Serpin targets in hemostasis/kinin formation.
Ontology highlight
ABSTRACT:
SUBMITTER: Schmaier AH
PROVIDER: S-EPMC6871307 | biostudies-literature | 2019 Nov
REPOSITORIES: biostudies-literature
Publications
Similar Datasets
Project description:BackgroundOcclusive thrombi are not homogeneous in composition. The core of a thrombus is rich in activated platelets and fibrin while the outer shell contains resting platelets. This core is inaccessible to plasma proteins. We produced a fusion protein (targeted SERPIN-TaSER), consisting of a function-blocking VH H against glycoprotein Ibα (GPIbα) and a thrombin-inhibiting serine protease inhibitor (SERPIN; α1-antitrypsin 355 AIAR358 ) to interfere with platelet-driven thrombin formation.AimTo evaluate the antithrombotic properties of TaSER.MethodsBesides TaSER, we generated three analogous control variants with either a wild-type antitrypsin subunit, a non-targeting control VH H, or their combination. We investigated TaSER and controls in protease activity assays, (platelet-dependent) thrombin generation assays, and by western blotting. The effects of TaSER on platelet activation and von Willebrand factor (VWF) binding were studied by fluorescence-activated cell sorting, in agglutination studies, and in ATP secretion experiments. We studied the influence of TaSER in whole blood (1) on platelet adhesion on VWF, (2) aggregate formation on collagen, and (3) thrombus formation (after recalcification) on collagen and tissue factor.ResultsTaSER binds platelets and inhibits thrombin activity on the platelet surface. It blocks VWF binding and disassembles platelet agglutinates. TaSER delays tissue factor-triggered thrombin generation and ATP secretion in platelet-rich plasma in a targeted manner. In flow studies, TaSER interferes with platelet adhesion and aggregate formation due to GPIbα blockade and limits thrombus formation due to targeted inhibition of platelet-dependent thrombin activity.ConclusionThe synergy between the individual properties of TaSER makes it a highly effective antithrombotic agent with possible clinical implications.
| S-EPMC9298318 | biostudies-literature
Project description:Hemophilia A and B, diseases caused by the lack of factor VIII (FVIII) and factor IX (FIX) respectively, lead to insufficient thrombin production, and therefore to bleeding. New therapeutic strategies for hemophilia treatment that do not rely on clotting factor replacement, but imply the neutralization of natural anticoagulant proteins, have recently emerged. We propose an innovative approach consisting of targeting a natural and potent thrombin inhibitor, expressed by platelets, called protease nexin-1 (PN-1). By using the calibrated automated thrombin generation assay, we showed that a PN-1-neutralizing antibody could significantly shorten the thrombin burst in response to tissue factor in platelet-rich plasma (PRP) from patients with mild or moderate hemophilia. In contrast, in PRP from patients with severe hemophilia, PN-1 neutralization did not improve thrombin generation. However, after collagen-induced platelet activation, PN-1 deficiency in F8-/-mice or PN-1 blocking in patients with severe disease led to a significantly improved thrombin production in PRP, underlining the regulatory role of PN-1 released from platelet granules. In various bleeding models, F8-/-/PN-1-/- mice displayed significantly reduced blood loss and bleeding time compared with F8-/-mice. Moreover, platelet recruitment and fibrin(ogen) accumulation were significantly higher in F8-/-/PN-1-/- mice than in F8-/-mice in the ferric chloride-induced mesenteric vessel injury model. Thromboelastometry studies showed enhanced clot stability and lengthened clot lysis time in blood from F8-/-/PN-1-/- and from patients with hemophilia A incubated with a PN-1-neutralizing antibody compared with their respective controls. Our study thus provides proof of concept that PN-1 neutralization can be a novel approach for future clinical care in hemophilia.
| S-EPMC9635585 | biostudies-literature
Project description:Steinernema carpocapsae is an entomopathogenic nematode widely used for the control of insect pests due to its virulence, which is mainly attributed to the ability the parasitic stage has to overcome insect defences. To identify the mechanisms underlying such a characteristic, we studied a novel serpin-like inhibitor (sc-srp-6) that was detected in a transcriptome analysis. Recombinant Sc-SRP-6 produced in Escherichia coli had a native fold of serpins belonging to the α-1-peptidase family and exhibited inhibitory activity against trypsin and α-chymotrypsin with Ki of 0.42 × 10(-7) M and 1.22 × 10(-7) M, respectively. Functional analysis revealed that Sc-SRP-6 inhibits insect digestive enzymes, thus preventing the hydrolysis of ingested particles. Moreover, Sc-SRP-6 impaired the formation of hard clots at the injury site, a major insect defence mechanism against invasive pathogens. Sc-SRP-6 does not prevent the formation of clot fibres and the activation of prophenoloxidases but impairs the incorporation of the melanin into the clot. Binding assays showed a complex formation between Sc-SRP-6 and three proteins in the hemolymph of lepidopteran required for clotting, apolipophorin, hexamerin and trypsin-like, although the catalytic inhibition occurred exclusively in trypsin-like. This data allowed the conclusion that Sc-SRP-6 promotes nematode virulence by inhibiting insect gut juices and by impairing immune clot reaction.
| S-EPMC3712955 | biostudies-literature
Project description:Prime regulation over hematopoietic progenitor cell (HPC) production is exerted by hematopoietins (HPs) and their Janus kinase-coupled receptors (HP-Rs). For HP/HP-R studies, one central challenge in determining specific effects involves the delineation of nonredundant signal transduction factors and their lineage restricted actions. Via loss-of-function studies, we define roles for an HP-regulated Serpina3g/Spi2A intracellular serpin during granulomyelocytic, B-cell, and hematopoietic stem cell (HSC) formation. In granulomyelocytic progenitors, granulocyte macrophage colony stimulating factor (GMCSF) strongly induced Serpina3g expression with Stat5 dependency. Spi2A-knockout (KO) led to 20-fold decreased CFU-GM formation, limited GMCSF-dependent granulocyte formation, and compromised neutrophil survival upon tumor necrosis factor alpha (TNF-α) exposure. In B-cell progenitors, Serpina3g was an interleukin-7 (IL7) target. Spi2A-KO elevated CFU-preB greater than sixfold and altered B-cell formation in competitive bone marrow transplant (BMT), and CpG challenge experiments. In HSCs, Serpina3g/Spi2A expression was also elevated. Spi2A-KO compromised LT-HSC proliferation (as well as lineage(neg) Sca1(pos) Kit(pos) (LSK) cell lysosomal integrity), and skewed LSK recovery post 5-FU. Spi2A therefore functions to modulate HP-regulated immune cell and HSC formation post-5-FU challenge.
| S-EPMC4138266 | biostudies-literature
Project description:Coordinated reorganization of cytoskeletal structures is critical for key aspects of platelet physiology. While several studies have addressed the role of microtubules and filamentous actin in platelet production and function, the significance of their crosstalk in these processes has been poorly investigated. The microtubule-actin cross-linking factor 1 (MACF1; synonym: Actin cross-linking factor 7, ACF7) is a member of the spectraplakin family, and one of the few proteins expressed in platelets, which possess actin and microtubule binding domains thereby facilitating actin-microtubule interaction and regulation. We used megakaryocyte- and platelet-specific Macf1 knockout (Macf1fl/fl, Pf4-Cre) mice to study the role of MACF1 in platelet production and function. MACF1 deficient mice displayed comparable platelet counts to control mice. Analysis of the platelet cytoskeletal ultrastructure revealed a normal marginal band and actin network. Platelet spreading on fibrinogen was slightly delayed but platelet activation and clot traction was unaffected. Ex vivo thrombus formation and mouse tail bleeding responses were similar between control and mutant mice. These results suggest that MACF1 is dispensable for thrombopoiesis, platelet activation, thrombus formation and the hemostatic function in mice.
| S-EPMC6531446 | biostudies-literature
Project description:Protein disulfide isomerase (PDI) derived from intravascular cells is required for thrombus formation. However, it remains unclear whether platelet PDI contributes to the process. Using platelet-specific PDI-deficient mice, we demonstrate that PDI-null platelets have defects in aggregation and adenosine triphosphate secretion induced by thrombin, collagen, and adenosine diphosphate. Such defects were rescued by wild-type but not mutant PDI, indicating that the isomerase activity of platelet surface PDI is critical for the regulatory effect. PDI-deficient platelets expressed increased levels of intracellular ER protein 57 (ERp57) and ERp72. Platelet PDI regulated αIIbβ3 integrin activation but not P-selectin exposure, Ca(2+) mobilization, β3-talin1 interaction, or platelet spreading on immobilized fibrinogen. Inhibition of ERp57 further diminished αIIbβ3 integrin activation and aggregation of activated PDI-deficient platelets, suggesting distinct roles of PDI and ERp57 in platelet functions. We found that platelet PDI is important for thrombus formation on collagen-coated surfaces under shear. Intravital microscopy demonstrates that platelet PDI is important for platelet accumulation but not initial adhesion and fibrin generation following laser-induced arteriolar injury. Tail bleeding time in platelet-specific PDI-deficient mice were not significantly increased. Our results provide important evidence that platelet PDI is essential for thrombus formation but not for hemostasis in mice.
| S-EPMC3739031 | biostudies-literature
Project description: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 α6β1 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.
| S-EPMC6682619 | biostudies-literature
Project description:The human serine protease inhibitor (serpin) gene cluster at 14q32.1 is a useful model system to study cell-type-specific gene expression and chromatin structure. Activation of the serpin locus can be induced in vitro by transferring human chromosome 14 from non-expressing to expressing cells. Serpin gene activation in expressing cells is correlated with locus-wide alterations in chromatin structure, including the de novo formation of 17 expression-associated DNase I-hypersensitive sites (DHSs). In this study, we investigated histone acetylation throughout the proximal serpin subcluster. We report that gene activation is correlated with high levels of histone H3 and H4 acetylation at serpin gene promoters and other regulatory regions. However, the locus is not uniformly hyperacetylated, as there are regions of hypoacetylation between genes. Furthermore, genetic tests indicate that locus-wide controls regulate both gene expression and chromatin structure. For example, deletion of a previously identified serpin locus control region (LCR) upstream of the proximal subcluster reduces both gene expression and histone acetylation throughout the approximately 130 kb region. A similar down regulation phenotype is displayed by transactivator-deficient cell variants, but this phenotype can be rescued by transfecting the cells with expression cassettes encoding hepatocyte nuclear factor-1alpha (HNF-1alpha) or HNF-4. Taken together, these results suggest that histone acetylation depends on interactions between the HNF-1alpha/HNF-4 signaling cascade and the serpin LCR.
| S-EPMC1143699 | biostudies-literature
Project description:BackgroundKinins, with bradykinin and des-Arg(9)-bradykinin being the most important ones, are pro-inflammatory peptides released after tissue injury including stroke. Although the actions of bradykinin are in general well characterized; it remains controversial whether the effects of bradykinin are beneficial or not. Kinin-B2 receptor activation participates in various physiological processes including hypotension, neurotransmission and neuronal differentiation. The bradykinin metabolite des-Arg(9)-bradykinin as well as Lys-des-Arg(9)-bradykinin activates the kinin-B1 receptor known to be expressed under inflammatory conditions. We have investigated the effects of kinin-B1 and B2 receptor activation on N-methyl-D-aspartate (NMDA)-induced excitotoxicity measured as decreased capacity to produce synaptically evoked population spikes in the CA1 area of rat hippocampal slices.Principal findingsBradykinin at 10 nM and 1 µM concentrations triggered a neuroprotective cascade via kinin-B2 receptor activation which conferred protection against NMDA-induced excitotoxicity. Recovery of population spikes induced by 10 nM bradykinin was completely abolished when the peptide was co-applied with the selective kinin-B2 receptor antagonist HOE-140. Kinin-B2 receptor activation promoted survival of hippocampal neurons via phosphatidylinositol 3-kinase, while MEK/MAPK signaling was not involved in protection against NMDA-evoked excitotoxic effects. However, 100 nM Lys-des-Arg(9)-bradykinin, a potent kinin-B1 receptor agonist, reversed bradykinin-induced population spike recovery. The inhibition of population spikes recovery was reversed by PD98059, showing that MEK/MAPK was involved in the induction of apoptosis mediated by the B1 receptor.ConclusionsBradykinin exerted protection against NMDA-induced excitotoxicity which is reversed in the presence of a kinin-B1 receptor agonist. As bradykinin is converted to the kinin-B1 receptor metabolite des-Arg(9)-bradykinin by carboxypeptidases, present in different areas including in brain, our results provide a mechanism for the neuroprotective effect in vitro despite of the deleterious effect observed in vivo.
| S-EPMC3277507 | biostudies-literature
Project description:In the case of vascular injury, a complex process (of clotting) starts, involving mainly platelets and coagulation factors. This process in healthy humans is known as hemostasis, but when it is deregulated (thrombosis), it can be the cause of important cardiovascular diseases. Nowadays, the aging of the population and unhealthy lifestyles increase the impact of thrombosis, and therefore there is a need for tools to provide a better understanding of the hemostasis mechanisms, as well as more cost-effective diagnosis and control devices. This study proposes a novel microflow chamber, with interchangeable biomimetic surfaces to evaluate global hemostasis, using reduced amounts of blood sample and reagents, and also a minimized time required to do the test. To validate the performance of this novel device, a study on the new oral anticoagulant Apixaban (APIX) has been performed and compared to previous conventional techniques. The test shows an excellent agreement, while the amount of the required sample has been reduced (only 100 µL is used), and the amount of reagent as well. An imprinted electrode embedded in the chamber in order to measure the impedance during the coagulation process. This approach distinguishes the impedance behavior of plasma poor in platelets (PPP) and plasma rich in platelets (PRP) for the first time.
| S-EPMC6722990 | biostudies-literature