Summary
We recently reported that washed platelets (WP) activated with ADP and expressing
surface-bound vWF aggregated in flow through small tubes or in a cylindrical couette
device at physiological shear rates of G = 300 s–1-1000 s–1 in the absence of exogenous ligands, with GPIb-vWF partially, and activated GPIIb-IIIa
totally required for the aggregation. We have now extended these studies to aggregation
of platelets “activated” with ristocetin or thrombin. Washed platelet suspensions
with added soluble vWF and ristocetin (0.3-0.75 mg/ml), or activated with thrombin
(0.01-0.5 U/ml) but no added ligand, were sheared in a coaxial cylinder device at
uniform shear rate, G = 1000 s–1. The collision capture efficiency (αG) with which small aggregates form (= experimental/calculated initial rates of aggregation)
was correlated with vWF platelet binding assessed by flow cytometry. The vWF-GPIb
interaction was exclusively able to support ristocetin-mediated shear aggregation
of metabolically active platelets, with very few vWF monomer equivalents bound per
platelet (representing ≤10 molecules of 10 million Da) required to yield high capture
efficiencies (αG = 0.38 ± .02; n = 11), suggesting rapid and stable bond formations between vWF and
GPIb. However, platelet surface-expressed vWF, generated by addition of thrombin to
washed platelets, was found to mediate platelet aggregation with αG = 0.08 ± .01 (n = 6), surprisingly comparable to that previously reported for WP
and ADP activation. Blocking the GPIIb-IIIa receptor decreased αG by 95 ± 3% (n = 3), while a monoclonal antibody to the vWF site on GPIb caused a
49 ± 7% (n = 8) decrease in αG. The partial role for GPIb thus appears to reflect a facilitative function for increasing
contact time between flowing platelets, and allowing engagement of the GPIIb-IIIa
receptor to yield stable attachment.
