Protein Phosphorylation and Intracellular Free Calcium in Platelets of Patients With Essential Hypertension

Platelet intracellular free calcium concentration [Ca2+]i from patients with essential hypertension has been found to be elevated, but the intracellular effects of this increase are still unclear. As protein phosphorylation is an important regulatory step in cell activation and increased protein phosphorylation has been demonstrated in platelets from hypertensive animals, we investigated protein phosphorylation and [Ca2+]i in platelets from patients with essential hypertension and age-matched nor-motensives. We measured the 32P incorporation into a 20 kDa protein and a 47 kDa protein in 17 hypertensive patients and 20 normotensive, age-matched subjects. The [Ca2+]i was measured with the fluorescent dye fura-2. Protein phosphorylation and [Ca2+]i were assessed in unstimulated platelets and after exposure of the cells to 0.1 and 0.25 U/mL thrombin at 20, 60, and 300 sec. In addition we assessed the activity of protein kinase C by incubating the platelets with phorbolester TPA at 20, 60, and 300 sec. Basal phosphorylation of the two proteins was not different between the two groups. After exposure of the platelets to thrombin 32P, incorporation into the 20 kDa protein and the 47 kDa protein was significantly increased in platelets from hypertensive patients at all times. Furthermore, the specific stimulation of protein kinase C with TPA resulted in a significantly higher phosphorylation of the 47 kDa protein, whereas the 20 kDa protein was not phosphorylated after incubation with TPA for 1 min. Basal [Ca2+]i was higher in platelets from hypertensive patients (124 ± 7 nmol/ L v 104 ± 5 nmol/L, P < .05), although there was a wide overlap between the two groups. Exposure of platelets to 0.25 U/mL thrombin led to a sharp rise in [Ca2+]i to 434 ± 28 nmol/L in hypertensives and to 410 ± 34 nmol/L in normotensives (difference not significant). Basal [Ca2+]i was significantly correlated with the thrombin-induced changes of the 47 kDa protein (r = 0.343, P < .05, but not with that of the 20 kDa protein (r = 0.107). Our results show an increased agonist-induced protein phosphorylation in platelets from patients with essential hypertension that seems in part to be due to an increased activity of protein kinase C, as suggested by the stimulatory effects of TPA. The increased basal [Ca2+]i in hypertensives seems to be linked to the increased protein kinase C-dependent phosphorylation. Am J Hypertens 1992;5:117-124