©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Platelet Shape Change Induced by Thrombin Receptor Activation
RAPID STIMULATION OF TYROSINE PHOSPHORYLATION OF NOVEL PROTEIN SUBSTRATES THROUGH AN INTEGRIN- AND Ca-INDEPENDENT MECHANISM (*)

(Received for publication, August 3, 1994; and in revised form, October 17, 1994)

Emil V. Negrescu (§) Karin Luber de Quintana Wolfgang Siess (¶)

From the Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Pettenkoferstrasse 9, 80336 München, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 alphabeta(3) (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.


INTRODUCTION

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.


EXPERIMENTAL PROCEDURES

Materials

The partial thrombin receptor agonist YFLLRNP was synthesized by Dr. Arnold (Max-Planck-Institut, Martinsried, FRG). The tetrapeptide RGDS acetylsalicylic acid, apyrase, sodium orthovanadate, phenylmethylsulfonyl fluoride, leupeptin, Triton X-100, phosphotyrosine, phosphothreonine, and phosphoserine were from Sigma. BAPTA-AM (^1)was from Molecular Probes Inc. (Eugene, OR). Tween 20 and the reagents for gel electrophoresis were obtained from Bio-Rad. The monoclonal antibodies PY20 and Z027 against phosphotyrosine were from Leinco (St. Louis, MO) and Zymed (San Francisco, CA), respectively. The monoclonal antibodies against pp125 and vinculin were from UBI (Lake Placid, NY) and Boehringer Mannheim, respectively. The chemiluminescence-based Western blot detection system ECL and horseradish peroxidase-conjugated secondary antibodies were from Amersham. Iloprost and SIN-1 were kind gifts from Schering (Berlin, FRG) and Cassella-Riedel (Frankfurt, FRG), respectively. Herbimycin, tyrphostin A1, and tyrphostin A23 were from Biomol (Hamburg, FRG). Genistein and daidzein were from Biomol and Calbiochem.

Platelet Isolation

Human platelets were treated with acetylsalicylic acid and isolated as described(8, 9) . Platelets were resuspended at a concentration of 0.6 times 10^9 cells/ml in prewarmed buffer (pH 7.4; 20 mM HEPES, 138 mM NaCl, 2.9 mM KCl, 1 mM MgCl(2), 0.36 mM NaH(2)PO(4), 0.6 ADPase units/ml apyrase). In some experiments, platelet suspensions were incubated with EGTA (2 mM) and BAPTA-AM (20 µM) for 20 min at 37 °C.

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) .

Isolation of Platelet Cytoskeleton, Membrane Skeleton, and Soluble Fraction

Triton-soluble and insoluble fractions were prepared by modification of a method described previously(11) . Platelet suspensions (0.6 ml) were incubated at 37 °C in Eppendorf cups using an Eppendorf Thermostat 5320.

At various times after addition of YFLLRNP, platelets were lysed by adding equal volumes of ice-cold 2 times Triton lysis buffer (pH 7.5) containing 2% Triton X-100, 100 mM TrisbulletHCl, 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 times g for 15 min at 4 °C. The pellets (platelet cytoskeletons) were washed once without resuspension in 1 times 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 times g for 2.5 h at 4 °C in a Beckman ultracentrifuge (L5-50) with a 70.1 Ti rotor.

Separation of Platelet Proteins and Immunoblotting

Proteins from platelet suspensions or platelet subfractions were separated by SDS-polyacrylamide (10%) gel electrophoresis on 1.5 mm times 20 cm gels (Bio-Rad). Proteins were blotted to nitrocellulose membranes and detected as described(9, 10) . The dilutions of the mixed primary phosphotyrosine antibodies PY20 and Z027 were both 1:2000; the dilutions of anti-pp125 and anti-vinculin antibodies were also 1:2000. The dilution of the secondary anti-mouse antibody was 1:15,000. The specificity of the anti-phosphotyrosine antibodies was tested by competition using either phosphotyrosine (1 mM) or phosphothreonine (1 mM).

Analysis of Results

Tyrosine-phosphorylated protein bands were measured by laser densitometry (Ultroscan XL, Pharmacia). Absorption of proteins in unstimulated control samples was set to 100%. Data are presented as mean ± S.D. of individual experiments from different blood donors.


RESULTS

Stimulation of Protein Tyrosine Phosphorylation during Shape Change Induced by YFLLRNP

Preliminary experiments have shown that YFLLRNP (300 µM) induced shape change and a weak reversible aggregation of washed platelets but no detectable secretion of ATP from dense granules. In order to allow shape change and prevent platelet aggregation, suspensions of washed human platelets were either pretreated with RGDS for 2 min, which inhibits fibrinogen binding to the integrin alphabeta(3), or preincubated with EGTA for 20 min at 37° C, which dissociates the integrin alphabeta(3). EGTA pretreatment was, in some experiments, combined with BAPTA-AM in order to chelate any increase of cytosolic Ca eventually elicited by YFLLRNP, although this agonist has been reported not to enhance cytosolic Ca in fura-2-loaded human platelets(7) .

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 Me(2)SO (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 Me(2)SO (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. bullet, P62 with YFLLRNP; , P68 with YFLLRNP; , P130 with YFLLRNP; circle, P62 with genistein and YFLLRNP; box, P68 with genistein and YFLLRNP; up triangle, 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.





Effect of the Protein Tyrosine Kinase Inhibitors Genistein and Tyrphostin A23 on Tyrosine Phosphorylation of P62, P68, and P130 and Platelet Shape Change Induced by YFLLRNP

Preincubation of platelets with genistein (150 µM) reduced the basal tyrosine phosphorylation and inhibited tyrosine phosphorylation of P62, P68, and P130 and platelet shape change induced by YFLLRNP (Fig. 1A, Fig. 2, and Fig. 3). The structural inactive analog of genistein, daidzein, barely inhibited shape change and tyrosine phosphorylation of P62, P68, and P130 (Fig. 1A and Fig. 3). Concentration-response curves of genistein showed an ID value of 90 ± 4 µM for the inhibition of shape change and ID values of 61 ± 15, 60 ± 11, and 84 ± 7 µM for inhibition of tyrosine phosphorylation stimulated by YFLLRNP of P62, P68, and P130, respectively (values are the means ± S.D., n = 3). A different protein tyrosine kinase inhibitor, tyrphostin A23 (500 µM), also inhibited platelet shape change and tyrosine phosphorylation of P62, P68, and P130 stimulated by YFLLRNP, whereas the structural inactive analog tyrphostin A1 did not show these inhibitory effects (Fig. 1B and data not shown).

Effect of Increased cAMP and cGMP Levels on Tyrosine Phosphorylation of P62, P68, and P130 Induced by YFLLRNP

Platelet incubation with the prostacyclin analog iloprost (2 nM), which increased platelet cAMP levels 5-7-fold (data not shown), and the NO donor SIN-1 (10 µM), which increased platelet cGMP levels 5-fold (data not shown), prevented platelet shape change, and inhibited tyrosine phosphorylation of P62, P68, and P130 induced by YFLLRNP (Table 2).



The Proteins That Are Tyrosine-phosphorylated upon Platelet Stimulation with YFLLRNP Are Present in the Cytoskeleton

Platelets stimulated by YFLLRNP to change their shape were fractionated into the cytoskeleton, membrane skeleton, and supernatant. As can be seen in Fig. 4, P62, P68, and P130 and three further proteins with molecular masses of 50-60 kDa, all of which exhibited increased tyrosine phosphorylation upon stimulation with YFLLRNP were present in the cytoskeleton. P68 was also present in the membrane skeleton, but no increase of tyrosine phosphorylation upon stimulation with YFLLRNP was observed.


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.



P130 Is Not Identical to Platelet Vinculin or pp125

Proteins that are tyrosine-phosphorylated in aggregated platelets are pp125 and vinculin(13, 14, 15) . Vinculin is also present in the cytoskeleton of aggregated platelets (16) . Studies were carried out to compare the electrophoretic mobility of tyrosine-phosphorylated P130 with the electrophoretic mobility of platelet vinculin and pp125, which were identified by specific antibodies on immunoblots. We found that P130 had a slower electrophoretic mobility than vinculin or pp125 on SDS-polyacrylamide gel electrophoresis (Fig. 5). We estimated that 1.7 ± 0.7% of total vinculin was present in the cytoskeleton of resting platelets. The content of vinculin in the cytoskeleton increased slightly (1.4 fold) during shape change induced by YFLLRNP (p < 0.05). Interestingly, immunoprecipitates of vinculin from control and YFLLRNP-stimulated platelets showed a small increase of phosphotyrosine on anti-phosphotyrosine immunoblots (data not shown). Therefore, a small fraction of platelet vinculin might be tyrosine-phosphorylated during platelet shape change induced by YFLLRNP, but vinculin is not identical to P130.


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 alphabeta(3) integrin by fibrinogen (13, 14) or the alpha(2)beta(1) integrin by collagen(17) . In our studies, in which fibrinogen binding to and activation of the alphabeta(3) 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).


DISCUSSION

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 alphabeta(3) 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 alphabeta(3) (fibrinogen receptor) (13, 14) or the integrin alpha(2)beta(1) (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 alphabeta(3) 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.


FOOTNOTES

*
This study was supported by grants from the Deutsche Forschungsgemeinschaft (Si 274) and the August-Lenz-Stiftung. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
On leave of absence from the Institutul de Cercetari in Medicina Preventiva si Clinica, Chisinau, Republic of Moldova. Supported by a research fellowship awarded by the Alexander von Humboldt Foundation in Bonn, Germany.

To whom correspondence should be addressed. Tel.: 49-89-5160-4380; Fax: 49-89-5160-4382.

(^1)
The abbreviations used are: BAPTA-AM, acetoxymethyl ester of 5,5`-dimethyl-bis-(o-aminophenoxy)ethane-N,N,N`,N`-tetraacetic acid; SIN-1, 3-morpholino-syndnonime.


ACKNOWLEDGEMENTS

We thank U. Wielert for expert technical assistance and F. Haag for the skillful photographic reproduction of the autoradiographs.


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