Abstract
Platelet signal transduction involves not only reversible phosphorylation of proteins
on both tyrosine and serine/threonine residues, but also mechanisms of cross-talk
to coordinate different pathways. We have, therefore, investigated the effect of okadaic
acid, a potent inhibitor of serine/threonine protein phosphatases type 1 and type
2A (PP1 and PP2A), to better understand the interplay that must exist between serine/threonine
and tyrosine phosphorylations during platelet activation.
Okadaic acid drastically inhibits thrombin-induced platelet aggregation, secretion,
and thromboxane synthesis. The inhibition is accompanied by a marked increase in the
phosphorylation of at least 5 proteins (230, 210, 74, 57, and 50 to 52 kDa). However,
protein kinase C activity is not modified because thrombinand phorbol-12-myristate-13-acetate-induced
phosphorylation of pleckstrin is still occurring, although slightly decreased.
Inhibition of platelet function and extent of the phosphorylation of the 5 substrates
in the presence of okadaic acid are concentration and time dependent, suggesting a
relation between the accumulation of one or more phosphoproteins and the inhibitory
effect of okadaic acid.
Okadaic acid inhibits thrombin-induced tyrosine phosphorylation in a concentration-dependent
manner. According to Brautigan and Pinault, the inhibition of protein phosphatases
in kidney cells resulted in the activation of a 55-kDa-tyrosine phosphatase and the
tyrosine phosphatase activity was synergistically increased when okadaic acid acted
in concert with prostaglandin I2 (PGI2). Interestingly, in agreement with these results, the okadaic acid-induced phosphosphorylation
of the 50-kDa substrate, which occurs without a cyclic adenosine monophosphate increase
in platelets, has the same molecular weight as the platelet membrane tyrosine phosphatase
isolated by Dawicki and Steiner.
Furthermore, we also found that thrombin-induced tyrosine phosphorylation was markedly
inhibited in the presence of low concentrations of both okadaic acid and PGI2, therefore explaining the synergistic inhibition of platelet aggregation and secretion.
The results greatly support the notion of a cross-talk between stimulation of serine/threonine
kinases (in response to inhibition of serine/threonine PP) and inhibition of tyrosine
phosphorylations and emphasize the role of the 50-kDa substrate in regulating platelet
activation.
Keywords:
Serine/threonine phosphatase - okadaic acid - tyrosine phosphorylation - platelet
aggregation - thrombin
