Genetic factors contribute to the risk of thrombotic diseases. Recent genome wide association studies have identified genetic loci including SLC44A2 which may regulate thrombosis. Here we show that Slc44a2 controls platelet activation and thrombosis by regulating mitochondrial energetics. We find that Slc44a2 null mice (Slc44a2(KO)) have increased bleeding times and delayed thrombosis compared to wild-type (Slc44a2(WT)) controls. Platelets from Slc44a2(KO) mice have impaired activation in response to thrombin. We discover that Slc44a2 mediates choline transport into mitochondria, where choline metabolism leads to an increase in mitochondrial oxygen consumption and ATP production. Platelets lacking Slc44a2 contain less ATP at rest, release less ATP when activated, and have an activation defect that can be rescued by exogenous ADP. Taken together, our data suggest that mitochondria require choline for maximum function, demonstrate the importance of mitochondrial metabolism to platelet activation, and reveal a mechanism by which Slc44a2 influences thrombosis.

There are 37 million people globally infected with the Human Immunodeficiency Virus (HIV). People living with HIV can achieve nearly normal lifespans due to the use of antiretroviral drugs (ARVs). However, people living with HIV experience chronic inflammation and increased risk for cardiovascular diseases (CVD) relative to uninfected people. While the cause for this risk is unclear, some ARVs have been associated with CVD, and it is speculated that some ARVs potentiate inflammation in infected individuals. Platelets are a critical link between inflammation and the development and progression of CVD, but the effects of ARVs on platelets are largely understudied. In this study, we examined the effects of ARVs on human platelet function in vitro. Our data show that the ARV ritonavir, a protease inhibitor, severely altered human platelet lipid mediator production (prostaglandin E2 and thromboxane) in both resting and activated platelets. Further characterization revealed that ritonavir altered measures of platelet hemostatic and thrombotic function that included significantly decreased platelet spreading, increased platelet aggregation, and trended toward increased clot strength. These data provide proof-of-principle that ARVs can directly dysregulate human platelets, possibly contributing to inflammation-related comorbidities. These data may provide mechanistic insight into the factors contributing to increased risk of CVD in people living with HIV, and may help guide future development of new HIV agents and ARV regimens that mitigate platelet dysregulation by ARVs.

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In this document, we describe methods for the isolation, treatment, and functional testing of human blood platelets in vitro. Functional assays for inflammatory function include flow cytometry and immunoassays for platelet release of platelet factor 4, soluble CD40L, prostaglandin E , and thromboxane. Assays for platelet hemostatic function described here examine platelet spreading, aggregation using platelet-rich plasma, and thromboelastography. Also described here are methods for testing cigarette smoke on primary human platelets in vitro, which our lab developed to address a major knowledge gap regarding how cigarette smoke dysregulates platelets and how this platelet dysregulation contributes to cardiovascular disease. Some of these protocols may be repurposed for investigation of the toxicity potential of other tobacco products and environmental insults. © 2018 by John Wiley & Sons, Inc.

Human blood platelets are major hemostatic regulators in the circulation and important in the mediation of chronic inflammation and immunomodulation. They are key elements that promote cardiovascular pathogenesis that leads to atherosclerosis, thrombosis, myocardial infarction, and stroke. New information on tobacco use and platelet dysregulation shows that these highly understudied vascular cells are dysregulated by tobacco smoke. Thus, platelet function studies should be an important consideration for the evaluation of existing and next-generation tobacco and non-tobacco products. Novel in vitro approaches are being sought to investigate these products and their influence on platelet function. Platelets are ideally suited for product assessment, as robust and novel in vitro translational methods are available to assess platelet function. Furthermore, the use of human biological systems has the advantage that risk predictions will better reflect the human condition.