A synthesis approach of mouse studies to identify genes and proteins in arterial thrombosis and bleeding.

Antithrombotic therapies reduce cardiovascular diseases by preventing arterial thrombosis and thromboembolism, but at expense of increased bleeding risks. Arterial thrombosis studies using genetically modified mice have been invaluable for identification of new molecular targets. Because of low sample sizes and heterogeneity in approaches or methodologies, a formal meta-analysis to compare studies of mice with single-gene defects encountered major limitations. To overcome these, we developed a novel synthesis approach to quantitatively scale 1514 published studies of arterial thrombus formation (in vivo and in vitro), thromboembolism, and tail-bleeding of genetically modified mice. Using a newly defined consistency parameter (CP), indicating the strength of published data, comparisons were made of 431 mouse genes, of which 17 consistently contributed to thrombus formation without affecting hemostasis. Ranking analysis indicated high correlations between collagen-dependent thrombosis models in vivo (FeCl3 injury or ligation/compression) and in vitro. Integration of scores and CP values resulted in a network of protein interactions in thrombosis and hemostasis (PITH), which was combined with databases of genetically linked human bleeding and thrombotic disorders. The network contained 2946 nodes linked to modifying genes of thrombus formation, mostly with expression in megakaryocytes. Reactome pathway analysis and network characteristics revealed multiple novel genes with potential contribution to thrombosis/hemostasis. Studies with additional knockout mice revealed that 4 of 8 (Apoe, Fpr2, Ifnar1, Vps13a) new genes were modifying in thrombus formation. The PITH network further: (i) revealed a high similarity of murine and human hemostatic and thrombotic processes and (ii) identified multiple new candidate proteins regulating these processes.

Variable impairment of platelet functions in patients with severe, genetically linked immune deficiencies.

In patients with dysfunctions of the Ca2+ channel ORAI1, stromal interaction molecule 1 (STIM1) or integrin-regulating kindlin-3 (FERMT3), severe immunodeficiency is frequently linked to abnormal platelet activity. In this paper, we studied platelet responsiveness by multiparameter assessment of whole blood thrombus formation under high-shear flow conditions in 9 patients, including relatives, with confirmed rare genetic mutations of ORAI1, STIM1 or FERMT3. In platelets isolated from 5 out of 6 patients with ORAI1 or STIM1 mutations, store-operated Ca2+ entry (SOCE) was either completely or partially defective compared to control platelets. Parameters of platelet adhesion and aggregation on collagen microspots were impaired for 4 out of 6 patients, in part related to a low platelet count. For 4 patients, platelet adhesion/aggregation and procoagulant activity on von Willebrand Factor (VWF)/rhodocytin and VWF/fibrinogen microspots were impaired independently of platelet count, and were partly correlated with SOCE deficiency. Measurement of thrombus formation at low shear rate confirmed a greater impairment of platelet functionality in the ORAI1 patients than in the STIM1 patient. For 3 patients/relatives with a FERMT3 mutation, all parameters of thrombus formation were strongly reduced regardless of the microspot. Bone marrow transplantation, required by 2 patients, resulted in overall improvement of platelet function. We concluded that multiparameter assessment of whole blood thrombus formation in a surface-dependent way can detect: i) additive effects of low platelet count and impaired platelet functionality; ii) aberrant ORAI1-mediated Ca2+ entry; iii) differences in platelet activation between patients carrying the same ORAI1 mutation; iv) severe platelet function impairment linked to a FERMT3 mutation and bleeding history.

The proteasome antechamber maintains substrates in an unfolded state.

Eukaryotes and archaea use a protease called the proteasome that has an integral role in maintaining cellular function through the selective degradation of proteins. Proteolysis occurs in a barrel-shaped 20S core particle, which in Thermoplasma acidophilum is built from four stacked homoheptameric rings of subunits, α and ß, arranged α(7)ß(7)ß(7)α(7) (ref. 5). These rings form three interconnected cavities, including a pair of antechambers (formed by α(7)ß(7)) through which substrates are passed before degradation and a catalytic chamber (ß(7)ß(7)) where the peptide-bond hydrolysis reaction occurs. Although it is clear that substrates must be unfolded to enter through narrow, gated passageways (13 Å in diameter) located on the α-rings, the structural and dynamical properties of substrates inside the proteasome antechamber remain unclear. Confinement in the antechamber might be expected to promote folding and thus impede proteolysis. Here we investigate the folding, stability and dynamics of three small protein substrates in the antechamber by methyl transverse-relaxation-optimized NMR spectroscopy. We show that these substrates interact actively with the antechamber walls and have drastically altered kinetic and equilibrium properties that maintain them in unstructured states so as to be accessible for hydrolysis.

Gradual increase in thrombogenicity of juvenile platelets formed upon offset of prasugrel medication.

In patients with acute coronary syndrome, dual antiplatelet therapy with aspirin and a P2Y12 inhibitor like prasugrel is prescribed for one year. Here, we investigated how the hemostatic function of platelets recovers after discontinuation of prasugrel treatment. Therefore, 16 patients who suffered from ST-elevation myocardial infarction were investigated. Patients were treated with aspirin (100 mg/day, long-term) and stopped taking prasugrel (10 mg/day) after one year. Blood was collected at the last day of prasugrel intake and at 1, 2, 5, 12 and 30 days later. Platelet function in response to ADP was normalized between five and 30 days after treatment cessation and in vitro addition of the reversible P2Y12 receptor antagonist ticagrelor fully suppressed the regained activation response. Discontinuation of prasugrel resulted in the formation of an emerging subpopulation of ADP-responsive platelets, exhibiting high expression of active integrin αIIbß3. Two different mRNA probes, thiazole orange and the novel 5'Cy5-oligo-dT probe revealed that this subpopulation consisted of juvenile platelets, which progressively contributed to platelet aggregation and thrombus formation under flow. During offset, juvenile platelets were overall more reactive than older platelets. Interestingly, the responsiveness of both juvenile and older platelets increased in time, pointing towards a residual inhibitory effect of prasugrel on the megakaryocyte level. In conclusion, the gradual increase in thrombogenicity after cessation of prasugrel treatment is due to the increased activity of juvenile platelets.

Supporting roles of platelet thrombospondin-1 and CD36 in thrombus formation on collagen.

OBJECTIVE: Platelets abundantly express the membrane receptor CD36 and store its ligand thrombospondin-1 (TSP1) in the α-granules. We investigated whether released TSP1 can support platelet adhesion and thrombus formation via interaction with CD36. APPROACH AND RESULTS: Mouse platelets deficient in CD36 showed reduced adhesion to TSP1 and subsequent phosphatidylserine expression. Deficiency in either CD36 or TSP1 resulted in markedly increased dissolution of thrombi formed on collagen, although thrombus buildup was unchanged. In mesenteric vessels in vivo, deficiency in CD36 prolonged the time to occlusion and enhanced embolization, which was in agreement with earlier observations in TSP1-deficient mice. Thrombi formed using wild-type blood stained positively for secreted TSP1. Releasate from wild-type but not from TSP1-deficient platelets enhanced platelet activation, phosphatidylserine expression, and thrombus formation on collagen. The enhancement was dependent on CD36 because it was without effect on thrombus formation by CD36-deficient platelets. CONCLUSIONS: These results demonstrate an anchoring role of platelet-released TSP1 via CD36 in platelet adhesion and collagen-dependent thrombus stabilization. Thus, the TSP1-CD36 tandem is another platelet ligand-receptor axis contributing to the maintenance of a stable thrombus.

Distinct role of von Willebrand factor triplet bands in glycoprotein Ib-dependent platelet adhesion and thrombus formation under flow.

Multimeric glycoprotein von Willebrand factor (VWF) exhibits a unique triplet structure of individual oligomers, resulting from ADAMTS-13 (a disintegrin and metalloproteinase with thrombospondin type 1 motifs 13) cleavage. The faster and slower migrating triplet bands of a given VWF multimer have one shorter or longer N-terminal peptide sequence, respectively. Within this peptide sequence, the A1 domain regulates interaction of VWF with platelet glycoprotein (GP)Ib. Therefore, platelet-adhesive properties of two VWF preparations with similar multimeric distribution but different triplet composition were investigated for differential functional activities. Preparation A was enriched in intermediate triplet bands, whereas preparation B predominantly contained larger triplet bands. Binding studies revealed that preparation A displayed a reduced affinity for recombinant GPIb but an unchanged affinity for collagen type III when compared to preparation B. Under high-shear flow conditions, preparation A was less active in recruiting platelets to collagen type III. Furthermore, when added to blood from patients with von Willebrand disease (VWD), defective thrombus formation was less restored. Thus, VWF forms lacking larger-size triplet bands appear to have a decreased potential to recruit platelets to collagen-bound VWF under arterial flow conditions. By implication, changes in triplet band distribution observed in patients with VWD may result in altered platelet adhesion at high-shear flow.

Key role of glycoprotein Ib/V/IX and von Willebrand factor in platelet activation-dependent fibrin formation at low shear flow.

A microscopic method was developed to study the role of platelets in fibrin formation. Perfusion of adhered platelets with plasma under coagulating conditions at a low shear rate (250(-1)) resulted in the assembly of a star-like fibrin network at the platelet surface. The focal fibrin formation on platelets was preceded by rises in cytosolic Ca(2+), morphologic changes, and phosphatidylserine exposure. Fibrin formation was slightly affected by α(IIb)ß(3) blockage, but it was greatly delayed and reduced by the following: inhibition of thrombin or platelet activation; interference in the binding of von Willebrand factor (VWF) to glycoprotein Ib/V/IX (GpIb-V-IX); plasma or blood from patients with type 1 von Willebrand disease; and plasma from mice deficient in VWF or the extracellular domain of GpIbα. In this process, the GpIb-binding A1 domain of VWF was similarly effective as full-length VWF. Prestimulation of platelets enhanced the formation of fibrin, which was abrogated by blockage of phosphatidylserine. Together, these results show that, in the presence of thrombin and low shear flow, VWF-induced activation of GpIb-V-IX triggers platelet procoagulant activity and anchorage of a star-like fibrin network. This process can be relevant in hemostasis and the manifestation of von Willebrand disease.

TolC-AcrA complex formation monitored by time dependent single-channel electrophysiology.

Characterizing protein-protein interaction on a single molecular level is a challenge, experimentally as well as interpretation of the data. For example, Gram-negative bacteria contain protein complexes spanning the outer and inner cell wall devoted to efflux effectively cell toxic substances. Recent seminal work revealed the high-resolution structure of such a tripartic composition TolC-AcrA-AcrB suggesting to design inhibitors preventing efflux of antibiotics. To show that electrophysiology can provide supporting information here, we reconstitute single TolC homotrimer into a planar lipid membrane, apply a transmembrane voltage and follow the assembly of AcrA to TolC using the modulation of the ion current through TolC channel during binding. In particular, the presence of AcrA in solution increases the average ionic current through TolC and, moreover, reduces the ion-current fluctuations caused by flickering of TolC. Here, we show that statistical properties of ion-current fluctuations (the power spectral density) provide a complementary measure of the interaction of the TolC-AcrA complex in presence of putative efflux pump inhibitors. Both characteristics, the average ion current across TolC and the current noise, taken into consideration together, point to a stiffening of the tip of TolC which might reduce the formation of the complex.

Efflux Pump-Binding 4(3-Aminocyclobutyl)Pyrimidin-2-Amines Are Colloidal Aggregators.

Efflux pumps are a relevant factor in antimicrobial resistance. In E. coli, the tripartite efflux pump AcrAB-TolC removes a chemically diverse set of antibiotics from the bacterium. Therefore, small molecules interfering with efflux pump function are considered adjuvants for improving antimicrobial therapies. Several compounds targeting the periplasmic adapter protein AcrA and the efflux pump AcrB have been identified to act synergistically with different antibiotics. Among those, several 4(3-aminocyclobutyl)pyrimidin-2-amines have been shown to bind to both proteins. In this study, we intended to identify analogs of these substances with improved binding affinity to AcrA using virtual screening followed by experimental validation. While we succeeded in identifying several compounds showing a synergistic effect with erythromycin on E. coli, biophysical studies suggested that 4(3-aminocyclobutyl)pyrimidin-2-amines form colloidal aggregates that do not bind specifically to AcrA. Therefore, these substances are not suited for further development. Our study emphasizes the importance of implementing additional control experiments to identify aggregators among bioactive compounds.

A concise guide to choosing suitable gene expression systems for recombinant protein production.

This overview guides both novices and experienced researchers facing challenging targets to select the most appropriate gene expression system for producing a particular protein. By answering four key questions, readers can determine the most suitable gene expression system following a decision scheme. This guide addresses the most commonly used and accessible systems and provides brief descriptions of the main gene expression systems' key characteristics to assist decision making. Additionally, information has been included for selected less frequently used "exotic" gene expression systems.

Non-functional ubiquitin C-terminal hydrolase L1 drives podocyte injury through impairing proteasomes in autoimmune glomerulonephritis.

Little is known about the mechanistic significance of the ubiquitin proteasome system (UPS) in a kidney autoimmune environment. In membranous nephropathy (MN), autoantibodies target podocytes of the glomerular filter resulting in proteinuria. Converging biochemical, structural, mouse pathomechanistic, and clinical information we report that the deubiquitinase Ubiquitin C-terminal hydrolase L1 (UCH-L1) is induced by oxidative stress in podocytes and is directly involved in proteasome substrate accumulation. Mechanistically, this toxic gain-of-function is mediated by non-functional UCH-L1, which interacts with and thereby impairs proteasomes. In experimental MN, UCH-L1 becomes non-functional and MN patients with poor outcome exhibit autoantibodies with preferential reactivity to non-functional UCH-L1. Podocyte-specific deletion of UCH-L1 protects from experimental MN, whereas overexpression of non-functional UCH-L1 impairs podocyte proteostasis and drives injury in mice. In conclusion, the UPS is pathomechanistically linked to podocyte disease by aberrant proteasomal interactions of non-functional UCH-L1.

Monitoring in vitro thrombus formation with novel microfluidic devices.

Cardiovascular disease is a major cause of mortality globally and is subject to ongoing research to improve clinical treatment. It is established that activation of platelets and coagulation are central to thrombosis, yet at different extents in the arterial and venous system. In vitro perfusion chamber technology has contributed significant knowledge on the function of platelets in the thrombotic process under shear conditions. Recent efforts to downscale this technique with a variety of microfluidic devices has opened new possibilities to study this process under precisely controlled flow conditions. Such microfluidic devices possess the capability to execute platelet function tests more quickly than current assays, while using small blood samples. Gradually becoming available to the clinic now, they may provide a new means to manage the treatment of cardiovascular diseases, although accurate validation studies still are missing. This review highlights the progress that has been made in monitoring aspects of thrombus formation using microfluidic devices.

Biophysical Screening Pipeline for Cryo-EM Grid Preparation of Membrane Proteins.

Successful sample preparation is the foundation to any structural biology technique. Membrane proteins are of particular interest as these are important targets for drug design, but also notoriously difficult to work with. For electron cryo-microscopy (cryo-EM), the biophysical characterization of sample purity, homogeneity, and integrity as well as biochemical activity is the prerequisite for the preparation of good quality cryo-EM grids as these factors impact the result of the computational reconstruction. Here, we present a quality control pipeline prior to single particle cryo-EM grid preparation using a combination of biophysical techniques to address the integrity, purity, and oligomeric states of membrane proteins and its complexes to enable reproducible conditions for sample vitrification. Differential scanning fluorimetry following the intrinsic protein fluorescence (nDSF) is used for optimizing buffer and detergent conditions, whereas mass photometry and dynamic light scattering are used to assess aggregation behavior, reconstitution efficiency, and oligomerization. The data collected on nDSF and mass photometry instruments can be analyzed with web servers publicly available at spc.embl-hamburg.de. Case studies to optimize conditions prior to cryo-EM sample preparation of membrane proteins present an example quality assessment to corroborate the usefulness of our pipeline.

Force spectroscopy of substrate molecules en route to the proteasome's active sites.

We used an atomic force microscope to study the mechanism underlying the translocation of substrate molecules inside the proteasome. Our specific experimental setup allowed us to measure interaction forces between the 20S proteasome and its substrates. The substrate (ß-casein) was covalently bound either via a thiol-Au bond or by a PEG-based binding procedure to the atomic force microscope cantilever tip and offered as bait to proteasomes from Methanosarcina mazei. The proteasomes were immobilized densely in an upright orientation on mica, which made their upper pores accessible for substrates to enter. Besides performing conventional single-molecule force spectroscopy experiments, we developed a three-step procedure that allows the detection of specific proteasome-substrate single-molecule events without tip-sample contact. Using the active 20S wild type and an inactive active-site mutant, as well as two casein mutants bound with opposite termini to the microscope tip, we detected no directional preference of the proteasome-substrate interactions. By comparing the distribution of the measured forces for the proteasome-substrate interactions, were observed that a significant proportion of interaction events occurred at higher forces for the active versus the inactive proteasome. These forces can be attributed to the translocation of substrate en route to the active sites that are harbored deep inside the proteasome.

GroEL is an immunodominant surface-exposed antigen of Rickettsia typhi.

Rickettsioses are neglected and emerging potentially fatal febrile diseases that are caused by obligate intracellular bacteria, rickettsiae. Rickettsia (R.) typhi and R. prowazekii constitute the typhus group (TG) of rickettsiae and are the causative agents of endemic and epidemic typhus, respectively. We recently generated a monoclonal antibody (BNI52) against R. typhi. Characterization of BNI52 revealed that it specifically recognizes TG rickettsiae but not the members of the spotted fever group (SFG) rickettsiae. We further show that BNI52 binds to protein fragments of ±30 kDa that are exposed on the bacterial surface and also present in the periplasmic space. These protein fragments apparently derive from the cytosolic GroEL protein of R. typhi and are also recognized by antibodies in the sera from patients and infected mice. Furthermore, BNI52 opsonizes the bacteria for the uptake by antigen presenting cells (APC), indicating a contribution of GroEL-specific antibodies to protective immunity. Finally, it is interesting that the GroEL protein belongs to 32 proteins that are differentially downregulated by R. typhi after passage through immunodeficient BALB/c CB17 SCID mice. This could be a hint that the rickettsia GroEL protein may have immunomodulatory properties as shown for the homologous protein from several other bacteria, too. Overall, the results of this study provide evidence that GroEL represents an immunodominant antigen of TG rickettsiae that is recognized by the humoral immune response against these pathogens and that may be interesting as a vaccine candidate. Apart from that, the BNI52 antibody represents a new tool for specific detection of TG rickettsiae in various diagnostic and experimental setups.

Dissecting the Gene Expression, Localization, Membrane Topology, and Function of the Plasmodium falciparum STEVOR Protein Family.

During its intraerythrocytic development, the malaria parasite Plasmodium falciparum exposes variant surface antigens (VSAs) on infected erythrocytes to establish and maintain an infection. One family of small VSAs is the polymorphic STEVOR proteins, which are marked for export to the host cell surface through their PEXEL signal peptide. Interestingly, some STEVORs have also been reported to localize to the parasite plasma membrane and apical organelles, pointing toward a putative function in host cell egress or invasion. Using deep RNA sequencing analysis, we characterized P. falciparumstevor gene expression across the intraerythrocytic development cycle, including free merozoites, in detail and used the resulting stevor expression profiles for hierarchical clustering. We found that most stevor genes show biphasic expression oscillation, with maximum expression during trophozoite stages and a second peak in late schizonts. We selected four STEVOR variants, confirmed the expected export of these proteins to the host cell membrane, and tracked them to a secondary location, either to the parasite plasma membrane or the secretory organelles of merozoites in late schizont stages. We investigated the function of a particular STEVOR that showed rhoptry localization and demonstrated its role at the parasite-host interface during host cell invasion by specific antisera and targeted gene disruption. Experimentally determined membrane topology of this STEVOR revealed a single transmembrane domain exposing the semiconserved as well as variable protein regions to the cell surface.IMPORTANCE Malaria claims about half a million lives each year. Plasmodium falciparum, the causative agent of the most severe form of the disease, uses proteins that are translocated to the surface of infected erythrocytes for immune evasion. To circumvent the detection of these gene products by the immune system, the parasite evolved a complex strategy that includes gene duplications and elaborate sequence polymorphism. STEVORs are one family of these variant surface antigens and are encoded by about 40 genes. Using deep RNA sequencing of blood-stage parasites, including free merozoites, we first established stevor expression of the cultured isolate and compared it with published transcriptomes. We reveal a biphasic expression of most stevor genes and confirm this for individual STEVORs at the protein level. The membrane topology of a rhoptry-associated variant was experimentally elucidated and linked to host cell invasion, underlining the importance of this multifunctional protein family for parasite proliferation.

Comparative Analysis of Microfluidics Thrombus Formation in Multiple Genetically Modified Mice: Link to Thrombosis and Hemostasis.

Genetically modified mice are indispensable for establishing the roles of platelets in arterial thrombosis and hemostasis. Microfluidics assays using anticoagulated whole blood are commonly used as integrative proxy tests for platelet function in mice. In the present study, we quantified the changes in collagen-dependent thrombus formation for 38 different strains of (genetically) modified mice, all measured with the same microfluidics chamber. The mice included were deficient in platelet receptors, protein kinases or phosphatases, small GTPases or other signaling or scaffold proteins. By standardized re-analysis of high-resolution microscopic images, detailed information was obtained on altered platelet adhesion, aggregation and/or activation. For a subset of 11 mouse strains, these platelet functions were further evaluated in rhodocytin- and laminin-dependent thrombus formation, thus allowing a comparison of glycoprotein VI (GPVI), C-type lectin-like receptor 2 (CLEC2) and integrin α6ß1 pathways. High homogeneity was found between wild-type mice datasets concerning adhesion and aggregation parameters. Quantitative comparison for the 38 modified mouse strains resulted in a matrix visualizing the impact of the respective (genetic) deficiency on thrombus formation with detailed insight into the type and extent of altered thrombus signatures. Network analysis revealed strong clusters of genes involved in GPVI signaling and Ca2+ homeostasis. The majority of mice demonstrating an antithrombotic phenotype in vivo displayed with a larger or smaller reduction in multi-parameter analysis of collagen-dependent thrombus formation in vitro. Remarkably, in only approximately half of the mouse strains that displayed reduced arterial thrombosis in vivo, this was accompanied by impaired hemostasis. This was also reflected by comparing in vitro thrombus formation (by microfluidics) with alterations in in vivo bleeding time. In conclusion, the presently developed multi-parameter analysis of thrombus formation using microfluidics can be used to: (i) determine the severity of platelet abnormalities; (ii) distinguish between altered platelet adhesion, aggregation and activation; and (iii) elucidate both collagen and non-collagen dependent alterations of thrombus formation. This approach may thereby aid in the better understanding and better assessment of genetic variation that affect in vivo arterial thrombosis and hemostasis.

Crystallization and preliminary X-ray analysis of the Thermoplasma acidophilum 20S proteasome in complex with protein substrates.

The 20S proteasome is a 700 kDa barrel-shaped proteolytic complex that is traversed by an internal channel which widens into three cavities: two antechambers and one central chamber. Entrance to the complex is restricted by the narrow opening of the channel, which only allows unfolded substrates to reach the active sites located within the central cavity. The X-ray structures of 20S proteasomes from different organisms with and without inhibitors bound have led to a detailed knowledge of their structure and proteolytic function. Nevertheless, the mechanisms that underlie substrate translocation into the 20S proteasome and the role of the antechambers remain elusive. To investigate putative changes within the proteasome that occur during substrate translocation, ;host-guest' complexes between the Thermoplasma acidophilum 20S proteasomes and either cytochrome c (cyt c) or green fluorescent protein (GFP) were produced and crystallized. Orthorhombic crystals belonging to space group P2(1)2(1)2(1), with unit-cell parameters a = 116, b = 207, c = 310 A (cyt c) and a = 116, b = 206, c = 310 A (GFP), were formed and X-ray diffraction data were collected to 3.4 A (cyt c) and 3.8 A (GFP) resolution.

Proteasome assembly triggers a switch required for active-site maturation.

The processing of propeptides and the maturation of 20S proteasomes require the association of beta rings from two half proteasomes. We propose an assembly-dependent activation model in which interactions between helix (H3 and H4) residues of the opposing half proteasomes are prerequisite for appropriate positioning of the S2-S3 loop; such positioning enables correct coordination of the active-site residue needed for propeptide cleavage. Mutations of H3 or H4 residues that participate in the association of two half proteasomes inhibit activation and prevent, in nearly all cases, the formation of full proteasomes. In contrast, mutations affecting interactions with residues of the S2-S3 loop allow the assembly of full, but activity impacted, proteasomes. The crystal structure of the inactive H3 mutant, Phe145Ala, shows that the S2-S3 loop is displaced from the position observed in wild-type proteasomes. These data support the proposed assembly-dependent activation model in which the S2-S3 loop acts as an activation switch.