We have also had the experience of decreased PAC-1 binding in response
to increasing concentrations of platelet agonists.These cells were still
able
to increase binding of CD62 and CD63 antibodies at the same time.
According to Shattil (Blood, Vol 70, No 1 (July), 1987:pp 307-315)
PAC-1
and fibrinogen inhibit each others binding in a competitive manner and
bind
to the same RDG binding site. The PAC1 antibody has a much higher
affinity (Kd=5nmol/l) for the site than does fibrinogen (Kd=250nmol/l)
and
wins out. Shattil also mentions that he also sees a time dependent
decrease
in PAC-1 binding when the antibody was added after stimulation of the
cells.He surmises that this occurs because fibrinogen becomes
irreversibly
bound to the receptor and can not be displaced by PAC-1. It is also
known
that bound fibrinogen, attached to its receptor, can be internalized
into
alpha granules over time. Proteolysis of the receptor is also a
possibility.
Because of these concerns, measuring fibrinogen receptor activation with
PAC-1 must be interperted carefully.
Alternate methods of assessing fibrinogen receptor competency are to
use a LIBS antibody to a receptor which becomes expressed on IIbIIIa
when
it is RDG site is occupied or to use a RIBS antibody whose receptor
is expressed only on fibrinogen that has bound to its receptor.
Good luck and have fun,
Ann Byrne
abyrne@compucyte.com
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