Regulatory role of protein tyrosine phosphorylation in platelet activating factor-induced signal transduction in platelets.
OBJECTIVE
To study the role of protein tyrosine phosphorylation (PTP) in platelet activating factor (PAF)-induced platelet signal transduction cascade.


METHODS
Washed rabbit platelets were used to test the inhibitory effect of genistein (Gen) on platelet aggregation and serotonin secretion. Intracellular Ca2+ ([Ca2+]i) and pH (pHi) were measured by a dual wavelength fluorophotometer with Fura 2-AM and BCECF-AM. PTP was determined with a specific anti-phosphotyrosine monoclonal antibody by Western blotting.


RESULTS
Pretreatment with Gen (100 and 200 mumol.L-1) inhibited PAF (20 nmol.L-1)-stimulated platelet serotonin release by 23.7% +/- 2.0% and 41% +/- 8%, respectively. Similar inhibitory effects of Gen were observed on PAF-evoked increase of [Ca2+]i and intracellular alkalization. PAF also elicited a pronounced increase in PTP of several bands with M(r) 70,000, 60,000, 50,000, 42,000/40,000, and 34,000, which were suppressed markedly by Gen 200 and 400 mumol.L-1. Pretreatment with staurosporine (Sta) 20 nmol.L-1, BAPTA 200 mumol.L-1, and egtazic acid 2 mmol.L-1 to inhibit PKC activation, [Ca2+]i elevation, and Ca2+ influx respectively, also showed an inhibitory effects on the formation of PTP.


CONCLUSIONS
PTP is involved in multiple signal transduction pathways induced by PAF, on which PKC activation and calcium mobilization play a regulatory role.
Zeng
S
S
Laboratory of Molecular Pharmacology, Hu-nan Medical University, Changsha, China.
Yi
F X
FX
Guo
Z G
ZG
D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D002118:Calcium / Q000097:blood; D019833:Genistein / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D010972:Platelet Activating Factor / Q000494:pharmacology*; D010974:Platelet Aggregation / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000378:metabolism / Q000502:physiology*; D011817:Rabbits; D012701:Serotonin / Q000097:blood; D015398:Signal Transduction
D010972:Platelet Activating Factor; D012701:Serotonin; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D002118:Calcium