Anticoagulant System Research Articles

The modulation of coagulation by aptamers

Coagulation and anticoagulation system is kept in balance by the orchestrated action of a variety of biological factors, and the disruption of this balance leads to the risk of hemorrhage or thrombosis. Oligonucleotide aptamers are single-stranded DNA (ssDNA) or RNA ligands that are synthesized in vitro and bind to target molecules through dimensional structure with high specificity and affinity, and thus represent attractive candidates for the development of agents to maintain the balance of coagulation and anticoagulation. In this review, we summarize recent progress in aptamer-based application in the modulation of coagulation. The aptamers with specific chemical and biological characteristics have great potential to be explored as agents for the treatment of blood coagulation abnormalities.

Low Dose Oral Vitamin K Does Not Increase Time in Therapeutic Range: Results of a Multicentre Clinical Trial

Warfarin remains the most commonly used oral anticoagulant. Its efficacy is closely correlated with the time in therapeutic range (TTR) of the international normalized ratio (INR). Excellent anticoagulant management systems can achieve TTRs in excess of 80%: however, in day-to-day clinical practice TTR values in the range of 50 to 60% are more commonly seen. Strategies to increase TTR are widely sought; one such intervention is the daily administration of low dose vitamin K (VK) administered to reduce variations in VK intake and thus reduce fluctuations in the INR due to such variability. In this multicentre, blinded, randomized controlled trial we allocated patients on chronic warfarin therapy to receive either 0.150 mg of daily oral VK, or matching placebo. After a one month “run in period” to allow adjustment of the warfarin dose, patients were followed for a maximum of 9 months (mean 6 months), their INR was determined, their TTR calculated and their mean warfarin dose calculated. A total of 253 patients were enrolled at 4 clinical centres between Aug 19, 2010 and Aug 30, 2013; 18 (9 in each group) were excluded from this analysis due to inadequate follow-up data, mostly as a result of withdrawal from the study during the run in period. Of the 117 analyzable patients allocated to VK, 66 were male (average age 66.9 yrs). Of the 118 analyzable patients allocated to placebo 59 were male (average age 65.7 yrs). Indications for warfarin were similar between the two groups, as was the frequency of renal insufficiency and the presence of cancer. Patients allocated to placebo were more likely to report a history of bleeding (7% vs 3%). Mean TTR over the 6 months prior to enrollment to the study was 54.6% in the placebo group, and 51.8% in the VK group. Mean TTR over an average follow-up of 6 months after completion of the 1 month run-in period was 66% in the placebo group, and 65.1% in the VK group. We conclude that inclusion in this study resulted in a statistically (and likely clinically) significant improvement in the TTR. The improvement in the TTR we observed appeared to be predominately as a result of attentive clinical management, rather than administration of VK. Our findings do not support the use of daily, low dose oral vitamin K administered in an effort to improve TTR. DisclosuresCrowther:Portola: Consultancy; Leo: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy; AKP America: Consultancy; Heart and Stroke Foundation: Career Investigator award Other; Celgene: Honoraria; Shire: Honoraria; CSL Behring: Honoraria. Lazo Langer:Pfizer: Honoraria, Research Funding; Bayer: Honoraria, Research Funding; Leo Pharma: Honoraria, Research Funding; Boehringer Ingelheim: Honoraria; Alexion: Research Funding; Daichii Sankyo: Research Funding; Novartis: Research Funding; Celgene: Research Funding. Yeo:Bayer: Honoraria. Schulman:Boehringer Ingelheim: Consultancy, Honoraria, Research Funding; Bayer HealthCare: Consultancy, Honoraria, Research Funding.

Secretory group V phospholipase A2: a new player in thrombosis?

The protein C anticoagulant system plays a critical role in the regulation of haemostasis and inflammation. Binding of protein C to endothelial cell protein C receptor (EPCR) and the subsequent activation of EPCR-bound protein C by thrombin/thrombomodulin are the key steps in the protein C anticoagulant system. Activated protein C (APC) -mediated cell signalling, supported by its binding to EPCR or independent of EPCR, activates anti-inflammatory and cytoprotective pathways [1]. Therefore, pathophysiologic conditions that impair the interaction of protein C with EPCR have the potential to decrease APC generation. Such reduced binding to EPCR may not only contribute to thrombotic disorders, but also result in the loss of endogenous APC-mediated cytoprotection..

Anticoagulant and fibrinolytic blood systems in patients with arterial hypertension stage III

Aim. To measure the indicators of the anticoagulant and fibrinolytic blood systems activity in patients with arterial hypertension after suffering a hemorrhagic stroke. Methods. 82 patients with arterial hypertension were examined at different terms after suffering a hemorrhagic stroke. Antithrombin III activity, total blood fibrinolytic activity and D-dimer levels, as well as tissue plasminogen activator activity and antigen levels were studied. Patients were examined at admission before standard treatment initiation and at discharge. Tissue plasminogen activator activity and its antigen level were determined at admission. Statistical data processing was performed using Statistica 8 (StatSoft) integrated package for Windows. Results. Inhibited fibrinolytic activity and an imbalance of its components, as well as reduced levels and activity of antithrombin III and signs of fibrin formation were revealed in patients with arterial hypertension stage III at different periods after suffering hemorrhagic stroke. Changes were most evident in early and late recovery periods after suffered cerebral stroke. Subsequent follow-up for over-a-year term after suffered cerebral stroke revealed a trend for normalization of these indicators. The study of tissue plasminogen activator activity and levels of its antigen showed significant changes in these parameters. Analysis of the obtained results for all patients with hypertension, regardless of the period of hemorrhagic stroke, showed the reduction of the antigen tissue plasminogen activator level and its increased activity. After in-patient treatment, there was a trend for improvement in all studied parameters without their complete normalization. Conclusion. The results suggest an activation of intravascular clotting in parients with arterial hypertension who suffered a hemorrhagic stroke, increasing the risk for further cardiovascular complications.

GW25-e0503 The congenital and acquired thrombophilia in Chinese pulmonary thromboembolism patients

The genetic polymorphisms of coagulation factor V Leiden and prothrombin G20210A are common risk factors of inherited thrombophilia in Caucasians, but rare in Aisan. Factor V is an substrate of the activated protein C anticoagulant system. While in Asian population, dysfunction of APC system

0991. Nebulized heparin reduces pulmonary inflammatory responses in a rat model of acute lung injury

Sepsis is a mayor cause of acute lung injury (ALI). Pulmonary coagulopathy is intrinsic to ALI and in sepsis, anticoagulant system is impaired, due to consumption and downregulation by inflammatory mediators. Several studies, in ALI patients and in ALI experimental models, inconsistently suggest beneficial effects of systemic anticoagulants which affect systemic coagulation. Nebulization of anticoagulants might allow for higher pulmonary concentration and reduce the risk of systemic bleeding.

Differential expression of leukocyte receptors in disseminated intravascular coagulation: Prognostic value of low protein C receptor expression

BackgroundThe protein C receptor (PROCR), toll-like receptor (TLR) and L-selectin leukocyte receptor play roles in systemic inflammatory response including disseminated intravascular coagulation (DIC). Expression of these receptors, which mediate systemic immune or coagulation responses, is tightly regulated by physiologic or pathologic signals. We investigated whether the expressions of 3 leukocyte receptors (PROCR, TLR4, L-selectin) was related to clinical outcomes in patients suggestive of having DIC. MethodsRNA was extracted from the peripheral blood buffy coats of patients suggestive of having DIC. After reverse transcription, mRNA expression levels of PROCR, TLR4, and L-selectin were measured using Taqman Gene Expression Assays. The 28-day hospital mortality rate was used as a clinical outcome. ResultsThe expression level of PROCR mRNA in leukocytes was lower in those with overt-DIC as compared to those without overt-DIC, however, this difference was not statistically significant. As for TLR4 and L-selectin mRNA expression, there were no significant differences observed between those with and without overt-DIC. A Kaplan-Meier survival analysis revealed that patients with low PROCR mRNA expression levels showed significantly lower survival rates than those with high expression levels. On multivariate cox regression analysis, low levels of PROCR mRNA expression were an independent prognostic marker. However, expression levels of TLR4 and L-selectin mRNA were not associated with any prognostic value. ConclusionConsidering that the PROCR is an important anticoagulant receptor, low PROCR mRNA expression levels associated with a poor prognosis in patients with DIC represents an exhaustion of the natural anticoagulant system, and reflects the final decompensate stage of DIC. The leukocyte PROCR may contribute to a dampening of florid activation of coagulation reactions in vivo.

Procoagulant versus anticoagulant factors in cirrhotic patients

Background and study aimLiver cirrhosis leads to decreased production of clotting factors that are generally all produced in the liver except factor VIII and von Willebrand factor. However, cirrhotic patients are not protected from thrombosis. The present study aimed to assess the procoagulant and anticoagulant factors in cirrhotic patients with and without bleeding and/or thrombotic events. Patients and methodsA total of 102 adult subjects were enroled: 51 cirrhotic patients and 51 healthy controls. After full history taking with special attention given to thromboembolic and haemorrhagic events, platelet count, serum albumin, bilirubin, international normalised ratio (INR), PT, partial thromboplastin time (PTT), hepatitis B surface antigen (HBsAg), hepatitis B core (HBc) antibodies, hepatitis C virus (HCV) antibodies, factor VIII, protein C, Protac-induced coagulation inhibition percentage (PICI%) assay and abdominal ultrasound were performed for patients and controls. Upper gastrointestinal endoscopy was conducted for the patients. ResultsCompared with control subjects, factor VIII and factor VIII/protein C were significantly higher, while protein C and PICI% were significantly lower among patients. ConclusionPatients with liver cirrhosis may have a tendency for bleeding or thrombosis according to the balance of coagulant and anticoagulant status. PICI%, the assay that evaluated the functionality of the protein C anticoagulant system, was significantly lower in patients compared to control subjects. Accordingly, low PICI% and high factor VIII/protein C ratio can be taken as an index of hypercoagulability in cirrhotic patients.

Recent advances in the antiphospholipid antibody syndrome.

The antiphospholipid syndrome (APS) is a systemic autoimmune disorder characterized by recurrent thrombosis and/or obstetrical morbidity in the presence of persistently positive antiphospholipid antibodies. Recent insights into the pathogenesis of APS have begun to elucidate pathophysiology and led to the identification of potential therapeutic interventions. The objective of this review is to examine the advances in this field and highlight the areas of further investigation. Several mechanisms of thrombosis and pregnancy loss in APS have been proposed. These include activation of endothelial cells, monocytes, and platelets, and/or inhibition of natural anticoagulant and fibrinolytic systems by antiphospholipid antibodies. However, in many cases the underlying molecular mechanisms and their relevance to the human disorder remain uncertain. New therapeutic agents such as statins, hydroxychloroquine, rituximab, complement inhibitors, and interventions aimed at disruption of intracellular signaling pathways have shown promise in preclinical and clinical studies. Indefinite anticoagulation remains the mainstay of treatment for thrombotic APS. Despite advances in diagnostic techniques, it remains difficult to predict thrombotic risk in asymptomatic patients with antiphospholipid antibodies. Further mechanistic and clinical studies are needed to predict thrombotic risk and develop improved therapies for this devastating illness.

Necrosis cutánea por warfarina

La necrosis cutánea por warfarina aparece entre 0.01-0.1% de los pacientes, afecta más a mujeres con obesidad y antecedentes de enfermedad tromboembólica luego del quinto día de su inicio. Se debe a un imbalance transitorio de los sistemas anticoagulante y procoagulante, caracterizado por flictenas hemorrágicas en zonas con tejido graso. Reportamos el caso de una necrosis cutánea por warfarina con un desenlace fatal

Antithrombin-resistant prothrombin Yukuhashi mutation also causes thrombomodulin resistance in fibrinogen clotting but not in protein C activation

IntroductionProthrombin Yukuhashi (p.Arg596Leu) mutation can result in thrombophilia associated with antithrombin (AT) resistance. Mutant thrombin, an active form of prothrombin Yukuhashi, demonstrated moderately lower clotting activity than the wild-type but substantially impaired the formation of the complex with AT. However, the effects of the mutation on the thrombomodulin (TM)–protein C (PC) anticoagulant system have not been previously elucidated. Materials and MethodsWe prepared recombinant wild-type and mutant prothrombins, converted to thrombins using Oxyuranus scutellatus venom, and performed fibrinogen-clotting assays with or without recombinant soluble TM (rTM). We also evaluated activated PC (APC) generation activity of recombinant thrombins by measuring APC activity after incubation with human PC in the presence or absence of rTM. Result and ConclusionsrTM treatment reduced the relative fibrinogen-clotting activity of the wild-type down to 8.4% in a concentration-dependent manner, whereas the activity of the mutant was only decreased to 44%. In the absence of rTM, APC generation activity (∆A/min at 405nm) was fairly low (0.0089 for the wild-type and 0.0039 for the mutant). In the presence of rTM, however, APC generation activity was enhanced to 0.0907 (10.2-fold) for the wild-type and to 0.0492 (12.6-fold) for the mutant, and the relative activity of the mutant with rTM was 54% of that of the wild-type. These data suggested that the prothrombin Yukuhashi mutation may cause TM resistance in terms of inhibition of fibrinogen clotting; this may contribute to susceptibility to thrombosis, although the enhancing effect of APC generation can be maintained.

Thrombotic processes in multiple sclerosis as manifestation of innate immune activation.

Multiple sclerosis (MS) is a chronic demyelinating inflammatory disease of the central nervous system (CNS) affecting prevalently young adults (1, 2). The pathogenesis of MS has long been attributed to self-reactive T cells but recently the relevant role of B cells has also been recognized (2, 3). Furthermore, it has been demonstrated that innate immunity has a pivotal role in the beginning and in advanced stages of MS (4–6). Innate immunity represents the immediate non-specific defense against infections and dangerous agents acting through its essential arms such as inflammation and blood coagulation (7–10). The coagulant processes are normally balanced by the natural anti-coagulant system needed to limit the host damage, and their imbalance leads to venous thrombosis (7, 8). Many evidences reported below support a significant presence of local and systemic thrombotic events in MS confirming the global over-stimulation of innate immunity for both its inflammatory and coagulant components.

Normal saline influences coagulation and endothelial function after traumatic brain injury and hemorrhagic shock in pigs

Traumatic brain injury (TBI) and hemorrhagic shock (HS) are the leading causes of trauma-related deaths. These insults disrupt coagulation and endothelial systems. This study investigated whether previously reported differences in lesion size and brain swelling during normal saline (NS), colloids (Hextend [HEX]), and fresh frozen plasma (FFP) resuscitation are associated with differential effects on coagulation and endothelial systems. We subjected 15 Yorkshire swine to TBI and HS (40% blood volume), and kept in HS for 2hours before resuscitation with NS, HEX, or FFP. Markers of endothelial activation (E-selectin, Intercellular adhesion molecule [ICAM]-1), coagulation activation (prothrombin fragment 1+2), and natural anticoagulation (activated protein C [aPC]) were determined in serum and brain whole cell lysates. Serum levels of aPC were greater in the NS group (203±30 pg/mL) compared with HEX (77±28 pg/mL; P=.02) and FFP (110±28 pg/mL; P=.09), as was PF 1 + 2 in the brain when compared with FFP (PF 1 + 2, 89±46 vs 37±14ng/mL; P=.035). Brain E-selectin was greater in the NS group compared with FFP (3.36±0.02 vs 3.31±0.01ng/mL; P=.029). Circulating ICAM-1 levels were increased in the NS group (151±9ng/mL) compared with the HEX (100±9ng/mL; P<.01) and FFP (108±9ng/mL; P=.01). In this clinically realistic large animal model of TBI+HS, NS resuscitation was associated with an early activation of coagulation, natural anticoagulation, and endothelial systems, compared with HEX and FFP.

Characteristics of AIDS patients in Dutch intensive care units

Background: Infection with HIV enhances the risk to develop invasive bacterial infection, often resulting in sepsis. Both HIV infection and sepsis are associated with activation of the coagulation system. The influence of HIV infection on the sepsis-induced disturbance of the hemostatic balance is unknown. We aimed to determine the impact of HIV infection on coagulation activation during sepsis. Methods & Materials: We performed a prospective observational study in Lambarene, Gabon, and included all patients that were hospitalized with either fever or hypothermia and at least one other SIRS (Systemic Inflammatory Response Syndrome) criterion. Blood cultures, HIV testing and malaria diagnostics were performed. Patients with evidence of infection (based on adapted Centers for Disease Control and Prevention, and International Sepsis Forum Consensus Conference definitions) were classified as sepsis cases. Citrated plasma was obtained on admission, as well as in asymptomatic controls with or without HIV infection from the same region. Statistical analyses were done using the KruskalWallis test followed by a post-hoc Mann-Whitney U test. Results: We included113 sepsis patients (34 HIV positive), and 95 asymptomatic controls (60 HIV positive). Both asymptomatic HIV infection and sepsis were associated with activation of coagulation, as reflected by elevated levels of D-dimer and fibrinogen, and endothelial cell activation, as evidenced by elevated levels of Von Willebrand factor. Sepsis was associated with similar changes albeit to a greater extent; in addition, sepsiswas accompaniedwith downregulation of anticoagulant pathways, as reflected by reduced antithrombin and protein S levels. In asymptomatic HIV infection protein S was downregulated, whereas antithrombin levels were increased. Sepsis, but not HIV infection, resulted in prolonged prothrombin time and activated partial thromboplastin time. Coinfection with HIV did not impact on activation of coagulation during sepsis. Importantly, however, HIV co-infection did aggravate perturbations in VonWillebrand and protein S levels in sepsis patients. Conclusion: Both HIV infection and sepsis cause a net procoagulant state. HIV co-infection augments impairment of the anticoagulant system and activation of endothelium during sepsis. These mechanisms may play a role in enhanced sepsis related morbidity of HIV patients.

The influence of HIV infection on coagulation activation during sepsis

Background: Infection with HIV enhances the risk to develop invasive bacterial infection, often resulting in sepsis. Both HIV infection and sepsis are associated with activation of the coagulation system. The influence of HIV infection on the sepsis-induced disturbance of the hemostatic balance is unknown. We aimed to determine the impact of HIV infection on coagulation activation during sepsis. Methods & Materials: We performed a prospective observational study in Lambarene, Gabon, and included all patients that were hospitalized with either fever or hypothermia and at least one other SIRS (Systemic Inflammatory Response Syndrome) criterion. Blood cultures, HIV testing and malaria diagnostics were performed. Patients with evidence of infection (based on adapted Centers for Disease Control and Prevention, and International Sepsis Forum Consensus Conference definitions) were classified as sepsis cases. Citrated plasma was obtained on admission, as well as in asymptomatic controls with or without HIV infection from the same region. Statistical analyses were done using the KruskalWallis test followed by a post-hoc Mann-Whitney U test. Results: We included113 sepsis patients (34 HIV positive), and 95 asymptomatic controls (60 HIV positive). Both asymptomatic HIV infection and sepsis were associated with activation of coagulation, as reflected by elevated levels of D-dimer and fibrinogen, and endothelial cell activation, as evidenced by elevated levels of Von Willebrand factor. Sepsis was associated with similar changes albeit to a greater extent; in addition, sepsiswas accompaniedwith downregulation of anticoagulant pathways, as reflected by reduced antithrombin and protein S levels. In asymptomatic HIV infection protein S was downregulated, whereas antithrombin levels were increased. Sepsis, but not HIV infection, resulted in prolonged prothrombin time and activated partial thromboplastin time. Coinfection with HIV did not impact on activation of coagulation during sepsis. Importantly, however, HIV co-infection did aggravate perturbations in VonWillebrand and protein S levels in sepsis patients. Conclusion: Both HIV infection and sepsis cause a net procoagulant state. HIV co-infection augments impairment of the anticoagulant system and activation of endothelium during sepsis. These mechanisms may play a role in enhanced sepsis related morbidity of HIV patients.

Changes in Plasma Levels of Natural Anticoagulants in Disseminated Intravascular Coagulation: High Prognostic Value of Antithrombin and Protein C in Patients with Underlying Sepsis or Severe Infection

BackgroundDysfunctional natural anticoagulant systems enhance intravascular fibrin for mation in disseminated intravascular coagulation (DIC), and plasma levels of natural anti coagulants can be used in the diagnosis and prognosis of DIC. Herein, the diagnostic value of 4 natural anticoagulants was assessed, and the prognostic value of antithrombin and protein C were validated in a large population.MethodsPart 1 study included 126 patients with clinically suspected DIC and estimated plasma levels of 4 candidate anticoagulant proteins: antithrombin, protein C, protein S, and protein Z. Part 2 comprised 1,846 patients, in whom plasma antithrombin and protein C levels were compared with other well-known DIC markers according to the underlying dis eases. The 28-day mortality rate was used to assess prognostic outcome.ResultsAntithrombin and protein C showed higher areas under the ROC curve than pro tein S and protein Z. In part 2 of the study, antithrombin and protein C levels significantly correlated with DIC score, suggesting that these factors are good indicators of DIC severity. Antithrombin and protein C showed significant prognostic power in Kaplan-Meier analyses. In patients with sepsis/severe infection, antithrombin and protein C showed higher hazard ratios than D-dimer. Platelet count showed the highest hazard ratio in patients with hemato logic malignancy. In patients with liver disease, the hazard ratio for antithrombin levels was significantly high.ConclusionsDecreased plasma anticoagulant levels reflect florid consumption of the phys iologic defense system against DIC-induced hypercoagulation. Plasma antithrombin and protein C levels are powerful prognostic markers of DIC, especially in patients with sepsis/severe infection.

プロテインC・プロテインS 系とその臨床応用

プロテインC・プロテインS 系とその臨床応用

Activated protein C and thrombin participate in the regulation of astrocyte functions

Protein C anticoagulant system is a multifunctional cofactor-dependent system. In addition to anticoagulant function, activated protein C (APC) also exhibits neuroprotective activity in hypoxia and stroke, but there are no data on potential effects of APC on astrocytes. In the present work we have studied the influence of APC and thrombin on rat astrocytes in primary culture. It was found that thrombin at concentrations above 10 nM (1 U/mL) induced significant activation in the cultured astrocytes resulting in reactive astrogliosis. The cultures exposed to thrombin for 24 h demonstrated a significant increase in proliferation and the S100b protein expression. Thrombin at high concentrations produced visible changes in the cytoskeleton of astrocytes, in particular, an increase in the number of stress fibers in the cultured cells. Moreover, thrombin apparently affected astrocyte migration. Thus, the treatment of serum-starved astrocytes with thrombin resulted in changes in cell monolayer uniformity and formation of “free fields”. APC prevented thrombin-induced proliferation of astrocytes and the S100b protein expression, reducing the parameters under study to the control values. In addition, APC reduced thrombin-induced disorganization of fibrils and formation of “free fields”. The results have demonstrated a new aspect of the protective effect of APC, which suppresses astrocyte activation induced by the proinflammatory effect of thrombin. It suggests a potential application of APC as a regulator of astrogliosis in pathological brain conditions.

Is the neutrophil a 'prima donna' in the procoagulant process during sepsis?

Activation of the coagulation system is a fundamental host defense mechanism. Microorganisms that have invaded the body are trapped and disposed of in clots. Monocytes/macrophages are widely accepted as the main players in the procoagulant process; however, recent evidence suggests that neutrophils also play important roles. Tissue factor, which initiates the extrinsic coagulation cascade, is reportedly expressed on the surface of neutrophils, as well as on microparticles derived from neutrophils. Neutrophil extracellular traps (NETs) are another source of tissue factor. The components of NETs, such as DNA, histones, and granule proteins, also provide procoagulant activities. For instance, DNA initiates the intrinsic pathway, histones are a strong generator of thrombin, and granule proteins such as neutrophil elastase, cathepsin G and myeloperoxidase contribute to the suppression of the anticoagulation systems. Although understanding of the mechanisms that are involved in coagulation/fibrinolysis in sepsis has gradually progressed, the impact of neutrophils on thrombogenicity during sepsis remains to be addressed. Since the importance of the connection between coagulation and inflammation is advocated nowadays, further research on neutrophils is required.

Disorders of Hemostasis in Overt and Subclinical Hypercortisolism

Glucocorticoids are a group of hormones of a particular impact on hemostasis. Epidemiological studies show an approximately severalfold greater incidence of thromboembolic events in hypercortisolemic patients compared to those without hormonal disorders. The prothrombotic action of this steroid class is caused by both the direct impact of hypercortisolism on the activation of coagulation and the inhibition of fibrinolysis, as well as, the pathology of hemostasis due to metabolic disorders, which occur in this endocrinopathy. The aim of this study was to discuss the hemostasis abnormalities that occur in patients with overt and subclinical hypercortisolism with a particular emphasis on plasmatic coagulation, endogenous anticoagulation system, homocysteine and proinflammatory cytokines.