water , the most abundant molecule in living cells , is required for various vital life functions including the regulation of cell and organelle volume .
changes in cell volume are known to occur during cell secretion , in erythrocyte maturation , during cell growth and differentiation , and in cell migration and metabolism .
similarly , intracellular swelling of membrane - bound secretory vesicles [ 4 , 5 ] has been demonstrated in the regulated release of vesicular contents during cell secretion .
all these studies demonstrate that rapid swelling of cells or secretory vesicles occurs as a result of rapid water entry through membrane - associated water channels or aqps .
platelets are 23 m in diameter anuclear cells that contain lysosomes ,  granules and dense granules , and secrete a variety of essential molecules including growth factors .
impaired platelet function is causal to bleeding or clotting , resulting in adverse outcomes such as myocardial infarction , stroke or pulmonary embolism . because regulation of platelet volume significantly affects its function , this study was undertaken to determine the molecular mechanism of platelet volume regulation .
volume regulation in cells and secretory vesicles has previously been examined [ 4 , 5 ] .
live pancreatic acinar cells in near physiological buffer , when imaged using the atomic force microscope ( afm ) , revealed at nanometre resolution the presence of membrane - bound secretory vesicles called zymogen granules ( zgs ) lying immediately below the surface of the apical plasma membrane . within 2.5 min .
of exposure to a secretory stimulus , the majority of zgs within cells swell , followed by a decrease in size , and a concomitant discharge of intra - vesicular contents .
these studies directly demonstrate intracellular swelling of secretory vesicles following stimulation of cell secretion , and vesicle deflation following partial discharge of vesicular contents .
a similar mechanism of synaptic vesicle ( sv ) swelling for neurotransmitter release has also been reported .
this direct estimation of vesicle size dynamics at nanometre resolution under various experimental conditions has enabled determination of some of the molecular components participating in secretory vesicle swelling .
studies using isolated zgs demonstrate the presence of cl and atp - sensitive k selective ion channels at the zg membrane , whose activities are implicated in zg swelling .
these studies further demonstrate that secretion of zg contents from pancreatic acinar cells require the presence of both k and cl .
heterotrimeric gi3 protein has been implicated in the regulation of both k and cl ion channels in a number of tissues . analogous to the regulation of k and cl ion channels at the plasma membrane in cells , the regulation of these channels at the zg membrane by a gi3 protein has been demonstrated .
isolated zgs from exocrine pancreas swell rapidly in response to gtp and naf , suggesting the involvement of rapid water entry into zgs following gtp exposure .
as opposed to osmotic swelling , membrane - associated aqp channels are involved in rapid water entry into cells .
the presence of aqp1 and gi3 protein at the zg membrane [ 4 , 5 ] , and aqp6 and go protein at the sv membrane [ 4 , 5 ] , and their involvement in gtp - mediated vesicle water entry and swelling have previously been reported [ 4 , 5 ] .
mastoparan , an amphiphilic tetradecapeptide from wasp venom , potentiates the gtpase activity of gi / go proteins [ 13 - 15 ] .
stimulation of g proteins is believed to occur by the peptide inserting into the phospholipid membrane and forming a highly structured -helix resembling the intracellular loops of g - protein  coupled receptors .
analogous to receptor activation , mastoparan is thought to interact with the cooh - terminal domain of the g protein  subunit .
active mastoparan ( mas7 ) in the presence of [ -p]gtp demonstrate a significant increase in swelling and gtpase activity in zg , establishing the presence of gi proteins with zgs . similarly , mastoparan has also been demonstrated to stimulate sv swelling .
studies further report that vh - atpase present at the sv membrane   is responsible for the generation of electrochemical h gradient ( ph 5.25.5 ) within the sv   required for transport of neurotransmitters into the sv lumen .
in addition to the established role of vh - atpase in neurotransmitter transport into sv , vh - atpase has been suggested to participate in the secretion of stored neurotransmitters [ 20 , 21 ] . because sv swelling is go - mediated , and is required for cell secretion ,
the involvement of vh - atpase at the sv membrane   in go - mediated water gating through the aqp6 channel was hypothesized and tested in a recent study . in agreement
, results from the study demonstrated that sv - associated vh - atpase is required for gtp - go  mediated swelling of sv . in view of these earlier studies , the involvement of aquaporin and heterotrimeric g - proteins in the regulation of platelet volume was hypothesized and tested in this study .
mercuric chloride is known to inhibit most aqps except aqp6 , which is stimulated by the compound .
exposure of platelets to hgcl2-induced cell swelling in a dose - dependent manner , suggesting the presence of aqp6 in platelets .
immunoblot analysis of platelet protein confirmed the presence of aqp6 . to further determine if g proteins were involved in aqp6 function in platelets , g - protein  specific immunoblot analysis and mastoparan - stimulated swelling was examined .
exposure of platelets to mastoparan results in a dose - dependent swelling of platelets , demonstrating for the first time the involvement of g - proteins in platelet volume regulation .
immmunoblot analysis further confirm the presence of aqp6 in platelets , and the presence of go , gi1 and gi3 proteins in platelets as previously demonstrated [ 24 , 25 ] .
results from this study demonstrate for the first time that aqp6 , go , gi1 and gi3 proteins are involved in platelet volume regulation .
all animal studies were approved by the iacuc at wayne state university . to isolate red blood cells ( rbc ) ,
the blood sample was diluted in 10 volume of pbs ph 7.4 , and spun at 120  g for 10 min . at 4c .
the resulting pellet was resuspended in 2 volume of pbs , and washed three times , followed by their final resuspension in 10 volume of pbs prior to use .
the purity of rbc preparation was examined using light and scanning electron microscopy ( fig .
1a ) . to isolate platelets , blood was dispersed in acid citrate - dextrose solution ( acd ; 0.1 m citric acid , 0.2 m sodium citrate , 0.4 m dextrose , ph 6.8 ) , which functions as an anti - coagulant and anti - stimulant for platelets .
1a , i ) . whole blood with an equal amount of acd was centrifuged at 100  g for 15 min .
and platelet - rich supernatant was mixed with an equal amount of buffer a ( 20 mm hepes - naoh ph 7.4 , 3.3 mm nah2po4 , 2.9 mm kcl , 1 mm mgcl2 , 128 mm nacl , 5.5 mm d - glucose , ph 7.4 ) .
the supernatant was centrifuged at 1000  g for 10 min . to obtain a pure platelet pellet .
isolated platelet and rbc preparations for electron microscopy were fixed using 2% paraformaldehyde and 1% glutaraldehyde for 1 hr at room temperature followed by washing in pbs .
isolated pure platelet preparations were used in dynamic light scattering experiments , and both platelet and rbc preparations were solubilized using 1% triton / lubrol for immunoblot analysis ( fig .
light and scanning electron micrographs of isolated platelets from rat blood , and presence of aqp6 , go , gi1 and gi3 proteins in the preparation .
( a , ii ) light and ( a , iii ) scanning electron micrographs of purified platelet preparation .
( a , iv ) light and ( a , v ) scanning electron micrographs of purified rbc preparation .
( b , i ) immmunoblot analysis of platelet homogenate , demonstrates the presence of aqp6 , and ( b , ii ) go , gi1 and gi3 proteins .
total rbc homogenate demonstrates the presence of aqp1 and the absence of aqp6 . to perform light microscopy on isolated platelets ,
cells were allowed to settle and stick to the slide surface at room temperature , and the unattached cells rinsed gently three times using 200 l of pbs .
similarly , to perform scanning electron microscopy on isolated platelet preparations , 25 l of aldehyde fixed isolated platelets was spread on 13 mm thermonox cover slips coated with 0.2% poly - l - lysine .
cells were allowed to settle and stick to the surface of the cover slip overnight at 4c , and the excess sample rinsed gently by dipping into four changes of milli q dh2o .
, each in increasing concentrations of ethanol ( 35% , 50% , 70% , 85% and 95% ) followed by four times 5 min .
. the sample was dried and mounted onto one large aluminium specimen stub in puddles of liquid graphite , sputter coated with gold and palladium and imaged with a joel jsm 6060 sem operating at 25 kv , spot size 30 , wd810 mm at various magnifications up to 28,000 ( fig .
platelet size dynamics were determined using real - time , right angle light scattering in a hitachi f-2000 spectrofluorimeter .
real - time scattered light intensities at excitation and emission wavelengths of 530 nm were used to measure platelet size dynamics . in these experiments ,
isolated platelets were suspended in water , and rapid changes in their size was monitored before and after addition of hgcl2 ( an aqp1 inhibitor and an aqp6 stimulator ) , followed by mastoparan exposure .
student 's t - test was performed for comparison between groups , with significance established at p < 0.01 .
sample aliquots solubilized in laemmli sample preparation buffer were resolved using 10% sds - page .
the nitrocellulose membranes were incubated in blocking buffer ( 5% non - fat milk in pbs with 0.1% tween ) for 30 min . at room temperature , followed by immunoblotting for 1 hr at room temperature with specific antibodies .
primary polyclonal antibodies ( santa cruz biotechnology , santa cruz , ca , usa ) were used at a dilution of 1:200 ( aqp-1 , aqp-6 , go , gi1 and gi3 ) in blocking buffer .
immunoblotted nitrocellulose membranes were washed in pbs and incubated in horseradish peroxidase  conjugated secondary antibody ( santa cruz biotechnology ) at a dilution of 1:1000 for 1 hr at room temperature . after washing in pbs , the nitrocellulose membranes were processed for enhanced chemiluminescence ( amersham bioscience , piscataway , nj , usa ) and developed using a kodak 440 image station .
analogous to a number of other mammalian cell types , platelet volume significantly affects its function , such as platelet activation and the secretion of a variety of essential molecules .
impaired volume regulation in platelets negatively influences blood clotting , resulting in adverse outcomes such as myocardial infarction , stroke or pulmonary embolism . because water channels or aquaporins have previously been implicated in cellular and organelle volume regulation , their presence in platelets and their potential role in platelet volume regulation was hypothesized and investigated .
platelets were isolated from rat blood , and their purity determined by both light and scanning electron microscopy ( fig .
exposure of platelets to hgcl2 induced swelling in a dose - dependent manner , suggesting the presence of aqp6 in platelets ( fig .
hgcl2 induced a dose- and time - dependent increase in platelet size , as demonstrated using dynamic light scattering .
dynamic light scattering further demonstrated that 300 m hgcl2 was the optimal dose in stimulating platelet size increase . in the first 15 sec .
after addition of 300 m hgcl2 , increase of platelet swelling is a linear function of time ( fig .
2a , iii ) , suggesting that influx of water and ions is unrestricted and rapid at this initial period . after this point
, platelet swelling slows down , and changes in platelet volume assume a logarithmic form that can be expressed by a first - order equation , with a rate constant k = 2.3  10/sec .
similarly , analogous to hgcl2 , dynamic light scattering further demonstrated that mastoparan induces a dose- and time - dependent increase in platelet size ( fig .
dynamic light scattering demonstrated that 40 m mastoparan was the optimal dose in stimulating platelet size increase ( fig .
after addition of 40 m mastoparan , increase of platelet swelling is a linear function of time ( fig .
2b , iii ) , again suggesting that influx of water and ions is unrestricted and rapid at this initial period . after this point
, platelet swelling slows down , and changes in platelet volume assume a logarithmic form that can be expressed by a first - order equation , with a rate constant k = 24  10/sec .
these studies suggested that both aqp6 and gi / go proteins are present in platelets and participate in platelet volume regulation .
this is further confirmed , because immunoblot analysis performed on total platelet proteins using specific antibodies to aqp6 , and to gi and go proteins , demonstrate their presence in platelets ( fig .
immunoblot analysis further demonstrates that aqp6 is present in platelets ; however , the rbc water channel aqp1 is absent .
similarly , aqp6 is absent in rbc . to further determine if g proteins are present in platelets , g - protein 
these immunoblot studies confirm the presence of go , gi1 and gi3 proteins in platelets ( fig .
these results demonstrate for the first time that functional aqp6 , go , gi1 and gi3 proteins are present in platelets and are involved in cell volume regulation .
it is possible that one or all of the above g - proteins are involved in platelet volume regulation .
analogous to secretory vesicle volume dynamics , we have now demonstrated that the regulation of platelet volume involves both water channels and heterotrimeric g - proteins , driving the active and rapid transport of water through the aqp6 channels at the platelet plasma membrane .
hgcl2- and mastoparan - induced dose- and time - dependent increase in platelet size , demonstrated using dynamic light scattering .
( a , i ) dynamic light scattering , demonstrating hgcl2 dose - dependent increase in platelet size over control .
( a , ii ) exposure of 300 m hgcl2 to isolated platelets , demonstrates a time - dependent increase in platelet size .
( a , iv ) graph depicts the first - order kinetics of increase in platelet size following exposure to 300 m hgcl2 .
( b , i ) dynamic light scattering demonstrating the mastoparan dose - dependent increase in platelet size over control .
( b , ii ) exposure of 40 m mastoparan to isolated platelets , demonstrates a time - dependent increase in platelet size .
( b , iv ) graph depicts the first order kinetics of increase in platelet size following exposure to 40 m mastoparan .
, performed majority of the experiments , both contributing equally to the study . m.t . and d.j.t .
 abstractthe regulation of platelet volume significantly affects its function . because water is the major molecule in cells and its active transport via water channels called aquaporins ( aqps ) 
 have been implicated in cellular and organelle volume regulation , the presence of water channels in platelets and their potential role in platelet volume regulation was investigated . 
 g - protein  mediated aqp regulation in secretory vesicle swelling has previously been reported in neurons and in pancreatic acinar cells . 
 mercuric chloride has been demonstrated to inhibit most aqps except aqp6 , which is stimulated by the compound . 
 exposure of platelets to hgcl2-induced swelling in a dose - dependent manner , suggesting the presence of aqp6 in platelets . 
 immunoblot analysis of platelet protein confirmed the presence of aqp6 , and also of go , gi1 and gi3 proteins . 
 results from this study demonstrate for the first time that in platelets aqp6 is involved in cell volume regulation via a g - protein  mediated pathway . 
Introduction
Materials and methods
Results and discussion
Authors contribution
Conflict of interest