Search

Native circulating blood platelets present with a discoid flat morphology maintained by a submembranous peripheral ring of microtubules, named marginal band. The functional importance of this particular shape is still debated, but it was initially hypothesized to facilitate platelet interaction with the injured vessel wall and to contribute to hemostasis. The importance of the platelet discoid morphology has since been questioned on the absence of clear bleeding tendency in mice lacking the platelet-specific β1-tubulin isotype, which exhibits platelets with a thinner marginal band and an ovoid shape. Here, we generated a mouse model inactivated for β1-tubulin and α4A-tubulin, an α-tubulin isotype strongly enriched in platelets. These mice present with fully spherical platelets completely devoid of a marginal band. In contrast to the single knockouts, the double deletion resulted in a severe bleeding defect in a tail-clipping assay, which was not corrected by increasing the platelet count to normal values by the thrombopoietin-analog romiplostim. In vivo, thrombus formation was almost abolished in a ferric chloride–injury model, with only a thin layer of loosely packed platelets, and mice were protected against death in a model of thromboembolism. In vitro, platelets adhered less efficiently and formed smaller-sized and loosely assembled aggregates when perfused over von Willebrand factor and collagen matrices. In conclusion, this study shows that blood platelets require 2 unique α- and β-tubulin isotypes to acquire their characteristic discoid morphology. Lack of these 2 isotypes has a deleterious effect on flow-dependent aggregate formation and stability, leading to a severe bleeding disorder.

•Platelets lacking α4A- and β1-tubulins are devoid of marginal band and fully spherical and inefficiently interact with matrices under flow.•Mice deficient for α4A- and β1-tubulins exhibit severe defects in hemostatic and thrombotic responses in vivo.

Blood flow is a major regulator of hemostasis and arterial thrombosis. The current view is that low and intermediate flows occur in intact healthy vessels, whereas high shear levels (>2000 s−1) are reached in stenosed arteries, notably during thrombosis. To date, the shear rates occurring at the edge of a lesion in an otherwise healthy vessel are nevertheless unknown. The aim of this work was to measure the shear rates prevailing in wounds in a context relevant to hemostasis. Three models of vessel puncture and transection were developed and characterized for a study that was implemented in mice and humans. Doppler probe measurements supplemented by a computational model revealed that shear rates at the edge of a wound reached high values, with medians of 22 000 s−1, 25 000 s−1, and 7000 s−1 after puncture of the murine carotid artery, aorta, or saphenous vein, respectively. Similar shear levels were observed after transection of the mouse spermatic artery. These results were confirmed in a human venous puncture model, where shear rates in a catheter implanted in the cubital vein reached 2000 to 27 000 s−1. In all models, the high shear conditions were accompanied by elevated levels of elongational flow exceeding 1000 s−1. In the puncture model, the shear rates decreased steeply with increasing injury size. This phenomenon could be explained by the low hydrodynamic resistance of the injuries as compared with that of the downstream vessel network. These findings show that high shear rates (>3000 s−1) are relevant to hemostasis and not exclusive to arterial thrombosis.

Purpose: Assessing the rupture probability of intracranial aneurysms (IAs) remains challenging. Therefore, hemodynamic simulations are increasingly applied toward supporting physicians during treatment planning. However, due to several assumptions, the clinical acceptance of these methods remains limited. Methods: To provide an overview of state-of-the-art blood flow simulation capabilities, the Multiple Aneurysms AnaTomy CHallenge 2018 (MATCH) was conducted. Seventeen research groups from all over the world performed segmentations and hemodynamic simulations to identify the ruptured aneurysm in a patient harboring five IAs. Although simulation setups revealed good similarity, clear differences exist with respect to the analysis of aneurysm shape and blood flow results. Most groups (12/71%) included morphological and hemodynamic parameters in their analysis, with aspect ratio and wall shear stress as the most popular candidates, respectively. Results: The majority of groups (7/41%) selected the largest aneurysm as being the ruptured one. Four (24%) of the participating groups were able to correctly select the ruptured aneurysm, while three groups (18%) ranked the ruptured aneurysm as the second most probable. Successful selections were based on the integration of clinically relevant information such as the aneurysm site, as well as advanced rupture probability models considering multiple parameters. Additionally, flow characteristics such as the quantification of inflow jets and the identification of multiple vortices led to correct predictions. Conclusions: MATCH compares state-of-the-art image-based blood flow simulation approaches to assess the rupture risk of IAs. Furthermore, this challenge highlights the importance of multivariate analyses by combining clinically relevant metadata with advanced morphological and hemodynamic quantification.

We present a novel, conceptually simple approach to calculate the configurational entropy difference between two conformational ensembles of a molecular system. The method estimates the full-dimensional probability density function of the system by a Gaussian mixture, using an efficient greedy learning algorithm with a cross-validation-based stopping criterion. An evaluation of the method on conformational ensembles corresponding to substates of five small peptide systems shows that excellent agreement is found with the exact entropy differences obtained from a full enumeration of conformations. Compared with the quasiharmonic method and two other, more recently developed methods, the Gaussian mixture method yields more accurate results at smaller sample sizes. We illustrate the power of the method by calculating the backbone torsion angle entropy difference between disulfide-bonded and nondisulfide-bonded states of tachyplesin, a 17-residue antimicrobial peptide, and between two substates in the native ensemble of the 58-residue bovine pancreatic trypsin inhibitor.

A retrospective modeling was performed in a case with restenosis after 6 months of a DES implantation to investigate the effect of the step-down at the distal edge of the stent on the wall shear stress (WSS) using 3D coronary angiography reconstruction and the intracoronary pressure traces during a cardiac cycle. The kinetics of the WSS was calculated through the cardiac cycle along the reconstructed vessel by fluid dynamic analysis. Contrary to the previous reports the greatest amplitude and average of the arterial WSS was detected where the restenosis developed later at the site of the step-down.

The presence of collaterals and high thrombus permeability are associated with good functional outcomes after an acute ischaemic stroke. We aim to understand the combined effect of the collaterals and thrombus permeability on cerebral blood flow during an acute ischaemic stroke. A cerebral blood flow model including the leptomeningeal collateral circulation is used to simulate cerebral blood flow during an acute ischaemic stroke. The collateral circulation is varied to capture the collateral scores: absent, poor, moderate and good. Measurements of the transit time, void fraction and thrombus length in acute ischaemic stroke patients are used to estimate thrombus permeability. Estimated thrombus permeability ranges between 10−7and 10−4mm2. Measured flow rates through the thrombus are small and the effect of a permeable thrombus on brain perfusion during stroke is small compared with the effect of collaterals. Our simulations suggest that the collaterals are a dominant factor in the resulting infarct volume after a stroke.

Enzyme function depends on specific conformational motions. We show that the temperature dependence of enzyme kinetic parameters can provide insight into these functionally relevant motions. While investigating the catalytic properties of IPMDH from Escherichia coli, we found that its catalytic efficiency (kcat/KM,IPM) for the substrate IPM has an unusual temperature dependence, showing a local minimum at ∼35°C. In search of an explanation, we measured the individual constants kcat and KM,IPM as a function of temperature, and found that the van 't Hoff plot of KM,IPM shows sigmoid-like transition in the 20–40°C temperature range. By means of various measurements including hydrogen-deuterium exchange and fluorescence resonance energy transfer, we showed that the conformational fluctuations, including hinge-bending domain motions increase more steeply with temperatures >30°C. The thermodynamic parameters of ligand binding determined by isothermal titration calorimetry as a function of temperature were found to be strongly correlated to the conformational fluctuations of the enzyme. Because the binding of IPM is associated with a hinge-bending domain closure, the more intense hinge-bending fluctuations at higher temperatures increasingly interfere with IPM binding, thereby abruptly increasing its dissociation constant and leading to the observed unusual temperature dependence of the catalytic efficiency.

The amino acid composition of intrinsically disordered proteins and protein segments characteristically differs from that of ordered proteins. This observation forms the basis of several disorder prediction methods. These, however, usually perform worse for smaller proteins (or segments) than for larger ones. We show that the regions of amino acid composition space corresponding to ordered and disordered proteins overlap with each other, and the extent of the overlap (the “twilight zone”) is larger for short than for long chains. To explain this finding, we used two-dimensional lattice model proteins containing hydrophobic, polar, and charged monomers and revealed the relation among chain length, amino acid composition, and disorder. Because the number of chain configurations exponentially grows with chain length, a larger fraction of longer chains can reach a low-energy, ordered state than do shorter chains. The amount of information carried by the amino acid composition about whether a protein or segment is (dis)ordered grows with increasing chain length. Smaller proteins rely more on specific interactions for stability, which limits the possible accuracy of disorder prediction methods. For proteins in the “twilight zone”, size can determine order, as illustrated by the example of two-state homodimers.

Hydrogenases are redox metalloenzymes in bacteria that catalyze the uptake or production of molecular hydrogen. Two homologous nickel–iron hydrogenases, HupSL and HydSL from the photosynthetic purple sulfur bacterium Thiocapsa roseopersicina, differ substantially in their thermal stabilities despite the high sequence similarity between them. The optimum temperature of HydSL activity is estimated to be at least 50 °C higher than that of HupSL. In this work, homology models of both proteins were constructed and analyzed for a number of structural properties. The comparison of the models reveals that the higher stability of HydSL can be attributed to increased inter-subunit electrostatic interactions: the homology models reliably predict that HydSL contains at least five more inter-subunit ion pairs than HupSL. The subunit interface of HydSL is more polar than that of HupSL, and it contains a few extra inter-subunit hydrogen bonds. A more optimized cavity system and amino acid replacements resulting in increased conformational rigidity may also contribute to the higher stability of HydSL. The results are in accord with the general observation that with increasing temperature, the role of electrostatic interactions in protein stability increases. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00894-001-0071-8.

The annual wandering of Earth's pole takes place in an area about 200 m2. Within this area, the amplitudes of the displacements due to Earth tide and global meteorological processes do not exceed a few decimetres. Obviously, very precise measuring technique is necessary for the examination of the above phenomena. Nowadays, these are primarily the devices and the methods of cosmic geodesy. From them, on the basis of Ádám (1990) the VLBI (Very Long Baseline Interferometry), and the SLR (Satellite Laser Ranging) are able to show the variations of the pole with periods between one day and one year. The GPS (Global Positioning System) and the developed processing system of VLBI are suitable for the examination of processes within a day.

The catalytic properties of C1r, the protease that mediates activation of the C1 complex of complement, are mediated by its C-terminal region, comprising two complement control protein (CCP) modules followed by a serine protease (SP) domain. Baculovirus-mediated expression was used to produce fragments containing the SP domain and either 2 CCP modules (CCP1/2-SP) or only the second CCP module (CCP2-SP). In each case, the wild-type species and two mutants stabilized in the proenzyme form by mutations at the cleavage site (R446Q) or at the active site serine residue (S637A), were produced. Both wild-type fragments were recovered as two-chain, activated proteases, whereas all mutants retained a single-chain, proenzyme structure, providing the first experimental evidence that C1r activation is an autolytic process. As shown by sedimentation velocity analysis, all CCP1/2-SP fragments were dimers (5.5–5.6 S), and all CCP2-SP fragments were monomers (3.2–3.4 S). Thus, CCP1 is essential to the assembly of the dimer, but formation of a stable dimer is not a prerequisite for self-activation. Activation of the R446Q mutants could be achieved by extrinsic cleavage by thermolysin, which cleaved the CCP2-SP species more efficiently than the CCP1/2-SP species and yielded enzymes with C1s-cleaving activities similar to their active wild-type counterparts. C1r and its activated fragments all cleaved C1s, with relative efficiencies in the order C1r < CCP1/2-SP < CCP2-SP, indicating that CCP1 is not involved in C1s recognition.

3-isopropylmalate dehydrogenase (IPMDH) from the psychrotrophic bacterium Vibrio sp. I5 has been expressed in Escherichia coli and purified. This cold-adapted enzyme is highly homologous with IPMDHs from other organisms, including mesophilic E. coli and thermophilic Thermus thermophilus bacteria. Its molecular properties are similar to these counterparts. Whereas the E. coli and T. thermophilus enzymes are hardly active at room temperature, the Vibrio IPMDH has reasonable activity below room temperature. The thermal stabilities, conformational flexibilities (hydrogen-deuterium exchange), and kinetic parameters of these enzymes were compared. The temperature dependence of the catalytic parameters of the three enzymes show similar but shifted profiles. The Vibrio IPMDH is a much better enzyme at 25 degrees C than its counterparts. With decreasing temperature i.e. with decreasing conformational flexibility, the specific activity reduces, as well; however, in the case of the Vibrio enzyme, the residual activity is still high enough for normal physiological operation of the organism. The cold-adaptation strategy in this case is achieved by creation of an extremely efficient enzyme, which has reduced but still sufficient activity at low temperature.

The comparison of the three‐dimensional structures of thermophilic (Thermus thermophilus) and mesophilic (Escherichia coli) 3‐isopropylmalate dehydrogenases (IPMDH, EC 1.1.1.85) suggested that the existence of extra ion pairs in the thermophilic enzyme found in the intersubunit region may be an important factor for thermostability. As a test of our assumption, glutamine 200 in the E. colienzyme was turned into glutamate (Q200E mutant) to mimic the thermophilic enzyme at this site by creating an intersubunit ion pair which can join existing ion clusters. At the same site in the thermophilic enzyme we changed glutamate 190 into glutamine (E190Q), hereby removing the corresponding ion pair. These single amino acid replacements resulted in increased thermostability of the mesophilic and decreased thermostability of the thermophilic enzyme, as measured by spectropolarimetry and differential scanning microcalorimetry.

Blood flow is a major regulator of hemostasis and arterial thrombosis. The current view is that low and intermediate flows occur in intact healthy vessels, while high shear levels (>2,000 s-1) are reached in stenosed arteries, notably during thrombosis. To date, the shear rates occurring at the edge of a lesion in an otherwise healthy vessel are nevertheless unknown. The aim of this work was to measure the shear rates prevailing in wounds in a context relevant to hemostasis. Three models of vessel puncture and transection were developed and characterized for a study which was implemented in mice and humans. Doppler probe measurements supplemented by a computational model revealed that shear rates at the edge of a wound reached high values, with medians of 22,000 s-1, 25,000 s-1and 7,000 s-1after puncture of the murine carotid artery, aorta or saphenous vein, respectively. Similar shear levels were observed after transection of the mouse spermatic artery. These results were confirmed in a human venous puncture model, where shear rates in a catheter implanted in the cubital vein reached 2,000-27,000 s-1. In all models, the high shear conditions were accompanied by elevated levels of elongational flow exceeding 1,000 s-1. In the puncture model, the shear rates decreased steeply with increasing injury size. This phenomenon could be explained by the low hydrodynamic resistance of the injuries as compared to that of the downstream vessel network. These findings show that high shear rates (>3,000 s-1) are relevant to hemostasis and not exclusive to arterial thrombosis.

A review of some of the recent results in an effort to obtain electron–ion differential scattering cross sections using fast ion–atom collisions is given. In the projectile frame, if we neglect the effects from the target nucleus, the ion–atom collision can be described as an electron–ion scattering process where the energy distribution of the impinging quasi-free electrons is determined by the Compton-profile of the target. In this electron scattering model (ESM), in addition to the direct electron scattering, doubly excited state formation of the projectile ion is also possible. This is a resonant process in which the doubly excited states can subsequently decay by ejecting Auger-electrons. We have studied elastic, inelastic and superelastic electron scattering as a function of incoming electron energy by observing the outgoing electron energy in the ion–atom collision emission spectra.

A novel method was developed for the analysis of the interaction of large multivalent ligands with surfaces (matrices) to analyse the binding of complement subcomponent C1q to immune precipitates. Our new evaluation method provides quantitative data characteristic of the C1q-immune-complex interaction and of the structure of the immune complex as well. To reveal the functional role of domain-domain interactions in the Fc part of IgG the binding of C1q to different anti-ovalbumin IgG-ovalbumin immune complexes was studied. Immune-complex precipitates composed of rabbit IgG in which the non-covalent or covalent bonds between the heavy chains had been eliminated were used. Non-covalent bonds were abolished by splitting off the CH3 domains, i.e. by using Facb fragments, and the covalent contact was broken by reduction and alkylation of the single inter-heavy-chain disulphide bond. The quantitative analysis of the binding curves provides a dissociation constant (K) of 200 nM for the interaction between C1q and immune precipitate formed from native IgG. Surprisingly, for immune precipitates composed of Facb fragments or IgG in which the inter-heavy-chain disulphide bond had been selectively reduced and alkylated, stronger binding (K = 30 nM) was observed. In this case, however, changes in the structure of the immune-complex matrix were also detected. These structural changes may account for the strengthening of the C1q-immune-complex interaction, which can be strongly influenced by the flexibility and the binding-site pattern of the immune-complex precipitates. These results suggest that domain-domain interactions in the Fc part of IgG affect the segmental mobility of IgG molecules and the spatial arrangement of the immune-complex matrix rather than the affinity of individual C1q-binding sites on IgG.

The terminal regions of monomeric hook protein fromSalmonella typhimuriumare known to be highly mobile and exposed to the solvent. Although hook protein exhibits an unusual far UV circular dichroism spectrum, resembling that of random coil structures, our calorimetric experiments clearly demonstrate that the molecule has a compact ordered core. The compact part probably consists of three domains as suggested by deconvolution analysis of the calorimetric melting profiles. Secondary structure prediction, together with the analysis of fat-UV circular dichroism spectra, has shown that the domains of monomeric hook protein contain β-sheeted structures without significant α-helical content. The polymerization of hook protein is accompanied by the stabilization of its disordered terminal regions into a predominantly α-helical domain. Evaluation of circular dichroism data suggests that about 45 terminal residues are involved in helical segments. Coiled-coil prediction indicates that whereas the whole carboxy-terminal helical region of hook protein has a strong bundle-forming potential, there is only a single short amino-terminal segment exhibiting weak coiled-coil forming tendencies. The formation of α-helical bundles in commonly believed to be a key event during the polymerization of the axial structure of bacterial flagella. To clarify the role of helical bundle formation in hook assembly, proteolytic fragments of hook protein with truncations of various lengths in their carboxy-terminal disordered regions were generated, and their polymerizational behavior was investigated. We found that even fragments completely lacking the main helix-forming carboxy-terminal regions can polymerize into filamentsin vitrounder appropriately high salt concentrations. Our results suggest that, although helical bundle formation may occur during self-assembly, governing precise subunit packing and playing an important role in the stabilization of hook filaments, it is not the principal interaction mainly responsible for the development of their filamentous structure.

Life on earth is ubiquitous within the limits from −5 to 110°C for temperature, 0.1 to 120 MPa for hydrostatic pressure. 1.0 to 0.6 for water activity and pH 1 to 12. In general, mutative adaptation of proteins to changing environmental conditions tends to maintain ‘corresponding states’ regarding overall topology, flexibility and hydration. Due to the minute changes in the free energy of stabilization responsible for enhanced stability, nature provides a wide variety of different adaptive strategies. In the case of thermophilic proteins, improved packing densities are crucial. In halophilic proteins, decreased hydrophobicity and clustered surface charges serve to increase water and salt binding required for solubilization at high salt concentration. In the case of barophiles, high-pressure adaptation is expected to be less important than adaptation to low temperatures governing the deep sea. Nothing is known with respect to the mechanisms underlying psychrophilic and acidophilic alkalophilic adaptation.

Our protein engineering studies on human C1r and C1s revealed important characteristics of the individual domains of these multidomain serine-proteases, and supplied evidence about the cooperation of the domains to create binding sites, and to control the activation process. We expressed the recombinant subcomponents in the baculovirus-insect cell system and checked the biological activity. Deletions and point mutants of C1 r were constructed and C1r-C1s chimeras were also produced. Our deletion mutants demonstrated that the N-terminal CUB domain and the EGF-like domain of C1r together are responsible for the calcium dependent C1r-C1s interaction. It seems very likely that these two modules form the calcium-binding site of the C1r α-fragment and participate in the tetramer formation. The deletion mutants also demonstrated that the N-terminal region of the C1r molecule contains essential elements involved in the control of activation of the serine-protease module. The substrate specificity of the serine-protease is also determined by the five N-terminal noncatalytic domain of C1r/C1s chimera, which contains the catalytic domain of C1s preceded by the N-terminal region of C1r, could replace the C1r in the hemolytically active C1 complex. The C1s/C1r chimera, in which the α-fragment of the C1r was replaced for that of the C1s exibits both C1r- and C1s-like characteristics. We stabilized the zymogen form of human C1r by mutating the Arg(463)-Ile(464) bond. Using our stable zymogen C1r we showed that one active C 1r in the C1 complex is sufficient for the full activity of the entire complex. Further experiment with this mutant could provide us with important information about the structure of the C1 complex.

Life on earth is ubiquitous within the limits from −5 to 110°C for temperature, 0.1 to 120 MPa for hydrostatic pressure. 1.0 to 0.6 for water activity and pH 1 to 12. In general, mutative adaptation of proteins to changing environmental conditions tends to maintain ‘corresponding states’ regarding overall topology, flexibility and hydration. Due to the minute changes in the free energy of stabilization responsible for enhanced stability, nature provides a wide variety of different adaptive strategies. In the case of thermophilic proteins, improved packing densities are crucial. In halophilic proteins, decreased hydrophobicity and clustered surface charges serve to increase water and salt binding required for solubilization at high salt concentration. In the case of barophiles, high‐pressure adaptation is expected to be less important than adaptation to low temperatures governing the deep sea. Nothing is known with respect to the mechanisms underlying psychrophilic and acidophilic alkalophilic adaptation.

Spodoptera frugiperda cells are the hosts of wild type and recombinant virus in the baculovirus insect cell expression system. The expression of the foreign gene could be enhanced by the addition of ecdysteroids and increased amount of recombinant protein was secreted into the medium. The time and concentration dependence of this effect was followed in the case of 20-hydroxyecdysone, makisterone and ecdysone. 20-hydroxyecdysone proved to be the most efficient, producing a three fold increase in the level of recombinant protein secreted into the medium, as it was measured by ELISA. This effect was also confirmed by tracing the L-(35S)methionine incorporation into the gene product. Makisterone was also effective in stimulation, while ecdysone proved to be ineffective.

By injection of a liposomal complex containing baculovirus DNA directly into the hemocoel of insect larvae a polyhedrosis disease was induced. After cotransfection of insect larvae with circular viral DNA and transfer vector DNA recombinant viruses were generated with a frequency of about 2%, similar to what is obtained in vitro using insect cell cultures. Based on these results an alternative strategy for the generation of recombinants can be derived for baculoviruses for which susceptible cell lines are not readily available. This strategy involves the injection of baculovirus DNA into susceptible larvae followed by in vivo cloning.

The influence of the flow environment on platelet aggregation is not fully understood in high-shear thrombosis. The objective of this study is to investigate the role of a high shear rate in initial platelet aggregation. The haemodynamic conditions in a microfluidic device are studied using cell-based blood flow simulations. The results are compared with in vitroplatelet aggregation experiments performed with porcine whole blood (WB) and platelet-rich-plasma (PRP). We studied whether the cell-depleted layer in combination with high shear and high platelet flux can account for the distribution of platelet aggregates. High platelet fluxes at the wall were found in silico. In WB, the platelet flux was about twice as high as in PRP. Additionally, initial platelet aggregation and occlusion were observed in vitroin the stenotic region. In PRP, the position of the occlusive thrombus was located more downstream than in WB. Furthermore, the shear rates and stresses in cell-based and continuum simulations were studied. We found that a continuum simulation is a good approximation for PRP. For WB, it cannot predict the correct values near the wall.

We study the effect of pulsatile flow on the transport of red blood cells (RBCs) and platelets into aneurysm geometries with varying dome-to-neck aspect ratios (AR). We use a validated two-dimensional lattice Boltzmann model for blood plasma with a discrete element method for both RBCs and platelets coupled by the immersed boundary method. Flow velocities and vessel diameters were matched with measurements of cerebral perforating arteries and flow was driven by a synthetic heartbeat curve typical for such vessel sizes. We observe a flow regime change as the aspect ratio increases from a momentum-driven regime in the small aspect ratio to a shear-driven regime in the larger aspect ratios. In the small aspect ratio case, we see the development of a re-circulation zone that exhibits a layering of high (greater than or equal to 7 s) and low (less than 7 s) residence cells. In the shear-driven regime, we see high and low residence cells well mixed, with an increasing population of cells that are trapped inside the aneurysm as the aspect ratio increases. In all cases, we observe aneurysms that are platelet-rich and red blood cell-poor when compared with their respective parental vessel populations. Pulsatility also plays a role in the small aspect ratio as we observe a smaller population of older trapped cells along the aneurysm wall in the pulsatile case when compared with a steady flow case. Pulsatility does not have a significant effect in shear-driven regime aspect ratios.