Fibrinogen nitrotyrosination after ischemic stroke impairs thrombolysis and promotes neuronal death.

Ischemic stroke is an acute vascular event that compromises neuronal viability, and identification of the pathophysiological mechanisms is critical for its correct management. Ischemia produces increased nitric oxide synthesis to recover blood flow but also induces a free radical burst. Nitric oxide and superoxide anion react to generate peroxynitrite that nitrates tyrosines. We found that fibrinogen nitrotyrosination was detected in plasma after the initiation of ischemic stroke in human patients. Electron microscopy and protein intrinsic fluorescence showed that in vitro nitrotyrosination of fibrinogen affected its structure. Thromboelastography showed that initially fibrinogen nitrotyrosination retarded clot formation but later made the clot more resistant to fibrinolysis. This result was independent of any effect on thrombin production. Immunofluorescence analysis of affected human brain areas also showed that both fibrinogen and nitrotyrosinated fibrinogen spread into the brain parenchyma after ischemic stroke. Therefore, we assayed the toxicity of fibrinogen and nitrotyrosinated fibrinogen in a human neuroblastoma cell line. For that purpose we measured the activity of caspase-3, a key enzyme in the apoptotic pathway, and cell survival. We found that nitrotyrosinated fibrinogen induced higher activation of caspase 3. Accordingly, cell survival assays showed a more neurotoxic effect of nitrotyrosinated fibrinogen at all concentrations tested. In summary, nitrotyrosinated fibrinogen would be of pathophysiological interest in ischemic stroke due to both its impact on hemostasis - it impairs thrombolysis, the main target in stroke treatments - and its neurotoxicity that would contribute to the death of the brain tissue surrounding the infarcted area.

Internalization of Tissue Factor-Rich Microvesicles by Platelets Occurs Independently of GPIIb-IIIa, and Involves CD36 Receptor, Serotonin Transporter and Cytoskeletal Assembly.

Platelets are important in hemostasis, but also detect particles and pathogens in the circulation. Phagocytic and endocytic activities of platelets are widely recognized; however, receptors and mechanisms involved remain poorly understood. We previously demonstrated that platelets internalize and store phospholipid microvesicles enriched in human tissue factor (TF+MVs) and that platelet-associated TF enhances thrombus formation at sites of vascular damage. Here, we investigate the mechanisms implied in the interactions of TF+MVs with platelets and the effects of specific inhibitory strategies. Aggregometry and electron microscopy were used to assess platelet activation and TF+MVs uptake. Cytoskeletal assembly and activation of phosphoinositide 3-kinase (PI3K) and RhoA were analyzed by western blot and ELISA. Exposure of platelets to TF+MVs caused reversible platelet aggregation, actin polymerization and association of contractile proteins to the cytoskeleton being maximal at 1 min. The same kinetics were observed for activation of PI3K and translocation of RhoA to the cytoskeleton. Inhibitory strategies to block glycoprotein IIb-IIIa (GPIIb-IIIa), scavenger receptor CD36, serotonin transporter (SERT) and PI3K, fully prevented platelet aggregation by TF+MVs. Ultrastructural techniques revealed that uptake of TF+MVs was efficiently prevented by anti-CD36 and SERT inhibitor, but only moderately interfered by GPIIb-IIIa blockade. We conclude that internalization of TF+MVs by platelets occurs independently of receptors related to their main hemostatic function (GPIIb-IIIa), involves the scavenger receptor CD36, SERT and engages PI3-Kinase activation and cytoskeletal assembly. CD36 and SERT appear as potential therapeutic targets to interfere with the association of TF+MVs with platelets and possibly downregulate their prothrombotic phenotype.

TLR4 and NALP3 inflammasome in the development of endothelial dysfunction in uraemia.

BACKGROUND: The increased cardiovascular risk present in chronic kidney disease (CKD) is related to the development of endothelial dysfunction, whose mechanisms are still unclear. Accumulation of toxins and proinflammatory cytokines may constitute danger-associated molecular patterns (DAMP) to which endothelial cells are continuously exposed. Potential involvement of mechanisms recognizing DAMP, such as TLR and inflammasomes, has been explored. MATERIALS AND METHODS: Endothelial cells in culture were exposed to sera samples collected from patients with CKD: (i) stages 4-5 not on dialysis (PreD), (ii) on maintenance haemodialysis (HD) and (iii) peritoneal dialysis (PD). Changes in TLR4 and ICAM-1 expression, reactive oxygen species (ROS) production and TLR4 signalling were explored. Assembly of NALP3 inflammasome components was also investigated. RESULTS: TLR4 was expressed at the cell surface and increased significantly in response to PreD, HD and PD sera, paralleling with the activation of the cell stress protein Akt and the inflammation-related transcription factor NFκB, with elevated surface ICAM-1 expression and ROS production. TLR4 blockade partially decreased these effects. Exposure of cells to uraemic sera induced assembly of NALP3 components, with caspase-1 activation, especially in response to HD and PD sera. CONCLUSIONS: TLR4 and NALP3 inflammasomes, crucial elements of innate immunity, contribute to the development and perpetuation of endothelial dysfunction in response to the uraemic toxicity. These mechanisms constitute potential therapeutic targets to improve endothelial dysfunction and to reduce the increased cardiovascular risk in CKD.

Quantitative and qualitative analysis of coagulation factors in cryoprecipitate prepared from fresh-frozen plasma inactivated with amotosalen and ultraviolet A light.

BACKGROUND: There were no previous studies about the quality of cryoprecipitate prepared from fresh-frozen plasma (FFP) inactivated with amotosalen and ultraviolet A (UVA) light. The aim of this study was to analyze the quantity and quality of coagulation factors in cryoprecipitate prepared from FFP treated with amotosalen and UVA light. STUDY DESIGN AND METHODS: FFP was obtained from whole blood donations and inactivated with amotosalen and UVA light according to the manufacturer's instructions. Fibrinogen, factor VIII (FVIII), von Willebrand factor antigen (VWF:Ag) and activity (VWF:RCo), the von Willebrand factor cleavage protease activity (ADAMTS-13), and the multimeric structure of VWF were analyzed. RESULTS: The content of fibrinogen, FVIII, and ADAMTS-13 was lower in cryoprecipitates prepared from amotosalen-treated plasma when compared with cryoprecipitates prepared from nontreated plasma (35, 40, and 18% loss, respectively). The quantity and quality of VWF as well as VWF multimer patterns were not affected by the inactivation method. CONCLUSION: Cryoprecipitates prepared from amotosalen-treated FFP contained significantly reduced levels of fibrinogen, FVIII, and ADAMTS-13. However, the VWF quantity and quality was well preserved.

Role of sodium tungstate as a potential antiplatelet agent.

PURPOSE: Platelet inhibition is a key strategy in the management of atherothrombosis. However, the large variability in response to current strategies leads to the search for alternative inhibitors. The antiplatelet effect of the inorganic salt sodium tungstate (Na2O4W), a protein tyrosine phosphatase 1B (PTP1B) inhibitor, has been investigated in this study. METHODS: Wild-type (WT) and PTP1B knockout (PTP1B(-/-)) mice were treated for 1 week with Na2O4W to study platelet function with the platelet function analyzer PFA-100, a cone-and-plate analyzer, a flat perfusion chamber, and thrombus formation in vivo. Human blood aliquots were incubated with Na2O4W for 1 hour to measure platelet function using the PFA-100 and the annular perfusion chamber. Aggregometry and thromboelastometry were also performed. RESULTS: In WT mice, Na2O4W treatment prolonged closure times in the PFA-100 and decreased the surface covered (%SC) by platelets on collagen. Thrombi formed in a thrombosis mice model were smaller in animals treated with Na2O4W (4.6±0.7 mg vs 8.9±0.7 mg; P<0.001). Results with Na2O4W were similar to those in untreated PTP1B(-)/(-) mice (5.0±0.3 mg). Treatment of the PTP1B(-)/(-) mice with Na2O4W modified only slightly this response. In human blood, a dose-dependent effect was observed. At 200 µM, closure times in the PFA-100 were prolonged. On denuded vessels, %SC and thrombi formation (%T) decreased with Na2O4W. Neither the aggregating response nor the viscoelastic clot properties were affected. CONCLUSION: Na2O4W decreases consistently the hemostatic capacity of platelets, inhibiting their adhesive and cohesive properties under flow conditions in mice and in human blood, resulting in smaller thrombi. Although Na2O4W may be acting on platelet PTP1B, other potential targets should not be disregarded.

Impact of experimental haemodilution on platelet function, thrombin generation and clot firmness: effects of different coagulation factor concentrates.

BACKGROUND: Haemodilution during resuscitation after massive haemorrhage may worsen the coagulopathy and perpetuate bleeding. MATERIALS AND METHODS: Blood samples from healthy donors were diluted (30 and-60%) using crystalloids (saline, Ringer's lactate, Plasmalyte(TM)) or colloids (6% hydroxyethylstarch [HES130/0.4], 5% human albumin, and gelatin). The effects of haemodilution on platelet adhesion (Impact R), thrombin generation (TG), and thromboelastometry (TEM) parameters were analysed as were the effects of fibrinogen, prothrombin complex concentrates (PCC), activated recombinant factor VII (FVIIa), and cryoprecipates on haemodilution. RESULTS: Platelet interactions was already significantly reduced at 30% haemodilution. Platelet reactivity was not improved by addition of any of the concentrates tested. A decrease in TG and marked alterations of TEM parameters were noted at 60% haemodilution. HES130/0.4 was the expander with the most deleterious action. TG was significantly enhanced by PCC whereas rFVIIa only caused a mild acceleration of TG initiation. Fibrinogen restored the alterations of TEM parameters caused by haemodilution including those caused by HES 130/0.4. Cryoprecipitates significantly improved the alterations caused by haemodilution on TG and TEM parameters; the effects on TG disappeared after ultracentrifugation of the cryoprecipitates. DISCUSSION: The haemostatic alterations caused by haemodilution are multifactorial and affect both blood cells and coagulation. In our in vitro approach, HES 130/0.4 had the most deleterious effect on haemostasis parameters. Coagulation factor concentrates did not improve platelet interactions in the Impact R, but did have favourable effects on coagulation parameters measured by TG and TEM. Fibrinogen notably improved TEM parameters without increasing thrombin generation, suggesting that this concentrate may help to preserve blood clotting abilities during haemodilution without enhancing the prothrombotic risk.

Nitro-oxidative stress after neuronal ischemia induces protein nitrotyrosination and cell death.

Ischemic stroke is an acute vascular event that obstructs blood supply to the brain, producing irreversible damage that affects neurons but also glial and brain vessel cells. Immediately after the stroke, the ischemic tissue produces nitric oxide (NO) to recover blood perfusion but also produces superoxide anion. These compounds interact, producing peroxynitrite, which irreversibly nitrates protein tyrosines. The present study measured NO production in a human neuroblastoma (SH-SY5Y), a murine glial (BV2), a human endothelial cell line (HUVEC), and in primary cultures of human cerebral myocytes (HC-VSMCs) after experimental ischemia in vitro. Neuronal, endothelial, and inducible NO synthase (NOS) expression was also studied up to 24 h after ischemia, showing a different time course depending on the NOS type and the cells studied. Finally, we carried out cell viability experiments on SH-SY5Y cells with H2O2, a prooxidant agent, and with a NO donor to mimic ischemic conditions. We found that both compounds were highly toxic when they interacted, producing peroxynitrite. We obtained similar results when all cells were challenged with peroxynitrite. Our data suggest that peroxynitrite induces cell death and is a very harmful agent in brain ischemia.

NFκB in the development of endothelial activation and damage in uremia: an in vitro approach.

Impaired hemostasis coexists with accelerated atherosclerosis in patients with chronic kidney disease (CKD). The elevated frequency of atherothrombotic events has been associated with endothelial dysfunction. The relative contribution of the uremic state and the impact of the renal replacement therapies have been often disregarded. Plasma markers of endothelial activation and damage were evaluated in three groups of patients with CKD: under conservative treatment (predialysis), on hemodialysis, and on peritoneal dialysis. Activation of p38 MAPK and the transcription factor NFκB was assessed in endothelial cell (EC) cultures exposed to pooled sera from each group of patients. Most of the markers evaluated (VCAM-1, ICAM-1, VWF, circulating endothelial cells) were significantly higher in CDK patients than in controls, being significantly more increased in the group of peritoneal dialysis patients. These results correlated with the activation of both p38 MAPK and NFκB in EC cells exposed to the same sera samples, and also to the peritoneal dialysis fluids. Hemodialysis did not further contribute to the endothelial damage induced by the uremic state observed in predialysis patients, probably due to the improved biocompatibility of the hemodialysis technique in recent years, resulting in lower cellular activation. However, peritoneal dialysis seemed to exert a significant proinflammatory effect on the endothelium that could be related to the high glucose concentrations and glucose degradation products present in the dialysis fluid. Although peritoneal dialysis has been traditionally considered a more physiological technique, our results raise some doubts with respect to inflammation and EC damage.

Platelet-associated tissue factor enhances platelet reactivity and thrombin generation in experimental studies in vitro.

INTRODUCTION: The thrombogenic potential of tissue factor (TF) associated to platelets is controversial. We have investigated the in vitro contribution of platelet-associated TF to thrombus formation. MATERIALS AND METHODS: Platelets suspensions were exposed to human TF-rich microvesicles (TF-MV) from placental or recombinant origin. Platelet-associated TF was quantified through coagulometric assays. Adhesive and cohesive properties of platelets containing TF were assessed in perfusion models using two thrombogenic surfaces: 1) type-I collagen, or 2) damaged vascular segments. Perfusion studies were performed with heparinized blood enriched with a 30% of washed platelets exposed to TF-MV vs. washed control platelets. Thrombin generation and thromboelastometric properties of clots were also assessed using a fluorometric assay and ROTEM analysis, respectively. Inhibitory strategies with an antibody to TF were performed in some cases. RESULTS: The addition of 30% of platelets containing TF to blood perfusates resulted in a statistically significant increase in the platelet coverage (%CS) vs. non-exposed platelets on collagen surfaces (%CS: 19.7 ± 0.6 and 23.9 ± 0.7 respectively, vs.14.5 ± 1.4; p<0.01) and on the vascular subendothelium (%CS: 54.0 ± 1.5 and 47.2 ± 6.8 respectively vs. 38.0 ± 3.5, p<0.05), with a statistically significant increase in the size of large platelet aggregates (p<0.05) vs. control platelets. These effects on collagen surfaces were almost totally prevented by an antibody to TF. Platelet-associated TF significantly accelerated thrombin generation and clot formation (p<0.05), effects that were partially prevented by a neutralizing anti-TF. CONCLUSIONS: Platelet-associated TF potentiated adhesive and aggregating properties in in vitro studies with flowing blood and accelerated thrombin generation and clot formation time under steady conditions.