antibodies and reagents  anti - human gpvi monoclonal antibody 
  ( mab ) 204 - 11 has been described previously 
  ( 25 ) . anti - syk polyclonal ab 
  ( pab ) ( 26 ) was kindly provided 
  by j. b. bolen ( dnax , ca ) .
anti - phospho - syk ( tyr ) pab and 
  anti - myc mab were purchased from cell signaling technology ( new england 
  biolabs uk ltd . ,
anti - phosphotyrosine mab 4g10 , anti - fcr -chain pab , 
  and normal rabbit igg were purchased from upstate biotechnology ( milton 
  keynes , uk ) .
anti - pecam-1 mab ab468 was from autogen - bioclear 
  ( wiltshire , uk ) .
horseradish peroxidase - conjugated donkey anti - rabbit 
  secondary ab and enhanced chemiluminescence reagents ( ecl ) were purchased from 
  amersham biosciences .
fluorescein isothiocyanate - conjugated anti - mouse igg 
  secondary antibody was purchased from sigma .
the src kinase 
  inhibitors used were , pp1 , purchased from biosource europe ( nivelles , 
  belgium ) ; pp2 , purchased from calbiochem ( nottingham , uk ) ; and pd0173952 , a 
  gift from pfizer global research and development ( ann arbor , mi ) .
fcr -chain  knock - out  mice were bred as 
  heterozygotes as described 
  ( 28 ) .
10 mm 
  pervanadate was freshly prepared on the day for use by mixing sodium 
  orthovanadate and hydrogen peroxide in phosphate - buffered saline to final 
  concentrations 10 mm , then left for 5 min at room temperature and 
  kept on ice .
other reagents were from previously described sources 
  ( 6 , 
  8 , 
  24 ) .
constructs  the human prc / gpvi , pef6/fcr -chain , 
  pef6/clec-2 , and mutant clec-2 ( y7f ) expression plasmids have been previously 
  described ( 8) . the human 
  pcdna3/-g6b - b ( kindly given by prof r. d. campbell , oxford , uk ) has been 
  described ( 9 ) and the human 
  pcdna3/pecam-1 ( kindly given by c. d. buckley , birmingham , uk ) has also been 
  described ( 29 ) .
the nfat 
  luciferase reporter containing three copies of the distal nfat site from the 
  interleukin-2 promoter has been described 
  ( 30 ) . making myc - tagged fcr -chain
 the c - myc 
  epitope tag ( eqkliseedl ) was fused at the amino terminus of fcr -chain 
  by using pef6/fcr -chain as a template and the primers : forward 
  ( 5-gaa caa aaa ctc atc tca gaa gag gat ctg ctg gga gag cct cag 
  ctc-3 ) and reverse ( 5-cag atc ctc ttc tga gat gag ttt ttg ttc 
  ggc cgc tgc ttg ttc aac-3 ) , and subcloned into pef6 .
site - directed mutagenesis of g6b - b  site - directed mutagenesis 
  of g6b - b was performed by a quikchange site - directed mutagenesis kit 
  ( stratagene , cambridge , uk ) .
the primers g6b - b - y211f - forward ( 5-ccg agc 
  ctg ctc ttt gcg gat ctg gac-3 ) and g6b - b - y211f - reverse ( 5-gtc 
  cag atc cgc aaa gag cag gct cgg-3 ) were used for tyrosine to 
  phenylalanine mutation in g6b - b ( y211f ) using wild - type human pcdna / g6b - b as a 
  template .
the primers g6b - b - y237f - forward ( 5-gat gcc tcc acc atc ttt 
  gca gtt gta gtt tg-3 ) and g6b - b - y237f - reverse ( 5-caa act aca act 
  gca aag atg gtg gag gca tc-3 ) were used for tyrosine to phenylalanine 
  mutation in g6b - b ( y237f ) using wild - type human pcdna / g6b - b as a template and 
  they were also used for tyrosine to phenylalanine mutation in g6b - b 
  ( y211f / y237f ) using mutant pcdna / g6b - b ( y211f ) as a template .
cell culture  wild - type ( wt ) , syk - deficient 
  ( 31 ) , shp1 and shp2 
  double - deficient ( 32 ) ( kindly 
  donated by l. meyaard , utrecht , the netherlands ) , ship - deficient 
  ( 33 ) ( kindly donated by d. k. 
  newman , milwaukee , wi ) dt40 chicken b cells were grown in rpmi supplemented 
  with 10% fetal bovine serum , 1% chicken serum , 100 units / ml penicillin , 100 
  g / ml streptomycin , 50 m -mercaptoethanol , and 20 
  mm glutamine .
luciferase assay  the nfat reporter assay was performed as 
  described ( 8 , 
  24 ) .
the indicated amount of 
  dna of each construct and 15 g of nfat - luciferase reporter construct were 
  transfected by electroporation at 350 v and 500 microfarads into 2  
  10 cells of wt , syk - deficient , and shp1/shp2-deficient dt40 cells . 
 
twenty hours after transfection , live cells were counted by trypan blue 
  exclusion , and samples divided for luciferase assay ( 2  10 
  cells / ml ) , flow cytometry ( 5  10 cells / sample ) , and western 
  blotting ( 1  10 cells / sample ) .
collagen was used at 10 
  g / ml and rhodocytin was used at 50 nm .
luciferase activity was 
  measured with a centro lb960 microplate luminometer ( berthold technologies , 
  germany ) .
cell surface expression of transfected cells 
  was analyzed by flow cytometry using 1 g / ml , gpvi mab , pecam-1 mab , and 
  myc mab to detect clec-2 and fcr -chain , t7-tag mab to detect g6b - b , or 
  mouse igg followed by staining with 4 g / ml fluorescein 
  isothiocyanate - conjugated anti - mouse igg secondary antibody , and assessed on a 
  facscalibur ( becton dickinson , san jose , ca ) .
human and mouse platelets  washed preparations of human and 
  mouse platelets were prepared as previously described 
  ( 5 , 
  8) .
platelets were resuspended 
  in a modified tyrodes - hepes buffer at concentrations of 4  
  10/ml ( human ) or 2  10/ml ( mouse ) . platelets 
  were prewarmed to 37 c for 5 min and incubated with inhibitors or solvent 
  controls for up to 10 min .
immunoprecipitation and western blotting  transfected cells 
  ( 1  10/ml ) were incubated for 30 min in rpmi at 37 c 
  before stimulating .
after stimulation , transfected cells or platelets were 
  lysed with ice - cold 2 lysis buffer ( 2% triton x-100 , 2% dodecyl 
  maltoside , 4 mm 4-(2-aminoethyl)benzenesulfonyl fluoride , 20 
  g / ml aprotinin , 20 g / ml leupeptin , 2 g / ml pepstatin , 10 
  mm sodium orthovanadate , ph 7.5 ) and insoluble material was removed 
  by centrifugation .
for immunoprecipitation , lysates were precleared with 
  protein a ( g)-sepharose beads for 30 min at 4 c and mixed with 2 g of 
  the indicated antibodies and protein a - sepharose beads ( protein g - sepharose ) . 
 
whole cell lysates or 
  immunoprecipitated lysates were added to 2 laemmli sample buffer . 
 
samples were separated by sds - page on 10 or 412% bistris gels 
  ( invitrogen ) and transferred to polyvinylidene difluoride membrane .
statistical analysis  experiments were performed on at least 
  three occasions and results are shown as mean  s.e . with the exception 
  of the representative western blots and flow cytometry histograms .
constitutive signaling of gpvi - fcr -chain in dt40 
  cells  we set out to extend our previous characterization of the 
  gpvi - fcr -chain signaling pathway in the dt40 b cell line 
  ( 24 ) by testing whether 
  activation of gpvi by collagen is dependent on its associated fcr 
  -chain .
activation was monitored in the absence and presence of 
  collagen and compared with mock - transfected cells that had been allowed to 
  settle on fibronectin using a nfat - luciferase reporter assay .
adhesion to 
  fibronectin alone did not induce nfat activation ( not shown ) . as anticipated , 
  collagen stimulated nfat activation in dt40 cells expressing both gpvi and an 
  nh2-terminal myc - tagged version of fcr -chain , whereas it 
  had no effect when either subunit was transfected on its own 
  ( fig .
the myc - tagged version of fcr -chain supports a similar level 
  of activation to that of the wild - type protein ( not shown ) and has the 
  advantage that it can be used to monitor surface expression by flow cytometry , 
  which is not altered by expression of gpvi 
  ( fig .
, gpvi was not expressed on the surface of dt40 cells in 
  the absence of the fcr -chain ( not shown ) , as is the case for platelets 
  ( 34 ) .
these results are 
  consistent with a model in which cross - linking of gpvi by collagen leads to 
  tyrosine phosphorylation of the fcr -chain and activation of the 
  tyrosine kinase syk .
these studies further revealed the unexpected observation that expression 
  of the myc - tagged or wild - type fcr -chain caused a significant increase 
  in luciferase activity that approached almost 10% of the response to collagen 
  ( fig .
furthermore , the constitutive signal from the fcr 
  -chain was not altered by co - expression with gpvi 
  ( fig .
thus the increase in luciferase activity that was observed in the 
  absence of a stimulatory agonist reflects signaling by the itam - containing 
  protein . to investigate constitutive signaling in further detail ,
dt40 cells were 
  transfected with varying concentrations of the fcr -chain plasmid and 
  the level of nfat activation monitored .
increasing levels of transfection of 
  fcr -chain led to a corresponding increase in both nfat activation 
  ( fig .
1b , panel 
  i ) and fcr -chain expression as shown by western blotting 
  ( fig .
furthermore , western blotting of gpvi - fcr -chain - transfected 
  cells demonstrated an increase in tyrosine phosphorylation of a band of 72 kda 
  that comigrates with syk , along with constitutive tyrosine phosphorylation of 
  fcr -chain ( fig . 
 
confirmation that the 72-kda 
  protein corresponds to syk was confirmed using a phosphospecific antibody 
  ( phospho - syk tyr ) that binds to the activated tyrosine 
  kinase ( not shown ) .
tyrosine phosphorylation of syk and fcr -chain were 
  inhibited in the presence of the src family kinase inhibitor pd0173952 
  ( fig .
these results demonstrate that the fcr -chain 
  generates constitutive signals in dt40 cells that lead to tyrosine 
  phosphorylation of syk and nfat activation .
stimulation of gpvi - fcr 
  -chain - transfected cells with collagen leads to a further increase in 
  tyrosine phosphorylation of syk ( fig . 
  1c , panel ii ) , consistent with the activation of 
  syk by the collagen receptor complex , as described in earlier studies .
the above 
  studies were extended to clec-2 , which is a recently identified platelet 
  glycoprotein receptor that mediates activation through a single yxxl 
  motif in its cytosolic tail .
clec-2 is a receptor for the snake venom toxin , 
  rhodocytin , and the lymphatic marker podoplanin .
both agonists induce 
  activation of clec-2-transfected dt40 cells , with the response being dependent 
  on the conserved tyrosine in the cytoplasmic yxxl motif 
  ( 7 , 
  8) . in the present study
, we have confirmed that rhodocytin stimulates nfat 
  activation in clec-2-transfected dt40 cells , with the level of response 
  increasing with the level of surface expression of clec-2 
  ( fig .
moreover , as is the case with the fcr -chain , 
  expression of clec-2 was sufficient to increase nfat activity in the absence 
  of agonist stimulation , with the level of response increasing in parallel with 
  that of clec-2 ( fig . 
 
the level of 
  constitutive activity approached between 10 and 20% the response to 
  rhodocytin .
constitutive signaling by clec-2 is abolished following mutation 
  of the tyrosine in the clec-2 yxxl sequence to a phenylalanine ( y7f ) 
  ( fig .
2b , panel 
  i ) , as is the case for stimulation by rhodocytin 
  ( 8) .
flow cytometry confirmed a 
  similar level of expression of wild - type and the y7f mutant of clec-2 in these 
  studies ( fig .
these results demonstrate that constitutive and 
  agonist - induced signaling by clec-2 is dependent on the yxxl 
  motif .
a , panel i , constitutive and collagen - induced signaling in dt40 cells 
  transfected with plasmids ( 10 g ) encoding gpvi and/or myc - fcr 
  -chain ( fcr ) was analyzed using an nfat - luciferase reporter 
  assay .
cell lysates were analyzed for luciferase activity following incubation 
  with media or collagen ( 10 g / ml ) for 6 h. data are expressed as -fold 
  increase over the basal of mock - transfected cells .
panel ii , gpvi and fcr 
  surface expression was measured by flow cytometry following transfection with 
  10 g of plasmid of gpvi / myc - fcr or myc - fcr only 
  ( corresponding to a , panel i ) relative to a control igg .
data are 
  representative of three experiments.b , constitutive signaling by the 
  fcr alone .
panel ii , western blotting ( wb ) for 
  fcr -chain in dt40 cells transfected with increasing amounts of fcr 
  -chain plasmid .
c , 
  panel i , constitutive tyrosine phosphorylation of syk and fcr. 
  mock and gpvi - fcr-transfected dt40 cells were pre - treated with 
  pd0173952 ( 20 m ) for 5 min .
whole cell lysates were western 
  blotted for tyrosine phosphorylation using mab 4g10 and then reprobed for syk 
  and fcr. tyrosine phosphorylated bands that comigrate with syk and 
  fcr are shown .
panel ii , tyrosine phosphorylation of syk in 
  gpvi - fcr-transfected dt40 cells lysates by collagen ( 30 g / ml ) is 
  partially inhibited by cotransfection with g6b - b .
cells were stimulated by 
  collagen for 90 s. experimental conditions were as for panel i. data 
  are representative of three experiments .
a , panel i , constitutive and collagen - induced signaling in dt40 cells 
  transfected with plasmids ( 10 g ) encoding gpvi and/or myc - fcr 
  -chain ( fcr ) was analyzed using an nfat - luciferase reporter 
  assay .
cell lysates were analyzed for luciferase activity following incubation 
  with media or collagen ( 10 g / ml ) for 6 h. data are expressed as -fold 
  increase over the basal of mock - transfected cells .
panel ii , gpvi and fcr 
  surface expression was measured by flow cytometry following transfection with 
  10 g of plasmid of gpvi / myc - fcr or myc - fcr only 
  ( corresponding to a , panel i ) relative to a control igg .
data are 
  representative of three experiments.b , constitutive signaling by the 
  fcr alone .
panel ii , western blotting ( wb ) for 
  fcr -chain in dt40 cells transfected with increasing amounts of fcr 
  -chain plasmid .
c , 
  panel i , constitutive tyrosine phosphorylation of syk and fcr. 
  mock and gpvi - fcr-transfected dt40 cells were pre - treated with 
  pd0173952 ( 20 m ) for 5 min .
whole cell lysates were western 
  blotted for tyrosine phosphorylation using mab 4g10 and then reprobed for syk 
  and fcr. tyrosine phosphorylated bands that comigrate with syk and 
  fcr are shown .
panel ii , tyrosine phosphorylation of syk in 
  gpvi - fcr-transfected dt40 cells lysates by collagen ( 30 g / ml ) is 
  partially inhibited by cotransfection with g6b - b .
cells were stimulated by 
  collagen for 90 s. experimental conditions were as for panel i. data 
  are representative of three experiments .
constitutive signaling by gpvi - fcr -chain and clec-2 is 
  mediated by src and syk tyrosine kinases  we and others have 
  previously reported that collagen stimulates phosphorylation of the fcr 
  -chain itam in platelets through the src kinases , fyn and lyn , leading 
  to recruitment and tyrosine phosphorylation of syk 
  ( 3537 ) . 
 
similarly , phosphorylation of clec-2 and syk by rhodocytin in platelets is 
  mediated through a src kinase pathway , although the identity of the src kinase 
  is not known ( 5 ) .
consistent 
  with this , we observed constitutive and collagen - stimulated tyrosine 
  phosphorylation of syk in gpvi - fcr -chain - transfected dt40 cells 
  ( fig .
, we have not been able to detect constitutive phosphorylation of 
  clec-2 in our studies , possibly because of a lower level of expression 
  relative to that of fcr -chain , as shown using a myc - tagged version of 
  both proteins ( supplemental fig .
s1 ) , and because the c - type lectin receptor 
  has a single yxxl motif .
however , we have observed a small increase 
  in constitutive phosphorylation of syk in dt40 cells expressing clec-2 , which 
  was further increased in the presence of rhodocytin ( not shown ) .
experiments were designed to investigate whether nfat signaling by the 
  gpvi - fcr -chain complex and clec-2 in transfected dt40 cells is 
  mediated through src and syk kinases . as shown in 
  fig .
3a , constitutive 
  and agonist - induced nfat activation by both receptors is blocked in the 
  presence of the src family kinase inhibitor pd0173952 . furthermore , 
  constitutive and agonist - induced signaling by the two receptors was also 
  markedly inhibited in the absence of syk 
  ( fig .
thus , these results demonstrate that constitutive and agonist - induced 
  signaling by gpvi - fcr -chain and clec-2 are both dependent on src 
  family and syk tyrosine kinases .
g6b - b inhibits constitutive and agonist - induced signaling by 
  gpvi - fcr -chain and clec-2 through its two itims  a 
  series of experiments were undertaken to investigate the possible regulation 
  of constitutive and agonist - induced signaling through the gpvi - fcr 
  -chain complex and clec-2 by the platelet immunoglobulin receptor 
  g6b - b , which contains two itims in its cytosolic tail 
  ( 9 , 
  10 ) .
xpression of g6b - b 
  significantly inhibited constitutive signaling by both receptors by more than 
  60% ( fig .
4 , panel i ) , 
  as well as inhibiting the response to collagen and rhodocytin by 35 and 
  60% , respectively ( fig . 4 , 
 
. the greater degree of inhibition of the response to 
  rhodocytin may reflect the lower level of expression of clec-2 relative to 
  gpvi - fcr -chain ( supplemental fig .
s1 ) . expression of g6b - b had no 
  effect on the level of expression of either clec-2 or gpvi ( supplemental fig . 
 
expression of g6b - b also partially inhibited collagen - induced tyrosine 
  phosphorylation of syk ( fig . 
  1c , panel ii ) , demonstrating a molecular basis 
  for its inhibitory action .
these results demonstrate that g6b - b inhibits 
  constitutive and agonist - induced signaling induced by gpvi - fcr -chain 
  and clec-2 .
figure 2.constitutive signaling by clec-2 in dt40 cells is dependent on its 
  yxxl motif .
a , panel i , constitutive and 
  rhodocytin - induced signaling in dt40 cells transfected with varying amounts of 
  the clec-2 plasmid was analyzed by nfat - luciferase reporter assay .
cells were 
  stimulated with 50 nm rhodocytin for 6 h. values are mean  
  s.e .
panel ii , clec-2 surface 
  expression following transfection with varying amounts of clec-2 plasmid was 
  assessed by flow cytometry using anti - myc tag mab relative to a control igg . 
 
b , panel i , 
  constitutive and rhodocytin - induced signaling in cells transfected with 
  wild - type ( wt ) and mutant ( y7f ) clec-2 .
panel ii , clec-2 surface expression 
  ( corresponding to panel i ) was assessed by flow cytometry using 
  anti - myc tag mab relative to a control igg .
a , panel i , constitutive and 
  rhodocytin - induced signaling in dt40 cells transfected with varying amounts of 
  the clec-2 plasmid was analyzed by nfat - luciferase reporter assay .
cells were 
  stimulated with 50 nm rhodocytin for 6 h. values are mean  
  s.e .
panel ii , clec-2 surface 
  expression following transfection with varying amounts of clec-2 plasmid was 
  assessed by flow cytometry using anti - myc tag mab relative to a control igg . 
 
b , panel i , 
  constitutive and rhodocytin - induced signaling in cells transfected with 
  wild - type ( wt ) and mutant ( y7f ) clec-2 .
panel ii , clec-2 surface expression 
  ( corresponding to panel i ) was assessed by flow cytometry using 
  anti - myc tag mab relative to a control igg .
g6b - b contains two itims in its cytosolic tail , which independently mediate 
  binding to shp1 and shp2 following incubation with the protein - tyrosine 
  phosphatase inhibitor , pervanadate 
  ( 9 ) .
mutation of the two 
  conserved tyrosines at positions 211 and 237 to phenylalanine inhibits 
  association with the two sh2 domain - containing protein - tyrosine phosphatases 
  ( 9 ) . to investigate whether 
  either or both of the itims mediate the inhibitory effect of g6b - b , the 
  y211f / y237f double mutant was expressed in dt40 cells together with the 
  gpvi - fcr -chain complex and clec-2 .
flow cytometry was used to confirm 
  similar levels of expression of the g6b - b mutant , gpvi and clec-2 in the 
  transfected cell lines to that in cells transfected with wild - type g6b - b and 
  the two yxxl - containing receptors ( supplemental fig .
mutation of the two conserved tyrosines in the g6b - b itims abolished the 
  inhibitory effect of g6b - b against constitutive and agonist - induced activation 
  of nfat induced by gpvi - fcr -chain and clec-2 
  ( fig .
4 ) , whereas mutation of 
  either of the two tyrosines alone had a partial or neglible effect ( not 
  shown ) . in line with this
, we were able to detect weak tyrosine 
  phosphorylation of g6b - b in the dt40-transfected cells ( not shown ) .
these 
  results demonstrate that g6b - b inhibits constitutive and agonist - induced 
  signaling through the conserved tyrosines at positions 211 and 237 .
the ability of g6b - b to inhibit constitutive and agonist - induced responses 
  mediated through gpvi - fcr -chain and clec-2 was further investigated in 
  the combined absence of the two sh2 domain - containing tyrosine phosphatases , 
  shp1 and shp2 , or in the absence of the 5-inositol phosphatase , ship . 
 
unexpectedly , the absence of the two protein - tyrosine phosphatases led to an 
  increase in the level of constitutive and agonist - induced nfat activation 
  through gpvi - fcr -chain and clec-2 , suggesting that both receptors are 
  under dynamic inhibitory feedback through the two tyrosine phosphatases 
  ( fig .
nevertheless , g6b - b was still able to inhibit constitutive and agonist - induced 
  signaling through gpvi - fcr -chain and clec-2 in the absence of shp-1 
  and shp-2 ( fig .
5a ) . 
  indeed , the inhibitory action against rhodocytin was enhanced in the combined 
  absence of shp1 and shp2 ( fig . 
 
5a ) , although this may reflect the increased response to 
  rhodocytin that is observed in the absence of the two protein - tyrosine 
  phosphatases .
g6b - b also inhibits constitutive and agonist - induced nfat 
  activation by gpvi - fcr -chain and clec-2 in dt40 cells deficient in the 
  sh2-domain - containing inositol 5-phosphatase ( ship ) 
  ( fig .
indeed the 
  inhibitory action of g6b - b against clec-2 was also enhanced by the absence of 
  the 5-inositol phosphatase ( fig . 
 
5b ) , as was the case in the absence of shp1 and shp2 . 
 
these results demonstrate that g6b - b inhibits gpvi and clec-2 signaling in 
  dt40 cells by a shp1/shp2-independent and ship - independent mechanism .
figure 3.constitutive and agonist - induced signaling by gpvi - fcr -chain and 
  clec-2 is dependent on src and syk tyrosine kinases .
constitutive and 
  agonist - induced signaling in wild - type ( wt ) and syk - deficient dt40 
  cells transfected with gpvi - fcr-chain or clec-2 was analyzed using an 
  nfat - luciferase reporter assay .
cell lysates were analyzed for luciferase 
  activity following agonist stimulation for 6 h. a , wt dt40 cells were 
  either mock - transfected or transfected with ( panel i ) gpvi - fcr 
  -chain ( 10 g of each plasmid ) or ( panel ii ) clec-2 ( 10 
  g ) and stimulated with 10 g / ml collagen or 50 nm rhodocytin 
  in the presence and absence of the src kinase inhibitor , pd0173952 ( 20 
  m ) .
b , basal signaling in wt or syk - deficient dt40 
  cells transfected with mock or transfected with ( panel i ) gpvi - fcr 
  -chain ( 10 g of each plasmid ) or ( panel ii ) clec-2 ( 10 
  g ) and incubated for 6 h to monitor the degree of constitutive signaling . 
  values are mean  s.e .
constitutive and agonist - induced signaling by gpvi - fcr -chain and 
  clec-2 is dependent on src and syk tyrosine kinases .
constitutive and 
  agonist - induced signaling in wild - type ( wt ) and syk - deficient dt40 
  cells transfected with gpvi - fcr-chain or clec-2 was analyzed using an 
  nfat - luciferase reporter assay .
cell lysates were analyzed for luciferase 
  activity following agonist stimulation for 6 h. a , wt dt40 cells were 
  either mock - transfected or transfected with ( panel i ) gpvi - fcr 
  -chain ( 10 g of each plasmid ) or ( panel ii ) clec-2 ( 10 
  g ) and stimulated with 10 g / ml collagen or 50 nm rhodocytin 
  in the presence and absence of the src kinase inhibitor , pd0173952 ( 20 
  m ) .
b , basal signaling in wt or syk - deficient dt40 
  cells transfected with mock or transfected with ( panel i ) gpvi - fcr 
  -chain ( 10 g of each plasmid ) or ( panel ii ) clec-2 ( 10 
  g ) and incubated for 6 h to monitor the degree of constitutive signaling . 
 
figure 4.the novel platelet itim transmembrane protein g6b - b inhibits 
  constitutive and agonist - induced signaling through gpvi and clec-2 .
the 
  effect of the wild - type ( wt ) and double itim - mutant transmembrane 
  protein g6b - b ( y211f / y237f ) on ( panel i ) constitutive and ( panel 
  ii ) agonist - induced signaling by gpvi - fcr -chain and clec-2 in 
  transfected ( 10 g of each plasmid ) dt40 cells , measured using an 
  nfat - luciferase reporter assay .
cells were stimulated with 10 g / ml 
  collagen or 50 nm rhodocytin as appropriate .
the double itim mutant 
  contained inactivating point mutations ( y / f ) at tyrosines 211 and 237 .
the 
  results are mean  s.e . of between 4 and 7 independent experiments . 
 
* , 
  p < 0.05 ; and * * , p < 0.01 compared with the gpvi - fcr 
  -chain or clec-2-transfected samples .
the novel platelet itim transmembrane protein g6b - b inhibits 
  constitutive and agonist - induced signaling through gpvi and clec-2 .
the 
  effect of the wild - type ( wt ) and double itim - mutant transmembrane 
  protein g6b - b ( y211f / y237f ) on ( panel i ) constitutive and ( panel 
  ii ) agonist - induced signaling by gpvi - fcr -chain and clec-2 in 
  transfected ( 10 g of each plasmid ) dt40 cells , measured using an 
  nfat - luciferase reporter assay .
cells were stimulated with 10 g / ml 
  collagen or 50 nm rhodocytin as appropriate .
the double itim mutant 
  contained inactivating point mutations ( y / f ) at tyrosines 211 and 237 .
the 
  results are mean  s.e . of between 4 and 7 independent experiments . 
 
* , 
  p < 0.05 ; and * * , p < 0.01 compared with the gpvi - fcr 
  -chain or clec-2-transfected samples . figure 5.the inhibitory effect of g6b - b is independent of shp1 , shp2 , and 
  ship .
constitutive and agonist - induced signaling in shp1/shp2 
  double - deficient ( a ) and ship - deficient dt40 cells ( b ) 
  transfected with gpvi - fcr -chain , clec-2 , and g6b - b was analyzed using 
  a nfat - luciferase reporter assay .
cells were stimulated with 10 g / ml 
  collagen or 50 nm rhodocytin for 6 h. * , p < 0.05 
  relative to response in the absence of g6b - b .
the inhibitory effect of g6b - b is independent of shp1 , shp2 , and 
  ship .
constitutive and agonist - induced signaling in shp1/shp2 
  double - deficient ( a ) and ship - deficient dt40 cells ( b ) 
  transfected with gpvi - fcr -chain , clec-2 , and g6b - b was analyzed using 
  a nfat - luciferase reporter assay .
cells were stimulated with 10 g / ml 
  collagen or 50 nm rhodocytin for 6 h. * , p < 0.05 
  relative to response in the absence of g6b - b .
pecam-1 does not inhibit constitutive signaling in dt40 
  cells  a similar set of studies were undertaken to investigate 
  whether the major platelet itim receptor , pecam-1 , also inhibits constitutive 
  and agonist - induced activation of nfat downstream of gpvi - fcr -chain 
  and clec-2 .
s3a , pecam-1 had no effect 
  on constitutive or agonist - induced activation of either receptor in dt40 
  cells , possibly because of a low level of expression ( supplemental fig . 
 
a , human washed platelets were incubated at 37 
  c with the syk kinase inhibitor , r406 ( 1 m ) , and src 
  inhibitor pp2 ( 20 m ) for 1 min .
b , wild - type ( wt ) or 
  fcr -chain deficient mouse ( ko ) platelets ( pretreated with 
  egta to block aggregation ) were stimulated with 100 m 
  pervanadate ( pv ) for 90 s in the presence or absence of src inhibitor 
  pp2 ( 20 m ) .
c , human washed platelets were incubated 
  at 37 c with the src kinase inhibitors pp1 ( 10 m ) or 
  pd0173952 ( 10 m ) for 5 min .
cell lysates were 
  immunoprecipitated with anti - syk ab or an igg and probed for tyr(p ) . 
 
d , human 
  washed platelets were stimulated with 30 nm rhodocytin for 5 min in 
  the presence and absence of the src kinase inhibitor , pp2 ( 10 
  m ) .
cell lysates were immunoprecipitated ( ip ) with 
  anti - clec-2 ab and probed for tyr(p ) .
a , human washed platelets were incubated at 37 
  c with the syk kinase inhibitor , r406 ( 1 m ) , and src 
  inhibitor pp2 ( 20 m ) for 1 min .
b , wild - type ( wt ) or 
  fcr -chain deficient mouse ( ko ) platelets ( pretreated with 
  egta to block aggregation ) were stimulated with 100 m 
  pervanadate ( pv ) for 90 s in the presence or absence of src inhibitor 
  pp2 ( 20 m ) .
c , human washed platelets were incubated 
  at 37 c with the src kinase inhibitors pp1 ( 10 m ) or 
  pd0173952 ( 10 m ) for 5 min .
cell lysates were 
  immunoprecipitated with anti - syk ab or an igg and probed for tyr(p ) . 
 
d , human 
  washed platelets were stimulated with 30 nm rhodocytin for 5 min in 
  the presence and absence of the src kinase inhibitor , pp2 ( 10 
  m ) .
cell lysates were immunoprecipitated ( ip ) with 
  anti - clec-2 ab and probed for tyr(p ) .
src and syk kinase - dependent constitutive signaling in 
  platelets  experiments were designed to investigate whether there is 
  evidence for constitutive signaling through src and syk kinases in platelets . 
  in this context , it is important to emphasize that platelets express several 
  classes of receptor that signal through src and syk tyrosine kinases , 
  including the major platelet integrin , iib3 , and the gpib - ix - v 
  complex , the levels of which are over 1 order of magnitude greater than that 
  of gpvi - fcr -chain and clec-2 .
we first asked the question as to whether the tyrosine phosphorylation that 
  is seen in the absence of agonist stimulation in platelets is regulated by src 
  or syk tyrosine kinases .
western blotting for phosphotyrosine using the 
  monoclonal antibody 4g10 reveals the presence of a broad band of proteins in 
  non - stimulated platelets of between 50 and 60 kda 
  ( fig .
this region includes src 
  kinases , which are constitutively phosphorylated in resting platelets by the 
  src kinase - regulatory protein , csk , at an inhibitory site that promotes 
  intramolecular binding to the sh2 domain .
in addition , several other proteins , 
  which have been shown to be constitutively phosphorylated in nonstimulated 
  platelets , migrate in this region , including dok-2 
  ( 38 ) .
additional 
  phosphorylation bands that lie outside of this region can also be seen in 
  non - stimulated platelets in longer exposures , as illustrated in 
  fig .
tyrosine 
  phosphorylation of the majority of these bands is markedly inhibited in the 
  presence of the src family kinase inhibitors , pp1 and pp2 , and the syk family 
  kinase inhibitor , r406 ( 39 ) , 
  with the exception of the broad band of proteins that runs at 5060 kda , 
  which is only partially reduced ( fig . 
 
the residual phosphorylation that is 
  seen in this region is likely to reflect phosphorylation of src kinases on 
  their inhibitory site by csk .
significantly , csk is not known to be regulated 
  downstream of src and syk kinases .
the reduction in phosphorylation in this 
  region is presumably due to loss of phosphorylation of proteins such as dok-2 , 
  as discussed above .
the similar pattern of reduction in tyrosine 
  phosphorylation that is induced by r406 to that of pp2 can not be due to direct 
  inhibition of src kinases , as r406 does not inhibit collagen - stimulated 
  tyrosine phosphorylation of the fcr 
  -chain,5 which 
  is mediated through src kinase activation .
these results demonstrate that 
  constitutive signaling by src and syk family kinases underlies tyrosine 
  phosphorylation of the majority of proteins in non - stimulated platelets , 
  thereby providing evidence of constitutive signaling by src and syk 
  kinases .
we used the protein - tyrosine phosphatase inhibitor , pervanadate , to further 
  investigate the contribution of src and syk family kinases to constitutive 
  tyrosine phosphorylation in human and mouse platelets , on the grounds that 
  this would potentiate protein tyrosine phosphorylation by constitutively 
  active tyrosine kinases .
6b ) , with 
  similar observations made for human platelets ( not shown ) . as previously 
  reported 
  ( 4042 ) , 
  pervanadate induces a marked increase in tyrosine phosphorylation of a large 
  number of proteins in platelets ( fig . 
 
strikingly , protein tyrosine phosphorylation induced 
  by pervanadate was dramatically inhibited in the presence of the src kinase 
  inhibitor , pp2 , confirming that src family kinases are the major family of 
  kinases that contribute to constitutive tyrosine phosphorylation in platelets . 
 
a small number of tyrosine - phosphorylated proteins are seen in longer 
  exposures of pervanadate - treated platelets in the presence of pp2 , including a 
  band of 72 kda ( fig . 
 
6b ) that was identified as syk by immunoprecipitation of 
  the kinase and western blotting for phosphotyrosine 
  ( fig .
confirm that src kinases are constitutively active in nonstimulated platelets 
  and demonstrate that they underlie the major increase in tyrosine 
  phosphorylation that is induced in the presence of protein - tyrosine 
  phosphatase inhibition .
further experiments were designed to investigate the possible dependence of 
  constitutive signaling through src and syk family kinases on the gpvi - fcr 
  -chain complex . for these experiments ,
syk was immunoprecipitated from 
  resting human platelets and western blotted for phosphotyrosine in the absence 
  and presence of the src family kinase inhibitor , pd0173952 .
6c demonstrate 
  association of constitutively tyrosine - phosphorylated syk and constitutively 
  tyrosine - phosphorylated fcr -chain , which is dramatically reduced in 
  the presence of the src kinase inhibitor pd0173952 . on the other hand , 
  treatment of fcr -chain - deficient mouse platelets with
pervanadate 
  revealed that constitutive tyrosine phosphorylation in mouse platelets is 
  minimally altered in platelets deficient in the itam - containing protein 
  ( fig .
6b ) , 
  demonstrating that the gpvi - fcr -chain complex makes a relatively minor 
  contribution to the overall level of tyrosine phosphorylation .
constitutive phosphorylation of clec-2 is observed in human platelets and 
  is reduced in the presence of the src kinase inhibitor , pp2 
  ( fig .
the 
  contribution of clec-2 to constitutive tyrosine phosphorylation in mouse 
  platelets can not be assessed , as mice lacking the c - type lectin receptor have 
  not been made .
the present study demonstrates that the gpvi - fcr -chain complex and 
  clec-2 generate constitutive signals in transfected dt40 cells leading to nfat 
  activation with the degree of signaling corresponding to the level of 
  expression .
furthermore , the dependence on src and syk family kinases provides 
  strong evidence that constitutive signaling by both receptors is mediated 
  through the same pathway as used by agonists to mediate activation . in line 
  with this ,
constitutive signaling by clec-2 is inhibited by mutation of the 
  conserved tyrosine in the yxxl sequence in its cytosolic tail . 
 
furthermore , the response to the gpvi - fcr -chain complex is mediated by 
  fcr -chain , and is independent of gpvi , consistent with it being 
  generated through the tandem yxxl sequence of the fcr 
  -chain .
constitutive and agonist - induced signaling through gpvi - fcr -chain 
  and clec-2 is reduced by co - expression of the itim - containing platelet 
  protein , g6b - b ( 9 ) .
the 
  inhibitory effect of g6b - b is dependent on the conserved tyrosines in its two 
  itims , as shown by abolition of the inhibitory response upon mutation of the 
  itim tyrosines to phenylalanine .
however , unexpectedly , this inhibitory effect 
  is retained in dt40 cells in the combined absence of the sh2 domain - containing 
  tyrosine phosphatases , shp1 and shp2 , which have been shown to bind to g6b - b 
  ( 9 , 
  10 ) .
the inhibitory effect of 
  g6b - b is also retained in the absence of the sh2 domain - containing inositol 
  phosphatase , ship , which has been shown to inhibit b cell receptor signaling 
  through association with the fcriib itim 
  ( 43 , 
  44 ) .
an alternative pathway of 
  inhibition could be through recruitment of the sh2 domain - containing inhibitor 
  of src kinases , csk , to the two itims in g6b - b .
such a mechanism has been 
  proposed to underlie the inhibition of fcri signaling by the 
  itim - containing transmembrane protein , lair-1 , in rat rbl mast cells 
  ( 45 ) .
however , we were unable 
  to demonstrate association of csk with g6b - b in transfected dt40 cells ( not 
  shown ) , possibly because of the low level of constitutive phosphorylation of 
  the itim - containing protein .
access to csk - deficient dt40 cells is required to 
  directly test the role of the src kinase regulatory protein in mediating the 
  inhibitory effect of g6b - b .
it is also possible that the inhibitory effect of 
  g6b - b is mediated by association with several proteins , including a 
  combination of shp1 , shp2 , ship , and csk , such that loss of any one is 
  compensated by expression of the others .
such a mechanism would explain why 
  g6b - b is still able to mediate inhibition in dt40 cells deficient in shp1 and 
  shp2 , which have both been shown to bind to its cytosolic itims . 
 
interestingly , we observed a small reduction in tyrosine phosphorylation of 
  syk by g6b - b in dt40 cells stimulated by collagen , suggesting that this 
  underlies the inhibitory effect of the itim - containing protein .
the observation that the fcr -chain and clec-2 generate constitutive 
  signals is in line with reports that itam receptors generate weak , 
  constitutive signals in other hematopoietic cells .
for example , in mouse 
  thymocytes and peripheral t cells , there is limited constitutive 
  phosphorylation of tcr chains leading to association with the 
  syk - related tyrosine kinase zap-70 
  ( 46 , 
  47 ) .
this gives rise to 
  constitutive signaling in resting mouse thymocyte and jurkat t cell lines , as 
  shown by suppression of rag gene expression via extracellular 
  signal - regulated kinase ( erk ) and abl kinases , and prevention of further 
  recombination of t cell receptor genes 
  ( 48 ) .
the b cell antigen 
  receptor also signals independently of ligand engagement and this provides 
  functionally relevant signals in immature b lymphocytes 
  ( 49 ) and in diffuse large 
  b - cell lymphoma ( 50 ) . 
 
significantly , this constitutive signaling is mediated through the b cell 
  receptor itams ( 51 , 
  52 ) .
thus , constitutive 
  signaling appears to be a common feature of yxxl - containing 
  receptors .
evidence of constitutive signaling through src and syk tyrosine kinases in 
  platelets has been demonstrated in the present study in association with a low 
  level of tyrosine phosphorylation of the fcr -chain and clec-2 .
studies 
  in mice deficient in the fcr -chain demonstrate that the gpvi receptor 
  complex makes only a minimal contribution to this phosphorylation , most likely 
  because of the association of src kinases with other surface receptors , 
  including the iib3 complex that is expressed at over 20 times the 
  level of gpvi .
the demonstration of constitutive signaling via src and syk kinases in 
  platelets emphasizes the need for inhibitory signals to oppose activation 
  occurring in healthy vessels .
we speculate that this function may be mediated , 
  at least in part , by the itim receptor , g6b - b , which is markedly 
  phosphorylated in resting platelets 
  ( 10 ) .
the ligand for g6b - b is 
  not known , but if this is expressed on platelets , it is possible that this may 
  account for the high level of constitutive phosphorylation of g6b - b through a 
  cis - interaction .
a similar function to that of g6b - b may be mediated in platelets by the 
  itim - containing receptor , pecam-1 .
this is in line with studies that have 
  reported inhibition of agonist - induced platelet activation by the 
  immunoglobulin receptor 
  ( 2123 ) . 
 
however , unexpectedly , we were unable to observe inhibition of constitutive 
  and agonist - induced signaling by pecam-1 in the dt40 cell line , possibly 
  because of too low a level of expression .
in summary , the present study reports that both fcr -chain and 
  clec-2 generate weak , sustained constitutive signals that are inhibited by 
  co - expression with g6b - b .
we speculate that this could represent an important 
  physiological role of g6b - b and other platelet itim proteins in helping to 
  prevent platelet activation in vivo .
 platelets play an essential role in wound healing by forming thrombi that 
  plug holes in the walls of damaged blood vessels . to achieve this , platelets 
  express a diverse array of cell surface receptors and signaling proteins that 
  induce rapid platelet activation . in this study 
 we show that two platelet 
  glycoprotein receptors that signal via an immunoreceptor tyrosine - based 
  activation motif ( itam ) or an itam - like domain , namely the collagen receptor 
  complex glycoprotein vi ( gpvi)-fcr -chain and the c - type lectin - like 
  receptor 2 ( clec-2 ) , respectively , support constitutive ( i.e. 
  agonist - independent ) signaling in a cell line model using a nuclear factor of 
  activated t - cells ( nfat ) transcriptional reporter assay that can detect low 
  level activation of phospholipase c ( plc ) . 
 constitutive and 
  agonist signaling by both receptors is dependent on src and syk family 
  kinases , and is inhibited by g6b - b , a platelet immunoglobulin receptor that 
  has two immunoreceptor tyrosine - based inhibitory motifs in its cytosolic tail . 
  
 mutation of the conserved tyrosines in the two immunoreceptor tyrosine - based 
  inhibitory motifs prevents the inhibitory action of g6b - b . 
 interestingly , the 
  inhibitory activity of g6b - b is independent of the src homology 2 ( sh2)-domain 
  containing tyrosine phosphatases , shp1 and shp2 , and the inositol 
  5-phosphatase , ship . 
 constitutive signaling via src and syk tyrosine 
  kinases is observed in platelets and is associated with tyrosine 
  phosphorylation of gpvi - fcr -chain and clec-2 . 
 we speculate that 
  inhibition of constitutive signaling through src and syk tyrosine kinases by 
  g6b - b may help to prevent unwanted platelet activation . 