Target-sensitive immunoerythrocytes: interaction of biotinylated red blood cells with immobilized avidin induces their lysis by complement

AbstractRed blood cells (RBC) coated with antibody (immunoerythrocytes) may be useful for drug targeting. Previously we have developed a methodology for avidin (streptavidin)-mediated attachment of biotinylated antibodies (b-Ab) to biotinylated RBC (B-RBC). We have observed that binding of avidin to B-RBC in suspension leads to their complement-mediated lysis by autologous serum. In the present work we have studied the interaction of B-RBC, which are not complement susceptible, with immobilized avidin and their consequent susceptibility to lysis by complement. B-RBC adhered tightly to avidin-coated surfaces and were rendered susceptible to lysis by autologous serum. A long biotin ester provided more effective binding of the B-RBC to immobilized avidin and greater lysis by complement, than a short biotin ester. Based on these results, we have hypothesized that targeting of serum-stable drug-loaded B-RBC attained by step-wise administration of b-Ab and streptavidin may provide target-sensitive lysis of B-RBC. To confirm this hypothesis, we have studied b-Ab and streptavidin mediated targeting of B-RBC to immobilized antigen. Step-wise addition of biotinylated antibody, avidin or streptavidin and b-RBC caused specific binding of B-RBC to immobilized antigen and their subsequent lysis by autologous serum. Therefore, our results obtained in an in vitro model demonstrate that B-RBC might be used for targeting and local release of drug

STUDIES ON KINETICS OF INHIBITION AND BINDING OF XIIIa BY A CROSS-REACTING ANTIFIBRINOGEN ANTIBODY*

Coagulation factor XIIIa, plasma transglutaminase(endo-g-glutamine:e-lysine transferase EC 2.3.2.13) cata-lyzes isopeptide bond formation between glutamine andlysine residues and rapidly cross-links fibrin clots. Amonoclonal antibody (5A2) directed to a fibrinogen Aa-chain segment 529 –539 was previously observed fromanalysis of end-stage plasma clots to block fibrina-chaincross-linking. This prompted the study of its effect onnonfibrinogen substrates, with the prospect that 5A2was inhibiting XIIIa directly. It inhibited XIIIa-cata-lyzed incorporation of the amine donor substrate dan-sylcadaverine into the glutamine acceptor dimethylca-sein in an uncompetitive manner with respect todimethylcasein utilization and competitively with re-spect to dansylcadaverine. Uncompetitive inhibitionwas also observed with the synthetic glutamine sub-strate, LGPGQSKVIG. Theoretically, uncompetitive in-hibition arises from preferential interaction of the in-hibitor with the enzyme-substrate complex but is alsofound to inhibitg-chain cross-linking. The conjunctionof the uncompetitive and competitive modes of inhibi-tion indicates in theory that this bireactant system in-volves an ordered reaction in which docking of the glu-tamine substrate precedes the amine exchange. Thepresence of substrate enhanced binding of 5A2 to XIIIa,an interaction deemed to occur through a C-terminalsegment of the XIIIa A-chain (643– 658,GSDMTVTVQFT-NPLKE), 55% of which comprises sequences occurring inthe fibrinogen epitope Aa-(529 –540) (GSESGIFTNTKE).Removal of the C-terminal domain from XIIIa abolishesthe inhibitory effect of 5A2 on activity. Crystallographicstudies on recombinant XIIIa place the segment 643– 658in the region of the groove through which glutaminesubstrates access the active site and have predicted thatfor catalysis, a conformational change may accompanyglutamine-substrate binding. The uncompetitive inhibi-tion and the substrate-dependent binding of 5A2 pro-vide evidence for the conformational change

Coagulation Factor XIIIa Undergoes a Conformational Change Evoked by Glutamine Substrate

Coagulation factor XIIIa, plasma transglutaminase(endo-g-glutamine:e-lysine transferase EC 2.3.2.13) cata-lyzes isopeptide bond formation between glutamine andlysine residues and rapidly cross-links fibrin clots. Amonoclonal antibody (5A2) directed to a fibrinogen Aa-chain segment 529 –539 was previously observed fromanalysis of end-stage plasma clots to block fibrina-chaincross-linking. This prompted the study of its effect onnonfibrinogen substrates, with the prospect that 5A2was inhibiting XIIIa directly. It inhibited XIIIa-cata-lyzed incorporation of the amine donor substrate dan-sylcadaverine into the glutamine acceptor dimethylca-sein in an uncompetitive manner with respect todimethylcasein utilization and competitively with re-spect to dansylcadaverine. Uncompetitive inhibitionwas also observed with the synthetic glutamine sub-strate, LGPGQSKVIG. Theoretically, uncompetitive in-hibition arises from preferential interaction of the in-hibitor with the enzyme-substrate complex but is alsofound to inhibitg-chain cross-linking. The conjunctionof the uncompetitive and competitive modes of inhibi-tion indicates in theory that this bireactant system in-volves an ordered reaction in which docking of the glu-tamine substrate precedes the amine exchange. Thepresence of substrate enhanced binding of 5A2 to XIIIa,an interaction deemed to occur through a C-terminalsegment of the XIIIa A-chain (643– 658,GSDMTVTVQFT-NPLKE), 55% of which comprises sequences occurring inthe fibrinogen epitope Aa-(529 –540) (GSESGIFTNTKE).Removal of the C-terminal domain from XIIIa abolishesthe inhibitory effect of 5A2 on activity. Crystallographicstudies on recombinant XIIIa place the segment 643– 658in the region of the groove through which glutaminesubstrates access the active site and have predicted thatfor catalysis, a conformational change may accompanyglutamine-substrate binding. The uncompetitive inhibi-tion and the substrate-dependent binding of 5A2 pro-vide evidence for the conformational change