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CD40 ligand enhances monocyte tissue factor expression and thrombin generation via oxidative stress in patients with hypercholesterolemia

Valerio Sanguigni, MD^*, Domenico Ferro, MD, Pasquale Pignatelli, MD, Maria Del Ben, MD, Tini Nadia, MD, Mirella Saliola, PhD, Roberto Sorge, PhD^* and Francesco Violi, MD^,*

^*Department of Internal Medicine, University of Rome "Tor Vergata," Rome, Italy
Department of Experimental Medicine and Pathology, University of Rome "La Sapienza," Rome, Italy

View larger version (10K): [in a new window]   Figure 1 Plasma levels of soluble CD40 ligand (sCD40L) in patients with hypercholesterolemia (HC) and healthy subjects (HS). *p < 0.0005 for HC versus HS.

View larger version (10K): [in a new window]   Figure 2 Plasma levels of prothrombin fragment (F1+2) in patients with hypercholesterolemia (HC) and healthy subjects (HS). *p < 0.0005 for HC versus HS.

View larger version (10K): [in a new window]   Figure 3 Plasma levels of 8-hydroxy-2-deoxyguanosine (8-OHdG) in patients with hypercholesterolemia (HC) and healthy subjects (HS). *p < 0.005 for HC versus HS.

View larger version (13K): [in a new window]   Figure 4 (A) Correlation between low-density lipoprotein cholesterol (LDL-C) and soluble CD40 ligand (sCD40L). (B) Correlation between LDL-C and prothrombin fragment (F1+2). (C) Correlation between LDL-C and 8-hydroxy-2-deoxyguanosine (8-OHdG).

View larger version (12K): [in a new window]   Figure 5 (A) Correlation between soluble CD40 ligand (sCD40L) and prothrombin fragment (F1+2). (B) Correlation between 8-hydroxy-2-deoxyguanosine (8-OHdG) and sCD40L. (C) Correlation between prothrombin F1+2 and 8-OHdG.

View larger version (26K): [in a new window]   Figure 6 Expression of tissue factor (TF) (A) and generation of prothrombin fragment (F1+2) (B) in monocytes taken from healthy subjects (2 men and 2 women, mean age 50 years) and stimulated with scalar doses of CD40 ligand (CD40L) (5 to 50 ng/ml). UM = unstimulated monocytes; SM = stimulated monocytes. *p < 0.001 for CD40L (5 ng/ml) SM vs. UM. **p < 0.0001 for CD40L (25 ng/ml) SM versus UM and CD40L (50 ng/ml) SM versus UM.

View larger version (13K): [in a new window]   Figure 7 Generation of prothrombin fragment (F1+2) (A) and expression of tissue factor (TF) (B) by unstimulated monocytes (a) and CD40 ligand (CD40L)-stimulated monocytes taken from healthy subjects (b; n = 4) or patients with hypercholesterolemia (c; n = 4). *p < 0.0005 for b versus a. **p < 0.003 for c versus b.

View larger version (16K): [in a new window]   Figure 8 Expression of tissue factor (TF) (A) and generation of prothrombin fragment (F1+2) (B) by monocytes taken from healthy subjects (n = 4) and stimulated with CD40 ligand (CD40L) (50 ng/ml) or CD40L plus diphenyliodonium (DPI) (15 µmol/l), eicosatetraynoic acid (ETYA) (35 µmol/l), and wortmannin (0.1 µmol/l). *p < 0.0005 for CD40L (50 ng/ml) + DPI (15 µM) versus CD40L (50 ng/ml). **p < 0.0001 for CD40L (50 ng/ml) + wortmannin (0.1 µM) versus CD40L (50 ng/ml).

View larger version (9K): [in a new window]   Figure 9 Expression of tissue factor (TF) (A) and generation of prothrombin fragment (F1+2) (B) by 50 ng/ml CD40 ligand-stimulated monocytes taken from hereditary gp91 deficient-patients (X-GCD; four different determinations) or from four healthy subjects (HS) matched for age. *p < 0.001 for X-GCD versus HS.

View larger version (13K): [in a new window]   Figure 10 Generation of prothrombin fragment (F1+2) by monocytes stimulated with CD40 ligand (CD40L) (50 ng/ml) and CD40L-stimulated monocytes added with 50 or 100 µmol/l of vitamin C in healthy subjects (open bars; n = 4) (A) and patients with hypercholesterolemia (solid bars; n = 4) (B). *p < 0.003 for CD40L (50 ng/ml)-stimulated monocytes added with 50 µmol/l of vitamin C versus CD40L (50 ng/ml)-stimulated monocytes. **p < 0.002 for CD40L (50 ng/ml)-stimulated monocytes added with 100 µmol/l of vitamin C versus CD40L (50 ng/ml)-stimulated monocytes.

View larger version (14K): [in a new window]   Figure 11 Expression of tissue factor (TF) by monocytes stimulated with CD40 ligand (CD40L) (50 ng/ml) and CD40L-stimulated monocytes added with 50 or 100 µmol/l of vitamin C in healthy subjects (open bars; A) and patients with hypercholesterolemia (solid bars; B). *p < 0.003 for CD40L (50 ng/ml)-stimulated monocytes added with 50 µmol/l of vitamin C versus CD40L (50 ng/ml)-stimulated monocytes. **p < 0.002 for CD40L (50 ng/ml)-stimulated monocytes added with 100 µmol/l of vitamin C versus CD40L (50 ng/ml)-stimulated monocytes.