Thrombin Time Of Normal Plasma Research Articles

Characterization Of An Acquired IgG Autoantibody To Bβ and γ Chains Of Fibrinogen Resulting In Delayed Fibrin Polymerization and Severe Bleeding

A 88 year-old woman was referred to our center for 3 weeks of gastrointestinal bleeding and a spontaneous right rectus femoris hematoma. The prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT) and reptilase time (RT) were markedly prolonged (Table 1). Assays for coagulation factors V, VIII, IX and X were normal. A fibrinogen concentration of < 50 mg/dl was measured by Clauss method while fibrinogen concentration measured by radial immunodiffusion was 180 mg/dl. Following infusions of cryoprecipitate there was accelerated clearance of fibrinogen as measured by the Clauss method. An inhibitor of fibrinogen function was suspected.Table 1TestTime (sec)1:1 mix (sec)Normal Range (sec)PT>902212-15aPTT70.73923-36TT>120>12015-19Reptilase Time>120>12014.8-20.4When the patient's plasma was tested towards immobilized fibrinogen in ELISA plates, a concentration dependent specific binding of IgG was seen while under similar conditions control IgG had no significant binding (Figure 1). The IgG antibody was polyclonal comprising of both kappa and lambda subtypes. [Display omitted] The IgG fraction was purified on a Protein A column and the isolated IgG fraction prolonged the thrombin time of normal plasma in a concentration dependent manner. The effect of the isolated IgG fraction on fibrin polymerization was measured by monitoring the absorbance at λ390 in a spectrophotometer. The IgG inhibited fibrin polymerization in a concentration dependent manner. Control IgGs had no significant effect on polymerization under these conditions (Figure 2). [Display omitted] The immunologic specificity of antibody was tested in immunoblots of fibrinogen subjected to SDS-PAGE. The patients IgG reacted with fibrinogen in unreduced buffers. Under reducing conditions, the IgG reacted with Bβ and γ chains of fibrinogen while the control IgG did not (Figure 3). [Display omitted] The patient was treated with rituximab and steroids. After four, weekly doses of rituximab, fibrinogen levels by Clauss method normalized (450 mg/dl). There was no detectable antibody to fibrinogen and no further bleeding was noted. ConclusionThis is a description of a rare case of a polyclonal autoantibody to Bβ and γ chains of fibrinogen that inhibited fibrin polymerization leading to a severe bleeding diathesis. Disclosures:No relevant conflicts of interest to declare.

Fibrinogen Oviedo I. A new Spanish dysfibrinogenaemia

An abnormal fibrinogen was discovered in the plasma of a clinically asymptomatic woman. Laboratory evaluation of five members of the affected family showed low fibrinogen values in kinetic assays whereas the fibrinogen levels, tested by immunological procedures were normal. The patient's plasma had an inhibitory effect on the thrombin time of normal plasma. The calcium ions totally corrected the thrombin and reptilase times. Either low or high ionic strength prolonged the thrombin time of the proposita's purified fibrinogen. Kinetic analysis of clotting by monitoring transmission at 350 nm showed abnormally slow clotting with thrombin and reptilase. Assays were preformed in whole plasma as well as in purified fibrinogen. A delay in the rate of polymerization was evident when purified patient monomers were compared with those of normals. Immunoelectrophoretic, chromatofocusing, and isoelectrofusing experiments detected neither structural nor immunological abnormalities of fibrinogen. The rate of release of fibrinopeptide A by thrombin, measured by a specific immunoenzymatic method was also normal. HPLC analysis showed normal liberation of fibrinopeptides after prolonged thrombin action. Cross-linking of fibrin by factor XIII and lysis of fibrinogen by plasmin were normal. In view of these results, the defect of this dysfibrinogenemia, designated as Fibrinogen Oviedo I, probably could be due to conformational modifications in the D section of the molecule.

Fibrinogen Cleveland II. An abnormal fibrinogen with defective release of fibrinopeptide A.

An abnormal fibrinogen (fibrinogen Cleveland II) was detected in the plasma of a 23-yr-old white man with a mild bleeding diathesis. The one-stage prothrombin time, thrombin time, and Reptilase time were all prolonged. 16 of 24 tested relatives had the defect, which appeared to be transmitted as an autosomal dominant characteristic. The thrombin time of normal plasma was slightly inhibited by the proband's plasma. The abnormally long thrombin time of fibrinogen Cleveland II was partially corrected by addition of calcium ions. Fibrinogen Cleveland II was indistinguishable from normal fibrinogen by immunoelectrophoresis, DEAE-cellulose column chromatography, or polyacrylamide gel electrophoresis of reduced fibrinogen in sodium dodecyl sulfate. The major defect detected appeared to be impaired release of fibrinopeptide A when fibrinogen Cleveland II was incubated with thrombin. This defect was localized to the NH(2)-terminal disulfide knot portion of the molecule. An abnormality of polymerization of fibrin monomers was also present, but the abnormal fibrin demonstrated relatively normal crosslinking. Despite these defects, fibrinogen Cleveland II achieved a degree of coagulability similar to normal fibrinogen and appeared to incorporate some molecules of fibrin with intact fibrinopeptide A into the clot. The fibrin clot that was formed appeared to be abnormal by electron microscopy. These functional defects and other descriptive characteristics appear to distinguish fibrinogen Cleveland II from other inherited abnormal fibrinogens.

Fibrinogen Philadelphia. A hereditary hypodysfibrinogenemia characterized by fibrinogen hypercatabolism.

A new, autosomally inherited abnormal fibrinogen associated with hypofibrinogenemia has been described in several members of a family. Plasma fibrinogen measured either as thrombin-clottable protein or by immunodiffusion revealed a fibrinogen level ranging between 60 and 90 mg/100 ml. The thrombin time of plasma or purified fibrinogen was prolonged and only partially corrected by the addition of calcium. Purified fibrinogen prolonged the thrombin time of normal plasma. Fibrinopeptide release by thrombin was normal in rate and amount, but fibrin monomer aggregation was grossly disturbed, especially in a high ionic strength medium. We have designated this fibrinogen "fibrinogen Philadelphia." Acrylamide gel electrophoresis of mixtures of [121I]normal and [125I]abnormal fibrinogens revealed a slight increase in the anodal mobility of fibrinogen Philadelphia. Similarly, DEAE-cellulose chromatography showed slightly stronger binding of fibrinogen Philadelphia than normal. To elucidate the mechanism responsible for the low plasma fibrinogen concentration, simultaneous metabolic studies of autologous (patient) and homologous (normal) fibrinogen, labeled with 125I and 121I, respectively, were performed in two affected subjects. Autologous fibrinogen half-life was short and the fractional catabolic rate was markedly increased in both family members. In contrast, homologous fibrinogen half-life and fractional catabolic rate were normal. These metabolic studies demonstrate that rapid degradation of fibrinogen Philadelphia is largely responsible for the depressed levels of a plasma fibrinogen. This represents the first example of a mutant plasma protein in which the molecular defect is associated with an altered catabolism.