Fibrinolytic Cascade Research Articles

Thrombosis and Hemorrhage in Diabetic Retinopathy: A Perspective from an Inflammatory Standpoint.

Retinal ischemia and hemorrhage are hallmarks of worsening diabetic retinopathy, which can lead to neovascularization, macular edema, and severe vision loss. Although diabetes alters expression of clotting factors and their activities, and increases retinal microthromboses, the effects of thrombotic processes on the pathogenesis of diabetic retinopathy are not fully understood. In addition to the roles of coagulation and fibrinolytic cascades in thrombosis and hemostasis, components in these systems also mediate multiple effects on the vasculature that promote inflammation. Plasma kallikrein, thrombin, and urokinase are increased in diabetic retinopathy, and exert proinflammatory effects that contribute to retinal vascular dysfunction. The accumulation and activation of these and additional coagulation factors in the vitreous due to hemorrhage and chronic retinal injury in the diabetic retina may contribute to worsening of retinal inflammation and capillary dysfunction, which lead to retinal ischemia and edema. Further understanding of the role for specific coagulation factors in diabetic retinopathy may suggest new therapeutic opportunities for this vision-threatening disease.

Glioma-derived plasminogen activator inhibitor-1 (PAI-1) regulates the recruitment of LRP1 positive mast cells.

Glioblastoma (GBM) is a high-grade glioma with a complex microenvironment, including various inflammatory cells and mast cells (MCs) as one of them. Previously we had identified glioma grade-dependent MC recruitment. In the present study we investigated the role of plasminogen activator inhibitor 1 (PAI-1) in MC recruitment.PAI-1, a primary regulator in the fibrinolytic cascade is capable of forming a complex with fibrinolytic system proteins together with low-density lipoprotein receptor-related protein 1 (LRP1). We found that neutralizing PAI-1 attenuated infiltration of MCs. To address the potential implication of LRP1 in this process, we used a LRP1 antagonist, receptor-associated protein (RAP), and demonstrated the attenuation of MC migration. Moreover, a positive correlation between the number of MCs and the level of PAI-1 in a large cohort of human glioma samples was observed. Our study demonstrated the expression of LRP1 in human MC line LAD2 and in MCs in human high-grade glioma. The activation of potential PAI-1/LRP1 axis with purified PAI-1 promoted increased phosphorylation of STAT3 and subsequently exocytosis in MCs.These findings indicate the influence of the PAI-1/LRP1 axis on the recruitment of MCs in glioma. The connection between high-grade glioma and MC infiltration could contribute to patient tailored therapy and improve patient stratification in future therapeutic trials.

Activation of markers of inflammation, coagulation and fibrinolysis in musculoskeletal trauma.

BackgroundTraumatic injury induces changes in mediators of inflammation and coagulation, but the pivotal roles of inflammation and coagulation has not been precisely clarified. Therefore we have studied markers of inflammation and coagulation after a standardized musculoskeletal trauma like total hip replacement surgery.MethodsWe allocated 21 patients aged 50 to 84 years who underwent total hip replacement surgery. Releases of TNF-α, IL-1β, IL-6, IL-8 and IL-10 and protrombin fragment F1.2 and plasmin-antiplasmin complex (PAP) were examined during surgery and up 6 days postoperatively, and systemic releases were compared to pre-operative values. Surgery induced significant increments in serum levels of IL-6 at 6 hours and at 1 day after surgery and in levels of IL-8 at 6 hours after surgery. There were no significant changes in serum levels of TNF-α, IL-1β or IL-10. There were significant increments in blood levels of F1.2 and PAP up to 6 days postoperatively with highest levels at 6 hours after surgery. There were only week correlations between IL-6 and IL-8 and F1.2 and PAP.ConclusionMajor musculoskeletal surgery causes changes of the inflammatory, coagulatory and fibrinolytic cascades in stable patients, but with no correlations between inflammation and coagulation and fibrinolysis.

Abstract 38: Identification of Thrombomodulin Binding Sites on Thrombin Activatable Fibrinolysis Inhibitor that Mediate Accelerated Activation by Thrombin

Thrombin activatable fibrinolysis inhibitor (TAFI) is a human plasma zymogen that provides a molecular connection between coagulation and fibrinolysis. TAFI is activated through proteolytic cleavage by thrombin, thrombin in complex with the endothelial cell cofactor thrombomodulin (TM), or plasmin to generate the enzyme activated TAFI (TAFIa). TAFIa possesses basic carboxypeptidase activity which down-regulates fibrin clot lysis by removing carboxyl-terminal lysine residues from partially degraded fibrin thereby attenuating the positive feedback in the fibrinolytic cascade. Accumulating evidence from several studies suggests that TM and TAFI make direct contacts such that the enhancement of TAFI activation by TM is through binding at sites remote from the activation site (Arg92-Ala93). The elements of TAFI structure that allow accelerated activation of thrombin by TM are largely unknown. Therefore, alanine scanning mutagenesis of surface-exposed charged residues on TAFI was used to identify sites that mediate acceleration of activation by TM and which thus may indicate sites where TM binds to TAFI directly. The rates of activation by thrombin of the variants was measured in the presence or absence of TM. The variants R12A, E28A and R15A exhibited the lowest enhancement of catalytic efficiency in the presence of TM with decreases of 3.0-fold, 3.2-fold and 2.6-fold, respectively, compared to wild-type. The variants D75A/E77A/D78A, E106A, E112A/E116A, R12A/R15A and D54A/D56A showed about half the extent of rate enhancement of wild-type. On the other hand, the variants E99A and E116G exhibited approximately 2.0 to 2.5- fold increases in rate enhancement which may indicate increased binding between TAFI and TM. We determined the antifibrinolytic potential of each variant using an in vitro plasma clot lysis assay. The variants that showed reduced activation by thrombin/TM correspondingly showed impaired stimulation of antifibrinolytic potential by TM. Overall, the data indicate that the variants that showed the strongest TM dependence were focused around the activation peptide which would be near where the C-loop of EGF-3 of TM would contact TAFI. These residues contribute to the increased efficiency of TAFI activation by thrombin.

The multifunctional role of the pallilysin‐associated Treponema pallidum protein, Tp0750, in promoting fibrinolysis and extracellular matrix component degradation

The mechanisms that facilitate dissemination of the highly invasive spirochaete, Treponema pallidum, are incompletely understood. Previous studies showed the treponemal metalloprotease pallilysin (Tp0751) possesses fibrin clot degradation capability, suggesting a role in treponemal dissemination. In the current study we report characterization of the functionally linked protein Tp0750. Structural modelling predicts Tp0750 contains a von Willebrand factor type A (vWFA) domain, a protein-protein interaction domain commonly observed in extracellular matrix (ECM)-binding proteins. We report Tp0750 is a serine protease that degrades the major clot components fibrinogen and fibronectin. We also demonstrate Tp0750 cleaves a matrix metalloprotease (MMP) peptide substrate that is targeted by several MMPs, enzymes central to ECM remodelling. Through proteomic analyses we show Tp0750 binds the endothelial fibrinolytic receptor, annexin A2, in a specific and dose-dependent manner. These results suggest Tp0750 constitutes a multifunctional protein that is able to (1) degrade infection-limiting clots by both inhibiting clot formation through degradation of host coagulation cascade proteins and promoting clot dissolution by complexing with host proteins involved in the fibrinolytic cascade and (2) facilitate ECM degradation via MMP-like proteolysis of host components. We propose that through these activities Tp0750 functions in concert with pallilysin to enable T. pallidum dissemination.

Residual vein thrombosis and onset of post-thrombotic syndrome: Influence of the 4G/5G polymorphism of plasminogen activator inhibitor-1 gene

Plasminogen activator inhibitor-1 (PAI-1) is the most important inhibitor of plasminogen activator. The functional 4G/5G polymorphism of the gene coding for PAI-1 may affect PAI-1 plasmatic activity, influencing the imbalance between coagulation and fibrinolysis cascades. In this prospective cohort analytic study, we investigated the role of this single nucleotide polymorphism in the persistence of thrombotic lesion and the occurrence of post-thrombotic syndrome. In a group of 168 patients with post-surgical deep vein thrombosis of the legs, we analyzed the 4G/5G polymorphism in the promoter of PAI-1 gene and plasmatic PAI-1 activity. Enrolled patients were divided in two groups: patients with 4G/5G polymorphism and increased PAI-1 activity (n=85) and patients without 4G/5G polymorphism and normal PAI-1 activity (n=83). All patients were treated according to current protocols and re-examined after 3, 12 and 36 months in order to evaluate the persistence of thrombotic lesion and the occurrence of post-thrombotic syndrome. We found a significantly increased PAI activity in carrier of the 4G allele, who experienced much more frequently a persistence of thrombosis after 3, 12 and 36 months and/or the development of post-thrombosis syndrome, in spite of the anticoagulant treatment. These data not only confirm the role played by PAI-1 activity and by the 4G/5G SNP of the PAI-1 gene, but also suggest that current therapeutic protocols, recommending the administration of low weight molecular heparin and oral anticoagulant for the treatment of deep vein thrombosis, could be non sufficient for patients genetically predisposed to a less efficient clot lysis.

Lipoprotein (a): structure, pathophysiology and clinical implications.

The chemical structure of lipoprotein (a) is similar to that of LDL, from which it differs due to the presence of apolipoprotein (a) bound to apo B100 via one disulfide bridge. Lipoprotein (a) is synthesized in the liver and its plasma concentration, which can be determined by use of monoclonal antibody-based methods, ranges from < 1 mg to > 1,000 mg/dL. Lipoprotein (a) levels over 20-30 mg/dL are associated with a two-fold risk of developing coronary artery disease. Usually, black subjects have higher lipoprotein (a) levels that, differently from Caucasians and Orientals, are not related to coronary artery disease. However, the risk of black subjects must be considered. Sex and age have little influence on lipoprotein (a) levels. Lipoprotein (a) homology with plasminogen might lead to interference with the fibrinolytic cascade, accounting for an atherogenic mechanism of that lipoprotein. Nevertheless, direct deposition of lipoprotein (a) on arterial wall is also a possible mechanism, lipoprotein (a) being more prone to oxidation than LDL. Most prospective studies have confirmed lipoprotein (a) as a predisposing factor to atherosclerosis. Statin treatment does not lower lipoprotein (a) levels, differently from niacin and ezetimibe, which tend to reduce lipoprotein (a), although confirmation of ezetimibe effects is pending. The reduction in lipoprotein (a) concentrations has not been demonstrated to reduce the risk for coronary artery disease. Whenever higher lipoprotein (a) concentrations are found, and in the absence of more effective and well-tolerated drugs, a more strict and vigorous control of the other coronary artery disease risk factors should be sought.

Characterization of Chronic Subdural Hematoma Fluid Proteome

Chronic subdural hematoma (CSDH) is commonly caused by head injury, but the progression of CSDH is recognized as an inflammatory process. The protein composition of the CSDH fluid has not been fully elucidated, nor has the contribution of its components to the enlargement of the hematoma cavity and to its chronic manifestation. To characterize the protein content of CSDH fluid and study the differences between CSDH fluid and serum to identify proteins putatively involved in the pathogenesis of CSDH. CSDH fluid and serum of 5 patients were investigated with 2-dimensional gel electrophoresis followed by glycosylation-specific fluorescence staining and mass spectrometry. Two-dimensional gel electrophoresis revealed approximately 1100 protein spots in the CSDH fluid. We identified 213 spots representing 57 different proteins, most of which were glycosylated. The comparison with serum revealed 11 proteins with elevated levels in the CSDH fluid including carbonic anhydrase I, catalase, ferritin light chain, fibrinogen (α, β, γ), hemoglobin (α, β), malate dehydrogenase, peroxiredoxin 2, and transforming growth factor-β-induced protein ig-h3. The levels of haptoglobin and a fragment of complement C4 were decreased. Changes in spot positions were detected for apolipoprotein A1 and a fragment of complement C3. The hematoma fluid originates mainly from blood and the results suggest the involvement of coagulation and fibrinolysis cascades. However, proteins with a potential role in CSDH pathogenesis were detected including carbonic anhydrase I, transforming growth factor-β-induced protein ig-h3, and the altered components of the complement system. Inflammation and fibrosis indicate targets for further studies in the pathogenesis of CSDH.

Abstract 52: Early Elevation of Plasma Plasminogen Activator Inhibitor-1 is Associated with Vasospasm following Subarachnoid Hemorrhage

Background: While the mechanism of early brain injury following subarachnoid hemorrhage (SAH) remains unclear, animal studies suggest that SAH is associated with significant changes in the fibrinolytic cascade. Human data shows that SAH-related brain ischemia is associated with the polymorphism in plasminogen activator inhibitor 1 (PAI-1) promotor linked to higher plasma PAI-1 levels. However, human plasma PAI-1 has not been previously studied in SAH. We hypothesize that elevated plasma PAI-1 is associated with VSP and poor outcome in SAH. Methods: In a prospective cohort of 61 SAH subjects, we compared plasma PAI-1 levels by ELISA (R&amp;D Systems) on post-SAH days 1, 5, 7 and 14 with respect to angiographic VSP and to functional outcome at 3- and 6-months. Angiographic VSP was defined as &gt;50% reduction in vessel caliber on post-SAH day 6-8 angiography. Poor functional outcome was defined as a modified Rankin score &gt;2. Biomarker levels were compared using Wilcoxon rank sum test. Bonferroni correction was applied for multiple comparisons. Results: Subjects with (n=25) and without VSP (n=36) were comparable in gender, Fisher and Hunt and Hess (HH) grade distribution, aneurysm treatment modality, and incidence of hydrocephalus. Subjects with VSP had lower mean age (50 vs. 56). Elevated plasma PAI-1 on post-SAH day 1 was strongly association with VSP (3.28 vs. 1.82 ng/mL; p=.0058) and showed a trend towards association with VSP on post-SAH day 5 (2.65 vs 1.83 ng/mL, p=0.05). Plasma PAI-1 levels were not associated with functional outcome at 3 or 6 months. Elevated plasma PAI-1 on post-SAH day 1 remained significantly associated with VSP (p=0.0155) after adjustment for age and HH and Fisher grades by logistic regression. Conclusion: Early elevation of plasma PAI-1 on post-SAH day 1 is strongly and independently associated with angiographic VSP in this small prospective SAH cohort and may be a candidate biomarker for VSP. Future mechanistic studies with simultaneous measurements of tissue plasminogen activator (tPA) and tPA-PAI complex and larger clinical studies are necessary to understand the utility of PAI-1 as potential biomarker in SAH.

Anticoagulation in patients with liver cirrhosis: complication or therapeutic opportunity?

Like many physiological systems, the coagulation cascade is dependent upon a dynamic interplay, which is between multiple opposing procoagulant and anticoagulant drivers so as to maintain physiological haemostasis and prevent...

Decreasing blood loss and the need for transfusion after CABG surgery: a double-blind randomized clinical trial of topical tranexamic acid

Reopening sternotomy to control bleeding after coronary artery bypass grafting surgery (CABG) has been reported in 2%-7% of cases. Platelet dysfunction and activation of fibrinolytic cascade are the common causes of bleeding after on-pump CABG. Different antifibrinolytic drugs have been used to reduce bleeding. In this study, we aimed to investigate the efficacy of topical tranexamic acid in reducing postoperative mediastinal bleeding after CABG. Materials and methods: This was a double-blind placebo-controlled randomized clinical trial on 40 patients as the control and another 40 patients as the study group. On completion of CABG before sternotomy wound closure, tranexamic acid (2 g/20 mL) or placebo (20 mL of saline) was diluted in 500 mL of warm saline (37 °C), poured into the pericardial cavity, and left for 5 min. Results: There was no significant difference in baseline demographic data and laboratory results between the 2 groups (P > 0.05). Mediastinal bleeding and packed red cell transfusion requirements were significantly lower in the study group (P =< 0.01). There were no complications related to topical tranexamic such as mortality, myocardial infarction, cerebrovascular accident, seizure, or renal failure. Conclusion: Topical tranexamic acid can reduce mediastinal bleeding and packed red cell transfusion requirements after CABG.

A Preliminary Study of Platelet Activation After Embolization of Marrow Contents

Intravasation of bone marrow contents into venous circulation and pulmonary embolization after intramedullary nailing may be coupled with the activation of coagulation and fibrinolytic cascades. The objective of this study was to assess hemostatic response to pulmonary extravasated marrow contents. We hypothesize that activation of platelet activity and the coagulation cascade may occur after embolization of marrow contents in an experimental animal model of intramedullary nailing. Fifteen New Zealand white male rabbits were randomly assigned to control or fat embolism (FE) groups. In the FE group (n = 8), femurs were surgically instrumented with retrograde intramedullary nails and pressurized with bone cement. In the control group (n = 7), a sham knee incision was made that was immediately closed without drilling, reaming, or pressurization. Fibrinogen, D-dimer latex screen assay, 1 stage prothrombin time, and activated partial thromboplastin time were analyzed. As the main platelet activation indicators, the marker Annexin-V percent binding increased in the FE group at 2 hours (P = 0.04) and 4 hours (P = 0.04), and the marker CD62P percent expression increased in the FE group at 2 hours (P = 0.04). This preliminary study showed that pressurization of marrow and intravasation of fat and marrow products cause activation of platelets and the coagulation cascade, with or without tissue trauma. This may be relevant to the treatment of multiply injured patients with prior respiratory and coagulation abnormalities. A future larger study may be needed.

Transcriptome Temporal and Functional Analysis of Liver Regeneration Termination

Decades of liver regeneration studies still left the termination phase least elucidated. However regeneration ending mechanisms are clinicaly relevant. We aimed to analyse the timing and transcriptional control of the latest phase of liver regeneration, both controversial. Male Wistar rats were subjected to 2/3 partial hepatectomy with recovery lasting from 1 to 14 days. Time-series microarray data were assessed by innovative combination of hierarchical clustering and principal component analysis and validated by real-time RT-PCR. Hierarchical clustering and principal component analysis in agreement distinguished three temporal phases of liver regeneration. We found 359 genes specifically altered during late phase regeneration. Gene enrichment analysis and manual review of microarray data suggested five pathways worth further study: PPAR signalling pathway; lipid metabolism; complement, coagulation and fibrinolytic cascades; ECM remodelling and xenobiotic biotransformation. Microarray findings pertinent for termination phase were substantiated by real-time RT-PCR. In conclusion, transcriptional profiling mapped late phase of liver regeneration beyond 5(th) day of recovery and revealed 5 pathways specifically acting at this time. Inclusion of longer post-surgery intervals brought improved coverage of regeneration time dynamics and is advisable for further works. Investigation into the workings of suggested pathways might prove helpful in preventing and managing liver tumours.

Plasmin as a proinflammatory cell activator

The serine protease plasmin generated from its zymogen plasminogen is best known for its function as a key enzyme of the fibrinolytic cascade. However, beyond fibrinolysis, plasmin has a number of crucial functions in a variety of processes, including inflammation. Various cells can bind plasminogen and plasmin via plasminogen-binding sites exposing a C-terminal lysine. Plasmin, generated as a result of plasminogen activation at the cell surface, is protected from its physiological inhibitors. Apart from its ability to facilitate cell migration in tissues, plasmin is capable of triggering signaling, which depends on cellular binding via its lysine-binding sites and its proteolytic activity. Plasmin-induced signaling affects various functions of monocytes, macrophages, DCs, and others, with the list of affected cells still growing. In vitro and in vivo studies have demonstrated the ability of plasmin to stimulate the production of cytokines, ROS, and other mediators, thereby contributing to inflammation. Plasmin-induced chemotaxis of monocytes and DCs indicates that it is also a potent chemoattractant for immune cells. Therefore, excessive activation of plasmin in chronic inflammatory or autoimmune diseases might exacerbate the activation of inflammatory cells and the pathogenesis of the disease. This review focuses on the available evidence for physiological and pathophysiological roles the serine protease plasmin in inflammatory processes.

Chemerin Bioactivity Is Regulated by Factor XIa: A Novel Interface Linking Between Coagulation, Hemostasis and Immunity

Abstract 2258Proteolytic modulation of chemokines by extracellular proteases, either released upon leukocyte degranulation or activated by the coagulation and fibrinolytic cascades, plays an important role in inflammation and immunity. Chemerin is a novel chemoattractant involved in innate and adaptive immunity and adipogenic differentiation via its receptor, CMKLR1, expressed on dendritic cells, macrophages and adipocytes. Chemerin circulates as an inactive precursor (chem163S) in blood whose bioactivity is regulated through proteolytic processing at its C-terminus. The most potent form of chemerin (chem157S) can be generated from chem163S in vitro by elastase directly or by sequential cleavages by plasmin to chem158K and then plasma carboxypeptidase-2 (CPB2, also termed thrombin-activatable fibrinolysis inhibitor TAFIa) or CPN to chem157S. Thus proteolytic processing is essential for chemerin activation. We have recently developed specific ELISAs for chem163S, 158K and 157S and showed that chem158K is present in plasma at a low level compared to chem163S. In contrast chem158K is the dominant form in synovial fluids of patients with inflammatory arthritis, suggesting that cleavage of chem163S at position 158K is a major step in the activation of chemerin in vivo. Although chem158K is present in vivo, it is unknown if it is generated by plasmin with a second cleavage by CPB2 or CPN to generate the active isoform, chem157S, as CPB2 inhibits plasmin generation. In this study we screened a panel of 21 proteases involved in coagulation, fibrinolysis and inflammation to determine which one could cleave the chemerin precursor chem163S to modulate its bioactivity. We carried out in vitro enzyme assays using 100 nM enzyme and 10 μM chem163S as a substrate, determining the results by SDS-PAGE and mass spectroscopy. Among the 21 proteases tested, cathepsin G, elastase, tryptase, plasmin, chymase, protease 3 and factor XIa could cleave chem163S. Mass spectroscopy identified tryptase, plasmin and factor XIa as enzymes that can digest chem163S to generate chem158K. Based on our previous observation that platelets are a rich cellular source of chemerin and release partially cleaved chemerin upon thrombin activation, we focused on factor XIa and compared its potency to plasmin. Factor XIa converted chem163S into chem158K in a dose-dependent manner whereas incubation with high concentrations of plasmin (300 nM) resulted in production of a non-specific smaller fragment, showing that the specificity of factor XIa was better than that of plasmin. Time-course studies showed that factor XIa and plasmin cleaved chem163S at comparable rates. Finally, we found that the fraction of chem158K to levels of total chemerin as determined by ELISA in factor XI-deficient plasma was lower than that in factor II-, IX-, X-deficient plasmas as well as normal pooled plasma. These data demonstrated that factor XIa could be the enzyme responsible for the first step of chemerin activation in the plasma compartment, ultimately leading to downstream inflammatory responses and provide a novel molecular link between coagulation, hemostasis and immunity. Disclosures:No relevant conflicts of interest to declare.

A Comparison of Inhibitors for Thrombin, FXa, FXIa, and FXIIa in Whole Blood Coagulation and Fibrinolysis Using Thromboelastography

Abstract▪2256▪This icon denotes a clinically relevant abstractBlood coagulation is marked by sequential proteolytic activation of the coagulation factors and complex feedback modulation which is central to haemostasis. The fine balance between the coagulation cascade and the fibrinolytic system is also an integral component in haemostasis. Multiple coagulation factors, including FIIa (thrombin), FXa, FXIa, and FXIIa, are attractive targets for treating thrombosis, and novel inhibitors for several targets are already in late stage development or on the market. However, the responsiveness of these inhibitors to intrinsic vs. extrinsic cues warrants further study because in different disease settings the relative signaling intensities from the two pathways could be different in vivo. It is also poorly understood how these inhibitors might differentially modulate fibrinolytic activity, and a dysregulated fibrinolytic cascade is a potential contributing factor to both thrombosis and bleeding. We therefore sought to gain insight by comparing effects of four agents (dabigatran, apixaban, FXIa inhibitory antibody (FXIa Ab), and FXIIa inhibitory antibody (FXIIa Ab)) in whole blood coagulation and fibrinolysis in vitro using thromboelastography (TEG).We first established optimal clot-triggering conditions for TEG by using either a low concentration of kaolin (approximately 100-fold less kaolin than in standard aPTT assay), or a low concentration of tissue factor (TF) (1:8000 dilution of Recombiplastin in blood), along with recalcification. We then performed titration of tissue plasminogen-activator (tPA) with these clotting conditions and identified a tPA concentration of 125 ng/ml as optimal in inducing a fibrinolytic phase without significantly altering the clotting phase. Compound profiling was then pursued by dose titration with the individual agent in normal human donor’s blood and tested TEG responses to kaolin+/− tPA and TF+/−tPA. Key TEG parameters that were assessed include R (clotting time), reflecting anticoagulant effect, MA (maximal amplitude), representing clot strength, and Ly30 (percent lysis at 30 minutes after MA), reflecting fibrinolytic activity.For clotting time, dabigatran and apixaban both delayed R in response to both kaolin and TF. FXIa Ab and FXIIa Ab delayed R only in response to kaolin and not to TF. This is consistent with the understanding that thrombin and FXa are in the common pathway, and FXIa and FXIIa are in the intrinsic pathway. For agents that affected the response to kaolin, stack-ranking of the magnitude of effect was FXIa Ab > dabigatran > apixaban > FXIIa Ab. For agents that affected the response to TF, dabigatran was slightly more effective than apixaban. Regarding clot strength, none of the agents appreciably altered MA in the absence of tPA; in the presence of tPA, dabigatran, apixaban, and FXIa Ab reduced MA to a greater extent than FXIIa Ab. Regarding fibrinolytic activity, none of the agents modulated Ly30 in the absence of tPA, consistent with the notion that these inhibitors alone are not fibrinolytic agents. However, in the presence of tPA, dabigatran, apixaban, FXIa Ab, but not FXIIa Ab, enhanced Ly30, suggesting that inhibition of thrombin, FXa, or FXIa may potentiate tPA-induced fibrinolysis.In summary, each inhibitory agent examined with this whole blood spike-in paradigm exhibited a distinct profile of effects on clotting time, clot strength and fibrinolysis with intrinsic and extrinsic cues in the presence and absence of tPA. Because the thrombotic cues in different patient segments are highly complex, and the endogenous fibrinolytic activities in different tissues may vary considerably, our results provide a benchmarking dataset for these inhibitory agents and their respective targets and represent a step forward in differentiating specific agents and targets in our effort to optimize safety and efficacy with novel anticoagulant therapies. Disclosures:Chen:Merck Research Laboratories: Employment. Wu:Merck Research Laboratories: Employment. Xu:Merck Research Laboratories: Employment. Ogletree:Merck Research Laboratories: Employment. Plump:Merck Research Laboratories: Employment. Graziano:Merck Research Laboratories: Employment. Wang:Merck Research Laboratories: Employment.

Chemerin and its receptors in leukocyte trafficking, inflammation and metabolism

Chemerin was isolated as the natural ligand of the G protein-coupled receptor ChemR23. Chemerin acts as a chemotactic factor for leukocyte populations expressing ChemR23, particularly immature plasmacytoid dendritic cells, but also immature myeloid DCs, macrophages and natural killer cells. Chemerin is expressed by epithelial and non-epithelial cells as an inactive precursor, present at nanomolar concentrations in plasma. Processing of the precursor C-terminus is required for generating bioactive forms of chemerin. Various proteases mediate this processing, including neutrophil serine proteases and proteases from coagulation and fibrinolytic cascades. ChemR23-expressing cells are recruited in human inflammatory diseases, such as psoriasis and lupus. In animal models, both pro-inflammatory and anti-inflammatory roles of chemerin have been reported. Recently, two other receptors for chemerin were described, GPR1 and CCRL2, but their functional relevance is largely unknown. Both chemerin and ChemR23 are also expressed by adipocytes, and the emerging role of chemerin as an adipokine regulating lipid and carbohydrate metabolism is an area of intense research.

Leptin and resistin induce increased procoagulability in diabetes mellitus

BackgroundPatients with diabetes mellitus (DM) suffer from an increased risk of cardiovascular events caused by thrombotic conditions. Adipose tissue might play a crucial role in this pathogenesis by synthesis of procoagulant mediators. This study was performed to elucidate the role of the adipocytokines leptin and resistin in the development of hypercoagulability and hypofibrinolysis under diabetic conditions. MethodsSixty two patients with or without DM were included in our study to measure leptin, resistin and tissue factor (TF) plasma concentrations. Moreover, flow chamber experiments were performed to assess factor Xa and plasmin activity on the surface of HUVECs. Western blot and real-time PCR were performed to determine mRNA and protein expression of main factors of the coagulation and fibrinolytic system. ResultsPatients with diabetes showed increased levels of leptin and resistin (leptin: 25.69±13.9 vs. 15.98±17.5ng/mL, p<0.05; resistin: 2.61±0.6 vs. 1.19±0.7ng/mL, p<0.05), which were positively correlated with TF. In vitro, leptin and resistin induced increased factor Xa activity (leptin: 4.29±0.57-fold, p<0.05; resistin 4.19±0.7-fold, p<0.05 vs. control) on HUVECs as also reflected by elevated TF mRNA and protein expression. Moreover, stimulatory (plasminogen activator inhibitor 1) and inhibitory (tissue plasminogen activator) mediators of the fibrinolytic cascade were induced by leptin and resistin, leading to a balanced plasmin activity regulation. ConclusionsLeptin and resistin lead to a procoagulant state in HUVECs by inducing TF expression. This mechanism might be one explanation for the prothrombotic state observed under diabetic conditions.

SecretedBacillus anthracisproteases target the host fibrinolytic system

The fibrinolytic system is often the target for pathogenic bacteria, resulting in increased fibrinolysis, bacterial dissemination, and inflammation. The purpose of this study was to explore whether proteases NprB and InhA secreted by Bacillus anthracis could activate the host's fibrinolytic system. NprB efficiently activated human pro-urokinase plasminogen activator (pro-uPA), a key protein in the fibrinolytic cascade. Conversely, InhA had little effect on pro-uPA. Plasminogen activator inhibitors (PAI)-1, 2 and the uPA receptor were also targets for NprB in vitro. InhA efficiently degraded the thrombin-activatable fibrinolysis inhibitor (TAFI) in vitro. Mice infected with B. anthracis showed a significant decrease in blood TAFI levels. In another mouse experiment, animals infected with isogenic inhA deletion mutants restored TAFI levels, while the levels in the parent strain decreased. We propose that NprB and InhA may contribute to the activation of the fibrinolytic system in anthrax infection.

Fungal Proteases and Their Pathophysiological Effects

Proteolytic enzymes play an important role in fungal physiology and development. External digestion of protein substrates by secreted proteases is required for survival and growth of both saprophytic and pathogenic species. Extracellular serine, aspartic, and metalloproteases are considered virulence factors of many pathogenic species. New findings focus on novel membrane-associated proteases such as yapsins and ADAMs and their role in pathology. Proteases from fungi induce inflammatory responses by altering the permeability of epithelial barrier and by induction of proinflammatory cytokines through protease-activated receptors. Many fungal allergens possess proteolytic activity that appears to be essential in eliciting Th2 responses. Allergenic fungal proteases can act as adjuvants, potentiating responses to other allergens. Proteolytic enzymes from fungi contribute to inflammation through interactions with the kinin system as well as the coagulation and fibrinolytic cascades. Their effect on the host protease-antiprotease balance results from activation of endogenous proteases and degradation of protease inhibitors. Recent studies of the role of fungi in human health point to the growing importance of proteases not only as pathogenic agents in fungal infections but also in asthma, allergy, and damp building related illnesses. Proteolytic enzymes from fungi are widely used in biotechnology, mainly in food, leather, and detergent industries, in ecological bioremediation processes and to produce therapeutic peptides. The involvement of fungal proteases in diverse pathological mechanisms makes them potential targets of therapeutic intervention and candidates for biomarkers of disease and exposure.