Reduced Fibrin Deposition Research Articles

Recombinant factor VIIa enhances platelet deposition from flowing haemophilic blood but requires the contact pathway to promote fibrin deposition.

In prior microfluidic studies with haemophilic blood perfused over collagen, we found that a severe deficiency (<1% factor level) reduced platelet and fibrin deposition, while a moderate deficiency (1-5%) only reduced fibrin deposition. We investigated: (i) the differential effect of rFVIIa (0.04-20nm) on platelet and fibrin deposition, and (ii) the contribution of the contact pathway to rFVIIa-induced haemophilic blood clotting. Haemophilic or healthy blood with low and high corn trypsin inhibitor (CTI, 4 or 40μgmL(-1) ) was perfused over collagen at an initial venous wall shear rate of 100s(-1) . At 100s(-1) wall shear rate, where FXIIa leads to thrombin production without added tissue factor, FXI-deficient blood (3%) or severely FVIII-deficient blood (<1%) produced no fibrin at either CTI level. Whereas rFVIIa potently enhanced platelet deposition, fibrin generation was not rescued. Distinct from the high CTI condition, engagement of the contact pathway (low CTI) in moderately FVIII-deficient (3%) or moderately FIX-deficient blood (5%) resulted in enhanced platelet and fibrin deposition following 4nm rFVIIa supplementation. In mildly FVIII-deficient blood (15%) at <24h since haemostatic therapy, rFVIIa enhanced both platelet and fibrin generation in either CTI condition although fibrin was produced more quickly and abundantly in low CTI. For tissue factor-free conditions of severe haemophilic blood clotting, we conclude that rFVIIa reliably generates low levels of 'signaling' thrombin sufficient to enhance platelet deposition on collagen, but is insufficient to drive fibrin polymerization unless potentiated by the contact pathway.

Contact activation of blood coagulation on a defined kaolin/collagen surface in a microfluidic assay

Generation of active Factor XII (FXIIa) triggers blood clotting on artificial surfaces and may also enhance intravascular thrombosis. We developed a patterned kaolin (0 to 0.3pg/μm2)/type 1 collagen fibril surface for controlled microfluidic clotting assays. Perfusion of whole blood (treated only with a low level of 4μg/mL of the XIIa inhibitor, corn trypsin inhibitor) drove platelet deposition followed by fibrin formation. At venous wall shear rate (100s−1), kaolin accelerated onset of fibrin formation by ~100sec when compared to collagen alone (250sec vs. 350sec), with little effect on platelet deposition. Even with kaolin present, arterial wall shear rate (1000s−1) delayed and suppressed fibrin formation compared to venous wall shear rate. A comparison of surfaces for extrinsic activation (tissue factor TF/collagen) versus contact activation (kaolin/collagen) that each generated equal platelet deposition at 100s−1 revealed: (1) TF surfaces promoted much faster fibrin onset (at 100sec) and more endpoint fibrin at 600sec at either 100s−1 or 1000s−1, and (2) kaolin and TF surfaces had a similar sensitivity for reduced fibrin deposition at 1000s−1 (compared to fibrin formed at 100s−1) despite differing coagulation triggers. Anti-platelet drugs inhibiting P2Y1, P2Y12, cyclooxygenase-1 or activating IP-receptor or guanylate cyclase reduced platelet and fibrin deposition on kaolin/collagen. Since FXIIa or FXIa inhibition may offer safe antithrombotic therapy, especially for biomaterial thrombosis, these defined collagen/kaolin surfaces may prove useful in drug screening tests or in clinical diagnostic assays of blood under flow conditions.

In vitro and in vivo characterisation of the profibrinolytic effect of an inhibitory anti-rat TAFI nanobody

One of the main disadvantages of current t-PA thrombolytic treatment is the increased bleeding risk. Upon activation, thrombin activatable fibrinolysis inhibitor (TAFI) is a very powerful antifibrinolytic enzyme. Therefore, co-administration of a TAFI inhibitor during thrombolysis could reduce the required t-PA dose without compromising the thrombolytic efficacy. In this study we generated and characterised a nanobody that is inhibitory towards rat TAFI and evaluated its profibrinolytic property in vitro and in vivo. Nanobody VHH-rTAFI-i81 inhibits (at a 16-fold molar ratio nanobody over TAFI) the thrombin/thrombomodulin (T/TM)-mediated activation of rat TAFI (rTAFI) by 83 ± 1.8% with an IC50 of 0.46 (molar ratio nanobody over TAFI). The affinity (KA) of VHH-rTAFI-i81 for rTAFI, as determined by surface plasmon resonance (Biacore®), is 2.5 ± 0.2 x 10(10) M(-1) and illustrates a very strong binding. In an in vitro clot lysis assay, administration of VHH-rTAFI-i81 strongly enhances the degree of lysis and reduces time to reach full lysis of t-PA-mediated clot lysis. Epitope mapping discloses that Lys392 is of primary importance for the nanobody/rTAFI interaction besides minor contributions of Tyr175 and Glu183. In vivo application of VHH-rTAFI-i81 in a tissue factor-induced mouse thromboembolism model significantly decreases fibrin deposition in the lungs in the absence of exogenous administered t-PA. Nanobody VHH-rTAFI-i81 is a very potent inhibitor of T/TM-mediated TAFI activation. Co-administration of this nanobody and t-PA enhances the fibrinolytic efficacy. In an in vivo mouse thromboembolism model, VHH-rTAFI-i81 reduces fibrin deposition in the lungs.

Abstract 545: PLacental eXpanded (PLX) Cell Treatment Ameliorates Preeclampsia Induced by TLR3 or TLR7 Activation in Mice

PLacental eXpanded (PLX) cells (Pluristem Therapeutics Inc.) are human placenta-derived, mesenchymal-like adherent stromal cells that release proteins in response to the environment of the host. PLX cells are non-immunogenic and have been shown to decrease inflammation and increase angiogenesis in inflammatory and ischemic conditions. Therefore, we tested whether PLX cell treatment could attenuate symptoms of preeclampsia (PE) in mice. We hypothesized that one-time PLX cell treatment would decrease the pregnancy-dependent hypertension, proteinuria, endothelial dysfunction, splenomegaly, inflammation, and placental injury induced by Toll-like receptor (TLR) activation during pregnancy. Pregnant C57BL/6 mice were given ip injections of saline vehicle (P), the TLR3 agonist poly I:C (PPIC), or the TLR7 agonist R837 (PR) on days 13, 15, and 17 of gestation. P, PPIC, and PR mice were also given either plasmalyte A (PLA, vehicle) or PLX cells (1 million) by im injection in the right leg on gestational day 14 (n=8 in each group). PLX cell treatment progressively decreased SBP over 3 days in PPIC and PR mice and had no effect in P control mice (day 17 SBP in mmHg: P+PLA = 100±4, P+PLX = 96±4, PPIC+PLA = 144±3, PPIC+PLX = 111±1, PR+PLA = 145±2, PR+PLX = 106±3; PPIC+PLA and PR+PLA p&lt;0.05 vs. P+PLA). PLX cell treatment also normalized the urinary protein/creatinine ratio and aortic endothelium-dependent relaxation responses in PPIC and PR mice to that of P mice while having no significant effects on the number of fetuses or incidence of fetal demise per litter. Inflammation plays a central role in the development of TLR-induced PE and PLX cell treatment reduced spleen weight/body weight ratios, normalized splenic levels of gamma-delta T cells, decreased plasma IL-6 levels, and restored plasma IL-4 levels in PPIC and PR mice. Additionally, PLX cell treatment reduced fibrin deposition in the placental vasculature and significantly reduced placental HIF-1alpha protein levels. These data demonstrate that one-time PLX cell treatment after PE is induced was able to decrease inflammation, proteinuric hypertension, endothelial dysfunction, and placental injury in mice and may be beneficial in women with PE.

In vitro comparison of the novel, dual-acting FIIa/FXa-inhibitor EP217609C101, unfractionated heparin, enoxaparin, and fondaparinux in preventing cardiac catheter thrombosis

Efficient and safe anticoagulation is crucial in patients requiring percutaneous coronary intervention (PCI) or extracorporeal circulation during cardiac surgery. Although new anticoagulant strategies have emerged for PCI as alternatives to the established treatment with heparins, the development of new anticoagulants with an improved efficacy/safety ratio is still necessary. Our study compared the efficacy of the novel, dual-acting, neutralizable FIIa/FXa-inhibitor EP217609C101 (EP) at 2, 1.2, 0.9, and 0.6μg/ml to unfractionated heparin (UFH), enoxaparin, and fondaparinux in preventing cardiac catheter thrombosis under in vitro conditions. Blood drawn by venepunction from healthy male volunteers (n=10) pretreated with 500mg aspirin orally was treated with the anticoagulant to test and continuously circulated through a cardiac catheter for 60min or until the catheter became blocked by thrombotic debris. Anticoagulant efficacy was assessed by thrombus weight, electron microscopic features of the developing thrombi, and laboratory parameters. Whereas UFH, enoxaparin, EP 2, and EP 1.2μg/ml secured maximum circulation times, statistically significant premature catheter occlusions were observed for EP 0.9, EP 0.6μg/ml, and fondaparinux. The UFH group and both high-dose concentrations of EP showed significantly lower thrombus weights than the low-dose concentrations of EP and fondaparinux, (p≤0.05). On electron microscopic analysis of the thrombotic debris no differences were observed in erythrocyte deposition between UFH, enoxaparin, and all EP concentrations tested. A significant reduction in fibrin deposition was achieved by UFH and EP 2μg/ml but no significant differences in platelet deposition were found, except for a significant reduction for EP 0.6μg/ml. Our in vitro study showed that EP217609C101 is a promising new drug that is dose-dependently superior to classical (UFH, enoxaparin) and newer (fondaparinux) drugs in preventing heart catheter thrombosis.

Liposomes bearing fibrinogen could potentially interfere with platelet interaction and procoagulant activity

BackgroundThe contribution of fibrinogen (FBN) to hemostasis acting on platelet aggregation and clot formation is well established. It has been suggested that FBN-coated liposomes could be useful in restoring hemostasis. In the present study, we evaluated the modifications induced by multilamellar raw liposomes (MLV) or fibrinogen-coated liposomes (MLV-FBN) on hemostatic parameters.Materials and methodsDifferent experimental settings using whole blood or thrombocy-topenic blood were used. Thromboelastometry, aggregation studies, platelet function analyzer (PFA-100®) tests and studies under flow conditions were applied to detect the effect of MLV-FBN on hemostatic parameters.ResultsThe presence of MLV-FBN in whole blood modified its viscoelastic properties, prolonging clot formation time (CFT) (226.5 ± 26.1 mm versus 124.1 ± 9.4 mm; P < 0.01) but reducing clot firmness (45.4 ± 1.8 mm versus 35.5 ± 2.3 mm; P < 0.05). Under thrombocy-topenic conditions, FIBTEM analysis revealed that MLV-FBN shortened clotting time (CT) compared to MLV (153.3 ± 2.8 s versus 128.0 ± 4.6 s; P < 0.05). Addition of either liposome decreased fibrin formation on the subendothelium (MLV 8.1% ± 4.7% and MLV-FBN 0.8% ± 0.5% versus control 36.4% ± 6.7%; P < 0.01), whereas only MLV-FBN significantly reduced fibrin deposition in thrombocytopenic blood (14.4% ± 6.3% versus control 34.5% ± 5.2%; P < 0.05). MLV-FBN inhibited aggregation induced by arachidonic acid (52.1% ± 8.1% versus 88.0% ± 2.1% in control; P < 0.01) and ristocetin (40.3% ± 8.8% versus 94.3% ± 1.1%; P < 0.005), but it did not modify closure times in PFA-100® studies. In perfusion experiments using whole blood, MLV and MLV-FBN decreased the covered surface (13.25% ± 2.4% and 9.85% ± 2.41%, respectively, versus control 22.0% ± 2.0%; P < 0.01) and the percentage of large aggregates (8.4% ± 2.3% and 3.3% ± 1.01%, respectively, versus control 14.6% ± 1.8%; P < 0.01).ConclusionOur results reveal that, in addition to the main contribution of fibrinogen to hemostasis, MLV-FBN inhibits platelet-mediated hemostasis and coagulation mechanisms.

Roles of fibrin deposition and protease activated receptor-1 in renal cytokine/chemokine production and inflammatory cell infiltration in rats of different ages

The present study aimed to investigate the roles of fibrin deposition and protease activated receptor-1 (PAR-1) in renal cytokine/chemokine production and inflammatory cell infiltration in rats of different ages. Acute inflammation was induced by lipopolysaccharide (LPS) in rats which were then treated with tranexamic acid (TA), TA+urokinase (UK) or TA+low-molecular-weight heparin (HP). Fibrin deposition, inflammatory cells and expressions of PAR-1, monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule 1 (ICAM-1) were detected. A reduction in fibrin deposition and PAR-1 expression in the LPS+TA+HP group was associated with decreased infiltration of inflammatory cells and down-regulated expressions of MCP-1 and ICAM-1. In the LPS+TA+UK group, the fibrin deposition, but not the PAR-1 expression, was reduced, However, the infiltration of inflammatory cells decreased and the expressions of MCP-1 and ICAM-1 down-regulated. There were significant differences in the fibrin deposition, infiltration of inflammatory cells and expression of PAR-1, MCP-1 and ICAM-1 between young and old rats undergoing the same treatment. These findings demonstrated that fibrin deposition plays more important roles than PAR-1 dose in cytokine/chemokine production and inflammatory cell infiltration in vivo, and ageing may deteriorate the fibrin deposition-induced production of cytokines/chemokines and infiltration of inflammatory cells.

Critical Role of Immobilized Factor VIII In Solid-Phase Blood Coagulation During Mural Thrombogenesis Under Whole Blood Flow Conditions

AbstractAbstract 2199Coagulation factor VIII (FVIII), lacking in hemophilic blood, plays an essential role in mechanisms of fibrin plug formation to arrest bleeding at sites of injured vessel walls. Physiologic activity of FVIII circulating in bloodstream (soluble FVIII; S-FVIII) could be extensively evaluated so far by classic plasma coagulation assays such as activated partial thromboplastin time. However, the in vivo functional relevance of FVIII bound to von Willebrand factor (VWF) which is immobilized in subendothelium (immobilized FVIII; I-FVIII) is more complex and remains to be addressed. Using an in vitro perfusion chamber system, we have therefore evaluated the function of I-FVIII in the process of mural thrombus generation under whole blood flow conditions. FVIII-free VWF was purified in the presence of 0.35 M CaCl2 from cryoprecipitate, and coated on a glass plate. Various concentrations (0 as a control, 0.1, 0.3, 1, or 3 U/ml) of recombinant FVIII (Kogenate FS provided by Bayer Pharmaceutical Co.) were reacted with the FVIII-free VWF-coated glass plate. After non-adherent proteins were washed out, the amount of FVIII immobilized to a glass surface via VWF (I-FVIII) was measured by ELISA-based assay using a peroxidase-conjugated anti-FVIII polyclonal antibody. Whole blood was then perfused over a glass plate described above in a parallel plate flow chamber with various shear rates, and the thrombus generation process on a glass surface was observed in real time by confocal laser scanning microscopy. The development of intra-thrombus fibrin deposition was assessed by immune-staining of thrombi with a fluorescence-labeled anti-fibrin specific monoclonal antibody (NYB-T2G1; Accurate Chem.), reflecting solid-phase blood coagulation reaction during mural thrombogenesis. In perfusion of control blood with a high shear rate (1500 s-1), the intra-thrombus fibrin deposition was found to increase as a function of I-FVIII, resulting in the 2.5-fold greater fibrin deposition at the plateau as compared to control thrombi generated in the absence of I-FVIII. This I-FVIII effect on intra-thrombus fibrin deposition was also confirmed in perfusion of synthetic hemophilic blood (S-FVIII activity < 1%) which was prepared by the incubation of control blood with an anti-FVIII human IgG (final inhibitor titer in synthetic blood; 5, 10, or 20 Bethesda U/ml). Indeed, I-FVIII normalized in a dose-dependent manner the reduced fibrin deposition (20-35% of normal control) within synthetic hemophilic thrombi generated in the absence of S-FVIII under a high shear rate condition. The improvement of impaired fibrin deposition by I-FVIII was unvarying regardless of the anti-FVIII inhibitor titer in synthetic hemophilic blood. In contrast, the direct addition of recombinant FVIII into synthetic hemophilic blood was poorly effective in this regard, due to the immediate neutralization of S-FVIII by an inhibitor involved in synthetic blood. Thus, these results clearly indicate that I-FVIII, independent of S-FVIII, does play a considerable role on the intra-thrombus fibrin-network formation in the process of mural thrombus generation under whole blood flow conditions with high shear rate, most relevant physiologically for the in vivo hemostasis and thrombosis. Our results might imply a possibility of novel strategic design targeting I-FVIII against hemophilic patients with a high titer anti-FVIII inhibitor. Disclosures:No relevant conflicts of interest to declare.

Hypoxia-Induced Annexin A2 Expression Regulates Oxygen-Induced Retinopathy.

Abstract 471Annexin A2 (A2), a Ca2+-dependent membrane binding protein that usually complexes with a S100 protein, p11, regulates fibrinolysis by serving as an endothelial cell co-receptor for plasminogen and tissue plasminogen activator to promote plasmin generation. Compared to A2+/+ mice, A2-/- mice have reduced post-hyperoxic retinal neovascularization in the model of oxygen-induced retinopathy (OIR) that mimics human retinopathy of prematurity. Here, we sought to determine the mechanism by which A2 contributes to the pathogenesis of OIR. A2+/+ mouse pups (P7) were placed with their dams in a high O2 (75%) chamber for 5 consecutive days (until P12), and then rapidly shifted to room air (21% O2) for several more days. Mouse retinas were isolated over a range of time points for analysis of neovascularization, A2 protein and mRNA expression, fibrin deposition, cell proliferation and apoptosis assays. In post-hyperoxic A2+/+ mice, we noted extensive retinal neovascularization along with a more than 10-fold increase of VEGFA compared to retinas of mice maintained in room air. Both A2 protein and mRNA levels increased 2-4-fold in mouse retinas with OIR in comparison with expression of either CD31 or VE-cadherin 5. p11 and hypoxia-inducible factor-1α (HIF-1α) protein, but not mRNA levels, also increased 2-4-fold during OIR. Because increased A2 expression was predominantly associated with retinal neovessels, we examined the response of cultured endothelial cells to hypoxia, and observed a 2-4-fold increase in A2 protein and mRNA. Although HIF-1α stabilization was not affected by A2 deficiency, upregulation of A2 by hypoxia was HIF-1α dependent. Expression of A6, on the other hand, was not upregulated by hypoxia. Neither erythropoietin (EPO) nor VEGFA signaling changed A2 expression. In addition, hypoxic endothelial cells translocated 50% more A2 protein to the cell surface, generated 30% more plasmin, and migrated twice as efficiently. While stability of A2 mRNA was not changed by hypoxia-related stimuli, we observed direct binding between HIF-1α and the A2 promoter by electrophoretic mobility shift assay, and a 3-4-fold increased A2 promoter activity in the presence of HIF-1α and HIF-1β by luciferase reporter assay. Promoter activation was abrogated when the HIF-1α binding site within the A2 promoter was mutated. By immunofluorescent staining of post-hyperoxic P17 retinal sections, we found that infiltration of endothelial cells and pericytes was 2-fold greater in A2+/+ mice as compared to A2-/- mice. In addition, there was significantly less fibrin deposition, more macrophage recruitment, less apoptosis and greater cell proliferation in P17 post-hyperoxic retinas of A2+/+ mice as compared to A2-/- mice. In conclusion, annexin A2 is upregulated by hypoxia through direct HIF-1α-mediated transcriptional regulation. Upregulated A2 appears to enhance plasmin generation, facilitate directed migration of vascular endothelial cells, and enable the recruitment of supporting cells (e.g. pericytes) and inflammatory cells (e.g. macrophages). It also reduces fibrin deposition, inhibits apoptosis, and promotes cell proliferation, thereby promoting pathologic oxygen-induced retinal neovascularization. Disclosures:No relevant conflicts of interest to declare.

Reduction of Phosphatidylserine Exposure through Copper Chelation Leads to Reduced Fibrin Deposition After Laser Induced Vascular Injury In Vivo.

Abstract 3049Poster Board II-1025Colocalization and assembly of blood coagulation factors in the presence of negatively charged phospholipids leads to a 1,000-fold increase in the rate of thrombin generation compared to the solution reaction. We have established prothrombin fragment 1, the region of prothrombin containing the γ-carboxy-glutamic acid domain, as a probe for anionic phospholipids including phosphatidylserine. Prothrombin fragment 1 binds with high affinity to phosphatidylserine-containing membranes in vitro and identifies phosphatidylserine exposure relevant for the site of assembly of coagulation complexes in vivo. In order to determine the effect of phosphatidylserine exposure on thrombus formation during the laser injury model in vivo, we treated mice orally with the Cu2+ chelator tetrathiomolybdate for one week prior to study. This treatment has been shown to suppress phosphatidylserine exposure in rats (PNAS, 100: 6700-05, 2003). After copper chelator treatment, normal partial thromboplastin times (39 sec vs 42 sec, p=0.5) and whole blood counts in treated versus untreated mice were similar, suggesting that copper chelation did not affect the function of coagulation factors or total blood cell counts. Annexin V and Prothrombin fragment 1 were also used to measure phosphatidylserine exposure after thrombin (1 U/ml) stimulation of washed platelets using flow cytometric analysis. Platelets from untreated mice exhibited 2-fold increase in binding of both Annexin V and Prothrombin fragment 1 after thrombin stimulation; these values are similar to those previously reported. In contrast, the platelets of treated mice did not expose phosphatidylserine upon thrombin stimulation. Treatment with copper chelator did not affect platelet degranulation, as determined by surface exposure of P-selectin in flow cytometry. In addition, total phospholipid content and the ratio of outer to inner membrane phospholipids was not affected by treatment with copper chelator, suggesting that any reduction in detection of phosphatidylserine was due to reduction in exposure on the cell surface in response to an appropriate stimulus rather than reduced biosynthesis. Fluorescently conjugated Prothrombin fragment 1 or fluorescently conjugated antibody directed against phosphatidylserine were used as probes to follow the kinetics of phosphatidylserine exposure after the laser injury of cremaster muscle arterioles of a living mouse using high speed fluorescence intravital microscopy. Endogenous platelets were labeled with a fluorescently conjugated Fab fragment of an anti-CD41 antibody and fibrin deposition was measured using a fluorescently conjugated antibody that recognizes fibrin but not fibrinogen. We observed a 42% reduction (median of 18 thrombi, p=0.02) in Prothrombin fragment 1 binding and a 60% reduction (median of 27 thrombi, p=0.0002) in anti-phosphatidylserine binding after laser injury compared to untreated animals (n=58 thrombi). The accumulation of platelets during thrombus formation was not affected by the treatment when compared to untreated mice (p=0.4). On the other hand, fibrin deposition was reduced by 64% in treated mice (median of 38 thrombi, p=0.001) when compared to untreated animals (39 of thrombi). These data suggest that suppression of phosphatidylserine exposure reduces assembly of coagulation complexes resulting in a suboptimal concentration of thrombin for full fibrin generation but sufficient thrombin to activate platelets to yield a normal platelet thrombus. This emphasizes the importance of the exposure of anionic phospholipids as the surface for the colocalization of the coagulation complexes in vivo. DisclosuresNo relevant conflicts of interest to declare.

Sildenafil attenuates pulmonary inflammation and fibrin deposition, mortality and right ventricular hypertrophy in neonatal hyperoxic lung injury

BackgroundPhosphodiesterase-5 inhibition with sildenafil has been used to treat severe pulmonary hypertension and bronchopulmonary dysplasia (BPD), a chronic lung disease in very preterm infants who were mechanically ventilated for respiratory distress syndrome.MethodsSildenafil treatment was investigated in 2 models of experimental BPD: a lethal neonatal model, in which rat pups were continuously exposed to hyperoxia and treated daily with sildenafil (50–150 mg/kg body weight/day; injected subcutaneously) and a neonatal lung injury-recovery model in which rat pups were exposed to hyperoxia for 9 days, followed by 9 days of recovery in room air and started sildenafil treatment on day 6 of hyperoxia exposure. Parameters investigated include survival, histopathology, fibrin deposition, alveolar vascular leakage, right ventricular hypertrophy, and differential mRNA expression in lung and heart tissue.ResultsProphylactic treatment with an optimal dose of sildenafil (2 × 50 mg/kg/day) significantly increased lung cGMP levels, prolonged median survival, reduced fibrin deposition, total protein content in bronchoalveolar lavage fluid, inflammation and septum thickness. Treatment with sildenafil partially corrected the differential mRNA expression of amphiregulin, plasminogen activator inhibitor-1, fibroblast growth factor receptor-4 and vascular endothelial growth factor receptor-2 in the lung and of brain and c-type natriuretic peptides and the natriuretic peptide receptors NPR-A, -B, and -C in the right ventricle. In the lethal and injury-recovery model we demonstrated improved alveolarization and angiogenesis by attenuating mean linear intercept and arteriolar wall thickness and increasing pulmonary blood vessel density, and right ventricular hypertrophy (RVH).ConclusionSildenafil treatment, started simultaneously with exposure to hyperoxia after birth, prolongs survival, increases pulmonary cGMP levels, reduces the pulmonary inflammatory response, fibrin deposition and RVH, and stimulates alveolarization. Initiation of sildenafil treatment after hyperoxic lung injury and continued during room air recovery improves alveolarization and restores pulmonary angiogenesis and RVH in experimental BPD.

Anti-platelet drugs and outcome in severe infection: Clinical impact and underlying mechanisms

Platelet activation contributes to microvascular thrombosis and organ failure in systemic inflammation. We tested the hypothesis whether anti-platelet drugs might favourably affect outcome in patients at risk for organ failure as well as in a mouse model of endotoxin shock. Two hundred twenty-four consecutive patients who were admitted for community acquired pneumonia over a time period of 5 years to a University Hospital were enrolled; about 20% of whom received anti-platelet drugs (acetylsalicylic acid, thienopyridines) for secondary prevention of cardiovascular disease. Patients with anti-platelet drugs were about 12 years old but did not differ in SOFA score and routine laboratory parameters at admission. Logistic regression and 2 × 2 table analysis in age-matched subgroups indicated that anti-platelet drugs may reduce the need of intensive care treatment (odds ratio (OR) 0.32 [95% confidential interval: 0.10–1.00] and 0.19 [0.04–0.87], respectively). In age-matched subgroups, the use of anti-platelet drugs was also associated with a shorter stay in hospital (13.9 ± 6.2 vs. 18.2 ± 10.2 days; p < 0.02). In the animal model Balb/c mice were pre-treated with clopidogrel (added to drinking water) for 4 days prior to intraperitoneal (i.p.) administration of endotoxin (lipopolsaccharide (LPS) from Escherichia coli 0111:B4). Within the first 48 hours after LPS there were no differences between clopidogrel and control animals (n = 26 each) in macro-haemodynamics. However, clopidogrel abolished the LPS-induced drop in platelet count and reduced fibrin deposition in lung tissue. Using DNA microarray technology, we could show that clopidogrel suppressed endotoxin-induced up-regulation of inflammation-relevant genes, including arachidonate-5-lipoxygenase activating protein and leukotriene B4 receptor 1. According to our data a possible benefit of anti-platelet drugs in patients on risk for systemic inflammation and organ failure should be tested in a prospective trial.

Transgenic tissue-type plasminogen activator expression improves host defense during Klebsiella pneumonia.

Severe pneumonia is associated with a local inhibition of fibrinolysis in the lung as reflected by strongly reduced pulmonary plasminogen activator activity. To study the effect of elevation of local plasminogen activator activity during pneumonia caused by the common respiratory pathogen Klebsiella pneumoniae. Female C57Bl/6 mice were inoculated intranasally with a replication-defective adenoviral vector expressing human tissue-type plasminogen activator or a control vector 24 h before intranasal infection with K. pneumoniae. Mice infected with Klebsiella via the airways developed overt pneumonia, which was accompanied by a downregulation of pulmonary tissue-type plasminogen activator levels at protein and mRNA levels. Pulmonary overexpression of human tissue-type plasminogen activator resulted in increased fibrinolytic activity in the lungs during pneumonia, as indicated by higher D-dimer levels and reduced fibrin deposition. Interestingly, overexpression of tissue-type plasminogen activator markedly improved host defense against pneumonia: mice treated with the tissue-type plasminogen activator vector displayed less bacterial growth and dissemination, attenuated distant organ injury and a reduced mortality. These data demonstrate that local elevation of plasminogen activator activity in the lungs improves host defense against severe gram-negative pneumonia and sepsis.

Reduced Fibrin Deposition and Intravascular Thrombosis in hDAF Transgenic Pig Hearts Perfused With Tirofiban

Solid organ xenograft rejection is associated with vascular injury resulting at least in part in platelet activation, and rejected xenografts invariably demonstrate intravascular thrombosis and interstitial hemorrhage. Complement activation plays a prominent role in platelet-endothelial interaction. We tested the effects of platelet GPIIb/IIIa inhibitor tirofiban during perfusion of hDAF pig hearts. Using a working-heart model, nontransgenic and hDAF pig hearts were perfused with tirofiban or human blood only. Myocardial damage was determined by hemodynamic parameters (cardiac output, stroke work index) and creatine phosphokinase. Further monitoring included the assessment of complement factors (C3, C4), platelets, fibrinogen, ATIII, and graft histology. Tirofiban increased cardiac output (CO) and stroke work index (SWI) of nontransgenic pig hearts and improved superior CO and SWI of hDAF pig hearts. Although perfusion time of nontransgenic pig hearts was prolonged by tirofiban (196+/-65 min vs. 162+/-122 min), a similar effect in hDAF pig hearts (218+/-116 min vs. 222+/-30 min) could not be demonstrated. Tirofiban reduced consumption of C3 and C4 independently from hDAF. Depletion of fibrinogen was equally diminished by tirofiban and hDAF; the combination of both agents obtained no further reduction. ATIII consumption was most effectively inhibited by this combination. Intravascular fibrin deposition was reduced by tirofiban and hDAF, but particularly by the combination of the two agents. Improvement of heart performance and reduction of myocardial damage and intravascular thrombosis confirm a role of the GPIIb/IIIa inhibitor tirofiban for the prevention of hDAF pig heart rejection and xenograft function.

Scar Formation

Injured tissue produces fibrin, which makes a provisional extracellular matrix (ECM). Degradation of this fibrin ECM by plasmin is required for minimizing scar formation. Plasmin is produced by proteolytic cleavage of plasminogen by plasminogen activators (tPA and uPA), which are inhibited by plasminogen activator inhibitor-1 (PAI-1). Sachs et al . find that mice deficient in p75 neurotrophin receptor (p75 NTR ) exhibit reduced fibrin deposition after sciatic nerve crush injury or lipopolysaccharide-induced pulmonary fibrosis. The decrease in fibrin associated with sciatic nerve injury was due to increased expression and activity of tPA, whereas the decrease in fibrin associated with pulmonary fibrosis was due to decreased PAI-1 abundance. In wild-type mice after sciatic nerve injury, Schwann cells in which p75 NTR was abundant lacked tPA, which suggests that cells expressing p75 NTR did not express tPA. The increased proteolytic activity toward fibrin (measured by in situ zymography) associated with the nerve injury in p75 NTR −/− mice was prevented by application of a specific inhibitor of tPA or by crossing the mice with tPA −/− mice to produce mice deficient in both tPA and p75 NTR . When cultured on a fibrin gel, Schwann cells from the p75 NTR −/− mice degrade the fibrin, whereas wild-type Schwann cells form a monolayer. Studies with a transfected fibroblast cell line overexpressing p75 NTR showed increased abundance of transcripts for PAI-1 and decreased transcripts and activity of tPA. Transcriptional regulation of tPA required a cAMP (adenosine 3′,5′-monophosphate) pathway. Inhibition of tPA activity in the p75 NTR -overexpressing cells was prevented if cAMP concentrations were increased pharmacologically and cAMP concentrations were decreased in the p75 NTR -overexpressing cells. The injured sciatic nerves also showed increased cAMP in the p75 NTR −/− mice compared with injured nerves from wild-type mice. Coimmunoprecipitation studies and in vitro interaction studies indicated that p75 NTR and phosphodiesterase 4A5 (PDE4A5) interacted. The authors propose that recruitment of PDE4A5 to the membrane inhibited cAMP signaling, a hypothesis supported by decreased activation of a plasma membrane protein kinase A reporter in the p75 NTR -overexpressing cells. Tissue remodeling and scar formation appear to be additional ways that p75 NTR signaling is involved in response to injury. B. D. Sachs, G. S. Baillie, J. R. McCall, M. A. Passino, C. Schachtrup, D. A. Wallace, A. J. Dunlop, K. F. MacKenzie, E. Klussmann, M. J. Lynch, S. L. Sikorski, T. Nuriel, I. Tsigelny, J. Zhang, M. D. Houslay, M. V. Chao, K. Akassoglou, p75 neurotrophin receptor regulates tissue fibrosis through inhibition of plasminogen activation via a PDE4/cAMP/PKA pathway. J. Cell Biol. 177 , 1119-1132 (2007). [Abstract] [Full Text]

Neutrophil Interaction with the Hemostatic System Contributes to Liver Injury in Rats Cotreated with Lipopolysaccharide and Ranitidine

Cotreatment of rats with nontoxic doses of ranitidine (RAN) and lipopolysaccharide (LPS) causes liver injury, and this drug-inflammation interaction might be a model for idiosyncratic adverse drug responses in humans. Both polymorphonuclear neutrophils (PMNs) and the hemostatic system have been shown to be important in the injury. We tested the hypothesis that PMNs cause liver injury by interacting with the hemostatic system and producing subsequent hypoxia. In rats cotreated with LPS/RAN, PMN depletion by anti-PMN serum reduced fibrin deposition and hypoxia in the liver. PMN depletion also reduced the plasma concentration of active plasminogen activator inhibitor-1 (PAI-1), a major down-regulator of the fibrinolytic system. This suggests that PMNs promote fibrin deposition by increasing PAI-1 concentration. PMNs were activated in the livers of LPS/RAN-cotreated rats as evidenced by increased staining for hypochlorous acid-modified proteins generated by the myeloperoxidase-hydrogen peroxide-chloride system of activated phagocytes. Antiserum against the PMN adhesion molecule CD18 protected against LPS/RAN-induced liver injury. Because CD18 is important for PMN transmigration and activation, these results suggest that PMN activation is required for the liver injury. Furthermore, anti-CD18 serum reduced biomarkers of hemostasis and hypoxia, suggesting the necessity for PMN activation in the interaction between PMNs and the hemostatic system/hypoxia. Liver injury, liver fibrin, and plasma PAI-1 concentration were also reduced by eglin C, an inhibitor of proteases released by activated PMNs. In summary, PMNs are activated in LPS/RAN-cotreated rats and participate in the liver injury in part by contributing to hemostasis and hypoxia.

The diverse role of inhaled nitric oxide in experimental BPD: reduced fibrin deposition and improved lung growth

dramatic improvements in neonatal care over the past decade have improved the overall survival of premature infants; however, more than one-third of infants born at less than 1,250 g will ultimately be diagnosed with bronchopulmonary dysplasia (BPD), a complex, multifactorial disease ([21][1]).

Inhaled nitric oxide attenuates pulmonary inflammation and fibrin deposition and prolongs survival in neonatal hyperoxic lung injury

Administration of inhaled nitric oxide (iNO) is a potential therapeutic strategy to prevent bronchopulmonary dysplasia (BPD) in premature newborns with respiratory distress syndrome. We evaluated this approach in a rat model, in which premature pups were exposed to room air, hyperoxia, or a combination of hyperoxia and NO (8.5 and 17 ppm). We investigated the anti-inflammatory effects of prolonged iNO therapy by studying survival, histopathology, fibrin deposition, and differential mRNA expression (real-time RT-PCR) of key genes involved in the development of BPD. iNO therapy prolonged median survival 1.5 days (P = 0.0003), reduced fibrin deposition in a dosage-dependent way up to 4.3-fold (P < 0.001), improved alveolar development by reducing septal thickness, and reduced the influx of leukocytes. Analysis of mRNA expression revealed an iNO-induced downregulation of genes involved in inflammation (IL-6, cytokine-induced neutrophilic chemoattractant-1, and amphiregulin), coagulation, fibrinolysis (plasminogen activator inhibitor 1 and urokinase-type plasminogen activator receptor), cell cycle regulation (p21), and an upregulation of fibroblast growth factor receptor-4 (alveolar formation). We conclude that iNO therapy improves lung pathology and prolongs survival by reducing septum thickness, inhibiting inflammation, and reducing alveolar fibrin deposition in premature rat pups with neonatal hyperoxic lung injury.

Ancrod: A Potential Treatment for Acute, Ischemic Stroke from Snake Venom

The direct effect of ancrod is defibrinogenation. This results in several secondary effects that differentiate ancrod from typical thrombolytics such as rt-PA. The combined actions of indirect fibrinolysis, reduced fibrin deposition, and decreased blood viscosity represent a constellation of actions that should be beneficial in a variety of thrombotic conditions. The clinical condition explored most thoroughly in recent years with ancrod has been its acute application to ischemic stroke. Two clinical studies (one of them with a 6 h time-to-treatment window) have demonstrated significant benefit in increasing the proportion of patients alive and disability-free at 3 months, but higher doses of the drug appear to increase the risk of symptomatic intracranial hemorrhage and reduce efficacy. Although the evidence is not conclusive, it suggests that, with modified dosing, ancrod could yield a favorable risk–benefit ratio in stroke by limiting the risk of symptomatic intracranial hemorrhage.

Factor Xa inhibition by immobilized recombinant tissue factor pathway inhibitor

The recombinant form of the endogenous anticoagulant, tissue factor pathway inhibitor (rTFPI), is a potent inhibitor of Factor Xa (FXa) and the tissue factor–factor VIIa (TF:VIIa) complex. Surface-immobilized rTFPI reduces the thrombogenicity and intimal hyperplasia associated with synthetic vascular grafts in animal models and specifically reduces fibrin deposition on collagen-impregnated Dacron grafts from native blood in an in vitro flow model. The FXa inhibitory capacity of rTFPI in the bulk phase has been demonstrated in static systems and immobilized rTFPI reduces fibrin deposition in whole blood in vitro and animal studies; however, the specific mode of this anticoagulation has not been studied. Therefore, a comparison was made between the FXa binding capacity of two forms of immobilized rTFPI, i.e., passively adsorbed and covalently bound. The rTFPI-coated surfaces were evaluated using a parallel-plate flow reactor and comparing the amount of FXa exiting the flow chamber after exposure to an rTFPI-coated versus an uncoated plate. The results demonstrate that adsorbed rTFPI exhibits increased binding capacity (1.5–3.6 times) the expected stoichiometry via interactions with the C-terminus, whereas covalently-bound rTFPI interacts with FXa in a 1:1 stoichiometry. Thus, the results imply that specific FXa inhibition is a key component of the anticoagulant effect of rTFPI-coated surfaces and that passive adsorption of rTFPI to glass surfaces produces a more effective coating than covalent binding of rTFPI.