Clinical And Basic Study Of Fibrinogen Abnormalities

Fibrinogen, a 340-kDa glycoprotein, is composed of 2 identical molecular halves, each having an Aα, a Bβand aγchain joined by an extensive network of 29 intra- and inter-chain disulfide bonds. Fibrinogen is the product of three closely linked-genes ( FGA, FGB, and FGG) which are clustered in a 50-kb region on chromosome 4q28-4q31. Fibrinogen plays a crucial role in the hemostasis process. Fibrin monomers, formed by thrombin cleavage of fibrinogen molecules, polymerize to form a fibrin network and mediate platelet aggregation. However, its mechanism and location of the major catabolic pathway remain unknown. Studies of fibrinogen abnormalities contribute to understand the relationship between function and metabolism. Fibrinogen abnormalities comprise 5 types of plasma fibrinogen defects: afibrinogenemia, hypofibrinogenemia, dysfibrinogenemia, hypodysfibrinogenemia and hyperfibrinogenemia. In this study, we found three patients (pedigrees) with hyperfibrinogenemia, hypofibrinogenemia, and hypodysfibrinogenemia, repectively, and explored a preliminary study on etiology and pathogenesis.1. Hyperfibrinogenemia and prolonged clotting times in a Turner syndrome patient with hepatocellular carcinomaObject To diagnose a Turner syndrome (TS) patient complicated with well differentiated hepatocellular carcinoma (HCC) and to investigate the mechanisms underlying the hyperfibrinogenemia and its effects on coagulation tests. Methods Plasma fibrinogen was analyzed by Clauss, immunoturbidimetry and Western blotting methods. The fibrinogen genes were sequenced. Activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) were measured. Fibrinogen expression in tumor tissues was examined immunohistochemically. Plasma cortisone, interleukin 6 (IL-6) and soluble tissue factor (sTF) were measured by immunoassays. Results Abundant fibrinogen protein was detected in tumor cells. Plasma fibrinogen activity and antigen were 14.4±0.8 and 15.1±0.3 g/L, respectively. On SDS-PAGE, patient and control fibrinogen subunits migrated similarly. No mutations were found in the fibrinogen genes. APTT, PT and TT were significantly prolonged, but were normalized when fibrinogen was partially absorbed by an anti-fibrinogen antibody. Plasma IL-6 and sTF levels were increased compared with those of controls. After tumor resection, plasma fibrinogen level and other abnormal laboratory tests returned to normal. Conclusions Plasma fibrinogen level was increased in a TS patient with HCC. High levels of plasma cortisone and IL-6 also were detected, which may contribute to the hyperfibrinogenemia. The increased level of plasma fibrinogen prolonged APTT, PT and TT. Our studies showed hyperfibrinogenemia altered clotting times in a unique TS patient with HCC.2. Inherited dysfibrinogenemia caused by Arg16His in Aαchain of fibrinogenObjective To analyze the phenotype and genotype of a family with inherited dysfibrinogenemia. Method Assays of coagulation,including APTT, PT and TT,were carried out with Stago Compact in the proband and his family members. The activity and antigen of fibrinogen in plasma were determined by Clauss and immunoturbidimetry, respectively. Fibrinogen and its constituents were analyzed in western blot with nonreducing 4%~20% SDS-PAGE. All exons and exon-intron boundaries of FGA, FGB and FGG were analyzed by PCR and then direct sequencing. Results The proband had normal APTT and PT, but prolonged TT. The activity of fibrinogen in plasma was decreased while its antigen level was normal. These abnormalities were also found in his mother and sister. Genetic analysis revealed heterozygous G1233→A in the exon 2 of FGA originating from his mother, which causes Arg16His missense mutation. Conclusion Inherited dysfibrinogenemia was caused by Arg16His mutation in the exon 2 of FGA and it is the first case reported in a Chinese family.3. A novel 4-bp insertion mutation in fibrinogen B beta-chain gene causing severe hypodysfibrinogenemia Objective To identify etiology and pathogenesis underlying hypodysfibrinogenemia in a Chinese family by sequencing genetic DNA and analysing expression of fibrinogenγchain in patient's plasma. Method Assays of coagulation,including APTT, PT , TT and fibrinogen activity, were carried out in the proband and his family members. Fibrinogen antigen was determined by immunoturbidimetry and ELISA. The specificity of antibodies to fibrinogen and expression of fibrinogenγchain in patient's plasma were analyzed on SDS-PAGE. Genetic DNA of fibrinogen were analyzed by PCR and then direct sequencing. Results Coagulation routine showed prolonged clotting times, including APTT 76.5 sec (N 28-40 sec), PT 79.5 sec (N 11-14.5 sec), TT 79.1 sec (N 14-21 sec). Plasma fibrinogen activity was undetectable, but fibrinogen antigen level determined was 0.43 g/l (N 2.0-5.0 g/l). His parents, brother and his son had normal APTT, PT and TT, but approximately half the normal level of plasma fibrinogen. Genetic analysis revealed a novel GTTT insertion in homozygosity between nt 2819 and 2820 of FGB exon 2 in the proband and the insertion in codon 40 predicts a truncated 50 polypeptide containing 10 exceptional abnormal residues. This mutation was also present in the heterozygous state in the proband's parents, brother and his son. On SDS-PAGE, the band at ~ 42 kDa representingγchain under reducing condition were similar to those of the control, but the bands with mobilities comparable to ~148 kDa under nonreducing condition were more abundant than those of the control. Conclusion Inherited hypodysfibrinogenemia was caused by GTTT insertion in FGB exon 2 in a homozygous state giving a frameshift and then premature termination at codon 50.