Complement-mediated permeabilization of platelets by monoclonal antibodies to CD9: inhibition by leupeptin, and effects on the GP Ib-actin-binding protein system.
Two monoclonal antibodies to CD9 of the IgM and IgG2a categories (FN 52 and FN 99), reproducibly induced platelet alterations in platelet-rich plasma by activation of the complement system with membrane incorporation of the pore-forming C5b-9 complex. The permeabilization could be monitored by measurements of extracellular ATP and observed as a shape change followed by an increase in light transmission in the aggregometer, and was associated with formation of tiny platelet aggregates. This could be accomplished by only minor lysis observed as extracellular lactate dehydrogenase (LDH). When leupeptin was added prior to, or immediately after the antibody, a total inhibition of the platelet alterations could be obtained. When added soon after the shape change, leupeptin had little effect on the liberation of ATP. However, whereas the ability of the platelets to become agglutinated by ristocetin was lost during the complement-mediated platelet alterations, addition of leupeptin immediately after the shape change, prevented this loss. The lost ability of the permeabilized platelets to undergo ristocetin-induced agglutination is not ascribed to degradation of GP Ib as this was relatively little affected in these studies as compared to the actin-binding protein (ABP) which was profoundly degraded. This protein represents a link between GP Ib and the submembraneous cytoskeleton, and the inhibition of its degradation by leupeptin, was clearly demonstrated. Experiments with digitonin-induced permeabilization showed that leupeptin did not inhibit permeabilization as such, but it did prevent the loss of ristocetin-induced agglutination even with this inducer.
Solum
N O
NO
Research Institute for Internal Medicine, Rikshospitalet, Oslo, Norway.
Rubach-Dahlberg
E
E
Pedersen
T M
TM
Reisberg
T
T
Høgåsen
K
K
Funderud
S
S
D000199:Actins / Q000097:blood / Q000187:drug effects; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D015703:Antigens, CD / Q000276:immunology*; D001792:Blood Platelets / Q000187:drug effects / Q000276:immunology* / Q000648:ultrastructure; D002463:Cell Membrane Permeability / Q000276:immunology*; D003169:Complement Inactivator Proteins / Q000494:pharmacology*; D004072:Digitonin / Q000494:pharmacology; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology*; D008562:Membrane Glycoproteins / Q000276:immunology*; D008969:Molecular Sequence Data; D010974:Platelet Aggregation / Q000187:drug effects; D010980:Platelet Membrane Glycoproteins / Q000187:drug effects / Q000378:metabolism; D011485:Protein Binding; D060245:Tetraspanin 29
D000199:Actins; D000911:Antibodies, Monoclonal; D015703:Antigens, CD; C554869:CD9 protein, human; D003169:Complement Inactivator Proteins; D007976:Leupeptins; D008562:Membrane Glycoproteins; D010980:Platelet Membrane Glycoproteins; D060245:Tetraspanin 29; C032854:leupeptin; D004072:Digitonin