Background: Thrombotic thrombocytopenic purpura (TTP) is caused by the absence of ADAMTS-13 activity. Thrombocytopenia is presumably related to the formation of microthrombi rich in von Willebrand factor (VWF) and platelets. Obesity may be a risk factor for TTP; it is associated with abundance of macrophages that may phagocytose platelets. Objectives: To evaluate the role of obesity and ADAMTS-13 deficiency in TTP, and to establish whether macrophages contribute to thrombocytopenia. Methods: Lean or obese ADAMTS-13-deficient (Adamts-13(-/-)) and wild-type (WT) mice were injected with 250 U kg(-1) of recombinant human VWF (rVWF), and TTP characteristics were evaluated 24 h later. In separate experiments, macrophages were depleted in the liver and spleen of lean and obese WT or Adamts-13(-/-) mice by injection of clodronate-liposomes, 48 h before injection of rVWF. Results: Obese Adamts-13(-/-) mice had a lower platelet count than their lean counterparts, suggesting that they might be more susceptible to TTP development. Lean Adamts-13(-/-) mice triggered with a threshold dose of rVWF did not develop TTP, whereas typical TTP symptoms developed in obese Adamts-13(-/-) mice, including severe thrombocytopenia and higher lactate dehydrogenase (LDH) levels. Removal of hepatic and splenic macrophages by clodronate injection in obese Adamts-13(-/-) mice before treatment with rVWF preserved the platelet counts measured 24 h after the trigger. In vitro experiments with cultured macrophages confirmed a VWF dose-dependent increase of platelet phagocytosis. Conclusions: Obese Adamts-13(-/-) mice are more susceptible to the induction of TTP-related thrombocytopenia than lean mice. Phagocytosis of platelets by macrophages contributes to thrombocytopenia after rVWF injection in this model.

Background Thrombotic thrombocytopenic purpura (TTP) is an acute life-threatening pathology, caused by occlusive von Willebrand factor (VWF)-rich microthrombi that accumulate in the absence of ADAMTS-13. We previously demonstrated that plasmin can cleave VWF and that plasmin is generated in patients during acute TTP. However, the exact role of plasmin in TTP remains unclear. Objectives Investigate if endogenous plasmin-mediated proteolysis of VWF can influence acute TTP episodes. Results In mice with an acquired ADAMTS-13 deficiency, plasmin is generated during TTP as reflected by increased plasmin-2-antiplasmin (PAP)-complex levels. However, mice still developed TTP, suggesting that this increase is not sufficient to control the pathology. As mice with TTP also had increased plasminogen activator inhibitor 1 (PAI-1) levels, we investigated whether blocking the plasmin(ogen) inhibitors would result in the generation of sufficient plasmin to influence TTP outcome in mice. Interestingly, when amplified plasmin activity was allowed (2-antiplasmin(-/-) mice with inhibited PAI-1) in mice with an acquired ADAMTS-13 deficiency, a resolution of TTP signs was observed as a result of an increased proteolysis of VWF. In line with this, in patients with acute TTP, increased PAP-complex and PAI-1 levels were also observed. However, neither PAP-complex levels nor PAI-1 levels were related to TTP signs and outcome. Conclusions In conclusion, endogenous plasmin levels are increased during acute TTP, although limited via suppression through 2-antiplasmin and PAI-1. Only when amplified plasmin activity is allowed, plasmin can function as a back-up for ADAMTS-13 in mice and resolve TTP signs as a result of an increased proteolysis of VWF.

Background & Aims: Increased prevalence of obesity is paralleled by an increase in non-alcoholic steatohepatitis (NASH). We previously found that the expression of ADAMTS5 (A Disintegrin And Metalloproteinase with Thrombospondin type 1 motifs; member 5) is enhanced in expanding adipose tissue. However, no information is available on a potential role in liver pathology. We studied the effect of ADAMTS5 deficiency on NASH in mice. Methods: Wild-type (Adamts5(+/+)) and deficient (Adamts5(-/-)) mice were kept on a standard-or high-fat diet (HFD) for 15 weeks. Alternatively, steatohepatitis was induced with methionine/choline-deficient (MCD) diet. Results: HFD feeding resulted in comparable body weights for both genotypes, but Adamts5(-/-) mice had approximately 40% lower liver weight (P = 0.0004). In the Adamts5(-/-) mice, the HFD as well as the MCD diet consistently induced less NASH with less fibrosis. The deteriorating effect of ADAMTS5 on the liver during diet-induced obesity may be due, at least in part, to proteolytic cleavage of the matrix components syndecan-1 and versican, thereby enhancing hepatic triglyceride clearance from the circulation. Plasma lipid levels were elevated in obese Adamts5(-/-) mice. There was no clear effect of ADAMTS5 deficiency on glycaemia or glucose tolerance, whereas insulin sensitivity was somewhat improved. Furthermore, Adamts5(-/-) mice were protected from hepatic mitochondrial dysfunction, as indicated by increased mitochondrial respiratory chain complex activity, higher ATP levels and higher expression of antioxidant enzymes. Conclusions: Absence of ADAMTS5 preserves liver integrity in a diet-induced obesity model. Selective targeting of ADAMTS5 could provide a new therapeutic strategy for treatment/prevention of NASH.

The effect of cluster of differentiation (CD) 36 on regulatory T cells (T-reg) was investigated in gonadal (GN) adipose tissues and mesenteric lymph nodes (MLN) of wild-type (WT) and CD36 deficient (CD36(-/-)) mice kept on standard fat (SFD, lean) or on high fat diet (HFD, obese). GN adipose tissue mass was smaller, but MLN size larger for obese CD36(-/-) versus obese WT mice. Overall, the reduction of T-reg cells in GN adipose tissue and MLN after a HFD is much more prominent in WT than CD36(-/-) mice. Moreover, CD36(-/-) mice may be protected against obesity-related chronic inflammation. (C) 2015 Elsevier Inc. All rights reserved.

. Background: The development of a procoagulant state in sepsis, owing to aberrant expression of tissue factor (TF) and a sharp decrease in the level of its major inhibitor, TF pathway inhibitor (TFPI), could lead to microthrombotic organ failure. The mechanism for the decline in TFPI activity in the lung could involve plasmin-mediated cleavage of the inhibitor. Objective: To investigate the effect of plasmin generation on lung-associated TFPI activity, in normal conditions and during infusion of endotoxin (lipopolysaccharide [LPS]) in mice. Methods: Plasmin generation and TFPI activity were assayed in the lungs of mice deficient in tissue-type plasminogen (Plg) activator (t-PA) or Plg, at 2 h after LPS or saline injection. Results: The sharp loss of lung-associated TFPI activity at 2 h after LPS challenge paralleled the abrupt increase in plasmin generation. TFPI activity was significantly retained in both t-PA-/- and Plg-/- mice, which are unable to generate plasmin. Conclusion: The increased plasmin generation during the early stages of sepsis could cleave/inactivate TFPI and thus lead to thrombotic complications.

Background: Matrix metalloproteinases (MMPs) are known to play a role in adipose tissue development, but little information is available on the role of individual proteinases. Expansion of adipose tissue is associated with an increased macrophage content. Macrophage elastase (MMP-12) has an important role in macrophage infiltration, which induces pro-inflammatory effects in adipose tissue.Methods: The role of MMP-12 was investigated in adipose tissues of MMP-12 deficient and wild-type control mice kept on normal chow or on high fat diet for 15 weeks.Results: MMP-12 deficiency had no significant effect on total body weight or on subcutaneous (SC) or gonadal (GUN) adipose tissue mass. Adipocyte and blood vessel size and density in SC and CON adipose tissues of obese mice were also comparable in MMP-12 deficient and control mice. Macrophage infiltration in SC and GUN adipose tissues was not affected by MMP-12 deficiency, but the amount of crown-like structures (CLS) was significantly lower. MMP-12 deficiency did not affect elastin content in the extracellular matrix of SC or GUN adipose tissue.Conclusions: Adipose tissue mass and composition in mice with nutritionally induced obesity was not markedly affected by MMP-12 deficiency, except for an apparently lower degree of CLS.General Significance: MMP-12 does not seem to be essential for macrophage infiltration in adipose tissue, but contributes to the formation of CLS surrounding moribund adipocytes. (C) 2013 Elsevier B.V. All rights reserved.

Summary A potential role for the gelatinases in adipocyte differentiation in vitro and adipose tissue development in vivo was investigated using the gelatinase inhibitor tolylsam ((R)‐3‐methyl‐2‐[4‐(3‐p‐tolyl‐[1,2,4]oxadiazol‐5‐yl)‐benzenesulphonylamino]‐butyric acid). Differentiation of murine 3T3‐F442A preadipocytes (12 days after reaching confluence) into mature adipocytes in vitro was promoted in the presence of tolylsam (10–100 μmol/L). De novo development of fat tissue in nude mice injected with preadipocytes and kept on a high‐fat diet was significantly impaired following treatment with tolylsam (100 mg/kg per day for 4 weeks). Adipose tissue development in matrix metalloproteinase (MMP)‐2 deficient mice, kept on a high‐fat diet, was significantly impaired following administration of tolylsam (100 mg/kg per day for 15 weeks). This was associated with markedly enhanced metabolic rate. Treatment of MMP‐2‐deficient mice with tolylsam (100 mg/kg per day, 15 weeks) was associated with the preservation of collagen and a reduction in blood vessel size in adipose tissues in vivo. Furthermore, plasma levels of triglycerides and free fatty acids were reduced by tolylsam treatment of MMP‐2‐deficient mice (100 mg/kg per day, 15 weeks), whereas nutrient adsorption in the intestine was not affected. The results of the present study indicate that tolylsam promotes preadipocyte differentiation in vitro, but impairs adipose tissue development in vivo.

Summary 1. The potential of the matrix metalloproteinase (MMP) inhibitor ABT‐518 to affect pre‐adipocyte differentiation in vitro and adipose tissue development in vivo was investigated using mouse models of adipogenesis and obesity. 2. Differentiation of 3T3‐F442A pre‐adipocytes into mature adipocytes was enhanced in a dose‐dependent manner by the addition of ABT‐518 (0–100 μmol/L). This was associated with increased expression of the adipogenic markers adipocyte fatty acid‐binding protein 2 (AP2), peroxisome proliferator‐activated receptor γ and adiponectin. 3. Feeding 5‐week‐old male wild‐type mice with a high‐fat diet, with or without ABT‐518 (to achieve a dose of 100 mg/kg per day), for 16 weeks resulted in a significant reduction in bodyweight throughout the experimental period. Magnetic resonance spectroscopy revealed that the lipid : water ratio was significantly lower in ABT‐518‐treated mice. The total weight of isolated subcutaneous or gonadal fat depots did not differ significantly following ABT‐518 treatment, but adipocyte and blood vessel size were significantly reduced in the gonadal fat. 4. Administration of ABT‐518–2 (100 mg/kg per day for 10 weeks) to 5‐week‐old male wild‐type mice with established obesity maintained on a high‐fat diet had no effect on total bodyweight at the end of the experiment, but was associated with reduced blood vessel size in the fat depots. 5. Thus, the MMP inhibitor ABT‐518 stimulates differentiation of 3T3‐F442A pre‐adipocytes in vitro . It mildly reduces bodyweight gain in a murine model of diet‐induced obesity, but does not affect established obesity.

BACKGROUND:Mice with single gene deficiency of thrombin-activatable fibrinolysis inhibitor (TAFI) or plasminogen activator inhibitor-1 (PAI-1) have an enhanced fibrinolytic capacity.OBJECTIVES:To unravel the function and relevance of both antifibrinolytic proteins through the generation and characterization of mice with combined TAFI and PAI-1 gene deficiency.RESULTS:Mating of TAFI knockout (KO) mice with PAI-1 KO mice resulted in the production of TAFI/PAI-1 double-KO mice that were viable, were fertile, and developed normally. In a tail vein bleeding model, the bleeding time and hemoglobin content of the TAFI/PAI-1 double-KO mice did not deviate significantly from those of the single-KO mice or of the wild-type (WT) counterparts. Interestingly, in ex vivo rotational thromboelastometry measurements with whole blood samples, TAFI KO mice and TAFI/PAI-1 double-KO mice were more sensitive to fibrinolytic activation with tissue-type plasminogen activator than WT or PAI-1 KO mice. This enhanced fibrinolytic capacity was confirmed in vivo in a mouse thromboembolism model, as shown by decreased fibrin deposition in the lungs of TAFI KO mice and TAFI/PAI-1 double-KO mice as compared with WT or PAI-1 KO mice.CONCLUSIONS:TAFI gene inactivation predominantly contributes to the increased fibrinolytic capacity of TAFI and PAI-1 double-gene-deficient mice, as observed in some basic thrombosis models.

AIM:In an effort to assess the role of plasminogen activator inhibitor-1 (PAI-1) in oral squamous cancer development and progression, two different carcinogen treatment protocols were conducted.MATERIALS AND METHODS:Protocol I included mice from a PAI-1 transgenic (Tg) breed (n=56) and their wild-type (WT) counterparts (n=56), divided into one control group and two main experimental groups, treated with 7,12-dimethylbenz[a]anthracene (DMBA) for 8 and 16 weeks, respectively. Protocol II included the same number and types of animals and groups, which were similarly treated with 4-Nitroquinoline 1-oxide (4-NQO) in drinking water. Two drugs that affect plasma PAI-1 levels, enalapril and pravastatin, were administered to certain subgroups of animals in both protocols.RESULTS:None of the animals developed macroscopically-visible oral cancer lesions. Eleven animals under Protocol I and 52 animals under Protocol II died. Skin lesions were noted only in DMBA-treated animals (n=9). Almost all animals administered with 4-NQO developed alopecia and lost weight, while two of them developed stomach tumours, and one female mouse developed a large ovarian cyst.CONCLUSION:Transgenic mice may respond differently when used in well-established carcinogen models and oral carcinogenesis is hard to achieve in these rodents.

The effect of ageing on the morphology of veins, venous valves and arteries was investigated in male wild-type mice using an adapted procedure with injection of a silicone polymer Microfil(®) that preserves morphology of the vasculature. Throughout the hind limb the arterial, but not the venous, lumen area and wall thickness were significantly greater in 24-month as compared to 10-week-old C57BL/6 mice. Venous valves were most frequently located at the sapheno-femoral vein junction in the lower extremities, and appeared thicker at the base supported by structurally intact collagen fibers, and thinner towards the proximal end of the valve leaflet, with less organized collagen. Overall, valves were less supported by structurally intact collagen at 24 months as compared to 10 weeks. Endothelial expression of CD31, endothelial protein C receptor or von Willebrand factor (VWF) was not affected by age, while thrombomodulin expression was lower in aged versus young arteries. At both ages, expression of VWF was lower at venous valves versus veins. Evaluation of the blood coagulation profile revealed that aged mice had shortened prothrombin time, elevated plasma levels of factor (F)VII, FVIII and VWF and increased neutrophil and platelet counts. Thus, our data indicate that in mice with ageing, venous valves become more fragile, in association with a procoagulant and inflammatory blood phenotype. Taken together, we found that the procoagulant state in ageing, is accompanied by mild vascular changes.

Obesity is a common disorder, and related diseases such as diabetes, atherosclerosis, hypertension, cardiovascular disease and cancer are a major cause of mortality and morbidity in Westerntype societies. Development of obesity is associated with extensive modifications in adipose tissue involving adipogenesis, angiogenesis and extracellular matrix proteolysis. The fibrinolytic (plasminogen/plasmin) and matrix metalloproteinase (MMP) systems cooperate in these processes. Adipogenesis is tightly associated with angiogenesis, as shown by the findings that adipose tissue expiants trigger blood vessel formation, whereas in turn adipose tissue endothelial cells promote preadipocyte differentiation. A nutritionally induced obesity model in transgenic mice has been used extensively to study the role of the fibrinolytic and MMP systems and of angiogenesis in the development of obesity. Most studies support a role of these systems in adipogenesis and obesity, and suggest that their modulation may affect development of adipose tissue. Such models have also shown that treatment of obese female mice with estrogens has the potential to improve obesity, insulin resistance and glucose intolerance, via decreased expression of lipogenic genes. Thus, murine models of obesity have been very useful tools to study mechanisms of adipose tissue development, as well as effects of hormonal therapy.

Background: ADAMTS13 deficiency causes accumulation of unusually large von Willebrand factor molecules, which cross-link platelets in the circulation or on the endothelial surface. This process of intravascular agglutination leads to the microangiopathy thrombotic thrombocytopenic purpura (TTP). Most TTP patients have acquired anti-ADAMTS13 autoantibodies that inhibit enzyme function and/or clear it from the circulation. However, the reason for ADAMTS13 deficiency is not always easily identified in a subset of patients. Objectives: To determine the origin of ADAMTS13 deficiency in a case of acquired TTP. Methods: Western blotting of ADAMTS13 in plasmas from acute and remission phases was used. Results: The ADAMTS13 deficiency was not caused by mutations or (detectable) autoantibodies; however, an abnormal ADAMTS13 truncated fragment (100 kDa) was found in acute-phase but not remission-phase plasma. This fragment resulted from enzymatic proteolysis, as recombinant ADAMTS13 was also cleaved when in the presence of acute-phase but not remission-phase plasma. Inhibitor screening showed that ADAMTS13 was cleaved by a serine protease that could be dose-dependently inhibited by addition of exogenous alpha(2)-antiplasmin. Examination of the endogenous alpha(2)-antiplasmin antigen and activity confirmed deficiency of alpha(2)-antiplasmin function in acute-phase but not remission-phase plasma. To investigate the possibility of ADAMTS13 cleavage by plasmin in plasma, urokinase-type plasminogen activator was added to an (unrelated) congenital alpha(2)-antiplasmin-deficient plasma sample to activate plasminogen. This experiment confirmed cleavage of endogenous ADAMTS13 similar to that observed in our TTP patient. Conclusion: We report the first acquired TTP patient with cleaved ADAMTS13 and show that plasmin is involved.

Background: It is unknown whether venous thrombosis after long haul air travel is exclusively attributable to immobilization. Objectives: We determined whether the following mechanisms were involved: hypoxia, stress, inflammation or viral infection. Patients/Methods: In a case crossover setting in 71 healthy volunteers who were exposed to an 8-h flight, 8-h movie marathon and 8 h of regular activities, we compared markers for several hypothetical pathways: plasminogen activator inhibitor-1 (PAI-1), stress, plasma factor (F)VIII coagulant activity (FVIIIc), soluble P-selectine (sP-selectine), interleukin-8 (IL-8) and neutrophil elastase. We reported earlier an activated clotting system, as evidenced by thrombin generation, in 17% of volunteers after the flight. Results: PAI-1 increased by 4.2 ng mL-1 (CI95:-49.5 to 6.5) in volunteers with an activated clotting system whereas it decreased in those without (-20.0 ng mL-1, CI95:-33.2 to -14.0). FVIIIc levels rose more in individuals with clotting activation (18.0%, CI95:-1.0 to 33.0) than in those without (2.0%, CI95:-2.0 to 5.0). The increases in FVIIIc were not associated with stress, which appeared unrelated to clotting activation. sP-selectin increased in those with clotting activation (3.5 mu g L-1, CI95: -3.0 to 10.0), but decreased in those without (-0.5 mu g L-1, CI95: -2.0 to 2.0). Changes in levels of neutrophil elastase or IL-8 were not different between the subjects with and without clotting activation. Conclusions: Our results do not support the hypotheses that stress, infection or air pollution are involved in the development of a prothrombotic state in air travellers. After long haul air travel, this state is more pronounced in patients with risk factors and may be caused by hypoxia, triggering systemic inflammation and platelet activation, leading to coagulation induction and degranulation of platelets.