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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