Improved platelet survival after cold storage by prevention of glycoprotein Ib  clustering in lipid rafts

Abstract

The online version of this article has a Supplementary Appendix. BackgroundStoring platelets for transfusion at room temperature increases the risk of microbial infection and decreases platelet functionality, leading to out-date discard rates of up to 20%. Cold storage may be a better alternative, but this treatment leads to rapid platelet clearance after transfusion, initiated by changes in glycoprotein Ibα, the receptor for von Willebrand factor. Design and MethodsWe examined the change in glycoprotein Ibα distribution using Förster resonance energy transfer by time-gated fluorescence lifetime imaging microscopy. ResultsCold storage induced deglycosylation of glycoprotein Ibα ectodomain, exposing N-acetyl-Dglucosamine residues, which sequestered with GM1 gangliosides in lipid rafts. Raft-associated glycoprotein Ibα formed clusters upon binding of 14-3-3ζ adaptor proteins to its cytoplasmic tail, a process accompanied by mitochondrial injury and phosphatidyl serine exposure. Cold storage left glycoprotein Ibα surface expression unchanged and although glycoprotein V decreased, the fall did not affect glycoprotein Ibα clustering. Prevention of glycoprotein Ibα clustering by blockade of deglycosylation and 14-3-3ζ translocation increased the survival of cold-stored platelets to above the levels of platelets stored at room temperature without compromising hemostatic functions. ConclusionsWe conclude that glycoprotein Ibα translocates to lipid rafts upon cold-induced deglycosylation and forms clusters by associating with 14-3-3ζ. Interference with these steps provides a means to enable cold storage of platelet concentrates in the near future.Key words: platelets, cold storage, glycoprotein Ibα, 14-3-3ζ, lipid raft, clustering. Ibα clustering in lipid rafts. Haematologica 2012;97(12):1873-1881. doi:10.3324/haematol.2012 This is an open-access paper. Citation: Gitz E, Koekman CA, van den Heuvel DJ, Deckmyn H, Akkerman JW, Gerritsen HC and Urbanus RT. Improved platelet survival after cold storage by prevention of glycoprotein Improved platelet survival after cold storage by prevention of glycoprotein Ibα clustering in lipid rafts ABSTRACT© F e r r a t a S t o r t i F o u n d a t i o n

Protein kinase A determines platelet life span and survival by regulating apoptosis

“…Moreover, PKA activator effectively prevented apoptotic events in platelets incubated with S. aureus isolated from sepsis patients ( Figure 8, D and E). Forskolin also significantly inhibited ΔΨ m depolariza- vation of p38 MAPK and p38 MAPK-mediated cytosolic phospholipase A2, resulting in release of arachidonic acid from membrane phospholipids, which transfers 14-3-3 to the cytoplasmic domain of GPIbα (48,49). In the meantime, p38 MAPK activates ADAM17, leading to GPIbα ectodomain shedding (47), which enhances GPIbα clustering and association of 14-3-3 with the cytoplasmic domain of GPIbα (50).…”

Platelet interaction with von Willebrand factor is enhanced by shear-induced clustering of glycoprotein Ib

“…20 In brief, 6B4-Fab fragments conjugated to either Alexa Fluor-488 or Alexa Fluor-594 (6B4-488 and 6B4-594, respectively) were incubated with fixed platelet samples under conditions in which each Fab labeled ~50% of the total number of receptors. GPIbα translocation to lipid rafts was determined by labeling GPIbα with 6B4-488 and monosialo-tetrahexosylganglioside (GM1) with Cholera toxin subunit B conjugated to Alexa Fluor-594 (CTB-594; 5 μg/mL).…”

“…18,19 During efforts to optimize the storage conditions of platelet concentrates used for transfusion, we recently demonstrated that GPIbα clusters in lipid rafts when platelets are kept at low temperature. 20 . Shear-induced clustering was reversible, not accompanied by granule release or αIIbβ3 activation and improved glycoprotein Ibα-dependent platelet interaction with von Willebrand factor.…”

Recent developments in ex vivo platelet production

“…Isolated platelets are commonly stored at room temperature to avoid the decrease in function that can arise at lower temperatures. Consequently, isolated platelets are viable for a maximum of 5-7 days (Gitz et al 2012), making routine platelet isolation is the only option for maintaining a constant supply of platelets.…”