Modulators of intraplatelet calcium concentration affect the binding of thrombospondin to blood platelets in healthy donors and patients with type 2 diabetes mellitus.

UNLABELLED: Thrombospondin (TSP), which is secreted from alpha-granules of activated platelets, binds to its surface receptor (CD36) in the presence of Ca2+. OBJECTIVES: We monitored how the modulation of intraplatelet Ca2+ affects TSP binding to CD36 on platelets from healthy donors and patients with type 2 diabetes mellitus. We also aimed to verify whether the impaired Ca2+ mobilisation in diabetes influences TSP binding upon the pharmacological modulation of calcium transport. METHODS: Whole blood cytometry was used to monitor TSP release/binding and CD36 presentation in platelets from 28 type 2 patients and 33 healthy donors. RESULTS: No significant changes in TSP and CD36 levels were revealed between the groups in circulating platelets and TRAP-, collagen- or thrombin-activated platelets. In healthy donors, 1 microM thapsigargin (TG) elevated the TRAP-activated TSP binding (by up to 50%, p<0.001), 5 mM EGTA reversed the effect (by up to 85%, p<0.001), and overcame the effect of TG when used together. Less profoundly expressed effects occurred in the NIDDM group. In both groups TG increased the presentation of CD36 in TRAP-stimulated platelets (p<0.05), whereas EGTA lowered the TRAP-stimulated increase in CD36 (p<0.001). The inhibition of CD36 by EGTA was stronger in healthy volunteers (41% vs. 32%, respectively, p<0.05), whereas the activation by TG was higher in the NIDDM group (11% vs. 27%, p<0.05). When acting together the suppressive effects of EGTA on TG-dependent Ca2+ mobilisation were much attenuated in diabetic subjects (p<0.05). CONCLUSION: Both the release of TSP and CD36 presentation are under the influence of agents modulating intracellular Ca2+. Diabetic platelets seem more vulnerable to the releasers of cytosolic [Ca2+] and more resistant to the blockers of cytosolic [Ca2+] mobilisation.

Possible mechanisms of the altered platelet volume distribution in type 2 diabetes: does increased platelet activation contribute to platelet size heterogeneity?

A direct consequence of increased platelet sensitivity in diabetes mellitus might be augmented release of platelet granule contents, which, in turn, may lead to the formation of a platelet volume gradient, increased platelet turnover and reduced survival of platelets from diabetic individuals. In this study we addressed the question whether diabetes-induced and lipid fluidity-mediated changes in platelet receptor exposure and accessibility might be part of a general mechanism underlying the increased rate of platelet ageing and reduced platelet survival in diabetes. Diabetic individuals showed higher numbers of platelets of extreme dimensions: very small platelets and larger platelets were more frequent compared to controls ( P(chi(2))< 0.03). The shifts in platelet volume distributions were paralleled by decreased expression of the alpha subunit of glycoprotein Ib (by up to 17%, P < 0.01) in platelet membranes from diabetic patients, increased expression of P-selectin in thrombin-stimulated diabetic platelets (P< 0.02), an increased number of platelet microparticles in diabetic individuals (P< 0.05 or P< 0.03 for resting or stimulated platelets, respectively), and reduced platelet membrane fluidity (by 5.2 +/- 0.6%, P< 0.01). We suggest that the distinct bimodality of platelet distribution in diabetic patients might arise from accelerated thrombopoiesis in diabetic subjects, and this is supported by the demonstration of elevated fractions of reticulated (rich with residual RNA) platelets in diabetic patients (14.6 +/- 5.6% vs 8.1 + 2.1% p(u) < 0.025). Overall, our results point to a fluidity-mediated platelet hypersensitivity and accelerated rate of platelet production in subjects with type 2 diabetes mellitus, which results in a greater number of very large and hypersensitive younger platelets and a more abundant fraction of small exhausted platelets.

Platelet membrane lipid fluidity and intraplatelet calcium mobilization in type 2 diabetes mellitus.

The aim of the present study was to relate the impairments in calcium mobilization and/or release to the altered membrane dynamics in platelets from patients with type 2 diabetes mellitus. Higher expression of P-selectin (1.4-fold, NS) and the reduction in GPIb alpha expression (by 27.8+/-16.7%, p < 0.0002), as well as the increased fractions of platelet microparticles (p < 0.03), reflected more intensified platelet release reaction in diabetic platelets. Overall, diabetic platelets appeared more vulnerable to stimuli facilitating calcium mobilization (by 41%, p < 0.01) and less susceptible to preventive effects of the agents hampering calcium release from intraplatelet storage pools (by 38%, p < 0.01). Both the increased calcium mobilization from intraplatelet storage pools and higher levels of intracellular free calcium in the presence of procaine in diabetic platelets correlated with the reduced platelet membrane lipid fluidity (resp. pR < 0.03 and pR < 0.015). We conclude that the biophysical state of platelet membrane components in diabetes mellitus is the crucial determinant of platelet hyperfunction and probably contributes to the intensified calcium mobilization in diabetic platelets. The depressed preventive effects of procaine on platelet release reaction and calcium mobilization in diabetic platelets may result from the primary dislocations and/or distortions of membrane components caused by the diabetic state.

The effects of in vivo and in vitro non-enzymatic glycosylation and glycoxidation on physico-chemical properties of haemoglobin in control and diabetic patients.

The erythrocyte deformability, which is related to erythrocyte internal viscosity, was suggested to depend upon the physico-chemical properties of haemoglobin. In the present study we employed ESR spectroscopy on order to explore further the extent to which the in vivo or in vitro glycation and/or glycoxidation might affect haemoglobin structure on conformation. We revealed that under both in vivo and in vitro conditions the attachment of glucose induced a mobilization of thiol groups in the selected domains of haemoglobin molecules ( the increased h+1/h0 parameter of maleimide spin label, MSL; 0.277 +/- 0.021 in diabetics vs 0.338 +/- 0.017 in controls, n = 12, P < 0.0001). The relative rotational correlation time (tau c) of two spin labels, TEMPONE and TEMPAMINE, respectively, in erythrocyte insides (5.22 +/- 0.42 in diabetics, n = 21 vs 4.79 +/- 0.38, n = 16 in controls, P < 0.005) and in the solutions of in vitro glycated haemoglobin, were increased. Neither oxidation nor crosslinking of thiol groups was evidenced in glycated and/or oxidized haemoglobin. In addition, erythrocyte deformability was found to be reduced in type 2 diabetic patients (6.71 +/- 1.08, n = 28 vs 7.31 +/- 0.96, n = 21, P < 0.015). In conclusion, these observations suggest that: the attachment of glucose to haemoglobin might have decreased the mobility of the Lys-adjacent Cys residues, thus leading to the increased h+1/h0 parameter of MSL. Such structural changes in haemoglobin owing to non-enzymatic glycosylation may contribute to the increased viscosity of haemoglobin solutions (r = 0.497, P < 0.0035) and the enhanced internal viscosity of diabetic erythrocytes (r = 0.503, P < 0.003). We argue that such changes in haemoglobin, and consequently in red blood cells, might contribute to the handicapped oxygen release under tissue hypoxia in the diabetic state.

Diabetes mellitus alters the effect of peptide and protein ligands on membrane fluidity of blood platelets.

The increased nonenzymatic glycosylation of platelet membrane proteins has been suggested to underlie platelet hypersensitivity in diabetes and the relationship of this to the reduced membrane lipid fluidity has been reported. As the modulation in membrane fluidity may determine the degree of accessibility of membrane receptors, the consequent alterations in membrane lipid-protein interactions in diabetes mellitus may also underlie the differentiated effects of various thrombotic and fibrinolytic agents on platelet membrane lipid bilayer. In the present study we employed electron paramagnetic resonance and fluorescence spectroscopy to explore the ligand-induced platelet membrane fluidity changes in diabetic state, i.e. under conditions when the membrane architecture is considerably altered. The yield of the excimer formation of pyrenemaleimide (PM), which depends directly upon the collisional rate and distances between molecules, was elevated in diabetic platelet membranes, thus pointing to the occurrence of some constraints in the structure/conformation of platelet membrane proteins in diabetes mellitus. Such an immobilization of PM was accompanied by the significant elevation in membrane protein glycation in diabetic platelets. The effects of various interacting ligands on platelet membrane fluidity were significantly lower in diabetic platelets, and the differences were much more distinct at the lower depths of a lipid bilayer. Nevertheless, the alterations in membrane lipid fluidity observed upon the interaction of a given ligand occurred with an approximately equal frequency in control and diabetic platelets. Moreover, the probability that these alterations were less profound in diabetic platelets was the same for all types of ligands studied. In diabetic patients the interaction of RGDS and tissue-type plasminogen activator with platelet membranes resulted in much smaller reductions of the h+/h0 parameters in 5-DOXYL-Ste acid-labelled platelets, thus indicating a lesser rigidization of membrane lipid bilayer in diabetes. Likewise, the fluidizing effect of both fibrinogen itself and fibrinogen-derived peptides containing gamma-chain carboxy-terminal sequence H-12-V was less pronounced in diabetic platelet membranes.