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.

Reversal of rivaroxaban-induced alterations on hemostasis by different coagulation factor concentrates ­ in vitro studies with steady and circulating human blood.

BACKGROUND: Despite the good safety of rivaroxaban, there is limited information on strategies for urgent reversal of its antihemostatic effects. METHODS AND RESULTS: Alterations of hemostasis induced by rivaroxaban (230 ng/ml) were assessed by using several tests applied to steady and circulating human blood. Effects on thrombin generation (TG) and thromboelastometry (TEM) parameters were measured. Modifications in platelet adhesive, aggregating and procoagulant activities were evaluated in studies with circulating blood. The potential reversal of prothrombin complex concentrates (PCCs; 50 IU/kg), activated PCCs (aPCCs; 75 IU/kg), or recombinant factor VIIa (rFVIIa; 270 µg/kg) was evaluated. Impairment of TG parameters induced by rivaroxaban were corrected by the different concentrates (aPCC≥PCC>rFVIIa). Prolonged clotting times and reduced clot firmness caused by rivaroxaban on TEM tests were improved by different concentrates (rFVIIa≥aPCC>PCC). Rivaroxaban significantly reduced platelets and fibrin interactions with damaged vascular surfaces in perfusion studies. While alterations of platelet interactions were favourably counteracted by rFVIIa or aPCCs, reductions in fibrin formation were only partially restored by the different factor concentrates (rFVIIa>aPCC≥PCC). CONCLUSIONS: Rivaroxaban-induced alterations on coagulation parameters measured through assays performed under static conditions were easily reversed by the different concentrates. Studies under flow conditions revealed that these concentrates normalized the action of rivaroxaban on platelets, and significantly improved fibrin formation; although in the later case, levels were not restored to the pre-treatment value.

Automated preparation of whole blood-derived platelets suspended in two different platelet additive solutions and stored for 7 days.

BACKGROUND: The Atreus system (Terumo BCT) automates the preparation of blood components from whole blood donations. Intermediate platelet (PLT) products can be pooled manually or with the OrbiSac (Terumo BCT) and suspended in different PLT additive solutions (PASs) to obtain PLT concentrates (PCs). The aim of our study was to compare the in vitro PLT quality of PCs obtained with either the Atreus 2C+ and the OrbiSac or the Atreus 3C and suspended in PAS-II or PAS-IIIM during storage for up to 7 days. STUDY DESIGN AND METHODS: We prepared eight PCs from buffy coats obtained with Atreus 2C+, pooled with the OrbiSac, and suspended in PAS-II and eight PCs from interim PLT units obtained with the Atreus 3C and suspended either in PAS-II or in PAS-IIIM. We measured volume, PLT content, and mean PLT component and performed metabolic assays (pH, glucose, lactate, pO2, and pCO2) and flow cytometry analyses (GPIb, GPIIbIIIa, GPIV, CD62P, CD63, von Willebrand factor [vWF], fibrinogen, Factor V, and annexin V). RESULTS: PCs prepared with the Atreus 3C showed lower volume and higher PLT concentration when compared with PCs prepared with the Atreus 2C+ and the OrbiSac (p < 0.05). Glucose consumption rate and the expression of CD62P, CD63, and vWF were lower in PCs suspended in PAS-IIIM when compared with PCs suspended in PAS-II (p < 0.05). CONCLUSION: PCs prepared with the Atreus 3C and suspended in PAS-IIIM preserve satisfactorily the in vitro PLT quality during 7-day storage. PLT activation during a 7-day storage period was lower when the storage solution was PAS-IIIM in comparison with PAS-II.

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.

Redistribution and hemostatic action of recombinant activated factor VII associated with platelets.

Clinical evidence accumulated from hemophilic patients during prophylaxis with recombinant activated factor VII (rFVIIa) suggests that the duration of the hemostatic action of rFVIIa exceeds its predicted plasma half-life. Mechanisms involved in this outcome have not been elucidated. We have investigated in vitro the redistribution of rFVIIa in platelets from healthy donors, patients with FVII deficiency, and one patient with Bernard-Soulier syndrome. Platelet-rich plasma was exposed to rFVIIa (3 to 60 µg/mL). Flow cytometry, immunocytochemistry, and coagulation tests were applied to detect and quantify rFVIIa. The hemostatic effect of rFVIIa associated to platelets was evaluated using perfusion models. Our studies revealed a dose-dependent association of rFVIIa to the platelet cytoplasm with redistribution into the open canalicular system, and α granules. Mechanisms implicated in the internalization are multiple, involve GPIb and GPIV, and require phospholipids and cytoskeletal assembly. After platelet activation with thrombin, platelets exposed rFVIIa on their membrane. Perfusion studies revealed that the presence of 30% of platelets containing FVIIa improved platelet aggregate formation and enhanced fibrin generation (P < 0.01 versus control). Our results indicate that, at therapeutic concentrations, rFVIIa can be internalized into platelets, where it is protected from physiological clearance mechanisms and can still promote hemostatic activity. Redistribution of rFVIIa into platelets may explain the prolonged prophylactic effectiveness of rFVIIa in hemophilia.

Impact of pathogen reduction technology and storage in platelet additive solutions on platelet function.

BACKGROUND: There are limited studies on the effect of pathogen reduction technology (PRT) on platelet (PLT) products stored in PLT additive solutions (PASs). Our study has evaluated the effect of Mirasol PRT (CaridianBCT) followed by storage in PASs (PAS-III or PAS-IIIM) on analytical and functional characteristics of PLT concentrates (PCs). STUDY DESIGN AND METHODS: PCs obtained by apheresis were divided into: 1) untreated and stored in plasma (CON-PPP); 2) untreated and stored in PAS (CON-PASIII and CON-PASIIIM); and 3) subjected to PRT and stored in PAS (PRT-PASIII and PRT-PASIIIM). Cell quality variables (pH, swirl, lactate, and glucose), flow cytometry analysis, and adhesive and aggregating functions using a perfusion system were assessed on Days 0, 3, 5, and 7. RESULTS: All the groups showed a progressive reduction in cell quality variables during storage. Swirl was progressively reduced and better maintained in CON-PPP. Flow cytometry analysis revealed a moderate reduction in glycoprotein Ib for all study groups on Day 7, being less evident in CON-PPP. A progressive increase in P-selectin levels was observed during storage. Perfusion studies demonstrated that PRT-treated PCs stored in PAS presented similar reactivity with subendothelium than that observed for untreated PCs stored in plasma or in PAS for Day 5. Moderate reductions in cohesive properties in PRT-PASIII PCs, but not in PRT-PASIIIM PCs, were observed after 7 days. CONCLUSIONS: PLT properties were preserved in PRT-treated concentrates stored in PAS for 5 days. PASIIIM provides a better preservation of adhesive and cohesive functions of PRT-PCs than PASIII for 7 days of storage. The relevance of in vitro data deserves further clinical investigations.

Serotonergic mechanisms enhance platelet-mediated thrombogenicity.

Although it is generally acknowledged that serotonin (5-HT) is a weak agonist for human platelets, recent information suggests an association between serotonergic mechanisms and cardiovascular risk. We investigated the action of 5-HT on adhesive, cohesive and procoagulant properties of human platelets. Impact of 5-HT on whole blood coagulation and thrombin generation was measured by modified thromboelastometry (TEM) and specific fluorogenic assays. We evaluated the effects of 5-HT on thrombus formation in an in-vitro model of thrombosis using human flowing blood. In platelet-rich plasma (PRP), 5-HT favoured the expression of CD62-P, and procoagulant molecules on platelet membranes. These effects were potentiated in the presence of Ca(++) and/or ADP. Incubation with 5-HT accelerated clotting times and augmented clot strength in whole blood TEM, and enhanced thrombin generation in PRP. In perfusion studies, 5-HT significantly increased fibrin deposition at low shear (300s(-1)) and enhanced platelet thrombus formation on the damaged vascular surface at high shear (1,200s(-1)). Selective inhibition of serotonin reuptake (SSRI) attenuated effects of 5-HT on platelet activation and downregulated the prothrombotic tendencies observed in the previous experimental conditions. In general, reductions of thrombogenic patterns observed with SSRI were more evident under shear conditions (aggregation and perfusion systems) and less evident under steady conditions (TEM and thrombin generation assays). In conclusion, 5-HT is not a weak agonist for human platelets; instead it accentuates platelet activation, potentiates procoagulant responses on human blood and increases thrombogenesis on damaged vascular surfaces. The remarkable antithrombotic actions achieved through SSRI deserve further mechanistic and clinical investigations.

Internalization of tissue factor by platelets.

Tissue factor (TF) has been found associated with platelets. Mechanisms responsible for TF-platelet interactions and transport are not fully understood. We explored the response of isolated washed platelets to preparations of recombinant (rTF) or placental (pTF) human TF, exposed on lipid microvesicles (MVs). Sequential ultrastructural and immunocytochemical studies revealed trafficking of these TF preparations, being endocytosed by platelets into channels of the open canalicular system (OCS) and accumulating in the cytoplasm and occasionally in alpha-granules. The process of internalization of TF-MVs was accomplished in less than 30 min, being faster for the placental (pTF) than for the recombinant (rTF) preparation. Signs of mild platelet activation with pseudopodia formation were observed at early stages of internalization. Platelets returned to an apparent resting state after 5-10 min. All of these observations paralleled with modifications on patterns of tyrosine phosphorylation for several signaling proteins. Our studies demonstrate that platelets possess mechanisms to capture and incorporate TF-rich vesicles. These processes were accelerated by the presence of other contaminating cellular antigens in the vesicles (pTF). TF carried by platelets could play a potential role in platelet thrombus formation and by extension in the development of ischemic complications.

Tissue factor-enriched vesicles are taken up by platelets and induce platelet aggregation in the presence of factor VIIa.

We investigated the interactions of vesicles containing human tissue factor (TF) with platelets and evaluated responses induced by rFVIIa using standard aggregometry, ultrastructural and flow-cytometry techniques. Washed platelets were exposed to a preparation of placental human TF (pTF) or to a relipidated formulation of recombinant human TF (rTF). Under stirring conditions, pTF induced reversible aggregation with platelets returning to their resting state after 5 minutes. This reversible response to pTF was partially inhibited by antibodies against CD62-P, but not by antithrombin agents, and was not observed with rTF. Sequential ultrastructural studies revealed uptake of both TF preparations by platelets involving traffic of vesicles through channels of the open canalicular system (OCS). Immunocytochemical studies on cryosections identified TF in the OCS, and occasionally in the alpha-granules of the platelets. These processes were faster with pTF than with rTF, but both TF preparations accumulated in platelets at the end of incubation periods. Flow cytometry studies revealed the presence of other cellular antigens (CD62-P, CD14 and CD45) associated to the pTF. Addition of rFVIIa to washed platelets exposed to pTF or rTF, caused a thrombin dependent irreversible platelet aggregation. Our studies demonstrate that platelets possess mechanisms to capture and incorporate TF-rich vesicles. These processes are accelerated by the presence of other cellular antigens in the vesicles. Our findings may explain the hemostatic action of rFVIIa in severely hemodiluted patients, but are also relevant for the understanding of potential implications of TF-associated to platelets in the propagation of thrombus.

Coagulation Factor Concentrates Fail to Restore Alterations in Fibrin Formation Caused by Rivaroxaban or Dabigatran in Studies With Flowing Blood From Treated Healthy Volunteers.

We evaluated the hemostatic alterations in blood from healthy individuals treated for 5 days with direct oral anticoagulants (DOACs) rivaroxaban (20 mg/d) or dabigatran (150 mg/12 h) in a single-blind clinical trial with crossover assignment (NCT01478282). We assessed the potential of prothrombin complex concentrates, activated prothrombin complex concentrates, or recombinant activated factor VII, when added ex vivo, to reverse the alterations caused by these DOACs. Blood was drawn at maximum plasma concentration after the last dose of each DOAC, and modifications in coagulation biomarkers were evaluated using a series of tests performed under steady conditions including routine coagulation, thrombin generation, and thromboelastometry assays. Additional studies in standardized flow devices were applied to evaluate alterations on platelet deposition and fibrin formation on damaged vascular surfaces exposed to flowing blood. Both DOACs caused important modifications of all coagulation biomarkers and significantly reduced fibrin formation in flow studies. Alterations in biomarkers observed in steady laboratory tests were normalized and occasionally overcompensated by procoagulant strategies. In contrast, reductions in fibrin formation observed in studies with flowing blood were improved, although never completely restored to baseline levels. Effects of dabigatran in flow studies appeared more resistant to reversal strategies than those of rivaroxaban. Inconsistencies between results of coagulation studies in steady or flowing assays not only raise concerns about the adequacy of the earlier tests to predict the restoration of the coagulopathy induced by DOACs but also suggest limitations of nonspecific procoagulant strategies to control severe coagulopathy in patients inadvertently overexposed these agents.

Platelet membrane fragments enhance the procoagulant effect of recombinant factor VIIa in studies with circulating human blood under conditions of experimental thrombocytopenia.

The mechanism of action of recombinant factor VIIa (rFVIIa), which is being considered as an alternative treatment for the control of bleeding episodes in patients with thrombocytopenia, has not been fully characterized. This study was undertaken to explore the effects of rFVIIa and platelet microvesicles on hemostasis in an experimental model of thrombocytopenia. Damaged arterial segments were exposed to thrombocytopenic blood (shear rate 600 s(-1)) either with or without the addition of rFVIIa and/or platelet microvesicles. The presence of fibrin and platelets on the subendothelium were morphometrically quantified and immunolocalization techniques and electron microscopy were used for a more detailed analysis. Both rFVIIa and platelet microvesicles consistently improved fibrin formation on the damaged vascular subendothelium, and microvesicles were shown to be localized at different levels of the fibrin lattice. Further, under conditions of moderate thrombocytopenia, addition of platelet microvesicles potentiated the procoagulant action of rFVIIa. This effect may be due to the phospholipid surface provided by the platelet microvesicles. These studies support the concept that, under conditions of thrombocytopenia, both rFVIIa and platelet microvesicles enhance fibrin formation at sites of vascular damage.

The pharmacokinetics of edoxaban for the prevention and treatment of venous thromboembolism.

INTRODUCTION: Thromboembolic diseases will become the most important contributors to mortality and morbidity for modern societies. Current antithrombotic strategies using heparins or vitamin K antagonists are inconvenient, with limitations and inherent side effects. A series of new oral anticoagulants with powerful and reliable antithrombotic actions have been developed in the last decade. AREAS COVERED: Edoxaban is a direct and specific inhibitor of activated factor X, delivered orally. This article reviews literature from PubMed and articles referenced within. The text explores the pharmacological aspects of its antithrombotic action. Pharmacokinetics, metabolism and drug interactions are examined. The review places the results of recent clinical trials that have evaluated the antithrombotic potential of edoxaban versus standard antithrombotic therapies in the prophylaxis and treatment of venous thromboembolism into perspective. The possible relationship between the pharmacokinetic profile of edoxaban and the favorable results in clinical trials is discussed. EXPERT OPINION: Edoxaban is perceived as a major advance, compared to vitamin K antagonists, in the prevention and treatment of thromboembolic disease given its favorable efficacy, safety, pharmacokinetic profile and renal clearance. The results of ongoing large international trials exploring the prevention of thrombotic complications in patients in different clinical settings should ensure the approval of edoxaban to treat new indications.

Prothrombotic platelet phenotype in major depression: downregulation by antidepressant treatment.

BACKGROUND: Serotonergic mechanisms have been suggested as a link between major depression and cardiovascular risk. We investigated the existence of a prothrombotic condition in depressed patients and its possible modulation during treatment with a selective serotonin-reuptake inhibitor (SSRI). METHODS: Modifications in a series of biomarkers of platelet and coagulation activation were evaluated in blood from 19 patients with a major depression disorder (MDD) at the time of diagnosis, and at 8 and 24 weeks of treatment with escitalopram. Response of blood aliquots recirculated through a thrombogenic surface was assessed in a thrombosis model. Results were compared with those of 20 healthy-matched controls. RESULTS: In comparison with controls, platelets from MDD patients showed elevated volumes (p<0.01), significantly enhanced aggregating response to arachidonic acid and augmented expression of GPIb, fibrinogen, factor V, and anionic phospholipids by flow cytometry (p<0.05). Clot firmness and procoagulant activity of platelet-associated tissue factor were also significantly elevated (p<0.05). Studies with circulating blood revealed increased fibrin formation in early diagnosed patients (71.1±9.5% vs. 45.8±5.3%; p<0.05 vs. controls). After 24 weeks of treatment with escitalopram, the majority of the alterations observed were normalized, except for a residual increased expression of GPIIbIIIa (p<0.05) and persistent alterations in thromboelatometic parameters. LIMITATIONS: Despite the reduced number of followed-up patients our findings were consistent reaching statistical significance. CONCLUSIONS: Our results reveal a prothrombotic phenotype in MDD patients. While continuous treatment with an SSRI downregulated the majority of the biomarkers analyzed, alterations in viscoelastic parameters of clot formation remained unaffected by the antidepressant treatment.

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.

Liposomes bearing fibrinogen could potentially interfere with platelet interaction and procoagulant activity.

BACKGROUND: The contribution of fibrinogen (FBN) to hemostasis acting on platelet aggregation and clot formation is well established. It has been suggested that FBN-coated liposomes could be useful in restoring hemostasis. In the present study, we evaluated the modifications induced by multilamellar raw liposomes (MLV) or fibrinogen-coated liposomes (MLV-FBN) on hemostatic parameters. MATERIALS AND METHODS: Different experimental settings using whole blood or thrombocytopenic blood were used. Thromboelastometry, aggregation studies, platelet function analyzer (PFA-100(®)) tests and studies under flow conditions were applied to detect the effect of MLV-FBN on hemostatic parameters. RESULTS: The presence of MLV-FBN in whole blood modified its viscoelastic properties, prolonging clot formation time (CFT) (226.5 ± 26.1 mm versus 124.1 ± 9.4 mm; P < 0.01) but reducing clot firmness (45.4 ± 1.8 mm versus 35.5 ± 2.3 mm; P < 0.05). Under thrombocytopenic conditions, FIBTEM analysis revealed that MLV-FBN shortened clotting time (CT) compared to MLV (153.3 ± 2.8 s versus 128.0 ± 4.6 s; P < 0.05). Addition of either liposome decreased fibrin formation on the subendothelium (MLV 8.1% ± 4.7% and MLV-FBN 0.8% ± 0.5% versus control 36.4% ± 6.7%; P < 0.01), whereas only MLV-FBN significantly reduced fibrin deposition in thrombocytopenic blood (14.4% ± 6.3% versus control 34.5% ± 5.2%; P < 0.05). MLV-FBN inhibited aggregation induced by arachidonic acid (52.1% ± 8.1% versus 88.0% ± 2.1% in control; P < 0.01) and ristocetin (40.3% ± 8.8% versus 94.3% ± 1.1%; P < 0.005), but it did not modify closure times in PFA-100(®) studies. In perfusion experiments using whole blood, MLV and MLV-FBN decreased the covered surface (13.25% ± 2.4% and 9.85% ± 2.41%, respectively, versus control 22.0% ± 2.0%; P < 0.01) and the percentage of large aggregates (8.4% ± 2.3% and 3.3% ± 1.01%, respectively, versus control 14.6% ± 1.8%; P < 0.01). CONCLUSION: Our results reveal that, in addition to the main contribution of fibrinogen to hemostasis, MLV-FBN inhibits platelet-mediated hemostasis and coagulation mechanisms.

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.

Serotonin enhances platelet procoagulant properties and their activation induced during platelet tissue factor uptake.

AIMS: Circulating tissue factor (TF) has been linked to thrombus propagation. Our group demonstrated that platelets possess mechanisms to capture TF-rich microvesicles (TF-MVs). Serotonin facilitates the development of platelets with increased procoagulant activity. An enhanced platelet serotonin uptake has been identified with increased cardiovascular risk. We have investigated the involvement of serotonergic mechanisms facilitating the interaction of human platelets with TF-MVs. Inhibitory strategies aimed at blocking serotonin and coagulation mechanisms were also studied. METHODS AND RESULTS: Standard aggregometry, flow cytometry, electron microscopy, and thrombin generation assays were performed. TF-MVs induced platelet aggregation in heparinized platelet-rich plasma (PRP) samples; this aggregation was further accelerated by serotonin. In washed platelets, serotonin enhanced platelet aggregation to TF-MVs with a maximum peak of 55.9 +/- 1.8 vs. 48.7 +/- 2.1% (P < 0.05). Inhibitory strategies with a selective serotonin re-uptake inhibitor and with lepirudin decreased these aggregations. Ultrastructural analysis revealed that serotonin induced platelet pseudopodia formation, thus facilitating the engulfment of TF-MVs. In general, serotonin significantly enhanced (P < 0.05) thrombin generation and the expression of activation markers and procoagulant activity in platelets measured for TF-MVs alone. CONCLUSION: Serotonin enhances the interaction of platelets with TF-MVs, increases platelet activation, and potentiates their overall procoagulant activity. The present results could have significant implications in thrombus formation and in the thrombogenic profile of pathological situations with increased cardiovascular risk.