Platelet Membrane Microviscosity Research Articles

Changes in membrane microenvironment and signal transduction in platelets from NIDDM patients—a pilot study

Background: Non-insulin-dependent diabetes mellitus (NIDDM) is characterized by the presence of abnormally active platelets in the circulation, leading to increased incidence of thrombotic complications. In this study, we have attempted to understand the pathophysiology of the platelets in NIDDM. Methods: Platelet aggregation was induced by thrombin receptor-activating peptide or epinephrine. Membrane fluidity was derived from the steady-state fluorescence anisotropy of diphenylhexatriene incorporated in the membrane. The phosphotyrosine content of the platelet proteins was probed using specific monoclonal antibodies. The extent of calpain activity was assessed from the proteolysis of calpain substrates. Results: Aggregation was significantly enhanced ( p<0.001) in the platelets obtained from the cases of NIDDM. Anisotropy measurements reflected a significant increase in the microviscosity of platelet membranes from 3.315 (±0.103) in the control to 4.153 (±0.119) in NIDDM. Proteins of relative mobilities of 131, 100, 47 and 38 kDa were found to remain phosphorylated on tyrosine in the resting platelets obtained from NIDDM patients, while they were not phosphorylated in the control counterparts. This was associated with heightened activity of the calcium-dependent thiol protease, calpain, in NIDDM. Conclusions: Taken together, these data indicated significant changes in the signaling mechanism in the platelets obtained from NIDDM, which could lead to platelet hyperactivity in this disease.

Platelets from eclampsia patients have reduced membrane microviscosity and lower activities of the signalling enzymes

It has been shown that platelets from patients suffering from eclampsia are hyporesponsive to stimulation by agonists like thrombin and ADP. Although platelet hyporeactivity contributes to the pathogenesis of the disease process, the cause for this is still not known. Platelet aggregation and secretion are membrane-based phenomena initiated by the processes of cell signalling. Hence, to understand the mechanisms underlying platelet hyporeactivity in eclampsia, membrane microviscosity and activities of the signalling enzymes were measured in human platelets stimulated with thrombin. Membrane fluidity was determined from the steady-state fluorescence anisotropy of diphenylhexatriene incorporated in cell membranes. Activities of phospholipase C and protein kinase C in stimulated platelets were assessed from the extents of phosphatidic acid generation and pleckstrin phosphorylation, respectively. Platelet membrane microviscosity in eclampsia (2.3±0.2 SEM, n=5) was significantly lower ( P<0.05) than that in the matched gravid control subjects (3.1±0.2, n=4). In eclampsia, generation of phosphatidic acid and phosphorylation of pleckstrin were decreased by 25% ( P<0.05, n=3) and 35% ( P<0.05, n=3), respectively, after 60 sec of platelet stimulation. It was concluded that the hyporeactive platelets obtained from eclampsia have more fluid membranes and diminished activities of phospholipase C and protein kinase C. In summary, this study shows that alterations in membrane fluidity and activities of the signalling enzymes (phospholipase C and protein kinase C) may contribute to the diminished platelet responsiveness observed in the eclamptic condition.

Tolerance to nitrates and simultaneous upregulation of platelet activity prevented by enhancing antioxidant state

We analysed the induction of tolerance to nitrates both in the vasculature (in vivo) and platelets (ex vivo). Simultaneously, we tested mechanisms underlying the induction of tolerance and interventions to prevent or overcome this phenomenon. For this purpose nitroglycerin (GTN 1.5 micrograms/kg per min i.v.), alone or in combination with ascorbate (55 micrograms/kg per min i.v.) as antioxidant, was infused continuously for a period of 5 days into chronically instrumented dogs. Along with haemodynamic parameters, ex vivo platelet function was continuously monitored. Following the start of GTN infusions there was a maximal coronary dilator response (245 +/- 15 microm) and, as an index of venodilation, a fall of left ventricular end-diastolic pressure (by 2.3 +/- 0.4 mmHg). Both responses declined progressively and disappeared during the infusion period. However, in combination with ascorbate as antioxidant the dilator responses were maintained fully throughout the infusion period. With GTN alone there was a progressive, unexpected upregulation of platelet activity demonstrated by enhanced thrombin-stimulated intracellular Ca2+ levels and increases in the microviscosity of platelet membranes (indicating enhanced receptor expression) associated with a progressive impairment in basal, unstimulated cGMP levels. These changes could also be prevented completely by i.v. co-administration of ascorbate. From these results it is concluded that vascular tolerance is closely reflected by simultaneous changes in platelet function and further, that both can be prevented completely by appropriate antioxidants such as ascorbate.

Platelet responsiveness in migrainous children during headache-free period.

Results of this study confirm the link between migraine and alterations of platelet responsiveness. Our data suggest that in migraineurs the perturbated platelet microviscosity, analyzed by means of fluorescence polarization, appears responsible for the decrease of stimulation-induced influx of external calcium through the platelet membrane. These findings suggest that platelet membrane microviscosity may be considered as a more significant platelet marker of migraine rather than the well-known and nonspecific phenomenon of platelet hyperaggregation, evaluated by time-transmittance variations induced by adenosine diphosphate and collagen.

Plasma Lipids and Platelet Membrane Fluidity in Essential Hypertension

Essential hypertension is often associated with high levels of plasma cholesterol or triglycerides. The relationships between plasma lipids and platelet lipids, membrane fluidity and functions in untreated hypertensive patients were investigated by measuring the fluorescence anisotropies of two fluorescent dyes (DPH and its cationic derivative, TMA-DPH, with different subcellular localization), cytosolic Ca2+ and pH, cyclic AMP content and aggregation to ADP and collagen. Hypercholesterolemia was found to be accompanied by a rise in platelet cholesterol content without changes in TMA-DPH or DPH anisotropies whereas hypertriglyceridemia was associated with a decreased cholesterol to phospholipid molar ratio, a decreased DPH anisotropy and a tendency of the cytosol to alkalinize. These results point out the differences between the effects of an acute cholesterol load and those of chronic hypercholesterolemia on platelet membrane microviscosity and aggregation. They demonstrate a strong association between plasma triglyceride levels and platelet membrane structure.

Alterations of Platelet Membrane Microviscosity in Essential Hypertension

1. The metabolism of blood platelets, taken as an accessible model of excitable cells, has been reported to be altered in hypertension. Most of the identified alterations concern the functions of various plasma membrane constituents. 2. A possible modification of membrane microviscosity was investigated by 1,6-diphenyl-1,3,5-hexatriene and 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene fluorescence depolarization. In order to determine whether or not the membrane structures probed by these indicators were related to platelet physiological functions, the cytosolic free Ca2+ concentration was determined in parallel. 3. At physiological temperature, the fluorescence anisotropy of 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene was decreased in untreated hypertensive patients (0.276 +/- 0.002 versus 0.288 +/- 0.002, n = 23 and 22, P less than 0.001), indicating a lowered microviscosity at the lipid-water interface of cell membrane. It correlated inversely with blood pressure (P less than 0.001) and cytosolic free Ca2+ concentration (P less than 0.030). On the contrary, 1,6-diphenyl-1,3,5-hexatriene fluorescence anisotropy was observed to vary with sex but not with blood pressure. 4. These results suggest that structural membrane modifications may participate in the various functional abnormalities observed in platelets from hypertensive patients.

Effect of dietary linoleic acid content of platelet aggregation and calcium sensitivity and on microviscosity of platelet membranes in rats

Effect of dietary linoleic acid content of platelet aggregation and calcium sensitivity and on microviscosity of platelet membranes in rats

In vitro effects of psychotropic agents on the microviscosity of platelet membranes.

Normal platelet membranes were exposed in vitro to a variety of psychotropic agents, including chlorpromazine, fluphenazine, haloperidol, imipramine, and lithium. Changes in microviscosity of the hydrocarbon layer of the drug-exposed membranes were determined by steady-state fluorescence polarization measurements, employing the fluorescent probe 1,6-diphenyl 1,3,5-hexatriene (DPH). Concentrations of the phenothiazines chlorpromazine and fluphenazine above 200 nM produced significant increases in microviscosity, while haloperidol produced a small but consistent decrease in microviscosity in the concentration range of 200 nM to 200 microM. Imipramine and lithium were without effect. The phenothiazine-induced increase in microviscosity was apparently dependent upon the structure of the phenothiazine nucleus; side chain structure was less important to this effect.

Further analysis of cell membrane changes in genetic hypertension in rats by diphenylhexatriene fluorescence polarization.

Fluorescence polarization of diphenylhexatriene embedded in membranes was used as an index of 'microviscosity' in platelets and erythrocyte ghosts of spontaneously hypertensive rats of the Okamoto-Aoki strain (SHR), Wistar-Kyoto strain (WKY) and of the hypertension-prone and -resistant Sabra strains (SBH and SBN ), and the original Sabra strain (SB). Microviscosity was increased both in erythrocyte ghosts and platelet membranes of male but not female SHR rats compared with WKY rats and in hypertension-prone Sabra rats compared with the original Sabra rats. Acute and chronic salt loading increased the microviscosity of platelet membranes in all strains of rats but had no effect on the erythrocyte membranes. Microviscosities of vesicles made of lipids extracted from SHR and WKY erythrocyte ghosts were similar. This supports the hypothesis that membrane proteins play a major role in the differences in microviscosity observed in SHR rats.

Thrombin receptors define responsiveness of cholesterol-modified platelets.

The microviscosity of human platelet membranes was changed by incubating platelets with liposomes containing various ratios of cholesterol and lecithin. Binding of 125I-thrombin to the modified platelets was measured together with platelet aggregation and secretion. In cholesterol-normal platelets (mole ratio of cholesterol to phospholipid (C:PL) = 0.553; eta = 2.40 poise), weighted nonlinear least squares curve fitting indicated that a model involving two classes of sites was adequate to describe the binding isotherm (K1 = 8.3 X 10(8) M-1; R1 = 150 sites/platelet; K2 = 6.4 X 10(6) M-1; R2 = 16,000 sites/platelet). In cholesterol-enriched platelets (C:PL = 0.857; eta = 3.05 poise), the apparent affinities for the two classes of sites decreased to 55 and 53%, respectively, while the binding capacities increased to 170 and 160%, respectively. In contrast, in the cholesterol-depleted platelets (C:PL = 0.435; eta = 2.03 poise), the affinities increased to 220 and 180%, respectively, while the binding capacities decreased to 53 and 46%, respectively. In cholesterol-enriched, cholesterol-normal, and cholesterol-depleted platelets, the thrombin concentrations required for half-maximal aggregation were 0.17, 0.35, and 0.52 nM, respectively, while the values for half-maximal secretion of [14C]serotonin were 0.17, 0.40, and 0.55 nM, respectively. Plots of receptor occupancy versus biological response showed that maximum response in cholesterol-enriched, cholesterol-normal, and cholesterol-depleted platelets occurred with occupancy of 30, 50, and 70% of the high affinity sites, respectively. In all three treatment groups, occupancy of 40-50 high affinity sites results in 50% aggregation. These results show that (i) modification of platelet membrane microviscosity results in changes in the number and affinity of both high and low affinity thrombin receptors, (ii) the change in receptor number rather than affinity is the determinant for platelet responsiveness, and (iii) the changes in membrane microviscosity do not appear to alter the coupling between occupied receptor and subsequent bioresponse.

Aggregation of platelet membrane vesicles by ADP. (II)

Aggregation of isolated platelet membranes (PM) was observed under phase-contrastmicroscopy. ADP at concentration above 5mM induced an apparent aggregation of PM, while UDP, GDP or AMP at the same concentration was without effects. Light transmission in PM suspension increased by adding ADP (10mM), reaching maximum within 2 to 3 ninutes. This was unexpected observation since light transmission of intact platelet suspension usually decreases when the cells are aggregated. Decrease in light transmission of PM suspension induced by ADP occurred at any wave lengths between 350 and 800nm. There was no change in light transmission in the supernatant of suspension of PM once aggregated by ADP.Light scattering intensity of PM suspension determined by Lasernepherometer promptly increased with the addition of ADP (10mM) and reached plateau within 2 to 3 minutes. This indicates that increase in particle size, probably aggregation of PM can proceed in the suspension.Treatment of PM with neuraminidase (0.1u/ml), trypsin (20μg/ml) or LaCl3 (1mM) inhibited both the aggregation and light transmission change of PM.The addition of ADP (10mM) to PM suspension resulted in the reduction in electrophoretic mobility (0.301±0.057μ·cm/sec·V, control: 1.194±0.087) and the increase in microviscosity of PM (P value, 0.277, control: 0.255).Changes in protein profiles in aggregated PM by ADP (5mM) were examined by SDS polyacrylamide gel disc electrophoresis. One protein band (M. W. 200, 000) on the gel disappeared and instead, there observed increased staining intensity at the top of the gel.These results suggest that the following changes may be responsible for the aggregation of platelet membrane vesicles by ADP; 1) Reduction in surface negative charge of membranes, possibly via change in sialic acid, 2) Reduction in membrane fluidity, 3) Increase in membrane turbidity and 4) Polymerization of membrane protein with molecular weight of approximately 200, 000.

Platelet Function in Hyperbetalipoproteinemia

Individuals with familial hyperbetalipoproteinemia are at increased risk of premature atherosclerosis and thrombosis. Although there is controversy whether platelet survival is shortened or normal in this disease, several in vitro tests of platelet function are abnormal including a decreased threshold concentration for stimulation of aggregation by ADP, epinephrine and collagen and increased release of nucleotides to the same agents. These functional changes are accompanied by an increase of cholesterol to phospholipid ratio in the platelet membrane and in low density lipoprotein in individuals with type IIa hyperlipoproteinemia. Clofibrate and halofenate reverse some of the abnormalities in vitro and the former drug, when administered for 6 weeks to patients with type IIa hyperlipoproteinemia decreases platelet sensitivity to ADP and epinephrine. The platelet hypersensitivity to aggregating agents can be reproduced in vitro by increasing the cholesterol to phospholipid rather in normal platelets. These artificially hypersensitive platelets can be returned to normal by halofenate in vitro. Incorporation of cholesterol into platelet membranes increases the basal level of the membrane associated enzyme adenylate cyclase. However, the enzyme no longer responds to stimulation by prostaglandin E1, and this is associated with relative resistance of the platelet to inhibition by this pharmacologic agent. These functional alterations produced by cholesterol enrichment of platelet membranes occur is parallel with an increase in platelet membrane microviscosity suggesting that the more rigid membrane can alter the behavior of membrane associated enzymes and receptors. A correlation appears to exist between the ability of certain drugs to induce phase separation in model membranes and the potency in inhibitory platelet aggregation.