Adenosine Diphosphate-Induced Aggregation of Human Platelets in Flow Through Tubes: III. Shear and Extrinsic Fibrinogen-Dependent Effects

 

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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.

• References

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