I am looking to investigate the signalling pathway initiated by an intracellular receptor present in the platelet granule.
Upon activation of the platelet, the granule is released and receptor moves to the plasma membrane. However, I am uncertain as to how I can determine which proteins are involved in the signalling process without activating the platelet and consequently not being able to distinguish the receptor specific events with general platelet activation. Any advice would be appreciated.
Hi Shishir,
Thanks for the reply. I was aware of Platelet Web but unfortunately there isn't many proteins known to be associated!
You should specify your question and then determine which signaling pathway is more important for that. as you know there are so many agonist that can activate platelet (e.g collagen, thrombin) and this agonist have specific receptors (e.g PAR1-4 for human thrombin) and specific signaling pathway
I am looking at the LOX-1 receptor which is associated with granules. I want to determine the signalling events downstream of this receptor, however this receptor is only localised on the plasma membrane in degranulated platelets. Therefore, I need to trigger a degranulation event in order to probe the specific LOX-1 pathway, but do not want to activate the platelets to such a degree in which they begin to aggregate.
Hi Casey! Well, that's a tough one...
However, it may be possible. You have to activate platelets, as otherwise your receptor will not translocate the the surface. This would be my plan: Stimulate isolated platelets (probably diluted to a very low count) with the PAR4-activating peptide AYPGKF (which activate the receptor, but does not cleave it) under resting/non-stirring/non-shaking(!!!) conditions (like for FACS analysis). In that setting platelets degranulate, but don't 'find' each other to form aggregates. Then add cAMP and cGMP elevating substances such as prostacyclin/adenosine and a NO-donor like SNAP, centrifuge them immediatly, carefully wash the pellet, and then resuspend the platelets again. Under these conditions you should have the receptor on the surface, and also wash away all secodary mediators such as ADP/ATP etc as well as secreted fibrinogen. Before experimentation you should allow them to rest at 37°C for like 30-60 min, in order to let them get down/back 'in shape'.
Good luck! And keep me posted, Knut
In this respect, it just occurred to me, that you should activate platelets in the presence of 1 U/ml Apyrase, which will break-down ATP and ADP into adenosine, the latter which via an elevation of cAMP will add to taming the activated platelets.
Thanks Knut, this was exactly the kind of help I was looking for! I'll keep you informed!
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