Fig. 2: GPV alters fibrin formation and localizes to fibrin fibers outside the thrombus after thrombin cleavage.
From: Platelet glycoprotein V spatio-temporally controls fibrin formation
a, Time-dependent fibrin generation of Gp5−/− and WT mice was quantified. Mean ± s.e.m.; WT, n = 9; Gp5−/−, n = 8; three independent experiments. Two-tailed unpaired t-test with Welch’s correction. P values: 4.5 min, P = 0.0072; 5.5 min, P = 0.0296; 6 min, P = 0.0006. b, Representative images of thrombus formation (anti-GPIX–Alexa Fluor (AF)647 antibody, red) and fibrin formation (fibrin(ogen)–AF488, green) on collagen–TF spots after 6 min. Scale bar, 20 µm. c, Quantification of time to fibrin formation. Each dot represents one animal. Mean ± s.d., n = 4, two-tailed Mann–Whitney test. P = 0.0286. d–f, Fibrin formation on collagen–TF microspots of Gp5dThr and WT mice. Quantification of fibrin formation (d), time to fibrin formation (f) and representative images (e). Scale bar, 20 µm. For staining, see b. d, Mean ± s.e.m.; WT, n = 6; Gp5dThr, n = 8; two-tailed unpaired t-test with Welch’s correction. P values: 6 min, P = 0.0287. f, Mean ± s.d.; WT, n = 5; Gp5dThr, n = 4; two-tailed Mann–Whitney test. P = 0.0317. SAC, surface area coverage. Horizontal dashed line in c and f indicates the end of the experiment; every dot above it did not show any occlusion/fibrin formation within the observation period. g, Subtraction heatmap of parameters of thrombus (increased multilayer and contraction score) and fibrin formation in mutant mice compared to WT controls. Colors represent unchanged (black), decreased (green) or increased (red) parameters. h, Pulldown of sGPV from WT platelets using streptavidin beads after stimulation with biotinylated thrombin in the presence or absence of GM6001 (matrix metalloproteinase inhibitor). Pulldown of sGPV was not observed from Gp5dThr platelets or in the presence of hirudin, which prevents GPV cleavage by thrombin. Eluates were analyzed by western blotting using GPV-specific antibodies and streptavidin–horseradish peroxidase (HRP). Representative results of five independent experiments are shown. M, marker; mGPV, murine GPV; SN, supernatant; arrow indicates pulldown of sGPV. i,j, Recalcified blood was perfused over collagen–TF microspots. Samples were stained for platelets (anti-GPIX antibody, yellow), fibrin(ogen) (cyan) and GPV (magenta) and analyzed with a Zeiss Airyscan microscope. i, Representative WT image and magnified view. Scale bar, 4 µm. j, Quantification of GPV intensities inside fibrin-rich but non-platelet area. Background GPV intensity in Gp5−/− images is displayed as a dashed line. Two-way ANOVA followed by Tukey’s multiple-comparison test, n = 14 (WT), n = 12 (Gp5dThr), n = 4 (Gp5−/−) regions of interest representing n = 4 mice. Mean ± s.d., P = 0.0086. For detailed analysis, see Supplementary Fig. 1. *P < 0.05, **P < 0.01, ***P < 0.001.
