Surface-adherent Platelets Research Articles

Refining a numerical model for device-induced thrombosis and investigating the effects of non-Newtonian blood models

Thrombosis is one of the main causes of failure in device implantation. Computational thrombosis simulation is a convenient approach to evaluate the risk of thrombosis for a device. However, thrombosis is a complicated process involving multiple species and reactions. Application of a macroscopic, single-scale computational model for device-induced thrombosis is a cost-effective approach. The current study has refined an existing thrombosis model, which simulated thrombosis by tracing four species in blood: non-activated platelets, activated platelets, surface adherent platelets, and ADP. Platelets are activated mechanically by shear stress, and chemically by ADP. Platelet adhesion occurs on surfaces with low wall shear stress with platelet aggregation inhibited in regions of high shear stress. The study improves the existing thrombosis model by: 1) Modifying the chemical platelet activation function so that ADP activates platelets; 2) Modifying the function describing thrombus deposition and growth to distinguish between thrombus deposition on wall surfaces and thrombus growth on existing thrombus surfaces; 3) Modifying the thrombus breakdown function to allow for thrombus breakdown by shear stress; 4) Modeling blood flow as non-Newtonian. The results show that the inclusion of ADP and the use of a non-Newtonian model improve agreement with experimental data.

Effect of temperature on platelet adherence.

Thrombogenicity is one of the main parameters tested in vitro to evaluate the hemocompatibility of artificial surfaces. While the influence of the temperature on platelet aggregation has been addressed by several studies, the temperature influence on the adherence of platelets to body foreign surfaces as an important aspect of biomedical device handling has not yet been explored. Therefore, we analyzed the influence of two typically applied incubation-temperatures (22°C and 37°C) on the adhesion of platelets to biomaterials. Thrombogenicity of three different polymers - medical grade poly(dimethyl siloxane) (PDMS), polytetrafluoroethylene (PTFE) and polyethylene terephthalate (PET) - were studied in an in vitro static test. Platelet adhesion was studied with stringently characterized blood from apparently healthy subjects. Collection of whole blood and preparation of platelet rich plasma (PRP) was carried out at room temperature (22°C). PRP was incubated with the polymers either at 22°C or 37°C. Surface adherent platelets were fixed, fluorescently labelled and assessed by an image-based approach. Differences in the density of adherent platelets after incubation at 22°C and 37°C occurred on PDMS and PET. Similar levels of adherent platelets were observed on the very thrombogenic PTFE. The covered surface areas per single platelet were analyzed to measure the state of platelet activation and revealed no differences between the two incubation temperatures for any of the analyzed polymers. Irrespective of the observed differences between the low and medium thrombogenic PDMS and PET and the higher variability at 22°C, the thrombogenicity of the three investigated polymers was evaluated being comparable at both incubation temperatures.

Soluble CD40 Ligand Stimulates CD40-Dependent Activation of the β2 Integrin Mac-1 and Protein Kinase C Zeda (PKCζ) in Neutrophils: Implications for Neutrophil-Platelet Interactions and Neutrophil Oxidative Burst

Recent work has revealed an essential involvement of soluble CD40L (sCD40L) in inflammation and vascular disease. Activated platelets are the major source of sCD40L, which has been implicated in platelet and leukocyte activation, although its exact functional impact on leukocyte-platelet interactions and the underlying mechanisms remain undefined. We aimed to determine the impact and the mechanisms of sCD40L on neutrophils. We studied neutrophil interactions with activated, surface-adherent platelets as a model for leukocyte recruitment to the sites of injury. Our data show that CD40L contributes to neutrophil firm adhesion to and transmigration across activated surface-adherent platelets, possibly through two potential mechanisms. One involves the direct interaction of ligand-receptor (CD40L-CD40), i.e., platelet surface CD40L interaction with neutrophil CD40; another involves an indirect mechanism, i.e. soluble CD40L stimulates activation of the leukocyte-specific β2 integrin Mac-1 in neutrophils and thereby further promotes neutrophil adhesion and migration. Activation of the integrin Mac-1 is known to be critical for mediating neutrophil adhesion and migration. sCD40L activated Mac-1 in neutrophils and enhanced neutrophil-platelet interactions in wild-type neutrophils, but failed to elicit such responses in CD40-deficient neutrophils. Furthermore, our data show that the protein kinase C zeta (PKCζ) is critically required for sCD40L-induced Mac-1 activation and neutrophil adhesive function. sCD40L strongly stimulated the focal clustering of Mac-1 (CD11b) and the colocalization of Mac-1 with PKCζ in wild-type neutrophils, but had minimal effect in CD40-deficient neutrophils. Blocking PKCζ completely inhibited sCD40L-induced neutrophil firm adhesion. Moreover, sCD40L strongly stimulates neutrophil oxidative burst via CD40-dependent activation of PI3K/NF-KB, but independent of Mac-1 and PKCζ. These findings may contribute to a better understanding of the underlying mechanisms by which sCD40L/CD40 pathway contributes to inflammation and vascular diseases.

EMMPRIN (CD147/basigin) mediates platelet–monocyte interactions in vivo and augments monocyte recruitment to the vascular wall

Platelets play a central role in hemostasis, in inflammatory diseases such as atherosclerosis, and during thrombus formation following vascular injury. Thereby, platelets interact intensively with monocytes and enhance their recruitment to the vascular wall. To investigate the role of the extracellular matrix metalloproteinase inducer (EMMPRIN) in platelet-monocyte interactions. Isolated human monocytes were perfused in vitro over firmly adherent platelets to allow investigation of the role of EMMPRIN in platelet-monocyte interactions under flow conditions. Monocytes readily bound to surface-adherent platelets. Both antibody blockade and gene silencing of monocyte EMMPRIN substantially attenuated firm adhesion of monocytes to platelets at arterial and venous shear rates. In vivo, platelet interactions with the murine monocyte cell line ANA-1 were significantly decreased when ANA-1 cells were pretreated with EMMPRIN-silencing small interfering RNA prior to injection into wild-type mice. Using intravital microscopy, we showed that recruitment of EMMPRIN-silenced ANA-1 to the injured carotid artery was significantly reduced as compared with control cells. Further silencing of EMMPRIN resulted in significantly fewer ANA-1-platelet aggregates in the mouse circulation as determined by flow cytometry. Finally, we identified glycoprotein (GP)VI as a critical corresponding receptor on platelets that mediates interaction with monocyte EMMPRIN. Thus, blocking of GPVI inhibited the effect of EMMPRIN on firm monocyte adhesion to platelets under arterial flow conditions in vitro, and abrogated EMMPRIN-mediated platelet-monocyte aggregate formation in vivo. EMMPRIN supports platelet-monocyte interactions and promotes monocyte recruitment to the arterial wall. Therefore, EMMPRIN might represent a novel target to reduce vascular inflammation and atherosclerotic lesion development.

Neointima formation after vascular injury: Is it all about CD39?

Since the pioneering era of interventional cardiology, platelet deposition after vascular injury has been regarded of central importance for neointima formation and subsequent restenosis (1–3). Depending on the type of injury, i.e. how many layers of the vessel wall are affected, surface-adherent platelets can be distinguished from mural thrombus extending into the torn media (4). Whereas organised thrombi can sometimes cause occlusion and restenosis, platelets adherent to the subendothelial matrix constitute the main adhesive substrate for the recruitment of leukocytes and smooth muscle progenitor cells to the injury site driving neointimal growth (5–8). P-selectin expressed on activated platelets, rather than platelet aggregation, is decisively involved through mediating adhesive interactions and the deposition of pro-inflammatory chemokines in a PSGL-1-dependent manner (9–12). In contrast to the compelling experimental evidence, however, most antiplatelet compounds have hitherto failed to prevent neointimal hyperplasia in the clinical setting (13–15). This mandates a search for new antiplatelet strategies, which specifically target platelet functions involved in the response to vascular injury (16, 17). In the current issue of Thrombosis and Haemostasis, Drosopoulos et al. evaluated the role of a recombinant and soluble form of human CD39 (solCD39) in a murine model of neointima formation after wireinduced denudation of the femoral artery by targeting platelet function (18). CD39 is a cell surface ADPase expressed on leukocytes and endothelial cells, which degrades nucleotides triand/or diphosphates and thereby terminates the aggregation response of platelets to adenosine diphosphate (19). The isolation of a 439 amino acid polypeptide from the extracellular domain of CD39 yielded an injectable protein which retained ATPase activity in vivo (20). The most astonishing finding of Drosopoulos et al. is the complete suppression of neointima formation and the absence of vascular inflammation, as evident by VCAM-1 expression and macrophage recruitment, after 19 days in solCD39-treated mice, indicating a highly active pharmacological compound in this disease model (18). Moreover, Drosopoulos et al. found that solCD39 treatment basically preserved the vessel wall architecture enclosing re-endothelialisation. These findings are in line with reports demonstrating a protective role of CD39 locally over-expressed at the injury site in neointima formation (21, 22). The infusion of solCD39, however, obviates gene transfer as a therapeutic approach making it much more attractive. Of note, CD39-/mice have recently been shown to be also protected from neointimal hyperplasia (23), which might be explained by purinergic type P2Y1 receptor desensitisation responsible for platelet hypofunction in CD39-/mice (24). As one might have deduced from the well established antithrombotic activity of CD39, Drosopoulos et al. reported an absence of mural thombus formation in solCD39-treated mice, although platelets lining the luminal wall were still present. In addition, platelet P-selectin expression was markedly reduced by solCD39, which might account for the diminished recruitment of monocytes/macrophages to the injured artery. However, even in P-selectin-/mice, neointima formation is detectable at least to some degree, suggesting additional CD39-dependent mechanisms (10). Apoptosis of medial smooth muscle cells is a very Correspondence to: Prof. Andreas Schober Institute for Molecular Cardiovascular Research (IMCAR) University Hospital Aachen, RWTH Aachen University Pauwelsstr. 30 52074 Aachen, Germany Tel.: +49 241 80 855 28, Fax: +49 241 80 827 16 E-mail: aschober@ukaachen.de

Inflammatory biomarkers in coronary artery disease

Current evidence supports that inflammation is a major driving force underlying the initiation of coronary plaques, their unstable progression, and eventual disruption; patients with a more pronounced vascular inflammatory response have a poorer outcome. Biomarkers are generally considered to be proteins or enzymes - measured in serum, plasma, or blood - that provide independent diagnostic and prognostic value by reflecting an underlying disease state. In the case of coronary artery disease (CAD), inflammatory biomarkers, have been extensively investigated; more evidence exists for C-reactive protein (CRP). Using high sensitivity (hs) assays, epidemiologic data demonstrate an association between hs-CRP and risk for future cardiovascular morbidity and mortality among those at high risk or with documented CAD. Moreover, a series of prospective studies provide consistent data documenting that mild elevation of baseline levels of hs-CRP among apparently healthy individuals is associated with higher long-term risk for cardiovascular events. Yet, the predictive value of hs-CRP is found to be independent of traditional cardiovascular risk factors. Recent studies suggest that, besides CRP, other inflammatory biomarkers such as cytokines [interleukin (IL)-1, IL-6, IL-8, monocyte chemoattractant protein-1 (MCP-1)], soluble CD40 ligand, serum amyloid A (SAA), selectins (E-selectin, P-selectin), myeloperoxidase (MPO), matrix metalloproteinases (MMPs), cellular adhesion molecules [intercellular adhesion molecule 1 (ICAM-1), vascular adhesion molecule 1 (VCAM-1)], placental growth factor (PlGF) and A(2) phospholipases may have a potential role for the prediction of risk for developing CAD and may correlate with severity of CAD. Finally, indications suggest that the increased risk associated with inflammation may be modified with certain preventive therapies and biomarkers may help to identify the individuals who would benefit most from these interventions.

Microparticles adhere to collagen type I, fibrinogen, von Willebrand factor and surface immobilised platelets at physiological shear rates

Microparticles (MPs), shed during the storage of platelets, support blood coagulation and could be helpful in restoring the haemostatic system in thrombocytopenic patients. The mechanisms by which MPs support haemostasis under flow conditions were investigated. Fluorescent-labelled MPs were perfused at shear rates of 100 and 1000/s over surfaces coated with collagen, fibrinogen, von Willebrand factor (VWF) or surface-adherent platelets. Adhesion was monitored in real-time by fluorescence microscopy. In addition, thrombin-antithrombin (TAT) complex formation was measured in flowing thrombocytopenic blood. MPs attained the capacity to firmly adhere to collagen, VWF, fibrinogen and surface-adherent platelets at high and low shear rate. Antibodies against glycoprotein Ibalpha and alpha(IIb)beta(3) were used to demonstrate the specificities of these interactions. The addition of MPs to thrombocytopenic blood did not affect platelet adhesion. TAT complex formation was increased in the presence of MPs in capillaries coated with fibrinogen, but not on collagen fibres. We confirmed that MPs adhere to a damaged vascular bed in vivo after infusion in denuded arteries in a mouse model. MPs have platelet-like adhering properties and accelerate thrombin generation. These properties strongly support the notion that MPs can be beneficial in maintaining normal haemostasis when platelet function is impaired or reduced, as in thrombocytopenic patients.

Effects of platelet binding on whole blood flow cytometry assays of monocyte and neutrophil procoagulant activity

Monocytes and neutrophils form heterotypic aggregates with platelets initially via engagement of platelet surface P-selectin with leukocyte surface P-selectin glycoprotein ligand-1 (PSGL-1). The resultant intracellular signaling causes the leukocyte surface expression of tissue factor and activation of leukocyte surface Mac-1 (integrin alphaMbeta2, CD11b/CD18). The activation-dependent conformational change in monocyte surface Mac-1 results in the binding of coagulation factor Xa (FXa) and/or fibrinogen to Mac-1. The aim of this study was to develop whole blood flow cytometry assays of these procoagulant activities and to investigate the effects of platelet binding to monocytes and neutrophils. Citrate or D-Phe-Pro-Arg-chloromethylketone (PPACK) anticoagulated whole blood was incubated with monoclonal antibodies against CD14 (PECy5), CD42a (PE), FITC-conjugated test antibody and an agonist, and then fixed with FACS lyse. Appropriate isotype negative controls were prepared in parallel. A BD FACSCalibur was used to analyze monocytes and neutrophils, which were identified based on CD14 fluorescence, forward and 90 degrees light scatter. These populations were further gated into CD42a-positive (platelet-bound) and CD42a-negative (platelet-free). Geometric mean fluorescence and per cent positive data were collected for each subpopulation to measure the binding of test antibodies directed at CD42a, tissue factor, coagulation FXa, bound fibrinogen, activated Mac-1, and CD11b. Compensation controls were prepared on six normal donors prior to the study and these settings were used throughout the 10 donor study. Negative controls verified the lack of cross talk, particularly in the quantified FITC and PE parameters. The physiologic agonists collagen and ADP increased monocyte-platelet and neutrophil-platelet aggregates and increased leukocyte surface Mac-1/CD11b and surface-bound tissue factor, FXa and fibrinogen. Whereas the increases in Mac-1/CD11b were mainly independent of leukocyte-platelet binding, the increases in surface-bound tissue factor, FXa and fibrinogen were mainly dependent on leukocyte-platelet binding. (i) We have developed novel whole blood flow cytometry assays to measure bound tissue factor, coagulation FXa, fibrinogen, activated Mac-1 and CD11b on the surface of monocytes and neutrophils, allowing independent analysis of monocytes and neutrophils with and without surface-adherent platelets. (ii) The monocyte and neutrophil surface binding of tissue factor, FXa and fibrinogen is mainly dependent on platelet adherence to monocytes and neutrophils, whereas the monocyte and neutrophil surface expression of CD11b and activated Mac-1 is mainly independent of platelet adherence to monocytes and neutrophils.

Platelets and Chemokines in Atherosclerosis

It becomes increasingly evident that blood platelets do not only exert important functions in hemostasis and thrombus formation but are also involved in atherosclerotic vascular disease. A major portion of the underlying mechanisms is related to an intricate functional interaction of platelets with chemokines, which have also been implicated in atherogenesis and neointima formation: (1) Platelets can induce the secretion of chemokines in different cells of the vascular wall; (2) In combination with primary agonists, certain chemokines can potentiate platelet aggregation and adhesion; (3) Activated platelets can release and deposit chemokines and precursors on vascular cell surfaces, which trigger atherogenic recruitment of vascular cells or modulate crucial processes such as angiogenesis and lipoprotein metabolism; (4) Surface-adherent platelets can bind and present vascular cell-derived chemokines to trigger arrest of circulating mononuclear cells. The close linkage between platelets and chemokines as culprits in the pathogenesis of vascular diseases may provide a valuable target for selective interventions.

Moderate-intensity exercise suppresses platelet activation and polymorphonuclear leukocyte interaction with surface-adherent platelets under shear flow in men.

The reciprocal modulation of platelet and polymorphonuclear leukocyte (PMN) activities is important in the pathogenesis of thrombosis and inflammation. This study investigated how moderate exercise affects shear-induced platelet activation and subsequent PMNs interaction with platelet-related thrombi under shear flow. Sixteen sedentary healthy men engaged in moderate exercise (about 60% VO(2max)) on a bicycle ergometer. Platelet activation, PMNs interaction with surface-adherent platelets, and PMN-dependent inhibition of platelet activation under shear flow were measured both before and immediately after exercise. The results of this study can be summarized as follows: (1) moderate exercise was associated with lower extents of shear-induced platelet adhesion and aggregation, binding of von Willbrand factor (vWF) to platelets, and glyco-protein IIb/IIIa activation and P-selectin expression on platelet than at rest; (2) the velocity and percentage of rolling PMNs increased while the number of PMNs remaining bound to surface-adherent platelets decreased after moderate exercise; (3) although treating the PMNs with oxidized-low density lipoprotein (Ox-LDL) enhanced PMNs interaction with surface-adherent platelets, moderate exercise suppressed the enhancement of platelet-PMN interaction by Ox-LDL; (4) moderate exercise decreased platelet [Ca (2+)](i) elevation induced by ADP and platelet [Ca(2+)](i) levels mediated by PMN and Ox-LDL-treated PMN; and (5) plasma and PMN-derived nitric oxide metabolites and plasma vWF antigen and activity increased after moderate exercise, whereas plasma and platelet-derived soluble P-selectin levels remained unchanged in response to exercise. Therefore, we conclude that moderate-intensity exercise suppresses shear-induced platelet activation and subsequent PMNs adhesion to platelets deposited at sites of vascular injury under flow, thereby reducing the risks of vascular thrombosis and inflammation.

Strenuous, acute exercise suppresses polymorphonuclear leukocyte respiratory burst under adherence to surface-adherent platelets in men

Interaction between polymorphonuclear leukocyte (PMN) and platelets is important in the pathogenesis of thrombosis and inflammation. This study investigates how strenuous, acute exercise affects PMN oxidative burst activity under adherence to surface-adherent platelets. Thirty sedentary healthy men exercised strenuously (up to maximal oxygen consumption) on a bicycle ergometer. Before and immediately after exercise, the kinetics of oxidant production, phosphorylation of various protein kinase C (PKC) isoforms, and translocation of p47(phox) in PMNs under adherence to surface-adherent platelets were measured using fluorescence microscopy combined with computerized image analysis. Analytical results can be summarized as follows: (i) either treating the platelet with P-selectin (CD62P) and glycoprotein IIb/IIIa (CD41) antibodies or treating the PMN with beta 2-integrin (CD18) and Mac-1 (CD11b) anti-bodies and PKC zeta pseudosubstrate effectively inhibits platelet-promoted oxidant production of PMN; (ii) PMNs adhesion to surface-adherent platelets is associated with a higher amount of phospho-PKC zeta and a larger ratio of membrane to cytosolic p47(phox) than suspended PMNs; (iii) strenuous, acute exercise decreases platelet-promoted oxidant production of PMN and is accompanied by suppressed phosphorylation of PKC zeta, translocation of p47(phox), and inhibition of PKC zeta pseudosubstrate to oxidant production; (iv) no significant changes occur in PKC alpha/beta II and delta phosphorylation of adherent PMNs following this exercise. Therefore, we conclude that strenuous, acute exercise suppresses platelet-promoted oxidative burst of PMN, possibly by reducing phosphorylation of PKC zeta and translocation of the cytosolic p47(phox) to the plasma membrane, thus inhibiting the assembly and activation of NADPH oxidase in PMN.

High Molecular Weight Kininogen Regulates Platelet-Leukocyte Interactions by Bridging Mac-1 and Glycoprotein Ib

Leukocyte-platelet interaction is important in mediating leukocyte adhesion to a thrombus and leukocyte recruitment to a site of vascular injury. This interaction is mediated at least in part by the beta2-integrin Mac-1 (CD11b/CD18) and its counter-receptor on platelets, glycoprotein Ibalpha (GPIbalpha). High molecular weight kininogen (HK) was previously shown to interact with both GPIbalpha and Mac-1 through its domains 3 and 5, respectively. In this study we investigated the ability of HK to interfere with the leukocyte-platelet interaction. In a purified system, HK binding to GPIbalpha was inhibited by HK domain 3 and the monoclonal antibody (mAb) SZ2, directed against the epitope 269-282 of GPIbalpha, whereas mAb AP1, directed to the region 201-268 of GPIbalpha had no effect. In contrast, mAb AP1 inhibited the Mac-1-GPIbalpha interaction. Binding of GPIbalpha to Mac-1 was enhanced 2-fold by HK. This effect of HK was abrogated in the presence of HK domains 3 or 5 or peptides from the 475-497 region of the carboxyl terminus of domain 5 as well as in the presence of mAb SZ2 but not mAb AP1. Whereas no difference in the affinity of the Mac-1-GPIbalpha interaction was observed in the absence or presence of HK, maximal binding of GPIbalpha to Mac-1 doubled in the presence of HK. Moreover, HK/HKa increased the Mac-1-dependent adhesion of myelomonocytic U937 cells and K562 cells transfected with Mac-1 to immobilized GPIbalpha or to GPIbalpha-transfected Chinese hamster ovary cells. Finally, Mac-1-dependent adhesion of neutrophils to surface-adherent platelets was enhanced by HK. Thus, HK can bridge leukocytes with platelets by interacting via its domain 3 with GPIbalpha and via its domain 5 with Mac-1 thereby augmenting the Mac-1-GPIbalpha interaction. These distinct molecular interactions of HK with leukocytes and platelets contribute to the regulation of the adhesive behavior of vascular cells and provide novel molecular targets for reducing atherothrombotic pathologies.

Strenuous, acute exercise affects reciprocal modulation of platelet and polymorphonuclear leukocyte activities under shear flow in men

Vigorous exercise transiently increases the risk of primary cardiac arrest. The reciprocal modulation of platelet and polymorphonuclear leukocyte (PMN) activities is important in the pathogenesis of thrombosis. This study investigates how strenuous, acute exercise affects platelet-PMN reciprocal modulation by closely examining 18 sedentary men who exercised strenuously on a bicycle ergometer. Shear-induced platelet activation, PMN interaction with surface-adherent platelets under shear flow, and PMN-dependent inhibition of platelet activation were measured both before and immediately after exercise. Analytical results can be summarized as follows: (i) shear-induced platelet adhesion on fibronectin-coated surface as well as ADP-induced release of platelet soluble P-selectin release and elevation of [Ca2+]i significantly increases after strenuous exercise; (ii) strenuous exercise is associated with higher velocity and percentage of rolling PMNs and lower numbers of PMNs remaining bound to surface-adherent platelets under shear flow than at rest; (iii) PMN-dependent inhibition of platelet [Ca2+]i elevation and soluble P-selectin release after strenuous exercise is much greater than that at rest; and (iv) strenuous exercise increases PMN-derived nitric oxide metabolite level and reduces oxidized low-density lipoprotein-promoted interaction between platelets and PMNs. Therefore, we conclude that platelet activity may be sensitized by strenuous exercise. However, strenuous exercise can also simultaneously enhance the antiplatelet effect of PMNs. The finding provides a new insight into the negative feedback of PMNs against exercise-evoked platelet-related thrombotic risk.

RANTES deposition by platelets triggers monocyte arrest on inflamed and atherosclerotic endothelium.

Circulating platelets and chemoattractant proteins, such as the CC chemokine RANTES, contribute to the activation and interaction of monocytes and endothelium and may thereby play a pivotal role in the pathogenesis of inflammatory and atherosclerotic disease. The binding of RANTES to human endothelial cells was detected by ELISA or immunofluorescence after perfusion with platelets or exposure to their supernatants. Monocyte arrest on endothelial monolayers or surface-adherent platelets was studied with a parallel-wall flow chamber and video microscopy. We show that RANTES secreted by thrombin-stimulated platelets is immobilized on the surface of inflamed microvascular or aortic endothelium and triggers shear-resistant monocyte arrest under flow conditions, as shown by inhibition with the RANTES receptor antagonist Met-RANTES or a blocking RANTES antibody. Deposition of RANTES and its effects requires endothelial activation, eg, by interleukin-1beta, and is not supported by venous endothelium or adherent platelets. Immunohistochemistry revealed that RANTES is present on the luminal surface of carotid arteries of apolipoprotein E-deficient mice with early atherosclerotic lesions after wire-induced injury or cytokine exposure. In a mechanistic model of atherogenesis, monocyte adherence on endothelium covering such lesions was studied in murine carotid arteries perfused ex vivo, showing that the accumulation of monocytic cells in these carotid arteries involved RANTES receptors. The deposition of RANTES by platelets triggers shear-resistant monocyte arrest on inflamed or atherosclerotic endothelium. Delivery of RANTES by platelets may epitomize a novel principle relevant to inflammatory or atherogenic monocyte recruitment from the circulation.

Platelet-leukocyte interactions

Platelet-leukocyte interactions may contribute to the development of several pathological conditions, including myocardial ischemic disorders. Using an in vitro system that simulates vascular flow conditions, we investigated platelet-neutrophil adhesion. Collagen-adherent platelets were shown to express P-selectin and were able to mediate the binding of flowing neutrophils. Some of the adherent neutrophils displayed signs of cellular activation. Flow cytometric analyses revealed that activation induced a rapid and marked reduction of the P-selectin ligand, with levels decreased by 71% after 15 minutes of stimulation. Using a visual assay of platelet-neutrophil rosetting, we showed that the P-selectin ligand was translocated and clustered at the uropod of neutrophils after the shape changes and polarization induced by stimulation. Activated neutrophils that were bound to surface-adherent platelets also displayed the clustering of P-selectin ligand at the uropod, and these neutrophils detached from the platelets when a shear stress was applied through the adhesion chamber. These results indicate that stimulation of neutrophils induces changes in the surface expression and distribution of the ligand for P-selectin, and that these changes might influence the adhesive interactions occurring between neutrophils and activated platelets. (Am Heart J 1998;135: S146-S151.)

Neutrophil accumulation on activated, surface-adherent platelets in flow is mediated by interaction of Mac-1 with fibrinogen bound to alphaIIbbeta3 and stimulated by platelet-activating factor.

We have studied the pathways that lead to arrest and firm adhesion of rolling PMN on activated, surface-adherent platelets. Stable arrest and adhesion strengthening of PMN on thrombin-stimulated, surface-adherent platelets in flow required distinct Ca2+- and Mg2+-dependent regions of Mac-1 (alphaMbeta2), and involved interactions of Mac-1 with fibrinogen, which was bound to platelets via alphaIIbbeta3. Mac-1 also bound to other unidentified ligands on platelets, which were not intracellular adhesion molecule-2 (ICAM-2), heparin, or heparan-sulfate proteoglycans. This was shown by inhibition with mAbs or peptides, by treatment of platelets with heparitinase, and by using platelets with defective alphaIIbbeta3 from a patient with Glanzmann thrombasthenia. Tethering of PMN on platelet ICAM-2 via LFA-1 (alphaLbeta2) was observed, which may facilitate the transition between rolling on selectins and Mac-1-dependent arrest. Arrest and adhesion strengthening was pertussis toxin sensitive and in flow was mainly induced by platelet-activating factor but not through activation of the chemokine receptor CXCR2. In stasis, spreading occurred and the CXCR2 contributed to firm adhesion.

Imaging of surface-adherent human-blood platelets by atomic-force microscopy (AFM)

Studies in our laboratory over the past three decades have explored platelet ultrastructural morphology by light microscopy as well as transmission, scanning and low-voltage, high-resolution scanning electron microscopy. Unlike these optical microscopes, scanning probe microscopes measure real space images of cells on flat surfaces. In brief, the AFM works by scanning a very sharp conducting tip (SiN) over the surface of a conducting biological membrane. Cantilivers used for probing were short tipped with thin legs (0.22 nm spring constant). Scanning was done with the J size scanner (150 x 150 u scan area). The difficulty in achieving high resolution images of biological samples relates to the damage caused by the probing tip in spite of its minute force on delicate biological cell surface membranes. In the present study we have evaluated surface topography of fixed resting and activated platelets using AFM. Resting platelets were treated with cytochalasin B to prevent actin filament assembly shape change prior to being placed on polylysine-coated grid surfaces.

Neutrophil rolling, arrest, and transmigration across activated, surface-adherent platelets via sequential action of P-selectin and the beta 2-integrin CD11b/CD18

Platelets bound to thrombogenic surfaces have been shown to support activation-dependent firm adhesion of neutrophils in flow following selectin-mediated tethering and rolling. The specific receptor(s) responsible for mediating adhesion-strengthening interactions between neutrophils and platelets has not previously been identified. Furthermore, the ability of adherent platelets to support the migration of bound neutrophils has not been tested. We studied neutrophil interactions with activated, surface-adherent platelets as a model for leukocyte binding in vascular shear flow and emigration at thrombogenic sites. Our results demonstrate that the beta 2-integrin Mac-1 (CD11b/CD18) is required for both firm attachment to and transmigration of neutrophils across surface-adherent platelets. In flow assays, neutrophils from patients with leukocyte adhesion deficiency-1 (LAD-I), which lack beta 2-integrin receptors, formed P-selectin-mediated rolling interactions, but were unable to develop firm adhesion to activated platelets, in contrast to healthy neutrophils, which developed firm adhesion within 5 to 30 seconds after initiation of rolling. Furthermore, the adhesion-strengthening interaction observed for healthy neutrophils could be specifically inhibited by monoclonal antibodies (mAbs) to Mac-1, but not to lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18) or intercellular adhesion molecule-2 (ICAM-2; CD102). Further evidence for a beta 2-integrin-dependent neutrophil/platelet interaction is demonstrated by the complete inhibition of interleukin (IL)-8-induced neutrophil transmigration across platelets bound to fibronectin-coated polycarbonate filters by mAbs to Mac-1. Thus, Mac-1 is required for firm adhesion of neutrophils to activated, adherent platelets and may play an important role in promoting neutrophil accumulation on and migration across platelets deposited at sites of vascular injury.

Neutrophil rolling, arrest, and transmigration across activated, surface-adherent platelets via sequential action of P-selectin and the beta 2-integrin CD11b/CD18

Abstract Platelets bound to thrombogenic surfaces have been shown to support activation-dependent firm adhesion of neutrophils in flow following selectin-mediated tethering and rolling. The specific receptor(s) responsible for mediating adhesion-strengthening interactions between neutrophils and platelets has not previously been identified. Furthermore, the ability of adherent platelets to support the migration of bound neutrophils has not been tested. We studied neutrophil interactions with activated, surface-adherent platelets as a model for leukocyte binding in vascular shear flow and emigration at thrombogenic sites. Our results demonstrate that the beta 2-integrin Mac-1 (CD11b/CD18) is required for both firm attachment to and transmigration of neutrophils across surface-adherent platelets. In flow assays, neutrophils from patients with leukocyte adhesion deficiency-1 (LAD-I), which lack beta 2-integrin receptors, formed P-selectin-mediated rolling interactions, but were unable to develop firm adhesion to activated platelets, in contrast to healthy neutrophils, which developed firm adhesion within 5 to 30 seconds after initiation of rolling. Furthermore, the adhesion-strengthening interaction observed for healthy neutrophils could be specifically inhibited by monoclonal antibodies (mAbs) to Mac-1, but not to lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18) or intercellular adhesion molecule-2 (ICAM-2; CD102). Further evidence for a beta 2-integrin-dependent neutrophil/platelet interaction is demonstrated by the complete inhibition of interleukin (IL)-8-induced neutrophil transmigration across platelets bound to fibronectin-coated polycarbonate filters by mAbs to Mac-1. Thus, Mac-1 is required for firm adhesion of neutrophils to activated, adherent platelets and may play an important role in promoting neutrophil accumulation on and migration across platelets deposited at sites of vascular injury.

Chemoattractant-Induced Changes in Surface Expression and Redistribution of a Functional Ligand for P-Selectin on Neutrophils

Abstract Adhesion between platelets and neutrophils is mediated through the interaction of P-selectin on activated platelets with a carbohydrate- containing structure on neutrophils, and occurs under both static and shear conditions. Recent studies using flow chambers have shown that neutrophils become activated after binding to surface-adherent platelets expressing P-selectin. The objective of the present study was to investigate the effect of such activation on the interactions of platelet P-selectin with its ligand on neutrophils. Flow cytometric analyses using P-selectin chimeras revealed that activation induced a rapid and marked reduction in chimera binding, with levels of binding decreased by 71% after 15 minutes of stimulation with the chemotactic agent, FMLP. Using a visual assay of platelet-neutrophil rosetting, we showed that the P-selectin ligand was translocated and clustered at the uropod of neutrophils following the shape changes and polarization induced by chemotactic stimulation. Activated neutrophils bound to surface-adherent platelets also displayed the clustering of P-selectin ligand at the uropod, and these neutrophils detached from the platelets when a shear stress (2 dynes/cm2) was applied through the adhesion chamber. These results indicate that chemotactic stimulation of neutrophils induces changes in the surface expression and distribution of a biologically relevant ligand for P-selectin, and that these changes might influence the adhesive interactions occurring between neutrophils and activated platelets.