Structural features mediating fibrin selectivity of vampire bat plasminogen activators.
The distinguishing characteristic of vampire bat (Desmodus rotundus) salivary plasminogen activators (DSPAs) is their strict requirement for fibrin as a cofactor. DSPAs consist of structural modules known from urokinase (u-PA) and tissue-type plasminogen activator (t-PA) such as finger (F), epidermal growth factor (E), kringle (K), and protease (P), combining to four genetically and biochemically distinct isoenzymes, exhibiting the formulas FEKP (DSPA alpha 1 and alpha 2) and EKP and KP (DSPA beta and DSPA gamma). Only DSPA alpha 1 and alpha 2 bind to fibrin. All DSPAs are single-chain molecules, displaying substantial amidolytic activity. In a plasminogen activation assay, all four DSPAs are almost inactive in the absence of fibrin but strongly stimulated by fibrin addition. The catalytic efficiency (kcat/Km) of DSPA alpha 1 increases 10(5)-fold, whereas the corresponding value of t-PA is only 550. The ratio of the bimolecular rate constants of plasminogen activation in the presence of fibrin versus fibrinogen (fibrin selectivity) of DSPA alpha 1, alpha 2, beta, gamma, and t-PA was found to be 13,000, 6500, 250, 90, and 72, respectively. Whereas all DSPAs are therefore more fibrin dependent and fibrin selective than t-PA, the extent depends on the respective presence of the various domains. The introduction of a plasmin-sensitive cleavage site in a position akin to the one in t-PA partially obliterates fibrin cofactor requirement. Fibrin dependence and fibrin selectivity of DSPAs are accordingly mediated by fibrin binding, which involves the F domain, as yet undefined determinants within the K and P domains, and by the absence of a plasmin-sensitive activation site. These findings transcend the current understanding of fibrin-mediated stimulation of plasminogen activation: in addition to fibrin binding, specific protein-protein interactions come into play, which stabilize the enzyme in its active conformation.
Bringmann
P
P
Research Laboratories, Schering AG Berlin, Federal Republic of Germany.
Gruber
D
D
Liese
A
A
Toschi
L
L
Krätzchmar
J
J
Schleuning
W D
WD
Donner
P
P
D015119:Aminocaproic Acid / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002685:Chiroptera; D004789:Enzyme Activation; D005337:Fibrin / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism; D005343:Fibrinolytic Agents / Q000378:metabolism*; D006868:Hydrolysis; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D010958:Plasminogen / Q000378:metabolism; D010960:Plasminogen Activators / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D012463:Saliva / Q000201:enzymology; D013379:Substrate Specificity; D010959:Tissue Plasminogen Activator / Q000378:metabolism
D005343:Fibrinolytic Agents; D007527:Isoenzymes; D009842:Oligopeptides; D011994:Recombinant Proteins; C069588:salivary plasminogen activator alpha 1, Desmodus rotundus; C015608:benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide; D005337:Fibrin; D005340:Fibrinogen; D010958:Plasminogen; D010960:Plasminogen Activators; D010959:Tissue Plasminogen Activator; D015119:Aminocaproic Acid