Interaction of ristocetin and bovine plasma with guinea pig megakaryocytes:
a means to enrich megakaryocytes based on membrane rather than physical
characteristics
CW Jackson, SA Steward, NK Hutson and RA Ashmun
We have investigated whether megakaryocytes can be aggregated by ristocetin
and bovine plasma and whether such aggregation can be used as a step in the
purification of megakaryocytes from marrow cell suspensions. Guinea pig
marrow cell suspensions were first enriched for megakaryocytes by density
equilibrium centrifugation in continuous Percoll density gradients. The
megakaryocyte-enriched marrow was stirred in a platelet aggregometer to
which ristocetin or bovine plasma was added. Megakaryocytes were aggregated
by both ristocetin and bovine plasma with the proportion aggregated being
related to the concentration of ristocetin or bovine plasma. Maximal
aggregation (greater than 90% of megakaryocytes) was achieved with 2.0
mg/mL ristocetin or 5% bovine plasma and required five minutes. All
maturation stages of morphologically recognizable megakaryocytes were
aggregated. The megakaryocyte aggregates were separated from the marrow
suspension by sedimentation at 1 g and the megakaryocytes disaggregated by
dilution with media (ristocetin aggregated) or addition of dextran sulfate
(bovine plasma aggregated). Megakaryocyte purity and recovery were higher
with bovine plasma than with ristocetin. A mean of 92% of the
megakaryocytes in the bovine plasma aggregated cell suspensions were
recovered with megakaryocytes constituting an average of 76% of the final
cell suspensions. The viability as well as the diameters and DNA content
distribution of these megakaryocytes were similar to those of the starting
population. We conclude that guinea pig megakaryocytes behave like
platelets in that they can be aggregated with ristocetin or bovine plasma
and that megakaryocyte aggregation induced by ristocetin or bovine plasma
provides a means to enrich these cells based on membrane rather than
physical characteristics. This approach yields purified megakaryocyte
populations that are representative of those in unfractionated marrow.
Volume 69,
Issue 1,
pp. 173-179,
01/01/1987
Copyright © 1987 by The American Society of Hematology