The Use of Several Leakage Markers which Enable Platelet Membrane Damage and Platelet Activation to be Distinguished

Abstract

We have studied the effects of four differently charged detergents and sphingosine on platelet membrane integrity and platelet activation, using the leakage of pre-loaded ⁴³K⁺, pre-loaded [¹⁴C]-serotonin ([¹⁴C]-5-HT) and lactate dehydrogenase (LDH) as markers.

Sphingosine, at a concentration of 20μM, induced a release of [¹⁴C]-5-HT as well as ⁴³K⁺, without releasing LDH. The [¹⁴C]-5-HT7⁴³K⁺ ratio was about 1.5. A similar leakage pattern and the same [¹⁴C]-5-HT/⁴³K⁺ ratio was also induced by thrombin, a potent platelet agonist, indicating that the release induced by sphingosine is due to platelet activation and not to membrane damage. The detergents, on the other hand, induced a marked release of ⁴³K⁺ followed by an increased release of LDH. No pronounced leakage of [¹⁴C]-5-HT occured with the detergents; the [¹⁴C]-5-HT/⁴³K⁺ ratio was ≤ 0.2. In this manner, we can distinguish between marker-release caused by membrane-damage and that caused by platelet-activation, by using the combination of leakage markers presented here.

Our study also showed that fixation prior to centrifugation is crucial in order to avoid leakage of markers, which are released upon activation of the platelets. About 35% of the [¹⁴C]-5-HT content of the platelets was released when unfixed platelets were centrifuged, and, interestingly, the detergents, as well as ethanol, inhibited the centrifugation-induced release of [¹⁴C]-5-HT.