Idarucizumab, but not procoagulant concentrates, fully restores dabigatran-altered platelet and fibrin components of hemostasis.

BACKGROUND: Comparative studies on the restoration of hemostasis with different reversal agents after dabigatran therapy have not been performed. We compared the efficacy and prothrombotic potential of the specific antidote idarucizumab with that of previously recommended non-specific procoagulant concentrates. STUDY DESIGN AND METHODS: We explored the in vitro effects of dabigatran (184 ng/mL) on fibrin and platelet-aggregate formation onto a damaged vessel under flow conditions (600 s-1 ). The reversal mechanisms and efficacy of idarucizumab (0.3-3 mg/mL) were compared with that of the non-specific procoagulant concentrates aPCC (25-75 U/Kg), PCC (70 U/Kg), or rFVIIa (120 µg/Kg). Generation of thrombin and prothrombin fragment (F1 + 2), and thromboelastometry parameters of clot formation were measured. RESULTS: Dabigatran caused pronounced reductions in fibrin (87%) and platelet interactions (36%) with damaged vessels (p < 0.01) and significantly impaired thrombin generation and thromboelastometric parameters (delayed dynamics and reduced firmness). Idarucizumab completely normalized rates of fibrin and platelet coverage to baseline values in flow studies; and reversed the alterations in thrombin generation, F1 + 2 and thromboelastometry parameters produced by dabigatran. In comparison, aPCC and PCC only partially compensated for the dabigatran-induced alterations in fibrin deposition, but were unable to fully restore them to baseline values. Reversal with aPCC or PCC improved the majority of alterations in coagulation-related tests, but tended to overcompensate thrombin generation kinetics and significantly increased F1 + 2 levels. CONCLUSION: Idarucizumab antagonizes alterations of direct and indirect biomarkers of hemostasis caused by dabigatran. In our studies, idarucizumab was clearly more efficacious than strategies with non-specific procoagulant concentrates and devoid of the excessive procoagulant tendency observed with the latter.

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.

In vitro evaluation of the hemostatic effectiveness of cryopreserved platelets.

BACKGROUND: Cryopreserved platelet (CPP) concentrates exhibit a variety of morphologic and functional alterations that may affect the action of CPP with accelerated platelet (PLT) response and clotting. The objective of this study was to compare the in vitro hemostatic effect of CPP with fresh whole blood (WB) and standard 5-day PLT concentrates (PCs). STUDY DESIGN AND METHODS: WB collected from eight healthy donors was used to prepare fresh WB, PLT-depleted WB (TPN), and PLT-restored TPN using CPP (TPN-CPP) or PC (TPN-PC). Clot properties were evaluated with thromboelastometry (ROTEM); adhesion and aggregate formation under high shear (Impact-R); and PLT adhesion, aggregate formation, fibrin formation, and prothrombin activation under medium shear in a perfusion system. RESULTS: TPN-CPP had faster clot initiation (ROTEM clot time--TPN-CPP 115 sec, WB 194 sec, TPN-PC 161 sec), and CPP contributes to a strong clot with PLT involvement (maximum clot firmness--TPN-CPP 32 mm, WB 62 mm, TPN-PC 59 mm). The Impact-R PLT-covered area with TPN-CPP was less than those of WB and PCs, but aggregate size was the same as WB. PLT coverage in perfusion studies was observed with TPN-CPP, although generally less than both WB and PC. Fibrin was deposited with CPP-restored samples, but did not exceed the level of WB. CONCLUSION: CPPs present a phenotype supporting a moderate increase in the rate of clot formation, form stable PLT clots, and do not present a hypercoagulable phenotype during in vitro functional tests.

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.

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.

Effect of Melatonin Plus Zinc Supplementation on Fatigue Perception in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: A Randomized, Double-Blind, Placebo-Controlled Trial.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex, multisystem, and profoundly debilitating condition, probably of multifactorial etiology. No effective approved drugs are currently available for its treatment. Several studies have proposed symptomatic treatment with melatonin and zinc supplementation in chronic illnesses; however, little is known about the synergistic effect of this treatment on fatigue-related symptoms in ME/CFS. The primary endpoint of the study was to assess the effect of oral melatonin plus zinc supplementation on fatigue in ME/CFS. Secondary measures included participants' sleep disturbances, anxiety/depression and health-related quality of life. A proof-of-concept, 16-week, randomized, placebo-controlled, double-blind trial was conducted in 50 ME/CFS patients assigned to receive either oral melatonin (1 mg) plus zinc (10 mg) supplementation (n = 24) or matching placebo (n = 26) once daily. Endpoint outcomes were evaluated at baseline, and then reassessed at 8 and 16 weeks of treatment and 4 weeks after treatment cessation, using self-reported outcome measures. The most relevant results were the significant reduction in the perception of physical fatigue in the Mel-Zinc group at the final treatment follow-up versus placebo (p < 0.05), and the significant improvement in the physical component summary at all follow-up visits in the experimental group. Urinary 6-sulfatoxymelatonin levels were significantly elevated though the treatment in experimental group vs. placebo (p < 0.0001); however, no significantly differences were observed for zinc concentration among participants. Our findings suggest that oral melatonin plus zinc supplementation for 16 weeks is safe and potentially effective in reducing fatigue and improving the quality of life in ME/CFS. This clinical study was registered on ClinicalTrials.gov (NCT03000777).

Internalization of microparticles by platelets is partially mediated by toll-like receptor 4 and enhances platelet thrombogenicity.

BACKGROUND AND AIMS: Circulating platelet microparticles (PMP) are the most abundant in bloodstream, are highly procoagulant and contribute to cross-talk with inflammatory cells. The aim of the present study was to investigate the interactions of PMP with platelets and explore the involvement of toll-like receptor 4 (TLR-4). METHODS: PMP were separated by ultracentrifugation of expired platelet concentrates and added to: i) washed platelets, to confirm uptake, by flow cytometry and confocal and transmission electron microscopy, ii) platelet rich plasma (PRP), to assess changes in platelet function due to uptake by aggregometry in response to ADP; and iii) whole blood, to evaluate heterotypic aggregate (HA) formation by flow cytometry. Moreover, whole blood previously enriched with platelets with internalized PMP was used to explore modifications in thromboelastometry parameters (ROTEM). The inhibitory action of anti-TLR-4 was investigated. RESULTS: Confocal and ultrastructural microscopy studies revealed PMP internalization by platelets. Flow cytometry showed PMP-platelet association (p < 0.01 vs controls, at different PMP dilutions). PMP, at 1/20 dilution, increased HA (p < 0.05 vs controls), the percentage of maximal platelet aggregation to ADP (p < 0.05 vs controls), and accelerated clotting and clot formation times (p < 0.05 vs controls). Incubation of platelets with anti-TLR-4 prior to exposure to PMP reduced PMP-platelet association (p < 0.05 vs absence of the antibody), prevented HA formation, reduced maximal platelet aggregation and normalized ROTEM parameters. CONCLUSIONS: Platelets exhibit internalization ability towards their own PMP, a process that potentiates their thrombogenicity and is partially mediated by the innate immunity receptor TLR-4.

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.

Inhibition of tyrosine kinase activity prevents the adhesive and cohesive properties of platelets and the expression of procoagulant activity in response to collagen.

INTRODUCTION: Platelet activation leads to signal transduction mechanisms, in which phosphotyrosine proteins play a relevant role. MATERIAL AND METHODS: Platelet suspensions were independently activated by collagen and thrombin in the absence and in the presence of two tyrosine kinase inhibitors, tyrphostin 47 and genistein. Samples were processed to visualize morphological changes by electron microscopy, to evaluate changes in cytoskeletal assembly, to analyze modifications in the expression of activation dependent antigens, and the procoagulant activity at the surface level by flow cytometry. Additional experiments applying flow conditions were performed to assess the effect of inhibiting tyrosine phosphorylation on primary platelet adhesion and fibrin formation. RESULTS: Inhibition of tyrosine phosphorylation blocked shape change and cytoskeletal assembly induced by collagen, and inhibited, though partially, those effects due to thrombin. Both activating agents induced the expression of the intraplatelet antigens CD62P and CD63 at the surface, although only collagen promoted expression of anionic phospholipids. Both tyrphostin 47 and genistein prevented those effects. The extent of platelet adhesion on both collagen-coated and subendothelial surfaces was significantly diminished by the presence of the tyrosine kinase inhibitors assayed. Fibrin formation was also significantly reduced. CONCLUSIONS: Platelet shape change and secretion during platelet activation depends on tyrosine phosphorylation. In addition, primary adhesion of platelets induces signaling through tyrosine kinases to achieve full spreading, and results in the exposure of a procoagulant surface on platelets.

BDNF as a marker of response to cognitive remediation in patients with schizophrenia: A randomized and controlled trial.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) is considered to be a putative biomarker for cognitive recovery in schizophrenia. However, current evidence is still scarce for pharmacological treatments, and the use of BDNF as a biomarker has only been tested once with cognitive remediation treatment (CRT). METHODS: A randomized and controlled trial (NCT02341131) with 70 schizophrenia outpatients and 15 healthy volunteers was conducted. The participants with schizophrenia were randomly assigned to either CRT or the control group. All the participants were assessed in terms of cognition, quality of life, and their serum BDNF levels at both baseline and after the intervention. Additionally, comparisons of the effects of the different genotypes of the Val66Met polymorphism at the BDNF gene on the outcome variables were also performed. RESULTS: The patients in the CRT group presented with improvements in both cognition and quality of life. However, no significant changes were detected in the serum levels of BDNF. Interestingly, we found a significant positive interaction effect between the serum BDNF levels and the different BDNF genotypes. The Val/Val group showed significantly higher serum levels after the CRT treatment. However, the interaction among the serum BDNF levels, the BDNF genotypes and the treatment condition was not statistically significant. CONCLUSIONS: The replication of the previous finding of increased serum BDNF levels after cognitive remediation in clinically stable individuals with schizophrenia was not achieved. However, our data indicated that genetic variability may be mediating serum BDNF activity in the context of CRT.

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.

Differential inhibitory action of apixaban on platelet and fibrin components of forming thrombi: Studies with circulating blood and in a platelet-based model of thrombin generation.

INTRODUCTION: Mechanisms of action of direct oral anticoagulants (DOAC) suggest a potential therapeutic use in the prevention of thrombotic complications in arterial territories. However, effects of DOACs on platelet activation and aggregation have not been explored in detail. We have investigated the effects of apixaban on platelet and fibrin components of thrombus formation under static and flow conditions. METHODS: We assessed the effects of apixaban (10, 40 and 160 ng/mL) on: 1) platelet deposition and fibrin formation onto a thrombogenic surface, with blood circulating at arterial shear-rates; 2) viscoelastic properties of forming clots, and 3) thrombin generation in a cell-model of coagulation primed by platelets. RESULTS: In studies with flowing blood, only the highest concentration of apixaban, equivalent to the therapeutic Cmax, was capable to significantly reduce thrombus formation, fibrin association and platelet-aggregate formation. Apixaban significantly prolonged thromboelastometry parameters, but did not affect clot firmness. Interestingly, results in a platelet-based model of thrombin generation under more static conditions, revealed a dose dependent persistent inhibitory action by apixaban, with concentrations 4 to 16 times below the therapeutic Cmax significantly prolonging kinetic parameters and reducing the total amount of thrombin generated. CONCLUSIONS: Our studies demonstrate the critical impact of rheological conditions on the antithrombotic effects of apixaban. Studies under flow conditions combined with modified thrombin generation assays could help discriminating concentrations of apixaban that prevent excessive platelet accumulation, from those that deeply impair fibrin formation and may unnecessarily compromise hemostasis.

Escitalopram Impairs Thrombin-Induced Platelet Response, Cytoskeletal Assembly and Activation of Associated Signalling Pathways.

Background Serotonin reuptake inhibitors (SSRIs) may impair platelet function. Thrombin is a strong platelet agonist causing irreversible aggregation, release of granules' contents, cytoskeletal rearrangement and activation of signalling pathways. We investigated the effects of the SSRI escitalopram (SCIT) on thrombin-induced platelet response. Methods Isolated platelets were exposed to SCIT and activated with thrombin. We evaluated (1) platelet response by aggregometry and flow cytometry; (2) modifications in cytoskeleton proteins and signalling pathways by electrophoresis and Western blot; and (3) ultrastructural changes in platelets by electron microscopy. Results SCIT inhibited platelet response to thrombin, measured as platelet aggregation and expression of activation markers CD62-P and CD63 from platelet granules. Platelet aggregation decreased in a dose-dependent manner, reaching statistical significance with SCIT ≥32 µg/mL (65.4 ± 6.8% vs. 77.7 ± 2.5% for controls; p < 0.05). Expression of activation markers was statistically reduced with SCIT ≥20 µg/mL (p < 0.05). SCIT impaired the polymerization of the actin cytoskeleton and association of contractile proteins during activation with thrombin (p < 0.05 with SCIT ≥50 µg/mL). Resting platelets incubated with SCIT became most spherical, with increased platelet roundness (p < 0.01, SCIT 50 µg/mL vs. control). SCIT interfered with signalling pathways modulated by thrombin (RhoA, PKC, Erk1/2 and PI3K/AKT). Conclusions Our data indicate that SCIT inhibits thrombin-induced platelet response and interferes with cytoskeletal assembly and related signalling pathways, thus resulting in compromised release of granules' contents, reduced platelet activation and aggregation. These mechanisms may explain the antithrombotic benefits observed in patients treated with this SSRI, and could become new therapeutic targets for future antithrombotic strategies.

New strategy of tacrolimus administration in animal model based on tacrolimus-loaded microspheres.

New strategies for tacrolimus administration that conserve its immunosuppressive effect but avoiding fluctuations in tacrolimus circulating levels are needed. The aim was to analyze if subcutaneous biodegradable tacrolimus-loaded microspheres injection promoted a significant immunosuppressive response in rats. Rats received two subcutaneous tacrolimus-loaded microspheres injections at different days, the first injection was done at day 0 and the second injection was done 12 days after. Plasma circulating levels of tacrolimus, interleukin-2 (IL-2) and calcineurin phosphatase (PP2B) activity in mononuclear cells were measured. Tacrolimus plasma levels were significantly increased from the day after tacrolimus-loaded microspheres injection and remained increased during 10days. Compared to control, plasma IL-2 levels and PP2B activity in mononuclear cells were significantly decreased during ten days. At day 12, a new subcutaneous injection of tacrolimus-loaded microspheres was performed and two days after injection, tacrolimus plasma levels were again increased and both IL-2 plasma levels and PP2B activity decreased. A single subcutaneous tacrolimus-loaded microspheres injection was enough to reduce tacrolimus-related immunosuppressive parameters. These results open the possibility of new therapeutic strategies to administrate calcineurin inhibitors reducing the variability of their circulating levels related to gastrointestinal drug absorption/metabolism modifications.

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.

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.

Reversal of apixaban induced alterations in hemostasis by different coagulation factor concentrates: significance of studies in vitro with circulating human blood.

Apixaban is a new oral anticoagulant with a specific inhibitory action on FXa. No information is available on the reversal of the antihemostatic action of apixaban in experimental or clinical settings. We have evaluated the effectiveness of different factor concentrates at reversing modifications of hemostatic mechanisms induced by moderately elevated concentrations of apixaban (200 ng/ml) added in vitro to blood from healthy donors (n = 10). Effects on thrombin generation (TG) and thromboelastometry (TEM) parameters were assessed. Modifications in platelet adhesive, aggregating and procoagulant activities were evaluated in studies with blood circulating through damaged vascular surfaces, at a shear rate of 600 s(-1). The potential of prothrombin complex concentrates (PCCs; 50 IU/kg), activated prothrombin complex concentrates (aPCCs; 75 IU/kg), or activated recombinant factor VII (rFVIIa; 270 µg/kg), at reversing the antihemostatic actions of apixaban, were investigated. Apixaban interfered with TG kinetics. Delayed lag phase, prolonged time to peak and reduced peak values, were improved by the different concentrates, though modifications in TG patterns were diversely affected depending on the activating reagents. Apixaban significantly prolonged clotting times (CTs) in TEM studies. Prolongations in CTs were corrected by the different concentrates with variable efficacies (rFVIIa≥aPCC>PCC). Apixaban significantly reduced fibrin and platelet interactions with damaged vascular surfaces in perfusion studies (p<0.05 and p<0.01, respectively). Impairments in fibrin formation were normalized by the different concentrates. Only rFVIIa significantly restored levels of platelet deposition. Alterations in hemostasis induced by apixaban were variably compensated by the different factor concentrates investigated. However, effects of these concentrates were not homogeneous in all the tests, with PCCs showing more efficacy in TG, and rFVIIa being more effective on TEM and perfusion studies. Our results indicate that rFVIIa, PCCs and aPCCs have the potential to restore platelet and fibrin components of the hemostasis previously altered by apixaban.