Effects of cyclic AMP, cyclic GMP and protein kinase C on calcium homeostasis and mobilization in normal and thrombotic platelets

Date of Award




Degree Name

Doctor of Philosophy (Ph.D.)

First Committee Member

Duncan H. Haynes - Committee Chair


Ca$\sp{2+}$-handling, Ca$\sp{2+}$-dependent activation processes by normal and abnormal intact human platelets and their regulation by cAMP, cGMP and PKC activation have been studied in situ with fluorescent Ca$\sp{2+}$ indicators chlorotetracycline (CTC), quin2 and fluo3.The study started with the development of a method to probe the kinetics of Ca$\sp{2+}$ transport across the dense tubular membrane (DT) and the effects of cAMP, PKC on Ca$\sp{2+}$ transport across DT and plasma membranes (PM) of intact resting platelets. The experiments show the following: (1) The DT pump has a $\rm K\sb{m}$ of 180 nM and a Hill coefficient of 1.4, which is countered by a passive leak with a rate constant of ca. 1.7 min$\sp{-1}.$ (2) Cyclic AMP increases the rate of DT Ca$\sp{2+}$ uptake by raising $\rm V\sb{m}$ of the pump. (3) PKC stimulates DT Ca$\sp{2+}$ pump by rising $\rm V\sb{m}$ of the pump but has no effect on PM Ca$\sp{2+}$ pump.The effects of thapsigargin (Tg) on the Ca$\sp{2+}$ handling system of platelets were evaluated for their specificity and usefulness. The results show that Tg is not a completely specific agent. It incompletely inhibits DT Ca$\sp{2+}$ uptake (70%), partially inhibits the Ca$\sp{2+}$ extrusion pump and increases PM Ca$\sp{2+}$ permeability. The study also demonstrated a single class of dense tubules and that the total amount of Ca$\sp{2+}$ in the DT of the human platelet is $1.35\pm0.3$ mM.The resting cytoplasmic Ca$\sp{2+}$ concentration ($\rm\lbrack Ca\sp{2+}\rbrack\sb{cyt})$ and agonist-induced Ca$\sp{2+}$ responses of single platelets were determined by videomicroscopy to obtain a more detailed understanding of platelet Ca$\sp{2+}$ handling. The results showed that there is little variation in $\rm\lbrack Ca\sp{2+}\rbrack\sb{cyt}$ between individual platelets. In contrast, there is considerable heterogeneity in agonist-induced Ca$\sp{2+}$ response among individual human platelets. The study also suggested a large inter-platelet variability in the size of the DT Ca$\sp{2+}$ pool.Platelet Ca$\sp{2+}$-dependent activation processes such as platelet granule membrane protein 140 (GMP-140) expression and adhesion between human platelets and neutrophils were investigated using flow cytometry. Platelet granule membrane protein 140 expression and adhesion between human platelets and neutrophils, and their correlation with platelet $\rm\lbrack Ca\sp{2+}\rbrack\sb{cyt}$ were examined using fluorescent Ca$\sp{2+}$ indicator fluo3. In this study, fluo3 played the dual role of both adhesion and $\rm\lbrack Ca\sp{2+}\rbrack\sb{cyt}$ indicators. The effects of cAMP, cGMP on platelet GMP-140 expression and platelet-neutrophil interaction were examined both in the basal and thrombin-stimulated states. Both cAMP and cGMP decrease GMP-140 expression and adhesion of platelets to neutrophils by reducing platelet $\rm\lbrack Ca\sp{2+}\rbrack\sb{cyt}.$Finally Ca$\sp{2+}$ handling by platelets from patients with myocardial infarction and unstable angina and cGMP effect were studied. The results show that there is a significant elevation of $\rm\lbrack Ca\sp{2+}\rbrack\sb{cyt},\ \lbrack Ca\sp{2+}\rbrack\sb{dt},\ and\ the\ Ca\sp{2+}$ response to a subcritical dose of thrombin in platelets of these patients. Sodium nitroprusside (SNP) can reduce or normalize the abnormal platelet Ca$\sp{2+}$ handling via cGMP elevation.


Health Sciences, Pharmacology; Chemistry, Biochemistry

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