Measurement of platelet intracellular free calcium ion concentration by ratio fluorescence microscopy: a study of platelet activation induced by contact with biomaterials

Abstract

Ratio fluorescence microscopy was used to image and measure the intracellular concentration of free calcium ion, (Ca$\sp{++}\rbrack \sb{\rm i}$, in single human platelets during attachment and spreading on several fibrinogen-coated biomaterial surfaces. The calcium-sensitive fluorophore Fura Red$\sp{\rm TM}$ was characterized, loaded into platelets and a calibration of intracellular Fura Red$\sp{\rm TM}$ was achieved using the imaging system.Washed platelets settled to the surface from a static suspension. Initially, (Ca$\sp{++}\rbrack \sb{\rm i}$ in quiescent cells was observed to be 50-100nM. Following a variable lag period of up to five minutes, responding cells exhibited a rise in (Ca$\sp{++}\rbrack \sb{\rm i}$ of 100-300nM. Sporadic oscillations in (Ca$\sp{++}\rbrack \sb{\rm i}$ were seen as the (Ca$\sp{++}\rbrack \sb{\rm i}$ reached an elevated plateau. Other platelets showed little or no change in (Ca$\sp{++}\rbrack \sb{\rm i}$ while in contact with the surface. Platelets that demonstrated larger transient rises in (Ca$\sp{++}\rbrack \sb{\rm i}$, were found to have spread during the experiment, while the (Ca$\sp{++}\rbrack \sb{\rm i}$ in non-spread cells remained low.Platelet (Ca$\sp{++}\rbrack \sb{\rm i}$ was also measured in single cells that attached to the surfaces from a flowing suspension of platelet and red blood cells under a shear rate of 300 inverse seconds. During the first five minutes after attachment, pseudopodial formation was observed without a large change in (Ca$\sp{++}\rbrack \sb{\rm i}$.This study demonstrates the visualization and quantification of a important signal transduction step in single platelets activated by contact with biomaterial surfaces. Imaging of platelet (Ca$\sp{++}\rbrack \sb{\rm i}$ may be useful in relating the mobilization of intracellular calcium with other platelet activation events, such as the release of platelet granule contents, and the scrambling of membrane phospholipids that supports blood coagulation. The technique may also be useful in investigating the mechanisms of platelet activation at surfaces, and in evaluating the effects of surface modification on platelet activation.