UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Studies of structure-function relationships in two human coagulation proteins: factor XII and prothrombin Côté, Hélène C. F.

Abstract

The epitope(s) of three anti human factor XII monoclonal antibodies were localized by screening a factor XII cDNA expression library in kgt11. One antibody, B7C9, had been shown previously to inhibit the activation of FXII by negatively charged surfaces. The positive recombinant phage contained inserts that coded for the amino-terminal 31 amino acids of FXII. These results were confirmed by binding of B7C9 to synthetic peptides containing amino acids 1-28 and 1-14 of FXII. Two other mAbs, C6B7 and KOK5 were also mapped. The epitope(s) for C6B7 which had been shown to inhibit factor XII activation was localized to amino acids 336-364, while KOK5 which inhibited the clotting activity of FXII mapped to amino acids 27-73. Four human FXIIcDNA constructs were expressed in BHK cells, using the pNUT vector: FXII wild type,FXIIA1-20, FXIIA5-20 and FXIIA28-69. The FXII wild type and FXIIA28-69 were secreted into the media at -5-10 [tg/mL while FXII01-20 and 5-20 were expressed in the cells but not secreted into the culture media. Recombinant human FXII was partially purified, analyzed by N-terminal sequencing, and assayed for amidolytic and clotting activity. These results indicate that the N-terminus of FXII might be involved in the binding to negatively charged surfaces, and that B7C9 blocks that interaction thereby inhibiting activation. During activation of prothrombin by the prothrombinase complex (FXa, FVa, phospholipids and Ca++), transient activation intermediates are produced. The intermediate meizothrombin has enzymatic activity but very little coagulant activity while intermediate prethrombin-2 has no enzymatic activity. Because meizothrombin is very sensitive to further activation and autolysis (converting it to meizothrombin (desFl)and ultimately thrombin), the isolation of meizothrombin is possible only in the presence of active-site thrombin inhibitors. This complicates studies of the activities and functions of meizothrombin. As a model, a mutant human prothrombin cDNA (R155A, R271A,R284A) (hMZ) was expressed, with three of the cleavage sites modified so that they are no longer cleaved by factor Xa or thrombin. Other mutants mimicking prethrombin-2(hPRE2) and "non-activable" prothrombin (hQM) were also expressed using the pNUT expression vector in BHK cells. When cultured in roller bottles, the cell lines secreted between 20 and 400 pg/mL of protein, at various levels of y-carboxylation. The secreted recombinant hMZ was purified to homogeneity and fractionated by Ca++ gradient chromatography to select for Ca++-binding and phospholipid-binding populations. Once activated by the prothrombinase complex or by ecarin, the rhMZ is converted to a meizothrombin-like molecule. Electrophoretic analysis and N-terminal sequence analysis were consistent with cleavage of a single bond between Arg320-11e321 and proper processing of the prepro-peptide. No other proteolytic cleavage was observed and rhMZa was stable for weeks at 4°C. Compared with human plasma-derived prothrombin, rhMZa demonstrated —7% clotting activity and 100% TAME esterase activity. The amidolytic activity of rhMZ toward S-2238 was found to be Ca++-dependent, and was identical to that of thrombin in the presence of 2 mM Ca++. Analysis of rhQM under the same conditions showed no cleavage of the molecule and no generation of activity. Furthermore, rhQM inhibited activation of prothrombin by the prothrombinase complex. These results indicate that these prothrombin mutants offer good models for further studies on the activity and physiological function of meizothrombin, and on the kinetics of prothrombin activation and the interactions between the components of the prothrombinase complex.

Item Media

Item Citations and Data

Rights

For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.

Usage Statistics