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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).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.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.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.

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 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.

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

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.

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.

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.

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).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.In comparison with controls, platelets from MDD patients showed elevated volumes (p0.01), significantly enhanced aggregating response to arachidonic acid and augmented expression of GPIb, fibrinogen, factor V, and anionic phospholipids by flow cytometry (p0.05). Clot firmness and procoagulant activity of platelet-associated tissue factor were also significantly elevated (p0.05). Studies with circulating blood revealed increased fibrin formation in early diagnosed patients (71.1±9.5% vs. 45.8±5.3%; p0.05 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 (p0.05) and persistent alterations in thromboelatometic parameters.Despite the reduced number of followed-up patients our findings were consistent reaching statistical significance.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.

Background Brain-derived neurotrophic factor (BDNF) has been proposed as a biomarker of schizophrenia and, more specifically, as a biomarker of cognitive recovery. Unfortunately, it 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 cognition. 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. Discussion 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. All in all, the current consideration of BDNF as a biomarker of cognitive recovery in schizophrenia is promising but still premature.

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.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.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; p0.01) and on the vascular subendothelium (%CS: 54.0 ± 1.5 and 47.2 ± 6.8 respectively vs. 38.0 ± 3.5, p0.05), with a statistically significant increase in the size of large platelet aggregates (p0.05) 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 (p0.05), effects that were partially prevented by a neutralizing anti-TF.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.

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.

There is a link between depression, cardiovascular events and inflammation. We have explored this connection through endothelial dysfunction, using in vivo and in vitro approaches. We evaluated circulating biomarkers of endothelial dysfunction in patients with major depression at their diagnosis (MD-0) and during antidepressant treatment with the selective serotonin reuptake inhibitor escitalopram, for 8 and 24 weeks (MD-8 and MD-24). Results were always compared with matched healthy controls (CON). We measured in vivo circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs) in blood samples, and assessed plasma levels of soluble von Willebrand factor (VWF) and vascular cell adhesion molecule-1 (VCAM-1). CEC counts, soluble VWF and VCAM-1 were statistically elevated in MD-0 (PIn vitro studies were performed in human endothelial cells cultured in the presence of sera from each study group. Elevated expression of the inflammation marker intercellular adhesion molecule-1 and oxidative stress, with lower presence of endothelial nitric oxide synthase and higher reactive oxygen species production, were found in cells exposed to MD-0 sera (P

Neuroplastic processes are thought to be involved in the pathophysiology of major depression. It has been reported that serum brain-derived neurotrophic factor (BDNF) is decreased in depressed patients.Compare BDNF levels in depressed patients and healthy controls in platelet poor plasma and in washed platelets. Observe the effects of 8- and 24-week treatment with S-citalopram on these levels.We assessed the levels of BDNF in platelet poor plasma and in washed platelets from 18 major depression patients, and compared them with 14 healthy controls. Blood samples were obtained from patients before and during treatment (8 and 24 weeks) with a selective serotonin reuptake inhibitor, S-citalopram.A significant decrease in severity of depressive symptoms was observed from the first month of treatment with S-citalopram, and symptoms continued decreasing until the 6th month. Plasma BDNF levels in untreated patients appeared significantly increased (p0.01) but reached values similar to those of controls at the 24th week. In contrast, levels of platelet BDNF appeared significantly decreased (p0.05), but treatment also normalized levels so that values obtained were equivalent to those of controls.Untreated depressed patients showed increased plasma BDNF levels and decreased platelet BDNF levels, as compared with control subjects, and tend to normalize during treatment with S-citalopram for 24 weeks, with BDNF reaching levels similar to those in healthy controls at the 24th week in both samples. We observed that improvement in depressive symptoms was accompanied by normalization of plasma and platelet BDNF levels.

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.

BACKGROUND While procoagulant activities of Tissue Factor (TF) have been widely investigated, its possible pro-adhesive properties towards platelets have not been studied in detail. MATERIAL AND METHODS We explored the interaction of platelets with human Tissue Factor (hTF) firmly adsorbed on a synthetic surface of polyvinilidene difluoride (PVDF) using different shear rates. For studies at 250 and 600 s(-1), TF firmly adsorbed was exposed to flowing anticoagulated blood in flat perfusion devices. Deposition of platelets and fibrin were evaluated by morphometric, immunocytochemical and ultrastructural methods. Prothrombin fragment 1 + 2 (F1 + 2) levels were also measured. Experiments at 5000 s(-1), were performed on the Platelet Function Analyzer (PFA-100) with experimental cartridges with collagen (COL) or collagen-hTF (COL + TF). Haemostatic effect of recombinant activated FVIIa (rFVIIa) was assessed in the same experimental settings. RESULTS Platelet deposition on hTF reached 19.8 +/- 1.3% and 26.1 +/- 3.4% of the total surface, at 250 and 600 s(-1), respectively. Fibrin formation was significantly higher at 250 s(-1) than at 600 s(-1) (P < 0.05). The addition of rFVIIa did not influence platelet deposition but raised fibrin formation and thrombin generation at both shear rates (P < 0.05). At 5000 s(-1), closure times (CT) in the PFA-100 were significantly shortened in the presence of hTF (154.09 +/- 14.69 s vs. 191.45 +/- 16.09 s COL alone; P < 0.05). Addition of rFVIIa did not cause a further reduction of CT. CONCLUSIONS Our studies demonstrate that hTF is an adhesive substrate for platelets and suggest that the von Willebrand factor could mediate these interactions. At low and intermediate shear rates, rFVIIa enhanced the procoagulant action of hTF, but this effect was not observed at very high shear rates.

SummaryWe 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 humanTF (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 bothTF 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.

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.

Gerard Ill-Raga et al., © 2014. 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., This work was supported by the Spanish Ministry of Science and Innovation (SAF2012-38140; SAF 2009-10365); Fondo de Investigación Sanitaria (FIS PI13/00408, FIS PI13/00864, CP04-00112, PS09/00664 and Red HERACLES RD12/0042/0014, RD12/0042/0016 and RD12/0042/0020); FEDER Funds; Generalitat de Catalunya (SGR09-1369); and Fundació la Marató de TV3 (100310)

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.

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. (Circ J 2015; 79: 331-338).

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.

Brain-derived neurotrophic factor (BDNF) has been proposed as a biomarker of schizophrenia and, more specifically, as a biomarker of cognitive recovery. Evidence collected in this review indicates that BDNF is relevant in the pathophysiology of schizophrenia and could play a role as a marker of clinical response. BDNF has been shown to play a positive role as a marker in antipsychotic treatment, and it has been demonstrated that typical antipsychotics decrease BDNF levels while atypical antipsychotics maintain or increase serum BDNF levels. Furthermore, BDNF levels have been associated with severe cognitive impairments in patients with schizophrenia. Consequently, BDNF has been proposed as a candidate target of strategies to aid the cognitive recovery process. There is some evidence suggesting that BDNF could be mediating neurobiological processes underlying cognitive recovery. Thus, serum BDNF levels seem to be involved in some synaptic plasticity and neurotransmission processes. Additionally, serum BDNF levels significantly increased in schizophrenia subjects after neuroplasticity-based cognitive training. If positive replications of those findings are published in the future then serum BDNF levels could be definitely postulated as a peripheral biomarker for the effects of intensive cognitive training or any sort of cognitive recovery in schizophrenia. All in all, the current consideration of BDNF as a biomarker of cognitive recovery in schizophrenia is promising but still premature.

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.

SummaryAlthough 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.

Thrombogenicity of ruptured atherosclerotic plaques is mainly attributed to the exposure of collagen (Col) and tissue factor (TF). Using in-vitro approaches, we explored the relative contribution of Col and TF to local thrombogenesis.Surfaces coated with Col and human TF, alone or in combinations (diluted Col on TF, and diluted TF on Col), were exposed to flowing blood at a shear rate of 600/s. Platelet and fibrin deposition were analyzed morphometrically. Generation of prothrombin fragments (F1+2) was determined as a measure of global activation of coagulation.Col and TF alone behaved as adhesive substrata to platelets supporting similar platelet coverage around 22%. Col induced tight aggregates, whereas TF promoted adhesive events. In studies with combinations of Col and TF, platelet aggregation was always enhanced with statistical elevations of the thrombus area (P0.05). Generation of F1+2 was surface-dependent and was at its highest levels when both proteins were combined (P0.05). However, local fibrin formation was statistically increased in surfaces containing lower concentrations of TF on Col (P0.01 vs. overall surfaces assessed).Our studies show that combinations of Col and TF always enhance thrombogenesis, but small amounts of TF on Col surfaces resulted in the most procoagulant combination. The present results suggest that plaques exposing high contents of Col, with small amounts of TF, would provide the most occlusive combination.

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.

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.

BACKGROUND: Despite the proven efficacy and safety profile of dabigatran as compared to warfarin, bleeding remains a concern as with all anticoagulants and the reversal of dabigatran’s anticoagulant effect for emergency procedures remains controversial. Recently, idarucizumab, a specific antidote for dabigatran, has been functionally characterized and its efficacy demonstrated in animal models and healthy volunteer studies. AIMS: We explored the effects of dabigatran on hemostasis in human blood focusing on possible interference with platelet and coagulation responses to vessel injury under flow conditions. We also compared the potential efficacy of idarucizumab with procoagulant strategies such as prothrombin complex concentrates (PCC), activated PCC (aPCC) or rFVIIa at reversing the antithrombotic action of dabigatran to better understand local processes in response to injury. METHODS: Concentrations of dabigatran equivalent to the Cmax reported at steady state after therapy with 150 mg twice daily (184 ng/mL) were added in vitro to blood aliquots from 11 healthy donors. Whole blood samples were used to evaluate modifications in different coagulation biomarkers: 1) fibrin and platelet deposition on damaged vascular segments with whole blood under flow conditions at a shear rate of 600 s-1, 2) dynamics of thrombin generation (TG) in plasma using a fluorogenic assay (Technothrombin TGA) and 3) viscoelastic parameters of clot formation in whole blood using by thromboelastometry (ROTEM) The efficacy of specific reversal with idarucizumab 0.3, 1 and 3 mg/mL was compared with that of non specific procoagulant concentrates such as aPCC 25 and 75 IU/kg, PCC 70 IU/kg, or rFVIIa 120 µg/kg. RESULTS: Dabigatran (184 ng/mL) caused a pronounced 85% reduction of fibrin coverage on the damaged vessel from 67.2±9.8 to 9.5±1.3 % (p CONCLUSIONS: Dabigatran (184 ng/mL) added to blood from healthy volunteers caused evident alterations in hemostasis parameters related to its recognized anticoagulant action. Procoagulant concentrates significantly compensated for the overall anti-hemostastic action of dabigatran. Overall, 75 U/kg aPCC seemed the more efficient nonspecific reversal therapy. In clear contrast with non specific procoagulant strategies, idarucizumab, the specific antidote to dabigatran completely reversed all alterations in coagulation parameters evaluated in circulating human blood and in assay systems. (Supported by SAF 2011-2814 and PI13/00517, Spanish Gov & FEDER) Disclosures van Ryn: Boehringer Ingelheim Pharma: Employment. Escolar:Boehringer Ingelheim Pharma: Investigator Sponsored Research Funding Other.

BACKGROUND: Several strategies are being developed to reduce the risk of pathogen transmission associated with platelet (PLT) transfusion. STUDY DESIGN AND METHODS: The impact of a new technology for pathogen reduction based on riboflavin plus illumination (Mirasol PRT, Navigant Biotechnologies, Inc.) at 6.2 and 12.3 J per mL on functional and biochemical characteristics of PLTs was evaluated. PLT concentrates (PCs) obtained by apheresis were treated with Mirasol PRT and stored at 22°C. Modifications in major PLT glycoproteins (GPIbα, GPIV, and GPIIb-IIIa), adhesive ligands (von Willebrand factor [VWF], fibrinogen [Fg], and fibronectin), activation antigens (P-selectin and LIMP), and apoptotic markers (annexin V binding and factor [F]Va) were analyzed by flow cytometry. Adhesive and cohesive PLT functions were evaluated with well-established perfusion models. Studies were performed on the preparation day (Day 0) and during PCs storage (Days 3 and 5). RESULTS: Levels of glycoproteins remained stable during storage in PCs treated with 6.2 J per mL pathogen reduction technology (PRT) and similar to those observed in nontreated PCs. When 12.3 J per mL PRT was applied, however, levels of GPIbα moderately decreased on Days 3 and 5. VWF, Fg, and FVa were not modified in their expression levels, either by treatment or by storage period. Fibronectin appeared more elevated in all PRT samples. A progressive increase in P-selectin and LIMP expression and in annexin V binding was observed during storage of PRT-treated PCs. Functional studies indicated that 6.2 J per mL Mirasol PRT–treated PLTs preserved adhesive and cohesive functions to levels compatible with those observed in the respective control PCs. CONCLUSION: PLT function was well preserved in PCs treated with 6.2 J per mL Mirasol PRT and stored for 5 days.

Abstract 3264 TF associated with platelets may play a critical role in the development of ischemic complications at sites of vascular damage. Platelets can internalize and store tissue factor-rich microvesicles from human origin (hTF), a process that can be potentiated by serotonin (5-HT) (Galan et al, Thromb Haemost. 102:511, 2009). There is limited knowledge on the mechanisms involved in the uptake, redistribution and functional expression of TF by platelets. We have investigated the mechanisms implied in the uptake and traffic of hTF with especial attention to CD36 and the 5HT transporter receptor (SERT), but also to the involvement of PI3-kinase, small GTPases (RhoA) and modifications of cytoskeletal organization during vesicular trafficking. Isolated platelets were exposed to hTF for up to 10 min. Cytoskeletal assembly and activation of RhoA were analyzed by electrophoresis and ELISA, respectively. Aggregometry and ultrastructural microscopy were used to assess platelet activation and hTF uptake. Protein arrays were used to evaluate changes in signaling molecules induced by hTF. In addition, inhibitory strategies to block GPIIbIIIa (Reopro), the scavenger receptor GPIV (anti-CD36), the SERT (S-Citalopram, SCit), and PI3-kinase (Wortmannin, Wo) were applied to platelets prior exposure to hTF. Internalization of hTF resulted in reversible actin polymerization and association of contractile proteins (alpha-actinin, actin binding protein and myosin) to the cytoskeleton, being maximal at 1 min. Maximal activation of RhoA occurred at 1 min, decreasing gradually afterwards, while activation of PI3-kinase occurred later (at 5 min). Screening of signaling proteins revealed activation of Akt, ERK1/2, and SNAP-25, among others. All inhibitory strategies fully prevented platelet aggregation to hTF. Uptake of hTF was slightly prevented by Reopro, while both anti-CD36 and Wo partially inhibited hTF uptake and, interestingly, SCit was the most efficient. We conclude that uptake of hTF by platelets promotes activation of specific signaling molecules with reversible cytoskeletal assembly and membrane fusion. While it may be difficult to design specific strategies to prevent the activation of small GTPases, cytoskeletal arrangements or membrane fusion specifically for platelets, our results indicate that the CD36 scavenger receptor and the 5-HT transporter (SERT) may become realistic therapeutic targets to prevent or reduce the association of TF to platelets and hence its possible prothrombotic action. Grants: SAF2009-10365, Red HERACLES RD06/0009/1003 Disclosures: No relevant conflicts of interest to declare.

Abstract 4349 BACKGROUND: Massive bleeding and subsequent coagulopathy are responsible for 35% of deaths in trauma patients. Hemodilution during resuscitation may worsen the coagulopathy and perpetuate bleeding. STUDY DESIGN AND METHODS: Blood samples from healthy donors were diluted (30–60%) using crystalloids (saline, Ringer’s lactate, Plasmalyte™) or colloids (6%hydroxyethylstarch (HES130/0.4), 5% human albumin, and gelatin). The impact of hemodilution on platelet adhesion, thrombin generation (TG), and clot viscoelastic properties by thromboelastometry (TEM) was analyzed. Effects of fibrinogen (Fbn), prothrombin complex concentrates (PCCs), rFVIIa, or cryoprecipates (cryo) on hemodilution were also assessed. RESULTS: Hemodilution caused a significant decrease in platelet interaction that was not improved by the addition of any of the plasma derivatives. A decrease in TG and important alterations of TEM were also observed. HES130/0.4 was the expander with the most deleterious action. TG was significantly enhanced by PCCs and their combination with Fbn whereas rFVIIa only slightly accelerated it. Fbn restored the alterations of TEM caused by hemodilution including those more deeply altered by HES 130/0.4. The combination of Fbn with PCC or rFVIIa did not have an additional effect in TEM. Cryo significantly improved the alterations caused by hemodilution on TG and TEM parameters. Effects of cryo on TG disappeared after ultracentrifugation, suggesting that contaminating microvesicular material could account for this effect. CONCLUSION: Hemostatic alterations caused by hemodilution are multifactorial and affect both blood cells and coagulation. In our in vitro approach, HES 130/0.4 seemed to exert a more deleterious effect on hemostasis. None of the concentrates improved platelet-mediated hemostasis, although they always showed variable beneficial effects on coagulation parameters. Our data indicate that PCC, rFVIIa and cryo enhance or accelerate thrombin generation. Fbn concentrates could be useful to preserve blood clotting abilities during fluid resuscitation of critically ill patients without exposing them to enhanced thrombin generation. Grants: PET(2008_0231), FIS(CP04-00112, PS09/00664), SAF2009-10365, RD06/0009 Disclosures: No relevant conflicts of interest to declare.

Background: Clinical evidence suggests that hemostatic action of recombinant activated factor VII (rFVIIa) exceeds its predicted plasma half life (around 3 hours). Mechanisms involved in the long lasting effects of rFVIIa for prophylactic treatment of patients with hemophilia and inhibitors have not been fully elucidated. Previous studies from our group have demonstrated that platelets internalize tissue factor preparations containing small amounts of FVII. We have investigated the possible redistribution of rFVIIa in different intracellular compartments focussing more specifically on platelets, endothelium and subendothelial matrix. Methods: We exposed platelet rich plasma, isolated platelets, human umbilical veins and endothelial cells in culture (HUVECs) to rFVIIa. Samples were incubated for up to 2 hours at rFVIIa concentrations from 2-60 mg/ml (normal plasma concentration: 0.5 mg/ml). Flow cytometry techniques were applied to detect surface and intracellular antigens, including FVIIa, in platelets before and after exposure to membrane permeabilizing agents. Immunocytochemical techniques were applied to detect FVIIa in cross sections of umbilical veins and confocal microscopy was used to detect FVIIa in HUVECs. In additional studies, aliquots of washed platelets previously exposed to rFVIIa were incorporated into whole blood anticoagulated with low molecular weight heparin (LMWH) and perfused through a collagen rich damaged vascular surface at a shear rate equivalent to 600/sec. Results: Flow cytometry studies revealed a significantly enhanced presence of intracellular FVIIa in platelets previously exposed to rFVIIa. Fluorescence intensity related to FVIIa was dependent on the concentration of rFVIIa used during the incubation. A more intense labeling for FVIIa was observed in the subendothelial matrix of umbilical veins exposed to rFVIIa. In perfusion studies, presence of platelets previously exposed to rFVIIa improved platelet thrombus formation and enhanced fibrin generation with respect to parallel experiments using platelets not exposed to rFVIIa. Conclusion: Our results indicate that rFVIIa can be internalized into platelets and redistributed into subendothelial compartments. Redistribution of rFVIIa into other cellular compartments may explain the prolonged prophylactic action of rFVIIa in some clinical conditions. The existence of extravascular sources of FVIIa may provide new insights into the physiological and pathological implications of FVIIa on hemostasis mechanisms.

Serotonergic mechanisms implied in both neuronal and platelet functions have been pointed out as a possible link between depressive disorders and cardiovascular risk. Recent studies suggest serotonin (5-HT) as a key element in the generation of a subpopulation of platelets with elevated procoagulant properties (coated-platelets). We have investigated the implication of serotonergic mechanisms in platelet and coagulation activation in samples from healthy donors or from patients suffering major depression. Furthermore, we evaluated the possible prothrombotic role of serotonergic mechanisms and the potential antithrombotic effect of selective serotonin reuptake inhibitors (SSRI) in vitro and compared results with those observed in a group of patients with major depression. Different experimental strategies were used to evaluate platelet function and the activation of coagulation system including standard aggregometry, flow cytometry, perfusion studies with circulating blood to evaluate platelet interaction and fibrin deposition on damaged vessels. Levels of tissue factor (TF) in whole blood and prothrombin fragment F1+2 in plasma samples were also determined. Aggregation studies did not reveal marked differences between control and patients with major depression. The presence of SSRI in plasma decreased up to 30% of the aggregation induced by ADP potentiated with 5-HT. Flow cytometry studies revealed that platelets from patients with major depression showed an enhanced expression of platelet antigens: FV, fibrinogen, CD63, GPIV, GPIb, as well as elevated exposure of anionic phospholipids (p

While procoagulant activity of tissue factor (TF) has been widely investigated, its possible proadhesive properties towards platelets have not been studied in detail. We explored the interaction of platelets with human TF (hTF) firmly attached to a surface using anticoagulated blood with low molecular weight heparin (20 U/ml) at different shear rates. For studies at 250 s−1 and 600 s−1, TF adsorbed on a synthetic surface was exposed to circulating blood in flat perfusion devices. Deposition of platelets and fibrin formation were evaluated by morphometric, immunocytochemical and ultrastructural methods. For experiments at 5000 s−1, we used the PFA-100™ with experimental cartridges with collagen or collagen-hTF. Effect of rFVIIa was assessed in all experimental settings. Prothrombin fragment F1+2 levels were also measured. At 250 and 600 s−1 platelet interaction was 19.84±1.33% and 26.12±3.42% of the total surface respectively. Our inmunocytochemical results suggest that von Willebrand factor could mediate these interactions. Fibrin formation was significantly higher at 250 s−1 than at 600 s−1 (p Our studies demonstrate that hTF is reactive for platelets. von Willebrand factor could mediate these interactions. Recombinant FVIIa enhances the procoagulant action of hTF at low and intermediate shear rates, but has no impact on the hemostatic performance at very elevated shear rates.

Hemophilic patients suffer bleeding episodes despite having a normal bleeding time. A possible platelet dysfunction in these patients has not been deeply investigated. rFVIIa improves hemostasis of hemophilic patients, even in those who develop inhibitors. Clinical efficacy of this drug has been widely confirmed, though, its mechanism of action is not fully understood. We used the PFA-100® with specially devised cartridges whose membrane apertures were coated with collagen alone (COL) or collagen-tissue factor (COL-TF). Blood samples from normal donors or from a group of patients with severe hemophilia A, were anticoagulated with low molecular weight heparin (LMWH). We tested the ability of rFVIIa to shorten the closure times under the previous conditions. The structure of the hemostatic plugs formed on the membrane apertures were further analyzed using light microscopy on thin cross-sections. Closure times were statistically prolonged in blood samples from hemophilic patients tested with COL cartridges (255±22 s.vs.187±15 s in normal donors; p Patients with severe hemophilia showed platelet dysfunction that could be detected with the PFA-100® using specific cartridges. It is likely that the platelet dysfunction observed in these patients could be related to concurrent reductions in VWF that could affect platelet adhesion in these patients revealed at the very elevated shear rates used in the PFA-100®. Under these conditions, TF deposited onto the collagen-coated apertures proved to play a significant role in the initiation of hemostasis. rFVIIa improved the recruitment of platelets on COL-TF and contributed to a partial correction of the platelet dysfunction observed in patients with hemophilia A as further confirmed by the formation of more efficient aggregates in the PFA-100. In essence, rFVIIa circumvented a pre-existent platelet adhesion defect in hemophiliac patients. The pro-hemostatic action of rFVIIa was not observed in parallel studies with blood from healthy donors, indirectly suggesting a good safety profile for this agent when hemostasis is well preserved. PFA-100 could be considered as a possible monitoring system of FVIIa when hemostasis is impaired.

The favorable action of FVIIa in critically deterred hemostasis is not completely elucidated though it has been suggested that FVIIa could activate platelets through mechanisms independent of TF. Standard aggregometry, ultrastructural, and immunocytochemical methods were combined with other techniques to assess the interaction of human TF (hTF) enriched microvesicles (MVs) and rFVIIa in a system with repeatedly washed platelets in absence of plasma proteins. Sequential analysis of morphology and immunolocalization of TF on platelet cryosections were also performed. Modifications in the expression of activation antigens and exposure of anionic phospholipids (Annexin V binding) were evaluated by flow cytometry. Activation of tyrosine phosphorylation mediated mechanisms was also monitored. Several inhibitory strategies were used to characterize mechanisms involved in these interactions. After exposure to hTF bearing MVs (hTF-MVs), platelets became reversibly activated reaching a maximum peak in the first minute (41 ± 3% maximal aggregation). Ultrastructural studies revealed that hTF-MVs became rapidly internalized by platelets. This process induced changes in platelet shape, organelle centralization and enhanced platelet-platelet interactions, but did not result in the formation of compact aggregates. Immunocytochemical studies at ultrastructural level demonstrated hTF-MVs trafficking from the external membranes to the alpha granules via the open canalicular system. Flow cytometry studies detected a moderate increase in the expression of platelet activation antigens. In the absence of rFVIIa platelets always return to an apparent resting state in 5 min, recovering their discoid shape, though immunocytochemical studies still demonstrate TF presence in platelets. When rFVIIa was present together with hTF-MVs, platelets became irreversibly aggregated (81% ± 6%). Ultrastructural studies confirmed internal contraction, degranulation and formation of perfectly structured platelet aggregates. Flow cytometry confirmed a marked expression of activation dependent antigens and a significant enhancement of annexin V binding which was never detected in studies with hTF-MVs alone. Antibodies to GPIIb-IIIa blocked reversible and irreversible aggregation steps. Heparins or hirudin inhibited the irreversible aggregation that occurred in the presence of rFVIIa, but did not prevent the initial reversible aggregation caused by hTF-MVs. Antibodies to P-selectin reduced the initial reversible activation of platelets in response to hTF-MVs, but did completely block the aggregation that occurred in the presence of rFVIIa. This study demonstrates that platelets internalize TF when it is exposed on MVs and confirms the existence of TF trafficking from external sources into internal platelet storage pools. Our results prove that the complex rFVIIa-hTF activates platelets in the practical absence of plasma proteins. If TF can be stored in platelets, the hypothesis of a direct action of rFVIIa on platelets independent of TF should be reconsidered, since internal pools of TF could be exposed on the surface upon activation. Our studies indirectly suggest that TF stored in platelets could enhance the potential thrombogenic capacity of platelets under special pathologic situations.

Several strategies for pathogen reduction are being developed to reduce the risk of pathogen transmission associated with platelet transfusion. There is concern that these techniques could have a deleterious effect on platelet integrity and functions. We evaluated the impact of a new technology for pathogen reduction based on riboflavin + illumination at 6.2 and 12.3 J/ml (Mirasol PRT) on functional and biochemical characteristics of platelets. Platelet concentrates (PCs) obtained by apheresis (n=8) were treated with Mirasol PRT and then stored at 220 C. Modifications in major platelet glycoproteins (GPIb, GPIV, and GP IIbIIIa), adhesive ligands (vWF, Fg, and Fn), activation dependent antigens (P-selectin and LIMP), and apoptotic markers (Annexin V binding and factor Va) were analyzed by flow cytometry. Adhesive and cohesive functions of platelets in the concentrates were evaluated using well-established models with circulating human blood. Studies were performed the preparation day (day 0) and along storage (days 3 and 5) of PCs. Aliquots were taken for aerobic and anaerobic microbiologic cultures. No bacterial growth was observed. Exposure to PRT treatment results in very mild modifications in platelet counts. Levels of GPIb, GPIIb-IIIa and GPIV remained stable during storage in PCs treated with PRT 6.2J/ml and in the same range as those observed in non treated PCs. However, when PRT 12.3 J/ml was applied, levels of GPIb moderately decreased along days of storage 3 and 5. VWF, Fg, and factor Va did not modify their expression, neither by treatment nor by storage period. Fn appeared more elevated in all PRT samples when compared with non-treated samples. A progressive increase in P-selectin and LIMP expression and in Annexin V binding was observed during storage of PRT-treated PCs. Functional studies in perfusion models indicated that Mirasol PRT 6.2 J/ml treated platelets preserved adhesive and cohesive function to levels compatible with those observed in the respective control PCs. In conclusion, platelet function was well preserved in PCs treated with Mirasol PRT 6.2 J/ml and stored for 5 days.

Although exposure of tissue factor (TF) at sites of disrupted atherosclerotic plaques plays a critical role in platelet-triggered ischemic complications, a direct interaction of TF with platelets has not been yet demonstrated. We have investigated the effects of TF on platelets under different shear rate conditions. Exposure of isolated washed platelets to TF derived from human placenta resulted in reversible aggregation (35-50% max at 1 min) using standard platelet aggregometry. This response was αIIb-β3 dependent and occurred in the presence of residual concentrations of calcium. TF-induced reversible aggregation was enhanced and made irreversible to 90–100% by the addition of FVIIa (5μg/ml). This second phase of activation was blocked by the presence of serine-protease inhibitors. A combination of ultrastructural and flow cytometry studies revealed that although TF induced shape change and moderate expression of P-selectin an ANV positive phospholipids, internal contraction and full platelet degranulation only occurred after addition of FVIIa. All these transformations occurred together with tyrosine phosphorylation of proteins. Platelets adhered, spread and aggregated onto synthetic membranes coated exclusively with TF that had been exposed to circulating blood. Addition of FVIIa caused a 50% enhancement on platelet interactions and a two-fold increase in fibrin deposition. Serotonin enhanced overall TF induced platelet interactions under the different experimental approaches. A selective serotonin reuptake inhibitor (citalopram) reduced the intensity and extent of the effects of TF in the two experimental settings used in our investigations. Our results are consistent with a direct role of TF on platelet-triggered ischemic complications. We demonstrate that a lipidated from of TF activates isolated platelets in suspension. In studies conducted with flowing whole blood we show that TF firmly attached to a surface acts itself as an adhesive substrate, and promotes formation of aggregates. These TF-platelet interactions are enhanced through serotoninergic mechanism and markedly potentiated in the presence of FVIIa. The possible synergism between FVIIa and serotoninergic mechanisms may well contribute to the development of severe ischemic complications triggered on ruptured atherosclerotic plaques.

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