Variations in intraplatelet phospho-VASP expression due to pre-analytical sample preparations, illustration of a quality control issue in platelet pharmacology

ABSTRACT

Intraplatelet vasodilator-stimulated phosphoprotein [VASP] analysis is a commonly used laboratory approach for monitoring of the anti-platelet therapy with adenosine diphosphate [ADP] receptor blocking agents; however, it's testing in clinical laboratory needs a high level of experience and proficiency. The ability to recognize how the pre-analytical variations can change the results would be helpful for the interpretation of data from intraplatelet VASP analysis. The aim of this study was to describe the possible differences of intraplatelet phospho- VASP expression between washed and platelet rich plasma [PRP] samples, both at baseline levels and following experimentally induction of VASP phosphorylation. PRP and washed platelet samples were treated with different inducers of VASP phosphorylation, including forskolin [10 micro M], prostaglandin E1 [PGE1] [50 nM] and sodium nitro-prusside [SNP] [100 micro M]. Untreated PRP and washed platelet samples were also included in study as baseline controls. After labeling of platelets with either anti P-Serine157-VASP or anti P-Serine239-VASP, the samples were subjected to flow cytometric analysis to monitor the levels of intraplatelet phospho-VASP expression. Washed platelet samples tend to show increased expression of intraplatelet P-Serine[157]- VASP at baseline state and also more expression of P-Serine[157]-VASP and P-Serine[239]-VASP in response to forskolin and SNP, compared with PRP samples. Though, reduced levels of PGE1- induced VASP phosphorylation at both residues were detected for washed platelets. In this study we have provided some background information required for performing of intraplatelet VASP analysis on differently handled platelet samples and interpretation of the obtained results: