Critical role of von Willebrand factor and platelet interaction in venous thromboembolism

It has been generally considered that platelets are less important in venous thrombus formation. However, clinical studies have shown an association between venous thromboembolism (VTE) and von Willebrand factor (VWF). We therefore investigated the contribution of VWF and platelet interaction to the onset of VTE using tissues from autopsies and from an animal model. An immunohistochemical study revealed that glycoprotein (GP) IIb/IIIa, fibrin, glycophorin A (erythrocyte-specific protein) and VWF were consistently localized in ilio-femoral venous thrombi and in pulmonary thromboemboli from 8 autopsied cases who died of VTE, and VWF was closely associated with GPIIb/IIIa and fibrin. Venous thrombi and pulmonary emboli contained significant amounts of GPIIb/IIIa and VWF, in addition to glycophorin A and fibrin, and the factors did not significantly differ between them. A rabbit model of VTE was developed by inserting a polyethylene tube into the iliac vein. The constituents of the induced thrombi were quite similar to those of human VTE. An antibody against VWF (AJW200), which inhibits interactions between the VWF A1 domain and platelet GPIb, significantly reduced venous thrombus formation and pulmonary thromboembolism in the model. These results suggest that VWF A1-platelet GPIb interaction plays a significant role in venous thrombus formation

Increased Metabolite Levels of Glycolysis and Pentose Phosphate Pathway in Rabbit Atherosclerotic Arteries and Hypoxic Macrophage

<div><p>Aims</p><p>Inflammation and possibly hypoxia largely affect glucose utilization in atherosclerotic arteries, which could alter many metabolic systems. However, metabolic changes in atherosclerotic plaques remain unknown. The present study aims to identify changes in metabolic systems relative to glucose uptake and hypoxia in rabbit atherosclerotic arteries and cultured macrophages.</p><p>Methods</p><p>Macrophage-rich or smooth muscle cell (SMC)-rich neointima was created by balloon injury in the iliac-femoral arteries of rabbits fed with a 0.5% cholesterol diet or a conventional diet. THP-1 macrophages stimulated with lipopolysaccharides (LPS) and interferon-γ (INFγ) were cultured under normoxic and hypoxic conditions. We evaluated comprehensive arterial and macrophage metabolism by performing metabolomic analyses using capillary electrophoresis-time of flight mass spectrometry. We evaluated glucose uptake and its relationship to vascular hypoxia using ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) and pimonidazole, a marker of hypoxia.</p><p>Results</p><p>The levels of many metabolites increased in the iliac-femoral arteries with macrophage-rich neointima, compared with those that were not injured and those with SMC-rich neointima (glycolysis, 4 of 9; pentose phosphate pathway, 4 of 6; tricarboxylic acid cycle, 4 of 6; nucleotides, 10 of 20). The uptake of ¹⁸F-FDG in arterial walls measured by autoradiography positively correlated with macrophage- and pimonidazole-immunopositive areas (r = 0.76, and r = 0.59 respectively; n = 69 for both; p<0.0001). Pimonidazole immunoreactivity was closely localized with the nuclear translocation of hypoxia inducible factor-1α and hexokinase II expression in macrophage-rich neointima. The levels of glycolytic (8 of 8) and pentose phosphate pathway (4 of 6) metabolites increased in LPS and INFγ stimulated macrophages under hypoxic but not normoxic condition. Plasminogen activator inhibitor-1 protein levels in the supernatant were closely associated with metabolic pathways in the macrophages.</p><p>Conclusion</p><p>Infiltrative macrophages in atherosclerotic arteries might affect metabolic systems, and hypoxia but not classical activation might augment glycolytic and pentose phosphate pathways in macrophages.</p></div

Levels of metabolites of glycolysis, the pentose phosphate pathway, tricarboxylic acid cycle and glyconeogenesis/glycogenolysis in rabbit iliac-femoral arteries.

<p>Gray, blue and red bars: iliac-femoral arteries that were not injured (conventional diet), and those with SMC-rich (conventional diet) and macrophage-rich (0.5% cholesterol diet) neointima, respectively (n = 3 for all). Metabolite levels are expressed as nmol/g. *p<0.05 vs. other groups, ^†p<0.05 vs. non-injured femoral artery. 1,3BPG, 1,3-bisphosphoglycerate; 2PG, 2-phosphoglyceric acid; 3PG, 3-phosphoglyceric acid; 2OG, 2-oxoglutaric acid; 6PG, 6-phosphogluconic acid; DHAP, dihydroxyacetone phosphate; E4P, erythrose 4-phosphate; F1-6P, fructose 1,6-diphosphate; F6P, fructose 6-phosphate; FFA, free fatty acid; G1P, glucose 1-phosphate; G3P, glyceraldehyde 3-phosphate; G6P, glucose 6-phosphate; Glu, glucose; Gly, glycogen; PEP, phosphoenolpyruvic acid; R5P, ribose 5-phosphate; Ru5P, ribulose 5-phosphate; S7P, sedoheptulose 7-phosphate; Xu5P, xylulose 5-phosphate.</p

Immunohistochemical findings of HIF-1α and HK-II in iliac-femoral artery with macrophage-rich neointima.

<p>Immunohistochemical staining for macrophages, pimonidazole and HIF-1α in paraffin sections (A) and for macrophages, pimonidazole and HK-II in frozen sections (B)</p

Levels of metabolites of glycolysis, the pentose phosphate pathway, tricarboxylic acid cycle and glyconeogenesis/glycogenolysis in cultured macrophages.

<p>For M1 polarization, THP-1 cells were treated with PMA (320 nM) for 6 hours and then cultured with PMA plus LPS (10 ng/ml) and INFγ (20 ng/ml,) for another 42 hours. After replacement of culture medium, PMA-treated control macrophages (gray bar, n = 6) and M1 macrophages (blue bar, n = 6) were incubated under normoxic (21% O₂) condition for 6 hours, or the M1 macrophages were incubated hypoxic (1% O₂) conditions for 6 hours (red bar, n = 6). Metabolite levels are expressed as pmol/10⁶ cell. *p<0.01 vs. PMA control, ^†p<0.05 vs. PMA control, ^§p<0.01 vs. M1 normoxia, ^¶p<0.05 vs. M1 normoxia. 1,3BPG, 1,3-bisphosphoglycerate; 2PG, 2-phosphoglyceric acid; 3PG, 3-phosphoglyceric acid; 2OG, 2-oxoglutaric acid; 6PG, 6-phosphogluconic acid; DHAP, dihydroxyacetone phosphate; E4P, erythrose 4-phosphate; F1-6P, fructose 1,6-diphosphate; F6P, fructose 6-phosphate; FFA, free fatty acid; G1P, glucose 1-phosphate; G3P, glyceraldehyde 3-phosphate; G6P, glucose 6-phosphate; Glu, glucose; Gly, glycogen; PEP, phosphoenolpyruvic acid; R5P, ribose 5-phosphate; Ru5P, ribulose 5-phosphate; S7P, sedoheptulose 7-phosphate; Xu5P, xylulose 5-phosphate.</p

Histological findings of iliac-femoral arteries that were not injured or those at three weeks after balloon injury in rabbits fed with conventional or 0.5% cholesterol diets.

<p>A. Representative histological images of arterial sections stained with hematoxylin eosin (HE), and antibodies for muscle actin or rabbit macrophages. N, neointima; BI, balloon injury. B. Vascular areas, as well as areas that are immunopositive for SMCs and macrophages, and numbers of Ki-67-immunopositive nuclei in arteries. White and black bars, conventional and 0.5% cholesterol diet, respectively; n = 12 each). BI, balloon injury. Arteries at three weeks after balloon injury shows neointimal formation rich in SMCs (conventional diet) or macrophages (0.5% cholesterol diet), compared with non-injured arteries. C. Representative double immunofluorescent images for Ki-67 and macrophage or SMC in arteries 3 weeks after balloon injury in rabbits fed with 0.5% cholesterol diet. Images stained with CF488A labeled anti-Ki-67 antibody (green), CF495-labeled anti-macrophage antibody (red) or anti-SMC antibody (red), and merged immunofluorescent images. There are Ki-67 immunopositive nuclei in macrophages (upper row). The cells with Ki-67-immunopositive nuclei don't have SMC-positive cytoplasm (arrows).</p

Arterial ¹⁸F-FDG uptake and its relationship to hypoxia.

<p>A. Radioactivity levels in iliac-femoral arteries and blood. White and black bars, conventional and 0.5% cholesterol diets, respectively; n = 5). BI, balloon injury. B. Representative findings of macrophages and pimonidazole in the iliac-femoral arteries at three weeks after balloon injury in rabbits fed with conventional (upper row) or 0.5% cholesterol diet (lower row). Autoradiographic images show increased ¹⁸F-FDG uptake in the artery with macrophage-rich neointima. Immunohistochemical staining shows hypoxic area (pimonidazole positive area) localized in macrophage-rich area in deep portion of wall. C. Uptake of ¹⁸F-FDG and immunopositive area for pimonidazole in arterial sections. White and black bars, conventional and 0.5% cholesterol diets, respectively. D. Correlations between ¹⁸F-FDG uptake and vascular area, SMC-, macrophage-, and pimonidazole-immunopositive areas in sections of arteries at three weeks after BI. E. Representative trace of macrophage-rich and/or pimonidazole immunopositive area in autoradiographic image. Areas that were rich in macrophages or pimonidazole were traced on immunohistochemical images, and then FDG uptake was measured in corresponding areas of autoradiographic images. F. Autoradiogram of arteries with macrophage-rich neointima shows ¹⁸F-FDG uptake in areas with or without macrophage-rich area or pimonidazole immunopositivity.</p