Coagulation and inflammation in experimental endotoxemia in vitro and in vivo : Monitoring method and effects of nicotinamide

Sammanfattning: In gram negative sepsis, endotoxin from the bacterial membrane elicits proinflammatory and procoagulant host responses. Sepsis and its frequent complication disseminated intravascular coagulation (DIC) are leading causes of morbidity and mortality in the intensive care. A pivotal mechanism in the pathogenesis of DIC is the expression of tissue factor (TF) on circulating monocytes after endotoxin encounter. Activation of the cascade systems of coagulation and inflammation can lead to a rapid deterioration in e.g. sepsis with procoagulant changes and microthromboses, resulting in DIC and multi organ failure. Monitoring of the degree of coagulation activation, to discover changes before clinical exacerbation, is critical. We have established a global monitoring method for coagulation activity, clotting onset time (COT), based on free oscillating rheometry. The assay design mimicks the natural blood milieu, as the method is based on CaC12 repletion of blood or plasma, adding no artificial activators or inhibitors. The COT method proved to be a quick and reliable test, able to detect hypoand hypercoagulation. It also showed promising results as a bedside monitoring method, able to detect the transient activation of coagulation caused by intravenous injection of endotoxin to healthy volunteers. We also demonstrate that the COT method is sensitive even to small changes in the amount of endotoxin induced monocyte surface TF. In neurotrauma patients, COT was a predictor of prognosis for the patients. This observation, however, is difficult to interpret and requires further investigation. The vitamin B derivative nicotinamide was assessed for its potential modulating effects on endotoxin induced activation of coagulation and inflammation. Nicotinamide is a known PARP inhibitor. PARP is necessary for activation of the transcription factor NFkappabeta, responsible for transcription of many genes involved in the response to endotoxin, e.g. proinflammatory cytokines, giving a rationale for a potential beneficial effect of nicotinamide in endotoxemia. We demonstrate that nicotinamide is a potent inhibitor of three major endotoxin induced proinflammatory cytokines, Il-1 beta, IL-6 and IL-8, in addition to the previously known TNFalpha inhibiting effect. However, the dose dependent inhibition of endotoxin induced proinflammatory responses was shown unlikely to be due to PARP inhibition. In endotoxin stimulated leukocyte suspensions as well as in whole blood, nicotinamide caused a dose dependent decrease of monocyte TF expression, describing a previously unknown inhibitory effect of nicotinamide on the procoagulant changes associated with endotoxemia. We have also demonstrated that the decrease of monocyte TF expression is at least partly caused by shedding from the monocyte surface. Our conclusion is that nicotinamide may have a therapeutic potential in modulating conditions associated with activation of coagulation and inflammation, such as in sepsis and DIC.