title: Effects of activation peptide bond cleavage and fragment 2 interactions on the pathway of exosite I expression during activation of human prethrombin 1 to thrombin.
abstract: Activation of prothrombin (Pro) by factor Xa to form thrombin occurs by proteolysis of Arg271-Thr272 and Arg320-Ile321, resulting in expression of regulatory exosites I and II. Cleavage of Pro by thrombin liberates fragment 1 and generates the zymogen analog, prethrombin 1 (Pre 1). The properties of exosite I on Pre 1 and its factor Xa activation intermediates were characterized in spectroscopic and equilibrium binding studies using the fluorescein-labeled probe, hirudin(54-65) ([5F]Hir(54-65)-(SO3-)). Prethrombin 2 (Pre 2), formed by factor Xa cleavage of Pre 1 at Arg271-Thr272, had the same affinity for hirudin(54-65) peptides as Pre 1 in the absence or presence of near-saturating fragment 2 (F2). Pre 2 and thrombin also had indistinguishable affinities for F2. By contrast, cleavage of Pre 1 at Arg320-Ile321, to form active meizothrombin des-fragment 1 MzT(-F1), showed a 11- to 20-fold increase in affinity for hirudin(54-65), indistinguishable from the 13- to 20-fold increase seen for conversion of Pre 2 to thrombin. Thus, factor Xa cleavage of Pre 1 at Arg271-Thr272 does not effect exosite I expression, whereas cleavage at Arg320-Ile321 results in concomitant activation of the catalytic site and exosite I. Furthermore, expression of exosite I on the Pre 1 activation intermediates is not modulated by F2, and exosite II is not activated conformationally. The differential expression of exosite I affinity on the Pre 1 activation intermediates and the previously demonstrated role of (pro)exosite I in factor Va-dependent substrate recognition suggest that changes in exosite I expression may regulate the rate and direction of the Pre 1 activation pathway.
authors.affiliation: Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
authors.forename: Patricia J
authors.initials: PJ
authors.lastname: Anderson
authors.forename: Anna
authors.initials: A
authors.lastname: Nesset
authors.forename: Paul E
authors.initials: PE
authors.lastname: Bock
mesh_terms: D001120:Arginine / Q000378:metabolism; D001665:Binding Sites; D004792:Enzyme Precursors / Q000737:chemistry / Q000378:metabolism*; D015951:Factor Xa / Q000378:metabolism; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006629:Hirudins / Q000378:metabolism; D006801:Humans; D010446:Peptide Fragments / Q000378:metabolism*; D011487:Protein Conformation; D011516:Prothrombin / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D013912:Threonine / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism*
chemical_list: D004792:Enzyme Precursors; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006629:Hirudins; D010446:Peptide Fragments; C091596:hirudin (54-65); D013912:Threonine; C045132:4-carboxyfluorescein; C044183:prothrombin fragment 2; D011516:Prothrombin; C012007:prethrombins; D001120:Arginine; D013917:Thrombin; C026812:meizothrombin; D015951:Factor Xa