Subject(s)
Carboxypeptidase B2/chemistry , Amino Acid Substitution , Animals , Carboxypeptidase B2/genetics , Carboxypeptidase B2/metabolism , Drug Stability , Humans , Models, Molecular , Mutagenesis, Site-Directed , Protein Conformation , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Species Specificity , ThermodynamicsABSTRACT
BACKGROUND: Activated thrombin activatable fibrinolysis inhibitor (TAFIa) plays a pivotal role in fibrinolysis. TAFIa activity is regulated by a temperature-dependent instability. This instability has not only complicated the study of structure-function relationships of TAFIa but has also prevented the crystallization of TAFIa. Furthermore, the TAFIa instability has severely compromised the development of activity inhibiting monoclonal antibodies. Recently, we combined all known stabilizing mutations (i.e. S305C, T325I, T329I, H333Y and H335Q) resulting in a synergistic (one hundred and eightyfold) stabilization of TAFIa at 37 degrees C. All these residues are located in an amino acid region (AA297-335) consisting of alpha-helix 9 and beta-sheet 11. OBJECTIVES: To provide a comparative evaluation of the characteristics of a panel of stable TAFIa mutants and an energy-minimized model of the most stable TAFI variant. RESULTS: The catalytic efficiency for activation of TAFI by thrombin/thrombomodulin was higher for all TAFI mutants compared with TAFI-wild type (wt). Except for TAFI variants carrying T325I-T329I, S305C-T325I or S305C-T325I-T329I mutations, the catalytic efficiency for Hip-Arg hydrolysis by TAFIa was similar for the TAFI mutants compared with the wild type. All TAFIa variants were equally well inhibited by potato tuber carboxypeptidase inhibitor (PTCI) and showed a significantly increased antifibrinolytic potential in accordance with their increased stability. Based on the intrinsic fluorescence decay of TAFIa, two independent structural transitions were found to be associated with the loss of functional activity. CONCLUSIONS: Using molecular dynamic calculations on both TAFI-wt and TAFI-S305C-T325I-T329I-H333Y-H335Q models, we were able to identify the molecular interactions that contribute to the increased stability of the mutants.
Subject(s)
Carboxypeptidase B2/chemistry , Carboxypeptidase B2/genetics , Animals , Carboxypeptidases/chemistry , Catalysis , Fibrinolysis , Humans , Mutation , Plant Proteins/chemistry , Protease Inhibitors , Protein Conformation , Protein Structure, Secondary , Rabbits , Structure-Activity Relationship , Temperature , Thrombin/chemistry , Thrombomodulin/chemistrySubject(s)
Carboxypeptidase B2/genetics , Mutation , Carboxypeptidase B2/chemistry , Fibrinolysis , Half-Life , Hemostasis , HumansABSTRACT
OBJECTIVE: To elucidate the mechanism and the binding regions of monoclonal antibodies (MA) that interfere with thrombin-activatable fibrinolysis inhibitor (TAFI)/activated thrombin-activatable fibrinolysis inhibitor (TAFIa) activity. RESULTS: Of 42 MA, 19 interfere with the TAFI activation/TAFIa activity resulting in an inhibition of up to 92%. Characterization of the mechanism of inhibition revealed that 14 MA blocked the activation of TAFI by thrombin/thrombomodulin completely whereas five MA interfered directly with the enzymatic activity of TAFIa. Surprisingly, the former, except one, induced a significant reduction of clot lysis time whereas the latter did not. Affinity studies using a human/murine TAFI chimer revealed that the binding region of the 14 activation blocking MA is located between AA1 and AA67. MA that inhibit exclusively the activation of TAFI by thrombin/thrombomodulin bind to Gly66. A MA that inhibits the activation of TAFI by both thrombin/thrombomodulin and plasmin binds to Val41. The MA that interfere with the enzymatic activity bind to the TAFIa moiety. CONCLUSIONS: The current study reveals at least three different putative molecular targets in the search for pharmacologically active compounds to modulate TAFIa activity.
Subject(s)
Antibodies, Monoclonal/pharmacology , Carboxypeptidase B2/metabolism , Protein Interaction Mapping , Animals , Binding Sites , Carboxypeptidase B2/antagonists & inhibitors , Carboxypeptidase B2/genetics , Fibrinolysin/metabolism , Genetic Variation , Humans , Mice , Thrombin/metabolism , Thrombomodulin/metabolismABSTRACT
We evaluated the prognostic role of peripheral blood polymerase chain reaction (PCR) assay for detection of the bcl-2(MBR)/J(H) rearrangement in 59 patients with follicular lymphoma (FL) treated at our centre since 1989. Thirty-five (59%) patients were bcl-2/J(H) positive and 24 (41%) were negative in the peripheral blood at diagnosis. Peripheral blood bcl-2/J(H) rearrangement detection at diagnosis had no relation to overall survival (OS) and time to progression (TTP). Peripheral blood PCR assay was performed post treatment in 17 patients who were bcl-2/J(H) positive at diagnosis. Fourteen of the patients (82%, 95% CI 56-96%) became bcl-2/J(H) negative. Nine of these patients were further analysed during follow-up and, after several months, circulating cells carrying the bcl-2/J(H) rearrangement reappeared in five of the nine patients. Peripheral blood clearance of bcl-2/J(H)-positive cells was correlated with better overall survival (log-rank P < 0.05) but not with TTP. Our data confirmed that bcl-2(MBR)/J(H) rearrangement detection by PCR at diagnosis is not a prognostic factor in follicular lymphoma. In our series, clearance of circulating bcl-2/J(H)-positive cells appeared to correlate with better overall survival. Post-treatment examination of the peripheral blood by PCR may have clinical relevance for prediction of the survival pattern of the patients.
Subject(s)
Genes, bcl-2 , Lymphoma, Follicular/blood , Lymphoma, Follicular/genetics , Neoplastic Cells, Circulating , Translocation, Genetic , Humans , PrognosisABSTRACT
BACKGROUND: Approximately one-fourth of diffuse large B-cell lymphomas (DLCL) carry the bcl-2(MBR)/JH rearrangement caused by the t(14;18) translocation. The clinical significance of this rearrangement in patients with DLCL remains controversial. By polymerase chain reaction (PCR) we prospectively evaluated the prognostic relevance of the bcl-2 (MBR)/JH rearrangement present in circulating B-cells at the time of diagnosis. MATERIALS AND METHODS: The bcl-2 (MBR)/JH rearrangement was analysed by a nested-PCR method in peripheral blood samples of 51 HIV-negative patients with previously untreated DLCL. RESULTS: The bcl-2 (MBR)/JH rearrangement was detected in 16 cases (31%). Peripheral blood bcl-2 (MBR)/JH rearrangement detection by PCR at diagnosis was correlated with poor overall survival, lymphoma-specific survival and time to progression (log-rank P < 0.05). There was no statistically significant difference between the clinical characteristics at presentation of bcl-2/JH-positive and negative patients. CONCLUSIONS: The peripheral blood is a readily accessible tissue for this type of analysis, and this study indicates that detection of the t(14;18) translocation at presentation in the blood of patients with DLCL may presage a poor prognosis.
