©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Anti-CD9 Monoclonal Antibody Activates p72 in Human Platelets (*)

Yukio Ozaki (1)(§), Kaneo Satoh (1), Kenji Kuroda (1), Ruomei Qi (1), Yutaka Yatomi (1), Shigeru Yanagi (2), Kiyonao Sada (2), Hirohei Yamamura (2), Mutsumasa Yanabu (3), Shosaku Nomura (3), Shoji Kume (1)

From the (1)Department of Clinical and Laboratory Medicine, Yamanashi Medical University, Shimokato 1110, Tamaho, Nakakoma, Yamanashi 409-38, the (2)Second Department of Biochemistry, Fukui Medical College, 23-3 Matuoka, Yoshida, Fukui, and the (3)First Department of Internal Medicine, Kansai Medical University, 1 Fumizono, Moriguchi, Osaka, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

NNKY 1-19, anti-CD9 monoclonal antibody (MoAb), induced protein tyrosine phosphorylation of 125-, 97-, 75-, 64-, and 40-kDa proteins in human platelets, whereas F(ab`) fragments of NNKY 1-19 did not, suggesting that the stimulation of FcII receptors is required for the induction of protein tyrosine phosphorylation. Tyrosine-phosphorylated proteins of 97 and 125 kDa were associated with aggregation, while NNKY 1-19-induced protein tyrosine phosphorylation was completely inhibited by prostaglandin I (PGI). The activity of p72 was assessed in immunoprecipitation kinase assays to determine at which step the signal transduction pathway leading to protein tyrosine phosphorylation was suspended. NNKY 1-19 induced a rapid and transient increase in the p72-associated tyrosine kinase activity that peaked at 10 s and subsided to the original level 2 min after stimulation. Coinciding with this time course, p60 transiently associated with p72. In platelets preexposed to GRGDS peptides or PGI, NNKY 1-19 also increased the p72-associated tyrosine kinase activity and led to the association of p60 with p72. However, in contrast to the control without any inhibitor, the elevated tyrosine kinase activity and the associated state of the two tyrosine kinases persisted as long as 5 min after stimulation. F(ab`) fragments of NNKY 1-19 induced changes similar to those observed with the effects of GRGDS peptides or PGI treatment on intact IgG NNKY 1-19 stimulation. F(ab`) fragments of another CD9 MoAb, PMA2, had effects on p72essentially similar to those of NNKY 1-19. These findings suggest that the binding of anti-CD9 MoAb to CD9 on the platelet membrane per se induces an increase in the p72-associated tyrosine kinase activity but that FcII receptor-mediated signal(s) is required for the full activation of platelets and the appearance of tyrosine-phosphorylated proteins. The elevated intracellular cAMP level induced by PGI acts at a step distal to the activation of p72 and inhibited the signal transduction pathway leading to protein tyrosine phosphorylation and aggregation. p72activation occurs in the absence of aggregation, but aggregation appears to reduce the elevated p72 activity induced by anti-CD9 MoAb.


INTRODUCTION

A number of antibodies directed against antigens on the platelet membrane activate platelets (Horsewood et al., 1991). Whereas the antigens involved are diverse, the known antibodies with stimulatory properties are predominantly against CD9, a 24-kDa cell surface glycoprotein (Higashihara et al., 1985; Rendu et al., 1987; Jennings et al., 1990; Carroll et al., 1990), which is confirmed by the fact that most of the monoclonal antibodies (MoAbs)()that induced rapid aggregation were directed against CD9, as presented at the platelet workshop of the IV Leucocyte Typing Conference in Vienna (Powling et al., 1989). Anti-CD9 MoAb is a potent activator of platelet function comparable to thrombin and induces the entire range of functions, including aggregation, granule secretion, protein phosphorylation, phosphoinositide hydrolysis with Ca mobilization, and arachidonic acid metabolism. These findings suggested that CD9 is unique among platelet membrane glycoproteins and that it is a signal transducer. Molecular cloning of CD9 revealed that it is a multiply inserted membrane protein containing four putative transmembrane domains, suggesting that it could indeed be involved in signal transduction pathways (Boucheix et al., 1991) Various attempts have been made to clarify the mechanism by which anti-CD9 MoAbs activate platelets. Phospholipase A activation plays an important role but is not a prerequisite (Ozaki et al., 1991). CD9 physically associates with GPIIb/IIIa in the course of anti-CD9 MoAb-induced platelet activation (Slupsky et al., 1989). These studies were based upon the concept that the effects of anti-CD9 MoAbs are exerted through CD9. However, this concept was challenged by recent findings that F(ab`) fragments of anti-CD9 MoAbs lack the ability of platelet activation and that the blocking of the FcII receptor by corresponding monoclonal antibodies inhibited aggregation and Ca mobilization induced by anti-CD9 MoAbs (Slupsky et al., 1989; Worthington et al., 1990). Moreover, stimulation of the FcII receptor by cross-linkage or by aggregated IgG presented a picture of platelet activation similar to that of MoAbs to CD9 (Henson and Spiegelberg, 1973; Anderson and Anderson, 1990). These findings imply that the signal transduction pathway is mediated entirely through the FcII receptor, independent of CD9.

There is an increasing body of evidence for an important role of protein tyrosine phosphorylation facilitated by tyrosine kinases in the regulation of cell functions, especially those related to cell growth and oncogenesis. While platelets lack the ability to proliferate, they possess a high level of tyrosine kinase activity. All platelet tyrosine kinases reported to date are non-receptor types, p60 being the most abundant (Rendu et al., 1989). In addition to p60, several tyrosine kinases including p59, p62, p54/58, p72, and p125 have been identified (Huang et al., 1991; Lipfert et al., 1992). Upon platelet activation induced by various stimulators, a set of proteins undergo tyrosine phosphorylation (Nakamura and Yamamura, 1989; Salari et al., 1990). Although it is not yet clear which tyrosine kinase is responsible for a particular tyrosine-phosphorylated protein, several kinases change their activities upon platelet stimulation. The activity of p72 is rapidly increased by 10-fold upon thrombin activation, reaching a maximum at 10 s (Taniguchi et al., 1993). Platelet activation also elevates the p60 activity, albeit to a lesser degree and with a slower time course (Wong et al., 1992). The activity of p125 appears to be modified even later by fibrinogen binding to glycoprotein IIb/IIIa on the platelet membrane (Lipfert et al., 1992). These lines of evidence suggest that protein tyrosine phosphorylation and tyrosine kinases are actively engaged in the regulation of platelet functions from the initial phase of activation to the late stage of aggregation.

Recently, we found that anti-CD9 MoAb induces protein tyrosine phosphorylation and that there is an increased level of 3`-phosphorylated polyphosphoinositides, the production of which is physiologically related to tyrosine kinase activity (Yatomi et al., 1993). In this study, we investigated the changes in p72 activity induced by anti-CD9 MoAb with reference to protein tyrosine phosphorylation.


EXPERIMENTAL PROCEDURES

Materials and Chemicals

MoAb against p72, p59, or p54/58 was obtained from Wako Chemicals (Tokyo). MoAbs against p60(GD11) and phosphotyrosine (4G10) were obtained from Upstate Biotechnology. Acetoxymethyl ester of fura 2 were from Dojin Chemicals (Kumamoto, Japan). GRGDS peptides were obtained from Peptide Institute (Osaka, Japan). EGTA, enolase, prostaglandin I (PGI), leupeptin, phenylmethylsulfonyl fluoride, and sodium orthovanadate were purchased from Sigma. Protein A-Sepharose and CNBr-activated Sepharose were from Pharmacia Japan (Tokyo). Hepes buffer containing 138 mM NaCl, 2.8 mM KCl, 0.8 mM KHPO, 0.8 mM MgCl, 10 mM Hepes (pH 7.2), and 5.5 mM glucose was sterilized by filtration and stored at 4 °C until use. NNKY 1-19, anti-CD9 MoAb, was raised by immunizing mice with human platelets (Nagata et al., 1990). Another anti-CD9 MoAb, PMA2, was a generous gift from Dr. Takaaki Hato (Ehime University, Japan) (Hato et al., 1990). F(ab`) fragments of NNKY 1-19 or PMA2 were prepared by pepsin digestion (Lamoyi and Nisonoff, 1983). Briefly, NNKY 1-19 was dialyzed overnight at 4 °C against 100 mM acetate buffer, pH 7.0. Pepsin (3% w/w) was added, and the mixture was incubated in acetate buffer, pH 4.2, for 12 h at 37 °C. The solution was fractionated on a Sephadex G150 column to obtain F(ab`) fragments, and the purity of the F(ab`) was confirmed by SDS-PAGE.

Platelet Separation

Citrated, anti-coagulated venous blood was obtained from healthy human donors who had not received any medication for a minimum of two weeks prior to the experiment. The blood was centrifuged at 160 g for 15 min to obtain platelet-rich plasma. Platelets were isolated by differential centrifugation as described (Golden and Brugge, 1989) and finally resuspended at a concentration of 10 cells/ml in Hepes buffer containing 1 mM Ca unless otherwise stated.

Preparation of Fura 2-loaded Platelets and Measurement of [Ca]

To platelet-rich plasma obtained as described above, fura 2-AM at a final concentration of 3 µM was added, and the mixture was incubated at 37 °C for 30 min. The platelets were then washed twice, and resuspended at a concentration of 1 10 cells/µl. Fura 2 fluorescence was measured with a Hitachi F-2000 fluorescence spectrophotometer, with an excitation wavelength alternating every 0.5 s from 340 to 380 nm, and the emission wavelength was set at 510 nm. The [Ca] values were determined from the ratio of fura 2 fluorescence intensity at 340 and 380 nm excitation (Grynkiewicz et al., 1985).

Immunoprecipitation Kinase Assay

The platelets were activated with anti-CD9 MoAb or its F(ab`) fragments with constant stirring unless otherwise stated. After the indicated periods of time, the reaction was terminated with an equal volume of ice-cold lysis buffer (2% Triton X-100, 100 mM Tris/HCl, pH 7.5, 50 mM NaCl, 5 mM EDTA, 2 mM vanadate, 1 mM phenylmethylsulfonyl fluoride, and 100 µg/ml leupeptin). The lysate was sonicated and separated by centrifugation at 16,000 g for 5 min. The supernatant was precleared with Sepharose beads twice and then mixed with anti-p72 antibody bound to protein A-Sepharose or CNBr-activated Sepharose. The mixture was rotated for 2 h at 4 °C. The Sepharose beads were washed three times with lysis buffer. The sample was then split into two portions. One was used for immunoblotting, described elsewhere, and the other was processed further for in vitro kinase assay. In vitro kinase assay was performed essentially as described (Clark and Brugge, 1993). The beads were washed once with low salt buffer (100 mM NaCl, 5 mM MnCl, 10 mM Tris, pH 7.4) incubated with 25 µl of kinase reaction buffer (20 mM Tris, pH 7.5, 10 mM MnCl) with or without 10 µg of acid-treated enolase. The reaction was initiated by the addition of 10 µCi of [-P]ATP and 2 µM ATP. After 10 min at 20 °C, the reaction was stopped by the addition of Laemmli buffer and then subjected to boiling for 3 min. The proteins were separated under reducing conditions by 8 or 12% SDS-PAGE and electrically transferred onto Clear Blot Membrane P (Atto, Tokyo). The membrane was treated with 1 M KOH for 60 min, dried, and quantified with a BAS-2000 Phosphorimager (Fuji Film, Japan).

Protein Analysis by Immunoblotting

Laemmli sample buffer was added to platelets activated with anti-CD9 MoAb for the indicated periods, and then the mixture was boiled for 3 min. In some experiments, proteins separated for immunoprecipitation kinase assay were similarly processed. Platelet proteins were separated by 8% SDS-PAGE and electroblotted onto Clear Blot Membrane P (Atto, Tokyo). The immunoblots were incubated with 1 µg/ml MoAb directed to phosphotyrosine or p60 for 3 h. Antibody binding was detected using peroxidase-conjugated goat anti-mouse IgG (Cappel, PA) and visualized with ECL detection reagents (Amersham, UK).


RESULTS

PTP Induced by NNKY 1-19, Anti-CD9 MoAb

In the presence of 1 mM extracellular Ca and without aspirin, NNKY 1-19 at concentrations as low as 1 µg/ml induced platelet aggregation, [Ca] elevation, and serotonin release (Nagata et al., 1990). The optimal concentration of NNKY 1-19 varied among individuals tested but usually fell in the range of 3 to 10 µg/ml. In experiments thereafter, NNKY 1-19 was used at a concentration of 10 µg/ml. Upon NNKY 1-19 stimulation, a set of tyrosine-phosphorylated proteins appeared with different profiles in the time course (Fig. 1A). A 75-kDa band appeared as early as 30 s after stimulation and tended to disappear after several minutes. The appearance of 97- and 125-kDa bands was a late event and probably corresponded to those alleged to be related to aggregation in thrombin-induced platelet activation (Golden et al., 1990), since these bands of PTP did not appear when platelets were treated with GRGDS (data not shown). F(ab`) fragments of NNKY 1-19 up to a concentration of 30 µg/ml had no effects on platelets in terms of the appearance of PTP (Fig. 1B), aggregation, or [Ca] elevation, confirming that stimulation of FcII receptors is required for the full picture of platelet activation (Worthington et al., 1990).


Figure 1: Protein tyrosine phosphorylation induced by anti-CD9 MoAb. Platelets were suspended in Hepes buffer containing 1 mM Ca. Platelets were activated either with 10 µg/ml of intact IgG NNKY 1-19, an anti-CD9 MoAb, or with 10 µg/ml F(ab`) fragments of NNKY 1-19 with constant stirring for the indicated periods, and reactions were terminated with Laemmli sample buffer. Platelet proteins were applied to SDS-PAGE, and tyrosine-phosphorylated proteins were detected by Western blotting using 4G10, anti-phosphotyrosine MoAb. A, intact IgG of NNKY 1-19; B, F(ab`) fragments.



Effects of Various Inhibitors on PTP Induced by NNKY 1-19

We previously found that extracellular Ca and the production of thromboxane A greatly facilitate platelet activation induced by a MoAb to CD9 (Ozaki et al., 1991). Thus, the effects of chelating extracellular Ca with EGTA and of aspirin were evaluated on NNKY 1-19-induced [Ca] elevation and PTP. Platelets in platelet-rich plasma were treated with 0.5 mM aspirin for 30 min and then washed and resuspended in Hepes buffer containing 200 µM EGTA and no Ca. Chelation of extracellular Ca and aspirin pretreatment markedly reduced [Ca] elevation induced by NNKY 1-19 (0.3 ± 0.2 versus 4.8 ± 1.1 in terms of Fura 2 fluorescence ratio). The appearance of 75-kDa PTP band was delayed, but in contrast to the control sample with extracellular Ca and no aspirin, the intensity of PTP persisted for up to 3 min (data not shown). The 97- and 125-kDa bands were barely, if at all, detectable.

We then evaluated the effect of PGI on NNKY 1-19-induced PTP. PGI raises the intracellular cAMP level, which attenuates PTP induced by thrombin (Pumiglia et al., 1990). Incubating platelets with 0.4 µM PGI for 5 min completely abrogated the appearance of PTP, aggregation, and [Ca] elevation induced by NNKY 1-19 (data not shown).

NNKY 1-19 Stimulation Induces a Transient Rise in p72-associated Tyrosine Kinase Activity with Concomitant Association of p60

NNKY 1-19 stimulation increased the level of p72 autophosphorylation, which peaked 10-60 s after stimulation and subsided to lower level thereafter (Fig. 2, upperpanel). Densitometry revealed a 1.5-3-fold increase, which was substantially lower than that for thrombin activation (Taniguchi et al., 1993). The autophosphorylated amino acid of p72 was exclusively tyrosine (Ohta et al., 1992 and data not shown). Concomitant with the change in autophosphorylation, in vitro kinase assays revealed that the tyrosine kinase activity for exogenous substrates was increased transiently along with the faint band of a 60-kDa tyrosine-phosphorylated protein (Fig. 2, lowerpanel). Western blotting using anti-p60 MoAb revealed that the 60-kDa band was p60 (Fig. 3), suggesting that p60 transiently associates with p72 upon NNKY 1-19-induced platelet activation. Unlike anti-p72, anti-p59 MoAb or anti-p54/58MoAb did not coprecipitate p60, suggesting that p60 specifically associates with p72 during platelet activation induced by anti-CD9 MoAb (data not shown).


Figure 2: p72-associated tyrosine kinase activity induced by anti-CD9 MoAb. Platelets suspended in a buffer containing 1 mM Ca were activated with 10 µg/ml NNKY 1-19 for the indicated periods. The reaction was terminated with lysis buffer, and p72 was isolated by immunoprecipitation with anti-p72 MoAb. Immunoprecipitates were either directly subjected to Western blotting using anti-phosphotyrosine MoAb (upper), or to in vitro kinase assay using enolase as exogenous substrate (lower). Arrowheads represent the bands presumably derived from IgG.




Figure 3: Association of p60 with p72 induced by anti-CD9 MoAb stimulation. Platelets were activated with NNKY 1-19, and p72-associated proteins were isolated by immunoprecipitation with anti- p72 MoAb. The sample was applied to SDS-PAGE, and Western blotting was performed using anti-p60 MoAb.



F(ab`)Fragment of NNKY 1-19 Induces a Persistent Increase in the p72-associated Tyrosine Kinase Activity and the Persistent Association of p60 with p72

The F(ab`) fragment of NNKY 1-19 at a concentration of 10 µg/ml also increased the level of p72 autophosphorylation and the association of tyrosine-phosphorylated 60-kDa protein with p72. However, in contrast to an early decay in the p72 activity and the early dissociation of the 60-kDa band from p72, the F(ab`)-induced process was persistent up to 5 min after stimulation (Fig. 4). At this time, the autophosphorylated level of p72 and the associated 60-kDa PTP was often greater than the maximum level attained by intact IgG of NNKY 1-19. Western blotting with anti-p60MoAb revealed the persistent p60association with p72 (data not shown). That the preparation of F(ab`) fragments was not contaminated with intact antibody was confirmed by SDS-PAGE under non-reducing conditions (data not shown). Furthermore, IV.3, an anti-FcII receptor MoAb, was used to block Fc receptor activation induced by residual, if any, intact antibody. The absence of inhibitory effects of IV.3 on the increased tyrosine kinase activity induced by F(ab`) fragments of NNKY1-19 suggests that F(ab`) fragments of NNKY1-19 are capable of activating p72 (Fig. 5). To reinforce the notion that F(ab`) fragments of anti-CD9 MoAb induce p72 activation in platelets, the effects of another CD9 MoAb, PMA2 (Hato et al., 1990), were evaluated on the changes in p72-associated tyrosine kinase activity and the association between p72 and p60. Fig. 6shows that the effects of intact IgG PMA2 or the F(ab`) fragments of PMA2 were essentially similar to those of NNKY 1-19. A transient increase in the autophosphorylated level of p72 and the association between p72 and p60 was observed with intact antibody, and these processes were persistent with the F(ab`) fragments.


Figure 4: The p72-associated tyrosine kinase activity induced by F(ab`) fragments of anti-CD9 MoAb compared with that by intact IgG. Platelets suspended in a buffer containing 1 mM Ca were activated with 10 µg/ml intact IgG or F(ab`) fragments of NNKY 1-19 for the indicated periods. The reaction was terminated with lysis buffer, and p72 was isolated by immunoprecipitation with anti-p72 MoAb. In vitro kinase assay was performed on the isolated sample as described under ``Experimental Procedures.'' A, intact IgG; B, F(ab`) fragments.




Figure 5: Effects of IV.3, anti-FcII receptor MoAb, on p72-associated tyrosine kinase activity induced by anti-CD9 MoAb F(ab`) fragments. Platelets were first incubated with 10 µg/ml IV.3 for 3 min, and the changes in p72-associated tyrosine kinase assay was assessed (A) at the indicated point of time. F(ab`) fragments, 10 µg/ml of NNKY1-19, were then added, and the changes in tyrosine kinase activity were evaluated for another 3 min (B).




Figure 6: p72-associated tyrosine kinase activity and p60 association with p72 induced by intact antibody or F(ab`) fragments of PMA2, another anti-CD9 MoAb. Platelets suspended in a buffer containing 1 mM Ca were activated with 10 µg/ml intact antibody or F(ab`) fragments of PMA2, an anti-CD9 MoAb for the indicated periods. The reaction was terminated with lysis buffer, and p72 was isolated by immunoprecipitation with anti-p72 MoAb. Immunoprecipitates were either directly subjected to Western blotting using anti-phosphotyrosine MoAb or to in vitro kinase assay using enolase as exogenous substrate. Arrowheads represent the bands presumably derived from IgG. A and C, in vitro kinase activity; B and D, Western blotting using anti-p60 MoAb. A and B, the changes induced by intact antibody; C and D, the changes induced by F(ab`) fragments of PMA2.



Effects of Various Inhibitors on p72-associated Tyrosine Kinase Activity Induced by NNKY 1-19

Aspirin slightly prolonged the elevated level of tyrosine kinase activity induced by NNKY 1-19. The overall tyrosine kinase activity associated with p72 was still detectable at 60 s after stimulation albeit to a lesser degree than that at 10 s (Fig. 7A). Western blotting using anti-p60 MoAb showed that the association of p60 with p72 was also slightly extended (Fig. 7B). However, the general profile of changes in p72-associated tyrosine kinase including a transient increase in p72autophosphorylation and the transient association of p60 with p72, followed by a marked decrease in kinase activity and the complete dissociation of p60, was essentially similar to that of the control sample without aspirin.


Figure 7: The effects of aspirin pretreatment on p72-associated tyrosine kinase activity and association between p72 and p60 induced by anti-CD9 MoAb. Platelet-rich plasma was incubated with or without 0.5 mM aspirin for 30 min. Platelets were then washed and resuspended in a buffer containing 200 µM EGTA. Platelets were activated with 10 µg/ml NNKY 1-19 for the indicated periods, and the reaction was terminated with lysis buffer. p72 was isolated by immunoprecipitation with anti-p72 MoAb. The sample was analyzed either by in vitro kinase assay or to SDS-PAGE followed by Western blotting using anti-p60 MoAb. A, in vitro kinase assay; B, detection of p60 with Western blotting. Arrowheads represent the band presumably derived from IgG.



We then asked whether aggregation modified the changes in p72 activation induced by anti-CD9 MoAb. Chelation of extracellular Ca with 2 mM EGTA and 200 µM GRGDS peptide to inhibit platelet aggregation did not suppress p72 activation in terms of its autophosphorylation and the in vitro kinase activity (Fig. 8, A and B). However, a decrease in the tyrosine kinase activity, which appears 3-5 min after stimulation, was not observed in the absence of aggregation.


Figure 8: Effects of GRGDS peptides on p72 autophosphorylation and p72-associated tyrosine kinase activity induced by anti-CD9 MoAb. Extracellular Ca was chelated with 2 mM EGTA, and 200 µM GRGDS peptides were added to inhibit platelet aggregation. Platelets were activated with 10 µg/ml NNKY1-19 for the indicated periods. The reaction was terminated with lysis buffer, and p72 was isolated by immunoprecipitation with anti-p72 MoAb. Immunoprecipitates were either directly subjected to Western blotting using anti-phosphotyrosine MoAb or to in vitro kinase assay using enolase as exogenous substrate. Arrowheads represent the bands presumably derived from IgG. A, Western blotting using anti-phosphotyrosine MoAb; B, in vitro kinase assay.



When platelets were incubated with 0.4 µM PGI for 5 min, the profile of changes in the p72-associated tyrosine kinase activity was similar to that observed without aggregation. The elevated level of tyrosine kinase activity persisted even at 5 min after activation, and the association of p60 with p72 as assessed by Western blotting showed no phase of dissociation (Figs. 9 and 10).


DISCUSSION

Previous studies have suggested that the platelet activation induced by anti-CD9 MoAb is largely dependent upon FcII receptor stimulation. F(ab`) fragments that lack the ability to stimulate the FcII receptor did not induce [Ca] elevation or release of intracellular granule contents (Worthington et al., 1990). PTP induced by anti-CD9 MoAb was inhibited by preincubation of the platelets with anti-FcRII MoAb (Huang et al., 1992). Based upon the fact that direct FcII receptor stimulation, such as that caused by the cross-linkage of the receptors, is sufficient to induce platelet activation, it has been doubted whether the binding of anti-CD9 MoAb to CD9 molecules on the platelet membrane by itself elicits any signal that leads to platelet activation. It could only serve to prompt the binding of the Fc portion of MoAb to the FcII receptor. This study confirmed, using NNKY1-19, an anti-CD9 MoAb (Nagata et al., 1990), the previous findings that the binding of MoAb to CD9 in the absence of FcII receptor stimulation did not induce protein tyrosine phosphorylation or platelet aggregation but also showed that F(ab`) fragments of NNKY1-19 per se elicited intracellular signals in terms of p72activation and the association of p60 with p72. The effect of residual intact antibody was ruled out by the use of IV.3, anti-FcII receptor MoAb. Furthermore, that the F(ab`) fragments of another anti-CD9 MoAb, PMA2 (Hato et al., 1990), are capable of activating p72 reinforces the notion that antibody binding to CD9 alone can elicit a signal(s) leading to p72 activation. Griffith et al.(1991) have proposed that CD9 molecules induce an activation signal in platelets, based upon their study with immobilized anti-CD9 MoAb F(ab`) fragments. The association between CD9 and glycoprotein IIb/IIIa in platelets (Slupsky et al., 1989) and that with CD9 and the diphtheria toxin receptor in Vero cells (Mitamura et al., 1992) implies that CD9 molecules exert a regulatory function for various cell surface receptors. Our findings may provide a biochemical basis for the proximal signal that CD9 molecules generate within platelets. Several non-receptor tyrosine kinases have been found to associate with cell surface molecules (Samelson et al., 1990; Hatakeyama et al., 1991). A more recent finding provides evidence for the close association between B-cell antigen receptor and p72 (Yamada et al., 1993). In platelets, p59, p62, and p54/58 associate with CD36 (Huang et al., 1991). We have found that p72 specifically associates with p60 during platelet activation induced by CD9 MoAb. By analogy to other cell types, it is tempting to postulate that CD9 or some other related membrane protein generates a signal(s) to recruit p72 and p60. Whether CD9 actually localizes these tyrosine kinases awaits further investigation.

An increase in the p72-associated tyrosine kinase activity along with association between p72 and p60 induced by F(ab`) fragments, however, does not suffice to induce protein tyrosine phosphorylation or other parameters of platelet activation. These findings suggest that an additional signal(s) mediated by FcII receptor occupancy is required for the full picture of platelet activation. PGI pretreatment, which elevates the intracellular cAMP content, completely inhibited the appearance of PTP and aggregation induced by intact IgG of anti-CD9 MoAb, whereas p72 activation and association between p72 and p60 was preserved. The reduction in p72-associated tyrosine kinase activity and the apparent dissociation of p60 from p72, which occur 1-3 min after activation with intact IgG of anti-CD9 MoAb, was suppressed by PGI treatment. Thus, PGI reverted the overall profile of intact IgG anti-CD9 MoAb-induced activation to one similar to that with F(ab`) fragments. Taken together, these findings suggest that cAMP acts at a step distal to the activation of p72 and association between p72 and p60 and that intracellular cAMP accumulation facilitated by PGI must act at a particular step of the signal transduction pathway that links FcII receptor activation to PTP and the dissociation of p72 and p60.

With intact IgG of anti-CD9 MoAb, which induces a full picture of platelet activation, an increase in the p72-associated tyrosine kinase activity and association between p72 and p60 are transient, lasting 30-60 s at most. F(ab`) fragments lacking the Fc portion led to the persistent activation of p72 and association between the two tyrosine kinases. These findings suggest that FcII receptor stimulation induces the apparent dissociation between p72and p60 and the reduction in p72-associated tyrosine kinase activity. Calcium influx or the thromboxane pathway do not play a decisive role in these processes, although they may support them to some extent; the exclusion of either or both of these pathways did not suppress p72 activation or the association between P72 and p60but slightly suspended the dissociation and the decay of p72-associated tyrosine kinase activity. On the other hand, aggregation appears to play a key role on the apparent dissociation between p72 and p60and the reduction in p72-associated tyrosine kinase activity. A number of kinases including p60and substrates colocalize to the cytoskeleton in an aggregation-dependent manner (Grondin et al., 1991; Clark and Brugge, 1993) and therein transmit signals to the target proteins. p72 also translocates to the cytoskeletal fractions upon platelet activation (Yanagi et al., 1994). It is plausible that the associated form of p60and p72 translocates to the cytoskeletal fraction, thus reducing the recovery of p60 associated with p72, although, to date, we are not able to demonstrate that the associated form of p72 and p60colocalizes with the cytoskeleton (data not shown).

Alternately, the process of aggregation may deactivate p72and terminate the association between p72 and p60. The recovery of p72 in anti-p72 MoAb immunoprecipitates after NNKY 1-19 stimulation was virtually the same as that of the resting state (data not shown). A portion of activated p72 associated with the cytoskeleton cannot totally explain the magnitude of the reduction in p72-associated tyrosine kinase activity, although we lack accurate stoichiometry. The rapid activation of p72 and subsequent deactivation within 60 s after stimulation has been noted with platelet activation induced by thrombin and thromboxane A mimetics (Taniguchi et al., 1993; Maeda et al., 1993). It is likely that p72 is actually deactivated during the course of NNKY 1-19 activation, which is mediated by FcII receptor stimulation and dependent upon aggregation.

Involvement of p72 has been suggested for Fc receptor signaling with HL60 and macrophages (Agarwal et al., 1993; Greenberg et al., 1994) and for Fc receptor stimulation with a mast cell line (Benhamou et al., 1993). Direct association between p72 and subunits of Fc receptors may play an important role in signal transduction mediated by FcI receptors (Shuie et al., 1995). With platelets, cross-linking of FcII receptors or aggregated IgG induces PTP and other parameters of platelet activation (Anderson and Anderson, 1990; Huang et al., 1992; Kang et al., 1993). We have found that cross-linking of FcII receptors induces tyrosine phosphorylation of p72 and elevates the p72in vitro kinase activity, which suggests that p72 is also involved in the direct stimulation of the FcII receptor. However, we have also found that a tyrosine kinase distinct from p72 copurifies with FcII receptors, which does not take place in platelet activation induced by anti-CD9 MoAb.()Thus, we are inclined to believe that FcII receptor cross-linking differs from anti-CD9 MoAb in the mode of platelet activation. Cross-linking of FcII receptors may provide a signal(s) other than simple Fc receptor occupancy by restricting the free movement of bound receptors and initiating cytoskeletal reorganization. Based upon these findings, we suggest that platelet activation induced by anti-CD9 MoAb involves two separate signals, one generated by the binding of anti-CD9 MoAb to CD9 molecules leading to the activation of p72 and the association between p72 and p60, and another that is mediated by the FcII receptor. The former alone results in persistent elevation of p72 activation and association between p72 and p60 but does not lead to the full picture of platelet activation. The latter generates a signal(s) that is inhibitable by intracellular cAMP elevation, and in synergy with CD9 activation it leads to platelet activation in terms of [Ca] elevation and aggregation. Whether the latter also involves p72 activation awaits to be clarified.


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. Tel.: 81-552-73-1111; Fax: 81-552-73-6713.

The abbreviations used are: MoAb, monoclonal antibody; PGI, prostaglandin I; PTP, protein tyrosine phosphorylation; PAGE, polyacrylamide gel electrophoresis.

Y. Ozaki, manuscript in preparation.


ACKNOWLEDGEMENTS

We gratefully acknowledge the kind donation of PMA2, an anti-CD9 MoAb, by Dr. Takaaki Hato (Ehime University, Japan).


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