Pathological coagulation parameters in as many as 54 patients with autoimmune acquired factor XIII deficiency due to anti-factor XIII autoantibodies.

INTRODUCTION: Autoimmune factor XIII (FXIII) deficiency (AiF13D) due to anti-FXIII autoantibodies is an extremely rare, life-threatening bleeding disorder that mostly occurs in the elderly. The number of patients diagnosed with AiF13D has been increasing in Japan, probably because of the nationwide survey on AiF13D supported by the Japanese Ministry of Health, Labour and Welfare. AIM: To explore the pathologic characteristics of coagulation parameters in AiF13D. METHODS: AiF13D-suspected cases were consulted, and underwent unified/integrated coagulation screening and were definitively diagnosed as AiF13D separately. RESULTS: AiF13D patients had lower FXIII antigen levels than non-AiF13D patients, but their values overlapped. Among a series of 22-item screening tests and their resulting parameters, the 'FXIII inhibitory potential' yielded by a 1:1 mixing test of the patient's and healthy control's plasma and its 'residual FXIII activity' in 54 AiF13D cases were most distinguishable from 139 non-AiF13D cases, followed by FXIII activity per se and FXIII-specific activity. While the cross-linked α2 -plasmin inhibitor level reduced, the levels of D-dimer, fibrin/fibrinogen degradation products and plasmin-plasmin inhibitor complex increased, probably because the patients' haematoma nonspecifically induced secondary fibrinolysis in both AiF13D and non-AiF13D patients. CONCLUSION: AiF13D appears to induce a hypocoagulopathy combined with a hyper-fibrinolytic state secondary to severe FXIII deficiency caused by anti-FXIII autoantibodies, and the consequent bleeding further modifies its pathological conditions. In addition, the 1:1 mixing test of FXIII activity was confirmed to be a reliable screening method for AiF13D, especially when its derivative parameter, such as the 'FXIII inhibitory potential' or 'FXIII inhibitory potential ratio', is employed.

A cluster of immunoresolvents links coagulation to innate host defense in human blood.

Blood coagulation is a protective response that prevents excessive bleeding upon blood vessel injury. We investigated the relationship between coagulation and the resolution of inflammation and infection by lipid mediators (LMs) through metabololipidomics-based profiling of human whole blood (WB) during coagulation. We identified temporal clusters of endogenously produced prothrombotic and proinflammatory LMs (eicosanoids), as well as specialized proresolving mediators (SPMs). In addition to eicosanoids, a specific SPM cluster was identified that consisted of resolvin E1 (RvE1), RvD1, RvD5, lipoxin B4, and maresin 1, each of which was present at bioactive concentrations (0.1 to 1 nM). Removal of adenosine from the coagulating blood markedly enhanced the amounts of SPMs produced and further increased the biosynthesis of RvD3, RvD4, and RvD6. The cyclooxygenase inhibitors celecoxib and indomethacin, which block the production of thromboxanes and prostanoids, did not block the production of clot-driven SPMs. Unbiased mass cytometry analysis demonstrated that the SPM cluster produced in human blood targeted leukocytes at the single-cell level, directly activating ERK and CREB signaling in neutrophils and CD14+ monocytes. Treatment of human WB with the components of this SPM cluster enhanced both the phagocytosis and killing of Escherichia coli by leukocytes. Together, these data identify a proresolving LM circuit, including endogenous molecular brakes and accelerators, which promoted host defense. These temporal LM-SPM clusters can provide accessible metabolomic profiles for precision and personalized medicine.

An atypical IgM class platelet cold agglutinin induces GPVI-dependent aggregation of human platelets.

Platelet cold agglutinins (PCA) cause pseudothrombocytopenia, spurious thrombocytopenia due to ex vivo platelet clumping, complicating clinical diagnosis, but mechanisms and consequences of PCA are not well defined. Here, we characterised an atypical immunoglobulin (Ig)M PCA in a 37-year-old woman with lifelong bleeding and chronic moderate thrombocytopenia, that induces activation and aggregation of autologous or allogeneic platelets via interaction with platelet glycoprotein (GP)VI. Patient temperature-dependent pseudothrombocytopenia was EDTA-independent, but was prevented by integrin αIIbß3 blockade. Unstimulated patient platelets revealed elevated levels of bound IgM, increased expression of activation markers (P-selectin and CD63), low GPVI levels and abnormally high thromboxane (TX)A2 production. Patient serum induced temperature- and αIIbß3-dependent decrease of platelet count in allogeneic donor citrated platelet-rich plasma (PRP), but not in PRP from Glanzmann's thrombasthenia or afibrinogenaemia patients. In allogeneic platelets, patient plasma induced shape change, P-selectin and CD63 expression, (14)C-serotonin release, and TXA2 production. Activation was not inhibited by aspirin, cangrelor or blocking anti-Fc receptor (FcγRIIA) antibody, but was abrogated by inhibitors of Src and Syk, and by a soluble GPVI-Fc fusion protein. GPVI-deficient platelets were not activated by patient plasma. These data provide the first evidence for an IgM PCA causing platelet activation/aggregation via GPVI. The PCA activity persisted over a five-year follow-up period, supporting a causative role in patient chronic thrombocytopenia and bleeding.

Late-onset hemorrhagic cystitis after haploidentical hematopoietic stem cell transplantation in patients with advanced leukemia: differences in ATG dosage are key.

Late-onset hemorrhagic cystitis (LOHC) is a common complication following allogeneic hematopoietic stem cell transplantation (HSCT), but its cause remains obscure. More attention to risk factors for LOHC is needed. We retrospectively analyzed patients with advanced leukemia who had been treated with ATG-containing conditioning regimens to evaluate the influence of different doses of ATG on LOHC after haploidentical HSCT. Seventy-five patients undergoing haploidentical HSCT from January 2003 to February 2011 were evaluated. A total of 39 patients receiving transplantation before June 2008 were treated with high-dose ATG (10 mg/kg), whereas 36 patients received low-dose ATG (6 mg/kg) thereafter. LOHC occurred in 16.7% of the patients with low-dose ATG, and in 38.5% of the patients with high-dose ATG (P = 0.027). Univariate analysis showed high-dose ATG, male gender and cytomegalovirus reactivation to be significant risk factors for LOHC. However, only high-dose ATG (HR 2.96, 95% CI 1.143-7.663, P = 0.025) and male gender (HR 4.033, 95% CI 1.355-12.008, P = 0.012) were independent risk factors, as shown by multivariate analysis. In the setting of haploidentical HSCT, we concluded that LOHC is more prevalent in recipients of high-dose ATG and male patients. Future studies should focus on immune reconstitution and virus infection after haploidentical HSCT with 6 mg/kg or 10 mg/kg ATG.

Aggressive fatal case of autoimmune hemorrhaphilia resulting from anti-Factor XIII antibodies.

Factor XIII (FXIII) is a fibrin-stabilizing factor consisting of catalytic A subunits (FXIII-A) and carrier B subunits (FXIII-B). Congenital FXIII deficiency is a rare bleeding disorder. Acquired FXIII deficiency resulting from FXIII hypo-synthesis and/or hyperconsumption is a relatively common disorder in which patients seldom bleed. On the contrary, 'autoimmune/acquired hemorrhaphilia XIII/13 due to anti-FXIII antibodies (AH13)' is a rare but life-threatening bleeding disorder. Through a nationwide survey of AH13, we diagnosed aggressive AH13 in a 66-year-old woman. She consulted our department because of a spontaneous hematoma in her hand. After 1.5 months, she also developed an intramuscular hematoma but retained approximately half (52%) of the normal FXIII activities. The patient's bleeding symptoms were aggravated to catastrophic massive bleedings in the large abdominal muscles and intrapelvic and intraperitoneal spaces. Two months after the bleeding onset, she died despite undergoing plasma exchange, which was performed because we were deeply suspicious of the presence of an anti-FXIII inhibitor. Seven days after her death, extremely low FXIII activity (6%) and positive data on anti-FXIII inhibitor were reported by a commercial laboratory. Our dot blot assay detected anti-FXIII-A autoantibodies, afterwards. Thus, the diagnosis of aggressive AH13 as early as possible is necessary to save patients' lives.

Acquired factor XIII inhibitor: clinical features, treatment, fibrin structure and epitope determination.

Acquired factor XIII (FXIII) deficiency, arising from an autoantibody against factor XIII, is a rare bleeding disorder. This autoimmune disorder most commonly occurs in the elderly. Patients who develop such acquired FXIII inhibitors may present with catastrophic bleeding events and are hard to be diagnosed with the normal general coagulation tests. Though the disease is relatively rare, it is known to cause significant mortality. In this article we briefly describe a patient who presented with extensive bleeding and a normal activated partial thromboplastin time and prothrombin time (PT), but had an acquired inhibitor to FXIII; her primary disease was systemic lupus erythematosus (SLE). Also, we will focus on the clinical features, treatment modalities, fibrin structure and epitope identification for acquired factor XIII inhibitor with a review of the literature.

Effect of desmopressin on immune-mediated haemorrhagic disorders due to canine monocytic ehrlichiosis: a preliminary study.

To evaluate the possible use of desmopressin acetate (DDAVP) in haemorrhagic disorders consequent to canine monocytic ehrlichiosis (CME), three dogs infected by Ehrlichia canis, with a history of thrombocytopenia and recent bleeding, were studied. The dogs were administered desmopressin (1 µg/kg b.w. s.c.) every 24 h on three occasions. Blood samples were collected immediately before, and after 2 and 48 h the first DDAVP administration, to assess haematological, clinical chemistry and clotting time parameters. Spontaneous bleeding stopped within 1 h after the first DDAVP injection. Buccal mucosa bleeding time (BMBT) was shortened from 9.6 to 2.3 min within 2 h after the treatment. A statistically significant increase in platelet PLT count and fibrinogen, and a statistically significant decrease of PT and aPTT were observed after DDAVP administration. The haemorrhagic disorders caused by CME appear to be quickly corrected by DDAVP administration, giving the clinician the time necessary to administer appropriate chemotherapy.

What role do coagulation disorders play in the pathogenesis of leptospirosis?

Leptospirosis is a zoonosis of worldwide distribution, spread by the urine of infected animals. It is a major public health problem, especially in developing countries, where circumstances for transmission are most favourable. The clinical picture varies from mild disease to a severe illness with haemostatic derangements and multiorgan failure eventually leading to death. Although the haemorrhagic complications of severe disease are serious, the pathophysiology is scarcely elucidated. The complex mechanisms involved in inflammation-induced coagulation activation are extensively studied in various infectious diseases, i.e. Gram-negative sepsis. Tissue factor-mediated coagulation activation, impairment of anticoagulant and fibrinolytic pathways in close concert with the cytokine network are thought to be important. But for human leptospirosis, data are limited. Because of the growing interest in this field, the impact of leptospirosis, and the availability of new therapeutic strategies, we reviewed the evidence regarding the role of coagulation in leptospirosis and provide suggestions for future research.

Lupus anticoagulant and anticardiolipin antibodies in polytransfused beta thalassemia major.

The currently used therapeutic strategies in beta thalassemia have prolonged the survival for many patients; this longer survival has been accompanied by the development of a number of unexpected complications, these include hemostatic derangements. The presence of anti-phospholipid antibodies (APA) (lupus anticoagulant, LA and anti-cardiolipid antibody, ACA) has recently been reported in polytransfused patients of beta thalassemia. In this study 50 patients with beta thalassemia major (beta-TM) who had received at least 20 transfusions were evaluated for presence of antiphospholipid antibodies and correlated with a number of clinical and hemostatic parameters. About 32% cases had developed a minor bleeding tendency and one also showed ecchymotic patch. None of the patients had thromboembolic episodes. LA was seen in 16% of cases. The mean age and number of transfusions were higher in LA positive patients as compared to LA negative patients however the results were not statistically significant. IgM ACA were detected in 6% of patients but no statistically significant correlation was found with age, number of transfusions, platelet count and coagulation parameters. IgG ACA were detected in 30% of cases and showed a significant correlation with number of blood transfusions (p = 0.016) and age (p = 0.031). Anti HCV antibodies were detected in 30% of patients out of which 1 had IgM ACA and 10 had IgG ACA. The latter showed a significant correlation with HCV infection. An increased incidence of LA and IgG ACA is found in polytransfused patients with beta-TM when compared to the normal healthy population but their clinical significance is yet not clearly understood.

Catastrophic microangiopathy induced by high-titre factor VIII inhibitors after liver transplantation for haemophilia A with cirrhosis.

Liver transplantation may induce immune tolerance to factor VIII inhibitors but de novo development of inhibitors after transplantation may cause intractable haemorrhage. We report a patient with mild haemophilia A and high-titre FVIII inhibitors who received an orthotopic liver transplantation for complications of hepatitis C virus cirrhosis. Recombinant activated FVII was used in addition to routine haemostatic agents. Conventional immunosuppression was supplemented with antithymocyte globulin and cyclophosphamide. FVIII inhibitors disappeared from the circulation with liver transplantation but they were found to have bound to the graft endothelium, which became activated and induced catastrophic microangiopathy. A subsequent anamnestic response resulted in FVIII inhibitor titres of 1000 Bethesda Units. Uncontrollable haemorrhage persisted until the recipient's death. In patients with high-titre FVIII inhibitors resilient desensitization is required before liver transplantation.

[Simultaneous onset of pemphigoid and factor VIII antibody hemophilia]. / Simultanes Auftreten eines Pemphigoids und einer Hemmkörperhämophilie.

A 47-year old patient who had been suffering from hypertension and chronic renal failure for many years developed progressive extensive haemorrhagic erosions of the mouth within 3 months and less severe erosions of the genital and nasal mucosa. Additionally, subcutaneous haematomas developed spontaneously. Laboratory investigations demonstrated circulating antibodies against factor VIII while direct and indirect immunofluorescent microscopy showed discrete tissue-bound and circulating IgG reactive with the epidermal basement membrane in a pemphigoid-like fashion. Immunoblot analysis of the patient's serum revealed an "atypical" IgG reactivity against a central portion of the extracellular domain of the BP180 antigen. These findings were unexpected, since the clinical aspect showed striking resemblance to (paraneoplastic) pemphigus. The patient developed life-threatening complications. Eventually, reduction of circulating autoantibodies by a combination of plasmapheresis and subsequent immunosuppressive therapy led to a stable remission of both autoimmune bullous skin disorder and acquired haemophilia.

Subproteomics analysis of phosphorylated proteins: application to the study of B-lymphoblasts from a patient with Scott syndrome.

Proteomics based approaches, which examine the expressed proteins of a tissue or cell type, complement the genome initiatives and are increasingly used to address biomedical questions. Proteins are the main functional output, and post-translational modifications such as phosphorylation are very important in determining protein function. To address this question, we developed a method for specific immunoprecipitation using anti-phosphotyrosine antibodies. This method is directly compatible with two-dimensional gel electrophoresis (2-DE). In this report data are presented on B-lymphoblasts from a patient suffering of Scott syndrome. Scott syndrome is an orphan inherited hemorrhagic disorder due to a lack of exposure of procoagulant phosphatidylserine at the exoplasmic leaflet of plasma membrane of blood cells. We hypothesized that a consequence of the mutation is to alter phosphorylation of proteins involved in signal transduction leading to breakdown in cellular signaling pathways mediating phosphatidylserine exposure. An immunoprecipitation method combined with 2-DE was applied to search for modifications in the expression of phosphorylated polypeptides related to Scott syndrome phenotype. We report here the construction of a B-lymphoblast subproteomic map comprising of polypeptides observed after immunoprecipitation using antibodies to phosphotyrosine. The polypeptides were identified either by mass fingerprinting, by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and/or by matching with various lymphoid cell 2-DE maps included in the Laboratoire de Biochimie des Protéines et Protéomique 2-DE database. A differential analysis was further performed to explore several hundred proteins in Scott B-lymphoblasts in comparison with control B-lymphoblasts. Then, image analysis allowed detection of variations between control and Scott syndrome phenotype lymphoblasts. Five spots were specifically found on 2-DE from Scott syndrome phenotype lymphoblasts, and four only appeared on 2-DE from control cells. Protein identification was achieved using a combination of mass fingerprinting and peptide identification using LC-MS/MS.

Evaluation of hemostasis disorders and anticardiolipin antibody in patients with severe leptospirosis.

A prospective study was designed to evaluate disorders of hemostasis and levels of anticardiolipin antibodies (ACL) in 30 patients with severe leptospirosis and acute renal failure (ARF) (ARF was defined as serum creatinine > or =1.5 mg/dL). The patients had been admitted to the Walter Cantídio University Hospital, São José Infectious Diseases Hospital and General Hospital of Fortaleza, Ceará, from August 1999 to July 2001. They all were male, with a mean age of 32 +/- 14 years and with clinical and laboratory diagnoses of ARF leptospirosis. The time elapsed between onset of symptoms and the first hemorrhagic manifestation was 9 +/- 4 days. Bleeding was observed in 86% of the patients. Laboratory tests showed significantly high levels of urea (181 +/-95 mg/dl), fibrinogen, (515 +/- 220 mg/dl), prothrombin time (13.3 +/- 0.9 seconds) and low platelet counts (69 +/- 65 x 10(3)/mm3) on admission. There was no elevation in activated partial thromboplastin time or thrombin time. Levels of IgM and IgG ACL concentrations were significantly increased (p < 0.05) in leptospirosis patients when compared to control patients (28.5 +/- 32.4 vs. 11.5 +/- 7.9MPL U/ml and 36.7 +/- 36.1 vs. 6.5 +/- 2.5 GPL U/ml), respectively. Vasculitis, thrombocytopenia and uremia should be considered important factors for the pathogenesis of hemorrhagic disturbances and the main cause of death in severe leptospirosis.

Molecular biological aspects of inhibitor development.

Mutation genotype studies have led to a number of interesting observations about the role of the different types of mutations in inhibitor development. Searchable information on mutations, phenotype data, models, etc., are available on the internet at http://europium.mrc.rpms.ac.uk. A relatively high incidence of inhibitors occurs in patients with deletion mutations. Stop mutations are also associated with a high incidence of inhibitors, although there is anomalous distribution of the inhibitors associated with different stop codons. Inhibitors have been associated with a small number of missense mutations. Missense mutations are of great interest from the structure/function viewpoint; in many instances, a missense mutation which results in reduced function of a circulating protein can be mapped onto a model structure and inferences can be made about the effects on the functionality of the protein. A model of the A domains of FVIII is available, but as yet, no structure is available on which to model the C domain of FVIII. Stop codons appear to have a different incidence of inhibitor formation compared to other types of mutations. It is concluded that while genotype mutation studies have led to a number of interesting observations about the role of the different types of mutations in inhibitor development, though more questions have been raised than answers provided to date.

Inhibitory anti-factor V antibodies bind to the factor V C2 domain and are associated with hemorrhagic manifestations.

Factor V inhibitors may develop as spontaneous autoantibodies, as alloantibodies after exposure to bovine thrombin preparations, or in factor V-deficient patients after plasma therapy. Clinical manifestations range from asymptomatic laboratory abnormalities to life-threatening hemorrhage. We have characterized the anti-factor V antibodies from 12 patients diagnosed with factor V inhibitors. In 8 patients, hemorrhagic complications (5 autoantibodies and 3 bovine thrombin-induced alloantibodies) developed, and 4 were asymptomatic (2 autoantibodies and 2 alloantibodies). The IgG fractions from all 12 patients immunoprecipitated the factor Va light chain, but only the 8 IgG fractions associated with hemorrhage inhibited factor V activity in a prothrombinase assay. Nine IgG fractions, including the 8 patients with hemorrhage, immunoprecipitated the isolated second C-type domain (C2). The 8 IgG fractions from the symptomatic patients also immunoprecipitated recombinant chimeras containing only the N-terminal third of the factor V C2 domain, and isolated recombinant C2 domain abrogated the inhibitory effect of the antibodies. Five of the inhibitory IgG fractions blocked binding of factor V to phosphatidylserine. These results suggest that inhibitory anti-factor V antibodies are associated with hemorrhagic manifestations and frequently bind to a common region within the C2 domain, whether originating spontaneously or after exposure to bovine thrombin.

Acquired haemophilia.

Acquired haemophilia is a rare but life-threatening acquired bleeding diathesis caused by autoimmune depletion of factor VIII. This occurs most frequently in elderly patients who lack disease associations. Acquired haemophilia may also arise in association with SLE rheumatoid arthritis, Sjögren's syndrome, other autoimmune conditions, lymphoproliferative malignancy, pregnancy and as a drug reaction. Acquired haemophilia has an equal sex distribution. The aims of treatment are to eliminate the inhibitor by immunosuppression and to treat the bleeding, which is the most common cause of death in patients with acquired haemophilia. The inhibitor is abolished in up to 70% of patients using prednisolone and cyclophosphamide, although other immunosuppressive regimens may also be used. These include azathioprine, vincristine and other cytotoxic agents, high-dose immunoglobulin and cyclosporin A. Bleeding may be controlled using porcine factor VIII or recombinant factor VIIa, although human factor VIII and prothrombin complex concentrates also have a limited role as haemostatic agents in this condition.