Evidence that the thrombin-catalyzed feedback cleavage of fragment 1.2 at Arg154-Ser155 promotes the release of thrombin from the catalytic surface during the activation of bovine prothrombin.
During the course of prothrombin activation, as catalyzed by Factor Xa, Factor Va, Ca2+, and negatively-charged phospholipid vesicles, the three proteins distribute between the fluid phase and the vesicle surface. On the vesicle, efficient Factor Xa-catalyzed proteolysis yields thrombin plus Fragment 1.2. Further thrombin-catalyzed feedback cleavage of the latter then yields Fragment 1 plus Fragment 2. Prior to this cleavage Fragment 1.2 might retain thrombin at the site of catalysis since it binds both phospholipid and thrombin through its respective Fragment 1 and Fragment 2 domains. In order to study the role of the feedback cleavage, light scattering at right angles was used to deduce the nature of the components associated with the vesicle during prothrombin activation by continuous monitoring of the relative molecular weight of the vesicle-protein complex. When prothrombin (1.4 microM) was added to homogeneously sized phospholipid vesicles of phosphatidylcholine-phosphatidylserine (3:1) at a total phospholipid concentration of 20 microM, the scattering intensity doubled. Upon subsequent addition of Factor Xa and Factor Va (5.0 nM each) the scattering intensity smoothly decreased to a value about 1.25-fold greater than that of the vesicles alone. Analysis of the composition of the reaction mixture at intervals during the course of the reaction by gel electrophoresis and laser densitometry, provided a good correlation between the mass of the vesicle-protein complex measured by light scattering and its mass inferred by composition. In addition, the decrease in mass of the vesicle-protein complex measured by light scattering correlated temporally with cleavage of Fragment 1.2. When the reaction was initiated in the presence of the reversible thrombin inhibitor dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide no cleavage of Fragment 1.2 occurred, as indicated by gel electrophoresis, and no change in the mass of the vesicle-protein complex occurred as indicated by light scattering. The absence of change in scattering intensity in the presence of dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide suggests a 1:1 replacement of prothrombin at the catalytic surface by components of equivalent mass (Fragment 1.2 plus thrombin), whereas the decrease in scattering in the absence of dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide suggests replacement of prothrombin by Fragment 1 only. Together these results indicate that the thrombin-catalyzed cleavage of Fragment 1.2 promotes release of thrombin from the catalytic surface.
Department of Medicine, Queen's University, Kingston, Ontario, Canada.
M E
ME
Nesheim
T
T
Abbott
R
R
Jenny
K G
KG
Mann
D000465:Algorithms; D001120:Arginine / Q000031:analogs & derivatives / Q000494:pharmacology; D003619:Dansyl Compounds / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005165:Factor V / Q000378:metabolism; D015943:Factor Va; D015951:Factor Xa; D006801:Humans; D008027:Light; D010446:Peptide Fragments / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism; D011516:Prothrombin / Q000378:metabolism*; D012542:Scattering, Radiation; D012697:Serine Endopeptidases / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology*
D003619:Dansyl Compounds; D010446:Peptide Fragments; D011498:Protein Precursors; C045879:prothrombin fragment 1.2; C019162:dansylarginine N-(3-ethyl-1,5-pentanediyl)amide; D015943:Factor Va; C029733:prothrombin fragment 1; D005165:Factor V; D011516:Prothrombin; D001120:Arginine; D012697:Serine Endopeptidases; D013917:Thrombin; D015951:Factor Xa