RESUMO
The plasmin-antiplasmin system plays a key role in blood coagulation and fibrinolysis. Plasmin and α(2)-antiplasmin are primarily responsible for a controlled and regulated dissolution of the fibrin polymers into soluble fragments. However, besides plasmin(ogen) and α(2)-antiplasmin the system contains a series of specific activators and inhibitors. The main physiological activators of plasminogen are tissue-type plasminogen activator, which is mainly involved in the dissolution of the fibrin polymers by plasmin, and urokinase-type plasminogen activator, which is primarily responsible for the generation of plasmin activity in the intercellular space. Both activators are multidomain serine proteases. Besides the main physiological inhibitor α(2)-antiplasmin, the plasmin-antiplasmin system is also regulated by the general protease inhibitor α(2)-macroglobulin, a member of the protease inhibitor I39 family. The activity of the plasminogen activators is primarily regulated by the plasminogen activator inhibitors 1 and 2, members of the serine protease inhibitor superfamily.
Assuntos
Antifibrinolíticos/metabolismo , Plasminogênio/fisiologia , Antifibrinolíticos/química , Sítios de Ligação , Coagulação Sanguínea/fisiologia , Fibrinólise/fisiologia , Humanos , Modelos Biológicos , Modelos Moleculares , Plasminogênio/química , Ativadores de Plasminogênio/química , Ativadores de Plasminogênio/fisiologia , Inativadores de Plasminogênio/química , Inativadores de Plasminogênio/fisiologia , Estrutura Terciária de Proteína , Serina Proteases/química , Serina Proteases/fisiologia , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/fisiologia , alfa-Macroglobulinas/química , alfa-Macroglobulinas/fisiologiaRESUMO
The human alpha(2)-plasmin inhibitor (A2PI) possesses unique N- and C-terminal extensions that significantly influence its biological activities. The C-terminal segment, A2PIC (Asn(398)-Lys(452)), contains six lysines thought to be involved in the binding to lysine-binding sites in the kringle domains of human plasminogen, of which four (Lys(422), Lys(429), Lys(436), Lys(452)) are completely and two (Lys(406), Lys(415)) are partially conserved. Multiple Lys to Ala mutants of A2PIC were expressed in Escherichia coli and used in intrinsic fluorescence titrations with kringle domains K1, K4, K4 + 5, and K1 + 2 + 3 of human plasminogen. We were able to identify the C-terminal Lys(452) as the main binding partner in recombinant A2PIC (rA2PIC) constructs with isolated kringles. We could show a cooperative, zipper-like enhancement of the interaction between C-terminal Lys(452) and internal Lys(436) of rA2PIC and isolated K1 + 2 + 3, whereas the other internal lysine residues contribute only to a minor extent to the binding process. Sulfated Tyr(445) in the unique C-terminal segment revealed no influence on the binding affinity to kringle domains.