FLUORESCENT INDICATORS GIVE BIASED ESTIMATES OF INTRACELLULAR FREE CALCIUM CHANGE IN AGGREGATING PLATELETS - IMPLICATION FOR STUDIES WITH HUMAN VON-WILLEBRAND-FACTOR

The ratiometric fluorescent indicators Fura-2 and Indo-1 are considere d optimal probes for monitoring intracellular free calcium concentrati on ([Ca2+](i)). Unique problems arise, however, in studying [Ca2+](i) changes induced in platelets by von Willebrand factor (vWF), Binding o f native multimeric vWF causes extensive platelet aggregation, and is reported to evoke a gradual [Ca2+](i) increase, The present investigat ion examined the reliability of platelet [Ca2+](i) measurements in the se circumstances. Ristocetin-mediated binding of vWF to human platelet s promoted a slow rise in Fura-2 fluorescence ratio, Fura-2 extrusion contributed substantially to this rise, unless blocked by probenecid. Despite this precaution, the platelets were invariably contaminated sl ightly with extracellular indicator, As aggregation progressively redu ced the number of platelets in the spectrofluorometer beam, through se ttling of the larger aggregates, such extracellular Fura-2 contributed proportionately more to the observed fluorescence, This extraneous si gnal accounted completely for the fluorescence ratio increase, and app arent [Ca2+](i) rise, in response to native multimeric vWF, The same p roblem arose with Indo-1, whereas the single wavelength indicator Fluo -3 showed the opposite pattern of apparent [Ca2+](i) changes, Thus, no ne of these indicators provides reliable data on [Ca2+](i) signals in aggregating platelets, Use of a dimeric form of vWF eliminated the pro blem of platelet aggregates settling out of suspension, but also virtu ally abolished the [Ca2+](i) increase, These observations may explain. some of the inconsistencies among previous investigations of vWF-indu ced calcium signaling, Moreover similar problems may arise in studies with other adhesive proteins.