Journal: Journal of thrombosis and haemostasis : JTH

Article Title: Protease activated receptors and glycoprotein VI cooperatively drive the platelet component in thromboelastography

doi: 10.1016/j.jtha.2023.04.008

Figure Lengend Snippet: Role of PAR1/PAR4 and Syk in human blood TEG. Healthy volunteer blood samples were analyzed with addition of DMSO (n=4), vorapaxar (Vora, 5 μM, n=3), BMS-986120 (BMS, 10 μM, n=3) and PRT-2607 (PRT, 20 μM, n=4) to inhibit PAR1, PAR4 and Syk, respectively, 10 mins prior to TEG assay. (A-C) TEG parameters: R time (A), α-angle (B) and MA (C). (D) Representative TEG traces. Data shown as mean ± SD. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparison test. Symbols directly over bars represent significance compared to control. *P < .05, **P< .01, *** P<0.001, ****P < .0001.

Article Snippet: For inhibition of αIIbβ3, PAR1, PAR4, or Syk, samples were treated with abciximab (20 μg/ml, provided by Dr. Rick Stouffer), vorapaxar (5 μM, Med Chem Express), BMS-986120 (10 μM, Cayman Chemical), or PRT-062607 (20 μM), respectively.

Techniques: Comparison, Control

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: Protease activated receptors and glycoprotein VI cooperatively drive the platelet component in thromboelastography

doi: 10.1016/j.jtha.2023.04.008

Figure Lengend Snippet: PAR1/4 and GPVI play redundant roles in platelet activation and contraction in TEG. (1) Analysis of clot formation speed and clot strength in TEG is performed by recalcifying citrated whole blood and activating with kaolin to initiate coagulation. (2) Within several minutes, thrombin generation initiates fibrinogen cleavage to fibrin, which polymerizes and crosslinks. Additionally, thrombin activates platelets through protease activated receptors (PARs) (PAR4 on mouse platelets, PAR1 and PAR4 on human platelets). Both fibrin polymerization and platelet activation contribute to the speed of clot formation (α-angle). (3) As more fibrin is generated, platelets can also bind fibrin via GPVI for additional activation signaling, resulting in robust αIIbβ3 integrin activation and platelet-mediated contraction of the fibrin clot. Clot strength (MA) is entirely dependent on platelet contraction. (4) The end result is a tightly contracted whole blood clot with contracting platelets bound to fibrin. While loss of either platelet PARs or GPVI alone has limited impact on TEG parameters, loss of both leads to TEG traces similar to platelet-depleted blood samples. Created with BioRender.com.

Article Snippet: For inhibition of αIIbβ3, PAR1, PAR4, or Syk, samples were treated with abciximab (20 μg/ml, provided by Dr. Rick Stouffer), vorapaxar (5 μM, Med Chem Express), BMS-986120 (10 μM, Cayman Chemical), or PRT-062607 (20 μM), respectively.

Techniques: Activation Assay, Coagulation, Generated

Journal: Current eye research

Article Title: Upregulation of Thrombin/Matrix Metalloproteinase-1/Protease-Activated Receptor-1 Chain in Proliferative Diabetic Retinopathy.

doi: 10.3109/02713683.2016.1141964

Figure Lengend Snippet: Figure 3. Proliferative diabetic retinopathy epiretinal membranes. Negative control slide that was treated identically with an irrelevant antibody showing no labeling (A). Immunohistochemical staining for CD31 showing blood vessels positive for CD31 (B). Immunohistochemical staining for protease-activated receptor-1 (PAR1) showing immunoreactivity in the vascular endothelium (arrows), in stromal cells (arrowheads) (C), in intravascular leukocytes (arrowheads) (D), and in stromal spindle-shaped cells (E). Immunohistochemical staining for CD45 showing stromal cells positive for CD45 (F). Immunohistochemical staining for α-smooth muscle actin showing immunoreactivity in spindle-shaped myofibroblasts (G). Double immunohistochemistry for CD45 (brown) and PAR1 (red) showing stromal cells and intravascular leukocytes (arrowheads) co-expressing CD45 and PAR1 (H) (original magnification X40).

Article Snippet: Subsequently, the sections were incubated for 60 min with mouse monoclonal anti-CD31 (ready-touse; clone JC70A; Dako, Glostrup, Denmark), mouse monoclonal anti-CD45 (ready-to-use; clones 2B11 + PD7/26; Dako), mouse monoclonal anti-α-smooth muscle actin (ready-to-use; clone 1A4; Dako), and rabbit polyclonal anti-PAR1 antibody (1:200, APR-031, Alomone Labs, Jerusalem, Israel).

Techniques: Negative Control, Labeling, Immunohistochemical staining, Staining, Immunohistochemistry, Expressing

Journal: Current eye research

Article Title: Upregulation of Thrombin/Matrix Metalloproteinase-1/Protease-Activated Receptor-1 Chain in Proliferative Diabetic Retinopathy.

doi: 10.3109/02713683.2016.1141964

Figure Lengend Snippet: Figure 5. Western blot analysis of protease-activated receptor-1 (PAR1) (A) and thrombin (B) in rat retinas. Significant increases in the ratios of the cleaved PAR1 over the intact protein and of the proteolytically active thrombin over the zymogen prothrombin in the retinas of diabetic rats (D) compared to the nondiabetic control rats (C). Each experiment was repeated three times with fresh samples (n = 7). *p < 0.05 compared to control.

Article Snippet: Subsequently, the sections were incubated for 60 min with mouse monoclonal anti-CD31 (ready-touse; clone JC70A; Dako, Glostrup, Denmark), mouse monoclonal anti-CD45 (ready-to-use; clones 2B11 + PD7/26; Dako), mouse monoclonal anti-α-smooth muscle actin (ready-to-use; clone 1A4; Dako), and rabbit polyclonal anti-PAR1 antibody (1:200, APR-031, Alomone Labs, Jerusalem, Israel).

Techniques: Western Blot, Control

Journal: Current eye research

Article Title: Upregulation of Thrombin/Matrix Metalloproteinase-1/Protease-Activated Receptor-1 Chain in Proliferative Diabetic Retinopathy.

doi: 10.3109/02713683.2016.1141964

Figure Lengend Snippet: Figure 4. Human retinal microvascular endothelial cells were left untreated or were treated with either IL-1β (10 ng/ml), TNF-α (30 ng/ml), or a combination of cytokines containing IL-1β (10 ng/ml) plus TNF-α (30 ng/ml) for 24 h. The levels of expression of protease-activated receptor-1 (PAR1) were compared by Western blot analysis. Western blots are representative of at least three independent experiments, each is performed in duplicate and bar graphs are representative of all three experiments. *p < 0.05 compared to untreated control. #p < 0.05 compared to IL-1β.

Article Snippet: Subsequently, the sections were incubated for 60 min with mouse monoclonal anti-CD31 (ready-touse; clone JC70A; Dako, Glostrup, Denmark), mouse monoclonal anti-CD45 (ready-to-use; clones 2B11 + PD7/26; Dako), mouse monoclonal anti-α-smooth muscle actin (ready-to-use; clone 1A4; Dako), and rabbit polyclonal anti-PAR1 antibody (1:200, APR-031, Alomone Labs, Jerusalem, Israel).

Techniques: Expressing, Western Blot, Control

Journal: Current eye research

Article Title: Upregulation of Thrombin/Matrix Metalloproteinase-1/Protease-Activated Receptor-1 Chain in Proliferative Diabetic Retinopathy.

doi: 10.3109/02713683.2016.1141964

Figure Lengend Snippet: Figure 7. Effects of thrombin (A) and matrix metalloproteinase-1 (MMP-1) (B) on the migration of human retinal microvascular endothelial cells (HRMEC). Thrombin- induced migration of HRMEC was inhibited by the protease-activated receptor-1 (PAR1) inhibitor vorapaxar. HRMECs were left untreated or treated either with 1 nM recombinant thrombin, with 10 µM PAR1 inhibitor vorapaxar for 30 min before addition of 1 nM recombinant thrombin or with 5 nM recombinant MMP-1. Cells were visualized using inverted microscope (Olympus 1X 81, Olympus Corporation, Tokyo, Japan). Three independent experiments were performed. Six independent field images were taken from each treated group for migration analysis, which was performed using Image J software. One image from each group is illustrated and the bar graphs show the analysis of all six images from each group. *p < 0.05 compared to untreated control. #p < 0.05 compared to vorapaxar + thrombin.

Article Snippet: Subsequently, the sections were incubated for 60 min with mouse monoclonal anti-CD31 (ready-touse; clone JC70A; Dako, Glostrup, Denmark), mouse monoclonal anti-CD45 (ready-to-use; clones 2B11 + PD7/26; Dako), mouse monoclonal anti-α-smooth muscle actin (ready-to-use; clone 1A4; Dako), and rabbit polyclonal anti-PAR1 antibody (1:200, APR-031, Alomone Labs, Jerusalem, Israel).

Techniques: Migration, Recombinant, Inverted Microscopy, Software, Control