Glycoprotein Ibα clustering in platelet storage and function

Abstract

Platelets are anucleated, discoid-shaped cells that play an essential role in the formation of a hemostatic plug to prevent blood loss from injured vessels. Initial platelet arrest at the damaged arterial vessel wall is mediated through the interaction between the platelet receptor glycoprotein (GP) Ibα and von Willebrand factor (VWF), a plasma protein that binds to subendothelial-exposed collagen. Platelets screen the integrity of the vascular system and circulate for approximately 10 days. The bleeding tendency observed in thrombocytopenic patients emphasizes the importance of maintaining sufficient peripheral platelet counts. Several disorders are associated with thrombocytopenia, including sepsis, leukemia, hereditary and auto-immune diseases. Decreased platelet counts are also observed in patients undergoing chemotherapy, which increased the demand for platelet transfusions substantially since its introduction for cancer treatment. The increased need has challenged blood banks to optimize the storage conditions of platelet concentrates. Platelets are currently kept at room temperature, which limits their shelf life to seven days due to increased risk of bacterial infection. Cold storage may be a better alternative, but this introduces changes in GPIbα, leading to platelet apoptosis and early clearance in vivo. This thesis aims to increase our knowledge on GPIbα in relation to platelet storage and function. We have shown that cold storage triggers deglycosylation of GPIbα, leading to increased exposure of N-acetyl-D-glucosamine residues. Analysis of GPIbα surface distribution by Förster resonance energy transfer using time-gated fluorescence lifetime imaging microscopy revealed that the receptor translocates to lipid rafts upon cooling through carbohydrate-carbohydrate interactions between GPIbα and raft-associated gangliosides. Cold storage concurrently triggers the release and accumulation of arachidonic acid. Liberated arachidonic acid binds to and transfers 14-3-3ζ to the cytoplasmic tail of GPIbα, resulting in its clustering in lipid rafts. This leads to dissociation of [14-3-3ζ-Bad] complexes and activation of the apoptotic machinery. Interference with these cold-induced signaling events markedly improves the survival times of transfused cold-stored platelets, without affecting hemostatic functions. A similar mechanism regulates GPIbα clustering under physiologic conditions. Perfusion at a shear rate of 1,600 s-1 induces GPIbα clusters on platelets adhered to VWF, while clustering does not require VWF contact at 10,000 s-1. Shear-induced clustering is reversible, not accompanied by granule release or αIIbβ3 activation and improves GPIbα-dependent platelet interaction with VWF. This newly identified mechanism provides new insights in how changes in hemodynamics influence arterial thrombus formation. GPIbα is also a target for autoantibody formation in patients with immune thrombocytopenia (ITP). We have found that patient autoantibodies induce GPIbα clustering and surface expression of both P-selectin and phosphatidylserine. These clearance signals are generated by autoantibody-induced GPIbα translocation to lipid rafts, where the receptor clusters and associates with the low-affinity Fc-receptor FcγRIIa. Blocking of GPIbα translocation to lipid rafts or FcγRIIa neutralization prevents generation of destruction signals and may be explored therapeutically for the treatment of ITP caused by anti-GPIbα antibodies. The final chapter of this thesis evaluates these findings in relation to current views on the regulatory mechanisms of GPIbα function.