Fibrinolysis is amplified by converting alpha-antiplasmin from a plasmin inhibitor to a substrate.
alpha(2)-Antiplasmin (alpha(2)-AP) is the fast serpin inhibitor of plasmin and appears to limit the success of treatment for thrombosis. We examined the mechanisms through which monoclonal antibodies (mAbs) against alpha(2)-AP amplify fibrinolysis. The mAbs RWR, 49 and 77 interfered with the ability of alpha(2)-AP to inhibit plasmin, microplasmin and trypsin. In solution, mAbs 49 and 77 bound to alpha(2)-AP with 5-fold to 10-fold higher relative affinity than mAb-RWR, while mAb-RWR bound with greater avidity to immobilized or denatured alpha(2)-AP. Binding studies with chimeric alpha(2)-APs revealed that none of the mAbs bound to sites in alpha(2)-AP that form putative contacts with plasmin, namely the carboxy terminal lysines of alpha(2)-AP, or the reactive center loop in the serpin domain of alpha(2)-AP. Rather, mAb-RWR recognized an epitope in the amino-terminus of alpha(2)-AP (L(13)GNQEPGGQTALKSPPGVCS(32)) near the site at which alpha(2)-AP cross-links to fibrin. mAbs 49 and 77 bound to another conformational epitope in the serpin domain of alpha(2)-AP. mAbs 49 and 77 markedly increased the stoichiometry of plasmin inhibition by alpha(2)-AP (from 1.1 +/- 0.1 to 51 +/- 4 and 67 +/- 7) indicating that they convert alpha(2)-AP from an inhibitor to a substrate of plasmin. This was confirmed by sodium dodecylsulfate polyacrylamide gel electrophoresis analysis showing cleavage of alpha(2)-AP by plasmin in the presence of these mAbs. In summary, these mAbs appear to act at sites distinct from known alpha(2)-AP-plasmin contacts to enhance fibrinolysis by converting alpha(2)-AP from an inhibitor to a plasmin substrate.
Sazonova
I Y
IY
Medical College of Georgia, Augusta, GA, USA.
Thomas
B M
BM
Gladysheva
I P
IP
Houng
A K
AK
Reed
G L
GL
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry; D000939:Epitopes / Q000737:chemistry; D005341:Fibrinolysin / Q000037:antagonists & inhibitors* / Q000737:chemistry; D005342:Fibrinolysis; D006801:Humans; D008969:Molecular Sequence Data; D010958:Plasminogen / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013379:Substrate Specificity; D010959:Tissue Plasminogen Activator / Q000737:chemistry; D000979:alpha-2-Antiplasmin / Q000096:biosynthesis*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011994:Recombinant Proteins; D000979:alpha-2-Antiplasmin; D010958:Plasminogen; D010959:Tissue Plasminogen Activator; D005341:Fibrinolysin