Ristocetin-mediated interaction of human von Willebrand factor with platelet glycoprotein Ib evokes a transient calcium signal: Observations with Fura-PE3

High shear stress in narrowed arteries causes von Willebrand factor (vWf) to bind to its platelet receptor, glycoprotein Ib (Gplb). This binding is reported to promote an increase in intracellular free calcium concentration ((Ca2+)(i)), which may be responsible for platelet activation. The present study examined the platelet (Ca2+)(i) signal that arises when ristocetin mediates vWf-Gplb binding. Platelet (Ca2+)(i) was monitored with Fura-PE3 (Vorndran C, Minta A, Poenie M. Biophys J 1995;69:2112-24), a new ratiometric calcium indicator. Fura-PE3 has calcium-binding characteristics (K-d = 146 nmol/L) and fluorescent properties similar to those of Fura-P. However, its zwitterionic nature ensured much slower extrusion from the platelet (0.2% per minute) than that for Fura-5. This eliminated one of the technical problems that seriously distorted previous measurements of vWf-induced changes in platelets (Ca2+)(i). Design of a novel stirring arrangement avoided the other major problem, which is the tendency of platelet aggregates to settle to the bottom of the cuvette, beneath the detection zone of the spectrofluorometer. With Fura-PE3 and the new stirrer used in the present study, vWf-induced changes in (Ca2+)(i) could be measured reliably in aggregating platelets. Ristocetin-mediated vWf-Gplb binding induced a transient increase in platelet (Ca2+)(i). This increase occurred after a significant lag phase; platelet (Ca2+)(i) rose gradually, followed by a decline to almost the resting level. Binding of vWf to platelet Gplb was responsible for the (Ca2+)(i) signal. A similar signal was found in the absence of extracellular calcium. These characteristics differ substantially from those described in previous reports, in which the vWf-induced rise in (Ca2+)(i) was attributed to calcium influx through channels in the plasma membrane. Data from those earlier studies, however, were severely distorted by indicator extrusion and loss of platelet aggregates. The present findings are a more accurate representation of the vWf-induced platelet (Ca2+)(i) signal.