Nanoparticle-induced platelet aggregation and vascular thrombosis.

Ever increasing use of engineered carbon nanoparticles in nanopharmacology for selective imaging, sensor or drug delivery systems has increased the potential for blood platelet-nanoparticle interactions. We studied the effects of engineered and combustion-derived carbon nanoparticles on human platelet aggregation in vitro and rat vascular thrombosis in vivo. Multiplewall (MWNT), singlewall (SWNT) nanotubes, C60 fullerenes (C60CS) and mixed carbon nanoparticles (MCN) (0.2-300 microg ml(-1)) were investigated. Nanoparticles were compared with standard urban particulate matter (SRM1648, average size 1.4 microm). Platelet function was studied using lumi aggregometry, phase-contrast, immunofluorescence and transmission electron microscopy, flow cytometry, zymography and pharmacological inhibitors of platelet aggregation. Vascular thrombosis was induced by ferric chloride and the rate of thrombosis was measured, in the presence of carbon particles, with an ultrasonic flow probe. Carbon particles, except C60CS, stimulated platelet aggregation (MCN>or=SWNT>MWNT>SRM1648) and accelerated the rate of vascular thrombosis in rat carotid arteries with a similar rank order of efficacy. All particles resulted in upregulation of GPIIb/IIIa in platelets. In contrast, particles differentially affected the release of platelet granules, as well as the activity of thromboxane-, ADP, matrix metalloproteinase- and protein kinase C-dependent pathways of aggregation. Furthermore, particle-induced aggregation was inhibited by prostacyclin and S-nitroso-glutathione, but not by aspirin. Thus, some carbon nanoparticles and microparticles have the ability to activate platelets and enhance vascular thrombosis. These observations are of importance for the pharmacological use of carbon nanoparticles and pathology of urban particulate matter.

Identification, regulation and role of tissue inhibitor of metalloproteinases-4 (TIMP-4) in human platelets.

1. Matrix metalloproteinase-2 (MMP-2) released during activation of human platelets by aggregating agents and cancer cells is known to stimulate platelet aggregation. 2. The expression, activity and role of tissue inhibitors of metalloproteinases (TIMPs), natural inhibitors of MMPs, in isolated human platelets were investigated. 3. Western blot, reverse zymography, immunogold electron microscopy, aggregometry (collagen-, thrombin and HT-1080 human fibrosarcoma cells-induced aggregation), flow cytometry and the release of (14)C-serotonin from labelled platelets recruited to the aggregate were used to characterize the presence and function of platelet TIMPs. 4. TIMP-4 (23 kDa) has been identified as the major MMP inhibitor (12-16 ng per 10(8) platelets) in human platelets. Platelets expressed lower (<1 ng per 10(8) platelets) amounts of TIMP-1. No other TIMPs were detected using Western blot analysis. 5. TIMP-4 co-localized with MMP-2 in resting platelets and was released upon platelet aggregation induced by collagen and thrombin. 6. Collagen resulted also in the release of higher molecular weight (60 kDa) complexes of TIMP-4. 7. The release of TIMP-4 was reduced by prostacyclin and S-nitroso-glutathione (GSNO), an NO donor. 8. Human recombinant TIMP-4 (rTIMP-4), but not human rTIMP-1, inhibited partially both platelet aggregation and recruitment. 9. The recombinant TIMP-4 potentiated the recruitment inhibitor effects of GSNO. 10. TIMP-4 was not released during platelet aggregation induced by HT-1080 cells. 11. Human rTIMP-4 exerted a biphasic effect on HT-1080 cells-induced aggregation. 12. Thus, TIMP-4 is the major intraplatelet MMP inhibitor and it is involved in regulation of platelet aggregation and recruitment.

Platelet-leukocyte aggregation induced by PAR agonists: regulation by nitric oxide and matrix metalloproteinases.

Platelet-leukocyte aggregation (PLA) links haemostasis to inflammation. The role of nitric oxide (NO) and matrix metalloproteinases (MMP-1, -2, -3, -9) in PLA regulation was studied. Homologous human platelet-leukocyte suspensions were stimulated with thrombin (0.1-3 nM) and other proteinase activated receptor-activating peptides (PAR-AP), including PAR1AP (0.5-10 microM), PAR4AP (10-70 microM), and thrombin receptor-activating peptide (1-35 microM). PLA was studied using light aggregometry with simultaneous measurement of oxygen-derived free radicals, dual colour flow cytometry, and phase-contrast microscopy. The release of NO was measured using a porphyrinic nanosensor, while MMPs were investigated by Western blot, substrate degradation assays, immunofluorescence microscopy, and flow cytometry. The levels of P-selectin and microparticles (MP) in PLA were measured by flow cytometry. PLA was also characterized using pharmacological agents: S-nitroso-glutathione (GSNO, 0.01-10 microM), 1H-Oxadiazole quinoxalin-1-one (ODQ, 1 microM), N(G)-L-nitro-L-arginine methyl ester (L-NAME, 100 microM) and compounds that modulate the actions of MMPs such as phenanthroline (100 microM), monoclonal anti-MMP antibodies, and purified MMPs. PAR agonists concentration-dependently induced PLA, an effect associated with the release of microparticles (MP) and the translocation of P-selectin to the platelet surface. NO and radicals were also released during PLA. Inhibition of NO bioactivity by the concomitant release of free radicals or by the treatment with L-NAME or ODQ stimulated PLA, while pharmacological administration of GSNO decreased PLA. PAR agonist-induced PLA resulted in the liberation of MMP-1, -2, -3, and -9. During PLA, MMPs were present on the cell surface, as shown by flow cytometry and immunofluorescence. PLA led to the activation of latent MMPs to active MMPs, as shown by Western blot and substrate degradation assays. Inhibition of MMPs actions by phenanthroline and by the antibodies attenuated PLA. In contrast, purified active, but not latent, MMPs amplified thrombin-induced PLA. It is concluded that NO and MMP-1, -2, -3, and -9 play an important role in regulation of PAR agonist-induced PLA.

Increased matrix metalloproteinase activity after canine cardiopulmonary bypass is suppressed by a nitric oxide scavenger.

OBJECTIVES: We tested whether nitric oxide scavenging with a ruthenium-based compound (AMD6221) would improve hemodynamics and alter nitric oxide synthase and matrix metalloproteinase activities in a canine model of cardiopulmonary bypass. METHODS: Dogs were randomized to either cardiopulmonary bypass (n = 12) or control (n = 12) groups. They were further randomized to receive a continuous infusion of AMD6221 or placebo. Cardiopulmonary bypass was maintained for 90 minutes, and then, 4 hours later, dogs were killed. Cardiac, lung, and brain sections were snap frozen in liquid nitrogen for determination of nitric oxide synthase, matrix metalloproteinase 2, and matrix metalloproteinase 9 activities. RESULTS: After cardiopulmonary bypass, 3 of 6 placebo-treated (cardiopulmonary bypass-placebo) and 0 of 6 AMD6221-treated (cardiopulmonary bypass-6221) animals required phenylephrine infusion to maintain a predetermined blood pressure (P <.05). Total fluid administration was lower in the cardiopulmonary bypass-6221 group compared with that in the cardiopulmonary bypass-placebo group (983 +/- 134 vs 1617 +/- 254 mL, respectively; P <.005). After cardiopulmonary bypass, matrix metalloproteinase 2 and matrix metalloproteinase 9 activities in the lung, left ventricle, and left atrium were decreased in the cardiopulmonary bypass-6221 group compared with that in the cardiopulmonary bypass-placebo group (P <.05). Ca(2+)-independent nitric oxide synthase activity and matrix metalloproteinase 2 activity in the brain were also lower (P <.05) in the cardiopulmonary bypass-SCV group. Finally, neutrophil expression of CD18, an adhesion complex, was lower at 4 hours after cardiopulmonary bypass in the cardiopulmonary bypass-6221 group compared with that in the cardiopulmonary bypass-placebo group (38 +/- 27 vs 81 +/- 11; P <.05). CONCLUSIONS: We found that (1) infusion of an nitric oxide scavenger, AMD6221, was associated with improved predefined hemodynamics; (2) cardiopulmonary bypass increased activities of Ca(2+)-independent nitric oxide synthase and matrix metalloproteinases in multiple organs; and (3) AMD6221 could ameliorate the increased generation of nitric oxide and increased matrix metalloproteinase activities.

Release of matrix metalloproteinase-9 during balloon angioplasty in patients with stable angina. A preliminary study.

BACKGROUND: Vascular wall remodeling is a major factor contributing to restenosis after angioplasty that involves migration and proliferation of vascular smooth muscle cells. The release of matrix-degrading metalloproteinases, including metalloproteinase-2 and metalloproteinase-9, facilitates remodeling. Experimental data suggest that nitric oxide (NO) decreases the activity of metalloproteinases and this may attenuate arterial remodeling after balloon injury. We investigated whether metalloproteinase-2, metalloproteinase-9 and NO are released into the coronary sinus blood during angioplasty in coronary patients. METHODS: In 10 patients with stable angina undergoing elective percutaneous transluminal coronary angioplasty of an isolated stenosis of the proximal left anterior descending coronary artery, blood was sampled from the coronary sinus at baseline, immediately and 1 min after each balloon deflation. Plasma release of metalloproteinase-2 and metalloproteinase-9 was assayed by their gelatinolytic activity using zymography, while the liberation of NO metabolites was measured by high-performance liquid chromatography. RESULTS: Two consecutive balloon inflations each of 60 s duration, resulted in an immediate increase (P<0.05) of metalloproteinase-9, but not metalloproteinase-2 activity, followed by normalization of metalloproteinase-9 levels to the baseline within 1 min. Plasma levels of NO metabolites remained unchanged. CONCLUSIONS: Rapid release of metalloproteinase-9 after balloon inflation may both contribute to remodeling and protect the vascular wall from post-angioplasty thrombosis.

Wii-habilitation as balance therapy for children with acquired brain injury.

PURPOSE: To evaluate the effectiveness of the Nintendo Wii compared to traditional balance therapy in improving balance, motivation, and functional ability in children undergoing acute rehabilitation after brain injury. METHODS: A non-concurrent, randomized multiple baseline single-subject research design was used with three participants. Data were analyzed by visual inspection of trend lines. RESULTS: Daily Wii balance training was equally motivating to traditional balance therapy for two participants and more motivating for one participant. While improvements in dynamic balance were observed, the results for static balance remain inconclusive. All participants demonstrated improvements in functional ability. CONCLUSION: Wii balance therapy is a safe, feasible, and motivating intervention for children undergoing acute rehabilitation after an acquired brain injury. Further research to examine the effectiveness of Wii balance therapy in this population is warranted.

A differential release of matrix metalloproteinases 9 and 2 during coronary artery bypass grafting and off-pump coronary artery bypass surgery.

OBJECTIVES: Extracorporeal circulation is associated with the systemic inflammatory response syndrome. The objective of this study was to measure plasma and myocardial matrix metalloproteinase 2 and 9 levels in patients undergoing off-pump coronary artery bypass and coronary artery bypass grafting. METHODS: Twenty patients subjected to coronary artery bypass grafting and 20 subjected to off-pump coronary artery bypass surgery were included in this study. In both procedures blood was collected in 7 equivalent time points up to 12 hours after grafting. The myocardial biopsy specimens were collected before and after extracorporeal circulation in the coronary artery bypass grafting group and after harvesting and completion of proximal anastomoses in the off-pump coronary artery bypass group. Matrix metalloproteinase levels were measured by means of zymography. Myeloperoxidase and tissue inhibitor of metalloproteinase 1 and 2 levels were measured with an enzyme-linked immunosorbent assay. RESULTS: Coronary artery bypass grafting but not off-pump coronary artery bypass led to a 700- to 900-fold increase of plasma matrix metalloproteinase 9 levels. A small but significant increase in matrix metalloproteinase 2 levels was detected in both procedures. Myocardial matrix metalloproteinase 9 levels significantly increased at the end of coronary artery bypass grafting and off-pump coronary artery bypass. Increased matrix metalloproteinase 9 activity at the end of extracorporeal circulation was accompanied by augmentation of the endogenous matrix metalloproteinase inhibitors tissue inhibitor of metalloproteinase 1 and 2 in plasma, but its magnitude was unable to balance the plasma matrix metalloproteinase 9 increase. The matrix metalloproteinase 9 content in plasma at the end of extracorporeal circulation correlated with the myeloperoxidase plasma concentration (r(2) = 0.8212, P < 0.05). CONCLUSION: We propose that release of matrix metalloproteinase 9 might contribute to the extracorporeal circulation-induced inflammatory reactions.

Platelet compatibility of PLGA, chitosan and PLGA-chitosan nanoparticles.

AIM: The increasing interest in biodegradable nanoparticles containing biomaterials such as poly(D,L-lactide-co-glycolide) (PLGA) and chitosan for drug delivery raises issues regarding the blood compatibility of these nanoparticles, since some nanoparticles, including carbon nanoparticles, can affect human platelet aggregation and cause vascular thrombosis. Therefore, the aim of this work was to investigate the effect of polymeric nanoparticles on human platelet function by measuring aggregation and receptor expression in vitro. MATERIALS & METHOD: PLGA, chitosan-PLGA and a series of chitosan nanoparticles were prepared by the single emulsion technique and ionotropic gelation method. The effects of these nanoparticles (0.01-100 microg/ml) on resting platelets, as well as on platelet aggregation and expression of receptors (GPIIb/IIIa and P-selectin) induced by agonists in platelet-rich plasma were examined using light aggregometry and flow cytometry. RESULTS & CONCLUSION: All tested nanoparticles at concentrations below 10 microg/ml did not modify platelet aggregation, showing that they may be used for the delivery of active molecules to the bloodstream.

Isosorbide-based aspirin prodrugs: integration of nitric oxide releasing groups.

Aspirin prodrugs and related nitric oxide releasing compounds hold significant therapeutic promise, but they are hard to design because aspirin esterification renders its acetate group very susceptible to plasma esterase mediated hydrolysis. Isosorbide-2-aspirinate-5-salicylate is a true aspirin prodrug in human blood because it can be effectively hydrolyzed to aspirin upon interaction with plasma BuChE. We show that the identity of the remote 5-ester dictates whether aspirin is among the products of plasma-mediated hydrolysis. By observing the requirements for aspirin release from an initial panel of isosorbide-based esters, we were able to introduce nitroxymethyl groups at the 5-position while maintaining ability to release aspirin. Several of these compounds are potent inhibitors of platelet aggregation. The design of these compounds will allow better exploration of cross-talk between COX inhibition and nitric oxide release and potentially lead to the development of selective COX-1 acetylating drugs without gastric toxicity.

Increased alveolar and plasma gelatinases activity during postpump syndrome: Inhibition by inhaled nitric oxide.

Postpump syndrome is associated with systemic inflammation. Matrix metalloproteinases (MMP)-2 and -9 contribute to proinflammatory and platelet-activator reactions. Nitric oxide (NO) is involved in the regulation of MMPs. The objectives of our study were to investigate the intensity of inflammation induced by 3 different surgical procedures, the effects of inflammation on the activity of MMPs, and the regulation of inflammation by inhaled NO (20 ppm). Inhaled NO was initiated immediately after tracheal intubation and maintained for the total duration of the experiments. Thirty pigs were equally randomized into 6 groups [sham; sham + NO; cardiopulmonary bypass; bypass + NO; bypass + lipopolysaccharide (1 microg/kg for 50 min); bypass + lipopolysaccharide + NO] and animals were subjected to anesthesia and mechanical ventilation up to 24 h. The levels of MMP-2 and MMP-9 in plasma and bronchoalveolar lavage were measured using zymography. Bypass resulted in a time-dependent rise in MMP activity, an effect potentiated by lipopolysaccharide. Inhaled NO attenuated the effects of bypass + lipopolysaccharide. These results confirm that MMP-2 and MMP-9 are associated with the inflammatory process causing the postpump syndrome. Preemptive and continuous administration of inhaled NO helps to prevent increased MMP-2 and MMP-9 activity.

Protein kinase C delta mediates platelet-induced breast cancer cell invasion.

Platelets play an important role in carcinogenesis, but the underlying molecular mechanisms remain poorly understood. To investigate the effects of platelets on in vitro invasion of MCF7 human breast cancer cells, human MCF7 cells were used to study their interactions with platelets using aggregometry and cell invasion chambers. Zymography and quantitative polymerase chain reaction (PCR) were used to study matrix metalloproteinases (MMPs), whereas Western blot was used to study protein kinase C (PKC) delta in MCF7 cells. We observed that platelets promoted invasion of MCF7 cells (3-fold increase, p<0.05, n=3) and that this process correlated with a dramatic increase in MMP-9 (8 fold-increase, p<0.001, n=3), which is known to facilitate cancer cell invasion. Because both platelets and MCF7 cells have been shown to release MMP-9, we investigated the cellular source that accounted for this increase. The time course and the use of specific protein synthesis inhibitors demonstrated that most of the increase in MMP-9 levels derived from de novo synthesis of this protease by cancer cells. Furthermore, platelets activated PKCdelta in MCF7 cells after 1 h of incubation (18.45+/-4.75% increase, p<0.05, n=4-7), which, in turn, led to an up-regulation of MMP-9 mRNA (from 60+/-20 to 1040+/-100 pg, p<0.001, n=3) and protein levels (18-fold increase, p<0.001, n=3), with the subsequent cell invasion-promoting effects. PKCdelta plays a crucial role in transducing the invasion-promoting effects of platelets in breast cancer cells, and the specific inhibition of PKCdelta may be a strategy to decrease platelet-mediated cancer cell invasion.

Increased activity and expression of matrix metalloproteinase-9 in a rat model of distal colitis.

Matrix metalloproteinases may play a role in tissue remodelling and destruction associated with inflammation. We investigated activity and expression of matrix metalloproteinases in a rat model of colitis and tested the therapeutic potential of a synthetic inhibitor (CGS-27023-A). Colitis was induced by dextran sulphate sodium (at 5% in drinking water for 5 days) in a group of eight rats, whereas a matched control group received plain water. Activity and expression of matrix metalloproteinases were measured in colonic tissue homogenates using zymography and Western blot on days 3 and 5 after induction of colitis. In another set of experiments, two groups of colitic rats (20 per group) were treated with CGS-27023-A (20 mg/kg) or vehicle, respectively. On days 5 and 14, colonic mucosal lesions were blindly scored by microscopic examination. Induction of colitis led to a significant upregulation of matrix metalloproteinase-9 protein and its activity, but no change in matrix metalloproteinase-2 activity was observed. Treatment with CGS-27023-A significantly decreased the extent and severity of epithelial injury but did not influence mucosal repair. We conclude that increased activity of matrix metalloproteinases may contribute to epithelial damage in this model of colitis.