©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Differential Activation of Cytosolic Phospholipase A (cPLA) by Thrombin and Thrombin Receptor Agonist Peptide in Human Platelets
EVIDENCE FOR ACTIVATION OF cPLA INDEPENDENT OF THE MITOGEN-ACTIVATED PROTEIN KINASES ERK1/2 (*)

Ruth M. Kramer (§) , Edda F. Roberts , Paul A. Hyslop , Barbara G. Utterback , Kwan Y. Hui , Joseph A. Jakubowski

From the (1)From Lilly Research Laboratories, Indianapolis, Indiana 46285-0444

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The thrombin receptor agonist peptide SFLLRN was less effective than thrombin in eliciting the liberation of arachidonic acid and the generation of thromboxane A by human platelets. We found that while SFLLRN evokes an initial transient increase in cytosolic free calcium concentration ([Ca]) of similar magnitude as that caused by thrombin, the SFLLRN-induced elevation of [Ca]declines more rapidly to near resting levels than that evoked by thrombin, suggesting that disparate levels of [Ca] may contribute to the attenuated arachidonic acid release. Furthermore, we observed that SFLLRN is less effective than thrombin in mediating the ``activating'' phosphorylation of cytosolic phospholipase A (cPLA). Both thrombin and SFLLRN rapidly and transiently activated kinases that phosphorylate the 21-residue synthetic peptide Thr derived from the epidermal growth factor receptor, but the maximal activation of proline-directed kinases by SFLLRN was less pronounced than that by thrombin. MonoQ chromatography and immunoblot analysis of extracts from stimulated platelets revealed that while thrombin induced a prominent activation of the mitogen-activated protein kinases ERK1 and ERK2, SFLLRN completely failed to do so. On the other hand, SFLLRN, like thrombin, stimulated the activity of a proline-directed kinase distinct from ERK1/2, but the activation of this kinase was less pronounced following stimulation of platelets with SFLLRN compared with thrombin. We conclude 1) that the partial activation of cPLA and the subsequent attenuated mobilization of arachidonic acid in response to SFLLRN may be the consequence of a less prolonged elevation of [Ca] and insufficient activation of proline-directed kinase(s) by SFLLRN and 2) that the ability of SFLLRN to mediate the activating phosphorylation of cPLA in the absence of ERK1/2 stimulation suggests that, at least in human platelets, proline-directed kinases other than ERK1/2 may phosphorylate and activate cPLA.


INTRODUCTION

Thrombin, a serine protease, is a potent agonist for platelets, eliciting shape change, secretion of granular contents, and aggregation. Thrombin also evokes the release of arachidonic acid from platelet membrane phospholipids and induces the generation of prostaglandin endoperoxides, TXA,()and other metabolites of arachidonic acid(1) . These thrombin-induced platelet responses are mediated by cell-surface receptors that belong to the family of seven-transmembrane domain receptors coupling to G proteins, but that are activated by a novel mechanism(2) . Thus, receptor activation occurs when thrombin cleaves the extracellular amino-terminal portion of the thrombin receptor, exposing a new amino terminus that functions as a tethered peptide ligand for the receptor(2) . SFLLRN peptides, containing the first six residues of the new amino terminus, have been shown to mimic the effect of thrombin, causing platelets to secrete and aggregate(2, 3, 4) . Like thrombin, they activate phospholipase C, phosphatidylinositol 3-kinase, and protein kinase C(5, 6, 7) ; inhibit adenylate cyclase(6, 8) ; and induce phosphorylation on tyrosine residues of multiple platelet proteins(7, 9) . However, there is increasing evidence that thrombin receptor peptides are not full agonists for activation of platelets (10, 11) and other cells(12, 13, 14) .

We have shown recently that the Ca-sensitive cytosolic phospholipase A (cPLA) in human platelets is responsible for thrombin-stimulated mobilization of arachidonic acid for the synthesis of TXA. Incubation of platelets with thrombin induced the phosphorylation of cPLA, thereby increasing its intrinsic catalytic activity(15) . In the present study, we have examined the effect of the thrombin receptor agonist peptide SFLLRN on the phosphorylation and activation of cPLA. We found that SFLLRN does not match the substantial mobilization of arachidonic acid and TXA generation induced by thrombin and activates cPLA only partially compared with thrombin. We further discovered that SFLLRN is unable to evoke a sustained increase in [Ca] or to mediate activation of the MAP kinases ERK1 and ERK2. These results demonstrate that in human platelets, both [Ca] transients and activation of proline-directed kinases may play a critical role in thrombin-induced activation of cPLA.


EXPERIMENTAL PROCEDURES

Platelet Isolation and Incubation

Blood from healthy volunteers was drawn into 0.16 volume of acid/citrate/dextrose (85 mM trisodium citrate, 64 mM citric acid, and 111 mM glucose) containing a 10M concentration of a stable prostacyclin analog(15, 16) . Platelets were isolated as described previously (15) and suspended in 140 mM NaCl, 27 mM KCl, 1 mM MgCl, 5.5 mM glucose, 0.5 mM EGTA, 10 mM Hepes, pH 7.4. After the addition of CaCl and the glycoprotein IIb-IIIa antagonist cyclo-(S,S)-Mpr(Har)GDWPPen-NH (kindly provided by Dr. Robert Scarborough, COR Therapeutics) (17) to final concentrations of 3 mM and 30 µM, respectively, platelet suspensions (final concentration of 0.5 10/ml or as indicated) were incubated at 37 °C with -thrombin (3500 NIH units/mg; Enzyme Research Laboratories) or SFLLRN (C-terminal amide) synthesized as detailed before(3) . Incubations were terminated, and platelet sonicates were prepared as detailed previously(15) . Labeling of Platelet Phospholipids with [H]Arachidonic Acid and Agonist-induced Release of [H]Arachidonic Acid-Platelet-rich plasma (20 ml) was prepared from blood of donors who had taken 325 mg of aspirin the night before and again on the morning of phlebotomy and incubated for 1 h at 37 °C with 10 µCi of [5,6,8,9,11,12,14,15-H]arachidonic acid (60 Ci/mmol; DuPont NEN) added in 1 ml of 5 mM Tris/HCl, pH 8, bound to fatty acid-free bovine serum albumin (100 mg/ml). Free [H]arachidonic acid was removed by washing platelets as detailed above. The incorporation of [H]arachidonic acid into platelet phospholipids was 25%, and the content of esterified [H]arachidonic acid was 2 10 dpm/10 platelets. [H]Arachidonic acid-labeled platelets suspended at 0.5 10/ml in 140 mM NaCl, 27 mM KCl, 1 mM MgCl, 3 mM CaCl, 5.5 mM glucose, 0.5 mM EGTA, 10 mM Hepes, pH 7.4, were incubated with thrombin (5 units/ml) or SFLLRN (80 µM). Reactions were stopped by adding 2 ml of Dole's reagent (2-propanol, heptane, 0.5 M sulfuric acid (40:10:1)), and the released [H]arachidonic acid plus 12-lipoxygenase metabolites were extracted and quantified as described(18) .

Intracellular CaMobilization

[Ca] was measured using the fluorescent Ca indicator indo-1/AM (Molecular Probes, Inc.). Platelet-rich plasma was incubated for 45 min at room temperature with 3.3 µM indo-1/AM. The platelets were then pelleted and washed as detailed above; suspended at 0.5 10/ml in 140 mM NaCl, 27 mM KCl, 1 mM MgCl, 3 mM CaCl, 5.5 mM glucose, 0.5 mM EGTA, 10 mM Hepes, pH 7.4, containing 30 µM glycoprotein IIb-IIIa antagonist; and transferred to a glass cuvette, which was placed in a thermostatically controlled chamber at 37 °C in an SLM-AMINCO Model 48000 spectrofluorometer and stirred magnetically. After 2 min, thrombin or SFLLRN was added directly to the cuvette at the indicated concentrations. Indo-1 was excited at 355 nm, and emission was detected at 405 and 485 nm, the fluorescence maxima for Ca-bound and Ca-free indo-1, respectively. Changes of the fluorescence intensities at these two emission wavelengths were continuously monitored during stimulation. The platelet suspension was then centrifuged, and the fluorescence of the supernatants was assessed and subtracted from the suspension readings to correct for extracellular probe. The relative changes in the ratio of the fluorescence at 405 nm to that at 485 nm (i.e. ratio = 405 nm/485 nm) were used to quantitate the changes in [Ca] upon stimulation with thrombin or SFLLRN. [Ca] was calculated assuming a K of 250 nM for indo-1(19) .

MonoQ Chromatography

Soluble extracts were diluted 5-fold with 1 mM EGTA, 1 mM DTT, 100 µM NaVO, 50 mM -glycerophosphate, pH 7.2, and then applied to a MonoQ HR 5/5 column equilibrated in 25 mM NaCl, 1 mM EGTA, 1 mM DTT, 100 µM NaVO, 50 mM -glycerophosphate, pH 7.2, using a Pharmacia Biotech fast protein liquid chromatography system. After extensive washing, the column was eluted with a 40-ml linear salt gradient from 25 to 500 mM NaCl at a flow rate of 1.5 ml/min. Fractions of 1 ml were collected and assayed for kinase activity.

Assay for Proline-directed Kinases

Platelets were prepared and exposed to thrombin or SFLLRN as detailed above. The reaction was stopped by adding (final concentrations) 1% Triton X-100, 10 mM EGTA, 1 mM EDTA, 1 mM DTT, 0.2 mM NaVO, 1 µM microcystin, 100 µM leupeptin, 0.2 mg/ml aprotinin, 10 µM pepstatin A, 1 mM phenylmethylsulfonyl fluoride, 50 mM -glycerophosphate, pH 7.5. The suspension was centrifuged at 10,000 g for 15 min at 4 °C (for kinase assays in lysates) or at 100,000 g for 60 min at 4 °C (for MonoQ chromatography). Kinase assays were performed as described by Ahn et al.(20) with modifications as follows. The final assay volume was 25 µl and contained (final concentrations) 2 mM synthetic peptide Thr or Ala (KRELVEP/ALTPSGEAPNQALLR; Macromolecular Resources, Colorado State University), 0.3 mg/ml myelin basic protein (Sigma), 1.5 mg/ml human cPLA or S505A cPLA (modified by site-directed mutagenesis and produced and purified from a baculovirus/insect cell expression system as described previously (21)), and 8.33 µl of platelet extract (20 µg of protein) or MonoQ column fraction. Reactions were initiated by the addition of 8.33 µl of 3 ATP reaction buffer to provide (final concentrations) 0.1 mM NaVO, 1 mM DTT, 10 mM MgCl, 2 µM protein kinase A inhibitor peptide (Sigma), 1 mM EGTA, 50 mM -glycerophosphate, pH 7.5, 0.1 mM ATP (containing 40 µCi/ml [-P]ATP, 2000 Ci/mmol; DuPont NEN) and were allowed to proceed for 20 min at 30 °C. The reactions were stopped by the addition of 10 µl of 90% formic acid containing 50 mM ATP. Aliquots of 25 µl were applied to phosphocellulose membranes in SpinZyme separation units (Pierce). The membranes were washed four times with 0.5 ml of 1% phosphoric acid using centrifugation, retrieved from the unit, and transferred to scintillation vials containing 1 ml of 1% SDS. After the addition of 10 ml of Ready Protein (Beckman Instruments), the radioactivity was counted by liquid scintillation counting. Nonspecific incorporation of radioactivity was determined in the absence of peptide/protein substrate.

SDS-PAGE and Immunoblotting

After the addition of SDS sample buffer, platelet lysates were incubated at 60 °C for 15 min and electrophoresed at 4 °C on 10% Tris/glycine gels at 35 mA for 3.5 h (for cPLA detection) or at 30 mA for 2.5 h (for MAP kinase detection) using the Novex system. Standard proteins included rainbow markers (range of 14.3-200 kDa; Amersham Corp.), human cPLA, and MAP kinase ERK1 (Upstate Biotechnology, Inc.). After electroblotting, the nitrocellulose sheets were probed with mouse M3-1/horseradish peroxidase conjugate or rabbit anti-MAP kinase ERK1/2 antibodies (erk1-CT (Upstate Biotechnology, Inc.) and ERK 2 (C-14) (Santa Cruz Biotechnology)) and developed using the ECL detection system (Amersham Corp.) as detailed before(15) .

PLabeling, Immunoprecipitation, and Phosphoamino Acid Analysis

Human platelets from donors that had taken aspirin were labeled with carrier-free [P]HPO as described(15) . Platelets were incubated at 1 10/ml for 5 min at 37 °C without or with 5 units/ml thrombin as detailed above. The reaction was stopped by adding EDTA to a final concentration of 10 mM and by pelleting platelets for 1 min at 8500 g at 4 °C. The platelets were solubilized, and cPLA was immunoprecipitated with rabbit anti-cPLA IgG as described(15) . Immunoprecipitated cPLA (200 ng) derived from 4 10 platelets was subjected to SDS-PAGE (10% gel, 1.5 mm thick, 10 wells), electroblotted onto Immobilon-P (Millipore Corp.), and visualized by Coomassie Blue staining. The band containing the P-labeled cPLA was excised and subjected to acid hydrolysis with 5.7 N HCl for 1 h at 110 °C as described(22) . The samples were dried in a Speed Vac (Savant Instruments, Inc.); suspended in 5 µl of water containing 10 µg each of phosphoserine, phosphothreonine, and phosphotyrosine; and subjected to ascending thin-layer chromatography on cellulose plates (Merck) with butanol/acetic acid/ethanol/HO (1:1:1:1) as the mobile phase. The plates were dried, sprayed with ENHANCE (DuPont NEN), and exposed to x-ray film. The positions of standard phosphoamino acids were identified by staining with ninhydrin.

Assay of PLAActivity

PLA activity was assayed using sonicated liposomes containing 1-palmitoyl-2-[C]arachidonoyl-sn-glycero-3-phosphocholine (50 mCi/mmol; DuPont NEN) and sn-1,2-dioleoylglycerol (Avanti Polar Lipids, Inc.) at a molar ratio of 2:1 as described previously (23) with modifications as follows. The assay buffer consisted of 1 mM DTT, 150 mM NaCl, 50 mM Hepes, pH 7.5, containing 1 mg/ml bovine serum albumin, 1 mM CaCl, 2 µM [C]arachidonoylphosphatidylcholine (50,000 dpm), 1 µM dioleoylglycerol, and incubations were carried out at 37 °C for 15 min. The results are presented as disintegrations/minute [C]arachidonic acid released.

Other Methods

Protein measurements were made in the presence of 0.05% SDS using Coomassie Plus protein assay reagent (Pierce) with bovine serum albumin as a reference standard. The concentration of thromboxane B (reflecting TXA generation) was determined by radioimmunoassay as described previously(24) . Platelet aggregation and ATP release were assayed as described previously(25) .


RESULTS

Functional Responses of Platelets

In agreement with previous studies, we found that SFLLRN, like thrombin, induced platelet aggregation and ATP secretion(2, 3, 9) . We determined that the concentrations of thrombin and SFLLRN for half-maximal release of ATP were 0.33 ± 0.12 units/ml and 3.8 ± 1.7 µM (mean ± S.E. of three independent experiments using different donors), respectively. The concentrations causing half-maximal aggregation of platelets induced by thrombin and SFLLRN were 0.15 ± 0.05 units/ml and 2.6 ± 2.0 µM (mean ± S.E. of three independent experiments using different donors), respectively. In agreement with previous work, the molar concentration of peptide required to produce a response equivalent to that of thrombin was 1000 times that of thrombin (1 unit/ml thrombin taken to be 10 nM).

Release of Arachidonic Acid and TXAProduction

We observed that human platelets stimulated with the peptide SFLLRN produce less TXA than platelets stimulated with thrombin (Fig. 1). As shown with a dose dependence and time course study, the generation of TXA by platelets was maximal with 25 µM SFLLRN and stopped after 1 min of activation. In contrast, thrombin-induced TXA formation continued to increase up to 10 min. We next examined whether the lower amount of TXA formed in response to SFLLRN was the result of decreased mobilization of arachidonic acid. We prelabeled aspirinized platelets with [H]arachidonic acid and measured the release of radiolabeled arachidonic acid (including 12-lipoxygenase metabolites) from platelet phospholipids after stimulation with thrombin or SFLLRN. As shown in Fig. 2, the liberation of arachidonic acid was significantly lower when platelets were stimulated with SFLLRN compared with thrombin. These findings suggest that the enzyme responsible for thrombin-induced arachidonic acid release, cPLA, may not be fully activated by SFLLRN. Kinetics of Elevation of [Ca] by Thrombin and SFLLRN-Previous studies using cell-free systems and purified cPLA have demonstrated that Ca plays a critical role in the activation of cPLA as it promotes association of cPLA with its membrane phospholipid substrate(26, 27) . We therefore questioned whether dissimilar [Ca] responses may be the reason for the differential mobilization of arachidonic acid in platelets stimulated with SFLLRN and thrombin. SFLLRN peptides were reported to stimulate a transient increase in [Ca] similar to thrombin(2, 28, 29) . To verify that this was also true for human platelets, we loaded platelets with indo-1 and compared SFLLRN- and thrombin-induced changes in [Ca] by spectrofluorometry. As shown in Fig. 3, SFLLRN and thrombin elevated [Ca] from resting levels of 50 nM to 1 and 0.6 µM, respectively, with two apparent differences in the time course of the [Ca] transients. First, the maximal increase in [Ca] induced by SFLLRN was greater than and preceded that induced by thrombin. Second, the [Ca] transient evoked by SFLLRN declined more rapidly with time than that induced by thrombin. These results demonstrate that although SFLLRN reproduces the thrombin-induced initial rise of [Ca] to 600 nM, it is unable to maintain the prolonged elevation of [Ca] at 300 nM caused by thrombin. Hence, the differential release and metabolism of arachidonic acid observed with SFLLRN and thrombin could be a consequence of dissimilar [Ca] responses.


Figure 1: Thrombin- and SFLLRN-induced TXA production in human platelets. A, dose dependence. Platelets at 0.5 10/ml were incubated for 5 min at 37 °C with thrombin and SFLLRN as indicated. B, time course. Platelets were incubated at 37 °C with thrombin (5 units/ml) or SFLLRN (80 µM) for various times as indicated. The reaction was stopped by adding indomethacin to a final concentration of 10 µM; platelets were pelleted; and supernatants were assayed for thromboxane B (the stable metabolite of TXA) as described under ``Experimental Procedures.'' The data are representative of two different experiments, and values shown are the means ± range from two duplicate incubations.




Figure 2: Time course of thrombin- and SFLLRN-induced release of [H]arachidonic acid in platelets. Aspirinized platelets prelabeled with [H]arachidonic acid were suspended at 0.5 10/ml and incubated with thrombin (5 units/ml) or SFLLRN (80 µM) at 37 °C. The reaction was stopped, and released [H]arachidonic acid ([H]AA; including 12-lipoxygenase products) was determined as described under ``Experimental Procedures.'' The data are representative of two different experiments, and values shown are the means ± range of two separate incubations.




Figure 3: Effect of thrombin and SFLLRN on changes in [Ca] in human platelets. Indo-1-loaded human platelets (0.5 10/ml) were exposed to thrombin (2 units/ml) or SFLLRN (80 µM) for 5 min. The change in indo-1 fluorescence was continuously monitored at 37 °C in the fluorometer, and Ca transients were quantitated as described under ``Experimental Procedures.'' After 5 min, the [Ca] in thrombin- and SFLLRN-stimulated platelets in this experiment was 211 and 73 nM, respectively. The data are representative of three separate experiments with platelets obtained from different donors.



Phosphorylation and Activation of cPLA

Thrombin induces phosphorylation of platelet cPLA, improving its catalytic activity 2-3-fold(15) . Phosphoamino acid analysis of P-labeled cPLA immunoprecipitated from platelets revealed the presence of phosphoserine after thrombin stimulation (Fig. 4), indicating that a serine protein kinase may be responsible for the activating phosphorylation of cPLA in platelets. Thrombin-mediated phosphorylation converts cPLA to a form with decreased electrophoretic mobility, thereby providing a convenient means to monitor stimulus-induced phosphorylation and activation of cPLA (15, 30). As demonstrated with a dose dependence experiment (Fig. 5) and a time course study (Fig. 6), SFLLRN induced an incomplete gel shift and hence only partial activating phosphorylation of cPLA compared with thrombin (Fig. 5A and 6A). Accordingly, the SFLLRN-induced enhancement of cPLA activity in lysates derived from stimulated platelets was lower than that observed with thrombin (Fig. 5B and 6B). These results suggest that the liberation of arachidonic acid and the production of TXA in platelets may not be sustained in response to SFLLRN compared with thrombin due to incomplete phosphorylation and activation of cPLA.


Figure 4: Phosphoamino acid analysis of cPLA from control and thrombin-stimulated platelets. P-Labeled human platelets (10/ml) were incubated for 5 min without (-THR) or with (+THR) 5 units/ml thrombin as detailed under ``Experimental Procedures.'' cPLA was immunoprecipitated with anti-cPLA IgG, subjected to SDS-PAGE, and electroblotted onto Immobilon-P. The strips containing cPLA were subjected to acid hydrolysis and phosphoamino acid analysis by thin-layer chromatography on cellulose plates. Shown here is the fluorogram of the thin-layer plate on which phosphoamino acids were resolved by ascending chromatography as detailed under ``Experimental Procedures.'' The positions of the phosphoamino acids were determined by ninhydrin staining of standards added to each extract. P, free phosphate; P-Y, phosphotyrosine; P-T, phosphothreonine; P-S, phosphoserine.




Figure 5: Dose dependence of the effect of thrombin and SFLLRN on cPLA activity in lysates from human platelets. Platelets (0.5 10/ml) were incubated at 37 °C for 5 min in the presence of thrombin or SFLLRN as indicated and lysed as detailed under ``Experimental Procedures.'' A, SDS-PAGE/immunoblot probing with mouse M3-1/horseradish peroxidase conjugate. Solubilized lysates (20 µg of protein) from platelets stimulated with SFLLRN (lanes 1-5) or thrombin (lanes 6-10) were electrophoresed on a 10% gel. B, determination of cPLA activity in lysates (20 µg) from platelets stimulated with thrombin or SFLLRN. The data are representative of two different experiments; values shown for cPLA activity are the means ± range of two separate platelet incubations.




Figure 6: Time course of activation of cPLA in lysates from thrombin- and SFLLRN-stimulated platelets. Platelets (0.5 10/ml) were incubated at 37 °C in the presence of thrombin (1 unit/ml), SFLLRN (30 µM), or buffer for the indicated times and lysed as detailed under ``Experimental Procedures.'' A, SDS-PAGE/immunoblot probing with mouse M3-1/horseradish peroxidase conjugate. Solubilized lysates (20 µg of protein) from platelets incubated for 10 min with buffer (lane1) or stimulated for 1-10 min with SFLLRN (lanes2-5) or thrombin (lanes 6-9) were electrophoresed on a 10% gel. B, determination of cPLA activity in lysates (20 µg) from platelets stimulated with thrombin or SFLLRN. The data are representative of two different experiments; values shown for cPLA activity are the means ± range of two separate platelet incubations.



Activation of Proline-directed Kinases

Phosphorylation by MAP kinases is thought to be a prerequisite for activation of cPLA and subsequent mobilization of arachidonic acid in Chinese hamster ovary cells that overexpress cPLA(30) . We compared the kinetics of thrombin and SFLLRN-induced activation of these kinases using as substrate the 21-residue synthetic epidermal growth factor receptor peptide Thr known to contain the consensus sequence Pro-Leu-Thr-Pro for MAP kinases(31) . As shown in Fig. 7A, thrombin induced the activation of kinases that phosphorylate Thr in a time-dependent manner, reaching peak levels at 2 min and remaining significantly elevated after 10 min. In contrast, SFLLRN caused an early transient kinase activation within 1 min of stimulation that rapidly declined thereafter and remained only slightly elevated over basal levels. On average, the maximal activation of kinases that phosphorylate Thr by SFLLRN (after 1 min) was reduced significantly (53 ± 13%, mean ± S.D. of five independent experiments) compared with that achieved by thrombin (after 2 min). Immunoblot analysis using antibodies directed against ERK1 or ERK2 (Fig. 7B) revealed that after treatment of platelets with thrombin, two new immunoreactive bands of lower electrophoretic mobility became visible with the concomitant disappearance of the corresponding 42- and 44-kDa bands. This shift in electrophoretic mobility was most pronounced after 2-5 min. In contrast, no such mobility shift of the ERK1/2 bands indicative of activation (32) could be detected when platelets were stimulated with SFLLRN. Upon MonoQ chromatography of soluble platelet extracts, the Thr kinase activity eluted as two peaks after exposure of platelets to thrombin and as one peak after stimulation with SFLLRN (Fig. 8B). No kinase activity could be detected in MonoQ fractions from control platelets (data not shown). The first peak of kinase activity eluting with 150 mM NaCl (Peak I) was only observed after stimulation with thrombin and coincided with the elution of ERK1 and ERK2 as detected by immunoblotting (Fig. 8C). The second peak of Thr kinase activity eluting with 350 mM NaCl (Peak II) was apparent after both thrombin and SFLLRN treatment of platelets and comprised two-thirds and all of the Thr kinase activity induced by thrombin and SFLLRN, respectively. Preliminary characterization of the Peak II kinase activity is summarized in , showing that it readily phosphorylated myelin basic protein and cPLA. Furthermore, the kinase in Peak II, like ERK1/2, exhibited reduced activity with the synthetic peptide Ala, indicating that the amino-terminal proline is necessary for maximal kinase activity. More important, S505A cPLA was a poor substrate, suggesting that the Peak II kinase, like ERK1/2, targets Ser within the MAP kinase phosphorylation site of cPLA. In summary, the above findings demonstrate that the magnitude and time course of proline-directed kinase activation by SFLLRN are distinct from those observed with thrombin. Furthermore, they show that while SFLLRN fails to stimulate the MAP kinases ERK1/2, it activates, like thrombin, another proline-directed kinase that may phosphorylate cPLA at Ser.


Figure 7: Activation of proline-directed kinases in human platelets stimulated by thrombin and SFLLRN. A, time course of thrombin- and SFLLRN-stimulated kinase activities. Platelets (0.5 10/ml) were incubated at 37 °C with thrombin (5 units/ml), SFLLRN (80 µM), or buffer and solubilized with 1% Triton X-100, and kinase activities were determined using the Thr (EGFR) peptide substrate as detailed under ``Experimental Procedures.'' B, immunoblot analysis of platelet lysates, probing with anti-ERK1/2 antibodies (erk1-CT, anti-ERK1; or ERK 2 (C-14), anti-ERK2). Platelets (0.5 10/ml) were incubated at 37 °C with buffer (lanes1 and 2) or were stimulated with thrombin (5 units/ml; lanes 3-8) or SFLLRN (80 µM; lanes 9-14) for 0-10 min. Cell lysates (2 µg) were analyzed by SDS-PAGE/immunoblotting as detailed under ``Experimental Procedures.'' Agonist-induced phosphorylation and activation of the MAP kinases ERK1/2 are reflected by an upward mobility shift on SDS-PAGE. The data are representative of three independent experiments yielding similar results, and values shown are the means ± range of duplicate determinations.




Figure 8: MonoQ chromatography of thrombin- and SFLLRN-stimulated Thr kinase activity. Soluble extracts derived from 2 10 platelets stimulated at 1 10/ml with thrombin (5 units/ml) for 2 min or with SFLLRN (80 µM) for 1 min were subjected to chromatography on MonoQ as detailed under ``Experimental Procedures.'' The column was developed with a 40-ml linear gradient of 25-500 mM NaCl, and 1-ml fractions were collected at a flow rate of 1.5 ml/min. A, elution of proteins monitored by absorbance at 280 nm. B, determination of kinase activity in 8.3-µl aliquots of fractions as indicated using the synthetic peptide substrate Thr (EGFR). The data shown are the means ± range of two separate assays. Incorporation of radioactive phosphate was <250 dpm for flow-through and high-salt (0.5-1 M NaCl) fractions. Likewise, assays using MonoQ fractions from control platelets or assays performed in the absence of the Thr peptide yielded <250 dpm P radioactivity. C, 25-µl aliquots of fractions containing kinase activity (fractions 12-17 and 26-30) as well as 10 µg of control platelet lysate (laneC) and 20 ng of purified MAP kinase (laneErk1) were electrophoresed on 10% gels and immunoblotted with anti-ERK1/2 antibodies (erk1-CT). The migration position of the molecular mass marker ovalbumin is indicated on the right. The data are representative of three independent experiments.




DISCUSSION

Our study shows that in human platelets, the thrombin receptor agonist peptide SFLLRN causes only partial phosphorylation and activation of cPLA and is less effective than thrombin in promoting the release of arachidonic acid and production of TXA. We present evidence to indicate that this differential activation may be attributed to differences in [Ca] signaling and attenuated activation of proline-directed kinases by SFLLRN compared with thrombin. Our results further demonstrate that in SFLLRN-stimulated platelets, the activating phosphorylation of cPLA is mediated by a proline-directed kinase distinct from the MAP kinases ERK1/2, suggesting that phosphorylation and activation of cPLA may occur independent of ERK1/2.

The structure determination of the thrombin receptor by cloning has provided a new insight into the mechanism by which this receptor is activated(2) . The peptide SFLLRN was found to be an agonist for the thrombin receptor and to mimic many cellular effects of thrombin(2, 3, 4, 28, 29) . However, in human platelets, SFLLRN fails to match the production of TXA induced by thrombin, as also reported by others (10, 11) while this present work was in progress. Our studies indicate that this can be attributed to the inability of SFLLRN to sustain the activation of cPLA, the enzyme responsible for supplying free arachidonic acid for the synthesis of TXA in human platelets. It is of interest to note that SFLLRN peptides failed to reproduce other characteristic cellular effects of thrombin. Thus, in endothelial cells, thrombin receptor agonist peptides were unable to induce expression of the intracellular adhesion molecule ICAM-1(12) , to increase the mRNA levels of thrombomodulin(33) , and to promote secretion of platelet-derived growth factor(34) . The extent to which thrombin receptor agonist peptides fail to mediate mitogenesis in hamster fibroblast cells remains controversial(35, 36) .

As previously demonstrated, increasing [Ca]from levels of resting cells (80 nM) to levels typically found in stimulated cells (300 nM) is necessary to promote association of cPLA with its membrane phospholipid substrate via the CaLB domain(27) . In oocytes expressing functional human thrombin receptor(2) , HEL cells(28) , and endothelial cells(13, 29) , SFLLRN peptides promoted a transient increase in [Ca] that was similar to the [Ca] response elicited by thrombin. It is noteworthy that in endothelial cells, thrombin and SFLLRN peptides achieve equivalent formation of lysophosphatidylcholine and/or production of prostacyclin, suggesting a comparable activation of PLA(29, 37, 38) . On the other hand, it was reported that in endothelial cells, thrombin receptor agonist peptides failed to induce the translocation of protein kinase C from the cytosol to the plasma membrane, indicative of protein kinase C activation(13) . We observed that in human platelets, the decrease of elevated [Ca] was accelerated with SFLLRN compared with thrombin. Thus, the SFLLRN-induced increase in [Ca] declined to near resting levels within 1-2 min of stimulation, while the rise of [Ca] after thrombin treatment remained elevated around 300 nM for at least 3 min. As the prolonged elevation of [Ca] after the addition of thrombin in platelets was found to be due to continuing influx of external Ca(39) , it appears that SFLLRN does not mediate the later phase of external Ca entry in stimulated platelets. In this regard, the SFLLRN-induced [Ca] transient resembles the one induced by thrombin in the absence of external Ca. It is well known that thrombin-induced mobilization of arachidonic acid in human platelets is diminished in the absence of extracellular Ca(40) . Given our previous observation that the activating phosphorylation of cPLA in human platelets occurs independent of external Ca(15) , we conclude that the discrepancy in the late Ca signaling induced by SFLLRN compared with thrombin is not likely to be responsible for the differential activation of proline-directed kinases, but may compromise the association of cPLA with its membrane phospholipid substrate in the later phase of platelet activation.

Studies on the involvement of MAP kinases in functional properties of platelets, a terminally differentiated and highly specialized cell, have only recently been initiated. In agreement with our studies, Nozawa and co-workers (41) observed that thrombin stimulates the MAP kinases ERK1/2 in human platelets with optimal activation occurring after 2 min. On the other hand, Papkoff et al.(42) reported that in human platelets, thrombin activates the MAP kinase ERK2 but not ERK1 since they could not detect either enzymatic activity or a shift in the electrophoretic mobility of ERK1. Clearly, in the present study, thrombin induced the activation of both ERK1 and ERK2 as new bands with altered reduced mobilities appeared with the concomitant disappearance of the faster migrating forms. Conversely, SFLLRN failed to activate the MAP kinases ERK1 and ERK2 as demonstrated by gel shift analysis (Fig. 7B) and assay of Thr kinase activity after MonoQ chromatography (Fig. 8B).

The MAP kinases ERK1/2 are currently thought to be responsible for the activating phosphorylation of cPLA in stimulated cells. First, it was shown that purified ERK2 phosphorylates cPLAin vitro, increasing its activity and altering its electrophoretic mobility, thereby mimicking the agonist-induced activating phosphorylation of cPLA observed in many cell systems(30, 43) . Second, phosphorylation by the MAP kinase ERK2 in vitro was identical to the phorbol ester-stimulated phosphorylation of cPLA overexpressed in Chinese hamster ovary cells occurring at the consensus phosphorylation site for MAP kinase (Pro-Leu-Ser-Pro). Third, studies with macrophages stimulated with physiological agents demonstrated that the activation of ERK1/2 correlated closely with the activation of cPLA cells, suggesting that ERK1/2 may not only be responsible for the activation of overexpressed, but also endogenous cPLA(44, 45) . On the other hand, phosphorylation and activation of cPLA can take place independent of the MAP kinases ERK1/2. First, in macrophages treated with ionophore A23187, the activity of cPLA in lysates was increased despite the fact that A23187 was unable to promote stimulation of ERK1/2(44) . Second, it was recently reported that heparin suppresses endothelin-induced stimulation of the MAP kinases ERK1/2 in glomerular mesangial cells without affecting the activation of cPLA (46). Third, we show here that in SFLLRN-stimulated platelets, cPLA exhibits an electrophoretic mobility shift indicative of the activating phosphorylation despite the fact that both ERK1 and ERK2 are inactive. However, SFLLRN, like thrombin, activates another proline-directed kinase (Peak II in Fig. 8B) that phosphorylates cPLA. The maximal activation of this kinase by SFLLRN is approximately half of that induced by thrombin and thus correlates with the half-maximal activation of cPLA by SFLLRN compared with thrombin.

In summary, the differential activation of cPLA by thrombin and the thrombin receptor agonist peptide SFLLRN appears to be the result of a difference in the prolonged elevation of [Ca] that may affect the association of cPLA with its membrane phospholipid substrate and a distinction in the stimulation of proline-directed kinases that may activate cPLA by phosphorylation. SFLLRN fails to stimulate the MAP kinases ERK1/2, but induces, at least partly, the activating phosphorylation of cPLA, suggesting that a proline-directed kinase distinct from ERK1/2 may activate cPLA by phosphorylation. Hence, the thrombin receptor agonist peptide provides a useful tool to dissect and elucidate the biochemical events associated with thrombin-induced activation of cPLA. It will be of great interest to further define the signaling pathway(s) connecting the thrombin receptor to cPLA and to identify the proline-directed kinase(s) responsible for cPLA activation in human platelets.

  
Table: Comparison of the substrate specificity of thrombin-activated proline-directed kinases resolved by MonoQ chromatography

Aliquots (8.3 µl; derived from 2 10 platelets) of MonoQ fractions from thrombin-stimulated platelets (as in Fig. 8) comprising Peaks I and II (eluting with 150 and 350 mM NaCl, respectively) were incubated with substrates as detailed under ``Experimental Procedures.'' Incorporation of radioactive phosphate into various substrates was normalized with respect to phosphorylation of the Thr peptide (7000 and 12,000 dpm for Peak I and II fractions, respectively). The values represent the average of two separate MonoQ chromatographies, each assayed in duplicate. Identical results were obtained with Peak II fractions from SFLLRN-stimulated platelets. In control assays performed in the absence of substrate, the P radioactivity recovered on phosphocellulose membranes was <2%.



FOOTNOTES

*
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.

§
To whom correspondence should be addressed: Lilly Research Laboratories, Cardiovascular Research, Indianapolis, IN 46285-0444. Tel.: 317-276-1264; Fax: 317-276-1417; E-mail: kramer_ruth_m@lilly.com.

The abbreviations used are: TXA, thromboxane A; cPLA, cytosolic phospholipase A; [Ca], cytosolic free calcium concentration; MAP, mitogen-activated protein; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We gratefully acknowledge the synthesis of SFLLRN by Eddie Angleton and thank Larry Froelich, Chi Lin, and Bob Shuman for contributions to this work. Our special thanks go to Beth Strifler, Todd Pickard, and John Sharp for providing purified cPLA and S505A cPLA and to Natalie Ahn for helpful advice on the MAP kinase assay.


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