alongside antithrombin and c1-esterase inhibitor , activated protein c ( apc ) represents an important coagulatory inhibitor in humans .
the zymogen protein c is activated by the thrombin  thrombomodulin complex to give apc , whose anticoagulatory activity is conferred primarily by its ability to inactivate factor va and viiia .
disruption of the normal balance between coagulation and fibrinolysis has been characterized by its correlation with organ dysfunction and increased mortality .
thus , acquired protein c deficiency is associated with increased morbidity and mortality in patients with severe sepsis and septic shock .
direct anti - inflammatory properties of apc that have been characterized in vitro were considered independent from coagulatory inhibition [ 7 - 9 ] .
thus , an experimental apc application is capable of blocking the lethal effects of escherichia coli - induced septic disseminated intravascular coagulation , but also improves outcome in meningococcal - induced septic shock .
conversely , the application of antiprotein - c antibodies produces higher sensitivity towards defined endotoxin stimuli and increases mortality .
because organ dysfunction and animal survival are not improved by the administration of other substances with comparable anticoagulant effects , it has been suggested that the action of apc is not solely dependent upon its anticoagulatory properties .
although there is in vitro evidence from cell - culture studies to show that apc blocks leukocytic adhesion to the endothelium , the in vivo mechanisms of apc - related anti - inflammatory and microcirculatory action have not been completely elucidated thus far . before discussing the role of apc on the microcirculation in sepsis
, we will provide some information on the role of the microcirculation in the development of organ dysfunction and coagulatory failure .
leukocyte  endothelial - cell interaction is essential for an effective defense against bacterial invasion .
however , leukocytic activation is associated with an increased proinflammatory immune response , which is caused by an increased release of inflammatory cytokines , including tumor necrosis factor- , interleukin-1 and interleukin-8 , which initiate generalized neutrophil ( polymorphonucleocyte ) activation .
this release of mediators , together with the generalized activation and sequestration of neutrophils , may contribute to the widespread microvascular injury and subsequent endothelial damage observed in sepsis .
the migration of polymorphonucleocytes to inflamed tissue is primarily dependent on the induction of secondary adhesion molecules that are expressed on leukocytes and activated vascular endothelium .
this migration , along with the subsequent release of oxygen radicals and other toxic substances , represents a central step in the development of multiple organ failure . the endothelial switch from anticoagulant to procoagulant during sepsis
is thought to be related to the cytokine - mediated expression of endothelial adhesion molecules ( e.g. e - selectin , p - selectin , intercellular adhesion molecule-1 )   and increased tissue factor production by monocytes and endothelial cells .
additionally , endotoxins are , per se , known to directly generate an environment favoring coagulation processes by activating the extrinsic coagulation pathway via monocytic tissue factor expression .
thus , the concept of exogenous application of natural coagulatory inhibitors has been evaluated in terms of reducing systemic activation of leukocytes and endothelial cells .
although the anticoagulatory effects of apc have been extensively investigated during the last two decades , only a few studies have analyzed the actions of apc on the microcirculation during ischemia  reperfusion . to study ischemia 
reperfusion injury in a clinically relevant setting , models of warm ischemia have been extensively used in rodents . in a rat model
interestingly , the histological changes in renal tissue observed in control animals after noncrushing microvascular clamping for 60 min were almost completely absent after prophylactic apc administration in this model .
these apc effects on organ structure corresponded to a highly effective preservation of renal tissue blood flow that was significantly decreased after ischemia  reperfusion in controls .
since these postischemic alterations were only significantly inhibited by apc and not a specific factor xa inhibitor or heparin , the authors suggested that apc protects against ischemia  reperfusion - induced renal injury not by inhibition of coagulation abnormalities but rather by direct inhibition of leukocyte activation .
another group has also described a combination of anti - inflammatory and anticoagulatory actions of apc during renal ischemia reperfusion that provided microvascular protection . in this model ,
since leukocytes were , therefore , thought to be of central importance in the development of organ injury , the authors hypothesized that apc inhibits neutrophil adhesion , thereby attenuating the release of neutrophil elastase and oxygen radicals , both of which increase vascular permeability .
however , since neither group had the opportunity to evaluate the actions of apc on the microcirculation in vivo , their conclusions remain speculative .
the effects of apc on ischemia  reperfusion - induced organ damage have also been evaluated in hepatic tissue : yamaguchi and colleagues investigated the role of apc in a model of ischemia  reperfusion - induced injury in rat livers . before inducing liver ischemia by portal vein cross - clamping for 30 min ,
the serum concentrations of cytokine - induced neutrophil chemoattractants ( cinc ) were significantly reduced by apc application . simultaneously , an observed lowering of myeloperoxidase activity in hepatic tissue during apc application indicated a lesser accumulation of neutrophils in the liver . of interest ,
an inactive derivative of factor xa , which represents a selective inhibitor of thrombin generation , was also effective in decreasing cinc concentrations and could prevent leukocyte accumulation .
this indicated that , in this case , postischemic hepatic microcirculatory disturbances were induced by microthrombotic occlusion in a thrombin - dependent mechanism .
apc has been used in several sepsis models to modulate the sepsis - induced proinflammatory response .
thus , a prospective , blinded , placebo - controlled trial has been performed in a rabbit model of meningococcal endotoxin - induced shock .
strikingly , apc administration could not prevent an endotoxin - mediated decrease in mean arterial pressure and an increase in heart rate .
however , despite lacking any effect on macrohemodynamics , survival was significantly improved from 45% to 75% .
the authors hypothesized that apc had an effect on the microcirculation since the expected macrohemodynamic changes could not be characterized in their experiment . in a model of endotoxin - induced acute respiratory distress syndrome , apc attenuated endotoxin - induced pulmonary vascular injury , probably by the significant reduction of endotoxin - induced pulmonary leukocyte infiltration . in light of these findings ,
it is highly likely that apc also has a direct effect on the microcirculation during endotoxemia .
however , none of these models allowed the direct visualization of the microcirculation and , thus , the study of apc effects on microcirculatory dysfunction . to evaluate the effects of apc on the microcirculation in vivo during endotoxemia , our group tested apc in a sepsis model that permitted observation of the microcirculation under non - invasive , normotensive conditions .
endotoxin induced a massive increase in leukocyte accumulation in precapillary arterioles and postcapillary venules , with a maximal effect observed after 8 hours ( fig .
macrohemodynamic disturbances could be excluded by careful analysis of macrohemodynamic and microhemodynamic parameters in this model .
moreover , the next step in the leukocyte  endothelial - cell interaction ( namely , leukocyte adherence ) was also significantly inhibited in arterioles and venules ( fig .
in addition , endotoxin - mediated deterioration of microvascular perfusion was counteracted effectively by intravenous apc administration . since leukocyte rolling
is predominantly mediated by adhesion molecules of the selectin family , the reduction of leukocyte rolling by apc may indicate a modulation of selectin expression on leukocytes and the endothelium .
visualization of leukocyte sticking in the arteriole by intravital fluorescence microscopy ( performed using a charge - coupled device camera before and after lipopolysaccharide administration ) .
representative arterioles ( a ) before and ( b ) 8 hours after endotoxin application .
note that leukocytes also adhere to the endothelial lining of arterioles during endotoxemia despite high flow conditions .
effect of activated protein c ( apc ) on lipopolysaccharide ( lps)-induced ( a ) arteriolar and ( b ) venular leukocyte rolling .
the apc group contained animals ( n = 8) that received lps ( 2 mg / kg ) and apc ( 24 g / kg per 8 hours intravenously ) .
the control group contained vehicle - treated animals ( n = 7 ) that received lps ( 2 mg / kg ) only . * p < 0.05 , apc animals versus lipopolysaccharide - treated controls ( unpaired student 's t - test ) .
p < 0.05 , intergroup differences from baseline ( paired student 's t - test ) .
effect of activated protein c ( apc ) on lipopolysaccharide ( lps)-induced ( a ) arteriolar and ( b ) venular leukocyte adhesion .
the apc group contained animals ( n = 8) that received lps ( 2 mg / kg ) and apc ( 24 g / kg per 8 hours intravenously ) . the control group contained vehicle - treated animals ( n = 7 ) that received lps ( 2 mg / kg ) only . * p < 0.05 , apc animals versus lipopolysaccharide - treated controls ( unpaired student 's t - test ) .
p < 0.05 , intergroup differences from baseline ( paired student 's t - test ) .
this view corresponds to in vitro data obtained by vascular endothelial cell culturing , where human plasma - derived and human cell - produced protein c blocked e - selectin - mediated cell adhesion to the endothelium . from our in vivo study , we concluded that it is highly likely that a specific anti - inflammatory interaction between apc and the endothelium is responsible for microcirculatory protection .
since thrombin inhibition alone obviously does not reduce endotoxin - mediated leukocyte rolling and adhesion , anticoagulatory apc activities ( namely , inhibition of factors va and viiia that would result in less thrombin formation ) are probably unlikely to be responsible for the protective effects of apc on the microcirculation during endotoxemia .
table 1 shows an overview of the differential effects of the anticoagulants apc , antithrombin , and hirudin ( a specific thrombin inhibitor ) on different steps in the leukocyte  endothelial - cell interaction and capillary perfusion in septic disorders .
differential regulation of endotoxic microcirculatory disorders by coagulatory inhibitors note that hirudin is not antiadhesive and does not protect against endotoxic capillary dysfunction .
the protein c and antithrombin anticoagulant pathways represent major mechanisms in controlling microvascular dysfunction during ischemia  reperfusion injury and sepsis . local and systemic antithrombin and apc
apc and antithrombin have been used in experimental models to reduce ischemia  reperfusion injury and to improve septic disorders with comparable efficacy .
the exact molecular mechanisms of action of apc and antithrombin during ischemia  reperfusion and endotoxemia remain unclear . however , it is highly likely that a specific anti - inflammatory interaction with the endothelium and leukocytes is responsible for apc - related microcirculatory protection .
thus , apc treatment using a dose corresponding to that used in the recombinant human activated protein c worldwide evaluation in severe sepsis ( prowess ) study in vivo reduced the extent of transient leukocyte  endothelial - cell interactions ( leukocyte rolling ) and inhibited subsequent firm leukocyte adherence ( leukocyte sticking ) to the endothelium . both mechanisms lessened endotoxin - mediated microvascular perfusion injury .
these microcirculatory apc actions may also be used under clinical situations involving microcirculatory disorders . from the majority of experimental studies presented here
, there is , however , no evidence to support antithrombotic ( anticoagulatory ) therapy specifically targeting thrombin inhibition , since thrombin inhibitors ( e.g. hirudin and heparin ) do not provide comparable protection of the microcirculation , but , on the contrary , may even aggravate sepsis - mediated disorders .
apc = activated protein c ; cinc = cytokine - induced neutrophil chemoattractants ; prowess = recombinant human activated protein c worldwide evaluation in severe sepsis .
 experimental studies in ischemia  reperfusion and sepsis indicate that activated protein c ( apc ) has direct anti - inflammatory effects at a cellular level . in vivo , however , the mechanisms of action have not been characterized thus far . 
 intravital multifluorescence microscopy represents an elegant way of studying the effect of apc on endotoxin - induced leukocyte  endothelial - cell interaction and nutritive capillary perfusion failure . 
 these studies have clarified that apc effectively reduces leukocyte rolling and leukocyte firm adhesion in systemic endotoxemia . 
 protection from leukocytic inflammation is probably mediated by a modulation of adhesion molecule expression on the surface of leukocytes and endothelial cells . of interest , the action of apc and antithrombin in endotoxin - induced leukocyte  endothelial - cell interaction differs in that apc inhibits both rolling and subsequent firm adhesion , whereas antithrombin exclusively reduces the firm adhesion step . 
 the biological significance of this differential regulation of inflammation remains unclear , since both proteins are capable of reducing sepsis - induced capillary perfusion failure . to elucidate whether the action of apc and antithrombin is mediated by inhibition of thrombin 
 , the specific thrombin inhibitor hirudin has been examined in a sepsis microcirculation model . strikingly , hirudin was not capable of protecting from sepsis - induced microcirculatory dysfunction , but induced a further increase of leukocyte  endothelial - cell interactions and aggravated capillary perfusion failure when compared with nontreated controls . 
 thus , the action of apc on the microcirculatory level in systemic endotoxemia is unlikely to be caused by a thrombin inhibition - associated anticoagulatory action . 