COMPLEMENT-MEDIATED PERMEABILIZATION OF PLATELETS BY MONOCLONAL-ANTIBODIES TO CD9 - INHIBITION BY LEUPEPTIN, AND EFFECTS ON THE GP IB ACTIN-BINDING PROTEIN SYSTEM
No. Solum et al., COMPLEMENT-MEDIATED PERMEABILIZATION OF PLATELETS BY MONOCLONAL-ANTIBODIES TO CD9 - INHIBITION BY LEUPEPTIN, AND EFFECTS ON THE GP IB ACTIN-BINDING PROTEIN SYSTEM, Thrombosis research, 75(4), 1994, pp. 437-452
Two monoclonal antibodies to CD9 of the IgM and IgG2a categories (FN 5
2 and FN 99), reproducibly induced platelet alterations in platelet-ri
ch plasma by activation of the complement system with membrane incorpo
ration of the pore-forming C5b-9 complex. The permeabilization could b
e monitored by measurements of extracellular ATP and observed as a sha
pe change followed by an increase in light transmission in the aggrego
meter, and was associated with formation of tiny platelet aggregates.
This could be accomplished by only minor lysis observed as extracellul
ar lactate dehydrogenase (LDH). When leupeptin was added prior to, or
immediately after the antibody, a total inhibition of the platelet alt
erations could be obtained. When added soon after the shape change, le
upeptin had little effect on the liberation of ATP. However, whereas t
he ability of the platelets to become agglutinated by ristocetin was l
ost during the complement-mediated platelet alterations, addition of l
eupeptin immediately after the shape change, prevented this loss. The
lost ability of the permeabilized platelets to undergo ristocetin-indu
ced agglutination is not ascribed to degradation of GP Ib as this was
relatively little affected in these studies as compared to the actin-b
inding protein (ABP) which was profoundly degraded. This protein repre
sents a link between GP Ib and the submembraneous cytoskeleton, and th
e inhibition of its degradation by leupeptin, was clearly demonstrated
. Experiments with digitonin-induced permeabilization showed that leup
eptin did not inhibit permeabilization as such, but it did prevent the
loss of ristocetin-induced agglutination even with this inducer.