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Background In the early postoperative period after pancreas-kidney transplantation, pancreatic venous thrombosis is a major complication that leads to allograft dysfunction and graft loss. Beside ischemia and reperfusion injury, immunosuppressive drugs have been accused of supporting thrombogenicity. The aim of this study was to evaluate the effect of commonly applied immunosuppressants on microvascular thrombus formation in normal and postischemic tissue in vivo. Methods In the skin fold chambers of tacrolimus-, cyclosporine A-, antithymocyte globulin-, rapamycine-, or saline-treated mice, light/dye-induced microvascular thrombus formation was studied. Additional mice underwent ischemia and reperfusion of the skin fold chamber tissue and received tacrolimus, antithymocyte globulin, or saline before reperfusion. Additionally, the effect of prednisolone was tested in animals with ischemia and reperfusion. Concentrations of sP-selectin, soluble vascular cell adhesion molecule-1, and asymmetric dimethylarginine were assessed by enzyme-linked immunosorbent assay. Immunohistochemistry of the skin fold chamber tissue served for analysis of vascular endothelial nitric oxide synthase and inducible nitric oxide synthase expression. Results In normal tissue, tacrolimus, cyclosporine A, antithymocyte globulin, and rapamycine accelerated microvascular thrombus formation significantly when compared with saline. Whereas ischemia and reperfusion in saline-treated mice enhanced thrombus formation, thrombogenicity was not further increased by ischemia and reperfusion in tacrolimus - or antithymocyte globulin-treated animals. Application of prednisolone reversed the tacrolimus- and antithymocyte globulin-induced prothrombotic effect. Antithymocyte globulin increased sP-selectin and soluble vascular cell adhesion molecule-1, whereas tacrolimus induced asymmetric dimethylarginine production significantly. While tacrolimus and antithymocyte globulin additionally induced endothelial nitric oxide synthase and inducible nitric oxide synthase expression, cyclosporine A influenced only endothelial inducible nitric oxide synthase expression. Conclusion Immunosuppressants enhance thrombus formation in vivo. Although antithymocyte globulin activates the microvascular endothelium, we show for the first time that tacrolimus increases asymmetric dimethylarginine plasma levels. Thus, impaired nitric oxide availability might be the underlying mechanism for the tacrolimus-associated increased thrombogenicity. The efficacy of prednisolone to reverse the tacrolimus-associated and antithymocyte globulin-associated acceleration of thrombus formation underlines the application of this anti-inflammatory drug prior to reperfusion in immunosuppressive regimens.

Thrombotic microangiopathy (TMA) is a fatal complication that occurs after combined pancreaskidney transplantation leading to allograft dysfunction and graft loss. Immunosuppressive drugs, such as calcineurin inhibitors, have been implicated in the development of nonimmune TMA. Tacrolimus has been shown to be associated with decreased NO-production, thus contributing to thrombus formation. ADMA acts as an endogenous NO-synthase inhibitor and is considered to be a marker of endothelial dysfunction. Aim of this study was to analyse the influence of different immunosuppressants on microvascular thrombus formation in vivo and to further examine the underlying endothelial function. Using the skin fold chamber in C57BL/6J mice, microvascular thrombus formation was induced photochemically and was quantitatively analyzed by intravital fluorescence microscopy. Mice were treated with tacrolimus (TAC: 10 mg/kg/d; n = 7), cyclosporine (CYA: 5 mg/kg/d) or sirolimus (RAPA: 1,5 mg/kg/d ip) on 3 consecutive days. Control mice received NaCl 0,9 % (10 ml/kg/d ip; n = 5). Drug plasma levels were examined to ensure therapeutic doses. Additionally, the systemic circulation of sPand sE-selectin as well as ADMA plasma levels were measured by ELISA. Application of immunosuppressants produced clinically relevant plasma levels (TAC: 8 ± 2 ng/ml, CYA: 214 ± 16 ng/ml; RAPA: 9 ± 1 ng/ml). Microvascular thrombus formation was significantly accelerated in mice receiving TAC compared to control mice (arteriolar and venular occlusion: 251 ± 101s and 179 ± 27 s vs. control 818 ± 221 s and 749 ± 231 s; p < 0,05). Application of CYA significantly increased thrombus formation only in venules (276 ± 49s; p < 0,05 vs. control), whereas RAPA had no significant effect on thrombosis induction. Plasma concentrations of sP-Selectin and sE-selectin were slightly reduced after TAC and CYA application. ADMA-levels however were significantly increased in mice receiving TAC (1,28 ± 0,16 μmol/l vs. control: 0,74 ± 0,13 μmol/l; p < 0,05). The present results show that clinically relevant plasma levels of cyclosporine enhanced thrombus formation only in venules whereas tacrolimus significantly accelerates thrombus formation in arterioles and venules. This effect could be explained by increased ADMA-levels in plasma resulting in impaired NO-bioavailability and increased thrombogenicity, thus presenting a new molecular mechanism in the development of transplant-thrombosis.