Arterial thrombosis in the context of HCV-associated vascular disease can be prevented by protein C

Abstract

Hepatitis C virus (HCV) infection is a major problem worldwide. HCV is not limited to liver disease but is frequently complicated by immune-mediated extrahepatic manifestations such as glomerulonephritis or vasculitis. A fatal complication of HCV-associated vascular disease is thrombosis. Polyriboinosinic:polyribocytidylic acid (poly (I:C)), a synthetic analog of viral RNA, induces a Toll-like receptor 3 (TLR3)-dependent arteriolar thrombosis without significant thrombus formation in venules in vivo. These procoagulant effects are caused by increased endothelial synthesis of tissue factor and PAI-1 without platelet activation. In addition to human umbilical endothelial cells (HUVEC), human mesangial cells (HMC) produce procoagulatory factors, cytokines and adhesion molecules after stimulation with poly (I:C) or HCV-containing cryoprecipitates from a patient with a HCV infection as well. Activated protein C (APC) is able to prevent the induction of procoagulatory factors in HUVEC and HMC in vitro and blocks the effects of poly (I:C) and HCV-RNA on the expression of cytokines and adhesion molecules in HMC but not in HUVEC. In vivo, protein C inhibits poly (I:C)-induced arteriolar thrombosis. Thus, endothelial cells are de facto able to actively participate in immune-mediated vascular thrombosis caused by viral infections. Finally, we provide evidence for the ability of protein C to inhibit TLR3-mediated arteriolar thrombosis caused by HCV infection.

Figure 1

Poly (I:C) accelerated thrombus formation and the occlusion time in arterioles in vivo. C57/Bl6wt mice were treated with 200 μg poly (I:C) i.p. About 24 h before the light dye injury model of the cremaster muscle was performed as described in the Materials and methods section. The onset of thrombus formation and the occlusion time was measured in minutes in the arterioles (a and b) and venules (c and d) in the poly (I:C)-treated and control (ct) groups (n=11–12, **P<0,01. Mean±s.e., statistics with t-test (sigma plot)). (e and f) The onset of thrombus formation and the occlusion time was measured in the arterioles of TLR3^−/− mice (n=4, **P<0.01. Mean±s.e., statistics with t-test (sigma plot)). The extrinsic (EXTEM) or intrinsic (INTEM) coagulation cascades were activated under control or poly (I:C)-stimulated conditions as described in the Materials and methods section and the clotting time (g) and clot-formation time (h) were analyzed. (n=4, statistics with t-test (sigma plot)). Comparable results were obtained in two series of independent experiments. Poly (I:C), polyriboinosinic:polyribocytidylic acid.

Figure 2

Effect of poly (I:C) on the endothelial expression of procoagulatory factors and clotting time. HMEC were stimulated with poly (I:C) (10 μg/ml) for 12 h and the expression of tissue factor (a) and PAI-1 (b) was analyzed by RT–PCR (n=4, *P<0.05. mean±s.e., statistics with t-test (sigma plot); rel. to ct, relative to control). Comparable results were obtained in two series of independent experiments. (c) HMEC were stimulated with poly (I:C) (10 μg/ml) or TNFα (5 ng/ml) as a positive control for 24 h and then lysed. Whole blood samples were stimulated with cell lysates and the clotting time was analyzed as described in the Materials and methods section (n=5–6, *P<0.05. mean±s.e., statistics with t-test (sigma plot); rel. to ct, relative to control). Comparable results were obtained in two series of independent experiments. HMEC, human microvascular endothelial cell; poly (I:C), polyriboinosinic:polyribocytidylic acid; RT–PCR, reverse transcription–PCR. **P<0.01.

Figure 3

Poly (I:C) did not influence platelet aggregation and activation. Light transmission aggregometry (the method by Born) was performed in platelet-rich-plasma (PRP) from healthy human volunteers, as described in the Materials and methods section. The percent light transmission of platelet-rich plasma (PRP) was compared with platelet poor plasma (PPP) on stimulation with poly (I:C) (10 μg/ml) or ADP (10 μM) (a and b). PRP was incubated with poly (I:C) (10 μg/ml) for different time intervals (10, 20, 30, 45 min) and ADP-dependent (5 μM) platelet aggregation was analyzed (c). PRP was incubated with poly (I:C) (10 μg/ml) for 15 min in the presence of ADP at a low concentration (5 μM), ADP at a high concentration (10 μM), thrombin-receptor-activated peptide (TRAP) (20 μM) or collagen (10 μg/ml) and platelet aggregation was analyzed (n=4, P>0.05, mean±s.e., statistics with t-test (sigma plot)) Comparable results were obtained in two series of independent experiments. (d) Human platelets were isolated as described in the Materials and methods section. The platelets were stimulated with poly (I:C) (10 μg/ml) for different time intervals (10, 60 min) alone or in the presence of thrombin (2 U/ml) and FACS analysis with a monoclonal antibody against P-selectin (e and f) and GPIIbIIIa (g and h) was performed (n=3–4, P>0.05, mean±s.e., statistics with one-way ANOVA (sigma plot)). Comparable results were obtained in two series of independent experiments. ANOVA, analysis of variance; HMEC, human microvascular endothelial cell; poly (I:C), polyriboinosinic:polyribocytidylic acid.

Figure 4

Activated Protein C reduces the expression of procoagulatory factors in human endothelial and mesangial cells. Human umbilical vein endothelial cells (HUVEC) were stimulated without and with poly (I:C) (10 μg/ml) or HCV-RNA-containing cryoprecipitates (HCV) as described for 24 h in the presence or absence of activated Protein C (APC) (100 nM) and the expression of tissue factor (a), PAI-1 (b and c), IL-6 (g and h) and ICAM-1 (j) was analyzed by RT–PCR. Human mesangial cells (HMC) were stimulated without and with poly (I:C) (10 μg/ml) or HCV-RNA-containing cryoprecipitates (HCV) for 24 h in the presence or absence of activated protein C (APC) (100 nM) and the expression of tissue factor (d and f), PAI-1 (e), IL-6 (i) and ICAM-1 (k) was analyzed by RT–PCR (n=4, *P<0.05. mean±s.e., statistics with t-test (sigma plot); rel. to ct, relative to control). Comparable results were obtained in two series of independent experiments. RT–PCR, reverse transcription–PCR. **P<0.01.

Figure 5

Protein C is able to prevent the poly (I:C)-induced effects on thrombus formation and vessel occlusion in arterioles in vivo. C57/Bl6wt mice were treated with 200 μg poly (I:C) i.p. for 24 h before the light dye injury of the cremaster muscle was performed as described in the Materials and methods section. Protein C (100 U/kg) was given i.a. 30 min before the start of the injury. The onset of thrombus formation (a) and the occlusion time (b) was measured in minutes in the arterioles (n=5, *P<0.05, mean±s.e., statistics with one-way ANOVA (sigma plot)). (c) Representative images of thrombus formation in cremaster muscle arterioles on light dye injury. White arrows indicate the sites where thrombus material hinders blood flow and leads to vessel occlusion. ANOVA, analysis of variance; poly (I:C), polyriboinosinic:polyribocytidylic acid.