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Molecular Medicine Reports Dec 2021Label‑free quantitative mass spectrometry was used to analyze the differences in the granulation tissue protein expression profiles of patients with diabetic foot...
Label‑free quantitative mass spectrometry was used to analyze the differences in the granulation tissue protein expression profiles of patients with diabetic foot ulcers (DFUs) before and after negative‑pressure wound therapy (NPWT) to understand how NPWT promotes the healing of diabetic foot wounds. A total of three patients with DFUs hospitalized for Wagner grade 3 were enrolled. The patients received NPWT for one week. The granulation tissue samples of the patients prior to and following NPWT for one week were collected. The protein expression profiles were analyzed with label‑free quantitative mass spectrometry and the differentially expressed proteins (DEPs) in the DFU patients prior to and following NPWT for one week were identified. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were conducted to annotate the DEPs and DEP‑associated signaling pathways. Western blotting and ELISA were performed to validate the results. By comparing the differences in the protein profiles of granulation tissue samples prior to and following NPWT for one week, 36 proteins with significant differences were identified (P<0.05); 33 of these proteins were upregulated and three proteins were downregulated. NPWT altered proteins mainly associated with antioxidation and detoxification, the cytoskeleton, regulation of the inflammatory response, complement and coagulation cascades and lipid metabolism. The functional validation of the DEPs demonstrated that the levels of cathepsin S in peripheral blood and granulation tissue were significantly lower than those prior to NPWT (P<0.05), while the levels of protein S isoform 1, inter α‑trypsin inhibitor heavy chain H4 and peroxiredoxin‑2 in peripheral blood and granulation tissue were significantly higher than those prior to NPWT (P<0.05). The present study identified multiple novel proteins altered by NPWT and laid a foundation for further studies investigating the mechanism of action of NPWT.
Topics: Aged; Cathepsins; Diabetic Foot; Female; Foot Ulcer; Granulation Tissue; Humans; Male; Mass Spectrometry; Middle Aged; Negative-Pressure Wound Therapy; Peroxiredoxins; Protein S; Proteinase Inhibitory Proteins, Secretory; Proteome; Proteomics; Signal Transduction; Wound Healing
PubMed: 34608502
DOI: 10.3892/mmr.2021.12474 -
Journal of Thrombosis and Haemostasis :... Dec 2023For maximal TFPIα functionality, 2 synergistic cofactors, protein S and FV-short, are required. Both interact with TFPIα, protein S through Kunitz 3 residues...
BACKGROUND
For maximal TFPIα functionality, 2 synergistic cofactors, protein S and FV-short, are required. Both interact with TFPIα, protein S through Kunitz 3 residues Arg199/Glu226 and FV-short with the C-terminus. How these interactions impact the synergistic enhancement remains unclear.
OBJECTIVES
To determine the importance of the TFPIα-protein S and TFPIα-FV-short interactions for TFPIα enhancement.
METHODS
TFPIα variants unable to bind protein S (K3m [R199Q/E226Q]) or FV-short (ΔCT [aa 1-249]) were generated. TFPIα-FV-short binding was studied by plate-binding and co-immunoprecipitation assays; functional TFPIα enhancement by FXa inhibition and prothrombin activation.
RESULTS
While WT TFPIα and TFPIα K3m bound FV-short with high affinity (K∼2nM), TFPIα ΔCT did not. K3m, in contrast to WT, did not incorporate protein S in a TFPIα-FV-short-protein S complex while TFPIα ΔCT bound neither FV-short nor protein S. Protein S enhanced WT TFPIα-mediated FXa inhibition, but not K3m, in the absence of FV-short. However, once FV-short was present, protein S efficiently enhanced TFPIα K3m (EC50: 4.7nM vs 2.0nM for WT). FXa inhibition by ΔCT was not enhanced by protein S alone or combined with FV-short. In FXa-catalyzed prothrombin activation assays, FV-short enhanced TFPIα K3m function in the presence of protein S (5.5 vs 10.4-fold enhancement of WT) whereas ΔCT showed reduced or lack of enhancement by FV-short and protein S, respectively.
CONCLUSION
Full TFPIα function requires the presence of both cofactors. While synergistic enhancement can be achieved in the absence of TFPIα-protein S interaction, only TFPIα with an intact C-terminus can be synergistically enhanced by protein S and FV-short.
Topics: Humans; Blood Coagulation; Blood Coagulation Tests; Factor V; Factor Xa; Prothrombin
PubMed: 37739040
DOI: 10.1016/j.jtha.2023.09.003 -
Molecular Biology of the Cell Nov 2021The protein Slk19 has been shown to localize to kinetochores throughout mitosis and to the spindle midzone in anaphase. However, Slk19 clearly also has an important...
The protein Slk19 has been shown to localize to kinetochores throughout mitosis and to the spindle midzone in anaphase. However, Slk19 clearly also has an important role for spindle formation and stabilization in prometaphase and metaphase, albeit this role is unresolved. Here we show that Slk19's localization to metaphase spindles in vivo and to microtubules (MTs) in vitro depends on the MT cross-linking protein Ase1 and the MT cross-linking and stabilizing protein Stu1. By analyzing a mutant that specifically fails to localize to spindles and MTs, we surprisingly found that the presence of Slk19 amplified the amount of Ase1 strongly and that of Stu1 moderately at the metaphase spindle in vivo and at MTs in vitro. Furthermore, Slk19 markedly enhanced the cross-linking of MTs in vitro when added together with Ase1 or Stu1. We therefore suggest that Slk19 recruits additional Ase1 and Stu1 to the interpolar MTs (ipMTs) of metaphase spindles and thus increases their cross-linking and stabilization. This is in agreement with our observation that cells with defective Slk19 localization exhibit shorter metaphase spindles, an increased number of unaligned nuclear MTs, and most likely reduced ipMT overlaps.
Topics: Cell Cycle; Cell Nucleus; Kinetochores; Metaphase; Microtubule-Associated Proteins; Microtubules; Mitosis; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Spindle Apparatus
PubMed: 34495712
DOI: 10.1091/mbc.E21-05-0279 -
Hamostaseologie Oct 2022Hemostasis is a complex and tightly regulated system that attempts to maintain a homeostatic balance to permit normal blood flow, without bleeding or thrombosis.... (Review)
Review
Hemostasis is a complex and tightly regulated system that attempts to maintain a homeostatic balance to permit normal blood flow, without bleeding or thrombosis. Hemostasis reflects the subtle balance between procoagulant and anticoagulant factors in the pathways of primary hemostasis, secondary hemostasis, and fibrinolysis. The major components in this interplay include the vascular endothelium, platelets, coagulation factors, and fibrinolytic factors. After vessel wall injury, the subendothelium is exposed to the blood stream, followed by rapid activation of platelets via collagen binding and von Willebrand factor-mediated platelet adhesion to the damaged vessel wall through platelet glycoprotein receptor Ib/IX/V. Activated platelets change their shape, release bioactive molecules from their granules, and expose negatively charged phospholipids on their surface. For a proper function of this process, an adequate number of functional platelets are required. Subsequently, a rapid generation of sufficient amounts of thrombin begins; followed by activation of the coagulation system and its coagulation factors (secondary hemostasis), generating fibrin that consolidates the platelet plug. To maintain equilibrium between coagulation and anticoagulation, the naturally occurring anticoagulants such as protein C, protein S, and antithrombin keep this process in balance. Deficiencies (inherited or acquired) at any level of this fine-tuned system result in pathologic bleedings or increased hypercoagulability states leading to thrombosis. This review will focus on genetic diagnosis of inherited bleeding, thrombotic, and platelet disorders, discussing strengths and limitations of existing diagnostic settings and genetic tools and highlight some important considerations necessary for clinical application.
Topics: Humans; Protein S; von Willebrand Factor; Thrombin; Protein C; Hemostasis; Blood Platelet Disorders; Thrombosis; Blood Platelets; Blood Coagulation Factors; Hemorrhage; Fibrin; Anticoagulants; Platelet Membrane Glycoproteins; Antithrombins; Phospholipids; Collagen
PubMed: 35226963
DOI: 10.1055/a-1726-4793 -
DNA Repair Aug 2022Multiple eukaryotic SWI2/SNF2 DNA translocases safeguard genome integrity, mostly by remodelling nucleosomes, but also by fine-tuning mechanisms of DNA repair, such as... (Review)
Review
Multiple eukaryotic SWI2/SNF2 DNA translocases safeguard genome integrity, mostly by remodelling nucleosomes, but also by fine-tuning mechanisms of DNA repair, such as homologous recombination. Among this large family there is a unique class of Rad5/16-like enzymes, including Saccharomyces cerevisiae Uls1 and its Schizosaccharomyces pombe orthologues Rrp1 and Rrp2, that have both translocase and E3 ubiquitin ligase activities, and are often directed towards their substrates by SUMOylation. Here we summarize recent advances in understanding how different activities of these yeast proteins jointly contribute to their important roles in replication stress response particularly at centromeres and telomeres. This extends the possible range of functions performed by this class of SNF2 enzymes in human cells involving both their translocase and ubiquitin ligase activities and related to SUMOylation pathways within the nucleus.
Topics: Adenosine Triphosphatases; DNA; DNA Damage; DNA Helicases; DNA-Binding Proteins; Genomic Instability; Humans; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Schizosaccharomyces; Transcription Factors
PubMed: 35716431
DOI: 10.1016/j.dnarep.2022.103356 -
International Journal of Molecular... Jun 2024Among the myriad of existing tyrosine kinase receptors, the TAM family-abbreviated from Tyro3, Axl, and Mer tyrosine kinase (MerTK)-has been extensively studied with an...
Among the myriad of existing tyrosine kinase receptors, the TAM family-abbreviated from Tyro3, Axl, and Mer tyrosine kinase (MerTK)-has been extensively studied with an outstanding contribution from the team of Prof. Greg Lemke. MerTK activity is implicated in a wide variety of functions involving the elimination of apoptotic cells and has recently been linked to cancers, auto-immune diseases, and atherosclerosis/stroke. In the retina, MerTK is required for the circadian phagocytosis of oxidized photoreceptor outer segments by the retinal-pigment epithelial cells, a function crucial for the long-term maintenance of vision. We previously showed that MerTK ligands carry the opposite role in vitro, with Gas6 inhibiting the internalization of photoreceptor outer segments while Protein S acts conversely. Using site-directed mutagenesis and ligand-stimulated phagocytosis assays on transfected cells, we presently demonstrate, for the first time, that Gas6 and Protein S recognize different amino acids on MerTK Ig-like domains. In addition, MerTK's function in retinal-pigment epithelial cells is rhythmic and might thus rely on the respective stoichiometry of both ligands at different times of the day. Accordingly, we show that ligand bioavailability varies during the circadian cycle using RT-qPCR and immunoblots on retinal and retinal-pigment epithelial samples from control and beta5 integrin knockout mice where retinal phagocytosis is arrhythmic. Taken together, our results suggest that Gas6 and Protein S might both contribute to refine the acute regulation of MerTK in time for the daily phagocytic peak.
Topics: c-Mer Tyrosine Kinase; Phagocytosis; Animals; Intercellular Signaling Peptides and Proteins; Protein S; Humans; Retina; Mice; Circadian Rhythm; Ligands; Retinal Pigment Epithelium; Receptor Protein-Tyrosine Kinases
PubMed: 38928335
DOI: 10.3390/ijms25126630 -
Journal of Thrombosis and Haemostasis :... Jan 2022Factor V-Short (FV756-1458) is a natural splice variant in which 702 residues are deleted from the B domain. It exposes an acid region (AR2; 1493-1537) that binds tissue...
The preAR2 region (1458-1492) in factor V-Short is crucial for the synergistic TFPIα-cofactor activity with protein S and the assembly of a trimolecular factor Xa-inhibitory complex comprising FV-Short, protein S, and TFPIα.
BACKGROUND
Factor V-Short (FV756-1458) is a natural splice variant in which 702 residues are deleted from the B domain. It exposes an acid region (AR2; 1493-1537) that binds tissue factor pathway inhibitor alpha (TFPIα). Protein S also interacts with TFPIα and serves as TFPIα-cofactor in factor Xa (FXa) inhibition. FV-Short and protein S function as synergistic TFPIα-cofactors in inhibition of FXa. FV810-1492 is an artificial FV-Short variant that cannot synergize with protein S as TFPIα cofactor even though it contains AR2 and binds TFPIα.
OBJECTIVE
To elucidate the mechanisms for the synergism between FV756-1458 and protein S as TFPIα cofactors.
METHODS
Four FV-Short variants were created, FV756-1458 and FV712-1458 contained the preAR2 region (1458-1492), whereas FV810-1492 and FV713-1492 lacked this region. The synergistic TFPIα cofactor activity between FV-Short variants and protein S was analyzed by FXa-inhibition. A microtiter-based assay tested binding between FV-Short variants, protein S, and TFPIα.
RESULTS
The two preAR2-containing FV-Short variants were active as synergistic TFPIα cofactors, whereas the other two were inactive. All variants bound to TFPIα. None of the FV-Short variants bound directly to protein S. The combination of TFPIα and preAR2-containing FV-Short variants bound protein S, whereas TFPIα together with the preAR2-minus variants did not. Protein S potentiated TFPIα-binding to the preAR2-containing variants and binding between TFPIα and protein S was stimulated only by the preAR2-containing variants.
CONCLUSION
The preAR2 region is demonstrated to be crucial for the synergistic TFPIα-cofactor activity between FV-Short and protein S and for the assembly of a trimolecular FXa-inhibitory complex comprising FV-Short, protein S, and TFPIα.
Topics: Blood Coagulation; Factor V; Factor Xa; Humans; Lipoproteins; Protein S; Thrombin
PubMed: 34623729
DOI: 10.1111/jth.15547 -
Diagnostics (Basel, Switzerland) Apr 2022The deficiency of natural anticoagulants—antithrombin (AT), protein C (PC), and protein S (PS)—is a highly predisposing factor for thrombosis, which is still...
The deficiency of natural anticoagulants—antithrombin (AT), protein C (PC), and protein S (PS)—is a highly predisposing factor for thrombosis, which is still underdiagnosed at the genetic level. We aimed to establish and evaluate an optimal diagnostic approach based on a high-throughput sequencing platform suitable for testing a small number of genes. A fast, flexible, and efficient method involving automated amplicon library preparation and target sequencing on the Ion Torrent platform was optimized. The cohort consisted of a group of 31 unrelated patients selected for sequencing due to repeatedly low levels of one of the anticoagulant proteins (11 AT-deficient, 13 PC-deficient, and 7 PS-deficient patients). The overall mutation detection rate was 67.7%, highest in PC deficiency (76.9%), and six variants were newly detected—SERPINC1 c.398A > T (p.Gln133Leu), PROC c.450C > A (p.Tyr150Ter), c.715G > C (p.Gly239Arg) and c.866C > G (p.Pro289Arg), and PROS1 c.1468delA (p.Ile490fs) and c.1931T > A (p.Ile644Asn). Our data are consistent with those of previous studies, which mostly used time-consuming Sanger sequencing for genotyping, and the indication criteria for molecular genetic testing were adapted to this process in the past. Our promising results allow for a wider application of the described methodology in clinical practice, which will enable a suitable expansion of the group of indicated patients to include individuals with severe clinical findings of thrombosis at a young age. Moreover, this approach is flexible and applicable to other oligogenic panels.
PubMed: 35626216
DOI: 10.3390/diagnostics12051060 -
International Review of Cell and... 2022Mertk, a type I Receptor Tyrosine Kinase (RTK) and member of the TAM (Tyro3, Axl, and Mertk) family of homologous tyrosine kinases, has important roles in signal...
Mertk, a type I Receptor Tyrosine Kinase (RTK) and member of the TAM (Tyro3, Axl, and Mertk) family of homologous tyrosine kinases, has important roles in signal transduction both homeostatically on normal cells as well as patho-physiologically on both tumor-associated macrophages and malignant cells by its overexpression in a wide array of cancers. The main ligands of Mertk are Vitamin K-modified endogenous proteins Gas6 and Protein S (ProS1), heterobifunctional modular proteins that bind Mertk via two carboxyl-terminal laminin-like globular (LG) domains, and an N-terminal Gla domain that binds anionic phospholipids, whereby externalized phosphatidylserine (PS) on stressed viable and caspase-activated apoptotic cells is most emblematic. Recent studies indicate that Vitamin K-dependent γ-carboxylation on the N-terminal Gla domain of Gas6 and Protein S is necessary for PS binding and Mertk activation, implying that Mertk is preferentially active in tissues where there is high externalized PS, such as the tumor microenvironment (TME) and acute virally infected tissues. Once stimulated, activated Mertk can provide a survival advantage for cancer cells as well as drive compensatory proliferation. On monocytes and tumor-associated macrophages, Mertk promotes efferocytosis and acts as an inhibitory receptor that impairs host anti-tumor immunity, functioning akin to a myeloid checkpoint inhibitor. In recent years, inhibition of Mertk has been implicated in a dual role to enhance the sensitivity of cancer cells to cytotoxic agents along with improving host anti-tumor immunity with anti-PD-1/PD-L1 immunotherapy. Here, we examine the rationale of Mertk-targeted immunotherapies, the current and potential therapeutic strategies, the clinical status of Mertk-specific therapies, and potential challenges and obstacles for Mertk-focused therapies.
Topics: Biology; Humans; Neoplasms; Protein S; Proto-Oncogene Proteins; Tumor Microenvironment; Vitamin K; c-Mer Tyrosine Kinase
PubMed: 35636929
DOI: 10.1016/bs.ircmb.2022.04.004 -
The Tohoku Journal of Experimental... Sep 2019A defect in clearance of apoptotic materials is pivotal in the pathogenesis of systemic lupus erythematosus (SLE). Protein S participates in the removal of apoptotic...
Plasma Free Protein S Is Correlated with Disease Activity, but not with Subclinical Atherosclerosis among Patients with Systemic Lupus Erythematosus: A Cross-Sectional Study.
A defect in clearance of apoptotic materials is pivotal in the pathogenesis of systemic lupus erythematosus (SLE). Protein S participates in the removal of apoptotic remnants and the anticoagulation pathway. The aim of the study was to clarify the relationship between plasma levels of free protein S and the disease activity or subclinical atherosclerosis in SLE. Free protein S was measured by an enzyme-linked immunosorbent assay, and patients were classified into two groups of free protein S levels: low (< 50%) and normal (≥ 50%). One hundred-eleven Korean female patients with SLE were enrolled, and the levels of free protein S were 67.4 ± 19.7%. Carotid plaque was detected in 25 (22.5%) patients. Twenty-one patients with low free protein S had lower hemoglobin (11.4 ± 1.4 vs. 12.5 ± 1.4 g/dL) and lymphocytes (1,221 ± 609 vs. 1,720 ± 1,097/µL), higher erythrocyte sedimentation rate (30.1 ± 20.6 vs. 20.8 ± 17.8 mm/h), and lower complement 3 (80.8 ± 27.6 vs. 103.4 ± 25.8 mg/dL) and complement 4 (15.6 ± 10.4 vs. 21.5 ± 7.6 mg/dL) than those with normal protein S. There was no significant difference in the proportion of patients with increased carotid artery intima-media thickness (> 4.6 mm) or with carotid artery plaque between two groups. The low levels of free protein S were associated with hemoglobin (OR = 0.64, p = 0.04) and complement 3 (OR = 0.96, p = 0.005). Free protein S is correlated with disease activity, but not with subclinical atherosclerosis in SLE.
Topics: Adult; Atherosclerosis; Cross-Sectional Studies; Female; Humans; Logistic Models; Lupus Erythematosus, Systemic; Multivariate Analysis; Protein S
PubMed: 31511453
DOI: 10.1620/tjem.249.1