Background/Purpose: Patients with systemic lupus erythematosus (SLE) are at increased risk of premature atherosclerosis and thrombosis. Hydroxychloroquine (HCQ) is widely used in the treatment of SLE and has been considered of benefit for overall vascular health albeit studies to address this benefit at the cellular level have been limited.  Accordingly, this study was initiated to investigate the relationship between HCQ use and dose with platelet activity, the platelet transcriptome, and vascular functional readouts.

Methods: Patients fulfilling ACR or SLICC criteria for SLE were consecutively recruited for platelet evaluation with the only exclusion being on nonsteroidal anti-inflammatory medications, aspirin or anticoagulants.  At enrollment, blood was collected for hematology analysis using the Sysmex XN-1000 analyzer, platelet aggregation via the Helena AggRAM™ system, and platelet RNA isolation and storage. Microvascular function was assessed via sublingual sidestream darkfield imaging. Brachial artery reactivity testing was used to evaluate large vessel function. Stored platelet RNA was isolated and analyzed by RNA sequencing (Illumina HiSeq4000 Sequencing).

Results: Among 132 SLE subjects, 108 were on HCQ. Mean age was 39.9 ± 13.0 and 97% were female. Lupus disease activity at the time of blood draw assessed by the SELENA-SLEDAI activity index was 3.44 (range 0-20). Demographics and SLE disease activity did not differ between those on versus off HCQ (Table 1). Platelet count and size were not different between groups (Figure 1A). Platelet aggregation in response to submaximal ADP at multiple concentrations was lower in participants on HCQ (Figure 1B). Consistently, there was an inverse relationship between HCQ dosing and platelet aggregation in response to ADP (2uM: R=-0.213, P=0.037; 1uM: R=-0.310, P=0.0025; 0.4uM: R=-0.376, P=0.00018; Figure 1C).  Since no subjects were on aspirin (or any other antiplatelet therapy at enrollment), aggregation in response to arachidonic acid (AA) was robust and similar between groups. However, after incubating platelets with aspirin (3mM) in vitro, platelet aggregation in response to AA was lower in the HCQ group compared to non-HCQ group (P=0.035, Figure 1B). To investigate the potential mechanisms of HCQ induced lower platelet aggregation, we evaluated platelet RNA sequencing in 49 subjects (8 no HCQ, 41 on HCQ). Positive regulation of pathways related to platelet activation (and in particular, P-selectin expression) was inversely related to HCQ, especially with higher doses (Figure 1E). In terms of vascular function, subjects on HCQ had improved microvascular function as noted by an increased proportion of sublingual capillaries filled with RBCs (P=0.011) and smaller perfused boundary region (PBR, P=0.010). HCQ dosing correlated with PBR (R=-0.599, P=0.002, Figure 1H) and RBC Filling (R=-0.592, P=0.002, Figure 1I). BART also trended positively with HCQ dose (R=0.385, P=0.094; Figure 1J).

Conclusion: These findings suggest that HCQ may provide benefit for vascular health in SLE as supported by ex vivo experiments demonstrating decreased platelet aggregation and downregulation of platelet functional pathways as well as improved vascular readouts.

Figure 1: Associations between HCQ use and dose and platelet function, transcriptomics, and vascular function. Comparison of HCQ and non-HCQ groups of SLE subjects examining (A) platelet count and size. (B) Aggregation in response to ADP and AA with and without in vitro aspirin. (C) Pearson correlations of daily HCQ dosing and aggregation in response to 1µM ADP. (D) Heatmap depicting the correlation and p value between the sample eigengene values associated with platelet pathways and HCQ use, dose, and dose adjusted for weight. (E) Boxplots showing the eigengene values for the GO term Positive Regulation of Platelet Activation across different binned ranges of HCQ dose/weight. (F) Scatterplot showing the normalized transcript level of SELP compared against HCQ dose/weight for each patient. (G) Comparison of p-selectin surface expression fold change in subjects on and off HCQ as measured by flow cytometry and a Pearson correlation of HCQ dose and p-selectin fold change. Platelet activity and p-selectin surface expression comparisons made using the Mann-Whitney rank-sum test show median and IQR; for platelet aggregation the HCQ group ranged from 71 to 81 subjects and non-HCQ group ranged from 13 to 15 depending on the measure. For p-selectin, HCQ N=36, No HCQ N=5. Pearson correlations of daily HCQ dosing and PBR (H), RBC Filling (I), and BART (J) were also examined, shown here with 95% CI bands.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/hydroxychloroquine-is-associated-with-lower-platelet-activity-and-improved-vascular-health-in-systemic-lupus-erythematosus/