Tyrosine Phosphorylation of Human Platelet Plasma Membrane Ca ²⁺ -ATPase in Hypertension

Abstract

Abstract-IntracellularCa 2ϩ is increased in the platelets of hypertensive individuals. Previously, we demonstrated that platelet plasma membrane Ca 2ϩ -ATPase (PMCA) activity inversely correlates with diastolic blood pressure and that inhibition of this Ca 2ϩ pump could explain the elevation of cytosolic Ca 2ϩ in hypertension. More recently, we discovered that PMCA is phosphorylated on tyrosine residues during thrombin-stimulated platelet aggregation and that this phosphorylation causes inhibition of PMCA activity. In the present work, we tested the hypothesis that tyrosine phosphorylation of PMCA in hypertensive patients could account for the observed inhibition of the Ca 2ϩ pump. Platelets were obtained from untreated hypertensive and normotensive volunteers. PMCA was immunoprecipitated from solubilized platelets, and tyrosine phosphorylation was quantified by chemiluminescence of immunoblots treated with anti-phosphotyrosine. PMCA content was measured on the same immunoblots by stripping and reprobing with anti-PMCA. Phosphorylation was reported as normalized phosphotyrosine chemiluminescence per nanogram PMCA (meanϮSE). The average PMCA tyrosine phosphorylation for 15 normotensive subjects was 0.53Ϯ0.09, whereas the average for 8 hypertensive individuals was 1.82Ϯ0.25 (PϽ0.0005, Mann-Whitney U test). Age, gender, and systolic blood pressure did not correlate with PMCA phosphorylation. These results suggest that PMCA in platelets of hypertensive individuals is inhibited because of tyrosine phosphorylation, resulting in increased platelet intracellular Ca 2ϩ , hyperactive platelets, and increased risk of heart attack and stroke.

Plasma Membrane Ca2+-ATPase Associates with the Cytoskeleton in Activated Platelets through a PDZ-binding Domain

“…Changes in the activity and/or distribution of PMCA might have a crucial impact on platelet functions. Our earlier finding that platelet PMCA is phosphorylated on tyrosine residues and inhibited in hypertensive individuals resulting in increased cytosolic Ca 2ϩ demonstrates the importance of this protein for proper platelet function (14).…”

“…Isolation and Analysis of Platelet Subcellular Fractions-Platelet concentrates were obtained from the American Red Cross (Louisville, KY) and prepared as described previously (14). Briefly, platelets were isolated by centrifugation and resuspended in Tyrode's-HEPES buffer containing 138 mM sodium chloride, 2.9 mM potassium chloride, 12 mM sodium bicarbonate, 0.36 mM sodium phosphate, 5.5 mM glucose, 1.8 mM calcium chloride, 0.4 mM magnesium chloride, 10 mM HEPES, pH 7.4, 0.1 mM aspirin, and 0.2 unit/ml apyrase.…”

Cellular phenotypes and the genetics of hypertension

“…Alternatively, mechanisms relevant to dysfunctional Ca 2+ homeostasis in hypertension have also been investigated. Ca 2+ pump-specific tyrosine phosphorylation may effect pump inhibition and elevate intracellular Ca 2+ levels in hypertensive individuals [14]. In support of this concept, thrombin-stimulated Ca 2+ -ATPase phosphorylation levels were 2.5-fold greater in platelets from hypertensive than normotensive subjects.…”

“…In support of this concept, thrombin-stimulated Ca 2+ -ATPase phosphorylation levels were 2.5-fold greater in platelets from hypertensive than normotensive subjects. Blackenship et al [14] suggested Ca 2+ pump inhibition increased intracellular Ca 2+ and placed these individuals at greater risk for thrombotic events.…”

Cinnamtannin B-1 as an antioxidant and platelet aggregation inhibitor

“…Removal of cytosolic Ca 2+ and maintenance of a low [Ca 2+ ] c is mediated by Ca 2+ extrusion, mainly by the plasma membrane Ca 2+ -ATPase (PMCA), and sequestration into the intracellular stores (Rosado and Sage, 2000a). Ca 2+ mobilization in platelets is altered in a number of pathologies, such as type 2 diabetes mellitus (Saavedra et al, 2004) or hypertension (Blankenship et al, 2000), leading to cardiovascular disorders associated to platelet hyperaggregability. In platelets from type 2 diabetic subjects Ca 2+ mobilization and extrusion are altered (Saavedra et al, 2004;Rosado et al, 2004b).…”