Patricia Molina

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.

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

BACKGROUND: There are limited studies on the effect of pathogen reduction technology (PRT) on platelet (PLT) products stored in PLT additive solutions (PASs). Our study has evaluated the effect of Mirasol PRT (CaridianBCT) followed by storage in PASs (PAS‐III or PAS‐IIIM) on analytical and functional characteristics of PLT concentrates (PCs).

Distinct Deleterious Effects of Cyclosporine and Tacrolimus and Combined Tacrolimus–Sirolimus on Endothelial Cells: Protective Effect of Defibrotide

Endothelial dysfunction seems to be a key factor in the development of several complications observed early after hematopoietic stem cell transplantation (HSCT). The conditioning regimen and many other factors associated with the procedure are responsible for this endothelial damage. The effects of immunosuppressive agents on endothelial function have not been explored in detail. We evaluated the effects of 3 drugs commonly used in HSCT: 2 calcineurin inhibitors, cyclosporine A (CSA) and tacrolimus (TAC), and an inhibitor of mTOR, sirolimus (SIR). We also evaluated the effect of the combination of TAC and SIR (TAC+SIR), which is used increasingly in clinical practice. Microvascular endothelial cells (HMEC-1) were exposed to these drugs to evaluate changes in (1) intercellular adhesion molecule (ICAM)-1 expression on the cell surface, assessed by immunofluorescence labeling and expressed as the mean gray value (MGV); (2) reactivity of the extracellular matrix (ECM) toward platelets, upon exposure of the ECM to circulating blood; and (3) whole-blood clot formation, assessed by thromboelastometry. Studies were conducted in the absence and presence of defibrotide (DF) to assess its possible protective effect. The exposure of HMEC-1 to CSA and TAC+SIR significantly increased the expression of ICAM-1 (157.5 ± 11.6 and 153.4 ± 9.5 MGV, respectively, versus 105.7 ± 6.5 MGV in controls [both P < .05]). TAC applied alone increased ICAM-1 slightly (120.3 ± 8.2 MGV), and SIR had no effect (108.9 ± 7.4 MGV). ECM reactivity increased significantly only in response to CSA (surface covered by platelets of 41.2% ± 5.4% versus 30.1% ± 2.0%, P < .05). DF attenuated all these changes. No significant changes in the viscoelastic properties of clot formation were observed in any condition with blood samples incubated in vitro. In conclusion, CSA and TAC+SIR had a proinflammatory effect, but only CSA exhibited an additional prothrombotic effect. Interestingly, DF exerted clear protective anti-inflammatory and antithrombotic effects on the endothelium.

D225G mutation in the hemagglutinin protein found in 3 severe cases of 2009 pandemic influenza A (H1N1) in Spain.

From 27 April to 16 December 2009, we analyzed the hemagglutinin gene sequence of 2009 pandemic influenza A (H1N1) virus in 189 respiratory specimens. We only found the D225G mutation in 3 severe cases. However, it was not found in samples from other cases with or without clinical criteria of severity. The biologic significance of this mutation remains still unclear.

NFκB in the Development of Endothelial Activation and Damage in Uremia: An In Vitro Approach

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

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.

Influenza C virus surveillance during the first influenza A (H1N1) 2009 pandemic wave in Catalonia, Spain

Although particular attention is paid to influenza A and B virus isolates during influenza surveillance, influenza C virus (FLUCV) coexisted during the first influenza A (H1N1) 2009 pandemic wave during the 2009-2010 season. From 27 April 2009 to 9 May 2010, 12 strains of FLUCV were detected in specimens collected from 1713 nonhospitalized patients with upper respiratory tract illness using a molecular method. Half of the patients with FLUCV infection were older than 14 years. The most frequent symptoms were cough and fever, similar to other viral respiratory infections. Phylogenetic analysis of the hemagglutinin-esterase gene revealed that the strains belonged to the C/Kanagawa/1/76-related and C/Sao Paulo/378/82-related lineages, demonstrating their co-circulation in Catalonia. In addition to regular virological surveillance that provides information about the incidence and the exact role of FLUCV in acute viral respiratory infections in the general population, the genetic lineage identification offers additional data for epidemiological purposes.

In vitro evaluation of the hemostatic effectiveness of cryopreserved platelets

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).

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.

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. J. Cell. Biochem. 117: 448–457, 2016.