Thrombin-induced platelet microparticles improved the aggregability of cryopreserved platelets.
Platelets were activated with freezing/thawing and thrombin stimulation, and platelet microparticles generated following platelet activation were isolated with ultracentrifugation. The effects of platelet microparticles on platelet activation were studied with annexin V assay, protein tyrosine phosphorylation, and platelet aggregation. Freezing-induced platelet microparticles decreased but thrombin-induced platelet microparticles increased platelet annexin V binding and aggregation. Freshly washed platelets were cryopreserved using epinephrine and dimethyl sulfoxide (Me(2)SO) as combined cryoprotectants, and stimulated with thrombin-induced platelet microparticles. Following incubation of thrombin-induced platelet microparticles, the reaction time of platelets to agonists decreased but the percentages of aggregation increased, such as washed platelets from 44% +/- 30 to 92% +/- 7, p < 0.001, and cryopreserved platelets from 66% +/- 10 to 77% +/- 7, p < 0.02. By increasing platelet aggregability, platelet microparticles recovered after thrombin stimulation improved platelet function for transfusion. A 53-kDa platelet microparticle protein showed little phosphorylation if it was released from resting platelets or platelets stimulated with ADP, epinephrine, propyl gallate or dephosphorylation if it was derived from ionophore A 23187-stimulated platelets. However, the same protein released from frozen platelets showed significant tyrosine phosphorylation. Since a microparticle protein with 53 kDa was compatible with protein tyrosine phosphatase-1B (PTP-1B), its phosphorylation suggests the inhibition of enzyme activity. The microparticle proteins derived from thrombin-stimulated platelets were significantly phosphorylated at 64 kDa and pp60c-src, suggesting that the activation of tyrosine kinases represents a possible mechanism of thrombin-induced platelet microparticles to improve platelet aggregation.
Xiao
Hongyan
H
Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109-0602, USA. hxiao@umich.edu
Jepkorir
Carolyne J
CJ
Harvey
Kevin
K
Remick
Daniel G
DG
D000328:Adult; D000818:Animals; D017304:Annexin A5 / Q000097:blood; D001792:Blood Platelets / Q000187:drug effects; D001793:Blood Preservation / Q000379:methods*; D002417:Cattle; D015925:Cryopreservation / Q000379:methods*; D006801:Humans; D066298:In Vitro Techniques; D010766:Phosphorylation; D015539:Platelet Activation; D010974:Platelet Aggregation / Q000187:drug effects*; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000097:blood; D013917:Thrombin / Q000494:pharmacology*; D014443:Tyrosine / Q000097:blood
D017304:Annexin A5; D014443:Tyrosine; C516607:PTPN1 protein, human; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases; D013917:Thrombin