To examine the possible receptor-ligand pairs mediating adhesion of activated and “unactivated” platelets to leukocytes and the kinetics of leukocyte-platelet binding, we developed a flow cytometric assay using isolated cell fractions to accurately measure heterotypic cell adhesion, including both total leukocyte-platelet conjugate formation as well as the number of platelets bound per leukocyte. We have shown that (1) activated platelet binding to both polymorphonuclear leukocytes (PMN) and monocytes is dependent on both a specific epitope (blocked by monoclonal antibody G1) of granule membrane protein-140 (GMP-140) and the presence of divalent cations; (2) unactivated platelets bind to 87% of viable, resting monocytes but to only 34% of PMN; (3) the receptor(s) on unactivated platelets that mediate adhesion to PMN and monocytes do not require divalent cations and become nonfunctional after thrombin activation; and (4) the kinetics of platelet adhesion to monocytes and PMN indicate that monocyte adhesion is favored over neutrophil adhesion. We conclude that platelet- heterotypic cell adhesion is a dynamic process reflecting the activation status of the platelet and differential binding abilities of leukocytes.
Cardiopulmonary bypass (CPB) has been demonstrated to activate platelets, producing an increased number of circulating platelets that have undergone alpha-granule release and express granule membrane protein-140 (GMP-140) on their surface. In vitro, GMP-140 mediates activated platelet adhesion to neutrophils (PMN) and monocytes, causing the formation of leukocyte-platelet conjugates. Using a newly developed assay that measures the percentage of circulating leukocyte-platelet conjugates in whole blood, we studied 17 patients undergoing CPB and have determined that (1) monocyte-platelet conjugates increased significantly during CPB, from 18% +/- 1.5% to 44% +/- 4.5% (mean +/- SEM) by the end of CPB, while PMN-platelet conjugates increased only slightly and lymphocyte-platelet conjugates decreased; (2) the time course of the increase in monocyte- and PMN-platelet conjugates paralleled that of the increase in circulating activated platelets, as determined by the presence of surface GMP-140; and (3) monocyte activation, as assessed by increased surface expression of CD11b, showed a gradual increase similar to the increase in monocyte-platelet conjugates, while PMN surface CD11b peaked immediately after the start of CPB. We conclude that CPB, through increased platelet GMP-140 expression, causes formation of monocyte-platelet, and to a lesser extent, PMN-platelet conjugates. The activation of monocytes and PMN on CPB, as evidenced by CD11b expression, occurs with differing time courses.
The morphological development of larval cobia Rachycentron canadum from 3 days post hatch (dph) until weaning (27 dph) was examined using S.E.M. Two groups of fish were studied: a control group (CF), reared under standard feeding protocol, and a group in which prey items were enriched with supplemental taurine (4 g l(-1) day(-1) ; TF). TF fish grew faster (P < 0·001), attained greater size (mean ±s.e. 55·1 ± 1·5 v. 33·9 ± 1·0 mm total length) and had better survival (mean ±s.e. 29·3 ± 0·4 v. 7·1 ± 1·2 %) than CF fish. Canonical variance analysis confirmed findings with respect to differences in growth between the treatment groups with separation being explained by two cranial measurements. S.E.M. revealed that 3 dph larvae of R. canadum (in both groups) possess preopercular spines, superficial neuromasts on the head and body, taste buds in the mouth, an olfactory epithelium which takes the form of simple concave depressions, and primordial gill arches. Gill filaments start to form as early as 6 dph and lamellae buds are visible at 8 dph in both groups. In CF fish, the cephalic lateral line system continues its development at 12-14 dph with invagination of both supra- and infraorbital canals. At the same time, a thorn-like or acanthoid crest forms above the eye. At 14 dph, invaginations of the mandibular and preopercular canals are visible and around 22 dph enclosure of all cranial canals nears completion. In CF larvae, however, completely enclosed cranial canals were not observed within the course of the trial, i.e. 27 dph. In TF larvae, grooves of the cephalic lateral line system form 4 days earlier than observed in CF larvae of R. canadum (i.e. at 8 dph), with enclosure commencing at 16 dph, and completed by 27 dph. Along the flanks of 6 dph larvae of either treatment, four to five equally spaced neuromasts delineate the future position of the trunk lateral line. As myomeres are added to the growing larvae, new neuromasts appear such that at 16 dph a neuromast is associated with each myomere. By 27 dph, the trunk lateral line starts to invaginate in CF larvae, while it initiates closure in TF larvae. These findings elucidate important features of the larval development of R. canadum and show that dietary taurine supplementation benefits larval development, growth and survival in this species. Moreover, they suggest a conditional requirement for taurine in larval R. canadum.
The dynamics of leukocyte-platelet adhesion and platelet-platelet interaction in whole blood are not well understood. Using different platelet agonists, we have studied the whole blood kinetics of these heterotypic and homotypic interactions, the relative abilities of different leukocyte subsets to participate in platelet adhesion, and the ligands responsible for adhesion. When platelet aggregation was inhibited by the Arg-Gly-Asp-Ser (RGDS) peptide, thrombin stimulation of whole blood resulted in platelet expression of granule membrane protein 140 (GMP-140) and, simultaneously, a marked increase in the percentage of monocytes and neutrophils (PMN) binding platelets, as well as an increase in the number of platelets bound per monocyte and PMN. Lymphocytes were unaffected. Monocytes bound more platelets and at an initially faster rate than PMN. This increase in monocyte and PMN adhesion to platelets was completely inhibited by the blocking monoclonal antibody (MoAb), G1, to GMP-140. When the combination of epinephrine and adenosine diphosphate (epi/ADP) was used as a less potent agonist in the presence of RGDS, GMP-140 expression per platelet was less, and while monocyte-platelet conjugates formed, PMN-platelet conjugates did not. With epi/ADP in the absence of RGDS, there was an immediate, marked decrease in the percentage of all leukocytes with bound platelets, simultaneous with an increase in the percentage of unbound platelet aggregates. As these platelet aggregates dissociated, the percentage of monocytes and PMN with adherent platelets increased, with monocytes again binding at a faster initial rate than PMN. This recovery of monocyte and PMN adhesion to platelets was also inhibited by the G1 MoAb. We conclude that: (1) monocytes and PMN bind activated platelets in whole blood through GMP-140; (2) monocytes have a competitive advantage over PMN in binding activated platelets, particularly when less potent platelet agonists are used; and (3) platelet aggregate formation initially competes unactivated platelets off leukocytes; subsequent aggregate dissociation allows the now activated platelets to readhere to monocytes and PMN through GMP-140. These studies further elucidate the dynamic interaction of blood cells and possible links between coagulative and inflammatory processes.
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