Page 89 - Haematologica May 2020

Platelet vesicles and monocyte interaction
role in leukocyte recruitment in other vascular beds.5 Thus, the integrated function of the thrombotic and inflammato- ry systems results in recruitment of leukocytes to arteri- oles in models of ischaemic injury of the liver and other tissues.6-10 Moreover, there is substantial evidence support- ing a role for platelets in the preferential recruitment of monocytes to the artery wall during atherogenesis. For example, inhibition of platelet adhesion to the artery wall, or induction of thrombocytopenia, significantly reduces monocyte trafficking and the burden of atherosclerotic disease in genetically susceptible strains of mice.11-14 In addition, instillation of activated platelets exacerbates the formation of atherosclerotic plaques in such models.11-14 There is also direct evidence that platelet P-selectin plays a role in plaque formation in the ApoE-/- mouse.11-14 Other studies demonstrate that platelet derived chemokines such as CCL5 (RANTES) and CX3CL1 (fractalkine), once deposited on vascular endothelial cells, can selectively recruit monocytes in these models.11-15
The examples described above require platelet activa- tion at the vessel wall to facilitate leukocyte recruitment and trafficking. However, interactions between platelets and leukocytes also occur in circulating blood under pathological conditions. Indeed, formation of platelet- leukocyte aggregates has been described in diseases as diverse as bacterial infection, rheumatoid arthritis, dia- betes and inflammatory bowel disease.16-22 In cardiovascu- lar disease (CVD) the number of platelet-leukocyte aggre- gates increases significantly, and one can measure an increased incidence of such heterotypic aggregates in individuals with independent risk factors for CVD, such as hypertension.23-25 Indeed, it has been proposed that an increase in the incidence of platelet-leukocyte aggregates may in itself, be an independent risk factor for CVD.26 The formation of platelet leukocyte aggregates may also play an important role in acute and severe inflammatory responses. Thus, in patients with acute trauma or trauma associated sepsis, an enhanced capacity for platelet acti- vation and platelet interaction with monocytes and neu- trophils has been reported in response to exogenous acti- vation of their blood with the ionophore, ionomycin.27,28
Extracellular vesicles which can be detected in the blood, urine and other bodily fluids are heterogeneous particles 40-1,500 nm in diameter that are derived from the plasma membrane (microvesicles) or by exocytosis of multi-vesicular bodies (exosomes).29 They are released from cells of the vasculature, including platelets, endothe- lial cells (EC) and leukocytes, and specific populations can be identified using appropriate methodology (e.g. flow cytometry), as they express surface markers derived from their cell of origin. There is now mounting evidence that platelet-derived extracellular vesicles (PEV) (otherwise and often referred to as microparticles or microvesicles) are heterogeneous in nature. For example, in vitro, PEV have been generated in response to shear stress, throm- bin, calcium ionophore, adenosine diphosphate (ADP), collagen and collagen related peptide.30-33 Interestingly, these studies show that PEV derive by using different platelet agonists and differ in abundance, as well as the cargo that they convey. Indeed, there is now good evi- dence that platelets can shed large vesicles which contain organelles such as mitochondria.34 Until recent technolog- ical advancements it had been impossible to analyse the concentration and composition of vesicles using a single platform. Flow cytometry does not detect vesicles <200-
300 nm and does not accurately measure larger vesicles due to the disparity in the refractive index of biological vesicles and the latex beads used as size standards on this platform.35 However, electron microscopy studies show that the majority of PEV are small. Thus, although Ponomereva et al. described calcium ionophore derived PEV as large as 1,500 nm, particles were predominantly in the range of 50-130 nm.36 Similarly, Aatonen et al. described the main population of PEV as being 100-250 nm, with in excess of 90% of all vesicles being smaller than 500 nm irrespective of the platelet agonist used for PEV biogenesis.35 Mitochondria containing vesicles, referred to above, were in the range of 500-1,500 nm. Importantly, the study of the functions of distinct subsets of PEV is not a well-developed field, however, bearing in mind the diversity of the PEV generated upon platelet activation, vesicles with discrete functional roles cannot be ruled out. The diversity of platelet microparticles has recently been reviewed.37
There is mounting evidence that PEV play a pathophys- iological role in inflammation.38 An increased concentra- tion of circulating PEV is associated with a number of dis- eases. In diabetic retinopathy, the number of PEV was associated with the severity of disease,39 while the levels of PEV circulating in patients with type-1 diabetes corre- lated with the degree of pro-atherogenic dyslipidaemia.40 There was a correlation with vascular dysfunction (assessed by measuring arterial elasticity and flow-depen- dent vasodilatation of the brachial artery) in patients with type-2 diabetes.41 Interestingly, the number of PEV was higher in patients with acute coronary syndromes than those with stable angina,42 implying an association with the onset of athero-thrombotic disease. The roles of PEV in inflammation and pathogenesis of inflammatory dis- ease are not well understood. However, they possess adhesion receptors such as glycoprotein (GP)Ibα, αIIbβ3- integrin and P-selectin, meaning that they could interact with the vessel wall and circulating leukocytes to promote recruitment of the later. Importantly, as these receptors ordinarily regulate the process of haemostasis, PEV might provide an avenue of leukocyte recruitment to the disease environment which falls outside of regulatory pathways which ordinarily limit the duration and magnitude of the inflammatory response.
Here, using assays of heterotypic aggregate formation we have characterised the adhesive interactions between leukocytes and PEV in whole blood and identified a novel route by which the platelet adhesion receptor, GPIbα, pro- motes monocyte recruitment in both in vitro and in vivo models of vascular inflammation.
Methods
Full Methods can be found in the Online Supplementary Materials and Methods.
Blood donors
Blood was obtained from healthy donors with informed con- sent and with local ethical approval (ERN_07-058). Blood from the Golden Hour cohort (drawn within 1 hour of suffering traumatic injury) was obtained under the National Research Ethics Committee (reference 13/WA/0399). Specimen collection and informed consent procedures were approved and permission granted by the Biomedical Science Ethic Committee.
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