Characterization of the calcium(II) ion extrusion systems of human platelets

Date of Award




Degree Name

Doctor of Philosophy (Ph.D.)



First Committee Member

Duncan H. Haynes - Committee Chair


Deliberate overload of human platelets with the high-affinity Ca$\sp{2+}$ indicator quin2 was used for the in situ characterization of active Ca$\sp{2+}$ extrusion systems and cytoplasmic Ca$\sp{2+}$ binding. Platelets were (i) overloaded with quin2 to cytoplasmic concentrations of 3 mM and (ii) exposed to 2.0 mM external Ca$\sp{2+}$ and 1.0 $\mu$M ionomycin to rapidly achieve a cytoplasmic Ca$\sp{2+}$ activity ( (Ca$\sp{2+}$) $\sb{\rm cyt}$) of 1.5 $\mu$M. (iii) The external Ca$\sp{2+}$ is removed by EGTA, and (iv) the active Ca$\sp{2+}$ extrusion monitored as a function of time.The kinetics of decline of quin2 fluorescence (resulting from active Ca$\sp{2+}$ extrusion) were analyzed as a function of (Ca$\sp{2+}$) $\sb{\rm cyt}$ using a mathematical model involving the probability that an exported Ca$\sp{2+}$ was removed from a quin2 complex versus a cytoplasmic binding element). The rates of decline of quin2 fluorescence at a particular (Ca$\sp{2+}$) $\sb{\rm cyt}$ are dependent upon (i) the absolute rate of the extrusion system (a function of its K$\sb{\rm m}$, V$\sb{\rm m}$ and Hill coefficient (n)), (ii) the intrinsic Ca$\sp{2+}$ buffer capacity of the cytoplasm (a function of the total site concentration ( (B) $\sb{\rm T}$ and its K$\sb{\rm d}$) and (iii) the buffer capacity of the quin2 (a function of its concentration and K$\sb{\rm d}$). The contribution of (iii) was known and varied and was used to determine (i) and (ii) as a function of (Ca$\sp{2+}$) $\sb{\rm cyt}$.The Ca$\sp{2+}$ binding data were verified by $\sp{45}$Ca$\sp{2+}$ experiments. The data fit a single binding site model ( (B) $\sb{\rm T}$ = 730 $\pm$ 200 $\mu$M) with an average K$\sb{\rm d}$ = 140 $\pm$ 10 nM. The rate of the Ca$\sp{2+}$ extrusion as a function of (Ca$\sp{2+}$) $\sb{\rm cyt}$ can best be described by a model with two components: A saturable one with V$\sb{\rm m}$ = 2.3 $\pm$ 0.2 nmol min$\sp{-1}$mg$\sp{-1}$min$\sp{-1}$, K$\sb{\rm m}$ = 80 $\pm$ 10 nM and n = 1.7 $\pm$ 0.3 (probably a Ca$\sp{2+}$-Mg$\sp{2+}$-ATPase pump) and a linear one (probably a Na$\sp{+}$-Ca$\sp{2+}$-exchanger).Cyclic nucleotides stimulated the V$\sb{\rm m}$ of the saturable component of Ca$\sp{2+}$ extrusion, without affecting the K$\sb{\rm m}$ or Hill coefficient. Sodium nitroprusside (which stimulates guanylate cyclase), Bt$\sb2$-cGMP, forskolin (which stimulated adenylate cyclase), and Bt$\sb2$-cAMP, increased the V$\sb{\rm m}$ by factors of 1.6, 2.2, 1.6, and 2.0, respectively.


Biophysics, Medical; Biophysics, General

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