Cryofibrinogenemia triggered by a monoclonal paraprotein successfully treated with cyclophosphamide.

Cryofibrinogenemia is a rare clinical finding with a yet unknown physiopathogenic mechanism. We describe the case of a woman with cold-induced extensive necrotic lesions that responded poorly to initial corticosteroid and anticoagulant therapies. Serum cryoglobulin determinations were persistently negative. After several years of evolution, she developed severe cold-related skin lesions that required left-leg amputation. Further analysis disclosed the presence of cryofibrinogen and an apparently insignificant serum monoclonal immunoglobulin Gκ peak. We additionally demonstrate that the cold precipitation of fibrinogen was directly related to the monoclonal paraprotein presence. The lesions responded dramatically to a B cell-targeted therapy with intravenous cyclophosphamide and dexamethasone.

[Pre-operative care for cardiac surgery patients with cold antibody disorder, cryoglobulinaemia and cryofibrinogenemia]. / Péce a pacienty s nemocí chladovych protilátek, kryoglobulinemií a kryofibrinogenemií pred kardiochirurgickými zákroky.

We present an example of a patient with confirmed cold agglutinin disease who underwent cardiac surgery in hypothermia to illustrate a known fact that, when exposed to cold, cold agglutinins induce haemolysis of erythrocytes and that cryoglobulins and cryofibrinogens may, upon exposition to cold during a surgery under hypothermia, precipitate or gelify and thus increase plasma viscosity and damage microcirculation. Detailed immunological and haematological investigations in all patients awaiting cardiac surgery with a risk of developing hypothermia is not advantageous considering the low number of patients with clinical and laboratory signs of cold agglutinin disease, autoimmune haemolytic anaemia or paroxysmal cold haemoglobinuria and considering that these investigations, in addition, might not detect cryoglobulinaemia and cryofibrinogenemia. Identification of in-risk patients from the warning signs in the medical history, physical or basal laboratory testing who would subsequently undergo confirmatory investigations to verify the presence of these entities and define them accurately might be a potential solution to this clinical issue. Cardiac surgery strategy and peri-operative care should be tailored to the results of these investigations. Well-structured, practiced and functional cooperation between clinicians and laboratory personnel is a prerequisite for success in these circumstances.

Fibrinogen Guarenas, an abnormal fibrinogen with an Aalpha-chain truncation due to a nonsense mutation at Aalpha 467 Glu (GAA)-->stop (TAA).

Fibrinogen Guarenas is a dysfibrinogenemia with a nonsense mutation at G4731T that causes an Aalpha-chain truncation at Ser 466. This abnormal fibrinogen is associated with a bleeding diathesis, severe in the proposita and mild in one brother, even though the fibrinogen levels in plasma are normal. All other family members are asymptomatic. Fibrinogens from the proposita and one family member, the mother of the proposita, both heterozygous for the mutation, were studied. Turbidity curves of fibrin polymerization showed that the lateral association of protofibrils was impaired and the maximum rate of polymerization was slightly diminished. The binding of albumin to fibrinogen was increased compared to control due to the presence of a free sulfhydryl group because of the missing disulphide bridge between Aalpha-Cys 442-472 in the mutated molecules. The abnormal fibrinogen formed much less alpha-polymer, and gamma-dimer formation was delayed compared to the control. Plasminogen activation by t-PA in the presence of fibrin was decreased. When Guarenas clots were perfused with fibrinolytic enzymes, clot degradation was retarded. Clot structure studied by confocal 3D microscopy showed that the fibrin network was dense, made up of thin and highly branched fibers, which accounted for the decreased flow rates by buffer permeation and increased rigidity of the fibrin clots, measured using a torsion pendulum. It seems that the increased clot rigidity, decreased porosity, hypofibrinolysis and t-PA induced fibrinolysis, by itself are not necessarily associated with thrombotic disorders in dysfibrinogenemia.

In vitro fibrin clot formation and fibrinolysis using heterozygous plasma fibrinogen from gammaAsn319, Asp320 deletion dysfibrinogen, Otsu I.

INTRODUCTION: We have reported a heterozygous dysfibrinogenemia, fibrinogen Otsu I, caused by the deletion of gammaAsn319 and gammaAsp320, which was originally identified in the dysfibrinogen Vlissingen/Frankfurt IV (V/FIV) associated with thrombosis. Unlike the V/FIV family, the Otsu propositus showed no thrombotic tendencies. To analyze the relationship between thrombosis and the heterozygous plasma variant fibrinogen, we used purified plasma fibrinogen from the Otsu patient and compared it with a normal control. MATERIALS AND METHODS: Thrombin-induced fibrin clot formation and clot structure were observed by fibrin polymerization and scanning electron microscopy, respectively. For in vitro observation of fibrinolysis, plasmin generation and clot lysis assays were performed by the addition of tissue type plasminogen activation (tPA) and plasminogen. RESULTS AND CONCLUSIONS: Polymerization of Otsu was markedly impaired, while fibrin fibers were much thicker and the density of the bundles of fibrin fibers was less and porous compared with normal. Lysis of the Otsu clot was not significantly different from normal when a tPA and plasminogen mixture was overlaid onto the clots. For Otsu, the penetration of the tPA/plasminogen mixture into the clot was much faster than normal and the protection against plasmin cleavage was impaired; however, tPA-induced plasmin activation of the Otsu fibrin was slower than that of normal fibrin, resulting in a clot lysis of Otsu similar to normal.

Binding of a new monoclonal antibody against N-terminal heptapeptide of fibrin alpha-chain to fibrin polymerization site 'A': effects of fibrinogen and fibrinogen derivatives, and pretreatment of samples with NaSCN.

A novel murine monoclonal antibody against the fibrin alpha-chain N-terminus is presented, which reacts with desAA- and desAABB-fibrin. In immunoblot procedures, the antibody reacted with fibrin degradation products X and Y of non-crosslinked fibrin, and fragment E. No binding was observed to the fibrin fragment D-dimer, and fibrinogen fragments D and E. Minor binding to fibrinogen fragments X, and Y, and desBB-fibrin were presumably due to minor contamination with (desAA)-fibrin. A prerequisite for binding was release of fibrinopeptides A (FpA), the binding site being a fibrin-specific neo-epitope. No binding was observed to fibrinogen or to thrombin-treated dysfibrinogen MANNHEIM III (A alpha 16 Arg-->Cys) molecules, which do not release FpA. The antibody bound to abnormal fibrin molecules prepared from dysfibrinogen MANNHEIM I (A alpha 19 Arg-->Gly), albeit to a lesser extent than to normal fibrin. Binding of the antibody to the fibrin epitope was greatly enhanced by denaturation, e.g. by heat, or by treatment with chaotropic ions. Soluble fibrin in clinical samples is generally found as a complex with fibrinogen, since polymerization sites 'A' exposed by release of FpA react with complementary binding sites 'a' on the D-domains of other fibrin and fibrinogen molecules. Treatment of samples with NaSCN caused dissociation of fibrin monomer complexes. Reassociation was prevented by denaturation of both polymerization sites 'A' and 'a'. The antibody in combination with NaSCN-treatment of samples was useful for specific detection of fibrin monomer in plasma samples. Measurement was not influenced by fibrinogen degradation products, whereas fibrin degradation products at very high concentration caused some underestimation of fibrin monomer concentration.

Fibrinogens Bern IV, Bern V and Milano XI: three dysfunctional variants with amino acid substitutions in the thrombin cleavage site of the Aalpha-chain.

Thrombin-induced cleavage of fibrinopeptide A is the initial step in the conversion of fibrinogen to fibrin. Three dysfunctional fibrinogen variants are described with an amino acid substitution at position 16 of the Aalpha-chain: the fibrinogen variants Bern IV and Milano XI having an Arg-->His substitution and the variant Bern V having an Arg-->Cys substitution. Routine coagulation studies revealed prolonged plasma thrombin and reptilase clotting times in all patients, and a discrepancy between the plasma levels of fibrinogen determined by the clotting assay and electroimmunoassay. The defect was localized by high-performance liquid chromatography analysis of fibrinopeptide release and confirmed by polymerase chain reaction and sequencing of exon 2 of the Aalpha-chain. Immunoblotting analysis with a rabbit antiserum against human serum albumin indicated that albumin was linked to the additional sulfhydryl group of fibrinogen Bern V. The assay of tissue-plasminogen-activator-induced plasmic degradation revealed that the fibrinolysis of fibrin Bern V was delayed, whereas fibrin Bern IV was digested in the same way as normal fibrin.

[Cryofibrinogenemia caused by monoclonal anti-fibrinogen antibodies]. / Kryofibrinogenämie durch monoklonale Anti-Fibrinogen-Antikörper.

HISTORY AND CLINICAL FINDINGS: For nine years a 54-year-old woman had been suffering from worsening treatment-resistant cold-dependent purpura of the limbs as well as cutaneous ulcerations and arthralgia, which recently had occurred even at a even slight decrease in room temperature. INVESTIGATIONS: A special form of cryofibrinogenemia was identified by affinity-chromatographic separation of a plasma cryoprecipitate. From this cryoprecipitate a monoclonal antifibrinogen antibody (IgG-kappa) was isolated which, in the cold, formed a precipitating complex with fibrinogen. Paraproteinaemia was not demonstrated by conventional serum and plasma electrophoresis. There was no evidence of neoplasma. TREATMENT AND COURSE: Attempted treatment with steroids, fibrinolytic agents and intravenous cyclophosphamide was unsuccessful. But long-term repeated plasmaphereses and anti-immunoglobulin adsorption improved the symptoms. After 5 years of this treatment-14 years after onset of symptoms-the patient died of the consequences of fulminant pulmonary embolism. CONCLUSION: To establish the diagnosis of monoclonal cryofibrinogenemia it is necessary, first, to identify the cryoprecipitate in plasma; secondly, to undertake affinity-chromatographic separation of the cryoprecipitate with subsequent analysis of its components.

Structural and vascular permeability abnormalities associated with lacunes of the human brain.

Histopathological and immune-histochemical studies were carried out in four cases with multiple lacunes of the central grey matter and in control cases without such lesions. Routine light microscopic techniques were applied on paraffin-embedded material to identify lesions that may represent developing lacunes. In addition, polyclonal antisera to human albumin, IgG, fibrinogen and fibronectin were chosen as markers for extravasated plasma proteins. The brain tissue between lacunes contained several forms of focal injuries which may represent precursors of lacunes. Such lesions included foci of status spongiosus and status cribrosus, regions with dilated extracellular spaces and astrocytic gliosis, and multi-locular cysts. These "lacune-associated lesions" often included albumin immune-reactivity in extracellular spaces, nerve cell bodies and astrocytes. Less frequently signs of extravasated IgG, fibrinogen and fibronectin were identified. Thus, lacunes of the human brain frequently showed signs of antigenic sites to albumin. The extracellular deposits probably represent extravasated material from the blood. Vasogenic and cytotoxic oedema combined with other factors probably play important roles during the formation of some of the lacunes.