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 µM), prostaglandin E1 (PGE1) (50 nM) and sodium nitro-prusside (SNP) (100 µM). Untreated PRP and washed platelet samples were also included in study as baseline controls. After labeling of platelets with either anti P-Serine(157)-VASP or anti P-Serine(239)-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.

Keywords: Flow cytometry; PRP; Platelet; Pre-analytical variation; Quality control; VASP.

Figure1

A) Washed platelets treated with either anti-P-ser¹⁵⁷-VASP or anti-P-ser²³⁹-VASP antibodies in the presence of PGE1 (50 nM), were labeled by FITC conjugated secondary antibody. To analyze VASP phosphorylation, platelets were then subjected to flow cytometry. Platelets were detected by FSC/SSC characteristics. FL1-FSC dot plots demonstrate two distinct platelet populations (S1 and S2) which then gated separately to be analyzed in FL1 histograms. B) The overlaying histograms display the levels of VASP phosphorylation in platelets treated with different agonists including; forskolin (10 µM), SNP (100 µM) and PGE1 (50 nM). The histograms represented the whole population of platelets in FL1-FSC dot plots (S1+S2).

Figure 2

The histograms have been generated from flow cytometry analysis on platelets which were labeled with either anti-P-ser¹⁵⁷-VASP or anti-P-ser²³⁹-VASP as primary antibodies and then FITC-conjugated secondary antibody. The platelet samples labeled only with secondary antibody have been used as negative control. The histograms represented the S2 population of platelets in FL1-FSC dot plots. Comparing the histograms obtained for PRP and washed samples suggests some variation in the levels of intraplatelet P-VASP expression at baseline (control) and/or in response to forskolin (10 µM), PGE1 (50 nM), or SNP (100 µM).

Figure 3

Median fluorescence intensity (MFI) values indicating the levels of intraplatelet P-Ser¹⁵⁷-VASP and P-Ser²³⁹-VASP have been presented in graphs (A) and (B), respectively. The average percent values of MFI shifts from baseline (control), induced by forskolin, PGE1 and SNP, have been presented below the associated columns. From the results on graph (A), significant increase in baseline expression of P-Ser¹⁵⁷-VASP in washed platelets could be noted; however, the results presented in graph (B) indicate no significant differences in baseline expression of P-ser²³⁹-VASP between washed and PRP platelets. As can be seen from the data in graphs, washed platelet samples have shown more levels of intraplatelet P-Ser¹⁵⁷-VASP and P-Ser²³⁹-VASP induction in response to forskolin and SNP, in comparison with PRP samples; however these samples have expressed reduced levels of P-VASP at both residues in response to PGE1. Data shown are from six runs of experiments using platelet samples obtained from six different donors (n=6).