Table 1.
Characteristics of patients with coronary artery diseases.
Table 2.
Characteristics of patients with systemic inflammatory syndromes.
Table 3.
Characteristics of septic patients.
Table 4.
Neutrophil myeloperoxidase (MPO) content.
Figure 1.
Heterogeneity of myeloperoxidase content in circulating neutrophils from patients with acute myocardial infarction is associated to platelet adhesion and activation.
Intracellular MPO content in circulating neutrophils and platelet-neutrophil heterotypic aggregates were evaluated in whole blood samples by four-color flow cytometry. Venous blood samples were obtained within the first 6 hours of onset of symptoms. For confocal determinations, monoclonal antibodies against MPO were labeled with Alexa Fluor 488 (green) and against platelet glycoprotein Ib with Alexa Fluor 546 (red). Hoechst (blue) was used for counterstaining of nuclei. Panel A. Representative confocal images illustrate the MPO (green) content observed in circulating neutrophils of a patient with chronic stable angina. Panel B. Representative confocal images illustrate the heterogeneity in the MPO (green) content observed in circulating neutrophils of a patient with acute myocardial infarction. Panel C-D. The cytofluorimetric histogram shows the trimodal pattern MPO-associated distribution (black) in AMI but not in CSA, vs isotype control (white). Panel E: confocal images of neutrophils with complete MPO depletion, low or normal MPO content. Results are the mean value ± SEM of heterotypic aggregates observed in each neutrophil subpopulation. Panel F: confocal microscopy of aggregates between platelet (red) and degranulated neutrophils from a representative patient with AMI. Panel H. Correlation analysis indicates the association between platelet activation and neutrophil myeloperoxidase content in patients with AMI: platelet P-selectin expression (% of CD61+ cells, y axis) are plotted against the neutrophil intracellular MPO content (MFI-within CD66b+ cells, x axis; R = 0.6, P<0.0001).
Table 5.
Markers of cellular activation.
Figure 2.
Time course of leukocyte and platelet activation in patients and controls.
36 patients with acute myocardial infarction were studied before any therapeutic treatment and re-studied at different times on PCI. Results (expressed as mean ± SEM) were compared with 69 patients with chronic stable angina candidates (CSA) for PCI and 138 healthy donors. Determinations were performed by flow cytometry, with the exception of troponin I, as described in material and methods. Y axis: serum troponin (Panel A), granulocyte cell counts (panel B), fraction of circulating neutrophils with complete myeloperoxidase (MPO) depletion (Panel C), average of neutrophil MPO content (Panel D), fraction of platelet expressing P-selectin (Panel E), the fraction of platelet-neutrophils heterotypic aggregates (Panel F), fraction of platelet-monocyte heterotypic aggregates (Panel G). * = P<0.05; ** = P<0.01 (both respect to CSA and healthy donors) were determined by ANOVA followed Bonferroni test.
Figure 3.
In vivo evidence that circulating activated platelets cause neutrophil degranulation.
Purified platelets from wild type or Psel −/− mice were activated (or not) and injected in the tail vein of wild type C57BL/6 mice. Platelet activation was achieved with thrombin. The molecule was then inactivated by addition of hyrudin. Neutrophil myeloperoxidase content, platelet P-selectin expression and platelet-neutrophil heterotypic aggregates were assessed by flow cytometry as described in the Methods. Panel A shows the effect of the in vivo injection of the buffer used for platelet activation, containing both thrombin and hyrudin. Blood was retrieved at different times after injection (hours, x axis) and neutrophils with adherent (filled symbols) or internalized (grey symbols) platelets were identified by flow cytometry. >70% of circulating neutrophils were engaged in the internalization and adhesion of activated, but not resting, platelets after 1 hour. The fraction of circulating neutrophils with internalized and adherent platelets returned to background levels 24 hours after the injection. Results are expressed as mean±S.E.M. 5–7 animals were assessed per each point.