1) Platelet-derived microvesicles (PMV) preferentially bind to monocytes in blood and induce their aggregation. 2) PMV derive from platelets and express glycoprotein Ib (GPIb) on their surface. 3) GPIb on PMV promotes monocyte recruitment by supporting their adhesion and transmigration through endothelial cells under flow via binding von Willebrand factor exposed on stimulated endothelial cells. This represents a novel pathway of leukocyte recruitment in vascular inflammation and disease.

1. A novel thrombo-inflammatory pathway for monocyte recruitment
mediated by platelet-derived microvesicles
INTRODUCTION
Aigli Evryviadou, Clare L Box, Myriam Chimen, Matthew J Harrison, Sahithi Kuravi,
Steven G Thomas, Steve P Watson Paul Harrison, Gerard Nash, G Ed Rainger
Institute of Cardiovascular Science, University of Birmingham, Birmingham, UK
• Platelet-derived Microvesicle (PMV) Isolation: Platelets were isolated from whole blood and were suspended in
Dulbecco’s phosphate buffered saline (PBS) (with Ca2+ / Mg2+) and incubated at 37°C for 30 minutes with 1 μg / ml
CRP-XL to induce the shedding of PMV. After centrifugation to remove platelets, supernatant containing pure PMV
was collected and PMV numbers and purity was assessed by flow cytometry and NanoSight Tracking Analysis (NTA)
(Fig. 1A and 1B).
• Leukocyte-PMV aggregation in whole blood: Anticoagulated whole blood was treated with 333μM / ml TRAP
(thrombin receptor activating peptide) or blood was treated with 1.0 x 109 PMV / ml whole blood and incubated at
37°C for 30 minutes under low shear stress conditions. Following incubation, blood was fixed and stained for CD14,
CD16 and glycoprotein Ib (GPIb). Samples were analysed by flow cytometry.
• Monocyte-PMV aggregation: Monocytes were isolated from whole blood and were suspended in (PBS) (with Ca2+
/ Mg2+) containing 1 x 109 PMV per 1 x 106 monocytes and incubated at 37°C for 30 minutes under low shear stress
conditions. Following incubation, unbound PMV were removed by centrifugation and cells were stained for CD14,
CD16 and GPIb. Samples were analysed by flow cytometry and confocal microscopy.
• Flow-based adhesion assays: Capillary tubes were coated with 0.1 mg / ml human, purified VWF (von Willebrand
factor). Alternatively, ibidi channels were coated with endothelial cells (EC) and stimulated with TGF-β1 to induce
expression of VWF. Coated slides were connected to a flow adhesion assay and monocytes ± PMV were perfused
over the adhesive substrates. Adhesion and/or transmigration were monitored by phase-contrast video-
microscopy (Fig. 1C).
There is now accumulating evidence to suggest that platelets contribute to the progression of atherosclerosis by
acting as an adhesive substrate that leads to the recruitment of monocytes to the vessel wall1-3. Platelets also form
circulating aggregates with monocytes in inflammatory diseases such as diabetes, rheumatoid arthritis, inflammatory
bowel disease, 4-6 and these heterotypic aggregates can exacerbate atherosclerosis in ApoE -/- mice8. Platelets also
shed extracellular microvesicles when they get activated, the levels of which are high in atherosclerosis patients9. We
wondered whether these could also interact with monocytes in an atherogenic manner.
Hypothesis: Platelet-derived microvesicles (PMV) are involved in the recruitment of monocytes at sites of
inflammation.
METHODS
RESULTS
Platelet activation in whole blood induces the release of platelet-derived
microvesicles that form aggregates preferentially with monocytes
Figure 2. Aggregation between leukocytes and PMV in stimulated whole blood
(A) Percentage of GPIb-positive monocytes, neutrophils and lymphocytes in whole blood stimulated for 30 minutes with 100 μM TRAP at 37°C under shear,
as determined by flow cytometry, n=3. (B) GPIb staining on monocytes in TRAP-stimulated whole blood by time and on unstimulated platelets, as measured
by flow cytometry, showing accumulation of GPIb in small quanta at all time points, implying that monocytes are binding PMV. Data are from three
independent experiments using three donours and are shown as mean ± S.E.M. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 compared to the 0 min control by
Dunnet post-test.
Addition of pre-formed platelet-derived microvesicles to whole blood
causes their preferential adherence to monocytes
Platelet microvesicle-derived GPIb supports monocyte recruitment both on
TGF-β1 stimulated endothelial cells and human purified VWF
CONCLUSION
Figure 3. Aggregation between leukocytes in whole blood and exogenous PMV
(A) Percentage of GPIb-positive monocytes, neutrophils and lymphocytes in whole blood upon incubation with 1.0 x 109 PMV / ml whole blood for 20
minutes at 37°C under shear, as determined by flow cytometry, n=3. (C) Representative microscopy images of GPIb labelling (in green) on monocytes
upon incubation with purified PMV for 30 minutes at 37°C under shear.
Figure 4. Monocyte recruitment on TGF-β1- stimulated endothelial cells and on purified human VWF in the presence of PMV
(A) Total adhesion of monocytes with or without PMV and GPIb blockade on TGF-β1-stimulated endothelial cells in flow conditions, n=3-5. and (B)
Percentage of transmigrated monocytes with or without PMV and GPIb blockade on n TGF-β1-stimulated endothelial cells in flow conditions, n=3-5. (C)
Total adhesion of monocytes with or without PMV and GPIb blockade on purified human VWF, n=3. Data are shown as mean ± S.E.M. * P ≤ 0.05, ** P ≤
0.01, *** P ≤ 0.001 compared to the 0 min control by ANOVA and Bonferroni post-test.
References
(1) Bahra P, Nash GB. Sparsely adherent platelets support capture and immobilization of flowing neutrophils. Journal of Laboratory and Clinical Medicine. 1998; 132: 223-228. (2) Diakovo TG, Roth SJ, Buccola JM, Bainton DF, Springer
TA. Neutrophil rolling, arrest and transmigration across activated, surface-adherent platelets via sequential action of p-selectin and the beta 2-integrin cd11b/cd18. Blood. 1996; 88: 146-157. (3) Kuckleburg CJ, Yates CM, Kalia N, Zhao
Y, Nash GB, Watson SP, Rainger GE. Endothelial cell-borne bridges selectively recruit monocytes in human and mouse models of vascular inflammation. Cardiovascular Research. 2011; 91: 134-141. (4) Harding SA, Sommerfield AJ,
Sarma J, Twomey PJ, Newby DE, Frier BM, Fox KA. Increased cd40 ligand and platelet-monocyte aggregates in patients with type 1 diabetes mellitus. Atherosclerosis. 2004; 176: 321-325. (5) Joseph JE, Harrison P, Mackie IJ, Isenberg
DA, Machin SJ. Increased circulating platelet-leucocyte complexes and platelet activation in patients with antiphospholipid syndrome, systemic lupus erythematosus and rheumatoid arthritis. British Journal of Haematology. 2001;
115: 451-459. (6) Tekelioglu Y, Uzun H, Gucer H. Circulating platelet-leukocyte aggregates in patients with inflammatory bowel disease. Journal of Chinese Medical Association. 2013; 76: 182-185. (7) Huo Y, Schober A, Forlow SB,
Smith DF, Hyman MC, Jung S, Littman DR, Weber C, Ley K. Circulating activated platelets exacerbate atherosclerosis in mice deficient in apolipoprotein e. Nature Medicine. 2003; 9: 61-67. (8) Michelsen AE, Brodin E, Brosstad F,
Hansen JB. Increased level of platelet microparticles in survivors of myocardial infarction. Scand J Clin Lab Invest. 2008; 68: 386–392.
Figure 5. Schematic of paradigm
PMV preferentially bind monocytes in blood and the GPIb from PMV promotes adhesion of monocytes to
endothelial cells stimulated by TGF-β1 by binding to released VWF, enabling trans-endothelial migration (Fig. 5).
Importantly, this pathway of leukocyte recruitment falls outside of the usual regulatory mechanisms that control
the process of inflammation and may represent a novel pathway by which monocyte trafficking in vascular disease
can be moderated.
Figure 1. (A) Gating strategy for detecting PMV by flow cytometry. PMV are defined by having slightly smaller size and granularity than whole platelets. (B)
Total number of PMV generated from 3 x 108 platelets, counted by NTA. (C) Flow-based Adhesion assay to measure monocyte adhesion to immobilised
substrates.
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