Correlation between structural changes and acute thrombogenicity in transcatheter pericardium valves after crimping and balloon deployment.

INTRODUCTION: Transcathether heart valve replacement has gained considerable acceptance during the last decades. It is now part of the armamentarium for aortic valve replacement. The procedure proved to be highly efficient. However the issues of the blood compatibility and tissue durability were not raised and the adverse events were probably under-reported, according to observations of thrombosis after deployment. MATERIAL AND METHOD: Bovine pericardium leaflets were sewn inside a 26mm diameter stainless steel stent to manufacture these valves (one control and two experimental). The correlation between the trauma and the acute thombogenicity of bovine pericardium leaflets, after crimping and ballooning, was investigated via an in vitro blood flow with labeled platelets. These leaflets were processed for histology: scanning electron microscopy, light microscopy, and transmission electron microscopy. RESULTS: The control specimens showed a regular pericardium structure with some blood cells deposited on the collagen fibrous surface (inflow) and scarce blood cells deposited on the serous surface (outflow). After crimping and ballooning, the structure of the pericardium was severely injured, eventually with delaminations and ruptures. The blood cell uptake was considerably increased compared to the control. CONCLUSION: It would therefore be appropriate to pay more attention to the design of the valves. Specifically, the incorporation of a buffer tissue or fabric between the pericardium and the metallic stent is suggested. The issue of ballooning deserves detailed and in depth investigation regarding the lifetime of the device.

Antiphospholipid antibody-mediated effects in an arterial model of thrombosis are dependent on Toll-like receptor 4.

Patients with antiphospholipid syndrome (APS) produce antiphospholipid antibodies (aPL) and develop vascular thrombosis that may occur in large or small vessels in the arterial or venous beds. On the other hand, many individuals produce aPL and yet never develop thrombotic events. Toll-like receptor 4 (TLR4) appears to be necessary for aPL-mediated prothrombotic effects in venous and microvascular models of thrombosis, but its role in arterial thrombosis has not been studied. Here, we propose that aPL alone are insufficient to cause thrombotic events in an arterial model of APS, and that a concomitant trigger of innate immunity (e.g. TLR4 activation) is required. We show specifically that anti-ß2-glycoprotein I (anti-ß2GPI) antibodies, a subset of aPL, accelerated thrombus formation in C57BL/6 wild-type, but not TLR4-deficient, mice in a ferric chloride-induced carotid artery injury model. These aPL bound to arterial and venous endothelial cells, particularly in the presence of ß2GPI, and to human TLR4 by enzyme-linked immunoassay. Arterial endothelium from aPL-treated mice had enhanced leukocyte adhesion, compared to control IgG-treated mice. In addition, aPL treatment of mice enhanced expression of tissue factor (TF) in leukocytes induced by the TLR4 ligand lipopolysaccharide (LPS). aPL also enhanced LPS-induced TF expression in human leukocytes in vitro. Our findings support a mechanism in which aPL enhance TF expression by leukocytes, as well as augment adhesion of leukocytes to the arterial endothelium. The activation of TLR4 in aPL-positive individuals may be required to trigger thrombotic events.

Protein Corona Formation on Magnetite Nanoparticles: Effects of Culture Medium Composition, and Its Consequences on Superparamagnetic Nanoparticle Cytotoxicity.

The physicochemical properties and potential cytotoxicity of nanoparticles (NPs) are significantly influenced by their inter- action with proteins, which results in corona formation. Here, we have determined whether corona formation, resulting from interactions between superparamagnetic iron oxide nanoparticles (SPIONs) and different cell culture media, may have consequences for driving NP toxic effects. To address this issue, complementary methods were used. The deter- mination of the hydrodynamic size distribution by ζ (zeta) potential measurement indicated that SPIONs were negatively charged under all conditions but that the actual charge was differed with the cell culture medium used. In vitro protein adsorption studies were carried out using the Bradford protein assay and Fourier transform infrared spectroscopy (FTIR). The Bradford assay revealed that the concentration of unadsorbed proteins and other biomolecules decreased when the SPION concentration increased. FTIR showed that the proteins were, indeed, adsorbed onto the NP surface. This was followed by matrix-assisted laser desorption/ionization time-of-flight secondary ion mass spectrometry (MALDI TOF-SIMS), to identify the adsorbed proteins. Ultimately, three different cell viability assays led to the conclusion that the SPIONs were not toxic for all the concentrations used here. In summary, we found that corona formation on the SPIONs depends on the composition of the culture media but has no consequence for nanotoxicity. We have shown that the application of complementary methods has provided novel insights into SPION/protein interactions.

A primary role for kinin B1 receptor in inflammation, organ damage, and lethal thrombosis in a rat model of septic shock in diabetes.

Diabetes mellitus and septic shock increase the incidence of mortality by thrombosis. Although kinin B1 receptor (B1R) is involved in both pathologies, its role in platelet function and thrombosis remains unknown. This study investigates the expression, the inflammatory, and pro-thrombotic effects of B1R in a model of septic shock in diabetic rats. Sprague-Dawley rats were made diabetic with streptozotocin (STZ) (65 mg/kg, i.p.). Four days later, control and STZ-diabetic rats were injected with lipopolysaccharide (LPS) (2 mg/kg, i.p.) or the vehicle. B1R antagonist (SSR240612, 10 mg/kg by gavage) was given either acutely (12 and 24 h prior to endpoint analysis) or daily for up to 7 days. Moreover, a 7-day treatment was given either with cyclooxygenase (COX)-2 inhibitor (niflumic acid, 5 mg/kg, i.p.), non-selective COX-1 and COX-2 inhibitor (indomethacin, 10 mg/kg, i.p.), non-selective nitric oxide synthase (NOS) inhibitor (L-NAME, 50 mg/kg by gavage), iNOS inhibitor (1400W, 5 mg/kg, i.p.), or heparin (100 IU/kg, s.c.). The following endpoints were measured: edema and vascular permeability (Evans blue dye), B1R expression (qRT-PCR, western blot, flow cytometry), aggregation in platelet-rich plasma (optical aggregometry), and organ damage (histology). Rats treated with STZ, LPS, and STZ plus LPS showed significant increases in edema and vascular permeability (heart, kidney, lung, and liver) and increased expression of B1R in heart and kidney (mRNA) and platelets (protein). Lethal septic shock induced by LPS was enhanced in STZ-diabetic rats and was associated with lung and kidney damage, including platelet micro-aggregate formation. SSR240612 prevented all these abnormalities as well as STZ-induced hyperglycemia and LPS-induced hyperthermia. Similarly to SSR240612, blockade of iNOS and COX-2 improved survival. Data provide the first evidence that kinin B1R plays a primary role in lethal thrombosis in a rat model of septic shock in diabetes. Pharmacological rescue was made possible with B1R antagonism or by inhibition of iNOS and COX-2, which may act as downstream mechanisms.

Soluble CD40 ligand impairs the anti-platelet function of peripheral blood angiogenic outgrowth cells via increased production of reactive oxygen species.

Adult peripheral blood angiogenic early outgrowth cells (EOCs), also known as early endothelial progenitor cells, interact with other blood and vascular cells and may regulate atherothrombosis. We have previously shown that endothelial progenitor cells inhibit platelet function and thrombus formation. The CD40L/CD40 axis is a thrombo- inflammatory mediator that affects platelet and endothelial functions. It has been shown that EOCs express CD40, whereas platelets represent the major source of its soluble ligand (sCD40L), which impairs EOC function.We aimed to test the hypothesis that the sCD40L/CD40 axis affects the anti-platelet function of EOCs. Human peripheral blood mononuclear cell-derived EOCs in culture inhibited platelet aggregation. Pre-treatment of EOCs with sCD40L reduced their inhibitory effect on platelet aggregation in a CD40-dependent manner. EOCs viability and release of the anti-aggregating agents, prostacyclin and nitric oxide, were not affected by sCD40L. However, production of reactive oxygen species (ROS) was increased in sCD40L-treated EOCs. Blockade of ROS reversed the effects of sCD40L-treated EOCs on platelet aggregation. This study reveals that the sCD40L/CD40 axis impairs the anti-platelet properties of EOCs through increased production of ROS. These data may explain the link between elevated levels of sCD40L, impaired activity of EOCs and enhanced platelet reactivity, and consequently the occurrence of atherothrombotic disease.

Platelet adhesion and human umbilical vein endothelial cell cytocompatibility of biodegradable segmented polyurethanes prepared with 4,4'-methylene bis(cyclohexyl isocyanate), poly(caprolactone) diol and butanediol or dithioerythritol as chain extenders.

Biodegradable segmented polyurethanes were prepared with poly(caprolactone) diol as a soft segment, 4,4'-methylene bis(cyclohexyl isocyanate) (HMDI) and either butanediol or dithioerythritol as chain extenders. Platelet adhesion was similar in all segmented polyurethanes studied and not different from Tecoflex® although an early stage of activation was observed on biodegradable segmented polyurethane prepared with dithioerythritol. Relative viability was higher than 80% on human umbilical vein endothelial cells in contact with biodegradable segmented polyurethane extracts after 1, 2 and 7 days. Furthermore, both biodegradable segmented polyurethane materials supported human umbilical vein endothelial cell adhesion, spreading, and viability similar to Tecoflex® medical-grade polyurethane. These biodegradable segmented polyurethanes represent promising materials for cardiovascular applications.

In vitro biocompatibility assessment of functionalized magnetite nanoparticles: biological and cytotoxicological effects.

In the biomedical field, nanomaterials have the potential for use in the targeted delivery of drugs in the human body and in the diagnosis and therapy of certain diseases. In the category of targeted delivery, magnetite (Fe(3)O(4)) nanoparticles have received much attention. As with any similar new therapy, when such nanoparticles are functionalized with chemical groups designed to permit the specific attachment of drugs, cytotoxicological testing is necessary before moving to animal models. Here, we consider several variously functionalized magnetite nanoparticles, including those prepared with (1) a monolayer of oleic acid (Fe(3)O(4)@OA), which is subsequently converted to (2) a shell of amine-containing silane (Fe(3)O(4)@NH(2)), (3) a shell of silica (Fe(3)O(4)@SiO(2)), and (4) a shell of amine-containing silane over a shell of silica (Fe(3)O(4)@SiO(2)@NH(2)). These latter three functionalities were evaluated for biocompatibility, cellular morphology, mitochondrial function (MTT assay), lactate dehydrogenase membrane leakage (LDH assay), and proinflammatory potential through enzyme linked immunosorbent assay (ELISA) for interleukin 6 (IL-6). Controlled tests were performed over a period of 72 h, with results showing LDH leakage and abnormal Il-6 secretion at high concentrations (>50 µg/mL). The tests showed that, in addition to the surface characteristics of the nanoparticles, both the nutrient medium and the time of suspension before exposure to cells also contribute to nanoparticle cytotoxicity.

Inhibition of von Willebrand factor-mediated platelet activation and thrombosis by the anti-von Willebrand factor A1-domain aptamer ARC1779.

BACKGROUND: von Willebrand factor (VWF) has a role in both hemostasis and thrombosis. Platelets adhere to damaged arteries by interactions between the VWF A1-domain and glycoprotein Ib receptors under conditions of high shear. This initial platelet binding event stimulates platelet activation, recruitment, and activation of the clotting cascade, promoting thrombus formation. OBJECTIVE: To characterize the inhibitory activity of a VWF inhibitory aptamer. METHODS: Using in vitro selection, aptamer stabilization, and conjugation to a 20-kDa poly(ethylene glycol), we generated a nuclease-resistant aptamer, ARC1779, that binds to the VWF A1-domain with high affinity (K(D) approximately 2 nM). The aptamer was assessed for inhibition of VWF-induced platelet aggregation. In vitro inhibition of platelet adhesion was assessed on collagen-coated slides and injured pig aortic segments. In vivo activity was assessed in a cynomolgus monkey carotid electrical injury thrombosis model. RESULTS AND CONCLUSION: ARC1779 inhibited botrocetin-induced platelet aggregation (IC90 approximately 300 nM) and shear force-induced platelet aggregation (IC95 approximately 400 nM). It reduced adhesion of platelets to collagen-coated matrices and formation of platelet thrombi on denuded porcine arteries. ARC1779 also inhibited the formation of occlusive thrombi in cynomolgus monkeys. We have discovered a novel anti-VWF aptamer that could have therapeutic use as an anti-VWF agent in the setting of VWF-mediated thrombosis.

Autoantibodies to heat shock protein 60 promote thrombus formation in a murine model of arterial thrombosis.

BACKGROUND AND OBJECTIVES: Anti-heat shock protein (HSP)60 autoantibodies are associated with atherosclerosis and are known to affect endothelial cells in vitro. However, their role in thrombus formation remains unclear. We hypothesized that anti-HSP60 autoantibodies could potentiate thrombosis, and evaluated the effect of anti-murine HSP60 antibodies in a ferric chloride (FeCl3)-induced murine model of carotid artery injury. METHODS: Anti-HSP60, or control, IgG was administered to BALB/c mice 48 h prior to inducing carotid artery injury, and blood flow was monitored using an ultrasound probe. RESULTS: Thrombus formation was more rapid and stable in anti-HSP60 IGG-treated mice than in controls (blood flow=1.7%+/-0.6% vs. 34%+/-12.6%, P=0.0157). Occlusion was complete in all anti-HSP60 IgG-treated mice (13/13), with no reperfusion being observed. In contrast, 64% (9/14) of control mice had complete occlusion, with reperfusion occurring in 6/9 mice. Thrombi were significantly larger in anti-HSP60 IgG-treated mice (P=0.0001), and contained four-fold more inflammatory cells (P=0.0281) than in controls. Non-injured contralateral arteries of anti-HSP60 IgG-treated mice were also affected, exhibiting abnormal endothelial cell morphology and significantly greater von Willebrand factor (VWF) and P-selectin expression than control mice (P=0.0024 and P=0.001, respectively). CONCLUSIONS: In summary, the presence of circulating anti-HSP60 autoantibodies resulted in increased P-selectin and VWF expression and altered cell morphology in endothelial cells lining uninjured carotid arteries, and promoted thrombosis and inflammatory cell recruitment in FeCl3-injured carotid arteries. These findings suggest that anti-HSP60 autoantibodies may constitute an important prothrombotic risk factor in cardiovascular disease in human vascular disease.

The influence of isocyanurate content on the bioperformance of hydrocarbon-based polyurethanes.

Bulk, surface and bioactivity of newly synthesized hydroxy telechelic polyisoprene-based (H-HTPI) polyurethane were investigated by means of ATR-FT-IR, contact-angle measurements, cell viability, calcification, and platelet and fibrinogen quantification. The influence of isophorone diisocyanates isocyanurate (I-IPDI) content on these properties was determined. Results generally showed a non-significant difference in these properties when they were compared with a commercially available biomedical polyurethane (PU), such as Tecoflex. Unexpectedly, where the increase of isocyanate content for commercial diisocyanate-based biocompatible PU significantly increases the surface contact angle, the new hydroxy telechelic polyisoprene-based PU showed a decrease of water contact angle with increasing I-IPDI content in the polymer. Nevertheless, the overall surface exhibited hydrophobic properties, i.e., theta > 85. Polymer cytotoxicity, assessed with L929 cell line in direct contact with the surface of the samples, showed no toxic effects on the cells. Interestingly, regardless of the I-IPDI content, platelet adhesion and fibrinogen adsorption, as well as the mineral deposition were fairly similar for all synthesized PUs. Our findings revealed that replacing diisocyanates by their isocyanurate homologues is a very relevant approach for preparation of polyurethanes with different mechanical properties while maintaining similar surface properties.

The effect of varying Al2O3 percentage in hydroxyapatite/Al2O3 composite materials: morphological, chemical and cytotoxic evaluation.

Hydroxyapatite (HA) and HA-alumina (HA/Al2O3) composites, with Al2O3 contents of 5, 10, 20, and 30%, were synthesized using a wet precipitation method and sintered at 900 and 1300 degrees C. We investigated the effect of sintering temperature and relative concentration of HA and Al2O3 on the chemical composition, surface morphology, and cytotoxicity of the composite powders. The XRD results show that in the 1300 degrees C composites, HA partially decomposed into CaO which combined with Al2O3 to form different calcium aluminates. For the 900 degrees C composites the CaO phase was not detected, though a Ca/P ratio larger than 1.67 measured by XPS suggests that CaO was present in trace amounts. SEM-EDX analysis indicated that the HA microstructure was affected by the sintering temperature, and this HA is present on the surface of Al2O3 particles. The cytotoxicity of the composites was assessed indirectly using the MTT assay. The short-term effect of leachables was quantified by exposing a L929 mouse fibroblast cell line to the degradation products released by the composites after immersion in the cell culture medium. Degradation products were less toxic to L-929 at lower extract concentrations (10, 50%) than at 100% concentration. Cell viability was also influenced by leachable size.

In vitro thrombogenicity investigation of new water-dispersible polyurethane anionomers bearing carboxylate groups.

New segmented polyurethane (PU) anionomers based on hydroxytelechelic polybutadiene were synthesized via an aqueous dispersion process. Incorporation of carboxylic groups was achieved using thioacids of different length. Surface properties were investigated by mean of water absorption analysis and static contact-angle measurements using water, diiodomethane, formamide and ethylene glycol. Blood compatibility of the PUs was evaluated by in vitro adhesion assays using 111In-radiolabeled platelet-rich plasma and [125I]fibrinogen. Morphology of the adhered platelets was examined by scanning electron microscopy (SEM). Results were compared to two biomedical-grade PUs, namely Pellethane and Tecoflex. Insertion of carboxylic groups increased surface hydrophilicity and limited water uptake ( < 8% for an ion content of 5% by weight). Surface energy of all synthesized PUs was between 40 and 45 mJ/m2. Platelet adhesion and fibrinogen adsorption on the PU anionomer surfaces were affected as a function to the increase of graft length; thiopropionic was the most haemocompatible, followed by thiosuccinic and then thioglycolic acid. SEM analyses of all ionic PU samples exhibited low platelet adhesion to surfaces with no morphological modification. In conclusion, increased hydrophily, dynamic mobility and charge repulsion are synergistic key factors for enhanced haemocompatibility.

Hemocompatibilty of new ionic polyurethanes: influence of carboxylic group insertion modes.

New segmented polyurethane (PU) anionomers based on hydroxytelechelic polybutadiene (HTPB) were synthesized via two environment-friendly chemical routes. The effects of carboxylic content and ion incorporation mode on the surface properties were investigated by mean of water absorption analysis and static contact angle measurements using water, diiodomethane, formamide and ethylene glycol. Blood compatibility of the PUs was evaluated by in vitro adhesion assay using 111In-radiolabeled platelet rich plasma and 125I-fibrinogen. The morphology of platelet adhesion was also observed by scanning electron microscopy (SEM). Results were compared with a biomedical-grade PU, Pellethane. Insertion of the carboxylic groups on the soft segments (S-alpha series), using thioglycolic acid (TGA), increases surface hydrophilicity, limits water uptake (5%, for an ion content of 3.6 wt%), and reduces platelet adhesion and fibrinogen adsorption on the PUs' surfaces. In contrast, the classical insertion onto the hard segment (H-alpha series), using dimethylolpropionate (DMPA) as chain extender, leads to high water uptake (18%, for an ion content of 3.6 wt%) and promotes platelet and fibrinogen adhesion. SEM analyses of the non-ionic PUs exhibited surfaces with adhered platelets which underwent morphological modification. Similarly, the H-alpha ionic PUs show adherent and activated platelets. On the contrary, no platelet morphology changes were observed on the S-alpha ionic surfaces. In conclusion, insertion of carboxyl groups on the soft segments of PUs reduces their thrombogenicity.

Nitinol versus stainless steel stents: acute thrombogenicity study in an ex vivo porcine model.

Acute and subacute stents thrombosis along with thrombus mediating neointimal proliferation within the stent struts remain major concerns in coronary stenting. Up to date, there is an obvious lack of data on the thrombogenicity of stent materials in physiological conditions. This study was performed to compare the relative thrombogenicity of nitinol versus stainless steel stents. Nitinol stents were laser cut to reproduce the exact geometry of the stainless steel Palmaz stents and tested in an ex vivo AV shunt porcine model under controlled conditions. Nitinol stents presented only small amounts of white and/or red thrombus principally located at the strut intersections while Palmaz stents clearly exhibited more thrombus. As a result, 125I-fibrin(ogen) adsorption and (111)I-platelets adhesion were significantly lower on nitinol than on stainless steel devices (36%, p = 0.03 for fibrin(ogen) and 63%, p = 0.01 for platelet). These results were confirmed by scanning electron observations showing different thrombus morphologies for nitinol and stainless steel. Along with the unique mechanical properties of nitinol, its promising haemocompatibility demonstrated in our study may promote their increasing use for both peripheral and coronary revascularization procedures.

P-selectin antagonism with recombinant p-selectin glycoprotein ligand-1 (rPSGL-Ig) inhibits circulating activated platelet binding to neutrophils induced by damaged arterial surfaces.

Neutrophil P-selectin glycoprotein ligand-1 (PSGL-1) mediates the initial rolling and adhesion of neutrophils to P-selectin on activated endothelium and platelets. Platelet-neutrophil activation and binding occur in the blood of patients with arterial diseases, suggesting that arterial damage leads to these phenomena. We investigated the influence of endothelial surface integrity on circulating platelet activation and binding to neutrophils and the mechanism involved in these interactions. Expression of P-selectin on human platelets and their binding to neutrophils was determined by flow cytometry at baseline after thrombin activation and after exposure for 15 min to intact and damaged arterial surfaces in flow chambers. Expression of platelet P-selectin at baseline and after perfusion over intact endothelium averaged 13.8 +/- 1.2 and 12.7 +/- 1.8%, respectively, and increased significantly to 19.7 +/- 1.8% (P < 0.05) after perfusion over damaged arteries. In mixed neutrophil/platelet suspensions, the percentage of neutrophils that bind platelets increased significantly also, from 10.8 +/- 1.6% at baseline to 39.7 +/- 2.9% (P < 0.05) after perfusion over damaged arteries compared with 69.7 +/- 2.5% with thrombin. This binding was completely inhibited by a recombinant soluble PSGL-1 (rPSGL-Ig) and anti-P-selectin and PSGL-1-blocking monoclonal antibodies. The inhibitory effect of rPSGL-Ig correlated well with its binding to platelets (r = 0.98, P < 0.001). Circulating platelets are activated upon contact with damaged arteries, thereby enhancing their adhesive interactions with neutrophils via P-selectin and PSGL-1. Inhibition of this binding with rPSGL-Ig may constitute a target in the treatment of inflammatory and thrombotic reactions.

Relationship between platelets and neutrophil adhesion and neointimal growth after repeated arterial wall injury induced by angioplasty in pigs.

Platelet and neutrophil interactions with injured vascular wall may contribute to restenosis. Their importance was mainly examined following balloon injury of intact arteries. However, dilation of diseased arteries is clinically more relevant and may elicit different responses. We investigated the relationship between platelets and neutrophil adhesion, neointima formation and P-selectin expression on damaged arteries after repeated balloon injury. In an acute single-injury model, 8 pigs were subjected to bilateral carotid angioplasty and sacrificed 1 h later. In a chronic model, 19 pigs were subjected to similar procedures and allowed to recover for 4 weeks; then 18 arteries were redilated at the same previously injured sites (double injury) while the remaining arteries were not redilated and used to investigate the extent and the adhesive properties of the neointima. After single injury, (51)Cr-platelet adhesion (x10(6)/cm(2)) increased significantly from 3.8 +/- 0.6 to 45.9 +/- 6.5 (p < 0.05) on mildly and deeply injured segments, respectively, and were statistically similar after double injury. After single injury, (111)In-neutrophil adhesion (x10(3)/cm(2)) increased from 226.6 +/- 45.5 to 512.5 +/- 70.3 (p < 0.05) on mildly and deeply injured segments, and were significantly higher (p < 0.05) after double injury (mild: 1,289.1 +/- 227.9 and deep: 2,411.8 +/- 333.9). As well, the neo-endothelium expresses P-selectin at 4 weeks and platelet and neutrophil adhesion was directly related to neointimal growth. These results, which indicate ongoing proinflammatory processes 1 month post-angioplasty, suggest that neutrophils may participate in the progression of restenosis.

Influence of stent expansion states on platelet deposition in an extracorporeal porcine arteriovenous shunt model using a multichannel perfusion chamber.

Limited data are available about incomplete stent expansion (SE) on platelet deposition (PD). We examined PD following different SE using an extracorporeal porcine arteriovenous shunt model to which a perfusion chamber with four parallel silastic tubes were connected. Blood flow was set at a 20 and 100 mL/min in 1.8 and 3.1 mm diameter tubes, respectively. P154 stents were deployed completely (Group A, n=15) or incompletely (Group B, n=15) in 1.8 mm (n=13) and 3.1 mm (n=17) tubes. 51Cr-labelled platelet autologous blood was injected 1 hr before the perfusion. After 15 min-perfusion, the testing tubes were assessed for radioactivity counts. In-stent cross sectional area was measured by intravascular ultrasound. There was a significant difference in PD between group A and B regardless of channel size (118+/-18.4 vs 261.4+/-52.1 pits x 10(6)/cm2, p<0.05). With adjusted shear rate and similar stenosis, PD was similar in both tubes. In smaller 1.8 mm tubes, a stenosis as subtle as 10% was associated with a significant PD difference (226.1+/-20 vs 112.9+/-20.5 plts x 10(6)/cm2, p<0.005). This model enabled a repetitive, simultaneous comparison of PD following different SE states. It seems that the quality of SE remains crucial in smaller channels.

Recombinant soluble p-selectin glycoprotein ligand-1-Ig reduces restenosis through inhibition of platelet-neutrophil adhesion after double angioplasty in swine.

BACKGROUND: P-selectin mediates leukocyte recruitment to activated platelets and endothelium through its high-affinity receptor P-selectin glycoprotein ligand-1 (PSGL-1). Platelet and leukocyte activation and binding have been reported after coronary angioplasty and were correlated with restenosis. We investigated the effect of a recombinant soluble PSGL-1 (rPSGL-Ig) on the adhesion of platelets and neutrophils and the development of restenosis after double arterial injury. METHODS AND RESULTS: Four weeks after angioplasty of both carotid arteries in pigs, a second angioplasty was performed at the same sites, 15 minutes after a single administration of vehicle or rPSGL-1 (1 mg/kg IV). Animals were euthanized 1 hour, 4 hours, 1 week, or 4 weeks later. Adhesion of autologous (51)Cr-platelets and (111)In-neutrophils was quantified and histological/morphometric analyses were performed. Although rPSGL-Ig did not affect adherence of these cells 1 hour after injury, it significantly reduced the adhesion of platelets (50% at 4 hours and 85% at 1 week) and neutrophils (50% at 4 hours and 78% at 1 week) to deeply injured arteries. At 4 weeks, the residual lumen was 63% larger in rPSGL-Ig-treated arteries as compared with control arteries (6.1+/-0.6 versus 3.8+/-0.1 mm(2); P:<0.002). The neointimal area was slightly reduced (0.5 in rPSGL-Ig versus 0.7 mm(2) in control). The ratio of the external elastic lamina of injured to uninjured reference segments was >1 in treated arteries and <1 in control arteries. CONCLUSIONS: P-selectin antagonism with rPSGL-Ig inhibits early platelet/leukocyte adhesion on injured arteries and reduces restenosis through a positive impact on vascular remodeling. Hence, rPSGL-Ig may have potential in the prevention of restenosis.

Intravenous aspirin at reperfusion does not reduce infarct size in the dog with a residual critical stenosis.

Platelet-related events being associated with the increment of infarct size at reperfusion in the presence of a residual stenosis, we tested in dogs whether intravenous aspirin (ASA) could limit infarct size. The left anterior descending coronary artery was occluded for 90 min and reperfused for 6 h in the presence of a residual critical stenosis. Controls received saline, and treated groups were given 2, 6, or 12 mg/kg ASA, i.v., 5 min before reperfusion. Infarct size did not differ significantly between groups (control, 43.80+/-6.28%; ASA, 2 mg/kg: 41.07+/-7.78%; ASA, 6 mg/kg: 37.55+/-3.44%; ASA, 12 mg/kg: 29.40+/-5.41%), as well as transmural collateral blood flow and [111In]-platelet accumulation in the infarcted myocardium (2.5-3.6 x 10(5) platelets/g). However, myocardial neutrophil accumulation was significantly reduced (p < 0.05) in groups given 6 (15.0+/-2.6 x 10(6)/g tissue) and 12 mg/kg (18.4 +/-3.8) ASA, but not in the 2-mg/kg group (21.0+/-5.2), as compared with control group (32.0+/-7.2). Ex vivo platelet aggregation to collagen was abolished during reperfusion in all treated groups (p < 0.05). Transcardiac arteriovenous differences in 6-keto-PGF1alpha were reduced significantly 1 h after reperfusion in groups given 6 or 12 mg/kg ASA (94.7+/-13.1 and 71.7+/-19.2 pg/ml, respectively) but not in the 2-mg/kg group (178.3+/-78.2 pg/ml), as compared with control (405.4+/-171.6 pg/ml). ASA-insensitive platelet activation at the site of stenosis or inhibition by ASA of prostacyclin production by jeopardized myocardium may explain the observed lack of benefit of ASA.

Inhibition of platelet-neutrophil interactions by Fucoidan reduces adhesion and vasoconstriction after acute arterial injury by angioplasty in pigs.

The selectin family of cell-adhesion molecules contributes to the interactions of leukocytes and platelets at the site of vascular injury. Such interactions enhance inflammatory reactions and thrombus formation during the arterial response to injury. In this study, we investigated the effects of a selectin inhibitor (Fucoidan) on platelet and neutrophil interactions after arterial injury produced by angioplasty in pigs. [51Cr]-platelet deposition and [111In]-neutrophil adhesion were quantified on intact, mildly, and deeply injured carotid arterial segments, produced by balloon dilation in control (saline, n = 7) and Fucoidan-treated (i.v.; 1 mg/kg, n = 6; 5 mg/kg, n = 5) pigs. In the control group, platelet deposition (x10(6)/cm2) was influenced by the severity of injury and increased significantly (p < 0.05) from 0.06+/-0.06 on intact endothelium to 3.8+/-0.6 and 33.6+/-4.9 on mildly and deeply injured segments, respectively. Fucoidan, 1 mg/kg, had no significant effect, although doses of 5 mg/kg reduced platelet deposition by 73% on deeply injured segments. The level of neutrophil adhesion (x10(3)/cm2) was also influenced by the severity of injury: it increased in the control group from 8.8+/-2.5 on intact endothelium to 226.6+/-45.5 and 397.4+/-61.3 on mildly and deeply injured arterial segments, respectively (p < 0.05). Again, 1 mg/kg Fucoidan had no effect, although doses of 5 mg/kg reduced neutrophil adhesion by 92% and by 84% on mildly and deeply injured segments, respectively. The effects of Fucoidan were associated with a 51% decrease in the vasoconstrictive response at the site of arterial injury. However, Fucoidan had no significant effect on either platelet aggregation or activated clotting time (ACT). In the in vitro perfusion experiments, Fucoidan inhibited both isolated platelet, and neutrophil, adhesion to damaged arterial surfaces. This inhibition was more pronounced in experiments using mixed cell preparations, indicating that Fucoidan interferes with platelet and neutrophil interactions. These results highlight the importance of selectins in the acute physiopathologic reactions related to platelet-neutrophil interactions after arterial injury.