(Received for publication, August 3, 1994; and in revised form, October 17, 1994)
From the
Activation of human platelets by the peptide YFLLRNP has been
shown to induce shape change but not secretion, Ca mobilization, or pleckstrin phosphorylation (Rasmussen, U. B.,
Gachet, C., Schlesinger, Y., Hanau, D., Ohlmann, P., Van
Obberghen-Schilling, E., Pouyssegur, J., Cazenave, J.-P., and Pavirani,
A.(1993) J. Biol. Chem. 268, 14322-14328). YFLLRNP was
added to washed human platelets that had been pretreated with EGTA at
37 °C or preincubated with the fibrinogen receptor antagonist RGDS
to preclude the activation of the integrin
(fibrinogen receptor). YFLLRNP
induced shape change and stimulated the tyrosine phosphorylation of
proteins of 62, 68, and 130 kDa within 7 s. Tyrosine phosphorylation of
these proteins reached maximum levels (2-3-fold) 15-30 s
after addition of YFLLRNP and decreased subsequently. The chelation of
intracellular Ca
by BAPTA-AM decreased basal tyrosine
protein phosphorylation but did not inhibit the increase of tyrosine
phosphorylation of P62, P68, and P130 or the shape change induced by
YFLLRNP. Preincubation of platelets with the tyrosine kinase inhibitors
genistein or tyrphostin A23 completely inhibited platelet shape change
and protein tyrosine phosphorylation induced by YFLLRNP. The inactive
structural analogs daidzein and tyrphostin A1 were barely inhibitory.
P62, P68, and P130, which exhibited increased tyrosine phosphorylation
upon stimulation with YFLLRNP, were found in the cytoskeleton. P130 was
not identical to vinculin or the focal adhesion kinase
pp125
. The results indicate that stimulation of
G-protein-coupled thrombin receptors rapidly induces protein tyrosine
kinase activation through a Ca
- and
integrin-independent mechanism. Protein tyrosine kinase activation and
tyrosine phosphorylation of novel protein substrates seem to play an
essential role in the induction of platelet shape change.
Platelet shape change, the earliest platelet response induced by
physiological agonists, is generally associated with phospholipase
C-stimulated phosphoinositide hydrolysis,
inositol-1,4,5-trisphosphate-mediated Ca mobilization
from intracellular stores(1, 2) ,
Ca
-stimulated myosin light chain (20 kDa)
phosphorylation, and protein kinase C-dependent pleckstrin (47 kDa)
phosphorylation. Whereas the Ca
-dependent myosin
light chain phosphorylation seems to precede shape change and might
play a role in triggering this early platelet response(3) , no
such function has been found for protein kinase C-dependent pleckstrin
phosphorylation(2) . However, physiological stimuli such as
thrombin and prostaglandin-endoperoxide analogs can induce shape change
without increasing cytosolic Ca
, and an increase of
cytosolic Ca
alone is not sufficient to induce shape
change and myosin light chain
phosphorylation(4, 5, 6) . Physiological
agonists apparently activate Ca
-independent pathways
that might synergize with Ca
to produce myosin light
chain phosphorylation and platelet shape change(6) .
Recently, a heptapeptide ligand of the cloned thrombin receptor
YFLLRNP has been found that induces shape change but not Ca mobilization or pleckstrin phosphorylation in human
platelets(7) . By using this partial thrombin receptor agonist,
we studied the function and regulation of protein tyrosine
phosphorylation during platelet shape change.
Samples (0.6-0.8 ml) of the platelet suspension were transferred into aggregometer cuvettes and incubated at 37 °C with stirring (1100 rpm). Following 5 min of preincubation, YFLLRNP was added. Inhibitors (RGDS, genistein, daidzein, and tyrphostin A1 and A23) were added 2-5 min before the stimulus as indicated in the figure legends. Shape change was measured by recording the light transmission in a LABOR aggregometer (Fresenius, Bad Homburg, FRG). For analysis of protein phosphorylation, aliquots (0.06 ml) were transferred to an equal volume of sample buffer(10) .
At various times after
addition of YFLLRNP, platelets were lysed by adding equal volumes of
ice-cold 2 Triton lysis buffer (pH 7.5) containing 2% Triton
X-100, 100 mM Tris
HCl, 10 mM EGTA, 10 mM EDTA, 2 mM sodium orthovanadate, 20 µM leupeptin, 2 mM phenylmethylsulfonyl fluoride, and 20
µM pepstatin A. Samples were vortexed for 2 s, kept on ice
for up to 60 min, and then centrifuged at 15,600
g for
15 min at 4 °C. The pellets (platelet cytoskeletons) were washed
once without resuspension in 1
Triton X-100 (1%) lysis buffer
and dissolved in sample buffer (10) . To obtain the membrane
skeletal fraction(12) , supernatants were further centrifuged
at 100,000
g for 2.5 h at 4 °C in a Beckman
ultracentrifuge (L5-50) with a 70.1 Ti rotor.
The addition of YFLLRNP (300 µM) rapidly stimulated tyrosine phosphorylation of three proteins with the molecular masses of 62, 68, and 130 kDa during platelet shape change (Fig. 1). Tyrosine phosphorylation of these proteins reached maximum levels (2-3-fold) 15-30 s after addition of YFLLRNP (Fig. 2). Initiation of shape change and the increase of tyrosine phosphorylation of P62, P68, and P130 showed a good temporal correlation. Protein tyrosine phosphorylation was reversible within 1 min, but platelet shape change persisted for up to 5 min (Fig. 3) indicating that protein tyrosine phosphorylation was not necessary to maintain this early platelet response. In platelets pretreated with RGDS, maximal protein tyrosine phosphorylation of P62, P68, and P130 and shape change upon stimulation with YFLLRNP were observed earlier than in platelets pretreated with BAPTA-AM/EGTA (data not shown). Interestingly, RGDS as well as BAPTA-AM/EGTA pretreatment affected the basal state of tyrosine phosphorylation of these proteins in resting platelets (Table 1). RGDS decreased the phosphorylation of P62 and P68 by 20% but seemed to increase the phosphorylation of P130. BAPTA-AM/EGTA pretreatment decreased the phosphorylation of P62 and P130 by 75% and decreased that of P68 by 25%. These changes were due to the loading of platelets with BAPTA-AM and not due to EGTA (data not shown). In BAPTA-AM/EGTA-pretreated platelets, YFLLRNP increased the phosphorylation of P130 more (up to 3.8-fold) than in RGDS-pretreated platelets (up to 1.7-fold). The experiments with BAPTA-AM/EGTA-preincubated platelets show that the levels of tyrosine phosphorylation in P62 and P130 after exposure to YFLLRNP were less than that in control platelets (in the absence of BAPTA-AM/EGTA). Therefore, it seems that the change of protein tyrosine phosphorylation induced by platelet exposure to YFLLRNP, but not the level of protein tyrosine phosphorylation, correlates with shape change.
Figure 1:
Stimulation of tyrosine phosphorylation
of P62, P68, and P130 during shape change induced by YFLLRNP and
inhibition by genistein and tyrphostin A23, but not by daidzein and
tyrphostin A1. Washed human platelets were pretreated with EGTA (A) or EGTA and BAPTA-AM (B) for 20 min at 37 °C.
Samples were transferred into aggregometer cuvettes and incubated at 37
°C while being stirred with MeSO (0.1%, control) or
daidzein (150 µM), or genistein (150 µM) for
5 min (A) or with tyrphostin A1 (500 µM) or
tyrphostin A23 (500 µM) for 2 min before addition of
YFLLRNP (300 µM) (B). Aliquots of the platelet
suspensions were removed before stimulation and at various times after
stimulation with YFLLRNP. Anti-phosphotyrosine immunoblots of two
experiments are
shown.
Figure 2:
Stimulation of tyrosine phosphorylation of
P62, P68, and P130 during platelet shape change induced by YFLLRNP and
effect of genistein. Suspensions of washed human platelets were
incubated with RGDS (2 mM) for 2 min at 37 °C and with
MeSO (0.1%) or genistein (150 µM) for a
further 2 min before addition of YFLLRNP (300 µM). Data
are mean ± S.D. of three experiments.
, P62 with YFLLRNP;
, P68 with YFLLRNP;
, P130 with YFLLRNP;
, P62 with
genistein and YFLLRNP;
, P68 with genistein and YFLLRNP;
, P130 with genistein and YFLLRNP.
Figure 3: Shape change induced by YFLLRNP is inhibited by genistein but not by daidzein. Suspensions of washed human platelets pretreated with EGTA were placed into aggregometer cuvettes and preincubated at 37 °C for 5 min while being stirred with genistein (150 µM) or daidzein (150 µM) before exposure to YFLLRNP (300 µM, arrow). The decrease in light transmission together with the disappearance of rapid oscillations is indicative of shape change. 1, YFLLRNP; 2, daidzein and YFLLRNP; 3, genistein and YFLLRNP.
Figure 4: The proteins P62, P68, and P130 that are tyrosine phosphorylated during shape change induced by YFLLRNP are associated with the cytoskeleton. Suspensions of washed human platelets pretreated with EGTA/BAPTA-AM were exposed to YFLLRNP (300 µM) for various times. Platelet cytoskeleton, membrane skeleton, and supernatant were isolated. An anti-phosphotyrosine immunoblot of one experiment representative of four is shown.
Figure 5:
P130 is neither vinculin nor
pp125. Proteins from resting platelets (C) or
from platelets activated by YFLLRNP (Y) for 30 s were blotted
and either probed with anti-phosphotyrosine (Anti-PY)
antibodies or antibodies against vinculin (Anti-Vinculin) or
pp125
(Anti-pp125
). ST, biotinylated molecular weight
standard.
Previously it was
observed that tyrosine phosphorylation of pp125 was
dependent on activation of either the
integrin by fibrinogen (13, 14) or the
integrin by collagen(17) .
In our studies, in which fibrinogen binding to and activation of the
integrin were prevented, tyrosine
phosphorylation of pp125
was not significantly increased
during platelet shape change elicited by YFLLRNP. However, when
platelets were stimulated to aggregate, tyrosine phosphorylation of
pp125
dramatically increased (data not shown).
The present study demonstrates that partial activation by
YFLLRNP of the G-protein-coupled cloned thrombin receptor rapidly
stimulates protein tyrosine phosphorylation in human platelets. The
early increase of protein tyrosine phosphorylation is independent of
cytosolic Ca increase and activation of the
integrin and also is not mediated
by the protein kinase C pathway, because no pleckstrin phosphorylation
has been observed during platelet shape change induced by
YFLLRNP(7) . Thus, the results are consistent with a pathway
leading from thrombin receptor activation to protein tyrosine kinases.
Previously in Swiss 3T3 cells, bombesin, vasopressin, and endothelin
have been shown to stimulate the rapid tyrosine phosphorylation of the
focal adhesion kinase pp125
and the cytoskeletal protein
paxillin(18, 19) . Interestingly and in agreement with
our results, increased tyrosine phosphorylation of these proteins was
found to occur through a pathway that was also independent of the
mobilization of Ca
from intracellular stores and
protein kinase C activation. However, the dependence of pp125
and paxillin phosphorylation on integrin activation was not
investigated in these studies. In platelets, stimulation of tyrosine
phosphorylation of pp125
is mediated by activation of
either the integrin
(fibrinogen
receptor) (13, 14) or the integrin
(collagen receptor)(17) .
Strikingly, we observed that P130, which was tyrosine phosphorylated
and present in the cytoskeleton during platelet shape change induced by
thrombin receptor activation, was not identical to pp125
.
P130 was also not identical to vinculin, which has been reported to
undergo Ca
-dependent tyrosine phosphorylation and to
associate with the cytoskeleton in platelets aggregated by
thrombin(15, 16) . Thus, our study presents evidence
for a novel pathway of G-protein-coupled thrombin receptors to protein
tyrosine kinases that phosphorylate specific, still unknown protein
substrates during shape change. Interestingly, thrombin receptor
activation has recently been shown to cause shape change of neural
cells (cell rounding) through a mechanism that was also independent on
the Ca
and protein kinase C pathways(20) .
Our results implicate a role of tyrosine kinases in the induction of
platelet shape change elicited by thrombin receptor activation. Two
structurally different tyrosine kinase inhibitors, genistein and
tyrphostin A23, inhibited platelet shape change, whereas the respective
inactive analogs had less (daidzein) or no (tyrphostin A1) inhibitory
activity. Also, iloprost and SIN-1 inhibited shape change and tyrosine
phosphorylation elicited by YFLLRNP, indicating that the pathway of
G-protein-coupled thrombin receptors to protein tyrosine kinases is
sensitive to inhibition by cAMP and cGMP. Because many studies have
shown a role of myosin light chain phosphorylation in the initiation of
platelet shape change ((3) ; for review see (2) ), it
will be interesting to study the relationship between tyrosine
phosphorylation of P62, P68, and P130 and myosin light chain
phosphorylation. A Ca-independent stimulation of
myosin light chain phosphorylation has been suggested in previous
studies(4, 5, 6) . Based on our results one
could speculate that the Ca
-independent activation of
protein tyrosine kinases is involved in the regulation of myosin light
chain phosphorylation during shape change. The presence of
tyrosine-phosphorylated P62, P68, and P130 in the cytoskeleton in our
study and the association of phosphorylated myosin with the platelet
cytoskeleton observed previously (21) raise the possibility
that these proteins regulate the cytoskeletal reorganization during
platelet shape change.
The identification of the protein tyrosine
kinases and tyrosine-phosphorylated proteins that are activated and
phosphorylated during platelet shape change is of obvious interest.
Possible candidates are pp60, the src-related protein
tyrosine kinases p61
, p58
, and
p62
, and the tyrosine kinase p72
, all of
which are present in platelets(22, 23, 24) .
Activation and translocation of pp60
to the cytoskeleton
has been reported in aggregated
platelets(25, 26, 27, 28) . However,
both processes were found to occur subsequent to protein kinase C
and/or integrin
activation. It is
therefore unlikely that pp60
is activated during shape
change. An interesting candidate is p72
, which apparently
is activated in thrombin-stimulated platelets through a
Ca
-independent mechanism(24) . It remains to
be seen, however, whether p72
activation occurs during
shape change and whether it is independent of protein kinase C and
integrin activation.