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Algis Grybauskas, Paulius V Kuprys, Jordan Hauck, William M Norkett, Kelsey Green, Paul A Knepper; Improved method for characterizing platelet status in glaucoma. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2419.
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© ARVO (1962-2015); The Authors (2016-present)
Platelet functional status changes dramatically in transient ischemic attacks, Alzheimer’s disease, traumatic brain injuries and primary open-angle glaucoma (POAG), all of which have higher levels of super-activated platelets (SAPs) that are prothrombogenic. The expression of multiple platelet receptors, release of alpha granule contents, and change of cell surface proteins are hallmarks of changing platelet status. In the present study, we developed a new ‘direct’ method for classifying three separate platelet activation states (resting, activated, and SAPs) using flow cytometry.
5-10mL of whole blood was collected by venipuncture from 5 control subjects, then anticoagulated with acid citrate dextrose. This blood was centrifuged to isolate a platelet rich plasma layer. Platelets were treated with peptide gly-pro-arg-pro-NH2 (Sigma); to prevent platelet coagulation, SB-Fibrinogen (generous gift of George Dale), and with three different platelet agonist treatments: no agonist, thrombin (Sigma-Aldrich) only, or thrombin+convulxin (Santa Cruz). Activation states were analyzed on a Beckman Coulter Cyan ADP flow cytometer using fluorophore-conjugated antibodies. CD41-Phycoerythrin (Molecular Probes) was used as a platelet marker, PAC-1-fluorescein isothiocyanate (BD Biosciences) was used to identify activated platelets, and streptavidin allophycocyanin (BD Pharmigen) was used to identify SAPs. Unpaired, two-tailed t-tests were used to analyze data.
We conducted two separate analyses using an older 'indirect' method, and a newer 'direct' method to test differences between the two. Using indirect analysis, activated platelets comprised of 61.13%±13.01% and SAPs/resting platelets made up 38.59%±12.57% of platelets studied. Using the direct method, activated platelets made up 68.45%±4.00%, SAPs made up 18.91%±2.92%, and resting platelets made up 12.64%±2.30% of studied platelets. A significant difference in SAP percentage (p=0.022) between the indirect and direct methods was observed.
With the direct method, three activation states of platelets, particularly SAPs, can be clearly defined. The indirect method is insufficient in separating the resting and SAP platelet populations. The direct method leads to improved evaluation of the functional status of platelets, allowing for a better understanding of platelet activation states in glaucoma and other vascular diseases.
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