Ratio fluorescence microscopy has been used to image and measure the intracellular free calcium concentration, [Ca++]i, in single platelets as they attach and spread on biomaterial substrates. The composition and chemistry of the substrates were characterized by ESCA. A novel Ca++-sensitive fluorophore, Fura Red(tm) was loaded into platelets and an in situ calibration of the system was achieved. The [Ca++]i in resting cells was observed to be 50-100nM. The [Ca++]i response to material contact was found to be heterogeneous. Following a variable lag period of up to 5 minutes, responding cells exhibited a rise in [Ca++]i of 100-300nM more. Other platelets showed little or no change in [Ca++]i in contact with the surface. Those platelets demonstrating peak [Ca++]i values greater than 350nM were found to have spread during the experiment, while those that did not spread were seen to have [Ca++]i values remaining below 250nM; suggesting that platelet spreading is associated with a rise in [Ca++]i. Calcium imaging was also performed on platelets adhering to surfaces from a flowing cell suspension. It was found that platelets attached and showed early signs of pseudopodia formation without significant increases [Ca++]i during the initial few minutes of contact. The results demonstrate a novel method of visualizing the process of signal transduction in platelets stimulated by contact with biomaterials, and lay the groundwork for further study of [Ca++]i signaling in platelets and of the possible correlation of the [Ca++]i response with other aspects of platelet activation such as granule release and the promotion of blood coagulation.
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