Pathogen activators of plasminogen.

Group A streptococci (GAS) express soluble and surface-bound virulence factors. Secreted streptokinase (SK) allelic variants exhibit varying abilities to activate host plasminogen (Pg), and GAS pathogenicity is associated with Pg activation and localization of the resulting plasmin (Pm) on the bacterial surface to promote dissemination. The various mechanisms by which GAS usurp the host proteolytic system are discussed, including the molecular sexuality mechanism of conformational activation of the Pg zymogen (Pg*) and subsequent proteolytic activation of substrate Pg by the S•KPg* and SK•Pm catalytic complexes. Substantial progress has been made to delineate both processes in a unified mechanism. Pm coats the bacteria by direct and indirect binding pathways involving plasminogen-binding group A streptococcal M-like (PAM) protein and host fibrin(ogen). Transgenic mouse models using human Pg are being optimized to mimic infections by SK variants in humans and to define in vivo combined mechanisms of these variants and PAM.

Exosites in the substrate specificity of blood coagulation reactions.

The specificity of blood coagulation proteinases for substrate, inhibitor, and effector recognition is mediated by exosites on the surfaces of the catalytic domains, physically separated from the catalytic site. Some thrombin ligands bind specifically to either exosite I or II, while others engage both exosites. The involvement of different, overlapping constellations of exosite residues enables binding of structurally diverse ligands. The flexibility of the thrombin structure is central to the mechanism of complex formation and the specificity of exosite interactions. Encounter complex formation is driven by electrostatic ligand-exosite interactions, followed by conformational rearrangement to a stable complex. Exosites on some zymogens are in low affinity proexosite states and are expressed concomitant with catalytic site activation. The requirement for exosite expression controls the specificity of assembly of catalytic complexes on the coagulation pathway, such as the membrane-bound factor Xa*factor Va (prothrombinase) complex, and prevents premature assembly. Substrate recognition by prothrombinase involves a two-step mechanism with initial docking of prothrombin to exosites, followed by a conformational change to engage the FXa catalytic site. Prothrombin and its activation intermediates bind prothrombinase in two alternative conformations determined by the zymogen to proteinase transition that are hypothesized to involve prothrombin (pro)exosite I interactions with FVa, which underpin the sequential activation pathway. The role of exosites as the major source of substrate specificity has stimulated development of exosite-targeted anticoagulants for treatment of thrombosis.

The staphylocoagulase family of zymogen activator and adhesion proteins.

Staphylocoagulase (SC) secreted by Staphylococcus aureus is a potent non-proteolytic activator of the blood coagulation zymogen prothrombin and the prototype of a newly established zymogen activator and adhesion protein (ZAAP) family. The conformationally activated SC.prothrombin complex specifically cleaves fibrinogen to fibrin, which propagates the growth of bacteria-fibrin-platelet vegetations in acute bacterial endocarditis. Our recent 2.2 A X-ray crystal structures of an active SC fragment [SC(1-325)] bound to the prothrombin zymogen catalytic domain, prethrombin 2, demonstrated that SC(1-325) represents a new type of non-proteolytic activator with a unique fold. The observed insertion of the SC(1-325) N-terminus into the 'Ile 16' cleft of prethrombin 2, which triggers the activating conformational change, provided the first unambiguous structural evidence for the 'molecular sexuality' mechanism of non-proteolytic zymogen activation. Based on the SC(1-325) fold, a new family of bifunctional zymogen activator and adhesion proteins was identified that possess N-terminal domains homologous to SC(1-325) and C-terminal domains that mediate adhesion to plasma or extracellular matrix proteins. Further investigation of the ZAAP family may lead to new insights into the mechanisms of bacterial factors that hijack zymogens of the human blood coagulation and fibrinolytic systems to promote and disseminate endocarditis and other infectious diseases.

Human sera possess a limited antibody repertoire to influenza neuraminidase antigenic variants selected in vitro.

Four antigenic variants of the neuraminidase (NA) of A/Texas/77 (H3N2) virus were selected using monoclonal antibody at a frequency of one variant in 10(5) parental virions. The antigenic variants failed to react serologically with the monoclonal antibody used for their selection in vitro. The antigenic variants failed also to react serologically with a proportion of sera from children and adults although all of the sera reacted with the parental A/Texas/77 virus. Thus, certain human sera have a restricted antibody repertoire to influenza NA antigen which might enable virus antigenic variants to avoid anti-NA antibody-mediated neutralization in nature.