Membrane and protein properties of freeze-dried mouse platelets

Membrane properties and the overall protein secondary structure of freeze-dried trehalose-loaded mouse platelets were studied using steady state fluorescence anisotropy and Fourier transform infrared spectroscopy (FTIR). FTIR results showed that fresh control mouse platelets have a main phase transition at approximately 14 degrees C, whereas, freeze-dried platelets exhibited a main phase transition approximately 12 degrees C. However, the cooperativity of the transition of the rehydrated platelets was greatly enhanced compared to that of control platelets. Anisotropy experiments performed with 1,6 diphenyl-1,3,5 hexatriene (DPH) complemented FTIR results and showed that the lipid order in the core of the membrane was affected by freeze-drying procedures. Similar experiments with trimethyl ammonium 1,6 diphenyl-1,3,5 hexatriene (TMA-DPH), a membrane surface probe, indicated that membrane properties at the membrane/water interface were less affected by freeze-drying procedures than the core of the membrane. Lyophilization did not result in massive protein denaturation, but the overall protein secondary structure was altered, based on in situ assessment of the amide-I and amide-II band profiles. Lyophilization-induced changes to endogenous platelet proteins were further investigated by studying the protein's heat stability. In fresh control platelets, proteins denatured at 42 degrees C, whereas proteins in the rehydrated platelets denatured at 48 degrees C.     

Physical properties of membrane fractions isolated from human platelets: implications for chilling induced platelet activation

In previous studies, it has been suggested that chilling induced activation of human platelets is related to a lipid phase transition seen in membrane lipids. Those studies showed a single, surprisingly cooperative transition in human platelets, as determined by Fourier transform infrared (FTIR) spectroscopy, findings that are confirmed here with calorimetric measurements. Such transitions have now been studied in membrane fractions obtained from the platelets and it is reported that all fractions and purified phospholipids show similar transitions. In order to obtain these data it was necessary to develop means for separating these fractions. Therefore, a novel method for isolation and separation of dense tubular system (DTS) and plasma membranes in human platelets is described here. Lipid analysis showed that phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were the dominant phospholipids in both fractions, whereas cholesterol and sphingomyelin (SM) were predominantly located in the plasma membranes. Thermotropic phase transitions in the two membrane fractions, determined by differential scanning calorimetry (DSC) and FTIR spectroscopy were found to occur at about 15 degrees C, similar to the Tm of intact human platelets. These data are discussed in relation to the role of the DTS and plasma membranes in the cold-induced activation of human platelets.     

An experimental study of the use of ultrasound to facilitate the loading of trehalose into platelets

Trehalose is widely used as a freeze-drying protectant in biomaterial preservation. For this purpose, trehalose has to be loaded into the cells but this is difficult and many methods have been tried. The application of ultrasound can temporarily permeabilize cell membranes, which offers a non-chemical, non-viral, and non-invasive method of cellular drug delivery. Ultrasound is employed here to enhance the loading of trehalose into human platelets. Two frequencies were used, 25 kHz and 800 kHz. The estimated intensity of ultrasound in the sample was varied from 0 to 1.5 W/cm². The trehalose concentration in the platelets was 11.27 ± 2.53 mmol/L when Wolkers et al.’s method was used without ultrasound. The application of 0.8 W/cm², 800 kHz ultrasound for 1 h increased the concentration of trehalose loaded by 54%. The application of 0.8 W/cm², 25 kHz ultrasound for 30 min increased the trehalose concentration that was loaded by 172%. The number and mean volume of the platelets following ultrasonic radiation in these two cases remained normal as compared with fresh untreated platelets. Morphological examination of the radiated platelets showed slight changes. Although further work is needed, ultrasound has been shown to be efficient for the loading of trehalose into platelets.     

Preparation and application of freeze-dried platelets

Clinical platelet infusion is primarily used to prevent or stop bleeding, but can also have a role in treating infections or promoting wound healing. The demand for platelets has increased in recent years. However, as platelets can only be stored for short periods, there is a substantial loss due to the products reaching their expiry date. Platelet lyophilization is a particularly valuable and important research field. The purpose of studying the freeze-drying preservation of platelets is to realize the long-term preservation of platelets at room temperature. It is very possible to prepare qualified freeze-dried platelets. However, there are still problems that have not been solved in the process of platelet lyophilization. This review mainly summarizes research progress in the preparation and application of freeze-dried platelets.     

Generation of megakaryocytes and platelets from human subcutaneous adipose tissues

Recent advances in regenerative medicine have created a broad spectrum of stem cell research. Among them, tissue stem cell regulations are important issues to clarify the molecular mechanism of differentiation. Adipose tissues have been shown to contain abundant preadipocytes, which are multipotent to differentiate into cells including adipocytes, chondrocytes, and osteoblasts. In this study, we have first shown that megakaryocytes and platelets can be generated from adipocyte precursor cells. Human adipocyte precursor cells were cultured in conditioned media for 12 days to differentiate adipocytes, followed by 12 days of culture in media containing thrombopoietin. The ultrastructures of adipocyte precursor cell- and bone marrow CD34-positive cell-derived megakaryocytes and platelets were similar. In addition, adipocyte precursor cell-derived platelets exhibited surface expression of P-selectin and bound fibrinogen upon stimulation with platelet agonists, suggesting that these platelets were functional. This is the first demonstration that human subcutaneous adipocyte precursor cells can generate megakaryocyte and functional platelets in an in vitro culture system.     

On the degradation of heparin by human blood platelets.

Human blood platelets are able to degrade heparin from different tissues and species. The main degradation product is an oligosaccharide. Low molecular weight components such as inorganic sulfate or monosaccharides, i.e. products released by exoenzymes are not detected. The in vitro degradation of heparin by the crude enzyme is observed at pHs below 6.5 with an optimum temperature around 37 degrees C. The presence of sulfate in the substrate structure is shown to be essential for the enzyme activity. Since the oligosaccharides formed have only 10 per cent of the anticoagulant activity of the heparins tested, it is conceivable that the platelet enzyme may play an important role in the inactivation of some of the biological properties of heparin.     

Long-term preservation of freeze-dried mouse spermatozoa

Many genetically engineered mice strains have been generated worldwide and sperm preservation is a valuable method for storing these strains as genetic resources. Freeze-drying is a useful sperm preservation method because it requires neither liquid nitrogen nor dry ice for preservation and transportation. We report here successful long-term preservation at 4 °C of mouse spermatozoa freeze-dried using a simple buffer solution (10mM Tris, 1mM EDTA, pH 8.0). Offspring with fertility were obtained from oocytes fertilized with freeze-dried spermatozoa from C57BL/6 and B6D2F1 mouse strains stored at 4 °C for 3 years. This freeze-drying method is a safe and economical tool for the biobanking of valuable mouse strains.     

Inhibition of the glycolytic pathway by methylglyoxal in human platelets

The incubation of human platelets with methylglyoxal and glucose produces a rapid transformation of the ketoaldehyde to D-lactate by the glyoxalase system and a partial reduction in GSH. Glucose utilization is affected at the level of the glycolytic pathway. No effect of the ketoaldehyde on glycogenolysis and glucose oxidation through the hexose monophosphate shunt was demonstrated. Phosphofructokinase, fructose 1,6 diphosphate (F1, 6DP) aldolase, glyceraldehyde 3-phosphate dehydrogenase and 3-phosphoglycerate mutase were mostly inhibited by methylglyoxal. A decrease in lactate and pyruvate formation and an accumulation of some glycolytic intermediates (fructose 1,6 diphosphate, dihydroxyacetone phosphate, 3-phosphoglycerate) was observed. Moreover methylglyoxal induced a fall in the metabolic ATP concentration. Since methylglyoxal is an intermediate of the glycolytic bypass system from dihydroxyacetone phosphate to D-lactate, it may be assumed that ketoaldehyde exerts a regulating effect on triose metabolism.     

Protein kinase C alpha enhances sodium-calcium exchange during store-operated calcium entry in mouse platelets

A rise in intracellular calcium concentration ([Ca(2+)](i)) is necessary for platelet activation. A major component of the [Ca(2+)](i) elevation occurs through store-operated Ca(2+) entry (SOCE). The aim of this study was to understand the contribution of the classical PKC isoform, PKCα to platelet SOCE, using platelets from PKCα-deficient mice. SOCE was reduced by approximately 50% in PKCα(-/-) platelets, or following treatment with bisindolylmaleimide I, a PKC inhibitor. However, TG-induced Mn(2+) entry was unaffected, which suggests that divalent cation entry through store-operated channels is not directly regulated. Blocking the autocrine action of secreted ADP or 5-HT on its receptors did not reproduce the effect of PKCα deficiency. In contrast, SN-6, a Na(+)/Ca(2+) exchanger inhibitor, did reduce SOCE to the same extent as loss of PKCα, as did replacing extracellular Na(+) with NMDG(+). These treatments had no further effect in PKCα(-/-) platelets. These data suggest that PKCα enhances the extent of SOCE in mouse platelets by regulating Ca(2+) entry through the Na(+)/Ca(2+) exchanger.     

Physiological characteristics of serotonin transporters on rat platelets

Physiological characteristics of serotonin (5-hydroxytryptamine, 5HT) transport through the platelet membrane was investigated in Wistar rats with our recently developed method permitting repetitive measurements of transporter kinetics in individual animals. Full kinetic analysis in the population of 91 animals revealed Michaelis constant (K_m) of 0.158±0.025 μM and maximal velocity (V_max) of 5HT uptake of 225±32 pmol per 10⁸platelets min⁻¹ (mean±S.D.). Both kinetic parameters demonstrated normal distribution curves, which for V_max were slightly skewed toward higher than average values. No gender effect was shown in frequency distributions, mean values and variability of kinetic parameters. A significant intraindividual correlation between kinetic parameters was found suggesting compensation at the level of the plasma membrane. Kinetic parameters were not influenced by age (until the middle age) or annual cycle (under laboratory conditions) and were shown to be fairly stable in time, supporting the view that platelet 5HT transport kinetics could be a useful biological trait marker.     

Stimulation of human platelets with concanavalin A involves phospholipase C activation.

In response to concanavalin A, cytoplasmic calcium movement was observed in human platelets, both in the presence of 1 mM Ca2+ or 1 mM EGTA in the medium. Concanavalin A also caused the activation of inositide turnover and the production of inositol phosphates, suggesting that activation of phospholipase C occurs. The mechanism by which concanavalin A stimulates phospholipase C does not depend on GTP-binding transducers, because it was not inhibited by GDP beta S, while experiments performed in the presence of cytochalasin B suggested a role for membrane glycoprotein IIb-IIIa-cytoskeleton interaction in this process. Ca(2+)-proteases and Na+/H+ antiport also seemed to be related to concanavalin A-induced phospholipase C activation, as suggested by experiments performed in the presence of leupeptin and amiloride.     

Modeling of an Aerobic Membrane Bioreactor for Wastewater Treatment

The goal of this work is to develop a mathematical model to describe a continuous aerobic membrane bioreactor (MBR) for the treatment of different kinds of wastewater. Firstly, the experimental setup and the materials and methods to obtain data for the model verification are described. Secondly, a black box model is developed and verified for size containing wastewater. The model calculates output streams and concentrations from input streams and compositions using distribution coefficients. These coefficients are derived from experimental data. In a third step, two shortcut models of different complexity are developed. Both shortcut models use the black box balancing as a basis. However, the component balances for carbon and oxygen are no longer modeled by constant distribution coefficients. The basic shortcut model introduces Monod kinetics to modify the carbon balance. The enhanced shortcut model introduces transport laws for dissolved oxygen supply and combines the Monod kinetics with an additional term for oxygen limitation to model biological growth. The models show an increasing degree of agreement.     

Loading solution prevents activation damage of human platelets before lyophilization

The current study aims to optimize the compositions of platelet activation-inhibitors for a loading solution of lyophilizing protectants and to establish a series of perfect pretreatment methods for platelet lyophilization. The optimal combination of six kinds of inhibitors and loading solutions of lyophilizing protectants, including prostaglandin E1 (PGE1), adenosine, l-arginine, phyticacid, bivalirudin, and cilostazol, was analyzed using the orthogonal experimental design. The values of the expression rates of p-selectin (CD62p) and platelet membrane glyeoprotein (PAC-1), as well as of platelet and mean platelet volume (MPV), were selected as indices of platelet activation. The values of CD62p and Pac-1 induced by thrombin were determined as indices of platelet reactivity. The maximal aggregation and slide platelet aggregation test (SPAT) induced by the inducer were calculated as indices of the aggregation function of platelets. Level I of the loading condition factor had no adverse action on MPV, CD62p, PAC-1, SPAT, and the maximum platelet aggregation rate. Level II of factors PGE1, l-arginine, phycicacid sodium, and Bivalirudin could inhibit the activation of platelets and enable them to retain their function. The results show that the optimal solution compounding was the third group. The loading solution, which includes plasma, 1 μM prostaglandin E1, 5 mM l-arginine, 0.5 mM phyticacid, and 0.5 μM bivalirudin, could prevent the activation damage of platelets before lyophilization.     

Rigorous Approach to Modeling of a Continuous Aerobic Membrane Bioreactor

A rigorous approach to mathematical modeling of a continuous aerobic membrane bioreactor (MBR) for the treatment of wastewater is reported. The idea is to apply the activated sludge model ASM3 to the special configuration of a membrane bioreactor. Therefore, the biochemical processes modeled by the ASM3 were implemented together with mass balances typical of a MBR running at constant TSS. The model parameters were adapted to the properties of an artificial wastewater by using a global search algorithm. The model could be validated by comparing effluent chemical oxygen demand (COD), sludge production and CO₂ concentration in the exhaust to the experimental data.     

The role of blood platelets in nucleoside metabolism: regulation of megakaryocyte development and platelet production

In higher vertebrates, different types of blood cells develop from common precursors. Mammals are unique in possessing two types of blood cells — erythrocytes and platelets — which lack nuclei. Although platelets display consistent and easily-recognisble morphological and ultrastructural characteristics and show exreme metabolic and functional versatility, they are not true cells, being produced by fragmentation of giant polyploid precursors called megakaryocytes. At present, the physiological mechanisms which regulate megakaryocyte development and platelet production are not well understood.

Platelets are actively involved in metabolism of purine derivatives and a significant platelet role in pyrimidine metabolism has also been demonstrated (see previous papers). Here an attempt is made to integrate information about platelet involvement in nucleic precursor metabolism with current concepts of haematopoiesis, particularly megakaryocyte development and platelet production.

It is concluded (i) that megakaryocytic cels are immediate descendents of haematopoietic stem cells which have become polyploid as a result of genetic damage or metabolic imbalances, (ii) megakaryocytes and platelets are the ultimate regulators of stem cell development because they control the availability of thymidine and (iii) that the production of megakaryocytes and platelets is a physiological safety mechanism which prevents fixation of genetic damage and protects other cells from potentially cytotoxic and genotoxic stimuli.