A fibrinogen deficiency accelerates the initiation of LDL cholesterol–driven atherosclerosis via thrombin generation and platelet activation in genetically predisposed mice by Takayuki Iwaki, Mayra J. Sandoval-Cooper, Markus Brechmann, Victoria A. Ploplis, and Francis J. Castellino Blood Volume 107(10):3883-3891 May 15, 2006 ©2006 by American Society of Hematology

Cholesterol content of various lipoprotein fractions. Cholesterol content of various lipoprotein fractions. FPLC analysis of lipid profiles of plasma from (A) WT (n = 17), (B) FG–/– (n = 6), (C) L–/–/A–/– (n = 91), and (D) L–/–/A–/–/FG–/– (n = 15) mice. h indicates HDL; l, LDL; and v, VLDL. (E) Plasma concentrations of total-C, VLDL-C, LDL-C, and HDL-C, along with triglyceride (TG) levels in WT ([▪), FG–/– (▦), L–/–/A–/– (□), and L–/–/A–/–/FG–/– (▤) mice. Takayuki Iwaki et al. Blood 2006;107:3883-3891 ©2006 by American Society of Hematology

Plaque formation in entire aortic trees. Plaque formation in entire aortic trees. Sudan IV staining of aortic trees of male (A) L–/–/A–/– and (B) L–/–/A–/–/FG–/– mice at 24, 36, and 48 weeks of age, showing the extent of lipid-containing plaque (red/orange stains) in these strains. (C) The percent of total surface occupied by plaque in the aorta between L–/–/A–/– (▪) and L–/–/A–/–/FG–/– (▦) mice. N = number of mice. *P < .001 between pairs. Takayuki Iwaki et al. Blood 2006;107:3883-3891 ©2006 by American Society of Hematology

Plaque formation in aortic sinuses of mice. Plaque formation in aortic sinuses of mice. H&E stains of aortic sinuses from L–/–/A–/– (A,C,E) and L–/–/A–/–/FG–/– (B,D,F) mice at 24, 36, and 48 weeks of age. Red arrows indicate the leaflets of aortic valves; yellow arrows, the vascular wall of aorta; and black arrows, the plaques. Original magnification, × 40. (G) Plaque sizes in aortic sinuses of L–/–/A–/– (▪) and L–/–/A–/–/FG–/– (▦) mice, as revealed by morphometric analysis of H&E-stained slides. *P < .05. Takayuki Iwaki et al. Blood 2006;107:3883-3891 ©2006 by American Society of Hematology

Histology of the aortic sinuses from mice at various ages. Histology of the aortic sinuses from mice at various ages. Sections were cut from 24-week (A,C,E) and 48-week (G,I,K) L–/–/A–/–, and from 24-week (B,D,F) and 48-week (H,J,L) L–/–/A–/–/FG–/– mice. H&E (A,B) and Masson's trichrome stains (C,D), and anti-SMC (E,F,G,H), antifibrin(ogen) (I,J), and antimacrophage immunostains (K,L). Original magnification, × 200. (A) A well-formed fibrous cap (red arrowhead) encapsulating a foam cell-laden core (green arrowhead). (B) An intermediate size lesion with a thinning cap (red arrowheads). Focal acellular patches (green arrowhead), cellular debris, extracellular lipids, foam-like cells, and cholesterol clefts (blue arrowhead) within the core. (C) The collagen-rich fibrous cap (red arrowheads) encapsulates a collagen-negative foam cell core (green arrowheads). (D) Collagen is associated within the subcapsular region (red arrowheads). (E) A cellular multilayered region of SMCs associated with the fibrous cap (red arrowheads), as well as the normally positive medial compartment (green arrowheads). (F) Numerous single-layered positive SMCs (red arrowheads) at the endothelium. (G) Positive (dark red) SMC cells in the subluminal region, which are intensely positive (red arrowheads) and diffusely scattered among negative bordering cells. A few positive SMCs within the core (green arrowheads). (H) A thin, faint SMC layer (red arrowheads) in the subluminal region diffusely scattered among negative bordering foci. (I) Fibrin deposits (red staining) in the underlying subcapsular region. Patchy areas of fibrin are associated with the endothelium (red arrowheads). Diffuse areas of fibrin deposition in the lipid core (green arrowheads). (J) Negative anti-Fg immunostaining. (K) Macrophages (brown stain) in the thinned cap, in the subendothelium (red arrowheads), and within the lipid core (green arrowheads). (L) Several macrophages (brown stain) diffusely scattered within the core and at the base (red arrowheads). Takayuki Iwaki et al. Blood 2006;107:3883-3891 ©2006 by American Society of Hematology

Coagulation parameters in WT, FG–/–, L–/–, and L–/–/A–/–/FG–/– mice. Coagulation parameters in WT, FG–/–, L–/–, and L–/–/A–/–/FG–/– mice. (A) PT, (B) aPTT, (C) plasma levels of fibrinogen, and (D) plasma levels of TAT. N = the number of mice used at each time point. ▪ indicates WT; ▦, FG–/–; □, L–/–/A–/–; and ▤, L–/–/A–/–/FG–/–. *P < .01. Takayuki Iwaki et al. Blood 2006;107:3883-3891 ©2006 by American Society of Hematology

Flow cytometric analyses of murine platelets. Flow cytometric analyses of murine platelets. CD61 and CD62P on platelets of WT (black), FG–/– (blue), L–/–/A–/– (orange), and L–/–/A–/–/FG– (red) mice. Dotted lines indicate isotype-specific negative controls. (A) Histogram for CD61, (B) histogram for CD62, (C) fluorescence intensity of CD61, and (D) fluorescent intensity of CD62. N = the number of mice used at each time point. *P < .05; **P < .001; ^P < .01. Takayuki Iwaki et al. Blood 2006;107:3883-3891 ©2006 by American Society of Hematology

Plasma assays of platelet activation markers. Plasma assays of platelet activation markers. (A) ADAMTS13 in L–/–/A–/– and L–/–/A–/–/FG–/– mice. (B) sP-Sel in L–/–/A–/– and L–/–/A–/–/FG–/– mice. (C) VWF-Ag in L–/–/A–/– and L–/–/A–/–/FG–/– mice. (D) VWF-CBA in L–/–/A–/– and L–/–/A–/–/FG–/– mice. N = the number of mice used at each time point. *P < .05; **P < .001; ^P < .01. Takayuki Iwaki et al. Blood 2006;107:3883-3891 ©2006 by American Society of Hematology