Glycoprotein Ibalpha signalling in platelet apoptosis and clearance

Storage of platelets at low temperature reduces bacterial growth and might better preserve the haemostatic function of platelets than current procedures. Incubation at 0C is known to expose ?-N-acetyl-D-glucosamine-residues on glycoprotein (GP)Ibalpha inducing receptor-clustering and platelet destruction by macrophages. Here we show that incubation at 0/37C (4 hrs at 0C, followed by 1 hr at 37C to mimic cold-storage and post-transfusion conditions) triggers a conformational change in the N-terminal flank (NTF, amino acids, aa 1-35) but not in aa 36-282 of GPIbalpha? as detected by antibody-binding. Addition of the sugar N-acetyl-D-glucosamine (GN) inhibits responses induced by 0/37C. Incubation at 0C shifts GPIbalpha from the membrane skeleton to the cytoskeleton. Arrest of NTF-change by GN interferes with agglutination and spreading on a VWF-coated surface under flow. Strikingly, incubation at 0/37C initiates thromboxane A2-formation through a VWF-independent and GPIbalpha-dependent mechanism, as confirmed in VWF- and GPIbalpha-deficient platelets. We conclude that the NTF-change induced by 0/37C-incubation reflects clustering of GPIbalpha? supports VWF/ristocetin-induced agglutination and spreading and is sufficient to initiate platelet activation in the absence of VWF. GPIbalpha is associated with 14-3-3 proteins, which contribute to GPIbalpha-signaling and in nucleated cells take part in apoptosis regulation. We investigated whether GPIbalpha-clustering induces platelet apoptosis through 14-3-3 proteins during cold-rewarming. During cold-rewarming, 14-3-3 proteins associate with GPIbalpha and dissociate from Bad inducing Bad-dephosphorylation. This initiates pro-apoptosis Bax-translocation to the mitochondria inducing cytochrome c release. The result is activation of caspase-9 which triggers phosphatidylserine-exposure and platelet phagocytosis. Responses are prevented by GN and by O-sialoglycoprotein endopeptidase, which removes extracellular GPIbalpha. Cold-rewarming triggers apoptosis through a GN-sensitive GPIbalpha-change indicative for receptor-clustering. We investigated whether during cold-incubation arachidonic acid (AA) contributes to GPIbalpha-induced apoptosis. The cold-induced change in mitochondrial potential was reduced by p38MAPK-blockade, increased by COX-1 inhibition and unaffected by thromboxane-receptor blockade. Cold-incubation reduced [14-3-3zeta-Bad] and [14-3-3zeta-COX-1], as did AA-addition. AA-depletion made platelets resistant against cold-induced apoptosis and improved platelet survival. Indomethacin released 14-3-3zeta from the [14-3-3zeta-COX-1] complex and enhanced AA-induced apoptosis. Cold-incubation triggers the release of AA which becomes a “sink” for the 14-3-3zeta-protein, inducing Bad-activation and apoptosis. Attempts to improve platelet transfusion by cold-storage should focus on transient AA-depletion. We report a 70-year-old patient with a bleeding time of >30 minutes. Her platelets aggregated spontaneously during collection in citrate, EDTA and heparin and peripheral blood smears showed micro-aggregates. Blood-collection in a citrate/prostacyclin-mixture suppressed aggregation. FACS-analysis of patient platelets in the range of single control-platelets showed increase in activated GPIIb-IIIa, bound fibrinogen and in P-selectin expression compared with controls. Also, change in mitochondrial inner membrane potential and surface-expressed phosphatidylserine were increased as was binding/phagocytosis by macrophages. In contrast, binding of the anti-GPIbalpha-antibody AN51 was lower than in controls. Reconstitution of normal platelets in patient plasma induced the same responses. These responses were inhibited by prior cleavage of GPIbalpha and by interference with GN. We conclude that the patient had developed an auto-antibody against GPIbalpha, which initiates haemostasis and apoptosis-responses in platelets, possibly through clustering of GPIbalpha