Fluorescent Indicators Give Biased Estimates of Intracellular Free Calcium Change in Aggregating Platelets: Implication for Studies with Human von Willebrand Factor

ABSTRACT: The ratiometric fluorescent indicators Fura-2 and Indo-1 are considered optimal probes for monitoring intracellular free calcium concentration (Ca²⁺]_i). Unique problems arise, however, in studying Ca²⁺]_ichanges induced in platelets by von Willebrand factor (vWF). Binding of native multimeric vWF causes extensive platelet aggregation, and is reported to evoke a gradual Ca²⁺]_iincrease. The present investigation examined the reliability of platelet Ca²⁺]_imeasurements in these circumstances. Ristocetin-mediated binding of vWF to human platelets 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 slightly with extracellular indicator. As aggregation progressively reduced the number of platelets in the spectrofluorometer beam, through settling of the larger aggregates, such extracellular Fura-2 contributed proportionately more to the observed fluorescence. This extraneous signal accounted completely for the fluorescence ratio increase, and apparent Ca²⁺]_irise, in response to native multimeric vWF. The same problem arose with Indo-1, whereas the single wavelength indicator Fluo-3 showed the opposite pattern of apparent Ca²⁺]_ichanges. Thus, none of these indicators provides reliable data on Ca²⁺]_isignals in aggregating platelets. Use of a dimeric form of vWF eliminated the problem of platelet aggregates settling out of suspension, but also virtually abolished the Ca²⁺]_iincrease. These observations may explain some of the inconsistencies among previous investigations of vWF-induced calcium signaling. Moreover, similar problems may arise in studies with other adhesive proteins.