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Sepsis-induced acute kidney injury (AKI) contributes to the high mortality and morbidity in patients. Although the pathogenesis of AKI during sepsis is poorly understood, it is well accepted that plasminogen activator inhibitor-1 (PAI-1) and vitronectin (Vn) are involved in AKI. However, the functional cooperation between PAI-1 and Vn in septic AKI has not been completely elucidated. To address this issue, mice were utilized lacking either PAI-1 (PAI-1-/-) or expressing a PAI-1-mutant (PAI-1R101A/Q123K) in which the interaction between PAI-1 and Vn is abrogated, while other functions of PAI-1 are retained. It was found that both PAI-1-/- and PAI-1R101A/Q123K mice are associated with decreased renal dysfunction, apoptosis, inflammation, and ERK activation as compared to wild-type (WT) mice after LPS challenge. Also, PAI-1-/- mice showed attenuated fibrin deposition in the kidneys. Furthermore, a lack of PAI-1 or PAI-1-Vn interaction was found to be associated with an increase in activated Protein C (aPC) in plasma. These results demonstrate that PAI-1, through its interaction with Vn, exerts multiple deleterious mechanisms to induce AKI. Therefore, targeting of the PAI-1-Vn interaction in kidney represents an appealing therapeutic strategy for the treatment of septic AKI by not only altering the fibrinolytic capacity but also regulating PC activity.

Plasminogen activator inhibitor-1 (PAI-1) is known to protect mice against cardiac fibrosis. It has been speculated that PAI-1 may regulate cardiac fibrosis by inactivating urokinase-type plasminogen activator (uPA) and ultimately plasmin (Pm) generation. However, the in vivo role of PAI-1 in inactivating uPA and limiting the generation of Pm during cardiac fibrosis remains to be established. The objective of this study was to determine if the cardioprotective effect of PAI-1 is mediated through its ability to directly regulate urokinase -mediated activation of plasminogen (Pg). An Angiotensin II (AngII)-aldosterone (Ald) infusion mouse model of hypertension was utilised in this study. Four weeks after AngII-Ald infusion, PAI-1-deficient (PAI-1 −/− ) mice developed severe cardiac fibrosis. However, a marked reduction in cardiac fibrosis was observed in PAI-1 −/− /uPA −/− double knockout mice that was associated with reduced inflammation, lower expression levels of TGF-β and proteases associated with tissue remodeling, and diminished Smad2 signaling. Moreover, total ablation of cardiac fibrosis was observed in PAI-1 −/− mice that express inactive plasmin (Pm) but normal levels of zymogen Pg (PAI-1 −/− /Pg S743A/S743A ). Our findings indicate that PAI-1 protects mice from hypertension-induced cardiac fibrosis by inhibiting the generation of active Pm.

Acute coagulopathy is a serious complication of traumatic brain injury (TBI) and is of uncertain etiology because of the complex nature of TBI. However, recent work has shown a correlation between mortality and abnormal hemostasis resulting from early platelet dysfunction. The aim of the current study was to develop and characterize a rodent model of TBI that mimics the human coagulopathic condition so that mechanisms of the early acute coagulopathy in TBI can be more readily assessed. Studies utilizing a highly reproducible constrained blunt-force brain injury in rats demonstrate a strong correlation with important postinjury pathological changes that are observed in human TBI patients, namely, diminished platelet responses to agonists, especially adenosine diphosphate (ADP), and subarachnoid bleeding. Additionally, administration of a direct thrombin inhibitor, preinjury, recovers platelet functionality to ADP stimulation, indicating a direct role for excess thrombin production in TBI-induced early platelet dysfunction.

The fibrinolytic system has been implicated in the genesis and progression of atherosclerosis. It has been reported that a plasminogen (Pg) deficiency (Plg−/−) exacerbates the progression of atherosclerosis in Apoe−/− mice. However, the manner in which Plg functions in a low-density lipoprotein-cholesterol (LDL-C)-driven model has not been evaluated. To characterize the effect of Pg in an LDL-C-driven model, mice with a triple deficiency of the LDL-receptor (LDLr), along with the active component (apobec1) of the apolipoprotein B editosome complex, and Pg (L−/−/A−/−/Plg−/−), were generated. Atherosclerotic plaque formation was severely retarded in the absence of Pg. In vitro studies demonstrated that LDL uptake by macrophages was enhanced by plasmin (Pm), whereas circulating levels of LDL were enhanced, relative to L−/−/A−/− mice, and VLDL synthesis was suppressed. These results indicated that clearance of lipoproteins in the absence of LDLr may be regulated by Pg/Pm. Conclusions: The results from this study indicate that Pg exacerbates atherosclerosis in an LDL-C model of atherosclerosis and also plays a role in lipoprotein modification and clearance. Therefore, controlling the Pg system on macrophages to prevent foam cell formation would be a novel therapeutic approach.

To delineate the critical features of platelets required for formation and stability of thrombi, thromboelastography and platelet aggregation measurements were employed on whole blood of normal patients and of those with Bernard-Soulier Syndrome (BSS) and Glanzmann's Thrombasthenia (GT). We found that separation of platelet activation, as assessed by platelet aggregation, from that needed to form viscoelastic stable whole blood thrombi, occurred. In normal human blood, ristocetin and collagen aggregated platelets, but did not induce strong viscoelastic thrombi. However, ADP, arachidonic acid, thrombin, and protease-activated-receptor-1 and -4 agonists, stimulated both processes. During this study, we identified the genetic basis of a very rare double heterozygous GP1b deficiency in a BSS patient, along with a new homozygous GP1b inactivating mutation in another BSS patient. In BSS whole blood, ADP responsiveness, as measured by thrombus strength, was diminished, while ADP-induced platelet aggregation was normal. Further, the platelets of 3 additional GT patients showed very weak whole blood platelet aggregation toward the above agonists and provided whole blood thrombi of very low viscoelastic strength. These results indicate that measurements of platelet counts and platelet aggregability do not necessarily correlate with generation of stable thrombi, a potentially significant feature in patient clinical outcomes.

In humans, maternal fibrinogen (Fg) is required to support pregnancies by maintaining hemostatic balance and stabilizing uteroplacental attachment at the fibrinoid layer found at the fetal-maternal junction. To examine relationships between low Fg levels and early fetal loss, a genetic model of afibrinogenemia was developed. Pregnant mice homozygous for a deletion of the Fg-γ chain, which results in a total Fg deficiency state ( FG −/− ), aborted the fetuses at the equivalent gestational stage seen in humans. Results obtained from timed matings of FG −/− mice showed that vaginal bleeding was initiated as early as embryonic day (E)6 to 7, a critical stage for maternal-fetal vascular development. The condition of afibrinogenemia retarded embryo-placental development, and consistently led to abortion and maternal death at E9.75. Lack of Fg did not alter the extent or distribution pattern of other putative factors of embryo-placental attachment, including laminin, fibronectin, and Factor XIII, indicating that the presence of fibrin(ogen) is required to confer sufficient stability at the placental-decidual interface. The results of these studies demonstrate that maternal Fg plays a critical role in maintenance of pregnancy in mice, both by supporting proper development of fetal-maternal vascular communication and stabilization of embryo implantation.

Coagulopathy after severe traumatic brain injury (TBI) has been extensively reported. Clinical studies have identified a strong relationship between diminished platelet-rich thrombus formation, responsiveness to adenosine diphosphate agonism, and severity of TBI. The mechanisms that lead to platelet dysfunction in the acute response to TBI are poorly understood. The development of a rodent model of TBI that mimics the coagulopathy observed clinically has recently been reported. Using immunohistochemical techniques and thromboelastography platelet mapping, the current study demonstrated that the expression of coagulation (tissue factor and fibrin) and platelet activation (P-selectin) markers in the injured brain paralleled the alteration in systemic platelet responsiveness to the agonists, adenosine diphosphate and arachodonic acid. Results of this study demonstrate that local procoagulant changes in the injured brain have profound effects on systemic platelet function.

Clinical studies have indicated that high plasma levels of fibrinogen, or decreased fibrinolytic potential, are conducive to an increased risk of cardiovascular disease. Other investigations have shown that insoluble fibrin promotes atherosclerotic lesion formation by affecting smooth muscle cell proliferation, collagen deposition, and cholesterol accumulation. To directly assess the physiological impact of an imbalanced fibrinolytic system on both early and late stages of this disease, mice deficient for plasminogen activator inhibitor-1 (PAI-1 −/−) were used in a model of vascular injury/repair, and the resulting phenotype compared to that of wild-type (WT) mice. A copper-induced arterial injury was found to generate a lesion with characteristics similar to many of the clinical features of atherosclerosis. Fibrin deposition in the injured arterial wall at early (7 days) and late (21 days) times after copper cuff placement was prevalent in WT mice, but was greatly diminished in PAI-1 −/− mice. A multilayered neointima with enhanced collagen deposition was evident at day 21 in WT mice. In contrast, only diffuse fibrin was identified in the adventitial compartments of arteries from PAI-1 −/− mice, with no evidence of a neointima. Neovascularization was observed in the adventitia and was more extensive in WT arteries, relative to PAI-1 −/− arteries. Additionally, enhanced PAI-1 expression and fat deposition were seen only in the arterial walls of WT mice. The results of this study emphasize the involvement of the fibrinolytic system in vascular repair processes after injury and indicate that alterations in the fibrinolytic balance in the vessel wall have a profound effect on the development and progression of vascular lesion formation.

Activated protein C functions directly as an anticoagulant and indirectly as a profibrinolytic enzyme. To determine whether the fibrin deposition previously observed in PC −/−murine embryos and neonates was mediated through the FXI pathway, PC +/− /FXI −/−mice were generated and crossbred to produce double-deficient progeny ( PC −/− /FXI −/−). PC −/− /FXI −/−mice survived the early lethality observed in the PC −/− /FXI +/+neonates, with the oldest PC −/− /FXI −/−animal living to 3 months of age. However, the majority of these animals was sedentary and significantly growth-retarded. On sacrifice or natural death, all of these PC −/− /FXI −/−mice demonstrated massive systemic fibrin deposition with concomitant hemorrhage and fibrosis, as confirmed through histological analyses. Several of these animals also presented with enlarged lymph nodes and extensive lymphatic fluid in the thoracic cavity. Thus, although a number of the PC −/− /FXI −/−mice survived the lethal perinatal coagulopathy seen in the PC −/− neonates, they nonetheless succumbed to overwhelming thrombotic disease in later life. This combined deficiency state provided the first clear indication that the course of a severe thrombotic disorder could be manipulated by blocking the intrinsic pathway and provided the first opportunity to study a total protein C deficiency in an adult animal.

The protein C (PC) pathway is a well-characterized coagulation system. Endothelial PC receptors and thrombomodulin mediate the conversion of PC to its activated form, a potent anticoagulant and anti-inflammatory molecule. Here we show that the PC pathway is expressed on intestinal epithelial cells. The epithelial expression of PC and endothelial PC receptor is down-regulated In patients with inflammatory bowel disease. PC-/-/PC(Tg) mice, expressing only 3% of WT PC, developed spontaneous intestinal inflammation and were prone to severe experimental colitis. These mice also demonstrated spontaneous elevated production of inflammatory cytokines and increased intestinal permeability. Structural analysis of epithelial tight junction molecules revealed that lack of PC leads to decreased JAM-A and claudin-3 expression and an altered pattern of ZO-1 expression. In vitro, treatment of epithelial cells with activated PC led to protection of tight junction disruption induced by TNF-α, and in vivo, topical treatment with activated PC led to mucosal healing and amelioration of colitis. Taken together, these findings demonstrate that the PC pathway is a unique system involved in controlling intestinal homeostasis and inflammation by regulating epithelial barrier function.