.alpha.-Thrombin-catalyzed activation of human platelet factor XIII: relationship between proteolysis and factor XIIIa activity

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Abstract
The kinetics of activation of platelet factor XIII, an a-subunit dimer, were characterized by determining rate constants for activation peptide (AP) release, generation of activity, and exposure of the active-site thiol group. The specificity constant (kcat/Km) for .alpha.-thrombin-catalyzed AP release, 1.2 .times. 105 M-1 s-1, was found to be similar to that for AP release from the tetramer plasma factor XIII (a2b2) [Janus, T. J., Lewis, S. D., Lorand, L., and Shafer, J. A. (1983) Biochemistry 22, 6269-6272], implying that the b subunits of plasma factor XIII do not hinder .alpha.-throbin-catalyzed cleavage of AP from the a subunit. Platelet factor XIIIa activity was generated at a rate approximately twice the rate of AP release. This difference in rates was shown to be consistent with a reaction pathway for activation of platelet factor XIII wherein full factor XIIIa activity is generated when one AP is removed from the dimeric zymogen so that removal of the second AP had no detectable effect on catalytic activity. In accord with this conclusion, the rate constant for exposure of the active-site thiol group, as measured by the incorporation of [1-14C]-iodoacetamide, was about twice that observed for the removal of AP. The observed stoichiometry of labeling with iodoacetamide of only one active-site thiol group per dimer for active factor XIIIa, together the the values of the rate constants for AP release, generation of activity, and exposure of the active-site thiol group, suggests that either (i) release of one molecule of AP per platelet factor XIII dimer triggers subunit interactions that cause both active-site thiol groups to become exposed with one thiol group becoming unreactive upon alkylation of the other or (ii) release of one molecule of AP per platelet factor XIII dimer triggers subunit interactions that expose one active-site thiol group and prevent simultaneous exposure of both active-site thiol groups even after the second molecule of AP is released.

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