Background: Human Immunodeficiency Virus (HIV) infection is associated with an increased risk of thrombosis, and treatment with antiretroviral therapy (ART) does not decrease this risk. While the mechanism leading to thrombosis is unclear, HIV infection is associated with decreased plasma anticoagulant proteins, most commonly protein S (PS), expression of tissue factor (TF) on monocytes, platelet hyperactivity, and thrombocytopenia. Despite the prevalence of PS deficiency, its pathologic consequences are unclear because PS concentration does not correlate with thrombin generation in the standard calibrated automated thrombography (CAT) assay. As increased plasma thrombin generation is a strong predictor of mortality in these patients, we sought to determine which of the described procoagulant effects of HIV is associated with thrombin generation in this population. Method: Blood was collected from 27 consenting HIV+ patients (16 naive samples collected from patients on first diagnosis and 11 samples from patients on ART) and 10 healthy controls. Results: 63% (17/27) of HIV+ plasma samples were deficient in PS, compared to the control samples, as determined by a total PS ELISA (p=0.034). PS functions as a cofactor for the anticoagulant enzyme activated protein C (APC), whose activation requires endothelial cell thrombomodulin (TM). Thus, to correlate plasma PS with thrombin generation, we established a modified CAT assay that is sensitive to PS concentration, by supplementing plasma samples with 20nM TM. Plasma PS did not correlate with thrombin generation in the absence of TM, consistent with previous reports. In the presence of TM, there was a negative correlation, with decreased PS being associated with increased thrombin peak height (p=0.004) and endogenous thrombin potential (p=0.003). Since 2.5% of total circulating PS is stored in platelets and released upon platelet stimulation, we quantified platelet PS by immunoblotting, and found no correlation with plasma PS concentration, consistent with the physiologically distinct origins of these two pools. HIV is reported to promote TF expression in monocytes, and monocytes can release TF-bearing microvesicles into plasma, so we also measured circulating TF activity, but found no difference between the patients and controls. As anticipated, TF correlated with a shorter lag time for thrombin generation, but only when no exogenous TF was added (p=0.0097). Treatment with ART resulted in undetectable viral load and also decreased plasma markers of inflammation, including TNF-α, IL-6, and IL-1β, to the levels observed in controls. However, there was no effect of ART on PS concentration (p=0.521), and platelet-leukocyte aggregates (PLA) remained elevated in patients on ART (19.66%) compared to controls (4.62%, p=0.0001). Platelet hyperactivity, as measured by PLA, was independent of platelet count, and did not correlate with platelet or plasma PS concentration. Interestingly, the use of ART had no impact on plasma PS or TF, but did correlate with greater thrombin generation in assays performed in the absence of TM, and thus the absence of PS activity (10% higher total thrombin, p=0.039), suggesting a PS-independent procoagulant effect of ART. Conclusion: PS deficiency in HIV patients correlates with higher thrombin generation and likely contributes to the thrombotic risk associated with this condition, while microvesicle TF, which does not differ from that seen in healthy controls, is unlikely to be a contributing factor. ART treatment decreases viral load and inflammation, but does not decrease platelet hyperactivity or restore plasma PS concentration and is not associated with any decrease in thrombin generation. Rather, one or more of the ART drugs appear to be further increasing thrombin generation through a PS-independent mechanism. In summary, it appears that multiple, independent factors contribute to the thrombotic risk associated with HIV, including platelet hyperactivity, plasma PS, and the ART treatment itself. Disclosures No relevant conflicts of interest to declare.
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Sep 8, 2020
Ioannis Toliopoulos 
Open Access