Inositolphospholipid-accelerated activation of prekallikrein by activated factor XII and its inhibition by β2-glycoprotein I

Authors

  • Inger SCHOUSBOE

    Corresponding author
    1. Department of Biochemistry C, Panum Institute, University of Copenhagen
      Correspondence to I. Schousboe, Biokemisk Institut C, Københavns Universitet, Panum Institutet, Blegdamsvej 3, DK-2200 København, Denmark
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Correspondence to I. Schousboe, Biokemisk Institut C, Københavns Universitet, Panum Institutet, Blegdamsvej 3, DK-2200 København, Denmark

Abstract

Inositolphospholipid-accelerated activation of prekallikrein by α-factor XIIa was determined by measuring the appearance of kallikrein amidolytic activity towards the chromogenic substrate, D-prolyl-phenylalanyl-arginyl p-nitroanilide (S-2302). The activation reaction did not exhibit normal Michaelis-Menten kinetics. The Hill coefficient was found to be 1.6 indicating that the activation followed an allosteric reaction mechanism. The temperature dependence of the reaction showed a thermal transition at 30°C, which in addition to the allosteric reaction mechanism is indicative of a conformational change of prekallikrein following binding to the inositolphospholipid. The reaction exhibited pH optimum at pH 7.2 and ionic strength optimum at 50 mM NaCl. At optimal conditions the apparent KA value and the kcat/KA value for factor XIIa on prekallikrein were calculated to be 73 nM and 9.3×106 s−1 M−1, respectively. Kinetic constants could not be calculated at salt concentrations higher than the optimal concentrations, as Lineweaver-Burk plots were curvilinear in agreement with the Hill coefficient greater than unity. The activation was inhibited competitively by β2-glycoprotein I with a Ki value of 77 nM as determined by the Dixon plot.

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