A signaling pathway contributing to platelet storage lesion development: targeting PI3-kinase-dependent Rap1 activation slows storage-induced platelet deterioration.
BACKGROUND
The term platelet storage lesion (PSL) describes the structural and biochemical changes in platelets (PLTs) during storage. These are typified by alterations of morphologic features and PLT metabolism leading to reduced functionality and hence reduced viability for transfusion. While the manifestations of the storage lesion are well characterized, the biochemical pathways involved in the initiation of this process are unknown.


METHODS
A complementary proteomic approach has recently been applied to analyze changes in the PLT proteome during storage. By employing stringent proteomic criteria, 12 proteins were identified as significantly and consistently changing in relative concentration over a 7-day storage period. Microscopy, Western blot analysis, flow cytometry, and PLT functionality analyses were used to unravel the involvement of a subset of these 12 proteins, which are connected through integrin signaling in one potential signaling pathway underlying storage lesion development.


RESULTS
Microscopic analysis revealed changes in localization of glycoprotein IIIa, Rap1, and talin during storage. Rap1 activation was observed to correlate with expression of the PLT activation marker CD62P. PLTs incubated for 7 days with the PI3-kinase inhibitor LY294002 showed diminished Rap1 activation as well as a moderate reduction in integrin alphaIIbbeta3 activation and release of alpha-granules. Furthermore, this inhibitor seemed to improve PLT integrity and quality during storage as several in vitro probes showed a deceleration of PLT activation.


CONCLUSIONS
These results provide the first evidence for a signaling pathway mediating PSL in which PI3-kinase-dependent Rap1 activation leads to integrin alphaIIbbeta3 activation and PLT degranulation.
Schubert
Peter
P
Canadian Blood Services, University of British Columbia, Vancouver, British Columbia, Canada.
Thon
Jonathan N
JN
Walsh
Geraldine M
GM
Chen
Cindy H I
CH
Moore
Edwin D
ED
Devine
Dana V
DV
Kast
Juergen
J
D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D001793:Blood Preservation / Q000009:adverse effects*; D015153:Blotting, Western; D002867:Chromones / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D008856:Microscopy, Fluorescence; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D015398:Signal Transduction / Q000187:drug effects; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism*
D002867:Chromones; D004791:Enzyme Inhibitors; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D020827:rap1 GTP-Binding Proteins; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one