Calcium mobilization in human platelets using indo-1 and flow cytometry

Abstract

Regulation of cytoplasmic free calcium concentration is believed to be important in the response of platelets to external stimuli. A relatively new fluorescent calcium indicator, indo-1, has properties by which alterations of cytoplasmic calcium can be evaluated in single platelets by flow cytometry. Activation of platelets at a temperature lower than 37 degrees C allows examination of the heterogeneity of intracellular free calcium levels and can distinguish variations among platelets in the initiation, duration, and magnitude of calcium fluxes. The clear advantage of flow cytometric analysis of platelet cytosolic calcium is that stimulus-response coupling can now be studied on a single cell basis. Platelets were activated by addition of human alpha- thrombin or ADP at 37 degrees C or at room temperature (22 degrees C). Activation at 37 degrees C approaches more closely an in vivo response and, as expected, increases in cytosolic calcium occurred within seconds of agonist addition. Transient increases in cytoplasmic calcium levels occurred when platelets were challenged with a low concentration of agonist. Heterogeneity in cytoplasmic calcium levels was also observed at 10(-5) mol/L ADP and 0.1 U/mL alpha-thrombin. Some of this heterogeneity was no longer observed at higher concentrations of agonist (10(-4) mol/L ADP and 0.5 U/mL thrombin), suggesting that a sufficient magnitude of signal is required to induce changes in platelet cytosolic calcium. Light-scatter properties of the activated platelets were also monitored simultaneously and showed changes in response to both agonists. The ability to measure changes in cytoplasmic free calcium by ratio flow cytofluorimetry provides a new approach to study of the role of alterations in intracellular calcium in response to agonists acting through different membrane receptors as well as providing a sensitive technique to detect functional subpopulations of platelets.

Flow Cytometric Analysis of Calcium Mobilization in Whole‐Blood Platelets

“…The clearest limitations of this technique results from the difficulty in calibrating [Ca 2+ ] i , for reasons similar to those described by other authors in other experimental models (Jennings et al, ). Such calibration in the intact cells demands assumptions that cannot be easily proved, namely that hydrolysis of fluo‐3 AM ester to the acid form is complete, that microviscosity and cytoplasm refractive index do not affect the K d of fluo‐3, and that the ionophore used in [Ca 2+ ] i measurement equilibrates totally with all intracellular Ca 2+ stores.…”

“…Several studies using fluorescent Ca 2+ ‐sensitive indicators and FCM in samples of washed or gel‐filtered platelets have been reported (Davies et al, , Jennings et al, ; Merrit et al, ; Fijnheer et al, ; Kyle et al, ), although the whole‐blood approach has obvious advantages. In the protocol presented in this unit, sample manipulation is minimal, in contrast to other methods involving separation procedures that cause artifactual platelet activation and eventual loss of platelet subpopulations.…”

Effect of cellular and receptor activation on the extent of integrin αIIbβ3 internalization

“…It is well known that following disruption of vascular integrity, blood platelets become activated by a variety of endogenously released and/or generated agonists including thrombin and ADP. These agonists induce a variety of activation‐dependent sequelle including calcium mobilization, shape change, secretion, cytoskeleton reorganization, and platelet aggregation [21–23]. Platelet aggregation requires conversion of α IIb β 3 to an activated form that is capable of binding its physiologic ligand fibrinogen [24,25], and subsequent cell–cell adhesion is mediated by fibrinogen–α IIb β 3 bridging between adjacent platelets [1].…”

Flow Cytometric Monitoring of Dynamic Cytosolic Calcium, Sodium, and Potassium Fluxes Following Platelet Activation

“…Besides analysis of baseline and activation end points, the introduction of the "Time" parameter in the early 1980s (22) allowed to measure changes of cellular parameters over time (23). Since then, publications using kinetic flow-cytometry started to be reported (24)(25)(26)(27)(28). Subsequently, the technique was improved and redefined as "continuous" (29) or "real-time flow cytometry" (30,31).…”