Analysis of plasmin generation and clot lysis of plasma fibrinogen purified from a heterozygous dysfibrinogenemia, B beta Gly15Cys (Hamamatsu II)

This is a non-final version of an article published in final form in Blood Coagulation & Fibrinolysis. 20(8):726-732, December 2009.We found a heterozygous dysfibrinogenemia caused by the substitution of B beta Gly15Cys and designated it fibrinogen Hamamatsu II (H-II). Although the propositus suffered an infarction of the medulla oblongata, other thrombotic risk factors, paradoxical cerebral infarction, and arterial dissection were not found. To determine whether the delayed lysis of fibrin clots or not in the context of the B beta Gly15Cys substitution, we examined the clot lysis and plasmin generation of propositus' fibrinogen. Fibrinogen was purified from the propositus' and normal control plasma by immunoaffinity chromatography and was used for the following experiments: sodium dodecyl sulfate-polyacrylamide gel electrophoresis, fibrin polymerization, scanning electron microscopic observation of fibrin clot and fibers, clot lysis, and tissue-type plasminogen activator-mediated plasminogen activation. The H-II plasma fibrinogen showed the presence of albumin-binding variant forms, a dimeric molecule of variant fibrinogen, and impairment of lateral aggregation during fibrin polymerization. The H-II fibrin clot showed lower density of bundles and thinner diameters of fibers than in the normal fibrin clot. In the clot lysis experiments with overlaid plasmin, H-II fibrin showed a similar lysis period and lysis rate to the normal control. Moreover, plasmin generation from a mixture of thrombin, tissue-type plasminogen activator, plasminogen, and H-II fibrinogen also showed a similar rate to normal fibrinogen. Although the propositus suffered an infarction, the present study did not observe delayed clot lysis, that is, the clot was not resistant to plasmin degradation. Therefore, we did not clarify an association between the B beta Gly15Cys dysfibrinogenemia and arterial thrombosis.ArticleBLOOD COAGULATION & FIBRINOLYSIS. 20(8):726-732 (2009)journal articl

Quantitative monitoring of single nucleotide mutations by allele-specific quantitative PCR can be used for the assessment of minimal residual disease in patients with hematological malignancies throughout their clinical course

BackgroundMonitoring of minimal residual disease (MRD) in patients with hematological malignancies is important for evaluating the patients\u27 therapeutic response and risk of relapse. Single nucleotide mutations associated with leukemogenesis can be considered as applicable MRD markers.MethodsWe developed an allele-specific quantitative polymerase chain reaction (AS-qPCR) for FLT3 2503G > T, KIT 2446G > T, and KIT 2447A > T and compared the change in the expression levels of the FLT3 or KIT mutations assessed by AS-qPCR to those of the RUNX1–RUNX1T1 fusion gene and WT1 by conventional quantitative PCR.ResultsThe AS-qPCR using primers including template-mismatched nucleotide or template-mismatched nucleotide plus locked nucleic acid substituted nucleotide provided higher selectivity for mutant nucleotides. The change in the expression levels of the FLT3 or KIT mutations at the time of relapse and just after hematopoietic stem cell transplantation correlated well with that of the RUNX1–RUNX1T1 fusion gene and WT1. Moreover, during complete remission, only AS-qPCR could detect low-level expression of residual mutations.ConclusionsThe AS-qPCR for analyzing single nucleotide mutations contributes to the monitoring of MRD in patients without recurrent fusion gene throughout the clinical course and thus broadens the spectrum of patients in whom MRD can be monitored

In vitro transcription of compound heterozygous hypofibrinogenemia Matsumoto IX; first identification of FGB IVS6 deletion of 4 nucleotides and FGG IVS3-2A > G causing abnormal RNA splicing

BackgroundWe reported a case of hypofibrinogenemia Matsumoto IX (M IX) caused by a novel compound heterozygous mutation involving an FGB IVS6 deletion of 4 nucleotides (Δ4b) (three T, one G; between FGB IVS6-10 and -16) and FGG IVS3-2A/G, which are both identified for the first time. To examine the transcription of mRNA from the M IX gene, we cloned the wild-type and mutant genes into expression vectors.MethodsThe vectors were transfected into CHO cells and transiently produced wild-type, Bβ- or γ-mRNA in the cells. The mRNAs amplified with RT-PCR were analyzed by agarose gel electrophoresis and nucleotide sequencing.ResultsThe RT-PCR product from FGB IVS6Δ4b showed aberrant mRNA that included both introns 6 and 7, and that from FGG IVS3-2G showed two aberrant mRNAs, a major one including intron 3 and a minor in which intron 3 was spliced by a cryptic splice site in exon 4. We speculated that the aberrant mRNAs are degraded before translation into proteins, and/or translated variant chains are subjected to quality control and degraded in the cytoplasm.ConclusionThe reduced plasma fibrinogen level of the M IX patient was caused by abnormal RNA splicing of one or both of the FGB and FGG genes