Platelet glycoproteins IIb and IIIa as a calcium channel in liposomes.
Human platelet membrane glycoproteins IIb and IIIa (GPIIb and IIIa) were incorporated into phospholipid vesicles by the reverse-phase technique to assess the ability of GPIIb and IIIa to function as a Ca2+ channel. Movement of Ca2+ across the lipid bilayer was quantitated by injection of proteoliposomes with encapsulated Fura-2 into Ca2+ buffers and measurement of Fura-2 fluorescence as an indicator of Ca2+ influx. Reciprocally, to assess the function of proteins in an inside-out orientation, Ca2+-loaded vesicles were injected into Ca2+-free buffer and Ca2+ efflux monitored by a calcium electrode. Incorporation of the IIb-IIIa complex produced significant facilitation of Ca2+ movement across the lipid bilayer. No net transmembrane Ca2+ movement was seen with dissociated IIb and IIIa. Movement of Ca2+ was proportional to the transmembrane Ca2+ gradient. Ca2+ movement into the vesicles was inversely proportional to extravesicular NaCl from 25 to 150 mmol/L, analogous to several studies in the intact platelet. Adenosine triphosphate had no effect on Ca2+ movement into or out of the vesicles. Specific inhibition of a Ca2+ shift into the vesicles was seen with M148, a monoclonal antibody to IIb/IIIa, while no inhibition was observed with a panel of other anti-IIb/IIIa monoclonal antibodies. This suggests that a specific site on the complex or orientation of the complex is essential for calcium channel function. These data demonstrate that the GPIIb/IIIa complex can serve as a passive Ca2+ channel across a phospholipid bilayer and has the potential to play a role in Ca2+ flux across the platelet plasma membrane.
Rybak
M E
ME
University of Massachusetts Medical Center, Worcester 01655.
Renzulli
L A
LA
Bruns
M J
MJ
Cahaly
D P
DP
D000911:Antibodies, Monoclonal / Q000276:immunology; D002118:Calcium / Q000378:metabolism*; D000939:Epitopes; D006801:Humans; D007473:Ion Channels / Q000502:physiology*; D008081:Liposomes / Q000378:metabolism*; D010980:Platelet Membrane Glycoproteins / Q000276:immunology / Q000502:physiology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007473:Ion Channels; D008081:Liposomes; D010980:Platelet Membrane Glycoproteins; D002118:Calcium