Platelet/polymorphonuclear leukocyte interaction: P-selectin triggers protein-tyrosine phosphorylation-dependent CD11b/CD18 adhesion: role of PSGL-1 as a signaling molecule

Polymorphonuclear leukocyte (PMN) adhesion to activated platelets is important for the recruitment of PMN at sites of vascular damage and thrombus formation. We have recently shown that binding of activated platelets to PMN in mixed cell suspensions under shear involves P-selectin and the activated beta2-integrin CD11b/CD18. Integrin activation required signaling mechanisms that were sensitive to tyrosine kinase inhibitors.1 Here we show that mixing activated, paraformaldehyde (PFA)-fixed platelets with PMNs under shear conditions leads to rapid and fully reversible tyrosine phosphorylation of a prominent protein of 110 kD (P approximately 110). Phosphorylation was both Ca2+ and Mg2+ dependent and was blocked by antibodies against P-selectin or CD11b/CD18, suggesting that both adhesion molecules need to engage with their respective ligands to trigger phosphorylation of P approximately 110. The inhibition of P approximately 110 phosphorylation by tyrosine kinase inhibitors correlates with the inhibition of platelet/PMN aggregation. Similar effects were observed when platelets were substituted by P-selectin-transfected Chinese hamster ovary (CHO-P) cells or when PMN were stimulated with P-selectin-IgG fusion protein. CHO-P/PMN mixed-cell aggregation and P-selectin-IgG-triggered PMN/PMN aggregation as well as P approximately 110 phosphorylation were all blocked by antibodies against P-selectin or CD18. In each case PMN adhesion was sensitive to the tyrosine kinase inhibitor genistein. The antibody PL-1 against P-selectin glycoprotein ligand-1 (PSGL-1) blocked platelet/PMN aggregation, indicating that PSGL-1 was the major tethering ligand for P-selectin in this experimental system. Moreover, engagement of PSGL-1 with a nonadhesion blocking antibody triggered beta2-integrin-dependent genistein-sensitive aggregation as well as tyrosine phosphorylation in PMN. This study shows that binding of P-selectin to PSGL-1 triggers tyrosine kinase-dependent mechanisms that lead to CD11b/CD18 activation in PMN. The availability of the beta2-integrin to engage with its ligands on the neighboring cells is necessary for the tyrosine phosphorylation of P approximately 110.     

P-Selectin and platelet clearance

P-selectin is an adhesion receptor for leukocytes expressed by activated platelets and endothelial cells. To assess a possible role of P-selectin in platelet clearance, we adapted an in vivo biotinylation technique in mice. Wild-type and P-selectin-deficient mice were infused with N-hydroxysuccinimido biotin. The survival of biotinylated platelets was followed by flow cytometry after labeling with fluorescent streptavidin. Both wild-type and P-selectin-deficient platelets presented identical life spans of about 4.7 days, suggesting that P-selectin does not play a role in platelet turnover. When biotinylated platelets were isolated, activated with thrombin, and reinjected into mice, the rate of platelet clearance was unchanged. In contrast, storage of platelets at 4 degreesC caused a significant reduction in their life span in vivo but again no significant differences were observed between the two genotypes. The infused thrombin-activated platelets rapidly lost their surface P-selectin in circulation, and this loss was accompanied by the simultaneous appearance of a 100-kD P-selectin fragment in the plasma. This observation suggests that the platelet membrane P-selectin was shed by cleavage. In conclusion, this study shows that P-selectin, despite its binding to leukocytes, does not mediate platelet clearance. However, the generation of a soluble form of P-selectin on platelet activation may have biological implications in modulating leukocyte recruitment or thrombus growth.     

Synergism between platelets and neutrophils in provoking cardiac dysfunction after ischemia and reperfusion: role of selectins

BACKGROUND: Neutrophils (PMNs) are known to contribute to both cardiac dysfunction and myocardial necrosis after reperfusion of an ischemic heart. Moreover, platelets are also important blood cells that can aggravate myocardial ischemic injury. This study was designed to test the effects of PMNs and platelets separately and together in provoking cardiac dysfunction in isolated perfused rat hearts after ischemia and reperfusion. METHODS AND RESULTS: Control rat hearts not subjected to ischemia were perfused without blood cells for 80 minutes. Additional control rat hearts were perfused with 75x106 PMNs, with 100x106 platelets, or with 75x106 PMNs+100x106 platelets over a 5-minute perfusion followed by a 75-minute observation period. No significant reduction in coronary flow, left ventricular developed pressure (LVDP), or the first derivative of LVDP (dP/dtmax) was observed at the end of the observation period in any nonischemic group. Similarly, global ischemia (I) for 20 minutes followed by 45 minutes of reperfusion (R) produced no sustained effects on the final recovery of any of these parameters in any group of hearts perfused in the absence of blood cells. However, I/R hearts perfused with either PMNs or platelets alone exhibited decreases in these variables of 10% to 12% (P<0.05 from control). Furthermore, I/R hearts perfused with both PMNs and platelets exhibited decreases of 50% to 60% in all measurements of cardiac function (P<0.001). These dual-cell-perfused I/R hearts also exhibited marked increases in cardiac myeloperoxidase (MPO) activity, indicating a significant PMN infiltration, and enhanced P-selectin expression on the coronary microvascular endothelium. All cardiodynamic effects as well as MPO accumulation and PMN infiltration were markedly attenuated by a sialyl LewisX-oligosaccharide or a recombinant soluble P-selectin ligand, which inhibits selectin-mediated cell adhesion. CONCLUSIONS: These results provide evidence that platelets and neutrophils act synergistically in provoking postreperfusion cardiac dysfunction and that this may be largely due to cell-to-cell interactions mediated by P-selectin. These findings may help explain the reperfusion injury phenomenon.     

Platelet/polymorphonuclear leukocyte interaction in dynamic conditions: evidence of adhesion cascade and cross talk between P-selectin and the beta 2 integrin CD11b/CD18

Adhesion between platelets and polymorphonuclear leukocytes (PMN) is a key event in thrombosis and inflammation. Double color fluorescence-activated cell sorter (FACS) analysis was used to determine the extent and kinetics of adhesion of thrombin-activated platelets to resting or activated PMN when mixed cell populations were incubated in dynamic conditions. Activated platelets bound very rapidly to PMN. Mixed cell conjugates reached a maximum at 1 minute and were reversible within 10 minutes. Platelet/PMN adhesion required both Ca2+ and Mg2+ and was markedly increased by the presence of Mn2+. The latter made mixed cell conjugates stable up to 10 minutes. Adhesion of platelets required metabolic activity of PMN and was abolished by tyrosine kinase inhibitors. Furthermore, adhesion of platelets to PMN resulted in binding of a monoclonal antibody (MoAb 24) known as beta 2 integrins "activation reporter." When PMN were activated by exogenous stimuli, the adhesion of platelets was markedly increased: fMLP induced a rapid and transient effect, while PMA resulted in a slower, but stable, increase in mixed conjugates formation. The hypothesis that activated PMN beta 2 integrins are able to bind a counter-receptor on platelets was directly demonstrated by the increase of mixed cell conjugates following PMN treatment with KIM127 and KIM185, two anti-CD18 antibodies able to induce the active conformation of beta 2 integrins. Consistently, two other anti-CD18, as well as an anti-CD11b inhibitory antibody abolished platelet/PMN adhesion. PMN beta 2 integrin activation was not the only mechanism for activated platelet/PMN adhesion to occur: indeed, this phenomenon could also be inhibited by two anti-P-selectin antibodies. Resting platelets did not adhere to resting PMN, but markedly adhered to fMLP- or PMA-activated PMN. Resting platelet/fMLP-activated PMN adhesion was abolished by anti-CD18 antibodies, but not by anti-P-selectin antibodies. In conclusion, activated platelet/PMN interaction can be modeled as an adhesion cascade involving a P-selectin-dependent recognition step and a functional signal. The latter proceeds through tyrosine kinase activation and enables a beta 2 integrin-dependent adhesion to a not yet identified counter-receptor constitutively expressed on platelet surface.     

Affinity and kinetic analysis of P-selectin binding to P-selectin glycoprotein ligand-1

Leukocytes use the cell-surface mucin P-selectin glycoprotein ligand-1 (PSGL-1) to tether to and roll on P-selectin on activated endothelial cells and platelets. By using surface plasmon resonance, we measured the affinity and kinetics of binding of soluble monomeric human P-selectin to immobilized PSGL-1 from human neutrophils. Binding was specific, as documented by its Ca2+-dependence, its inhibition by specific monoclonal antibodies to P-selectin and PSGL-1, and its abrogation by treating PSGL-1 with sialidase. Similar binding was observed for soluble P-selectin that contained the lectin and epidermal growth factor domains plus all nine consensus repeats, and for a soluble construct that contained only the lectin and epidermal growth factor domains. Soluble P-selectin bound saturably to a single class of sites on PSGL-1 with a dissociation constant (Kd) of 320 +/- 20 nM. The measured koff was 1.4 +/- 0.1 s-1, and the calculated kon was 4.4 x 10(6) M-1 s-1. We conclude that monomeric P-selectin binds to PSGL-1 with fast association and dissociation rates and relatively high affinity. These features may be important for efficient tethering and rolling of leukocytes at physiologic densities of PSGL-1 and P-selectin.     

Failure of penicillin treatment of yaws on Karkar Island, Papua New Guinea

We evaluated the plasma concentrations of soluble adhesion molecules and platelet-derived microparticles (PMP) in patients with non-insulin dependent diabetes mellitus (NIDDM) and studied the effect of cilostazol on PMP generation. There were differences in the levels of soluble adhesion molecules between NIDDM patients (N = 43) and the control subjects (N = 30) (soluble thrombomodulin: 11.5+/-5.3 vs. 7.0+/-1.2 TU/ml, p<0.0001; soluble vascular cell adhesion molecule-1: 708+/-203 vs. 492+/-113 ng/dl, p<0.0001; soluble intercellular cell adhesion molecules- 1: 274+/-65 vs. 206+/-48 ng/dl, p<0.0001; soluble P-selectin: 194+/-85 vs. 125+/-43 ng/dl, p<0.0001). There were also differences in the levels of PMP and platelet activation markers between NIDDM patients and the controls (PMP: 943+/-504 vs. 488+/-219/10(4) plt, p<0.0001; platelet CD62P: 9.2+/-4.6 vs. 4.4+/-4.3%, p<0.001; platelet CD63: 10.2+/-4.5 vs. 4.5+/-3.3%, p<0.0001; platelet annexin V: 9.1+/-3.9 vs. 5.3+/-3.8%, p<0.001). To study the release of PMP into plasma, a modified cone-and-plate viscometer was used. Increased release of PMP from platelets was observed in diabetic plasma compared to normal plasma under high shear stress conditions (2,672+/-645 vs. 1,498+/-386/10(4) plt, p<0.05). Therefore, one cause of PMP elevation in NIDDM may be high shear stress. The levels of PMP, activated platelets, and soluble adhesion molecules all decreased significantly after treatment with cilostazol. These results suggest that cilostazol may be useful for the inhibition of both PMP-dependent and -independent vascular damage in NIDDM.     

Intravenous cocaine induces platelet activation in the conscious dog

BACKGROUND: Cocaine consumption has been associated with thrombosis of coronary and peripheral arteries. Since cocaine has been found to induce platelet activation in vitro, we sought to establish whether cocaine induced platelet activation in vivo. METHODS AND RESULTS: Chronically instrumented, conscious dogs were infused with cocaine (1 mg/kg), norepinephrine (0.2 to 0.4 mg/kg), or saline intravenously over 1 minute. Activated canine platelets were identified in whole blood collected from an indwelling aortic catheter by flow cytometric detection of the binding of a monoclonal antibody directed against the activation-dependent antigen P-selectin. Infusion of cocaine resulted in an elevation of mean arterial pressure (91 +/- 3 to 128 +/- 7 mm Hg [P < .01]) and heart rate (87 +/- 9 to 125 +/- 11 beats per minute [P < .01]). A similar change (P = NS) in mean arterial pressure followed norepinephrine infusion (100 +/- 5 to 137 +/- 13 mm Hg [P < .04]), whereas saline infusion had no effect. Cocaine resulted in a substantial but delayed increase in platelet P-selectin expression (14 +/- 7% [P < .08], 31 +/- 12% [P < .04], and 55 +/- 22% [P < .04] at 17, 22, and 27 minutes after drug infusion, respectively). The magnitude of this increase was similar to that found in blood treated ex vivo with the agonists ADP or PAF (23 +/- 7% and 53 +/- 15%, respectively). No significant increase in P-selectin expression was detected in the blood of animals that received norepinephrine or saline. Serum cocaine concentrations were highest immediately after infusion (538 +/- 55 ng/mL at 2 minutes) but declined rapidly (185 +/- 22 and 110 +/- 25 ng/mL at 17 and 32 minutes after infusion); in contrast, the increase in benzoylecgonine concentrations was delayed (from < 25 ng/mL in all but one animal [34 ng/mL] at 2 minutes to 46 +/- 4 and 71 +/- 11 ng/mL at 17 and 32 minutes, respectively, after infusion). CONCLUSIONS: Intravenous cocaine induces activation of individual circulating platelets; this effect is not reproduced by infusion of norepinephrine at doses sufficient to exert similar hemodynamic effects. The delay in detection of activated platelets after treatment with cocaine may result from the adhesion and subsequent detachment of activated platelets; alternatively, cocaine metabolites, rather than the drug itself, may induce platelet activation.     

Extensive platelet activation in preeclampsia compared with normal pregnancy: enhanced expression of cell adhesion molecules

OBJECTIVES: Platelets play an important role in the pathophysiologic mechanisms of preeclampsia. Our purpose was to investigate by means of flow cytometry to what extent platelets circulate in an activated state during normal pregnancy and whether this activation is more extensive in preeclampsia. STUDY DESIGN: Platelets in whole blood from 10 preeclamptic third-trimester pregnant women (highest diastolic blood pressure range 100 to 130 mm Hg, proteinuria range 0.59 to 11.5 gm/24 hr) and from 10 normotensive third-trimester pregnant controls were analyzed with the following activation markers: anti-P-selectin (alpha-granule secretion), anti-CD63 (lysosomal secretion), PAC-1 (monoclonal antibody against fibrinogen receptor conformation of the glycoprotein IIb/IIIa complex), anti-platelet endothelial cell adhesion molecule-1, and annexin-V (a placental protein that binds to negatively charged phospholipids, present on the outside of the platelet plasma membrane after activation). The differences in surface antigen exposure between the two groups were determined by double-label flow cytometry. Flow cytometric data were analyzed in two ways: first, the percentages of activated platelets above a certain threshold compared with a nonpregnant control sample were determined, indicative for activation of a subpopulation of cells, and, second, the mean fluorescence intensities were determined, indicative of the mean surface antigen expression of the total platelet population. RESULTS: Analysis of the percentage of activated platelets proved most informative. With this analysis an enhanced platelet activation status was present in 4 of 10 normotensive patients and a more extensive platelet activation status in all 10 preeclamptic patients, as indicated by P-selectin (p = 0.008) and CD63 (p = 0.03) expression. Increased platelet endothelial cell adhesion molecule-1 (p = 0.005) expression was also observed in preeclampsia. CONCLUSIONS: Flow cytometric analysis clearly indicated that platelets circulate in a more extensively activated state during preeclampsia than during normal pregnancy. The increased platelet endothelial cell adhesion molecule-1 expression in preeclamptic patients demonstrates that, besides alpha-granular and lysosomal release, other hitherto unknown mechanisms are involved. Platelet endothelial cell adhesion molecule-1 appears to be the best marker to distinguish preeclamptic patients from normotensive pregnant women. Only a subpopulation of the platelets appears to be activated.     

Thrombin promotes platelet-mediated melanoma cell adhesion to endothelial cells under flow conditions: role of platelet glycoproteins P-selectin and GPIIb-IIIA

We investigated the role of platelets in human melanoma cell (line 397) interaction with vascular endothelial cells (ECs) under flow conditions. The ability of the tumour cells to adhere to the EC monolayer was significantly reduced by application of flow at a shear rate of 250 s(-1). A 2.2-fold increase in tumour cell adhesion to ECs under flow was observed upon addition of thrombin receptor agonist peptide (TRAP)-activated platelets but not resting platelets. A similar increase (2.5-fold) in tumour cell adhesion to ECs under flow was observed when the tumour cells were incubated with resting platelets on thrombin-treated ECs. However, thrombin treatment of the ECs alone had no effect on tumour cell adhesion in the absence of platelets. The enhancement of tumour cell adhesion to ECs by TRAP-activated platelets was virtually abolished by blockade of the platelet glycoproteins P-selectin and GPIIb-IIIa by monoclonal antibodies. Blockade of P-selectin also inhibited the direct adhesion of TRAP-activated platelets to ECs, but did not affect the interaction of the tumour cells with platelets immobilized on subendothelial extracellular matrix (ECM). Blockade of GPIIb-IIIa inhibited both platelet-EC and platelet-tumor cell interactions. Our results indicate that tumour cell adhesion to the endothelium under flow is enhanced by platelets under conditions that allow platelet adhesion to ECs. Inhibition studies suggest that activated platelet adhesion to ECs is mediated by P-selectin and GPIIb-IIIA, and tumour cell adhesion to EC-bound platelets--mainly by GPIIb-IIIa.     

Optimal antagonism of GPIIb/IIIa favors platelet adhesion by inhibiting thrombus growth. An ex vivo capillary perfusion chamber study in the guinea pig

To evaluate the involvement of the glycoprotein (GP) IIb/IIIa-dependent process in platelet deposition and thrombus growth on capillaries coated with human type III collagen, the effects of incremental doses of Lamifiban, a potent specific synthetic GPIIb/IIIa antagonist, were studied in ex vivo capillary perfusion chambers using guinea pig blood. In this model, nonanticoagulated blood was perfused for 4.5 minutes at three shear rates: 100, 650, and 1600 s-1. Platelet deposition was quantified by computer-assisted morphometry and expressed as platelet adhesion (percentage of capillary surface covered with spread and contact platelets and platelets implicated in thrombus), mean thrombus height, and total thrombus cross-sectional area. In control untreated guinea pigs, platelet adhesion and thrombus height were 63% and 2.5 microns at 100 s-1, 60.5% and 13.8 microns at 650 s-1, and 45% and 28.1 microns at 1600 s-1, respectively. At 100 s-1, Lamifiban had no effect on platelet deposition at any of the three doses administered to the guinea pigs (0.3, 1, and 3 mg/kg). At 0.3 mg/kg and shear rates of 650 and 1600 s-1, Lamifiban had no effect on platelet adhesion or thrombus size, but at 1 and 3 mg/kg and shear rates of 650 and 1600 s-1, it significantly reduced thrombus size. At 1600 s-1, 1 mg/kg Lamifiban significantly increased platelet adhesion from 45% to 62.5%, whereas at 3 mg/kg it induced a significant overall decrease from 45% to 25% and qualitatively increased the ratio of contact to spread platelets. These data suggest that at high shear rates, GPIIb/IIIa participates in platelet spreading and that there is a balance between platelet involvement in adhesion to the thrombogenic surface and the growth of the already formed thrombus. This indicates that important clinical implications of an optimal therapeutic degree of GPIIb/IIIa antagonism could be expected.     

Induction of cytokine expression in leukocytes by binding of thrombin-stimulated platelets

BACKGROUND: Activated platelets tether and activate myeloid leukocytes. To investigate the potential relevance of this mechanism in acute myocardial infarction (AMI), we examined cytokine induction by leukocyte-platelet adhesion and the occurrence of leukocyte-platelet conjugates in patients with AMI. METHODS AND RESULTS: We obtained peripheral venous blood samples in 20 patients with AMI before and daily for 5 days after direct percutaneous transluminal coronary angioplasty (PTCA) and in 20 patients undergoing elective PTCA. Throughout the study period, CD41 immunofluorescence of leukocytes (flow cytometry) revealed increased leukocyte-platelet adhesion in patients with AMI compared with control patients (mean +/- SE of fluorescence [channels] before PTCA: 77 +/- 16 versus 35 +/- 9; P = .003). In vitro, thrombin-stimulated fixed platelets bound to neutrophils and monocytes. Within 2 hours, this resulted in increased mRNA for interleukin (IL),1 beta, IL-8, and monocyte chemoattractant protein (MCP)-1 in unfractionated leukocytes. After 4 hours, IL-1 beta and IL-8 concentration of the cell-free supernatant had increased by 268 +/- 36% and 210 +/- 7%, respectively, and cellular MCP-1 content had increased by 170 +/- 8%. Addition of activated platelets to adherent monocytes had a similar effect and was associated with nuclear factor-kappa B activation. Inhibition of binding by anti-P selectin antibodies reduced the effect of activated platelets on cytokine production. CONCLUSIONS: In patients with AMI, leukocyte-platelet adhesion is increased. Binding of activated platelets induces IL-1 beta, IL-8, and MCP-1 in leukocytes. Our findings suggest that leukocyte-platelet adhesion contributes to the regulation of inflammatory responses in AMI.     

CD24 mediates rolling of breast carcinoma cells on P-selectin

P-selectin mediates rolling of neutrophils and other leukocytes on activated endothelial cells and platelets through binding to P-selectin glycoprotein ligand-1 (PSGL-1). Certain PSGL-1 negative tumor cell lines can bind P-selectin under static conditions through the GPI-linked surface mucin, CD24, but the physiological significance of this interaction and whether it can occur under flow conditions is not known. Here, we show that CD24+ PSGL-1- KS breast carcinoma cells attach to and roll on recombinant P-selectin under a continuous wall shear stress, although at a lower density and higher velocity than CD24+ PSGL-1+ cells, such as HL-60. Adding excess soluble CD24 or removing CD24 from the cell surface with phosphatidylinositol-phospholipase C (PI-PLC) significantly reduced KS cell rolling on P-selectin. The ability of KS cells to roll on P-selectin was positively correlated with the CD24 expression level. Comparison with three other CD24+ cell lines established that expression of sialyl-Lewis(x) antigen was also necessary for CD24-mediated rolling on P-selectin. CD24 purified from KS cells supported rolling of P-selectin transfectants, but not L-selectin transfectants. Finally, KS cells rolled on vascular endothelium in vivo in a P-selectin-dependent manner. Together our data show that CD24 serves as a ligand for P-selectin under physiological flow conditions. Interaction of tumor cells with P-selectin via CD24 may be an important adhesion pathway in cancer metastasis.     

A case of acquired immune deficiency syndrome presenting acute lumbosacral polyradiculopathy due to opportunistic infection of cytomegalovirus

The binding of antiphospholipid antibodies to circulating platelets and the potential association with thrombocytopenia and platelet activation was investigated in 25 patients with primary antiphospholipid syndrome (APS). Fourteen patients had a platelet count above 150 x 10(9)/l, and 11 patients had mild to moderate thrombocytopenia of 50-150 x 10(9)/l. The presence of platelet autoantibodies was investigated by immunofluorescent binding. No correlation between the presence of autoantibodies on platelets and thrombocytopenia was found. The binding of antibodies in patients' serum and platelet eluates was investigated by performing enzyme-linked immunosorbent assays with phospholipids as antigens. In seven patients antibodies to negatively charged phospholipids were present in platelet eluates. Platelet activation was measured by flow cytometry using a fluorescein isothiocyanate (FITC) labeled monoclonal antibody to P-selectin (CD62). The binding of anti-P-selectin to patients' platelet surface P-selectin was not increased, compared with the binding to platelets obtained from normal donors. Platelet serotonin concentration in APS patients was significantly lower than that found in the platelets of normal controls. More studies are necessary to determine the exact role of antiphospholipid antibodies in the pathogenesis of thrombocytopenia, and to elucidate the cause of low serotonin levels in platelets of APS patients.     

Extracellular matrix regulates apoptosis in human neutrophils

BACKGROUND: During inflammation, polymorphonuclear neutrophils (PMNs) migrate into the affected tissue interacting with extracellular matrix (ECM) proteins. We tested the hypothesis that PMN-matrix interaction affects PMN apoptosis. METHODS: Apoptosis of human PMNs was detected by DNA-fragmentation assay and was quantitated by flow cytometry, ultraviolet and light microscopy. Cell adhesion was assessed by a toluidine blue assay, and cell spreading was detected by phase contrast microscopy. Protein tyrosine phosphorylation was studied using Western blotting and confocal microscopy. RESULTS: PMN apoptosis was not different in unstimulated cultures on either surface-adherent fibronectin or on PolyHema, a surface that prevents cell adherence. However, tumor necrosis factor-alpha (TNF alpha) treatment significantly increased apoptosis on fibronectin (37 +/- 4%) compared with PolyHema (20 +/- 3%). Tests on other matrix substances revealed that the percentage of apoptotic PMNs in the presence of TNF alpha was 8 +/- 1% on PolyHema, 26 +/- 4% on fibronectin, 17 +/- 2% on collagen I, 16 +/- 2% on collagen IV, and 16 +/- 3% on laminin (P < 0.05 for all matrices compared with PolyHema). Preincubation with genistein (50 microM) significantly inhibited TNF alpha-mediated apoptosis on fibronectin (39 +/- 4% to 21 +/- 4%) but not on PolyHema (21 +/- 4% to 16 +/- 4%). Genistein also reduced PMN spreading on fibronectin. In contrast, inhibitors of mitogen-activated protein kinase and protein kinase C showed no effect on PMN apoptosis. Fibronectin strongly increased tyrosine phosphorylation of three 102, 63, and 54 kDa proteins. Five newly tyrosine-phosphorylated 185, 85, 66, 56, and 42 kDa bands were also visible. Using confocal microscopy, highest tyrosine phosphorylation was localized to sites of cell-matrix interaction. CONCLUSIONS: ECM influences apoptosis in TNF alpha-activated, adherent, spreading PMNs. The process is regulated by tyrosine phosphorylation. Acceleration of apoptosis may shorten the PMN lifespan and thereby locally regulate inflammation.     

The role of neutrophils in peritoneal adhesion formation

The most common cause of intraperitoneal adhesions is previous abdominal surgery. Postoperative adhesion formation results from a fibroproliferative inflammatory reaction that begins with an influx of polymorphonuclear leukocytes (PMNs) into the peritoneal cavity. Adherence of the PMNs to the endothelial cells (EC) is necessary for PMN migration into the tissue in response to a stimulus. Several receptor-counterreceptor pairs of ligands such as CD11/CD18 on the PMN and ICAM-1 (CD54) on EC have been identified. Monoclonal antibody against CD11/CD18 (R15.7) inhibits PMN adherence and migration and consequently protects against PMN-induced tissue injuries. We therefore studied the effect of preventing PMN-EC adherence, using anti-CD18 monoclonal antibody, on postoperative adhesion formation in rabbits. Group 1 was a control receiving physiologic saline, and group 2 received anti-CD18 antibody (R15.7, 2 mg/kg). The treatment was administered iv at the end of surgery and repeated on the first and second postoperative days. Peritoneal adhesions were induced at laparotomy by repairing two peritoneal defects, by oversewing the defect (model 1), and by resuturing the removed parietal peritoneum in its place as an ischemic graft (model 2). Adhesions were evaluated blindly at 10 days after operation by measuring the percentage of the suture line covered with adhesions (model 1) or by a scoring system (model 2). All control animals developed intraperitoneal adhesions and the percentage of the suture line covered with adhesions was 25 +/- 5.9% (mean +/- SEM) and the mean score in model 2 was 0.9 +/- 0.2. Anti-CD18 antibody, R15.7, increased the degree of postoperative adhesion formation in both models, but the results were significant only in model 2. Also, anti-CD18 antibody significantly decreased peritoneal neutrophils from 11.1 x 10(7) +/- 1.8 x 10(7) to 2.2 x 10(7) +/- 0.4 x 10(7) (P < 0.001) on the first postoperative day. It is concluded that inhibition of PMN-EC adherence does influence the postoperative adhesion formation. These results might suggest that PMNs have a role in modulating postoperative adhesion formation.     

Neutrophil and platelet activation at balloon-injured coronary artery plaque in patients undergoing angioplasty

OBJECTIVES: This study sought to investigate changes in the expression of activation-dependent adhesion receptors on neutrophils and platelets after exposure to the balloon-injured coronary artery plaque. BACKGROUND: Activation of blood cells at the balloon-injured coronary artery plaque may contribute to abrupt vessel closure and late restenosis after percutaneous transluminal coronary angioplasty. METHODS: In 30 patients undergoing elective coronary angioplasty, blood specimens were obtained through the balloon catheter proximal to the plaque before dilation and distal to the plaque after dilation. Simultaneous blood samples obtained through the guiding catheter served as control samples. Total surface expression of the inducible fibrinogen receptor (CD41) and surface expression of the activated fibrinogen receptor (LIBS1) on platelets as well as Mac-1 (CD11b) and L-selectin (CD62L) surface expression on neutrophils were assessed by flow cytometry. RESULTS: After exposure to the dilated coronary artery plaque, surface expression of LIBS1 on platelets increased by 40.5 +/- 11.0 mean (+/-SE) fluorescence (p=0.001) and that of CD11b on neutrophils increased by 20.1 +/- 4.4 mean fluorescence (p=0.018). Concomitantly, anti-CD62L binding on neutrophils decreased by 6.6 +/- 2.4 mean fluorescence (p=0.022). In contrast, surface expression of the adhesion receptors did not change significantly between the coronary ostium and the prestenotic coronary segment. CONCLUSIONS: The results of this study demonstrate neutrophil and platelet activation at the balloon-injured coronary artery plaque. This cellular activation may serve as a target for pharmacologic interventions to improve the outcome of coronary angioplasty.     

Ristocetin- and thrombin-induced platelet aggregation at physiological shear rates: differential roles for GPIb and GPIIb-IIIa receptor

We recently reported that washed platelets (WP) activated with ADP and expressing surface-bound vWF aggregated in flow through small tubes or in a cylindrical couette device at physiological shear rates of G = 300 s(-1)-1000 s(-1) in the absence of exogenous ligands, with GPIb-vWF partially, and activated GPIIb-IIIa totally required for the aggregation. We have now extended these studies to aggregation of platelets "activated" with ristocetin or thrombin. Washed platelet suspensions with added soluble vWF and ristocetin (0.3-0.75 mg/ml), or activated with thrombin (0.01-0.5 U/ml) but no added ligand, were sheared in a coaxial cylinder device at uniform shear rate, G = 1000 s(-1). The collision capture efficiency (alphaG) with which small aggregates form (= experimental/calculated initial rates of aggregation) was correlated with vWF platelet binding assessed by flow cytometry. The vWF-GPIb interaction was exclusively able to support ristocetin-mediated shear aggregation of metabolically active platelets, with very few vWF monomer equivalents bound per platelet (representing < or = 10 molecules of 10 million Da) required to yield high capture efficiencies (alphaG = 0.38+/-.02; n = 11), suggesting rapid and stable bond formations between vWF and GPIb. However, platelet surface-expressed vWF, generated by addition of thrombin to washed platelets, was found to mediate platelet aggregation with alphaG = 0.08+/-.01 (n = 6), surprisingly comparable to that previously reported for WP and ADP activation. Blocking the GPIIb-Illa receptor decreased alphaG by 95+/-3% (n =3), while a monoclonal antibody to the vWF site on GPIb caused a 49+/-7% (n = 8) decrease in alphaG. The partial role for GPIb thus appears to reflect a facilitative function for increasing contact time between flowing platelets, and allowing engagement of the GPIIb-IIa receptor to yield stable attachment.     

The impact of the Dialysis Outcomes Quality Initiative Guidelines on the care of the pediatric end-stage renal disease patient

OBJECTIVES: The purpose of this study was to monitor the effects of chimeric 7E3 Fab (ReoPro) on leukocyte and platelet activation and interaction during coronary angioplasty. BACKGROUND: Increased expression of CD11b on monocytes and neutrophils promotes their adhesion to endothelial cells, extracellular matrix and smooth muscle cells. Thrombin-activated platelets adhere via P-selectin to monocytes and neutrophils. These cell interactions may affect the outcome of coronary angioplasty. METHODS: During coronary angioplasty, venous blood was obtained for flow cytometric detection of leukocyte CD11b; platelet CD41a, CD61a and CD62P; the percentage of leukocytes with adherent platelets and the intensity of bound platelet fluorescence. RESULTS: Leukocyte CD11b expression increased after angioplasty in control patients (neutrophils 171+/-25 to 255+/-31 mean fluorescence intensity [MFI, mean+/-SEM], n=25, p < 0.0001; monocytes 200+/-40 to 248+/-36 MFI, n=17, p < 0.05) and decreased in the patients selected to receive chimeric 7E3 Fab (neutrophils 146+/-30 to 82+/-22 MFI, n=25, p < 0.0001; monocytes 256+/- 53 to 160+/-38 MFI, n= 17, p < 0.05). Neutrophil CD11b decreased after in vitro incubation of whole blood with chimeric 7E3 Fab (n=5, p=0.01), but fMLP-induced increases in CD11b were not prevented. The CD11b expression was unchanged and increased with fMLP stimulation after in vitro incubation of isolated neutrophils with chimeric 7E3 Fab. Direct-labeled chimeric 7E3 Fab was not detected bound to neutrophils in whole blood or isolated cells using flow cytometric techniques. Adhesion of isolated neutrophils to protein-coated glass was not prevented by in vitro incubation with chimeric 7E3 Fab. Platelet activation increased after angioplasty in control patients (CD62P 8.9+/-0.8 to 12.3+/-1.2 MFI, n=25, p < 0.05; CD41a 382+/-25 to 454+/-26 MFI, n=25, p < 0.05, CD61a 436+/-52 to 529+/-58 MFI, n=11, p < 0.05); it did not increase in the patients selected to receive chimeric 7E3 Fab (CD62P 13.2+/-1.0 to 9.0+/-0.9 MFI, n=25, p < 0.05; CD61a 398+/-32 to 410+/-38 MFI, n=7, p=NS). Leukocytes with adherent platelets tended to increase in the control group of patients and decrease after the procedure in patients selected to receive chimeric 7E3 Fab; individual and procedure-related variability were marked. CONCLUSIONS: Despite standard aspirin and heparin therapy, leukocyte and platelet activation with platelet adherence to leukocytes occurs after coronary angioplasty. Although chimeric 7E3 Fab does not bind to leukocytes directly, it influences CD11b expression in whole blood. Modulation of platelet and leukocyte activation and interaction by chimeric 7E3 Fab may contribute to an improved outcome after coronary angioplasty.     

A juxtacrine mechanism for neutrophil adhesion on platelets involves platelet-activating factor and a selectin-dependent activation process

The aim of this study was to identify the molecular mechanisms involved in neutrophil adhesion to immobilized platelets with particular focus on the possible existence of a juxtacrine system for neutrophil-platelet interactions. Platelets were immobilized onto collagen (type I)-coated coverslips that were placed in a flow chamber and neutrophils were perfused across these confluent monolayers at a shear stress of 1 to 4 dynes/cm2. Neutrophils rolled, and a significant proportion (25% to 50%) adhered to platelet monolayers. P-selectin was expressed in very large quantities on the surface of platelets and mediated all of the rolling, whereas the beta2-integrin mediated firm adhesion. An activation mechanism for adhesion was necessary inasmuch as fixed neutrophils continued to roll on immobilized platelets, but did not adhere. Platelets adherent to collagen produced significant levels of platelet-activating factor (PAF). Accordingly, the firm adhesion of neutrophils to platelets was significantly inhibited by a PAF receptor antagonist (WEB 2086). Treatment of only the platelets with acetylhydrolase, which converts membrane-associated PAF to lyso-PAF, prevented 60% of the adhesion. These data suggest that PAF, on the surface of platelets, mediated a significant portion of the adhesive interaction. Addition of some selectin-binding carbohydrates (fucoidan or soluble SLEx analogs but not dextran sulfate) to the platelets caused rolling neutrophils to immediately adhere, an event that was not observed on histamine or thrombin-treated endothelium or P-selectin transfectants. These data support the view that a juxtacrine activation process exists on immobilized platelets for neutrophils. This process can be greatly enhanced on platelets and may involve a signaling mechanism through P-selectin.     

beta1 integrins are critically involved in neutrophil locomotion in extravascular tissue In vivo

Recruitment of leukocytes from blood to tissue in inflammation requires the function of specific cell surface adhesion molecules. The objective of this study was to identify adhesion molecules that are involved in polymorphonuclear leukocyte (PMN) locomotion in extravascular tissue in vivo. Extravasation and interstitial tissue migration of PMNs was induced in the rat mesentery by chemotactic stimulation with platelet-activating factor (PAF; 10(-7) M). Intravital time-lapse videomicroscopy was used to analyze migration velocity of the activated PMNs, and the modulatory influence on locomotion of locally administered antibodies or peptides recognizing various integrin molecules was examined. Immunofluorescence flow cytometry revealed increased expression of alpha4, beta1, and beta2 integrins on extravasated PMNs compared with blood PMNs. Median migration velocity in response to PAF stimulation was 15.5 +/- 4.5 micron/min (mean +/- SD). Marked reduction (67 +/- 7%) in motility was observed after treatment with mAb blocking beta1 integrin function (VLA integrins), whereas there was little, although significant, reduction (22 +/- 13%) with beta2 integrin mAb. Antibodies or integrin-binding peptides recognizing alpha4beta1, alpha5beta1, or alphavbeta3 were ineffective in modulating migration velocity. Our data demonstrate that cell surface expression of beta1 integrins, although limited on blood PMNs, is induced in extravasated PMNs, and that members of the beta1 integrin family other than alpha4beta1 and alpha5beta1 are critically involved in the chemokinetic movement of PMNs in rat extravascular tissue in vivo.