Cardiovascular disease (CVD) is a leading cause of morbidity and mortality in HIV-infected individuals . Persistent inflammation is thought to be a major factor in this excess risk . Mounting evidence supports that platelets are important regulators of inflammation and are involved in both onset and progression of atherosclerosis, next to acute cardiovascular events [3,4]. Activated platelets release a wide range of inflammatory mediators and form aggregates with leukocytes, most notably via platelet P-selectin [4,5]. Platelet-monocyte aggregation (PMA) is a sensitive marker of in-vivo platelet activation  and renders monocytes more proinflammatory [7,8].
Different studies have shown that both antiretroviral naïve and treated HIV-infected individuals have more activated and reactive platelets and increased PMA compared with HIV-uninfected individuals [9–13], although other studies yielded opposite results [14,15]. In addition, HIV-infected patients with an acute coronary syndrome had higher platelet reactivity and residual platelet hyperreactivity on dual antiplatelet therapy compared with HIV-uninfected patients . Together, these data suggest that platelets are a potential target to reduce persistent inflammation and the excess CVD risk in HIV.
Our group recently found in a cross-sectional study that individuals on a raltegravir (RAL)-based regimen had reduced platelet reactivity and PMA compared with those on a nonintegrase inhibitor-based regimen . Therefore we performed a randomized controlled trial to study whether a switch to a RAL-based regimen leads to a reduction in platelet reactivity, platelet-leukocyte aggregation, inflammation and immune activation.
This investigator initiated, single centre, prospective randomized open-label, blinded end-point trial (NCT02383355) was performed at the Radboud university medical centre, a tertiary teaching hospital in the Netherlands. This study was conducted in accordance with the Declaration of Helsinki after approval of the ethics committee (CMO Arnhem-Nijmegen, The Netherlands; NL5068109114). Adult HIV-1-infected individuals receiving a nonintegrase inhibitor (INSTI) containing regimen with a standard backbone of two nucleoside or nucleotide reverse transcriptase inhibitors (NRTIs) for at least 6 months were included after providing written informed consent. Other inclusion criteria were a suppressed viral load (<50 copies/ml) for at least 6 months and a CD4+ cell count more than 300 cells/ml. Exclusion criteria included the use of platelet inhibitors, signs of an active infection other than HIV-1, estimated glomerular filtration rate below 50 ml/min, liver enzyme abnormalities, known genotypic resistance to any current antiretroviral therapy (ART) component, history of virological failure (RNA blips <500 copies/ml with subsequent suppression were allowed) and pregnancy or breastfeeding. Randomization was performed using a computer-generated block randomization algorithm (CASTOREDC, Amsterdam, The Netherlands). Participants were randomized 1 : 1 to either switch to RAL 400 mg twice daily continuing their current backbone therapy (RAL-group) or continuing their current regimen (CONT-group). Participants were followed for 10 weeks with study visits at weeks 4 and 10. At study visits, blood was obtained for platelet and inflammation markers and adherence and adverse events were recorded. The primary outcome was the change in platelet reactivity between weeks 0 and 10. This was defined as the expression of the platelet activation marker P-selectin, a marker for platelet degranulation, and the binding of fibrinogen to the activated fibrinogen receptor (integrin αIIbβ3), a marker for platelet aggregation, following ex-vivo stimulation. Secondary outcomes included changes in platelet-leukocyte aggregation, monocyte subsets, T-cell activation and soluble markers of inflammation, platelet activation and plasmatic coagulation. Platelet reactivity was determined in citrated whole blood using a flow-cytometry based assay as previously described  (Supplementary Fig. 1, https://links.lww.com/QAD/B353). Platelet-leukocyte aggregation, monocyte subsets and T-cell activation were measured at baseline and after 10 weeks in whole blood on a NAVIOS flow cytometer (Beckman Coulter, Brea, California, USA) as previously described  (Supplementary Fig. 2, https://links.lww.com/QAD/B353). Plasma concentrations of platelet factor 4 (PF4), beta-thromboglobulin (CXCL7), high-sensitive C-reactive protein (hsCRP) and thrombin antithrombin complexes were measured using ELISA as previously described . All laboratory procedures are described in detail in the supplementary information. The primary outcome was analysed by calculating the change in platelet reactivity from baseline to end-of-study time point (week 10) and comparing the groups using independent Student's t test. Secondary outcomes were analysed by independent t test or Mann–Whitney test depending distribution. Intention-to-treat analysis was performed and data are expressed as the mean with the SD or the median with the interquartile range (IQR) depending distribution. Sample size estimation was based on the data of previous cross-sectional study . We anticipated ADP-induced P-selectin expression in the RAL-group to decrease from a mean fluorescence intensity of 19 to 14 (SD 5.2). Demonstrating this difference with a power of 80% and α = 0.05 required 18 individuals per group. Consequently, we have included 20 per group. SPSS Statistics 22 (IBM, Corp., Armonk, New York, USA) and Graphpad Prism 5 (Graphpad Software, La Jolla, California, USA) were used for analyses.
Patient recruitment started in March 2015 and the last patient completed the study in March 2016. A flow diagram of the study is presented in Supplementary Fig. 3, https://links.lww.com/QAD/B353. Nineteen participants were randomized to switch to RAL without changing their NRTI backbone therapy (RAL-group) and 21 continued their current regimen (CONT-group). One patient started aspirin after informed consent, but before treatment allocation, and was therefore excluded. Thirty-eight participants completed the total follow-up period of 10 weeks. Two participants discontinued RAL at week 4 due to grade I adverse events (mild sleeplessness and headache) and this time point was included as the end-of-study time point. Overall, treatment was well tolerated and no grades III–IV adverse events were recorded.
Baseline characteristics, which are presented in Supplementary Table 1, https://links.lww.com/QAD/B353, were similar between the groups. Overall, 97.5% of participants were men with a median age of 48 years (IQR: 43–54) in the RAL-group and 49 years (IQR: 42–61) in the CONT-group. The ART backbone consisted of tenofovir difumarate/emtricitabine [RAL: 13 (68.8%), CONT: 17 (81%)] or abacavir/lamivudine [RAL: 5 (26.3%), CONT: 3 (14.3%)]. Seventy-nine per cent of participants in the RAL-group were using a nonnucleoside reverse transcriptase inhibitor (NNRTI)-based regimen at enrolment. All patients had an undetectable viral load (<50 copies/ml) at baseline. Two patients in the RAL-group had a detectable viral load during follow-up: one patient had a viral load of 90 copies/ml at week 4 with undetectable viral load at week 10; the second patient had a viral load of 2000 copies/ml at week 10 due to suboptimal adherence.
Platelet reactivity at baseline, as measured by P-selectin expression and fibrinogen binding after ex-vivo stimulation with ADP, CRP-XL and TRAP-6, was similar between groups (Fig. 1a and b). In addition, there were no differences observed in platelet reactivity at baseline between patients on abacavir or tenofovir as backbone therapy (Supplementary Table 2, https://links.lww.com/QAD/B353). At week 10, there was no difference between both groups in the primary outcome parameters, that is the change in platelet reactivity parameters (Fig. 1c and d). This was supported by results from the plasma markers. Plasma levels of the platelet activation markers beta-thromboglobulin/CXCL7 and PF4 were similar at baseline and did not change in either group at week 10 (Fig. 2a and b). Concentrations of the plasmatic coagulation marker thrombin antithrombin complexes and the inflammation marker hsCRP also remained unchanged at week 10 compared with baseline (Fig. 2a and b). Platelet-leukocyte aggregation in our study was most prominent for PMA [RAL: 18.2% CD41+ of CD14+cells (IQR: 9.2–43.26), CONT: 18.6% (IQR: 11.6–23.9)] compared with platelets aggregating with lymphocyte subsets [RAL: 2.5% (IQR: 1.5–5.8), CONT: 2.8% (IQR: 1.4–4.6)] (Fig. 2c). At baseline, no differences were observed between groups and platelet aggregation to monocyte and lymphocyte subsets did not differ significantly at week 10 (Fig. 2c and d). An increase in activated proinflammatory monocyte subsets (CD14+CD16+) is associated with CVD [19,20]. Circulating classical monocytes (CD14+CD16−), intermediate (CD14+CD16+) and nonclassical monocytes (CD14dimCD16+) were similar at baseline and were not influenced by a switch to RAL in this study (Fig. 2e and f). T-cell activation measured by CD38 and the MHC class II cell surface receptor human leukocyte antigen - antigen D related coexpression was also not statistically different between the RAL-group and CONT-group at baseline or after 10 weeks of treatment (Fig. 2e and f).
The current randomized controlled trial demonstrates that switching from a non-INSTI containing regimen to a RAL-based regimen in virologically suppressed HIV-infected individuals does not reduce platelet reactivity, platelet-leukocyte aggregation and markers of immune activation, such as activation of monocytes and T-lymphocytes and concentrations of CRP and thrombin–antithrombin. These findings are in accordance with a study by Martinez et al., which showed that switching from a boosted protease inhibitor to RAL for 48 weeks did not lead to a reduction of in-vivo platelet activation measured by soluble P-selectin. Yet, it is in apparent contrast to previous findings from a cross-sectional study performed by our group showing that individuals on a RAL-based regimen had reduced platelet reactivity compared with those on a NNRTI or protease inhibitor containing regimen . This may be explained in different ways. First, the follow-up period of 10 weeks in the current study is relatively short, whereas the majority of patients in the cross-sectional study were on the same combination antiretroviral therapy (cART) longer than 3 months. Although platelet life-span is short (<10 days) and RAL intensification has shown effects on viral replication and plasmatic coagulation within 3 months [22,23], effects on megakaryocytes may take longer. Second, cross-sectional studies are prone for selection bias. The latter is especially relevant as available data on platelet reactivity in cART-treated patients are contradictory, with some studies reporting increased platelet reactivity [9–13] while others reporting reduced reactivity [14,15].
Platelets are increasingly recognized as key effector cells in inflammation . In turn, inflammation may also influence platelet reactivity . Some studies have proposed that RAL may reduce inflammation more effectively than other treatment regimens [22,23]. Part of the inflammatory effects of platelets is mediated by their interaction with leukocytes [7,8]. Our data do not support the notion that switching to RAL reduces inflammation or immune activation. The effects of RAL on inflammation and immune activation in the literature are also controversial. In cART-intensification studies, addition of RAL reduced levels of the monocyte activation marker sCD14  and D-dimer in some studies , whereas no effect on innate immune activation or coagulation was observed in others [22,26,27]. In the study by Martinez et al., switching from a protease inhibitor-based to a RAL-based regimen decreased hsCRP and IL-6 levels, while D-dimer and soluble P-selectin levels remained similar. Conversely, a study in treatment-naive participants showed no consistent reduction of inflammation in RAL-treated patients compared with treatment with boosted protease inhibitors . Abacavir has been shown to influence platelet function . We did not find significant differences in platelet activation or reactivity between those on an abacavir or tenofovir-based backbone at enrolment. Also, the backbone was not changed during the trial, minimizing the possible confounding effects of abacavir on our outcome measures.
Our study has several limitations. Even though our study was correctly powered for the primary outcome (platelet reactivity), the sample size gave us limited statistical power to explore all secondary objectives in detail and to include subanalyses exploring possible confounders. Second, the inclusion of only long-term, virologically suppressed individuals could have masked the possible beneficial effects of RAL, but mirrors the current chronically infected HIV population. Finally, our study included mostly men limiting generalization of the findings to women. The same applies to generalizability of these data to other INSTIs.
In conclusion, the present study shows that switching to a RAL-based regimen does not reduce platelet reactivity, platelet-leukocyte aggregation, inflammation and immune activation in virologically suppressed HIV-infected individuals.
We are grateful to all study participants and would like to thank Bram van Cranenbroek for excellent support in flow-cytometry and, Karin Grintjes and Bert Zomer for their help with participant enrollment and follow-up.
W.A.v.d.H., A.J.v.d.V. and Q.d.M. conceived the study, interpreted the findings and drafted the article. W.A.v.d.H. and Q.d.M. performed statistical analyses. W.A.v.d.H., R.v.C., M.B., A.J.v.d.V. and M.K. contributed to subject inclusion and data collection. H.J.P.M.K. and R.T.U. provided laboratory support and interpreted the data. All authors critically revised the article for important intellectual content, read and approved the final article.
Supported in part by a research grant from the Investigator Initiated Studies Program of Merck Sharp & Dohme Corp. The opinions expressed in this article are those of the authors and do not necessarily represent those of Merck Sharp & Dohme Corp. W.A.v.d.H. received a travel grant from Merck Sharp & Dohme Corp.
Conflicts of interest
There are no conflicts of interest.
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