Neutrophils phagocytose activated platelets in vivo: a phosphatidylserine, P-selectin, and β2 integrin–dependent cell clearance program

Abstract

Activated platelets express ligands, which are recognized by counterreceptors on neutrophils. Here, we show that the ensuing cell-to-cell interaction programs neutrophil phagocytic function, resulting in activated platelet clearance. Neutrophils that have internalized platelets circulate in the blood of patients with acute myocardial infarction, and the extent of platelet clearance correlates with expression of platelet activation, including P-selectin. Activated platelets injected intravenously in experimental animals are detectable in circulating neutrophils 60 minutes after, and within 3 hours, more than 70% circulating neutrophils have internalized platelets. Platelet clearance comprises 2 events: adhesion to neutrophils, which requires divalent cations and depends on P-selectin, on the P-selectin glycoprotein ligand-1 (PSGL-1), and on the CD11b/CD18 ␤2 integrin; and internalization, which is abrogated by the phosphatidylserine-binding protein annexin A5. Adhesion to platelets causes neutrophil degranulation and is blocked by antibodies specific for P-selectin and PSGL-1, either in a synthetic medium in vitro or in the whole blood, therefore in the presence of a physiologic array of plasma cofactors and opsonins. The data suggest that the interaction between circulating platelets and neutrophils influences innate immune functions, possibly contributing to regulate vascular inflammation. IntroductionNeutrophils are recruited to inflamed sites, where they are required for microbial clearance. Neutrophil recruitment is a multistep process, which comprises initial tethering and rolling along the vessel wall, firm adhesion to endothelial cells, and eventual extravasation. It involves consecutively various adhesion molecules, including selectins and ␤2 integrins. [1][2][3][4] Granules of endothelial cells (Weibel-Palade bodies) and of platelets (␣-granules) contain P-selectin. Inflammatory stimuli cause its translocation at the endothelial cell surface. The interaction with the counterreceptor, PSGL-1, prompts leukocyte tethering and rolling and initiates the second phase of the process, firm adhesion. During these events, integrins shift to an active conformation. [5][6][7] Integrin activation depends on the signaling cascade downstream the P-selectin/PSGL-1 interaction. [8][9][10] Platelets adhere and are activated at sites of vascular injury: there, they produce a releasate, some contents of which penetrate and/or interact biochemically with neutrophils. This includes unprocessed free arachidonic acid which can then be transformed into leukotriene A4 and B4. 11-14 Furthermore, activated platelets express P-selectin. Platelet P-selectin guarantees the access of leukocytes to perivascular tissues even when dying or severely damaged endothelial cells fail to sustain leukocyte rolling and adhesion. 15,16 Indeed, endothelial cells surrounding the lesion release signals that amplify the expression of P-selectin on adhering platelets. 17 The P-selectin-dependent interaction of neutrophils and platelets amplifies mut...

Early and Transient Release of Leukocyte Pentraxin 3 during Acute Myocardial Infarction

“…For studies of neutrophil and platelet interaction, blood was carefully collected in tubes containing 0.2 ml sodium citrate (109 mM) and a mixture containing Na 2 EDTA (50 mM), N-ethylmaleimide (60 mM), and aprotinin (2000 kIU/ml) to limit in vitro cellular activation as much as possible. Aliquots were either immediately fixed with an equal volume of ThromboFix or treated with FACS lysing solution and processed for flow cytometry, immunofluorescence, and confocal microscopy (23,41).…”

“…Blood was drawn with an 18-gauge needle, dripping freely into open tubes, containing 3.8% sodium citrate (1:9, v/v). Neutrophils and platelets were isolated as described (23,38) and resuspended in HEPES-Tyrode buffer (pH 7.4) containing CaCl 2 (1 mM). Neutrophils were incubated (5 min at 37˚C) either alone or with resting or activated autologous platelets (neutrophil/platelet ratio, 1:20); with CHO-P or with CHO (neutrophil/CHO ratio, 5:1); with fMLF (0.5 mM) in the presence of cytochalasin B (2.5 mg/ml); or with purified Pselectin (5 mg/ml) (38).…”

“…Samples were then labeled with Alexa Fluor 488-biotin and with the anti-CD61 mAb; the fraction of platelets with bound PTX3 was then determined by flow cytometry (see below). To assess platelet-neutrophil and platelet-monocyte heterotypic aggregates, purified platelets were loaded with the BCECF-AM intracellular dye as previously described (23,38), resuspended at a final concentration of 200,000/ml, and stimulated with TRAP-6 (15 mM, 3 min at 37˚C) in the presence of increasing concentrations (5-40 mg/ml) of recombinant PTX3 or of human albumin. An equal volume of autologous whole blood was then added.…”

“…All samples were analyzed as described (23). Briefly, for ex vivo determinations, to assess neutrophil-platelet heterotypic aggregates, whole samples were fixed with ThromboFix and labeled with mAbs against CD45, CD14, CD66b, and CD61; neutrophils were identified within the CD45 + CD66b + and CD14 2 populations and by their orthogonal light scatter.…”

Co-existent Platelet Phagocytosis, Satellitism and Clumping Causing Spurious Thrombocytopenia

“…Platelet clumping in blood samples collected in EDTA occurs due to the chelating effect of EDTA, which alters the GPIIb-IIIa molecule on the platelet surface, thereby leading to binding of a naturally occurring IgG antibody to GPIIb [12]. Other anticoagulants, such as sodium citrate or heparin, do not affect GPIIb-IIIa, and therefore do not lead to platelet agglutination in these cases [13]. Enhanced platelet activation and subsequent aggregation has been reported in post renal transplant patients especially those on Cyclosporine and/or Azathioprine therapy [14].…”