Flow cytometric analysis of platelets type 2 diabetes mellitus reveals ‘angry’ platelets

Soma et al. Cardiovasc Diabetol (2016) 15:52 DOI 10.1186/s12933-016-0373-x ORIGINAL INVESTIGATION Cardiovascular Diabetology Open Access Flow cytometric analysis of platelets type 2 diabetes mellitus reveals ‘angry’ platelets Prashilla Soma, Albe Carina Swanepoel, Jeanette Noel du Plooy, Thandi Mqoco and Etheresia Pretorius* Abstract Background: The function of platelets have extended way beyond the horizon of haemostasis and thrombosis, and are recognised as active participants in vascular inflammation, as well as in prothrombotic complications of cardiovascular diseases. We describe and compare platelet function in type II diabetes (with and without cardiovascular manifestation) and healthy individuals using scanning electron microscopy and flow cytometry. Methods: Thirty subjects were recruited per group and informed consent was obtained from all participants. Diabetic patients were recruited from the diabetic clinic of the Steve Biko Academic Hospital (South Africa). Blood samples were drawn from all participants so that platelet specific antigens were analyzed in citrated whole blood. The platelet parameters used in the study were platelet identifiers (CD41 and CD42) and markers of platelet activation (CD62 and CD63). Results: Results show that, compared to healthy individuals, both diabetic groups showed a significant difference in both platelet identifiers (CD41-PE, CD42b-PE) as well as markers indicating platelet activation (CD62P-PE and CD63-PE). Interpretation: The flow cytometric data shows that the platelet surface receptors and platelet activation are statistically elevated. This is suggestive of enhanced platelet activation and it appears as if platelets are displaying ‘angry’ behaviour. The lysosomal granules may play a significant role in diabetes with cardiovascular complications. These results were confirmed by ultrastructural analysis. Background It is plausible to underestimate the impact of platelets in clinical medicine, when one considers that these blood cells are only 1.5–3 μm in size, survive for approximately 8–10 days, and are mere fragments of megakaryocyte cytoplasm [1–3]. The function of platelets have extended way beyond the horizon of haemostasis and thrombosis. In fact, they are now recognised as active participants in initiating and sustaining vascular inflammation as well as in prothrombotic complications of cardiovascular diseases [1]. Platelets have been assigned multiple attributes and have been described in inflammatory conditions such as atherosclerosis, arthritis and tumour metastasis [1]. Due to the multifunctional role of platelets, they *Correspondence: Department of Physiology, School of Medicine, Faculty of Health Sciences, University of Pretoria, Private Bag x323, Arcadia, Pretoria 0007, South Africa are an accessible and important inflammatory marker for disease pathophysiology [4, 5]. Platelets are activated when they are in contact with damaged vascular endothelium [5], and once activated, they are able to secrete a wide spectrum of inflammatory mediators that exert both local and systemic effects [6]. Platelet activation is also the mechanism implicated in the pathogenesis of chronic medical conditions such as atherosclerosis, coronary vascular disease and cerebrovascular disease [3]. Due to inflammation there is an imbalance between procoagulant and anticoagulant properties of the endothelium with subsequent local stimulation of the coagulation cascade [7]. Another feature of inflammation is a multitude of interactions between leukocytes, endothelial cells and platelets. More importantly, regardless of its aetiology, inflammation causes endothelial activation [7]. In diabetes mellitus, endothelial dysfunction is one of the mechanism ascribed to increased atherothrombotic risk [8]. With © 2016 Soma et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Soma et al. Cardiovasc Diabetol (2016) 15:52 Page 2 of 7 cigarette smoking, the endothelium becomes activated and induces the intrinsic coagulation pathway. This results in platelet activation and enhanced platelet aggregation, which in turn causes thrombin stimulation and fibrin formation [9]. Abnormal platelet activation, platelet count and volume have been implicated as risk factors of ischaemic stroke [10]. Once platelets are activated, they initiate reactions whereby changes in the level of expression of surface glycoproteins (GP) results, which act as receptors for platelet agonists and for adhesive proteins, involved in platelet aggregation. Platelet activity can be measured using various fluorescently labelled markers in flow cytometry. As flow cytometry allows the simultaneous detection of surface antigens in a sensitive and specific manner, it is therefore possible to examine aspects of the platelet membrane activity—see Table 1 for examples of available platelet markers. Platelets in inflammatory conditions There is a strong indication that platelets also have relevant functions in inflammation [11]. In fact, it was shown that thrombosis and inflammation share many key molecular mechanisms and that they are fundamentally linked processes [7]. It is now recognised that vascular inflammation is the key underlying mechanism in atherogenesis and atheroprogression. The evidence of platelets being fundamental mediators in the initiation and maintenance of a chronic proinflammatory milieu is provided by the direct interactions with inflammatory cells and secretion of autocrine and paracrine effector molecules [12]. Another emerging concept is the significant role of platelet-mediated recruitment of leucocytes in the propagation, progression and pathogenesis of atherosclerotic disease. Platelets can interact with leucocytes: (a) during haemostasis, when there is vascular damage and recruit leucocytes to the growing thrombus, (b) when endothelial cells are stimulated thereby adhering and activating platelets and then bridge blood-borne leucocytes to the vessel wall and (c) in the formation of heterotypic aggregates prior to contact with endothelial cells when adhesion between platelets and leucocytes occur in the blood [13]. It is well known that in subjects with type 2 diabetes mellitus, function of platelets is impaired. In fact, a subthreshold stimuli is needed to activate platelets which are constantly in activation despite the lack of a major plaque event and have thus been defined as ‘angry platelets’ [14]. This is significant as (...truncated)