Nascent ADAM17 synthesis potentiates GPIbα cleavage in resting and stimulated stored platelets

Platelet concentrates stored at room temperature have a shelf life of 5 to 7 days. During storage, platelets undergo glycoprotein cleavage by metalloproteases, notably cleavage of glycoprotein Ibα (GPIbα) by a disintegrin and metalloprotease 17 (ADAM17), which leads to decreased posttransfusion reactivity and recovery. To investigate the putative roles of nascent synthesis of ADAM17 in GPIbα shedding and platelet function during room temperature platelet storage. Human platelets maintained in autologous plasma were treated with naked endonuclease-resistant ADAM17 or control short inhibitory RNA (siRNA) and monitored for molecular and cellular effects during storage. Platelet-specific Adam17-deleted mice were generated, and the dynamics of GPIbα cleavage were assessed. Platelets translated nascent ADAM17 during storage, coinciding with progressive GPIbα ectodomain cleavage. siRNA treatment suppressed ADAM17 translation and rescued total but not surface levels of full-length GPIbα in resting platelets during storage. Flow cytometry and confocal microscopy in permeabilized platelets indicated an internal pool of GPIbα protected from agonist-induced cleavage by cell-permeable ADAM17 pharmacological blockade but not by non-cell-permeable blocking antibodies. ADAM17 siRNA did not alter stimulation-mediated decrease in surface GPIbα across 5 days in storage. Deletion of murine platelet Adam17 resulted in increased basal GpIbα in an inverse gene-dose-dependent manner, and protection from stimulation-mediated cleavage. Hemostasis was normal in platelet-specific Adam17-deleted mice. ADAM17 synthesis contributes to GPIbα cleavage in stored platelets, but extant siRNA-resistant ADAM17 is sufficient to cleave GPIbα upon platelet stimulation. An internal pool of GPIbα is exposed upon platelet stimulation but subject to rapid cleavage by ADAM17.