To clarify our first hypothesis, that NO differently affects aggregation mediated by PAR1 and PAR4, the NO-containing drug SNAP was added (in different concentrations) to platelet suspensions. PAR1 or PAR4 was stimulated and aggregation was measured. This experimental setup allowed us to see the effect of NO on aggregation, as well as to whether there was a difference between PAR1 and PAR4 in their resistance/sensitivity towards NO. The results are shown in Figure1.

To investigate our second hypothesis, that PAR1 and PAR4 differ in their ability to undergo desensitization, cumulative dose response studies were performed. For this, increasing doses of stimuli for PAR1 (X), or PAR4 (Y), was added to platelet suspensions.

The result revealed that platelets stimulated with increasing concentrations of stimuli activating PAR4, responded in a normal “cumulative dose dependent manner” (i.e. the magnitude of the aggregation response was not underestimated compared to the effect of a single, high, dose of stimuli (30Y, seen in Figure2)).

However, this cumulative dose-response effect was not possible to obtain when stimulating PAR1. Specifically it was found that low “non-aggregatory” concentrations of PAR1-stimuli fully desensitized platelets towards higher concentrations of stimuli. Another interesting feature found in this experiment was that addition of PAR4-stimuli (30Y) after the highest dose of PAR1 stimuli (30X), mediated a full aggregation response. This suggests that the desensitization of PAR1 does not affect PAR4.

The results from our study showed that:

• NO exerts similar inhibitory effects on PAR1 and PAR4.

• PAR1 and PAR4 differ in their ability to undergo desensitization.
  PAR1 is desensitized in cumulative dose-response studies. PAR4 is not.

• The desensitization of PAR1 does not seem to affect PAR4.

In a physiological point of view, this might imply:
A low concentration of thrombin circulating in our blood could desensitize PAR1 - make the receptors unresponsive toward re-exposure of thrombin. If these platelets would later be exposed to a higher level of thrombin - which is very common at sites of atherosclerotic plaques - the receptors would not respond (aggregate) to stimulation. This would thus avoid the formation of a plug inside the vessel.
However, if the concentration of thrombin is high enough, it might stimulate PAR4 and cause aggregation through this pathway.

Therefore, PAR4 might be a more suitable therapeutic target than PAR1, and special emphasis should be put on this receptor in future studies.