Summary
The effect of shear rate and fibrinogen concentration on adenosine diphosphate-induced
aggregation of suspensions of washed human platelets in Poiseuille flow at 23°C was
studied using a previously described double infusion technique and resistive particle
counter size analysis (1). Using suspensions of multiple-centrifuged and -washed cells
in Tyrodes-albumin [3 × 105 μl−1; (17)] with [fibrinogen] from 0 to 1.2μM, the, rate and extent of aggregation with
0.7 μM ADP in Tyrodes-albumin were measured over a range of mean transit times from
0.2 to 43 s, and at mean tube shear rates, Ḡ, = 41.9, 335 and 1,335 s−1. As measured by the decrease in singlet concentration, aggregation at 1.2 μM fibrinogen
increased with increasing Ḡ up to 1,335 s1, in contrast to that previously reported in citratcd plasma, in which aggregation
reached a maximum at Ḡ = 335 s−1. Without added fibrinogen, there was no aggregation at Ḡ = 41.9 s1; at Ḡ = 335 s1, there was significant aggregation but with an initial lag time, aggregation increasing
further at Ḡ = 1,335 s−1. Without added fibrinogen, aggregation was abolished at all Ḡ upon incubation with
the hexapeptide GRGDSP, but was almost unaffected by addition of an F(ab’)2 fragment of an antibody to human fibrinogen. Aggregation in the absence of added
fibrinogen was also observed at 37°C. The activation of the multiple-washed platelets
was tested using flow cytometry with the fluorescently labelled monoclonal antibodies
FITC-PAC1 and FITC-9F9. It was shown that 57% of single cells in unactivated PRT expressed
maximal GPIIb-IIIa fibrinogen receptors (MoAb PAC1) and 54% expressed pre-bound fibrinogen
(MoAb 9F9), with further increases on ADP activation. However, incubation with GRGDSP
and the F(ab’)2 fragment did not inhibit the prebound fibrinogen. Moreover, relatively unactivated
cells (8% expressing receptor, 14% prebound fibrinogen), prepared from acidified cPRP
by single centrifugation with 50 nM of the stable prostacyclin derivative, ZK 36 374,
and resuspension in Tyrodes-albumin at 5 × 104 μl−1, aggregated with 2 and 5 μM ADP at Ḡ = 335 and 1,335 s−1 in the absence of added fibrinogen. We therefore postulate that a protein such as
von Willebrand factor, secreted during platelet isolation or in flow at sufficiently
high shear rates, may yield the observed shear-rate dependent aggregation without
fibrinogen.
