Summary
Platelet aggregation and fibrinogen binding in whole blood, induced either by ADP
or by a 14 amino acid peptide mimicking an N-terminal region of the platelet thrombin
receptor (TRP, thrombin receptor activating peptide), have been studied with blood
from different species. Aggregation was assessed by counting the number of single
platelets in blood before und after addition of the agonist with an automated cell
counter. Both ADP (0.02-0.5 μM) and TRP (1-10 μM) were found to be potent agonists
of platelet aggregation in human, rhesus monkey and guinea-pig blood, causing a near-maximal
aggregatory response within 2 min of agonist addition. In contrast, hamster and rat
platelets were much less responsive to both ADP and TRP in terms of the whole blood
aggregation.
Echistatin, RGDW and a synthetic glycoprotein (GP) IIb/IIIa antagonist, Ro 43-8857,
inhibited fibrinogen binding to purified immobilized human GP-IIb/IIIa with IC50 ’s of 1.6, 88 and 11.4 nM, respectively. In whole human blood, the respective IC50 ’s (as determined by flow cytometric analysis of platelet fibrinogen binding) were
4.4, 1700 and 29.5 nM. The affinities of these three compounds for inhibiting fibrinogen
binding in whole blood from rhesus monkeys and guinea-pigs were similar to the affinities
for human platelets, but with rat blood echistatin, RGDW and Ro 43-8857 were all around
100-fold less potent. Ro 43-8857 was a potent inhibitor of ADP- or TRP-induced platelet
aggregation in human, rhesus monkey and guinea-pig whole blood (IC50 of 69-320 nM) but was much less active in hamster blood.
These results highlight important species differences in the response of platelets
to activation by two different agonists and also in their inhibition by GP-IIb/IIIa
antagonists. In particular, platelets from the rat and hamster were insensitive to
agonists and antagonists, whereas guinea-pig and rhesus monkey platelets responded
with an affinity similar to human platelets. Since these studies were performed in
whole blood, the results should be representative of those expected in animal experiments.
These recently developed methods for studying platelet responses in small aliquots
of whole blood are simple to perform and provide important information concerning
the optimal choice of species for subsequent in vivo studies with these compounds.
