Abstract

Background:

The degree of phosphorylation of vasodilator stimulated phosphoprotein (VASP) provides a convenient means of tracking changes in the levels of intracellular cyclic nucleotides. We have developed a simple and direct means of measuring phosphorylated VASP (VASP-P) using VASPFix (Platelet Solutions Ltd, UK) and demonstrated its performance in human platelets.

Methods:

We have measured the levels of VASP-P in platelets by adding VASPFix to whole blood or platelet rich plasma (PRP) samples after they had been treated with compounds that either increase (dibutyryl cAMP, adenosine, iloprost, PGE₁), or decrease (ADP, PGE₁) the level of cyclic adenosine monophosphate (cAMP). The level of VASP-P is assessed by flow cytometry and can be measured either immediately or after freezing, when treated samples are stable for at least 6 months.

Results:

VASPFix was used to measure VASP-P in platelets in both whole blood and PRP in response to agents that increase cAMP, such as dibutyryl cAMP, adenosine and iloprost. The effects of corresponding receptor antagonists (adenosine A_2Aand IP receptors) yielded predictable results.

The bi-directional effects of PGE₁and PGE₂on VASP-P were evaluated. The effect of PGE₁is mediated via its interaction with the IP receptor, leading to an increase in VASP-P, and the EP3 receptor, leading to a decrease in VASP-P. By contrast, the effect of PGE₂  is mediated via the EP4 receptor, leading to an increase in VASP-P, and the EP3 receptor , which leads to a decrease in VASP-P.

We have also demonstrated the ability of ADP to reduce previously raised levels of VASP-P in platelets, as well as the ability of three different ADP receptor (P2Y₁₂) antagonists to prevent this effect of ADP. It was also possible to detect a small increase in VASP-P in response to three different P2Y₁₂ antagonists, which might imply some constitutive activity of the P2Y₁₂ receptor on platelets.

Conclusions. VASPFix provides a simple means of measuring the level of phosphorylated VASP in platelets both in whole blood and platelet rich plasma. It is useful for evaluating the effects of various compounds on platelet function, which act on stimulatory and inhibitory  platelet receptors.