Thrombus-induced endothelial dysfunction: Hemoglobin and fibrin decrease nitric oxide bioactivity without altering eNOS.

Abstract

BACKGROUND: Arterial thrombosis is associated with endothelial dysfunction. The purpose of this study was to determine which components of thrombus induce endothelial dysfunction and to determine their effect on endothelial nitric oxide synthase (eNOS) activity and expression. MATERIALS AND METHODS: Human aortic endothelial cells were grown to confluence. Group 1 consisted of control cells exposed to media. Group 2 was exposed to cellular components of thrombus, including erythrocytes (RBCs) (1.5, 3, and 6 x 10(4) cells/ml) and platelets (0.5, 1, and 2 x 10(5) platelets/ml). Group 3 was exposed to extracellular thrombus components, including hemoglobin (1.25, 2.5, and 5.0 g/dl), thrombin (0.4, 4.0, and 10.0 units/ml), and fibrin. The exposure time was 20 h. Nitric oxide (NO) levels were measured following exposure. Global cellular integrity was evaluated by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) conversion. eNOS mRNA expression was measured using semiquantitative PCR and eNOS protein activity evaluated via a l-citrulline conversion assay. Studies were done in replicates of six and expressed as percentages of controls. RESULTS: NO levels decreased following exposure to hemoglobin (1.25 g/dl = 59 +/- 5%, 2.5 g/dl = 38 +/- 3%, 5 g/dl = 37 +/- 6%, P < 0.001). MTS metabolism was likewise suppressed (1.25 g/dl = 57 +/- 2%, 2.5 g/dl = 55 +/- 3%, 5 g/dl = 44 +/- 6%, P < 0.001). Fibrin diminished NO levels (37 +/- 5%, P < 0.01) and MTS metabolism (49 +/- 5%, P < 0.01). RBCs and platelets had no effect on NO production or MTS metabolism (P > 0.05). Thrombin's effect was concentration dependent, inhibiting nitric oxide production at low doses while stimulating it at high doses (P < 0.01). All thrombin doses enhanced MTS metabolism (P < 0.05). eNOS activity was not altered by fibrin, thrombin, or hemoglobin exposure (P > 0.05). Moreover, eNOS mRNA expression was unaffected (P > 0.05). CONCLUSIONS: Intact cellular components of thrombus do not cause endothelial dysfunction in vitro. However, free hemoglobin and fibrin diminish NO bioactivity, likely via scavenging. Thrombin affects NO levels in a concentration-dependent manner. Neither eNOS expression nor eNOS activity is diminished, indicating that thrombus does not cause permanent changes in NO generation capability.