Previous reports found clotting parameters (PT, aPTT, fibrinogen, and platelets) in MBL null mice are similar to WT mice, indicating that coagulation itself is not compromised in MBL deficiency(22). == Ferric chloride (FeCl3) coagulation model == We used a mouse model ML204 of localized thrombus formation as previously described(22,23,28). a key role in thrombus formationin vitroandin vivo. Keywords:complement, coagulation, thrombosis, MBL, MASP-1, rodent == INTRODUCTION == Occlusive thrombosis, resulting from atherosclerotic plaque rupture or restenosis plays an important role in the onset of two major causes of death in developed nations: myocardial infarction and ischemic stroke. Typically, acute thrombolytic therapy is the first step taken to reperfuse ischemic tissues downstream of the thrombus. In addition, most patients suffering from cardiovascular disease are prescribed chronic prophylactic thrombolytic drugs. Coagulation is usually a complex process and there is a delicate balance between bleeding disorders and clot formation. Considering the widespread and critical role of thrombosis in many diseases, significant research efforts have been aimed at the discovery and development of safe and effective antithrombotic (i.e., anti-platelet, anti-thrombin, etc.) drugs for acute and chronic clinical use. The complement cascade is usually part of the innate immune system responsible for the initiation of inflammation and elimination of invading foreign cells. There are three impartial pathways which can initiate the complement cascade: the classical, option, and lectin pathway. All three pathways converge at formation of the C3 convertase and follow a common pathway resulting in the formation of C5b-9 [the membrane attack complex (MAC)]. Lectin pathway activation is initiated via the binding of a multi-molecule complex to carbohydrate structures present on pathogens, or glycosylation patterns on apoptotic, necrotic, or ischemic cells(13). The MBL complex consists of MBL and three serine proteases, MASP-1, -2, and -3 (MBL-associated serine proteases 1, 2, and 3), and MAp19 and Map44/MAP-1, two nonenzymatic, truncated products of theMASP2gene, which may regulate lectin pathway activation by competing for the binding of MASPs to the carbohydrate recognition complexes(210). The system is usually slightly altered in mice, where two forms of MBL exist, MBL-A and MBL-C(2,7). Coagulation, like complement, is usually a highly conserved cascade-style system composed of multiple factors (F) that are activated in a sequential and amplified process, ultimately resulting in the formation of an insoluble fibrin clot. The coagulation cascade is usually primarily responsible for maintaining hemostasis following vascular injury. Activation of either the intrinsic or extrinsic pathways which make up the coagulation cascade ML204 leads to the formation of a prothrombinase complex composed of FVa and FXa, cleaving prothrombin to thrombin. Thrombin formation is usually a critical central step in coagulation, cleaving fibrinogen, FXIII ISGF-3 and activating platelets. Thrombin also plays ML204 important functions in the activation of protein C, an anti-coagulative protein with cellular protective actions (11,12). For many years, it has been recognized that this complement and coagulation systems interact(13). Complement activation is ML204 known to contribute to thrombotic tissue injury in systemic lupus erythematosus(14), biomaterial-associated thrombosis(15), and paroxysmal nocturnal hemoglobinuria(16), to name just a few. Additionally, reversal of heparinization with protamine, TPA and streptokinase activate complement(1719). An important study by Huber-Lang et al exhibited that thrombin can directly activate C5 to produce C5a and C5b-9 in C3 deficient mice(20). Further, mannose-binding lectin associated protease (MASP)-2 can activate prothrombin to thrombin and may explain the mechanism by which thrombin is usually produced from prothrombin in C3 deficient mice(21). More recently, work from our group indicated that MBL-MASP complexes are associated with thrombin-like activityin vitroand found that MBL null mice have prolonged bleeding timesin vivo(22). Endothelial injury and platelet aggregation are key events in the pathogenesis of thrombus formation. Our experimental approach was based on the hypothesis that occlusive thrombosis can be initiated by FeCl3-induced endothelial injury, likely mediated by oxidative radical formation and/or transported intraluminal ferric ion conversation with platelets, fibrin and other formed blood elements(23). Forin vivoanalysis, the FeCl3method is the optimal technique as it ML204 exhibits many features useful for studying the molecular determinants of arterial thrombosis in transgenic mice, including uniform injury from animal to.