Cellular immune responses to platelet factor 4 and heparin complexes in patients with heparin-induced thrombocytopenia.

Essentials The immunogenesis of Heparin-induced thrombocytopenia (HIT) is not well understood. Immunization to platelet factor 4 (PF4)-heparin occurs early in life, before any heparin exposure. PF4 and PF4-heparin complexes induce the proliferation of CD14+ cells. Reduced levels of regulatory cytokines contribute to immune dysregulation in HIT. SUMMARY: Background Heparin-induced thrombocytopenia (HIT) is an adverse reaction to heparin characterized by thrombocytopenia and thrombotic complications. HIT is caused by pathogenic antibodies that bind to complexes of platelet factor 4 (PF4) and heparin, leading to platelet activation and inducing a hypercoagulable state. Previous studies have shown immunity to PF4-heparin complexes occurs early in life, even before heparin exposure; however, the immunogenesis of HIT is not well characterized. Objectives To investigate cellular proliferation in response to PF4-heparin complexes in patients with HIT. Patients/Methods Peripheral blood mononuclear cells (PBMCs) from healthy controls (n = 30), postoperative cardiac surgery patients who had undergone cardiopulmonary bypass (CPB) (n = 17) and patients with confirmed HIT (n = 41) were cultured with PF4 and PF4-heparin complexes. Cellular proliferation was assessed by [3 H]thymidine uptake and 5-ethynyl-2'-deoxyuridine detection. Results and Conclusions PBMCs proliferated in the presence of PF4, and this was enhanced by the addition of heparin in all study groups. CPB and HIT patients showed significantly greater proliferative responses than healthy controls. PBMC proliferation was antigen-specific, depended on the presence of platelets, and only CD14+ cells were identified as proliferating cells. Culture supernatants were tested for the levels of regulatory cytokines, and both CPB and HIT patients produced significantly lower levels of interleukin-10 and transforming growth factor-ß1 than healthy controls. These findings further demonstrate cellular immune sensitization to PF4-heparin complexes occurs before heparin exposure, and suggests immune dysregulation can contribute to HIT.

Thrombogenic effect of high-dose aspirin in rabbits. Relationship to inhibition of vessel wall synthesis of prostaglandin I2-like activity.

Aspirin is a promising antithrombogenic agent. It inhibits the generation of thromboxane A(2) by acetylating platelet cyclo-oxygenase. Aspirin also inhibits vessel wall production of PGI(2) which is an inhibitor of platelet aggregation, and therefore is potentially thrombotic. To investigate these two opposing effects we studied the effects of aspirin upon fibrin accretion onto experimentally induced venous thrombi in rabbits and on the PGI(2)-like activity of vessel wall using the thrombin-induced [(14)C]serotonin release assay. A 200-mg/kg dose of aspirin significantly augmented thrombus size when compared to (a) sodium salicylate administered in equal doses, (b) aspirin in a 10-mg/kg dose or (c) controls (P < 0.001). A 200-mg/kg dose of aspirin totally inhibited vessel wall PGI(2)-like activity whereas aspirin in a 10-mg/kg dose produced less inhibition, and 200 mg/kg sodium salicylate had no effect. Local instillation of tranylcypromine, an inhibitor of PGI(2) formation, also significantly augmented thrombus size compared to saline-treated controls and totally inhibited the production of PGI(2)-like activity. The thrombogenic effect of high dose aspirin was lost if an interval of 2.5 h or longer elapsed between vessel damage and drug administration, indicating that in contrast to the platelet, the effect of aspirin on vessel wall prostaglandin synthesis is relatively short-lived. It is concluded that aspirin, in doses higher than those used clinically, can augment experimental thrombosis, presumably by inhibiting the synthesis of vessel wall PGI(2).

Multimerin is found in the alpha-granules of resting platelets and is synthesized by a megakaryocytic cell line.

In this report, we describe the intracellular localization of multimerin in platelets and its biosynthesis by Dami cells, a megakaryocytic cell line. Immunoelectron microscopy was used to examine frozen thin sections of resting and activated platelets. Multimerin was localized within the platelet alpha-granule in an eccentric position. Within activated platelets, multimerin was found in the surface-connected open cannalicular system and on the external plasma membrane. Light microscopic immunocytochemistry demonstrated multimerin in normal megakaryocytes and in Dami cells after stimulation with PMA. Confirmation of multimerin biosynthesis by Dami cells was obtained by metabolic labeling studies. Both platelet and Dami cell multimerin demonstrated several subunit sizes on reduced SDS-PAGE. However, peptide mapping confirmed structural homology between the different multimerin subunits. Glycosidase digestion demonstrated that multimerin is heavily glycosylated with mainly complex, N-linked carbohydrate. In contrast to the multimerin isolated from platelets, cultured Dami cells secreted mainly smaller multimers of the protein. Biosynthesis of multimerin by a megakaryocytic cell line supports endogenous biosynthesis by megakaryocytes as the origin of this platelet alpha-granule protein.

Immune-mediated thrombocytopenia of malaria.

Thrombocytopenia frequently complicates malarial infections but the mechanism has not been elucidated. We studied 28 patients with malarial infections and noted that 16 of 17 thrombocytopenic patients had elevated levels of platelet-associated IgG (PAIgG). In all thrombocytopenic patients studied, the level of PAIgG returned to normal as the platelet count rose to normal levels. To study the mechanism of the elevated platelet-bound IgG, IgG and F(ab')2 from patients with recurrent Plasmodium falciparum infections was purified and radiolabeled. Labeled and unlabeled P. falciparum antigen was also prepared. IgG did not nonspecifically bind to malaria-damaged platelets. Binding studies with 3H-malarial antigen demonstrated platelets have saturable binding sites for malarial antigen. Increasing concentrations of malarial antigen displaced the 125I-IgG antimalarial antibody from the platelets. The binding of 125I-IgG and 125I-F(ab')2 was similar and this excluded significant immune complex binding. The thrombocytopenia that complicates at least some malarial infections is caused by immune mechanisms; specific IgG binds to platelet-bound malaria antigen through the Fab portion of the immunoglobulin molecule.

Argatroban anticoagulant therapy in patients with heparin-induced thrombocytopenia.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is an immune-mediated syndrome caused by heparin. Complications range from thrombocytopenia to thrombocytopenia with thrombosis. We report a prospective, historical- controlled study evaluating the efficacy and safety of argatroban, a direct thrombin inhibitor, as anticoagulant therapy in patients with HIT or HIT with thrombosis syndrome (HITTS). METHODS AND RESULTS: Patients with HIT (isolated thrombocytopenia, n=160) or HITTS (n=144) received 2 microgram. kg(-1). min(-1) IV argatroban, adjusted to maintain the activated partial thromboplastin time 1.5 to 3.0 times baseline value. Treatment was maintained for 6 days, on average. Clinical outcomes over 37 days were compared with those of 193 historical control subjects with HIT (n=147) or HITTS (n=46). The incidence of the primary efficacy end point, a composite of all-cause death, all-cause amputation, or new thrombosis, was reduced significantly in argatroban-treated patients versus control subjects with HIT (25.6% versus 38.8%, P=0.014). In HITTS, the composite incidence in argatroban-treated patients was 43.8% versus 56.5% in control subjects (P=0.13). Significant between-group differences by time-to-event analysis of the composite end point favored argatroban treatment in HIT (P=0.010) and HITTS (P=0.014). Argatroban therapy, relative to control subjects, also significantly reduced new thrombosis and death caused by thrombosis (P<0.05). Argatroban-treated patients achieved therapeutic activated partial thromboplastin times generally within 4 to 5 hours of starting therapy and, compared with control subjects, had a significantly more rapid rise in platelet counts (P=0.0001). Bleeding events were similar between groups. CONCLUSIONS: Argatroban anticoagulation, compared with historical control subjects, improves clinical outcomes in patients who have heparin-induced thrombocytopenia, without increasing bleeding risk.

Inappropriate antidiuretic hormone complicating histiocytic lymphoma.

The syndrome of inappropriate antidiuretic hormone (IADH) often causes the hyponatremia that may be seen in patients with malignant disorders. Most physicians correctly associate IADH with small cell carcinoma of the lung. We describe two patients in whom IADH was caused by histiocytic lymphoma. One patient was thought to have small cell carcinoma of the lung on the basis of marrow infiltration and the IADH. When the proper diagnosis was made and therapy instituted, both patients responded, with rapid resolution of their disease and the IADH. The identification of the neoplasm that produces the IADH is important, since histiocytic lymphoma may mimic small cell carcinoma of the lung, yet may be very responsive with newer treatment regimens.

Distinguishing between anti-platelet factor 4/heparin antibodies that can and cannot cause heparin-induced thrombocytopenia.

BACKGROUND: Many patients exposed to heparin develop antibodies against platelet factor 4 (PF4) and heparin, yet only those antibodies that activate platelets cause heparin-induced thrombocytopenia (HIT). Patients who produce anti-PF4/heparin antibodies without developing HIT either have antibodies that do not cause platelet activation or produce pathogenic antibodies at levels that are insufficient to cause HIT. Understanding the differences between anti-PF4/heparin antibodies with and without HIT will improve test methods and reduce overdiagnosis. AIMS: To investigate the presence of low levels of platelet-activating antibodies in patients investigated for HIT who had anti-PF4/heparin antibodies but failed to cause platelet activation in the (14) C-serotonin release assay (SRA). MATERIALS/METHODS: We developed a platelet activation assay similar to the SRA using exogenous PF4 without added heparin (PF4-SRA). This assay was able to detect low levels of platelet-activating antibodies. We used this PF4-SRA to test for platelet-activating antibodies in patients investigated for HIT. RESULTS: The PF4-SRA detected platelet-activating antibodies in seven (100%) of seven SRA-positive sera even after the samples were diluted until they were no longer positive in the standard SRA. Platelet-activating antibodies were detected in 14 (36%) of 39 patients who had anti-PF4/heparin antibodies but tested negative in the SRA and did not have clinical HIT. The clinical diagnosis of HIT was confirmed by chart review and concordant with the SRA results. CONCLUSIONS: A subset of heparin-treated patients produce subthreshold levels of platelet-activating anti-PF4/heparin antibodies that do not cause HIT. An increase in the titer of these pathogenic antibodies, along with permissive clinical conditions, could lead to HIT.

Severe bleeding events in adults and children with primary immune thrombocytopenia: a systematic review.

BACKGROUND: The burden of severe bleeding in adults and children with immune thrombocytopenia (ITP) has not been established. OBJECTIVES: To describe the frequency and severity of bleeding events in patients with ITP, and the methods used to measure bleeding in ITP studies. PATIENTS/METHODS: We performed a systematic review of all prospective ITP studies that enrolled 20 or more patients. Two reviewers searched Medline, Embase, CINAHL and the Cochrane registry up to May 2014. Overall weighted proportions were estimated using a random effects model. Measurement properties of bleeding assessment tools were evaluated. RESULTS: We identified 118 studies that reported bleeding (n = 10 908 patients). Weighted proportions for intracerebral hemorrhage (ICH) were 1.4% for adults (95% confidence interval [CI], 0.9-2.1%) and 0.4% for children (95% CI, 0.2-0.7%; P < 0.01), most of whom had chronic ITP. The weighted proportion for severe (non-ICH) bleeding was 9.6% for adults (95% CI, 4.1-17.1%) and 20.2% for children (95% CI, 10.0-32.9%; P < 0.01) with newly-diagnosed or chronic ITP. Methods of reporting and definitions of severe bleeding were highly variable in primary studies. Two bleeding assessment tools (Buchanan 2002 for children; Page 2007 for adults) demonstrated adequate inter-rater reliability and validity in independent assessments. CONCLUSIONS: ICH was more common in adults and tended to occur during chronic ITP; other severe bleeds were more common in children and occurred at all stages of disease. Reporting of non-ICH bleeding was variable across studies. Further attention to ITP-specific bleeding measurement in clinical trials is needed to improve standardization of this important outcome for patients.

Heparin-induced thrombocytopenia: an overview.

Heparin-induced thrombocytopenia (HIT) is the most important immunological drug reaction that patients face today. HIT typically develops in patients 5 days after starting heparin therapy, but can occur sooner with recent heparin exposure or rarely have a delayed onset. The platelet count typically drops below 150 x 10(9)/L (average 60 x 10(9)/L), and patients may experience a thrombotic episode simultaneously or shortly after the onset of thrombocytopenia. The thrombocytopenia and the associated thrombotic episodes are now considered to be overlapping outcomes of the same syndrome. The pathophysiology of HIT has been characterized: immune complexes of IgG and heparin in association with a small platelet peptide, platelet factor 4 (PF4), activate platelets by binding to the Fc receptors (FcR) and releasing procoagulant-active, platelet-derived microparticles. The recognition that HIT is characterized by intense thrombin generation dictates the use of antithrombin agents in HIT therapy. Therapeutic approaches that are currently prevalent in the management of HIT will be discussed.

Methyldopa-induced autoantibodies against red blood cells.

Methyldopa therapy results in the formation of red cell autoantibodies in 10-20% of patients taking the drug for longer than 4 months. These red cell antibodies are true autoantibodies, that is they are directed against an autoantigen on the red blood cell membrane and not against the drug or against a drug-altered antigen. The target membrane antigen is usually within the Rhesus system, although often the antibody specificity cannot be defined. Red cell antibody is usually detectable in the patient's sera as well as on the red cells. The autoantibody is usually a warm reacting IgG antibody. Most patients who develop these autoantibodies do not go on to develop hemolytic anemia in spite of high titres of antibodies on their red cells. In addition, these patients do not tend to develop hemolysis if methyldopa therapy is continued. Rarely patients develop hemolytic anemia which can be severe. Differences in antibody characteristics, including subclass restriction, complement-binding ability, or titre do not explain why some patients with autoantibody hemolyze while most do not. One group of investigators found that hemolyzing patients had IgM on their red cells while those who did not had IgG only. But while this observation could explain why some patients (IgM-sensitized red cells) hemolyze, it does not explain why most patients with IgG-sensitized red cells do not hemolyze. Why the autoantibody forms is not known but some investigators have proposed that the drug may directly affect B or T cells with resulting impairment of immune tolerance.(ABSTRACT TRUNCATED AT 250 WORDS)

Thrombocytopenia in pregnancy: diagnosis, pathogenesis and management.

Maternal thrombocytopenia is a common find during pregnancy. The rapid determination of its cause can often be difficult, with the diagnosis only being made in retrospect once the course of the platelet count is known. The majority of patients with thrombocytopenia in pregnancy will have incidental thrombocytopenia of pregnancy which is of no clinical significance.

The clinical management of heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is an immunologic complication of drug therapy with potentially serious venous and arterial thrombotic sequelae. Recent studies have provided insight into the molecular and cellular basis of HIT underlying the diagnosis and treatment of this syndrome. Because various approaches to HIT treatment have resulted in mixed success, the use of heparin substitutes, other anticoagulant agents, and adjunctive therapy needs to be clarified. For patients with pre-existing conditions such as deep venous thrombosis or pulmonary embolism, new understanding of the clinicopathologic syndrome of HIT offers the promise of improved antithrombotic therapy. The participation of thrombin in HIT suggests that specific (hirudin and argatroban) thrombin inhibitors may be ideal agents for treating acute HIT.

Management of idiopathic thrombocytopenic purpura in pregnancy.

Idiopathic thrombocytopenic purpura (ITP) is a relatively common autoimmune disorder among women of child-bearing age. It has a frequency of approximately one to two per 1,000 live births, accounting for about 3% of all cases of maternal thrombocytopenia at delivery. ITP in pregnancy necessitates the management of two patients, the mother and her baby; hence, the close collaboration of a multidisciplinary group composed of a hematologist, obstetrician, and pediatrician is essential. Our understanding of thrombocytopenia in pregnancy has evolved considerably over the last decade, yet the optimal diagnostic and treatment strategies for ITP in pregnancy continues to create controversy. In reviewing the recent literature, there is resurgence in the trend towards treating these patients in a more conservative fashion. This review will summarize the current approach to the diagnosis of ITP in pregnancy, as well as explore the pertinent and controversial issues of investigation and management.

The use of electron microscopy in the investigation of the ultrastructural morphology of immune thrombocytopenic purpura platelets.

Ultrastructural studies have proven useful for the accurate identification and classification of certain lymphoid and hematopoietic disorders; however, the value of electron microscopy in the diagnosis and clinical management of platelet pathology is less well defined. Electron microscopy has been used to evaluate inherited platelet disorders. In these disorders, certain platelet structural defects can be characteristic. Recently, we investigated the ultrastructural morphology and immunogold localization of IgG in platelets from patients with idiopathic thrombocytopenic purpura (ITP). The accelerated platelet destruction of ITP is mediated by antiplatelet autoantibodies directed against platelet surface glycoproteins and by the functional capacity of the reticuloendothelial system. The basic dysregulation that occurs in these patients remains unexplained. Traditionally, laboratory investigation of ITP has focused on the development of serologic assays to measure the autoantibodies. As an alternative investigative approach, determination of the immunomorphologic characteristics of platelet-associated IgG (PAIgG) in ITP platelets may prove useful in the diagnosis or clinical management of patients with ITP. Our results showed that the ultrastructural morphology of ITP platelets is similar to that observed for normal platelets. No structural abnormalities are observed in ITP platelets. Immunogold labeling of IgG within alpha-granules of ITP platelets is significantly higher than that of normal platelets. Additionally, in some ITP platelets, immunogold labeling is also observed on the platelet surface and within channels of the open-cannalicular system. In comparison, immunogold labeling of these structures in normal platelets, is rare, or absent. In conclusion, electron microscopic studies should contribute to furthering our understanding of this common autoimmune disorder, and may provide possible biological explanations for the increased levels of PAIgG in platelets from patients with ITP.

Investigation of human platelet alloantigens and glycoproteins using non-radioactive immunoprecipitation.

Sensitive techniques to detect platelet antibodies are needed for the investigation of immune thrombocytopenic syndromes such as neonatal alloimmune thrombocytopenia and post-transfusion purpura. Radioimmunoprecipitation has proved useful in the investigation of platelet-antibody interactions; however, the requirement for a radioactive label is a disadvantage. We describe the immunoprecipitation of human platelet proteins labelled with nonradioactive NHSS-biotin and compare the results with proteins labelled with 125I. The efficiency of labelling was evaluated by immunoprecipitation using well-characterized human anti-platelet antisera and murine monoclonal antibodies. The immunoprecipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose and detected using streptavidin-horseradish peroxidase and a chemiluminescent substrate with exposure to X ray film. The biotinylation technique labelled glycoproteins Ia/IIa, Ib/IX, IIb/IIIa, IV, and p175 which carry all of the known platelet alloantigens and isoantigens. It was as sensitive as radiolabelling and had the advantage of labelling GPs Ib beta and IX, which were not labelled using radioiodine. Human sera containing alloantibodies to HPA-1a on GP IIIa, HPA-3a on GP IIb, HPA-5a and HPA-5b on GP Ia, Govb on p175, and the isoantibody Naka on GP IV precipitated the corresponding biotinylated proteins. Biotinylated proteins could be detected using a 30 s exposure compared to 2 days or longer for 125I. Immunoprecipitation of human platelet glycoproteins labeled with NHSS-biotin is a fast and sensitive alternative to conventional radioimmunoprecipitation for the study of human platelet antigens.

A 14-year study of heparin-induced thrombocytopenia.

PURPOSE: To determine the sites of thromboses (venous versus arterial circulation) that complicate the clinical course of immunemediated heparin-induced thrombocytopenia, and to determine the 30-day risk for thrombosis in patients who are initially recognized with isolated heparin-induced thrombocytopenia. PATIENTS AND METHODS: We analyzed objectively documented thrombotic events that complicated the clinical course of 127 patients with serologically confirmed heparin-induced thrombocytopenia identified in one medical community over a 14-year period. We classified heparin-induced thrombocytopenia patients into two groups: patients recognized with heparin-induced thrombocytopenia only after a new thrombosis had occurred, and patients initially recognized with isolated heparin-induced thrombocytopenia. We determined the subsequent 30-day risk for thrombosis for the cohort of patients initially recognized with isolated thrombocytopenia. RESULTS: Heparin-induced thrombocytopenia was associated with the development of new venous thrombotic events and arterial thrombotic events in 78 and 18 patients, respectively (ratio venous/arterial thrombosis = 4:1). Pulmonary embolism was the most common life-threatening thrombotic event, occurring in 25% of all patients. Approximately half of all heparin-induced thrombocytopenia patients were recognized only after they had a complicating thrombotic event. Of the remaining 62-patient cohort initially recognized with isolated thrombocytopenia, the subsequent 30-day risk of thrombosis was 52.8%. The risk of thrombosis did not differ whether the heparin had been discontinued alone or whether warfarin had been substituted for the heparin. CONCLUSIONS: Venous thrombosis complicates heparin-induced thrombocytopenia more frequently than does arterial thrombosis. The high risk of thrombosis in patients initially recognized with isolated thrombocytopenia suggests that conventional management approaches require reappraisal.

Correlation of a quinidine-induced platelet-specific antibody with development of thrombocytopenia.

The rapidity by which drug-dependent antiplatelet antibodies can develop is not known, since patients are only studied during or after the episode of thrombocytopenia. This report describes the development of quinidine-induced immune thrombocytopenia in a healthy volunteer during a drug study. The thrombocytopenia developed within two weeks of initiation of quinidine therapy. During the thrombocytopenic episode, but not before receiving the drug, the patient had an IgG antiplatelet antibody that bound to control platelets in the absence of the drug. This antibody was absent when the drug was discontinued and the platelet count rose. The patient's acute serum also induced the release of serotonin from control platelets, and the reaction was enhanced by quinidine. This indicates that drug-dependent antiplatelet antibodies can develop rapidly and supports the hypothesis that quinidine-induced thrombocytopenia is due to a quinidine-dependent platelet-specific IgG.

The serological investigation of patients with autoimmune thrombocytopenia.

Techniques for measuring the plasma factor responsible for idiopathic thrombocytopenic purpura (ITP) have been sought for some 40 years, but unlike red cell serological techniques, platelet antibody testing has proved difficult and, at times, generated controversial results. The difficulty in measuring platelet antibodies probably reflects the intrinsic nature of the platelet membrane--to act as a sponge and nonspecifically bind plasma proteins. There have now been three different general types of techniques used to measure platelet autoantibodies: phase I assays measuring a platelet-dependent endpoint after incubating test serum and control platelets. These assays lack sufficient sensitivity and specificity to be useful. Except for special instances (the diagnosis of heparin-induced thrombocytopenia), they are no longer used. Phase II assays measure IgG on the surface of platelet or within the platelets following lysis. The immunoglobulin detected is termed platelet associated IgG. PAIgG assays are falling out of favour because they lack the sensitivity and specificity to be useful diagnostic tools. Most recently, the ability to isolate individual platelet glycoproteins and measure the binding of IgG to these glycoproteins has dramatically increased our understanding of ITP. These Phase III assays may prove useful diagnostic tools for the investigation and classification of immune thrombocytopenic disorders.

Factor V Leiden and thrombotic complications in heparin-induced thrombocytopenia.

To determine whether factor V Leiden is associated with thrombotic events in patients with heparin-induced thrombocytopenia (HIT), we evaluated 165 patients with serologically confirmed HIT for the presence of factor V Leiden and determined the incidence of venous or arterial thrombosis during the period of HIT. Factor V Leiden was detected in 16 of 165 HIT patients (9.7%). HIT-associated venous thrombosis occurred in 11 of 16 factor V Leiden positive subjects and 94 of 149 factor V Leiden negative subjects (69% vs. 63%; p = 0.79). Arterial thrombosis occurred in 1 of 16 factor V Leiden positive subjects and 21 of 149 factor V Leiden negative subjects (6% vs. 14%; p = 0.70). There was no difference in the incidence of proximal limb DVT, pulmonary embolism, venous limb gangrene, local skin reactions, hemorrhagic adrenal infarction, stroke, or myocardial infarction between the groups. No difference in the severity of venous thrombosis between Leiden positive and negative subjects was detected. Our data suggest that in the acute prothrombotic milieu of HIT, heterozygous factor V Leiden is not an important additional risk factor for thrombosis.

Current concepts in the treatment of immune thrombocytopenia.

Platelet destruction by antibodies is a common cause of thrombocytopenia. In this review, various treatments for immune thrombocytopenia are discussed, with emphasis on corticosteroids, splenectomy, danazol, high-dose immunoglobulin G, anti-Rhesus globulin, colchicine and cytotoxic, immunosuppressive agents such as cyclophosphamide and azathioprine. An approach to the treatment of adult idiopathic thrombocytopenic purpura (ITP) is reviewed in detail and the treatments for several other immune thrombocytopenic disorders are summarised.