Study of the fibrinolytic process in a patient with antiphospholipid syndrome.

The antiphospholipid syndrome (APS) is an autoimmune disease characterized by the presence of antibodies against ß2-Glycoprotein I (aß2-GPI), cardiolipin (aCL) and lupus anticoagulant combined with venous or arterial thrombosis and/or foetal losses. A 28-year-old female was positive for aß2-GPI, aCL, aPT (antibodies against prothrombin) and lupus anticoagulant. She had two miscarriages and a deep vein thrombosis event. The patient plasma fibrinogen and IgG concentrations were two times higher than control. Fibrinolysis was induced in vitro adding tPA, before clotting plasma with 0.03 or 0.6 IU/ml thrombin, and in purified system with normal fibrinogen in the presence of 0.5 mg/ml of patient or normal IgG. The APS patient had 1.5 and 1.9 times higher clot rate formation (CRL) and maximum absorbance (MaxAbsL) at both thrombin concentrations. At 0.6 IU/ml of thrombin, the patient delay on fibrin polymerization onset was corrected. The patient Lys50MA (time needed for 50% clot dissolution) was slower (P < 0.05) at 0.03 IU/ml of thrombin; however, the lysis rate was faster at both thrombin concentrations. After adjusting the polymerization and fibrinolytic parameters according to the sample plasma fibrinogen concentration, there were almost no differences between patient and control at 0.6 IU/ml. In an IgG-fibrinogen purified system, fibrinolysis was equivalent in the presence of patient or normal IgG. In conclusion, the patient IgG fraction has no inhibitors against proteins of the fibrinolytic pathway. The differences observed between the APS patient and the control were more evident at low thrombin concentration due to the presence of aPT.

A novel mutation in the FGB: c.1105C>T turns the codon for amino acid Bß Q339 into a stop codon causing hypofibrinogenemia.

Routine coagulation tests on a 14year-old male with frequent epistaxis showed a prolonged thrombin time together with diminished functional (162mg/dl) and gravimetric (122mg/dl) fibrinogen concentrations. His father showed similar aberrant results and sequencing of the three fibrinogen genes revealed a novel heterozygous nonsense mutation in the FGB gene c.1105C>T, which converts the codon for residue Bß 339Q to stop, causing deletion of Bß chain residues 339-461. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and RP-HPLC (reverse-phase high-pressure liquid chromatography) of purified fibrinogen showed only normal Aα, Bß, and γ chains, indicating that molecules with the truncated 37,990Da ß chain were not secreted into plasma. Functional analysis showed impaired fibrin polymerization, fibrin porosity, and elasticity compared to controls. By laser scanning confocal microscopy the patient's fibers were slightly thinner than normal. Electrospray ionization mass spectrometry (ESI MS) presented normal sialylation of the oligosaccharide chains, and liver function tests showed no evidence of liver dysfunction that might explain the functional abnormalities.

Structure of fibrin network of two abnormal fibrinogens with mutations in the αC domain on the human dermal microvascular endothelial cells 1.

There is evidence that clot structure can be modulated by endothelial cells, wherein the fibrinogen αC domain plays a major role in the fibrin-cell interaction. The spatial distribution of fibrin fibers from fibrinogen Caracas V and Caracas I, with heterozygous mutation in the αC domain (Aα Ser432Cys and Aα Ser466stop, respectively) on human dermal microvasculature endothelial cells (HMEC-1), was studied by laser scanning confocal microscopy. In order to assess fibrin-cell interaction and the role of the αC domain, preliminary experiments were done with inert microspheres and RGD peptide included in the clotting reaction, and forming clots with fibrinogen fragment X (fibrinogen without αC domain). Groups of stressed fibers were observed near the cell surface and were related to fibrin-cell interactions, which were abolished by the RGD peptide, and by the absence of the αC domain. The fibrin network of fibrinogen Caracas V and Caracas I was very different from that of normal fibrinogen. In general, patient's clots were characterized by very thin, tightly packed fibrin fibers, with a substantially reduced network porosity. Near the cell's surface, both abnormal fibrinogens formed a very fine meshwork, with stressed fibers 'anchored' to the cell surface, a pattern that was lost far from the cell surface. The structure of normal and patient clots performed in the absence of cells resembled that observed far from the cell surface, concluding that Caracas V and Caracas I fibrin was modulated by the presence of endothelial cells.