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An Important Role of the Src Family Kinase Lyn in StimulatingPlatelet Granule Secretion*□S

Received for publication, December 23, 2009, and in revised form, February 25, 2010 Published, JBC Papers in Press, February 26, 2010, DOI 10.1074/jbc.M109.098756

Zhenyu Li‡§¶1, Guoying Zhang‡¶, Junling Liu‡�, Aleksandra Stojanovic‡, Changgeng Ruan§, Clifford A. Lowell**,and Xiaoping Du‡§

From the ‡Department of Pharmacology, College of Medicine, University of Illinois, Chicago, Illinois 60612, the §Jiangsu Institute ofHematology, First Affiliated Hospital of Soochow University, Suzhou 215006, China, the ¶Division of Cardiovascular Medicine, TheGill Heart Institute, University of Kentucky, Lexington, Kentucky 40536-0200, the �Department of Molecular and Cell Biology, JiaoTong University School of Medicine, Shanghai 200025, China, and the **Department of Laboratory Medicine, University ofCalifornia, San Francisco, California 94143

The Src family kinases (SFKs) have been proposed to playstimulatory and inhibitory roles in platelet activation. Themechanisms for these apparently contradictory roles areunclear. Here we show that SFK, mainly Lyn, is important instimulating a common signaling pathway leading to secretion ofplatelet granules. Lyn knock-out or an isoform-nonselectiveSFK inhibitor, PP2, inhibited platelet secretion of both denseand � granules and the secretion-dependent platelet aggrega-tion induced by thrombin, collagen, and thromboxane A2. Theinhibitory effect of Lyn knock-out on platelet aggregation wasreversed by supplementing granule content ADP, indicatingthat the primary role of Lyn is to stimulate granule secretion.Inhibitory effect of PP2 on platelet aggregation induced bythrombin and thromboxane A2 were also reversed by supple-menting ADP. Furthermore, PP2 treatment or Lyn knock-outdiminished agonist-inducedAkt activation and cyclicGMPpro-duction. The inhibitory effect of PP2 or Lyn knock-out on plate-let response can be corrected by supplementing cyclic GMP.These data indicate that Lyn stimulates platelet secretion byactivating the phosphoinositide 3-kinase-Akt-nitric oxide(NO)-cyclic GMP pathway and also provide an explanation whyLyn can both stimulate and inhibit platelet activation.

Platelets play a critical role in thrombosis and hemostasis. Atsites of vascular injury, platelets are activated by adhesive pro-teins, such as collagen and von Willebrand factor, and solubleplatelet agonists such as thrombin, thromboxane A2 (TXA2),2

and ADP (1). Collagen-induced platelet responses can beinduced by multiple collagen receptors, including glycoproteinVI (GPVI)/Fc receptor � (FcR�) complex, integrin �2�1, glyco-protein IV, etc. (2, 3). GPVI/FcR� signals via the immunorecep-tor tyrosine-based activationmotif (ITAM)-Syk signaling path-way, and GPVI signaling can be stimulated by GPVI-selectiveagonists, collagen-related peptide (CRP), and convulxin (4–6).von Willebrand factor activates platelets via the platelet glyco-protein Ib-IX (GPIb-IX)-mediated signaling. Soluble agonistsactivate platelets mainly via G-protein-coupled receptor(GPCR) signaling pathways. Among GPCRs, thrombin recep-tors (protease-activated receptors 1 and 4) and TXA2 receptorsignal mainly via the Gq and G13-coupled pathways (7–12).Platelet activation includes a series of rapid positive feedbackloops that greatly amplify activation signals and enable robustplatelet recruitment and stabilization of thrombi at the site ofvascular injury. An important mechanism of this responseamplification is the secretion of granule contents, which isrequired for full platelet responses induced by low concentra-tions of agonists or “weak” agonists. One of the important sub-stances secreted from dense granules is ADP, which inducesintegrin activation and platelet aggregationmainly through theP2Y1 (Gq-coupled) and P2Y12 (Gi-coupled) ADP receptors(13). The signaling pathways leading to platelet granule secre-tion are not totally clear but are believed to involve proteinkinase C and calcium-dependent signaling pathways andrequire the stimulation of formation of the SNARE complex,which mediates fusion between granules (vesicles) and plasmamembranes (14, 15). We have recently discovered that plateletgranule secretion can be stimulated via the phosphoinositide3-kinase (PI3K)-Akt-nitric oxide (NO)-cGMP pathway (16–20). It has also been shown that granule secretion in leukocytesand neuronal cells can also be stimulated via the NO-cGMPpathway (21–23).Src family kinases (SFKs) are a group of closely related non-

receptor protein tyrosine kinases. There are at least six differentSrc family tyrosine kinases that are expressed in platelets, Fgr,Fyn, Lck, Lyn, Src, and Yes (24). The roles of SFK in plateletactivation have been complex and controversial. For example,c-Src binds to the cytoplasmic domain of the integrin �3 sub-

* This work was supported in part by National Institutes of Health (NIH),NHLBI, Grants HL68819, HL62350, and 080264 (to X. D.) and by NIH/Na-tional Center for Research Resources Centers of Biomedical Research Excel-lence in Obesity and Cardiovascular Disease Grant P20 RR021954-01A1.This work was also supported by American Heart Association (AHA)National Scientist Development Grant 0430095N and AHA Midwest Affili-ate Grand-in-aid 0855698G (to Z. L.).

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Fig. 1.

1 To whom correspondence should be addressed: Division of CardiovascularMedicine, The Gill Heart Institute, 741 South Limestone St., BBSRB Bldg.,Rm. B251, University of Kentucky, Lexington, KY 40536-0200. Tel.: 859-257-0528; Fax: 859-257-3235; E-mail: [email protected].

2 The abbreviations used are: TXA2, thromboxane A2; GPVI, glycoprotein VI;FcR�, Fc receptor �; ITAM, immunoreceptor tyrosine-based activationmotif; CRP, collagen-related peptide; GPIb-IX, glycoprotein Ib-IX; GPCR,G-protein-coupled receptor; SNARE, soluble NSF attachment receptors;PI3K, phosphatidylinositol 3-kinase; cGMP, cyclic GMP; eNOS, endothelial

nitric-oxide synthase; SFK, Src family kinase; PKG, cGMP/cGMP-dependentprotein kinase; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 17, pp. 12559 –12570, April 23, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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unit and plays an important role in integrin �IIb�3-dependentoutside-in signaling (25–28). On the contrary, Lyn wasreported to inhibit integrin outside-in signals in platelets (29).In the GPIb-IX pathway, however, Lyn has been shown to playstimulatory roles in the GPIb-IX-mediated early signaling,leading to integrin activation and integrin-dependent stableadhesion to von Willebrand factor under shear stress (30–33).Lyn and Fyn have been shown to constitutively bind to thecytoplasmic domain of collagen receptor, GPVI (34, 35), andare involved in the collagen/GPVI-induced tyrosine phosphor-ylation of the FcR� ITAM, leading to activation of the tyrosinekinase Syk and its downstream enzymes (36). However, Lynknock-out platelets have also been shown to potentiate plateletaggregation and secretion induced by GPVI-selective agonists(37), suggesting the dual roles of Lyn inGPVI-mediated plateletactivation. Similarly controversial, although one study suggeststhat SFKs are not required in thrombin-induced platelet aggre-gation (38), other studies indicate that SFK stimulates plateletactivation induced by thrombin (39) and ADP (40, 41). Thecontroversies about the roles of Lyn in platelet activation are ina way similar to the controversies about the role of the NO-cGMP pathway in platelet activation in that both stimulatoryand inhibitory roles of this enzyme have been reported. How-ever, it is unclear what the mechanisms responsible for thesecontradictory roles of SFK are in different receptor-mediatedplatelet activation pathways.In this study, we demonstrate that SFK, mainly Lyn, plays an

important primary role in stimulating platelet secretion ofdense and �-granules, and this primary role of Lyn is responsi-ble for its stimulation and amplification of platelet aggregationduring platelet activation induced by low concentrations ofplatelet agonists or weak agonists. Furthermore, we have pro-vided data indicating that one mechanism by which SFK (Lyn)stimulates platelet secretion is its activation of the PI3K-Akt-eNOS-cGMP-PKG pathway. Because the role of the cGMPpathway in platelet activation is known to be biphasic (inhibi-tory and stimulatory), our results also provide a potential expla-nation for the controversy on the dual role of Lyn in stimulatingand inhibiting platelet activation.

EXPERIMENTAL PROCEDURES

Materials—The TXA2 analog U46619 and Src inhibitor PP2were purchased from Calbiochem. �-Thrombin was fromEnzyme Research Laboratories (South Bend, IN). Collagen andluciferase/luciferin reagentwere fromChrono-log (Havertown,PA). A rabbit polyclonal antibody against a recombinanthumanAkt 1 fragment (amino acid residues 345–480)was pur-chased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA),and a rabbit polyclonal antibody against phosphorylated Ser473of Akt was from Cell Signaling Technology. CRP was kindlyprovided by Dr. Debra K. Newman (Blood Research Institute,Blood Center of Milwaukee, WI). The TXB2 EIA kit was fromAssay Designs (Ann Arbor, MI). The cGMP enzyme immuno-assay kit was from Amersham Biosciences.Platelet Aggregation and Secretion—For studies using human

platelets, fresh blood was drawn by venipuncture from healthyvolunteers (performed in the General Clinical Research Cen-ters at the University of Illinois Medical Center and University

of Kentucky Medical Center). Institutional Review Boardapprovalwas obtained from theUniversity of Illinois at Chicagoand theUniversity of Kentucky, and informed consent was pro-vided to volunteers according to the Declaration of Helsinki.Bloodwas anticoagulatedwith one-seventh volume of ACD (85mM trisodium citrate, 110 mM dextrose, and 78 mM citric acid).PlateletswerewashedwithCGSbuffer (0.12M sodiumchloride,0.0129M trisodiumcitrate, and 0.03M D-glucose, pH6.5), resus-pended in modified Tyrode’s solution at 3 � 108/ml, andallowed to incubate at 22 °C for 1–2 h as described previously(42). Platelet aggregationwasmeasured by detecting changes inlight transmission. Platelet secretion was determined by mea-suring the release of ATP using luciferin/luciferase reagent(Chrono-lume). Luciferin/luciferase reagent (12 �l) was addedto 238 �l of washed platelet suspension within 1 min beforestimulation. Platelet aggregation and secretion were recordedin real time in a Chrono-log lumiaggregometer at 37 °C withstirring (1000 rpm). To examine the effects of PP2, washedplatelets were preincubated with PP2 (10 �M) or DMSO (0.1%)for 2 min prior to the addition of the agonists.Mouse Platelet Preparation—Lyn knock-out mice were gen-

erated as described previously (43) and were backcrossed toC57BL/6J background. C57BL/6J mice (obtained from JacksonLaboratory (BarHarbor,ME)) orwild type littermates fromLynheterozygote breeding were used as controls. Fyn knock-outmice were obtained from Jackson Laboratory. Mice were bredand maintained in the University of Illinois Animal Care Facilityand in the University of Kentucky Animal Care Facility followinginstitutional and National Institutes of Health guidelines afterapproval by the Animal Care Committee. Washed platelets fromLyn knock-out and wild type mice were prepared as describedpreviously (16). Briefly, male and female mice (6–8 weeks) wereanesthetized by intraperitoneal injection of pentobarbital. Wholeblood from homozygous Lyn knock-out and wild type mice wascollected from the inferior vena cava using one-seventh volume ofACD (85mM trisodium citrate, 83mM dextrose, and 21mM citricacid) as anticoagulant. For each experiment, blood was pooledfrom five or sixmice of each genotype. Platelets were thenwashedtwice with CGS, resuspended in modified Tyrode’s buffer at 3 �108/ml, and incubated for 2 h at 22 °C before use.P-selectin Expression—Washed platelets from healthy hu-

man donors were resuspended in Tyrode’s buffer and incu-bated at 37 °C for 2 h before use. Platelets were preincubatedwith or without PP2 or DMSO for 2 min at 37 °C. The plateletswere then incubated with different agonists for 5 min at 37 °Cand fixed by adding paraformaldehyde (final concentration of1%). The platelets were incubated with a monoclonal anti-hu-man P-selectin antibody, SZ51. After washing, the plateletswere further incubated with a fluorescein isothiocyanate-con-jugated goat anti-mouse Ig antibody. P-selectin expression wasanalyzed using a FACSCalibur flow cytometer. To determinethe role of Lyn in P-selectin expression induced by platelet ago-nists, washed platelets from Lyn knock-out mice or wild typecontrols were stimulated with platelet agonists for 5 min at37 °C and fixed by adding paraformaldehyde. Platelets werethen incubated with a fluorescein isothiocyanate-conjugatedanti-mouse P-selectin antibody at 22 °C for 30 min. P-selectinexpression was analyzed by flow cytometer.

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Measurement of cGMP Levels—Washed platelets (3 � 108/ml) in 400 �l of Tyrode’s buffer were stirred at 37 °C after theaddition of control buffer, thrombin (0.05 unit/ml), U46619(500 nM). To determine the effect of PP2 on cGMP production,platelets were preincubated with PP2 (10 �M) or DMSO at37 °C for 2 min and then exposed to the platelet agonists. Todetermine the role of Lyn in cGMP production induced byplatelet agonists, washed platelets from Lyn knock-out mice orwild type controls were stimulated with platelet agonists for 5min at 37 °C. The reactionwas stopped by the addition of 400�lof ice-cold 12% (w/v) trichloroacetic acid. Samples were mixedand centrifuged at 2000� g for 15min at 4 °C. The supernatantwas removed and washed four times with 5 volumes of water-saturated diethyl ether and then lyophilized. cGMP levels weremeasured using a cGMP enzyme immunoassay kit fromAmer-sham Biosciences.Western Blot Analysis of Akt Phosphorylation in Platelets—

Washed platelets (3 � 108/ml) were resuspended in modifiedTyrode’s buffer and incubated at room temperature for 1 hbefore use. Platelets were stimulated with collagen (0.5 �g/ml)or thrombin (0.05 unit/ml) in a platelet aggregometer at 37 °C

for 5 min and then solubilized inSDS-PAGE sample buffer. Toexamine the effect of PP2 on Aktphosphorylation, plateletswere pre-incubated with PP2 or DMSO at37 °C for 2 min prior to the additionof the platelet agonists. Plateletlysates were analyzed by SDS-PAGEon 4–15% gradient gel and electro-transferred to polyvinylidene difluo-ride membranes. The membraneswere blocked with 5% nonfat drymilk in TBS (20 mM Tris-HCl, 150mM NaCl, pH 7.5) and incubatedwith a polyclonal anti-Akt antibodyor a polyclonal antibody specific forthe phosphorylated Ser473 of Akt(Cell Signaling) for 2 h at 22 °C.After three washes in TBS contain-ing 0.05% Tween 20, the mem-branes were incubated with horse-radish peroxidase-conjugated goatanti-rabbit IgG (0.5 �g/ml) for 45min. After further washing, reac-tions were visualized using anAmersham Biosciences enhancedchemiluminescence kit.

RESULTS

Effect of an SFK Inhibitor, PP2,on Platelet Secretion and Aggrega-tion Induced by G-protein-coupledReceptor Agonists TXA2 and �-Thrombin—To investigate the roleof SFK inplatelet secretionandaggre-gation induced by GPCR-dependentagonists, we examined the effect of

an SFK inhibitor, PP2, on ATP release and platelet aggregationinduced by a stable TXA2 analog U46619 and �-thrombin. At alow concentration of U46619 (250 nM), human platelet aggre-gation and ATP secretion were both abolished by PP2 (Fig. 1,Aand E). However, at a higher concentration of U46619 (500 nM),platelet aggregation was partially inhibited by PP2, showingone-waveaggregationthat ischaracteristicofa lackof secretion-dependent secondary aggregation response. Indeed, ATPsecretion induced by this concentration of U46619 was inhib-ited by PP2 (Fig. 1, B and E). These results suggest that PP2inhibits platelet secretion and secretion-dependent plateletaggregation induced by U46619. Similarly, PP2 inhibited ATPrelease and the second wave of platelet aggregation induced bya low concentration of �-thrombin (0.05 unit/ml) (Fig. 1,C andF). However, at a higher concentration of thrombin (0.1 unit/ml), PP2 only partially inhibitedATP release, and consequently,platelet aggregation was not affected (Fig. 1, D and F), suggest-ing that the inhibitory effect of PP2 on platelet aggregation isalso secretion-dependent. To exclude possible nonspecificeffects of PP2 on platelet secretion and aggregation, we alsoobtained data indicating that a structural analogue of PP2, PP3,

FIGURE 1. Effects of the SFK inhibitor PP2 on GPCR-induced ATP secretion and platelet aggregation. A–D,washed human platelets in modified Tyrode’s buffer (3 � 108/ml) were preincubated with PP2 (10 �M) or DMSOcontrol (0.1%) at 37 °C for 2 min and then exposed to various concentrations of U46619 (A and B) or thrombin(C and D) and simultaneously recorded for ATP secretion and aggregation. The aggregation and ATP releasetraces are representatives of at least three different experiments. E and F, aggregation and secretion results inthe experiments described in A–D were quantitated. Secretion values were normalized with respect to plate-lets pretreated with DMSO and stimulated with the lowest concentration of U46619 (U) (E) or thrombin (Throm)(F).

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which does not inhibit SFK, did not affect platelet aggrega-tion and secretion induced by U46619 (Fig. 2A) or thrombin(data not shown). These data indicate that SFK is an impor-tant mediator but not the only mediator in platelet-densegranule secretion induced by GPCR-dependent agonists,TXA2 and thrombin.Inhibition of ADP Secretion Is Responsible for Decreased

Platelet Aggregation Induced by �-Thrombin and U46619 inPP2-treated Platelets—To further verify whether the inhibitoryeffect of PP2 on the second wave of platelet aggregationresulted from its inhibitory effect on secretion, we examinedwhether supplementing granule content ADP could rescue thesecond wave of platelet aggregation in PP2-treated humanplatelets. ExogenousADP (0.5�M) alone did not induce plateletaggregation in washed platelets (Fig. 2B). Thrombin-inducedplatelet aggregation was inhibited by PP2. Adding exogenousADP at a concentration (0.5 �M) below the concentrations ofADP released by low dose thrombin (44) significantly reversed

the inhibitory effect of PP2 andrestored platelet aggregation in-duced by thrombin (Fig. 2, B andC).Similarly, exogenous ADP alsorestored the second wave of aggre-gation of PP2-treated plateletsinduced by low dose U46619 (Fig.2D). These data suggest that theinhibitory effect of PP2 on throm-bin- and TXA2-induced plateletaggregation results from its inhibi-tion of platelet secretion of densegranule ADP.Role of SFK in Platelet Secretion of

�-Granules—To determine whetherSFK also plays a role in plateletsecretion of �-granules, we investi-gated the effect of PP2 on surfaceexpression of P-selectin induced byplatelet agonists. Expression of P-selectinwas examinedby flowcytom-etry using a monoclonal anti-humanP-selectin antibody SZ51. Fig. 3shows that PP2 abolished P-selectinexpression induced by U46619. P-se-lectin expression induced by throm-bin was partially but significantlyinhibited by PP2. Thus, SFK plays animportant role in platelet secretionfrom �-granules induced by TXA2and thrombin.Role of Lyn in Platelet Granule

Secretion—Lyn is one of the majorSFK members expressed in plate-lets. To investigate whether Lyn isresponsible for the role of SFKin platelet secretion induced byU46619 and thrombin, we com-pared agonist-induced platelet ATPrelease and aggregation between

Lyn-deficient platelets and wild type controls. Thrombin-in-duced ATP release and aggregation induced by low dose con-centrations of �-thrombin were significantly inhibited in theLyn-deficient platelets (Fig. 4,A andB), which is consistentwitha previous report showing inhibitory effect of Lyn knock-out on�-thrombin-induced platelet response (39). Also, P-selectinexpression induced by thrombin was significantly inhibited inLyn knock-out platelets compared with wild type platelets (Fig.4, C and D). To determine whether the defect in platelet secre-tion signaling is responsible for the defective integrin-depen-dent platelet aggregation response of Lyn knock-out, Lynknock-out platelets were stimulated with thrombin with thesupplementation of low concentrations of dense granule con-tent ADP. Indeed, ADP completely corrected the defect inplatelet aggregation (Fig. 4, E and F). These results indicate thatan important role of Lyn in platelet activation is to mediategranule secretion signaling and that Lyn is themajor SFKmem-ber responsible for mediating platelet secretion.

FIGURE 2. Specificity of the effect of PP2 on platelet secretion and aggregation and restoration of theplatelet aggregation by exogenous ADP in the PP2-treated platelets. A, washed human platelets in mod-ified Tyrode’s buffer (3 � 108/ml) were preincubated with DMSO (0.1%), PP2 (10 �M), or an analog control forPP2, PP3 (10 �M), which does not inhibit SFK at 37 °C for 2 min, and then exposed to U46619 (500 nM). B, washedhuman platelets were preincubated with DMSO or PP2 (10 �M) (PP2) for 2 min at 37 °C and then stimulated withthrombin (0.025 unit/ml). PP2-treated platelets were also stimulated with thrombin followed by 0.5 �M ADP(PP2�ADP). Platelets were also stimulated with ADP alone (0.5 �M) (ADP). C, quantitative data (mean � S.D.)from four experiments as described in B. D, washed human platelets were preincubated with DMSO or PP2 (10�M) for 2 min at 37 °C and then stimulated with U46619 (250 nM). PP2-treated platelets were also stimulatedwith U46619 followed by 0.5 �M ADP. Platelets were also stimulated with 0.5 �M ADP alone.

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Role of SFK in Collagen-induced Platelet Secretion—Unlikethe GPCR-coupled soluble agonists, adhesive protein collagenmediates platelet adhesion and activation via multiple plateletcollagen receptors and their signaling pathways, including theGPVI-ITAM pathway and integrin �2�1. Several members ofSFK have been suggested to play a role in early GPVI-ITAMsignaling (45). Indeed, PP2 totally abolished collagen-inducedATP release (Fig. 5A) and P-selectin expression (Fig. 5B) and

also totally abolished collagen-induced platelet aggregation.However, although collagen-induced FcR� phosphorylation inLyn knock-out platelets appeared slightly reduced at an earlytime point, as previously reported (37) (supplemental Fig. 1),platelet activation induced by GPVI-specific agonist CRP wasnot reduced (and was even enhanced) in Lyn knock-out plate-lets (37) (data not shown), suggesting that Lyn-independentITAM phosphorylation is sufficient for full-scale plateletresponse. In contrast, under our experimental conditions, lowdose collagen-induced ATP release and aggregation weremarkedly but partially reduced in the Lyn-deficient platelets(Fig. 5C). These data indicate that Lyn is important in collagen-induced platelet secretion and aggregation, and this role of Lynin collagen-induced platelet aggregation appears to be distinctfrom the role of SFK in the GPVI-ITAM pathway Consistentwith this notion, we show that, although inhibition of collagen-induced platelet aggregation by PP2 was not rescued by theaddition of exogenous ADP (data not shown), exogenous ADPreversed inhibition of Lyn deficiency on platelet aggregationinduced by collagen (Fig. 5,D and E). These results suggest thatan important role of Lyn in platelet activation induced by col-lagen is to mediate ADP secretion. Previously, it has beenshown that SFKs are important in stimulating TXA2 synthesis(40, 46), which is important in collagen-induced platelet aggre-gation. We found that collagen-induced TXA2 production wastotally abolished by PP2 in human platelets, which was not cor-rected by ADP supplementation (data not shown). Thus, ourdata suggest that although some SFK members are involved inthe proximal GPVI-dependent ITAM signaling leading toTXA2 synthesis and platelet activation, Lyn plays an importantrole in a distinct collagen signaling pathway mediating granulesecretion in response to low dose collagen.The Role of Fyn in Platelet Activation—Platelets also express

Fyn. However, Fyn is not required for thrombin-inducedplatelet aggregation and secretion (39) (data not shown). Toinvestigate whether Fyn is involved in platelet secretion andaggregation induced by U46619 and collagen, we comparedagonist-induced platelet ATP release and aggregation be-tween Fyn-deficient platelets and wild type controls. ATPrelease and aggregation induced by either low dose concentra-tions of U46619 (250 nM) or collagen (0.5 �g/ml) were not sig-nificantly affected in the Fyn-deficient platelets (Fig. 6), sug-gesting that Fyn is not required for platelet secretion andaggregation induced by these platelet agonists.Role of SFK in Platelet Agonist-induced Akt Phosphory-

lation and cGMP Production—In platelets and other cell types,SFKs have been suggested to be important in agonist-mediatedPI3K activation and signaling (39, 47). We have recentlyreported that the PI3K-Akt-eNOS-cGMP-PKG signaling path-way stimulates platelet secretion (18–20). Thus, we hypothe-sized that SFK mediates platelet secretion by activating thePI3K-Akt-NO-cGMP pathway. To test this hypothesis, weexamined the effects of PP2 on Akt phosphorylation and intra-cellular cGMP elevation induced by GPCR-dependent agonistthrombin and by collagen in human platelets. Akt phosphoryla-tion induced by either collagen (Fig. 7, A and B) or thrombin(Fig. 7, C and D) was nearly completely inhibited by PP2, indi-cating a critical role for SFK in the agonist stimulation of the

FIGURE 3. The inhibitory effect of PP2 on P-selectin expression. A and B,washed human platelets in modified Tyrode’s buffer were preincubated withPP2 (10 �M) or DMSO for 2 min at 37 °C and then stimulated with U46619 (U)(250 nM) or thrombin (T) (0.025 unit/ml) for 5 min at 37 °C with stirring andsubsequently fixed with paraformaldehyde. Fixed platelets were incubatedwith a monoclonal anti-human P-selectin antibody, SZ51, or negative controlmouse IgG (M IgG) for 30 min at 22 °C. After washing once with PBS, plateletswere further incubated with a fluorescein isothiocyanate-conjugated rabbitanti-mouse Ig antibody. Surface expression of P-selectin was analyzed usingflow cytometry. Data from a representative experiment are shown in A. Quan-titative results from three experiments are expressed as the P-selectin expres-sion index (fluorescence intensity of platelets stimulated with an agonist/fluorescence intensity of unstimulated platelets) (B).

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PI3K-Akt pathway. Interestingly,although collagen-induced cGMPelevation was completely inhibitedby PP2 (Fig. 7E), thrombin-inducedcGMP elevation was only partiallybut significantly blocked by PP2(Fig. 7F), suggesting that althoughSFK plays an important role in ago-nist-stimulated cGMP elevation,there is a SFK-independent signal-ing pathway leading to cGMP syn-thesis. These data indicate that SFKplays an important role in the acti-vation of the PI3K-Akt-NO-cGMPpathway in platelets, but thrombinmay also stimulate cGMP elevationvia an SFK-independent pathway.Role of Lyn in Platelet Agonist-

induced cGMP Elevation and AktPhosphorylation—To determinewhether Lyn is the major SFKresponsible for the activation ofcGMP-PKG pathway, we comparedagonist-induced cGMP elevationbetween Lyn-deficient platelets andwild type controls. The intracellularlevels of cGMP induced by throm-bin orU46619were significantly butpartially inhibited in Lyn-deficientplatelets (Fig. 8A). The collagen-in-duced platelet cGMP elevation wasalmost completely inhibited in Lynknock-out platelets. Similarly, Aktphosphorylation induced by eithercollagen or thrombin was reducedin the Lyn-deficient platelets (Fig. 8,B and C). These results are consis-tent with the results obtained withPP2, indicating that Lyn is a majorSFK responsible for agonist-in-duced activation of the cGMPpathway.Inhibition of Platelet Secretion

and Aggregation by PP2 and LynKnock-out Is Rescued by cGMP—IfSFK (Lyn)-stimulated platelet se-cretion is mediated via the cGMPpathway, the inhibitory effect of PP2or Lyn knock-out on platelet activa-tion should be reversed by supple-menting exogenous cGMP. Indeed,the inhibitory effect of PP2 onthrombin-induced aggregation wassignificantly reversed by adding alow concentration of membrane-permeable cGMP analog, 8-bromo-cGMP (5 �M) (Fig. 9, A and B), butnot the membrane-impermeable

FIGURE 4. The inhibitory effect of Lyn deficiency on platelet aggregation and secretion. A, washed plate-lets from Lyn�/� or Lyn�/� mice were stimulated with thrombin (0.025 units/ml) in a lumiaggregometer at37 °C. Real-time ATP secretion and platelet aggregation were simultaneously recorded. B, aggregation andsecretion results for the experiments described in A were quantitated (n � 3). Secretion values were normalizedwith respect to platelets from Lyn�/� mice. C and D, washed platelets from Lyn�/� (C) or Lyn�/� mice (D) wereincubated with various concentrations of thrombin for 5 min at 37 °C with stirring and then fixed withparaformaldehyde. Fixed platelets were incubated with a fluorescein isothiocyanate-labeled monoclonal anti-mouse P-selectin antibody, SZ51, for 30 min at 22 °C, and analyzed using flow cytometry. The P-selectin expressionindex (median of fluorescence intensity with a certain concentration of thrombin/median of fluorescence intensityof unstimulated platelets (control)) is also shown. E, aggregation traces of platelets from either Lyn knock-out mice(Lyn�/�) or wild type controls (Lyn�/�) stimulated with thrombin or thrombin plus 0.5 �M ADP (Lyn�/��ADP).F, aggregation results for the experiments described in E were quantitated (n � 3). WT, wild type.

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cGMP (data not shown). 8-Bromo-cGMP alone did not induceany platelet aggregation and secretion (data not shown) (17).8-Bromo-cGMP also partially but significantly reversed theinhibitory effect of PP2 on thrombin-induced platelet granulesecretion (Fig. 9C). Similarly, the defect of Lyn knock-out plate-lets in platelet secretion and aggregation was also partially cor-rected by supplementing 8-bromo-cGMP (Fig. 9D). These

results, together with the finding that inhibition of SFK by PP2or Lyn knock-out significantly diminished cGMP elevation,indicate that the cGMP-dependent signaling pathway is animportant signaling pathway that mediates the role of SFK instimulating platelet secretion.Inhibition of cGMPElevation by LynDeficiency Is Reversed by

Exogenous ADP—It has been previously reported that the ADPreceptor, P2Y12-mediated signaling pathway leads to activa-tion of Akt in platelets, which activates eNOS-dependent NOsynthesis and cGMP elevation. Therefore, we determinedwhether the inhibitory effect of Lyn deficiency on cGMP eleva-tion induced by collagen or thrombin could be reversed by theaddition of exogenousADP.We found that the addition ofADP(0.5 �M) partially reversed the inhibitory effect of Lyn knock-out on the cGMP elevation in the collagen- or thrombin-stim-ulatedmouse platelets. ADP alone at 0.5�Mdid not affect intra-cellular cGMP levels in the wild type or Lyn-deficient platelets(data not shown). These results suggest that ADP, possibly viathe P2Y12 pathway, induces a Lyn-independent positive feed-back loop amplifying cGMP elevation and platelet secretion(Fig. 10). Thus, ADP and cGMP signaling pathways mutuallyamplify each other, leading to promotion of platelet response tolow doses of agonists.

DISCUSSION

In this study, we demonstrate that SFK, mainly Lyn, plays animportant role in a GPCR- and collagen-induced common sig-naling pathway mediating platelet secretion from dense gran-ules and �-granules. We further demonstrate that Lyn-medi-ated stimulation of platelet granule secretion is the mechanismresponsible for the SFK role in promoting and stabilizing plate-let aggregation induced by Gq and G13-coupled agonists, suchas thrombin and TXA2. Furthermore, we show that the PI3K-Akt-NO-cGMPpathway is an important downstream signalingpathway of Lyn-mediated platelet granule secretion.Our data support the conclusion that SFK, mainly Lyn, plays

an important role inmediating platelet secretion of dense gran-ules and �-granules. These data are consistent with the resultsof Cho et al. who reported that �-thrombin-induced plateletaggregation and secretion were reduced by Lyn knock-out (39).However, the data of Cho et al. do not differentiate whether therole of Lyn is in the agonist-induced signaling leading to aggre-gation and thus subsequently affects aggregation-dependentgranule secretion or in the signaling leading to secretion, whichsubsequently affects the amplification of platelet aggregation.We show that platelet secretion induced by �-thrombin,U46619, or collagen was inhibited by PP2 and in Lyn knock-outplatelets and that the inhibitory effect of PP2 and Lyn-knock-out onplatelet aggregation induced by theseGPCRagonists canbe significantly reversed by supplementing low concentrationsof dense granule content ADP, indicating that inhibition by Lynknock-out on platelet aggregation is consequent to its inhibitoryeffect on granule secretion. The addition of ADP did not com-pletely reverse the inhibitory effect of PP2, suggesting that othersecreted granule contents and/or a secretion-independent path-waymay also be involved in SFK-mediated platelet activation.The role of SFK in platelet activation induced by Gq/G13-

coupled agonists, such as thrombin and TXA2, appears to be

FIGURE 5. Effects of PP2 and Lyn knock-out on collagen-induced plateletsecretion and aggregation. A, washed human platelets were preincubatedwith PP2 or DMSO for 2 min and then exposed to collagen in a lumiaggre-gometer at 37 °C. Real-time ATP secretion and platelet aggregation weresimultaneously recorded. B, washed human platelets were preincubated withPP2 or DMSO for 2 min and then stimulated with collagen (Coll) (0.5 �g/ml) for5 min at 37 °C with stirring and then analyzed for P-selectin expression by flowcytometric analysis of the binding of a monoclonal anti-human P-selectinantibody, SZ51. C, aggregation and ATP secretion traces of washed plateletsfrom Lyn�/� or Lyn�/� mice stimulated with collagen (0.5 �g/ml) in a lumi-aggregometer at 37 °C. D, aggregation traces of Lyn�/� or Lyn�/� mouseplatelets stimulated with collagen (0.5 �g/ml) or collagen plus 0.5 �M ADP(Lyn�/��ADP). E, quantitative data (mean � S.D.) from three experiments asdescribed in D. WT, wild type.

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distinct from that in adhesion protein (collagen or GPIb-IX)-induced platelet activation. The role of SFK (Lyn) as well as thecGMP pathway in GPIb-IX signaling appears to be proximal tothe receptor and is important in the stable platelet adhesion tovonWillebrand factor under shear stress, independent of ADPsecretion (17, 32, 33). It is recognized that SFK is important inthe collagen-induced activation of the ITAM pathway becauseSFK mediates tyrosine phosphorylation of the ITAM motif ofFcR� required for recruiting the downstream kinase Syk, whichis required for collagen-induced platelet secretion and aggrega-tion. Thus, the SFK inhibitor PP2 inhibited collagen receptor-proximal signaling responses, which can activate both ADPsecretion-dependent and -independent downstream signaling.Consequently, the inhibitory effect of PP2 cannot be correctedby supplementing ADP (data not shown). In contrast, Lyn isimportant in a collagen-induced signaling mechanism thatappears to be distinct from the early GPVI/ITAM pathwayinduced by theGPVI-specific agonist, CRP, because Lyn knock-out showed reduced platelet secretion and aggregation in

response to collagen but enhancedresponse to CRP (37) (data notshown). Furthermore, the de-creased collagen-induced plateletaggregation in Lyn knock-out plate-lets can be reversed by ADP supple-mentation, suggesting a granule se-cretion-dependent role for Lyn. Incontrast to collagen-induced plate-let activation, both PP2 and Lynknock-out had little effect on pri-mary platelet aggregation inducedby GPCR-coupled platelet agonists,such as thrombin and TXA2, butinhibited platelet granule secretionand thus the secretion-dependentsecond wave of platelet aggregation.The inhibitory effect of both PP2and Lyn knock-out on plateletaggregation induced by Gq/G13-coupled agonists, thrombin andTXA2, was rescued by supplement-ing ADP, which mainly activates Gisignaling pathway (in addition toGq, which is already activated bythrombin or TXA2). Also, the effectof ADP in correcting the inhibitionof platelet aggregation by PP2 wasabolished by the Gi-coupled P2Y12antagonist 2-methylthioadenosine5�-monophosphate triethylammo-nium salt (data not shown). Thus,Lyn appears to be a major SFKmember responsible for the role ofSFK in platelet activation inducedby low concentrations of Gq/G13-coupled agonists, such as thrombinand TXA2, which is to mediateplatelet granule secretion. Secreted

ADP, by activating theGi pathway, greatly amplifies the plateletactivation signals, including the cGMP signal, inducing the sec-ond wave of platelet aggregation and stabilizing the plateletthrombus. Our finding that SFK (Lyn) plays an important role inmediatingplatelet secretionrepresentsa significantadvance inourunderstanding of the roles of SFK in platelet activation and themechanism of platelet secretion signaling. It is important to notethat platelet aggregation induced by a high dose of thrombin doesnot require secretion of ADP (Fig. 1). Therefore, a low dose ofthrombin was used in this study. Due to various responses tothrombin indifferentdonors, variable concentrationsof thrombin(0.025–0.05 units/ml) were used to achieve an optimal secretion-dependent platelet aggregation inwhich the role of Lynmanifests.Platelet agonist-induced granule secretion involves both

aggregation-dependent and aggregation-independent path-ways. Aggregation-dependent platelet secretion involves inte-grin outside-in signaling. SFK, particularly c-Src, have beenshown to play critical roles in the outside-in signaling, leadingto platelet spreading. However, we believe that the main role of

FIGURE 6. The effect of Fyn deficiency on platelet aggregation and secretion. A, washed platelets fromFyn�/� or Fyn�/� mice were stimulated with collagen (0.5 �g/ml) in a lumiaggregometer at 37 °C. Real-timeATP secretion and platelet aggregation were simultaneously recorded. B, washed platelets from Fyn�/� orFyn�/� mice were stimulated with U46619 (250 nM) in a lumiaggregometer at 37 °C. Real-time ATP secretionand platelet aggregation were simultaneously recorded. C and D, aggregation (C) and secretion (D) results forthe experiments described A and B were quantitated (n � 3). Secretion values were normalized with respect towild type platelets stimulated with collagen. U, U46616; Coll, collagen.

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Lyn in platelet secretion is not due to integrin outside-in signal-ing for the following reasons. First, P-selectin expressioninduced by thrombin, U46619, or collagen is significantly butonly partially inhibited by the integrin inhibitor, RGDS peptide,

suggesting a partial dependence on integrin signaling. In con-trast, P-selectin expression is almost completely abolished byPP2, suggesting that SFK is important in mediating both integrin-dependent and integrin-independent granule secretion. Sec-ond, although Lyn is the major SFK member responsible formediating agonist-induced platelet secretion, Lyn has beenreported to negatively regulate integrin �IIb�3-mediated out-side-in signaling in platelets (29) and thus is unlikely to beresponsible for promoting integrin outside-in signaling leadingto platelet secretion. Instead, c-Src has been identified as theSFKmember that is critical for the outside-in signaling of inte-grin�IIb�3 in platelets (25–28). Therefore, although our data donot exclude the possibility that certain SFK isoforms such asc-Src are important in integrin outside-in signaling and conse-quent responses, our data indicate that the SFK Lyn stimulatesa platelet secretion signaling pathway that is distinct from theintegrin outside-in signaling.Despite the importance of granule secretion in platelet func-

tion, signaling pathways regulating platelet granule secretionare not well defined (48). Recently, we have discovered that theNO-cGMP-PKG pathway stimulates platelet secretion (17–

FIGURE 7. The effect of PP2 on Akt phosphorylation and cGMP produc-tion. A, washed human platelets were preincubated with PP2 (10 �M) orDMSO for 2 min at 37 °C. Platelets were then stimulated with collagen (Coll) inthe aggregometer for 5 min at 37 °C and solubilized with SDS-PAGE samplebuffer. Phosphorylation of Akt (p-Akt) was detected by Western blotting withan antibody specifically recognizing the phosphorylated Ser473 site in Akt.Equal sample loading was assessed by Western blotting using an anti-Aktantibody. B, densitometry measurements from results in A. Values were nor-malized with respect to sample with stimulation for each immunoblot and areexpressed as relative phosphorylation (mean � S.D. from three separateexperiments). Statistical significance was determined using Student’s t test.C, washed human platelets were preincubated with PP2 (10 �M) or DMSO(0.1%) for 2 min at 37 °C. Platelets were then stimulated with thrombin in theaggregometer for 5 min at 37 °C and solubilized with SDS-PAGE samplebuffer. Phosphorylation of Akt was detected by Western blotting. D, densi-tometry measurements from the results in C. Values were normalized withthe value obtained with stimulated platelets for each immunoblot and areexpressed as relative phosphorylation. E and F, washed human platelets werepreincubated for 2 min at 37 °C with DMSO or PP2 (10 �M). The platelets werethen stimulated with collagen (0.5 �g/ml) (E) or thrombin (0.05 units/ml) (F) ina platelet aggregometer for 5 min. cGMP concentrations were determinedusing a cGMP enzyme immunoassay kit. Results are expressed as mean � S.D.(n � 3) (p � 0.01).

FIGURE 8. The effect of Lyn deficiency on Akt phosphorylation and cGMPproduction. A, washed platelets from Lyn�/� or Lyn�/� mice were stimu-lated with collagen (0.5 �g/ml), thrombin (0.05 units/ml), or U46619 (500 nM)in a platelet aggregometer at 37 °C for 5 min. cGMP concentrations weredetermined using a cGMP enzyme immunoassay kit. Results are expressed asmean � S.D. (n � 3). B and C, washed platelets from Lyn�/� or Lyn�/� micewere stimulated with collagen (B) or thrombin (C) in the aggregometer for 5min at 37 °C and solubilized with SDS-PAGE sample buffer. Phosphorylationof Akt was detected by Western blotting. Values were normalized with thevalue of stimulated wild type platelets for each immunoblot and areexpressed as relative phosphorylation (mean � S.D. from three separateexperiments).

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19). These results are consistent with the report that insulinstimulates secretion of ATP from dense granule via the NO-cGMP-PKG pathway (49) and with the findings that the NO-cGMP-PKG pathway stimulates exocytosis in leukocytes andneuronal cells (21–23). The cGMP-dependent granule secre-tion is associatedwith the PKG-dependent association betweensyntaxin 2 VAMP3 (vesicle-associated membrane protein 3)(49) in platelets and phosphorylation of soluble SNAP (N-eth-ylmaleimide-sensitive factor-attached protein)-23, and syntax-ins 2 and 4 in leukocytes (21). However, the stimulatory effectcGMP in platelet activation was not repeated by some studies(50, 51). These discrepancies between our results and the datafromMarshall et al. (50) andMorrell et al. (51) could be attrib-uted to different experimental conditions because the plateletsused in these reports are insensitive to low concentrations ofplatelet agonists, such as thrombin (50, 51). Because the role ofcGMP in platelet activation is mainly to amplify low dose ago-nist-induced platelet activation, it is not surprising that theseinvestigators failed to show the stimulatory role of cGMP.

Although we are the first to systematically demonstrate thestimulatory roles and pathways of NO and cGMP in plateletaggregation and secretion, the phenomenon of stimulatoryeffects of cGMP analogs on platelet activation was reported asearly as in 1976 (52) but was submerged by the opposition andthus not publicly confirmed by other laboratories until ourreports in 2003 (17). Furthermore, PI3K and Akt are known topromote platelet secretion and secretion-dependent plateletaggregation (16, 20, 53, 54), and the effects of PI3K and Akt aremediated via theNO-sGC/cGMP-PKGpathway (20, 32). In thisstudy, we further show that 1) the SFK inhibitor, PP2, inhibitedagonist-induced Akt phosphorylation, indicating that SFK isimportant in GPCR-mediated activation of the PI3K-Akt sig-naling pathway; 2) PP2 and Lyn knock-out also inhibitedthrombin- and collagen-induced increases in the intracellularcGMP levels in platelets, indicating that SFK is upstream of theNO-cGMP signaling pathway; 3) supplementation of low con-centrations of 8-bromo-cGMP corrected the platelet aggrega-tion defect caused by PP2 and Lyn knock-out and partially butsignificantly reversed the inhibitory effect of PP2 on plateletgranule secretion, indicating that SFK (Lyn)-mediated activa-tion of the cGMP signaling pathway plays an important role inthe stimulatory effect of SFK (Lyn) on platelet secretion andaggregation; and 4) similar to PP2, the specific sGC inhibitorODQ inhibited platelet aggregation in response to low dosethrombin (19). ODQ also inhibited cGMP production inducedby thrombin in human platelets. Thus, although our data sug-

FIGURE 9. 8-Bromo-cGMP rescues platelet aggregation and secretion inPP2-treated platelets. A, washed human platelets in modified Tyrode’sbuffer (3 � 108/ml) were preincubated with DMSO (control) or PP2 (10 �M) for2 min at 37 °C and then stimulated with thrombin (0.025 units/ml), followedby the addition of 8-bromo-cGMP (5 �M) (PP2�cGMP) or buffer (PP2). B, quan-titative data (mean � S.D.) on the percentage of aggregation (light transmis-sion) from three experiments as described in A. C, 6 min after the addition ofthrombin, luciferase/luciferin reagent was added to the platelet suspensionto measure the total amount of ATP released. The data represent the mean �S.D. of ATP release from three experiments. D, platelets from Lyn knock-outmice were stimulated with thrombin (Lyn�/�) or thrombin followed by theaddition of 8-bromo-cGMP (Lyn�/��Br-cGMP). Platelets from wild type con-trols were also stimulated with thrombin (Lyn�/�).

FIGURE 10. Exogenous ADP rescues cGMP in Lyn-deficient platelets. A andB, washed platelets (3 � 108/ml) from Lyn�/� or Lyn�/� mice were stimulatedwith collagen in the aggregometer for 5 min at 37 °C. They were stimulatedwith collagen (0.5 �g/ml) (A) or thrombin (0.025 unit/ml) (B) with or withoutthe addition of ADP (0.5 �M). cGMP concentrations were determined using acGMP enzyme immunoassay kit. Results are expressed as mean � S.D. (n � 3).C, a positive feedback loop regulating platelet secretion. Platelet agonists,thrombin or collagen, bind to their receptors, which, by activating the SFKpathway, induce the activation of the PI3K/Akt pathway. PI3K/Akt inducesplatelet secretion via PKG. Secreted ADP activates the PI3K/Akt pathwaythrough its Gi-coupled P2Y12 receptor, which further induces cGMPelevation.

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gest that SFK is also important in other platelet signaling path-ways, our data reveal that SFK, mainly Lyn, sequentially acti-vates the PI3K-Akt pathway and NO-cGMP-PKG pathway,leading to platelet granule secretion and consequently the sec-ond wave of platelet aggregation. There have been reports thatsGC can be activated through Src kinase-dependent phosphor-ylation of the sGC �1 subunit in a nitric-oxide synthase/NO-independent manner (55, 56). Therefore, our data could notexclude the possibility that SFK (Lyn)-dependent phosphory-lation and activation of sGC might also be involved in the acti-vation of the cGMP/PKG pathway in response to plateletagonists.Interestingly, although the Lyn-dependent cGMP pathway

stimulates ADP secretion, ADP, via P2Y12, also stimulatesPI3K and Akt (16, 57) and partially reverses the defects incGMP production in Lyn knock-out platelets (Fig. 10D), sug-gesting that there is a positive feedback loop in which cGMPand ADP mutually amplify each other. The Lyn-independentactivation of cGMP synthesis is probably mediated by G��-mediated PI3K� and Akt activation induced via P2Y12 (16).Therefore, our results reveal not only a Lyn-dependent cGMPpathway leading to the platelet granule secretion pathway butalso a positive amplification mechanism in which cGMP pro-motesADP secretion andADP increases cGMP synthesis, lead-ing to greatly increased granule secretion and stabilization ofplatelet aggregation in the presence of low concentrations ofagonists.

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Src Family Kinases Stimulate Platelet Secretion

12570 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 17 • APRIL 23, 2010

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Page 13: AnImportantRoleoftheSrcFamilyKinaseLyninStimulating ... · kinase C and calcium-dependent signaling pathways and require the stimulation of formation of the SNARE complex, which mediates

Clifford A. Lowell and Xiaoping DuZhenyu Li, Guoying Zhang, Junling Liu, Aleksandra Stojanovic, Changgeng Ruan,

SecretionAn Important Role of the Src Family Kinase Lyn in Stimulating Platelet Granule

doi: 10.1074/jbc.M109.098756 originally published online February 26, 20102010, 285:12559-12570.J. Biol. Chem. 

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