Circulating but not immobilized N-deglycosylated von Willebrand factor increases platelet adhesion under flow conditions

The role of von Willebrand factor (VWF) as a shear stress activated platelet adhesive has been related to a coiled-elongated shape conformation. The forces dominating this transition have been suggested to be controlled by the proteins polymeric architecture. However, the fact that 20% of VWF molecular weight originates from glycan moieties has so far been neglected in these calculations. In this study, we present a systematic experimental investigation on the role of N-glycosylation for VWF mediated platelet adhesion under flow. A microfluidic flow chamber with a stenotic compartment that allows one to mimic various physiological flow conditions was designed for the efficient analysis of the adhesion spectrum. Surprisingly, we found an increase in platelet adhesion with elevated shear rate, both qualitatively and quantitatively fully conserved when N-deglycosylated VWF (N-deg-VWF) instead of VWF was immobilized in the microfluidic channel. This has been demonstrated consistently over four orders of magnitude in shear rate. In contrast, when N-deg-VWF was added to the supernatant, an increase in adhesion rate by a factor of two was detected compared to the addition of wild-type VWF. It appears that once immobilized, the role of glycans is at least modified if not—as found here for the case of adhesion—negated. These findings strengthen the physical impact of the circulating polymer on shear dependent platelet adhesion events. At present, there is no theoretical explanation for an increase in platelet adhesion to VWF in the absence of its N-glycans. However, our data indicate that the effective solubility of the protein and hence its shape or conformation may be altered by the degree of glycosylation and is therefore a good candidate for modifying the forces required to uncoil this biopolymer.     

Migration distance-based platelet function analysis in a microfluidic system

Aggregation and adhesion of platelets to the vascular wall are shear-dependent processes that play critical roles in hemostasis and thrombosis at vascular injury sites. In this study, we designed a simple and rapid assay of platelet aggregation and adhesion in a microfluidic system. A shearing mechanism using a rotating stirrer provided adjustable shear rate and shearing time and induced platelet activation. When sheared blood was driven through the microchannel under vacuum pressure, shear-activated platelets adhered to a collagen-coated surface, causing blood flow to significantly slow and eventually stop. To measure platelet adhesion and aggregation, the migration distance (MD) of blood through the microchannel was monitored. As the microstirrer speed increased, MD initially decreased exponentially but then increased beyond a critical rpm. For platelet-excluded blood samples, there were no changes in MD with increasing stirrer speed. These findings imply that the stirrer provided sufficiently high shear to activate platelets and that blood MD is a potentially valuable index for measuring the shear-dependence of platelet activation. Our microfluidic system is quick and simple, while providing a precise assay to measure the effects of shear on platelet aggregation and adhesion.     

Recent insights on biochemical and molecular basis for developing antihaemostatic agents: A review

The normal coagulation process is initiated by disruption and exposure of the subendothelial components of blood vessels. Platelets adhere to subendothelium-bound von Willebrand factor via glycoprotein (GP) Ib complex. This initial interaction per se and the release of platelet agonists transduce signals that leads to the rise in intracellular Ca2+ which induces shape change, prostaglandin synthesis, release of granular contents and conformational changes in platelet Gp IIb-IIIa. Gp IIb-IIIa in activated platelets binds fibrinogen and other adhesive proteins and mediates platelet cohesion the primary haemostatic plug. Furthermore, the activated platelets due to aggregation, result in the formation of fibrin (secondary hemostasis). Normally the haemostatic process plays a delicate balance between keeping the blood in the fluid state to maintain flow and rapidly forming an occluding plug following vessel injury. Thrombosis occurs because of alteration in this delicate balance. Arterial thrombosis occurs in the setting of previous vessel wall injury mostly because of atherosclerosis, while venous thrombosis occurs in areas of stasis. The recent advances in understanding of the haemostatic process have led to a better understanding of the mechanism of action of many antithrombotic drugs and identification of new targets for drug development. The molecular target of the ticlopidine has been identified. Large numbers of IIb-IIIa inhibitors have been developed. The mechanism of action of heparin has been defined at the molecular level. As a result, a synthetic pentasaccharide, based on antithrombin-binding domain of heparin, has been developed and tested successfully in clinical trials. New generation direct thrombin inhibitors are being developed. Factor Xa has a critical position at the convergence of intrinsic and extrinsic pathway. The clinical tolerability and the efficacy of low molecular weight heparins has established that inhibition of further thrombin generation, by blocking factor Xa alone can be an effective way of preventing thrombus growth without inactivating thrombin. A large number of specific factor Xa inhibitors are under development. Some of these are in preliminary clinical trials and appear to be promising. Future clinical trials will determine whether these new drugs will provide better risk-benefit ratio in treatment of thrombotic disorders. Similarly role of thrombolytics has been clearly established in many diseases including coronary artery disease.     

Interaction of LDL and platelets in ischaemic and ischaemic risk subjects

Platelets play a vital role in the progression of atherosclerosis and thrombosis, a major cause of death worldwide. Platelets are activated by many triggers like elevated LDL in blood resulting in aggregation and formation of plaque. The purpose of this study was to investigate the effect of LDL and signal transduction inhibitor on the activation of platelets in Ischaemic risk subjects. Platelets from IHD and hyperlipidemic subjects were hypersensitive to ADP, as higher levels of platelet aggregation were observed in these groups. LDL from IHD hyperlipidemic subjects was more effective in activating platelets from any other group. Ox-LDL was more effective in activating platelets than native-LDL as monitored by level of platelet aggregation induced by PAF and thrombin. Calcium channel blocker, nifedipine and verapamil inhibited platelet aggregation at micromolar level. Protein kinase inhibitor, staurosporine was effective in inhibiting ADP induced aggregation at nanomolar level.     

Covalently immobilized biomolecule gradient on hydrogel surface using a gradient generating microfluidic device for a quantitative mesenchymal stem cell study

Precisely controlling the spatial distribution of biomolecules on biomaterial surface is important for directing cellular activities in the controlled cell microenvironment. This paper describes a polydimethylsiloxane (PDMS) gradient-generating microfluidic device to immobilize the gradient of cellular adhesive Arg-Gly-Asp (RGD) peptide on poly (ethylene glycol) (PEG) hydrogel. Hydrogels are formed by exposing the mixture of PEG diacrylate (PEGDA), acryloyl-PEG-RGD, and photo-initiator with ultraviolet light. The microfluidic chip was simulated by a fluid dynamic model for the biomolecule diffusion process and gradient generation. PEG hydrogel covalently immobilized with RGD peptide gradient was fabricated in this microfluidic device by photo-polymerization. Bone marrow derived rat mesenchymal stem cells (MSCs) were then cultured on the surface of RGD gradient PEG hydrogel. Cell adhesion of rat MSCs on PEG hydrogel with various RGD gradients were then qualitatively and quantitatively analyzed by immunostaining method. MSCs cultured on PEG hydrogel surface with RGD gradient showed a grated fashion for cell adhesion and spreading that was proportional to RGD concentration. It was also found that 0.107–0.143 mM was the critical RGD concentration range for MSCs maximum adhesion on PEG hydrogel.     

Platelet satellitism in infectious disease?

Background

Platelet satellitism is a phenomenon of unknown etiology of aggregating platelets around polymorphonuclear neutrophils and other blood cells which causes pseudothrombocytopenia, visible by microscopic examination of blood smears. It has been observed so far in about a hundred cases in the world.

Case subject and methods

Our case involves a 73-year-old female patient with a urinary infection. Biochemical serum analysis (CRP, glucose, AST, ALT, ALP, GGT, bilirubin, sodium, potassium, chloride, urea, creatinine) and blood cell count were performed with standard methods on autoanalyzers. Serum protein fractions were examined by electrophoresis and urinalysis with standard methods on autoanalyzer together with microscopic examination of urine sediment. Erythrocyte sedimentation rate, blood culture and urine culture tests were performed with standard methods.

Results

Due to typical pathological values for bacterial urinary infection, the patient was admitted to the hospital. Blood smear examination revealed phenomenon, which has persisted for three weeks after the disease has been cured. Blood smears with EDTA as an anticoagulant had platelet satellitism whereas the phenomenon was not observed in tubes with different anticoagulants (Na, Li-heparin) and capillary blood.

Discussion

We hypothesize that satellitism was induced by some immunological mechanism through formation of antibodies which have mediated platelets binding to neutrophil membranes and vice versa. Unfortunately we were unable to determine the putative trigger for this phenomenon. To our knowledge this is the second case of platelet satellitism ever described in Croatia.Key words: blood platelets, thrombocytopenia, EDTA, urinary infection     

Immobilization of horseradish peroxidase on Fe3O4 magnetic nanoparticles

BackgroundIron magnetic nanoparticles have attracted much attention. They have been used in enzyme immobilization because of their properties such as product is easily separated from the medium by magnetic separation. The present work was designed to immobilize horseradish peroxidase on Fe₃O₄ magnetic nanopraticles without modification.ResultsIn the present study, horseradish peroxidase (HRP) was immobilized on non-modified Fe₃O₄ magnetic nanoparticles. The immobilized HRP was characterized by FT-IR spectroscopy, scanning electron microscopy, and energy dispersive X-ray. In addition, it retained 55% of its initial activity after 10 reuses. The optimal pH shifted from 7.0 for soluble HRP to 7.5 for the immobilized HRP, and the optimal temperature shifted from 40°C to 50°C. The immobilized HRP is more thermostable than soluble HRP. Various substrates were oxidized by the immobilized HRP with higher efficiencies than by soluble HRP. K_m values of the soluble and immobilized HRP were 31 and 45 mM for guaiacol and 5.0 and 7.0 mM for H₂O₂, respectively. The effect of metals on soluble and immobilized HRP was studied. Moreover, the immobilized HRP was more stable against high concentrations of urea, Triton X-100, and isopropanol.ConclusionsPhysical immobilization of HRP on iron magnetic nanoparticles improved the stability toward the denaturation induced by pH, heat, metal ions, urea, detergent, and water-miscible organic solvent.     

A tapered channel microfluidic device for comprehensive cell adhesion analysis, using measurements of detachment kinetics and shear stress-dependent motion

We have developed a method for studying cellular adhesion by using a custom-designed microfluidic device with parallel non-connected tapered channels. The design enables investigation of cellular responses to a large range of shear stress (ratio of 25) with a single input flow-rate. For each shear stress, a large number of cells are analyzed (500–1500 cells), providing statistically relevant data within a single experiment. Besides adhesion strength measurements, the microsystem presented in this paper enables in-depth analysis of cell detachment kinetics by real-time videomicroscopy. It offers the possibility to analyze adhesion-associated processes, such as migration or cell shape change, within the same experiment. To show the versatility of our device, we examined quantitatively cell adhesion by analyzing kinetics, adhesive strength and migration behaviour or cell shape modifications of the unicellular model cell organism Dictyostelium discoideum at 21 °C and of the human breast cancer cell line MDA-MB-231 at 37 °C. For both cell types, we found that the threshold stresses, which are necessary to detach the cells, follow lognormal distributions, and that the detachment process follows first order kinetics. In addition, for particular conditions’ cells are found to exhibit similar adhesion threshold stresses, but very different detachment kinetics, revealing the importance of dynamics analysis to fully describe cell adhesion. With its rapid implementation and potential for parallel sample processing, such microsystem offers a highly controllable platform for exploring cell adhesion characteristics in a large set of environmental conditions and cell types, and could have wide applications across cell biology, tissue engineering, and cell screening.     

Relationship between serum interleukin-1beta levels and acute phase response proteins in patients with familial Mediterranean fever

Introduction:

The aim of this study was to investigate whether serum levels of interleukin-1β (IL–1β) has any possible correlation on inflammatory parameters such as C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and fibrinogen concentration in patients with familial Mediterranean fever (FMF) patients during attack-free period.

Materials and methods:

The serum levels of IL-1β, as an indicator of cytokines status, and the acute phase response proteins, CRP, ESR and fibrinogen levels were evaluated in 35 attack-free patients with FMF and 25 healthy volunteers.

Results:

Serum IL-1β levels were significantly higher in patients with FMF than control subjects (P = 0.018). There was no statistically significant difference in the serum levels of ESR, CRP and fibrinogen between two groups (P = 0.181, P = 0.816, P = 0.686, respectively). There was a significant correlation between IL-1β and CRP (r = 0.513, P = 0.002) values of FMF group.

Conclusions:

In conclusion, our results confirm the presence of increased IL-1β levels in FMF patients during attack-free period. Serum IL-1β values seems to correlate with CRP levels. The elevation of IL-1β levels may be important in monitoring subclinical inflammation of attack free period in FMF patients.     

Circulating Thrombotic Risk Factors in Young Patients with Coronary Artery Disease Who Are on Statins and Anti-platelet Drugs

Thrombotic risk factors may contribute to premature coronary artery disease (CAD), in addition to the conventional risk factors. There is paucity of data on studies evaluating the role of thrombotic factors in premature CAD in Indian patients. Thus a case–control study was performed to evaluate the role of thrombotic and atherogenic factors in young patients with angiographically proven CAD who are on treatment with statins and anti-platelet drugs. 152 patients (≤55 years) with angiographically proven CAD and 102 asymptomatic controls were recruited. Clinical and biochemical data were obtained in both groups. Blood levels of thrombotic factors-fibrinogen, antithrombin-III, tissue-plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1), von-Willebrand factor (v-WF), lipoprotein(a) [Lp(a)] and homocysteine were analyzed. Patients had high levels of conventional CAD risk factors (diabetes mellitus, smoking, hypertension, dyslipidemia and positive family history) compared to controls. Logistic regression analysis revealed that low antithrombin-III (odds ratio/OR 11.2; 95 % confidence interval/CI 2.29–54.01), high fibrinogen (OR 6.04; 95 % CI 1.09–33.21) and high Lp(a) (OR 4.54; 95 % CI 0.92–22.56), as important, independent risk factors in patients. PAI-1(OR 0.15; 95 % CI 0.03–0.69) levels were significantly lower in patients. But other thrombotic risk factors studied (t-PA, v-WF and homocysteine) were comparable among patients and controls. The treatment using statins and anti-platelet drugs might be contributing to the control of some of the thrombotic risk factors. The strategies aiming at lowering the levels of thrombotic risk factors along with conventional risk factors may be useful in primary and secondary prevention of CAD.     

On-chip recalcification of citrated whole blood using a microfluidic herringbone mixer

In vitro assays of platelet function and coagulation are typically performed in the presence of an anticoagulant. The divalent cation chelator sodium citrate is among the most common because its effect on coagulation is reversible upon reintroduction of divalent cations. Adding divalent cations into citrated blood by batch mixing leads to platelet activation and initiation of coagulation after several minutes, thus limiting the time blood can be used before spontaneously clotting. In this work, we describe a herringbone microfluidic mixer to continuously introduce divalent cations into citrated blood. The mixing ratio, defined as the ratio of the volumetric flow rates of citrated blood and recalcification buffer, can be adjusted by changing the relative inlet pressures of these two solutions. This feature is useful in whole blood assays in order to account for differences in hematocrit, and thus viscosity. The recalcification process in the herringbone mixer does not activate platelets. The advantage of this continuous mixing approach is demonstrated in microfluidic vascular injury model in which platelets and fibrin accumulate on a collagen-tissue factor surface under flow. Continuous recalcification with the herringbone mixer allowed for flow assay times of up to 30 min, more than three times longer than the time achieved by batch recalcification. This continuous mixer allows for measurements of thrombus formation, remodeling, and fibrinolysis in vitro over time scales that are relevant to these physiological processes.     

Effect of vitamin E on the platelet xanthine oxidase and lipid peroxidation in the patients of myocardial infarction

Platelets play important role in precipitating ischaemic myocardial syndromes in many ways. One of the consequences of ischaemic diseases is excessive generation of oxygen derived free radicals that have numerous pathophysiological consequences. Platelet pro-oxidant enzyme, xanthine oxidase is one of the sources of generation of free radicals. In the present paper, we report the effect of administration of vitamin E along with aspirin on the levels of platelet xanthine oxidase and extent of free radical mediated damage in the patients reperfused after myocardial infarction. Our findings show that administration of 400 mg. vitamin E for six days along with 80 mg. aspirin has an excellent anti-oxidant effect as evidenced by reduced platelet xanthine oxidase activity and lowering of malondialdehdye (MDA) levels which is an index of the extent of free radical mediated damage.     

On-chip microfluidic systems for determination of L-glutamate based on enzymatic recycling of substrate

Two microfluidic systems have been developed for specific analysis of L-glutamate in food based on substrate recycling fluorescence detection. L-glutamate dehydrogenase and a novel enzyme, D-phenylglycine aminotransferase, were covalently immobilized on (i) the surface of silicon microchips containing 32 porous flow channels of 235 μm depth and 25 μm width and (ii) polystyrene Poros™ beads with a particle size of 20 μm. The immobilized enzymes recycle L-glutamate by oxidation to 2-oxoglutarate followed by the transfer of an amino group from D-4-hydroxyphenylglycine to 2-oxoglutarate. The reaction was accompanied by reduction of nicotinamide adenine dinucleotide (NAD⁺) to NADH, which was monitored by fluorescence detection (ε_ex=340 nm, ε_em=460 nm). First, the microchip-based system, L-glutamate was detected within a range of 3.1–50.0 mM. Second, to be automatically determined, sequential injection analysis (SIA) with the bead-based system was investigated. The bead-based system was evaluated by both flow injection analysis and SIA modes, where good reproducibility for L-glutamate calibrations was obtained (relative standard deviation of 3.3% and 6.6%, respectively). In the case of SIA, the beads were introduced and removed from the microchip automatically. The immobilized beads could be stored in a 20% glycerol and 0.5 mM ethylenediaminetetraacetic acid solution maintained at a pH of 7.0 using a phosphate buffer for at least 15 days with 72% of the activity remaining. The bead-based system demonstrated high selectivity, where L-glutamate recoveries were between 91% and 108% in the presence of six other L-amino acids tested.     

Label-free microfluidic characterization of temperature-dependent biomolecular interactions

We present a microfluidic approach to characterizing temperature-dependent biomolecular interactions. Solvated L-arginine vasopressin (AVP) and its immobilized RNA aptamer (spiegelmer) were allowed to achieve equilibrium binding in a microchip at a series of selected temperatures. Unbound AVP were collected and analyzed with matrix-assisted laser desorption∕ionization mass spectrometry (MALDI-MS), yielding melting curves that reveal highly temperature-dependent zones in which affinity binding (36–45 °C) or dissociation (25–33 °C and 50–65 °C) occurs. Additionally, temperature-dependent binding isotherms were constructed; from these, thermodynamic quantities involved in binding were extracted. The results illustrated a strong change in heat capacity of interaction for this system, suggesting a considerable thermodynamic influence controlling vasopressin-spiegelmer interaction.     

Asymmetry of red blood cell motions in a microchannel with a diverging and converging bifurcation

In microcirculation, red blood cells (RBCs) flowing through bifurcations may deform considerably due to combination of different phenomena that happen at the micro-scale level, such as: attraction effect, high shear, and extensional stress, all of which may influence the rheological properties and flow behavior of blood. Thus, it is important to investigate in detail the behavior of blood flow occurring at both bifurcations and confluences. In the present paper, by using a micro-PTV system, we investigated the variations of velocity profiles of two working fluids flowing through diverging and converging bifurcations, human red blood cells suspended in dextran 40 with about 14% of hematocrit level (14 Hct) and pure water seeded with fluorescent trace particles. All the measurements were performed in the center plane of rectangular microchannels using a constant flow rate of about 3.0 × 10⁻¹² m³/s. Moreover, the experimental data was compared with numerical results obtained for Newtonian incompressible fluid. The behavior of RBCs was asymmetric at the divergent and convergent side of the geometry, whereas the velocities of tracer particles suspended in pure water were symmetric and well described by numerical simulation. The formation of a red cell-depleted zone immediately downstream of the apex of the converging bifurcation was observed and its effect on velocity profiles of RBCs flow has been investigated. Conversely, a cell-depleted region was not formed around the apex of the diverging bifurcation and as a result the adhesion of RBCs to the wall surface was enhanced in this region.     

自然界最基本循环和微生物农业

为解决当前农业和环境面临的难题,文章讨论了有机物质在植物、动物、土壤和微生物这4个系统中的运动,指出这些运动形成了两个稳定的基本循环,我们称之为自然界的最基本循环。近100多年来,人类忽视了微生物在有机物质的转化和循环过程中的重要作用,结果使微生物系统受到伤害,最基本循环遭到严重破坏,导致农业和环境出现了一系列严重问题。文章提出了恢复微生物系统和最基本循环的具体方案,希望通过发展微生物农业使中国农业走上高效、可持续发展的道路。     

Platelet satellitism in a trauma patient

Platelet satellitism (PS) is a rare phenomenon observed in blood smears obtained from blood anticoagulated with EDTA. It is characterised by platelet rosetting around polymorphonuclear neutrophils and in rare cases around other blood cells. PS is a rare cause of pseudothrombocytopenia. References about the phenomenon of PS in medical literature are few. In this report we describe a case of PS fortunately noticed in one trauma patient. Furthermore, we discuss the possible pathophysiological mechanisms of PS proposed in the literature. To our knowledge this is the first case of PS reported in Croatia.     

Rapid bioparticle concentration and detection by combining a discharge driven vortex with surface enhanced Raman scattering

Rapid concentration and detection of bacteria in integrated chips and microfluidic devices is needed for the advancement of lab-on-a-chip devices because current detection methods require high concentrations of bacteria which render them impractical. We present a new chip-scale rapid bacteria concentration technique combined with surface-enhanced Raman scattering (SERS) to enhance the detection of low bacteria count samples. This concentration technique relies on convection by a long-range converging vortex to concentrate the bacteria into a packed mound of 200 μm in diameter within 15 min. Concentration of bioparticle samples as low as 10⁴ colony forming units (CFU)∕ml are presented using batch volumes as large as 150 μl. Mixtures of silver nanoparticles with Saccharomyces cerevisiae, Escherichia coli F-amp, and Bacillus subtilis produce distinct and noticeably different Raman spectra, illustrating that this technique can be used as a detection and identification tool.     

Numerical study of in situ preconcentration for rapid and sensitive nanoparticle detection

This paper presents a numerical study of a preconcentrator design that can effectively increase the binding rate at the sensor in a real time manner. The particle enrichment is realized by the ac electrothermal (ACET) effect, which induces fluid movement to carry nanoparticles toward the sensor. The ACET is the only electrical method to manipulate a biological sample of medium to high ionic strength (>0.1 S∕m, e.g., 0.06× phosphate buffered saline). The preconcentrator consists of a pair of electrodes striding over the sensor, simple to implement as it is electrically controlled. This preconcentrator design is compatible and can be readily integrated with many types of micro- to nanosensors. By applying an ac signal over the electrodes, local vortices will generate a large velocity perpendicular to the reaction surface, which enhances transport of analytes toward the sensor. Our simulation shows that the binding rate at the sensor surface is greatly enhanced. Our study also shows that the collection of analytes will be affected by various parameters such as channel height, inlet velocity, and sensor size, and our results will provide guidance in optimization of the preconcentrator design.     

Development and evaluation of realistic microbioassays in freely suspended droplets on a chip

A novel technique for biomolecular detection in microliter droplets floating on the surface of high density oil is presented. Each droplet was captured and manipulated dielectrophoretically and was used as a site for a microscopic bioassay based on agglutination of antibody-conjugated particles. The results were read out by the pattern of unagglomerated gold nanoparticles collected on the droplet surface. Two formats of bioassays, namely gold only agglutination and gold and latex agglutination, were investigated experimentally by varying analyte concentration, particle size and concentration, number of antigen binding sites per particle, time for incubation, and rate of particle collection on the droplet surface. The microbioassays performance was also evaluated with ricin antibodies and compared to the ricin assays in field use. It is estimated that the droplet based assays require 100× smaller sample volume and are ten times more sensitive, though they require longer times to complete. The experiments were interpreted by modeling the kinetics of particle agglutination and mass transfer processes inside the droplets. The incubation time and antigen concentration values calculated by the model correlate well with the experimental results. The results could allow for development of efficient immunoassays on a chip requiring even smaller sample volumes.