Hepatocyte tissue factor contributes to the hypercoagulable state in a mouse model of chronic liver injury

Patients with chronic liver disease and cirrhosis have a dysregulated coagulation system and are prone to thrombosis. The basis for this hypercoagulable state is not completely understood. Tissue factor (TF) is the primary initiator of coagulation in vivo. Patients with cirrhosis have increased TF activity in white blood cells and circulating microparticles. The aim of our study was to determine the contribution of TF to the hypercoagulable state in a mouse model of chronic liver injury

Plasma-based assays distinguish hyperfibrinolysis and shutdown subgroups in trauma-induced coagulopathy

BACKGROUND Trauma patients with abnormal fibrinolysis have increased morbidity and mortality. Knowledge of mechanisms differentiating fibrinolytic phenotypes is important to optimize treatment. We hypothesized that subjects with abnormal fibrinolysis identified by whole blood viscoelastometry can also be distinguished by plasma thrombin generation, clot structure, fibrin formation, and plasmin generation measurements. METHODS Platelet-poor plasma (PPP) from an observational cross-sectional trauma cohort with fibrinolysis shutdown (% lysis at 30 minutes [LY30] \u3c 0.9, n = 11) or hyperfibrinolysis (LY30 \u3e 3%, n = 9) defined by whole blood thromboelastography were studied. Noninjured control subjects provided comparative samples. Thrombin generation, fibrin structure and formation, and plasmin generation were measured by fluorescence, confocal microscopy, turbidity, and a fluorescence-calibrated plasmin assay, respectively, in the absence/presence of tissue factor or tissue plasminogen activator (tPA). RESULTS Whereas spontaneous thrombin generation was not detected in PPP from control subjects, PPP from hyperfibrinolysis or shutdown patients demonstrated spontaneous thrombin generation, and the lag time was shorter in hyperfibrinolysis versus shutdown. Addition of tissue factor masked this difference but revealed increased thrombin generation in hyperfibrinolysis samples. Compared with shutdown, hyperfibrinolysis PPP formed denser fibrin networks. In the absence of tPA, the fibrin formation rate was faster in shutdown than hyperfibrinolysis, but hyperfibrinolysis clots lysed spontaneously; these differences were masked by addition of tPA. Tissue plasminogen activator–stimulated plasmin generation was similar in hyperfibrinolysis and shutdown samples. Differences in LY30, fibrin structure, and lysis correlated with pH. CONCLUSION This exploratory study using PPP-based assays identified differences in thrombin generation, fibrin formation and structure, and lysis in hyperfibrinolysis and shutdown subgroups. These groups did not differ in their ability to promote tPA-triggered plasmin generation. The ability to characterize these activities in PPP facilitates studies to identify mechanisms that promote adverse outcomes in trauma

Cystamine Preparations Exhibit Anticoagulant Activity

Transglutaminases are a superfamily of isoenzymes found in cells and plasma. These enzymes catalyze the formation of ε-N-(γ-glutamyl)-lysyl crosslinks between proteins. Cystamine blocks transglutaminase activity and is used in vitro in human samples and in vivo in mice and rats in studies of coagulation, immune dysfunction, and inflammatory disease. These studies have suggested cystamine blocks fibrin crosslinking and has anti-inflammatory effects, implicating transglutaminase activity in the pathogenesis of several diseases. We measured the effects of cystamine on fibrin crosslinking, tissue factor-triggered plasma clot formation and thrombin generation, and coagulation factor enzymatic activity. At concentrations that blocked fibrin crosslinking, cystamine also inhibited plasma clot formation and reduced thrombin generation. Cystamine inhibited the amidolytic activity of coagulation factor XI and thrombin towards chromogenic substrates. These findings demonstrate that cystamine exhibits anticoagulant activity during coagulation. Given the close relationship between coagulation and inflammation, these findings suggest prior studies that used cystamine to implicate transglutaminase activity in disease pathogenesis warrant re-examination

Proceedings of the 3rd Biennial Conference of the Society for Implementation Research Collaboration (SIRC) 2015: advancing efficient methodologies through community partnerships and team science

It is well documented that the majority of adults, children and families in need of evidence-based behavioral health interventionsi do not receive them [1, 2] and that few robust empirically supported methods for implementing evidence-based practices (EBPs) exist. The Society for Implementation Research Collaboration (SIRC) represents a burgeoning effort to advance the innovation and rigor of implementation research and is uniquely focused on bringing together researchers and stakeholders committed to evaluating the implementation of complex evidence-based behavioral health interventions. Through its diverse activities and membership, SIRC aims to foster the promise of implementation research to better serve the behavioral health needs of the population by identifying rigorous, relevant, and efficient strategies that successfully transfer scientific evidence to clinical knowledge for use in real world settings [3]. SIRC began as a National Institute of Mental Health (NIMH)-funded conference series in 2010 (previously titled the “Seattle Implementation Research Conference”; $150,000 USD for 3 conferences in 2011, 2013, and 2015) with the recognition that there were multiple researchers and stakeholdersi working in parallel on innovative implementation science projects in behavioral health, but that formal channels for communicating and collaborating with one another were relatively unavailable. There was a significant need for a forum within which implementation researchers and stakeholders could learn from one another, refine approaches to science and practice, and develop an implementation research agenda using common measures, methods, and research principles to improve both the frequency and quality with which behavioral health treatment implementation is evaluated. SIRC’s membership growth is a testament to this identified need with more than 1000 members from 2011 to the present.ii SIRC’s primary objectives are to: (1) foster communication and collaboration across diverse groups, including implementation researchers, intermediariesi, as well as community stakeholders (SIRC uses the term “EBP champions” for these groups) – and to do so across multiple career levels (e.g., students, early career faculty, established investigators); and (2) enhance and disseminate rigorous measures and methodologies for implementing EBPs and evaluating EBP implementation efforts. These objectives are well aligned with Glasgow and colleagues’ [4] five core tenets deemed critical for advancing implementation science: collaboration, efficiency and speed, rigor and relevance, improved capacity, and cumulative knowledge. SIRC advances these objectives and tenets through in-person conferences, which bring together multidisciplinary implementation researchers and those implementing evidence-based behavioral health interventions in the community to share their work and create professional connections and collaborations

Abnormal plasma clot formation and fibrinolysis reveal bleeding tendency in patients with partial factor XI deficiency

Key Points Platelet-poor plasma clotting and fibrinolysis assays detect bleeding tendency in patients with factor XI deficiency. Contact pathway inhibition with corn trypsin inhibitor increases sensitivity of these assays to bleeding tendency.</jats:p

Cystamine inhibits fibrin crosslinking.

<p>Representative western blots and densitometry analysis showing the effects of cystamine (A, B) and T101 (C, D) on fibrin formation and crosslinking. Plasma was clotted by re-calcification and addition of lipidated tissue factor in the presence of cystamine or T101. Samples were reduced, boiled and separated by SDS-PAGE and identified by western blotting with anti-fibrinogen polyclonal antibody. MWM, molecular weight marker. NPP, un-clotted normal-pooled plasma. The upper high MW band (indicated by asterisk on the blot) was used for densitometry analysis of this species.</p

Cystamine inhibits plasma clot formation.

<p>Plasma clot formation was triggered by re-calcification and addition of lipidated tissue factor, in the presence of cystamine. Clot formation was monitored by turbidity. The data are representative of four experiments.</p

Clot formation and thrombin generation parameters.

<p>Mean ± standard deviation</p><p>*<i>P</i><0.05 vs 0 mM cystamine (untreated plasma)</p><p>Clot formation and thrombin generation parameters.</p

Cystamine inhibits the amidolytic activity of coagulation enzymes.

<p>Factors IXa (circles), Xa (squares), VIIa (diamonds), XIa (triangles), and thrombin (inverted triangles) amidolytic activity were assayed by incubating enzymes with cystamine (A) or T101 (B) and measuring the cleavage rate of chromogenic substrates. The data show means of 2–3 experiments per enzyme.</p