LOCATING THE UNPAIRED CYSTEINE OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR

Variants of tissue-type plasminogen activator (t-PA) were constructed with selected cysteines replaced by alanine to evaluate the role of an unpaired cysteine, which has been presumed to be in the growth factor module, C75A, C83A, C84A and CC83-84AA variants of t-PA were expresse d transiently in human embryonic kidney cells, The biochemical propert ies of these variants provided experimental evidence to identify the u npaired cysteine in t-PA, Assays of amidolytic activity, plasminogen a ctivation (in the presence or absence of fibrinogen or fibrin), plasma clot lysis, fibrin binding, clearance in mice, and interaction with a panel of monoclonal antibodies were performed as the basis for compar ing these variants with wild-type t-PA, In all assays, C83A t-PA was b iochemically equivalent to wild-type t-PA, C75A t-PA, C84A t-PA and CC 83-84AA t-PA variants exhibited reduced activities in a variety of fun ctional assays, These variants displayed two- to threefold lower activ ity in fibrinogen or fibrin stimulated plasminogen activation, and fiv efold reduced plasma clot lysis activity compared with that of wild-ty pe t-PA, The affinity of C75A t-PA and C84A t-PA for fibrin was decrea sed more than two orders of magnitude compared with C83A t-PA or wild- type t-PA, Plasma clearance of C75A t-PA and C84A t-PA was reduced 2-f old in mice, The C75A, C84A and CC83-84AA variants displayed significa ntly decreased reactivity with anti-tPA monoclonal antibodies specific for finger/growth factor domain epitopes, These data are consistent w ith a disulfide linkage of Cys75 with Cys84 and that Cys83 exists as a n unpaired sulfhydryl, The significance of the unpaired cysteine is as yet undetermined since C83A t-PA and wild-type t-PA are functionally equivalent.