills, common cold

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2005, The American Society of Hematology
2005
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