ADENOSINE DIPHOSPHATE-INDUCED AGGREGATION OF HUMAN PLATELETS IN FLOW-THROUGH TUBES .3. SHEAR AND EXTRINSIC FIBRINOGEN-DEPENDENT EFFECTS

The effect of shear rate and fibrinogen concentration on adenosine dip hosphate-induced aggregation of suspensions of washed human platelets in Poiseuille flow at 23-degrees-C was studied using a previously desc ribed double infusion technique and resistive particle counter size an alysis (1). Using suspensions of multiple-centrifuged and -washed cell s in Tyrodes-albumin [3 x 10(5) mul-1; (17)] with [fibrinogen] from 0 to 1.2 muM, the rate and extent of aggregation with 0.7 muM ADP in Tyr odes-albumin were measured over a range of mean transit times from 0.2 to 43 s, and at mean tube shear rates, GBAR, = 41.9, 335 and 1,335 s- 1. As measured by the decrease in singlet concentration, aggregation a t 1.2 muM fibrinogen increased with increasing GBAR up to 1,335 s-1, i n contrast to that previously reported in citrated plasma, in which ag gregation reached a maximum at GBAR = 335 s-1. Without added fibrinoge n, there was no aggregation at GBAR = 41.9 s-1; at GBAR = 335 s-1, the re was significant aggregation but with an initial lag time, aggregati on increasing further at GBAR = 1,335 s-1. Without added fibrinogen, a ggregation was abolished at all GBAR upon incubation with the hexapept ide GRGDSP, but was almost unaffected by addition of an F(ab')2 fragme nt of an antibody to human fibrinogen. Aggregation in the absence of a dded fibrinogen was also observed at 37-degrees-C. The activation of t he multiple-washed platelets was tested using flow cytometry with the fluorescently labelled monoclonal antibodies FITC-PAC1 and FITC-9F9. I t was shown that 57% of single cells in unactivated PRT expressed maxi mal 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 inhi bit the prebound fibrinogen. Moreover, relatively unactivated cells (8 % expressing receptor, 14% prebound fibrinogen), prepared from acidifi ed cPRP by single centrifugation with 50 nM of the stable prostacyclin derivative, ZK 36 374, and resuspension in Tyrodes-albumin at 5 x 10( 4) mul-1, aggregated with 2 and 5 muM ADP at GBAR = 335 and 1,335 s-1 in the absence of added fibrinogen. We therefore postulate that a prot ein such as von Willebrand factor, secreted during platelet isolation or in flow at sufficiently high shear rates, may yield the observed sh ear-rate dependent aggregation without fibrinogen.