The interaction of prothrombin with phospholipid membranes is independent of either kringle domain.
Prothrombin contains two kringle domains, a structural motif common to other plasma proteins involved in hemostasis and fibrinolysis. To determine the role of the kringle domains of prothrombin, we prepared three recombinant human prothrombin forms lacking the first kringle domain (residues 63-144; PT/delta K1), the second kringle domain (residues 144-249; PT/delta K2), or both kringle domains (63-249; PT/delta K1,2). The isolated prothrombin proteins were greater than 95% pure by SDS-polyacrylamide gel electrophoresis and were well carboxylated. PT/delta K1 displayed 50% of the specific coagulant activity of plasma prothrombin, PT/delta K2 had 10% of the specific coagulant activity, and PT/delta K1,2 was inactive. Polyclonal antibodies directed against the Ca(II)-specific conformer of prothrombin bound PT/delta K1 and PT/delta K2 with the same affinity as prothrombin, indicating that the Ca(II)-induced conformational transition does not involve sites on the prothrombin kringle domains. Gel filtration studies demonstrated that radiolabeled plasma prothrombin and all of the prothrombin kringle deletion mutants bound to phospholipid vesicles in the presence of Ca(II) but not in the presence of Mg(II) or EDTA. Relative dissociation constants of 1.10 +/- 0.75 and 0.49 +/- 0.18 microM were obtained by quasielastic light scattering for the interaction of phospholipid vesicles with plasma prothrombin and PT/delta K1, respectively. These data indicate that neither the first nor the second kringle domain contain unique sites for the interaction of prothrombin with phospholipid vesicles and are not required for prothrombin-phospholipid binding.
Center for Hemostasis and Thrombosis Research, New England Medical Center, Boston, Massachusetts.
K J
KJ
Kotkow
B
B
Furie
B C
BC
Furie
D015055:1-Carboxyglutamic Acid / Q000737:chemistry; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008563:Membrane Lipids / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D011487:Protein Conformation; D011516:Prothrombin / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D008563:Membrane Lipids; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D010743:Phospholipids; D011994:Recombinant Proteins; D015055:1-Carboxyglutamic Acid; D011516:Prothrombin; D002118:Calcium