Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
35
pubmed:dateCreated
1991-1-23
pubmed:abstractText
Active-site-blocked, fluorescent derivatives of tPA (Activase) and a variant (delta FEIX) which lacks the finger and epidermal growth factor-like domains and possesses Asn to Gln and Val to Met mutations at residues 117 and 245, respectively, were prepared. The binding of these to fibrin was studied by adding them at systematically varying concentrations to fibrinogen, at a fixed concentration, inducing clotting with thrombin, separating free and bound tPA or delta FEIX by centrifugation, and measuring the concentration of unbound material by extrinsic fluorescence. Similar studies were performed with Glu and Lys-plasminogen, using intrinsic fluorescence. epsilon-amino caproic acid (EACA) was utilized to distinguish kringle-dependent from finger-dependent binding. In the absence of EACA, delta FEIX-bound fibrin through a single class of sites with Kd = 0.69 microM and n = 1.34 delta FEIX/fibrin. The binding of delta FEIX was completely inhibited by EACA and 50% displacement occurred at [EACA] = 300 microM. Fibrin-bound tPA was only partially displaced with EACA. In the presence of 30 mM EACA, tPA binding reflected a single class of sites with Kd = 0.26 microM and n = 0.60 tPA/fibrin. In the absence of EACA, tPA binding was complex, typified by downwardly curved Scatchard plots, and was consistent with interactions of the two classes of sites, characterized by Kd = 0.13 microM, n = 0.60 and Kd = 0.61 microM, n = 1.23. These were attributed to finger and kringle-dependent interactions, respectively. Under the experimental conditions employed, Glu-plasminogen exhibited no binding to fibrin, whereas Lys-plasminogen bound to a single class of sites with Kd = 0.25 microM and n = 1.02 plasminogen/fibrin. This binding was completely inhibited by EACA and 50% displacement occurred at [EACA] = 28 microM. Competition experiments indicated that Lys-plasminogen does not displace either tPA or delta FEIX from fibrin. From these results the conclusions are drawn that tPA can interact with intact fibrin by two different and independent modes, involving, respectively, the finger and kringle 2 domains, and neither of these modes are competitive with the kringle-dependent binding of Lys-plasminogen.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Dec
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
15
pubmed:volume
265
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
21541-8
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
1990
pubmed:articleTitle
The dissociation constants and stoichiometries of the interactions of Lys-plasminogen and chloromethyl ketone derivatives of tissue plasminogen activator and the variant delta FEIX with intact fibrin.
pubmed:affiliation
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
pubmed:publicationType
Journal Article, In Vitro, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't