The value of flow cytometry in the measurement of platelet activation and aggregation in human immunodeficiency virus infection

Abstract

Human immunodeficiency deficiency virus (HIV) infection is associated with chronic inflammation and an increased risk of thrombotic events. Activated platelets (PLTs) play an important role in both thrombosis and inflammation, and HIV has been shown to induce PLT activation by both direct and indirect mechanisms. P-selectin (CD62P) is a well-described marker of PLT activation, and PLT glycoprotein (GP) IV (CD36) has been identified as a marker of PLT aggregation. Data on PLT function in the context of HIV infection remain inconclusive. Laboratory techniques, such as flow cytometry, enable the assessment of PLTs in their physiological state and environment, with minimal artifactual in vitro activation and aggregation. In this study, we describe a novel flow cytometry PLT assay, which enabled the measurement of PLT function in HIV infection. Forty-one antiretroviral-naïve HIV-positive individuals and 41 HIV-negative controls were recruited from a clinic in the Western Cape. Platelet function was evaluated by assessing the response of platelets to adenosine diphosphate (ADP) at two concentrations (0.04 mM, 0.2 mM). The percentage expression and mean fluorescence intensity (MFI) of CD62P and CD36 was used to evaluate platelet function. These were then correlated with platelet (PLT) count; CD4 count; % CD38/8; viral load and D-dimers. The % CD62P levels were higher in HIV-positive patients (HIV % CD62P 11.33[5.96–29.36] vs. control 2.48[1.56–6.04]; p < 0.0001). In addition, the HIV group showed higher CD62P MFI levels (HIV CD62P MFI 3.25 ± 7.23 vs. control 2.35 ± 1.31, p = 0.0292). Baseline levels of %CD36 expression were significantly higher in HIV-positive patients (%CD36 12.41[6.31–21.83] vs. control 6.04[1.34–13.15]; p = 0.0091). However, the baseline CD36MFI showed no significant difference between the two groups (HIV CD36 MFI 3.09 ± 0.64 vs. control 2.44 ± 0.11, p = 0.4591). The HIV group showed higher levels of % CD36 expression post stimulation with 0.04 mM ADP 43.32 ± 27.41 vs. control 27.47 ± 12.95; p < 0.0214) and no significant difference at 0.2 mM ADP (HIV % CD36 39.06 ± 17.91 vs. control 44.61 ± 18.76; p = 0.3277). Furthermore, the HIV group showed a single phase response to ADP as compared to the control group, which showed a normal biphasic response. We concluded that PLT flow cytometry is valuable in the assessment of levels of PLT activation, and further, that the addition of an endogenous agonist, such as ADP, enabled the measurement of PLT function in HIV infection. We were able to show that, although PLTs are significantly activated in HIV compared to uninfected controls, they retain their functional capacity.

• Flow cytometry
• HIV
• immune activation
• platelet function

Acknowledgements

We wish to thank the patients and staff of the Emavundleni Clinic in Crossroads Cape Town for their participation in this study as well as all members of the HAIG (HIV Activation and Inflammation Group) for their dedicated teamwork.

Declaration of interest

The authors declare that there are no financial, personal or professional competing interests that may interfere with this work.

This research was supported by research grants from the National research foundation (NRF), National health laboratory services research trust (NHLSRT), Medical Research council (MRC).