► hsp47 inhibitor  (MedChemExpress)

Journal: Journal of Thrombosis and Haemostasis

Article Title: The chaperone protein HSP 47: a platelet collagen binding protein that contributes to thrombosis and hemostasis

doi: 10.1111/jth.13998

Figure Lengend Snippet: Platelet HSP 47 binds to immobilized collagen fibrils and can modulate GPVI –collagen interaction. Isolated washed human platelets were lysed and incubated with either collagen sepharose or bovine serum albumin BSA ‐sepharose. For experiments with small molecule inhibitor of HSP47 (SMIH) , platelets were preincubated with 20 μ m SMIH or vehicle control dimethylsulfoxide (DMSO) (0.1%) prior to the addition of collagen sepharose. (Ai) The bound proteins were visualized using immunoblot analysis. (Aii) Data represent mean ± SD ( n = 3). Student's t ‐test, * P < 0.05. The basal level band intensity obtained from bovine serum albumin (BSA) sepharose was taken as 100% and data normalized to this value. For experiments with inhibitory anti‐ HSP 47, platelets were preincubated with control IgG (10 μg mL −1 ) or anti HSP 47 (10 μg mL −1 ) prior to the addition of collagen sepharose. (Bi) The bound HSP 47 was visualized using immunoblot analysis. (Bii) Data represent mean ± SD ( n = 4). Student's t ‐test, * P < 0.05. For experiments to evaluate bound GPVI , platelets were preincubated with control IgG (10 μg mL −1 ) or anti‐ HSP 47 (10 μg mL −1 ), SMIH (20 μ m ) or vehicle control (0.1% DMSO ) prior to the addition of collagen sepharose. (Ci) Bound GPVI was visualized by immunoblot analysis, (Cii) Cumulative data represent mean ± SD ( n = 4). Student's t ‐test, * P < 0.05.

Article Snippet: Washed platelets (4 × 10 8 cells) were incubated with either small molecule inhibitor of HSP47 (SMIH, compound IV, was identified from a large‐scale screen of compounds that interfered with HSP47 interaction with collagen and prevents its fibrillogenesis ; Maybridge, RH00007SC, 20 μ m , Thermo fisher scientific, Leicestershire, UK), vehicle (0.1% dimethylsulfoxide [DMSO]) or inhibitory rabbit polyclonal anti‐HSP47 (anti‐HSP47; the antibody was raised against synthetic peptide corresponding to the collagen binding site [amino acid residues 406–417] of human HSP47 [lyophilized‐azide free]; Life Span Biosciences, Nottingham, UK), control rabbit IgG for 2 min at 37 °C and lysed with 2% (v/v) Nonidet P40 buffer with protease inhibitors.

Techniques: Isolation, Incubation, Control, Western Blot

Journal: Journal of Thrombosis and Haemostasis

Article Title: The chaperone protein HSP 47: a platelet collagen binding protein that contributes to thrombosis and hemostasis

doi: 10.1111/jth.13998

Figure Lengend Snippet: Isolated, washed human platelets were stimulated with type I collagen fibrils in the presence of function blocking rabbit polyclonal Anti‐HSP47 or rabbit IgG (Control IgG). Aggregation responses were recorded following stimulation with 1 μg/mL collagen (Ai), 3 μg/mL (Bi), 0.3 μg/mL CRP‐XL (Ci) or 0.05U/mL thrombin (Di). Data (Aii, Bii, Cii, Dii) represent mean ± S.D ( n =3) one way ANOVA (** P < 0.01 and * P < 0.05) for collagen data and Student's t ‐test (* P < 0.05) for CRP‐XL or thrombin. Aggregation obtained with control IgG was taken as 100% and was normalised to this value.

Article Snippet: Washed platelets (4 × 10 8 cells) were incubated with either small molecule inhibitor of HSP47 (SMIH, compound IV, was identified from a large‐scale screen of compounds that interfered with HSP47 interaction with collagen and prevents its fibrillogenesis ; Maybridge, RH00007SC, 20 μ m , Thermo fisher scientific, Leicestershire, UK), vehicle (0.1% dimethylsulfoxide [DMSO]) or inhibitory rabbit polyclonal anti‐HSP47 (anti‐HSP47; the antibody was raised against synthetic peptide corresponding to the collagen binding site [amino acid residues 406–417] of human HSP47 [lyophilized‐azide free]; Life Span Biosciences, Nottingham, UK), control rabbit IgG for 2 min at 37 °C and lysed with 2% (v/v) Nonidet P40 buffer with protease inhibitors.

Techniques: Isolation, Blocking Assay, Control

Journal: Journal of Thrombosis and Haemostasis

Article Title: The chaperone protein HSP 47: a platelet collagen binding protein that contributes to thrombosis and hemostasis

doi: 10.1111/jth.13998

Figure Lengend Snippet: HSP 47‐deficient platelets display diminished response to collagen or CRP ‐ XL . Mice lacking platelet HSP 47(Pf4‐Cre; HSP47 flox/flox ) were generated using the recombination strategy mediated by expression of Cre‐recombinase in megakaryocytes. (A) PCR analysis of genomic DNA from Pf4‐Cre; HSP47 flox/flox and HSP47 flox/flox ear tissue samples. (B) Immunoblot analysis of mouse platelet lysates at concentration 4 × 10 8 cells/ml to detect HSP 47 protein. (C) The expression levels of α II bβ3, GPVI , α2β1 and GPI bα analyzed by flow cytometry using citrated mouse blood Pf4‐Cre; HSP47 flox/flox or HSP47 flox/flox mouse blood. Data represent mean ± SD ( n = 3). P ‐values calculated by Student's t ‐test ( P > 0.05). Washed platelets from HSP47 flox/flox and Pf4‐Cre; HSP47 flox/flox mice were stimulated with 0.5 μg mL −1 collagen (D, E) or 1.0 μg mL −1 collagen (F, G). Cumulative data are presented as mean ± SD ( n = 4). 100% aggregation is defined as the level of aggregation obtained with HSP47 flox/flox (control) (Student's t ‐test, ** P < 0.01 and * P < 0.05).

Article Snippet: Washed platelets (4 × 10 8 cells) were incubated with either small molecule inhibitor of HSP47 (SMIH, compound IV, was identified from a large‐scale screen of compounds that interfered with HSP47 interaction with collagen and prevents its fibrillogenesis ; Maybridge, RH00007SC, 20 μ m , Thermo fisher scientific, Leicestershire, UK), vehicle (0.1% dimethylsulfoxide [DMSO]) or inhibitory rabbit polyclonal anti‐HSP47 (anti‐HSP47; the antibody was raised against synthetic peptide corresponding to the collagen binding site [amino acid residues 406–417] of human HSP47 [lyophilized‐azide free]; Life Span Biosciences, Nottingham, UK), control rabbit IgG for 2 min at 37 °C and lysed with 2% (v/v) Nonidet P40 buffer with protease inhibitors.

Techniques: Generated, Expressing, Western Blot, Concentration Assay, Flow Cytometry, Control

Journal: Journal of Thrombosis and Haemostasis

Article Title: The chaperone protein HSP 47: a platelet collagen binding protein that contributes to thrombosis and hemostasis

doi: 10.1111/jth.13998

Figure Lengend Snippet: HSP 47 supports the initial adhesion of platelets to collagen, thus enhancing GPVI signaling. Blood drawn from HSP47 flox/flox and Pf4‐Cre; HSP47 flox/flox mice labeled with Di OC 6 was perfused over collagen‐coated surfaces at a wall shear rate of 1000 s −1 for 10 mins. (Ai) Representative images are shown. Captured images of adherent thrombi were analyzed by calculating the sum intensity of fluorescence as a measure of thrombus formation. (Aii) Surface coverage at 10 min is presented as mean ± SD ( n = 3); P = 0.02 by two‐way anova . (Bi) Anticoagulated human blood without eptifibatide was labeled with Di OC 6 incubated with small molecule inhibitor of HSP47 (SMIH) or controls (dimethylsulfoxide [ DMSO] ) at (1000 s ‐1 ) for 4 mins. Thrombus volume was measured by confocal microscopy following incubation of blood with 5 μ m , 10 μ m and 20 μ m SMIH (mean ± SD , n = 6, * P < 0.05). (Bii) Thrombus volume was measured following incubation of blood with function blocking anti‐ HSP 47 or control IgG (mean ± SD , n = 6, * P < 0.05). (Ci) Representative fields of thrombi formed under similar conditions with SMIH (20 μ m ) or vehicle control treatment in the presence of eptifibatide (4 μ m ). (Cii) The levels of platelet adhesion to collagen were measured by analysis of concentration of protein eluted from flow cells after perfusion (mean ± SD , n = 5, * P < 0.05). (Ciii) Platelet adhesion was measured after incubation of human blood with anti‐ HSP 47 or control IgG in the presence of eptifibatide (mean ± SD , n = 3, * P < 0.05). (D) Platelet adhesion to von Willebrand factor (VWF) in the presence of eptifibatide (4 μ m ) under arterial flow conditions using fluorescently labeled human blood in the presence or absence of anti‐ HSP 47 (mean ± SD , n = 3, P > 0.05). (Ei, Eii) Platelets from HSP47 flox/flox and Pf4‐Cre; HSP47 flox/flox mice were stimulated by the addition of 1 μg mL −1 CRP ‐ XL for 90 s. Immunoblot analysis was performed to detect total Syk phosphorylation using a phosphospecific Syk (Tyr 525/526) antibody. Data represent mean SYK 525/526 band intensity ± SD ( n = 3). Student's t ‐test, ** P < 0.01. The band intensity of actin and Syk was used to control for protein loading levels.

Article Snippet: Washed platelets (4 × 10 8 cells) were incubated with either small molecule inhibitor of HSP47 (SMIH, compound IV, was identified from a large‐scale screen of compounds that interfered with HSP47 interaction with collagen and prevents its fibrillogenesis ; Maybridge, RH00007SC, 20 μ m , Thermo fisher scientific, Leicestershire, UK), vehicle (0.1% dimethylsulfoxide [DMSO]) or inhibitory rabbit polyclonal anti‐HSP47 (anti‐HSP47; the antibody was raised against synthetic peptide corresponding to the collagen binding site [amino acid residues 406–417] of human HSP47 [lyophilized‐azide free]; Life Span Biosciences, Nottingham, UK), control rabbit IgG for 2 min at 37 °C and lysed with 2% (v/v) Nonidet P40 buffer with protease inhibitors.

Techniques: Labeling, Shear, Fluorescence, Incubation, Confocal Microscopy, Blocking Assay, Control, Concentration Assay, Western Blot, Phospho-proteomics

Journal: Journal of Thrombosis and Haemostasis

Article Title: The chaperone protein HSP 47: a platelet collagen binding protein that contributes to thrombosis and hemostasis

doi: 10.1111/jth.13998

Figure Lengend Snippet: Absence or inhibition of HSP 47 reduces thrombosis. (Ai) Thrombosis was measured following laser injury of cremaster muscle arterioles by intravital microscopy. C57 BL /6 mice were administered small molecule inhibitor of HSP47 (SMIH) or vehicle control via cannula prior to injury. Platelets were labeled by infusion of Alexa Fluor 488‐conjugated anti‐ GPI b antibody. Representative images of thrombi from vehicle control and SMIH (20 μ m in blood volume) following injury are shown. (Aii) Data represent mean maximum fluorescence, n = 27 thrombi from each group, P = 0.012. (Bi) Intravital microscopy was performed on Pf4‐Cre; HSP47 flox/flox and HSP47 flox/flox mice. (Bii) Mean of maximum fluorescence intensity was measured from 12 thrombi from HSP47 flox/flox mice and 15 thrombi from Pf4‐Cre; HSP47 flox/flox mice ( n = 3 mice) (Student's t ‐test, P = 0.031). Images are ×60 magnification. (Biii) The kinetics of the platelet accumulation during thrombus growth was analyzed in HSP47 flox/flox and Pf4‐Cre; HSP47 flox/flox mice. Each curve represents the median integrated platelet fluorescence from 12 thrombi from HSP47 flox/flox mice (black) and 15 thrombi from Pf4‐Cre; HSP47 flox/flox mice (red) (from three separate mice). Fluorescence intensity of platelets in arbitrary units is presented as a function of time. [Color figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Washed platelets (4 × 10 8 cells) were incubated with either small molecule inhibitor of HSP47 (SMIH, compound IV, was identified from a large‐scale screen of compounds that interfered with HSP47 interaction with collagen and prevents its fibrillogenesis ; Maybridge, RH00007SC, 20 μ m , Thermo fisher scientific, Leicestershire, UK), vehicle (0.1% dimethylsulfoxide [DMSO]) or inhibitory rabbit polyclonal anti‐HSP47 (anti‐HSP47; the antibody was raised against synthetic peptide corresponding to the collagen binding site [amino acid residues 406–417] of human HSP47 [lyophilized‐azide free]; Life Span Biosciences, Nottingham, UK), control rabbit IgG for 2 min at 37 °C and lysed with 2% (v/v) Nonidet P40 buffer with protease inhibitors.

Techniques: Inhibition, Intravital Microscopy, Control, Labeling, Fluorescence

Journal: Journal of Thrombosis and Haemostasis

Article Title: The chaperone protein HSP 47: a platelet collagen binding protein that contributes to thrombosis and hemostasis

doi: 10.1111/jth.13998

Figure Lengend Snippet: Inhibition of HSP 47 extends bleeding times. Tail bleeding assays were performed on anesthetized C57 BL /6 mice injected intravenously with small molecule inhibitor of HSP47 (SMIH) or vehicle control. (A) The graph represents bleeding time measured after mice were injected with SMIH (triangle) or vehicle control (circle) ( n = 13 of each group, Mann–Whitney test, * P < 0.05). Tail bleeding assays were performed on anesthetized platelet HSP 47‐deficient mice and control mice. (B) The graph represents bleeding time measured in HSP47 flox/flox (unshaded circle, n = 6) and Pf4‐cre; HSP47 flox/flox mice (square, n = 10) (Mann–Whitney test, * P < 0.05).

Article Snippet: Washed platelets (4 × 10 8 cells) were incubated with either small molecule inhibitor of HSP47 (SMIH, compound IV, was identified from a large‐scale screen of compounds that interfered with HSP47 interaction with collagen and prevents its fibrillogenesis ; Maybridge, RH00007SC, 20 μ m , Thermo fisher scientific, Leicestershire, UK), vehicle (0.1% dimethylsulfoxide [DMSO]) or inhibitory rabbit polyclonal anti‐HSP47 (anti‐HSP47; the antibody was raised against synthetic peptide corresponding to the collagen binding site [amino acid residues 406–417] of human HSP47 [lyophilized‐azide free]; Life Span Biosciences, Nottingham, UK), control rabbit IgG for 2 min at 37 °C and lysed with 2% (v/v) Nonidet P40 buffer with protease inhibitors.

Techniques: Inhibition, Injection, Control, MANN-WHITNEY

Journal: PLoS ONE

Article Title: The Collagen Chaperone HSP47 Is a New Interactor of APP that Affects the Levels of Extracellular Beta-Amyloid Peptides

doi: 10.1371/journal.pone.0022370

Figure Lengend Snippet: ( A ) Recruitment of endogenous HSP47 by overexpressed APP. HeLa cells were transfected with YFP –APP and HSP47 detected by a specific anti-HSP47 antibody. Arrows indicate some of the intracellular structures showing an obvious colocalization. ( B ) Primary rat cortical neurons (4 DIV) were exposed to DSP cross linking agent and total cell lysates were immunoprecipitated with control (MBP), with anti-HSP47 or anti-APP (6E10) antibodies. The immunoprecipitates and 40 µg of the total lysate were then immunoblotted with anti APP (C-Term) or with anti HSP47. ( C ) Colocalization of HSP47 and APP in 14 DIV neurons. A high magnification field of dendrites is shown in the right panel. The signals were in general justaxposed (white arrows) and in some cases were colocalized (yellow arrows).

Article Snippet: The HSP47 inhibitor compound IV was obtained from Calbiochem (code 385874).

Techniques: Transfection, Immunoprecipitation, Control

Journal: PLoS ONE

Article Title: The Collagen Chaperone HSP47 Is a New Interactor of APP that Affects the Levels of Extracellular Beta-Amyloid Peptides

doi: 10.1371/journal.pone.0022370

Figure Lengend Snippet: ( A ) Immunohistochemical analysis of Aβ and HSP47 on wild type (WT) and APPPS1 transgenic mice (12 months old). Arrows indicate HSP47 positive plaques. ( B–C ) Double immunostaining of APPPS1 brain slices with HSP47 and Aβ antibodies. The diffuse plaques (B) show a marked colocalization of the two signals. In dense core plaques (C), the HSP47 antibody stained the Aβ core and a surrounding coronal area. ( D–E ) Dense core plaques double-stained with HSP47 and Tau (D) or APP C-terminal (E) antibodies.

Article Snippet: The HSP47 inhibitor compound IV was obtained from Calbiochem (code 385874).

Techniques: Immunohistochemical staining, Transgenic Assay, Double Immunostaining, Staining

Journal: PLoS ONE

Article Title: The Collagen Chaperone HSP47 Is a New Interactor of APP that Affects the Levels of Extracellular Beta-Amyloid Peptides

doi: 10.1371/journal.pone.0022370

Figure Lengend Snippet: ( A ) Immunofluorescence analysis of HSP47 distribution (green) in normal human brain (Ctr). The arrowhead indicates a positive vessel. ( B ) Analysis of the distribution of HSP47 (green) and Aβ (red) in one of the III–IV stage AD cases. Arrows indicate two HSP47-positive amyloid plaques. ( C–D ) High magnification fields of AD brain sections analyzed as in B, showing diffuse (C) and dense-core (D) HSP47-positive plaques.

Article Snippet: The HSP47 inhibitor compound IV was obtained from Calbiochem (code 385874).

Techniques: Immunofluorescence

Journal: PLoS ONE

Article Title: The Collagen Chaperone HSP47 Is a New Interactor of APP that Affects the Levels of Extracellular Beta-Amyloid Peptides

doi: 10.1371/journal.pone.0022370

Figure Lengend Snippet: ( A ) APPsw cells were transiently transfected with two independent siRNA oligonucleotides (H2 and H3) designed against the human HSP47 sequence or with a mismatch control (C1). After additional 36 h in culture, total cell lysates were analyzed by western blotting (upper panel) with anti HSP47, APP and Vinculin (Vinc) antibodies. In parallel, the amount of A β peptide species was determined in the conditioned medium by ELISA (lower panel). ( B ) APPsw were infected with miRNA-based RNAi lentiviral vectors directed towards HSP47 or towards an unrelated sequence (RNAi Ctr), expressing also a GFP protein. Polyclonal populations were then analyzed as in (A) 36 h after the last medium change. ( C ) Extracellular (E-Aβ) and intracellular (I-Aβ) Aβ peptides were measured separately in APPsw cells treated as in panel A. ( D ) Primary rat cortical neurons were electroporated with two indipendent plKO plasmids (R1 and R2) encoding for shRNAs directed towards HSP47 or with a plKO containing a mismatched sequence (Ctr). 48 h after electroporation medium was changed and, after additional 72 hours, RNA was exctracted to evaluate Hsp47 knockdown by a Real Time PCR assay. In parallel, the amount of Aβ40 in the conditioned medium was determined by an ELISA assay directed against rodent Aβ40 (WAKO). ** = p<0.01; *** = p<0.001 (two tails Student T-Test).

Article Snippet: The HSP47 inhibitor compound IV was obtained from Calbiochem (code 385874).

Techniques: Transfection, Sequencing, Control, Western Blot, Enzyme-linked Immunosorbent Assay, Infection, Expressing, Electroporation, Knockdown, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: The Collagen Chaperone HSP47 Is a New Interactor of APP that Affects the Levels of Extracellular Beta-Amyloid Peptides

doi: 10.1371/journal.pone.0022370

Figure Lengend Snippet: ( A ) Dose dependent reduction of Aβ peptides levels by increasing concentrations of HSP47 inhibitors in 293 APPsw cells. The amount of Aβ peptides species in the conditioned medium of cells treated with the indicated inhibitors was determined by ELISA 24 hours after the last medium change and expressed as ratio on the vehicle control. ( B ) Primary rat cortical neurons were treated 24 h after plating with Compound IV (7.5 βµM) or Vehicle (DMSO). The amount of Aβ40 in conditioned medium was then evaluated as in . ( C ) 293 APPsw cells were transiently transfected with an unrelated control plasmid (Mock) or with an HSP47 overexpression construct. 12 hours later, cells were treated with vehicle only or with 7.5 µM Compound IV (IV). The amount of Aβ peptides in the conditioned medium was determined after additional 24 hours as in (A). ** = p<0.01 (two tails Student T-Test).

Article Snippet: The HSP47 inhibitor compound IV was obtained from Calbiochem (code 385874).

Techniques: Enzyme-linked Immunosorbent Assay, Control, Transfection, Plasmid Preparation, Over Expression, Construct