Hey Samarjit,

Platelet function is most truly best assessed in whole blood.  While washed platelet protocols have been around for many years, there is significant increases in activity markers found in the literature after isolation.  Unfortunately for various reasons it is sometimes impractical of even not feasible to use whole blood samples.  In these cases the best way to process samples for platelets is to make platelet rich plasma, which in theory is more of an enrichment than isolation.  If you provide more information on the applications you plan on running I may be able to provide more specific details.

Jason

Hi Samarjit, like Jason said, the more you manipulate platelets, more platelet activation will occur.

If you want enriched platelet fractions in 1-2 steps, then there are many methods available. 

I use this protocol (PMID: 15226531) with very nice results (paper attached)

Best

Pierre

Answer of  this problem has the diffence by species, such as human, rabbit, rat ..etc.,

and the status of platelet, such as PRP (platelet rich plasma) or WP (washed platelet).

For example, to isolae platelet  from rabbit, as below:

 " Blood collected directly into anticoagulant solution containing 0.8% citric acid, 2.2% trisodium citrate, and 2% dextrose (w/v). Briefly, PRP (platelet rich plasma) wasobtained by centrifugation of rabbit blood (230×g, 10 min). Platelets were sedimented  ycentrifugation of the PRP (800×g, 15 min) and washed with 4-(2-hydroxyethyl)-1-iperazineethanesulfonic acid (HEPES) buffer (137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 5.6 mM glucose, and 3.8 mM HEPES, pH 6.5) containing 0.35% bovine serum albumin (BSA) and 0.4 mM ethylene glycol-bis(2-aminoethylether)-tetraacetic acid (EGTA). The washed platelets were resuspended in HEPES buffer (pH 7.35)."

The method Dr. Pierre Fontana has recommended is about the best for washing platelets for functional testing afterwards. It also has a handy method for making up stock solutions of Tyrode's buffer. Dr. Lee's buffer is a modified Tyrode's I note.

A method I used to do some years ago was Stractan (arabinogalactan) density gradient centrifugation on PRP then washing. We used it to get rid of contaminating plasma to prepare platelet lysates for accurate quantitation of alpha granular proteins by ELISA. I think the arabinogalactan contains

The method should be in this paper - Gralnick et al., (1985). PNAS USA 82: 5968-72.I

It's available via PubMed.

There is a reference saying you can do aggregometry on platelets after using this method although the ADP response is altered but not thrombin and collagen.

I propose to prepare platelets by the gel-filtration procedure instead of centrifugation and reconstitution.

I studied formation of phosphatidylserine positive (PS+) murine platelets (these marker usually appears upon strong activation). Platelets were gel-filtered and a background level (without activator) of PS+ platelets were less than 2% (from total population). For comparison, Jobe et al. (2006) who prepared mice platelets by centrifugation got the background level up to 5%.

I would recommend the following papers: PMID: 6800196; 3113155; 2945282

If you want to get unactivated platelet in platelet rich plasma from whole blood you have to make sure that you do the following:

1.  Draw blood from healthy volunteers.

2.  Avoid using a tourniquet when drawing blood since tying a tourniquet itself may activate the blood.

3.  Use a butterfly needle (21 guage) to draw the blood.

4.  Follow a double syringe technique which means that the initial 1-2 ml of blood collected in the first syringe should be discarded for possible contamination with tissue factor which can activate the platelets.  Avoiding kinking of the tubing disconnect the first syringe and attach the second syringe to the needle.

5.  Slowly draw the blood in the second syringe.

6.  Slowly place the blood in a proportion of 1 part of sodium citrate solution to 9 parts of whole blood.

7.  Gently mix the contents of the tube.  Avoid vigorous shaking of the tube to mix.

8.  Centrifuge the blood at 800 rpm for 20 minutes.

9.  Avoid applying brakes when the centrifuge comes to stop.

10.  Using a pipette gently separate the platelet rich plasma avoiding admixture with the white or red cells.

If you follow these simple steps you may get platelets which have not been activated.

Good luck.

It depends on what you want to test them for afterwards. 

If you're looking to take them into an aggregometer to look at aggregation, the preparation of PRP from citrated whole blood by centrifugation (175 x g, 15 mins, low brake) will be sufficient.

To look at washed platelets, this PRP should be further spun in the presence of  apyrase plus prostacyclin and the pellet resuspended in modified Tyrode's buffer.

I would like to comment the answer of Dr. Chan. Sure, one can prepare washed platelets by spinning and resuspending - this method is very simple but can cause some background activation. The second method is gel filtration, as I noted above - it is more delicate for platelets but also it is technically more "complicated". So the method of choise is dependent on your experimental conditions.

 As mentioned in some of the preceding answers, at  this time there is no way to obtain platelets without activating them to some extent. One of my mentors used to say "platelets will activate just by looking at them", which is not actually true but carries the message. I did my EM rotation study, many years ago, (when the dinosaurs were arround!) on morphologic changes in platelets due to external stimuli.  The only thing that can be done is to let the platelets "rest" in an environment where the clotting cascade is not activated. Washing platelets with warm isotonic saline is a procedure that is done for patients who have IgA deficiency and / or have severe allergic reactions to platelet transfusion.  Platelets which get activated by centrifuge or manual washing, will emit psedopods in order to cover as large an area as possible. By letting the platelets sit in a  counter top withoug agitation for one hour will result on the platelets to become round again. Most of the activation markers will decrease or disappear. So, I hope this helps. 

Hi Samarjit

I think best method for platelet researches is whole blood. But if this method is not possible, the Omer Iqbal comments are very useful. I used this points on my own research and I got good results. Especially those related to centrifuge,s speed and duration and blood drawing. 

regards 

hiwa

Thank you Mr Rahmani.. i will try..

Arginine at 100-200 mM inhibits platelet activation.

Detailed protocol for Isolation of human platelets (Platelet-rich plasma) from whole blood, which including the reagents.

https://www.abcam.com/protocols/isolation-of-human-platelets-from-whole-blood

Please find the state.-of-the-art of washing platelets e