S-Nitrosylation of Tissue Transglutaminase in Modulating Glycolysis, Oxidative Stress, and Inflammatory Responses in Normal and Indoxyl-Sulfate-Induced Endothelial Cells.

Circulating uremic toxin indoxyl sulfate (IS), endothelial cell (EC) dysfunction, and decreased nitric oxide (NO) bioavailability are found in chronic kidney disease patients. NO nitrosylates/denitrosylates a specific protein's cysteine residue(s), forming S-nitrosothios (SNOs), and the decreased NO bioavailability could interfere with NO-mediated signaling events. We were interested in investigating the underlying mechanism(s) of the reduced NO and how it would regulate the S-nitrosylation of tissue transglutaminase (TG2) and its substrates on glycolytic, redox and inflammatory responses in normal and IS-induced EC injury. TG2, a therapeutic target for fibrosis, has a Ca2+-dependent transamidase (TGase) that is modulated by S-nitrosylation. We found IS increased oxidative stress, reduced NADPH and GSH levels, and uncoupled eNOS to generate NO. Immunoblot analysis demonstrated the upregulation of an angiotensin-converting enzyme (ACE) and significant downregulation of the beneficial ACE2 isoform that could contribute to oxidative stress in IS-induced injury. An in situ TGase assay demonstrated IS-activated TG2/TGase aminylated eNOS, NFkB, IkBα, PKM2, G6PD, GAPDH, and fibronectin (FN), leading to caspases activation. Except for FN, TGase substrates were all differentially S-nitrosylated either with or without IS but were denitrosylated in the presence of a specific, irreversible TG2/TGase inhibitor ZDON, suggesting ZDON-bound TG2 was not effectively transnitrosylating to TG2/TGase substrates. The data suggest novel roles of TG2 in the aminylation of its substrates and could also potentially function as a Cys-to-Cys S-nitrosylase to exert NO's bioactivity to its substrates and modulate glycolysis, redox, and inflammation in normal and IS-induced EC injury.

AGA Clinical Practice Update: Coagulation in Cirrhosis.

DESCRIPTION: This expert review was commissioned and approved by the AGA Institute Clinical Practice Updates Committee and the AGA Governing Board to provide timely guidance on a topic of high clinical importance to the AGA membership. The intent is to evaluate the current data on mechanism of altered coagulation in patients with cirrhosis, provide guidance on the use of currently available testing of the coagulation cascade, and help practitioners use anticoagulation and pro-coagulants appropriately in patients with cirrhosis. METHODS: This review is framed around the best practice points, which were derived from the most impactful publications in the area of coagulation in cirrhosis and agreed to by all authors. BEST PRACTICE ADVICE 1: Global tests of clot formation, such as rotational thromboelastometry, thromboelastography, sonorheometry, and thrombin generation, may eventually have a role in the evaluation of clotting in patients with cirrhosis, but currently lack validated target levels. BEST PRACTICE ADVICE 2: In general, clinicians should not routinely correct thrombocytopenia and coagulopathy before low-risk therapeutic paracentesis, thoracentesis, and routine upper endoscopy for variceal ligation in patients with hepatic synthetic dysfunction-induced coagulation abnormalities. BEST PRACTICE ADVICE 3: Blood products should be used sparingly because they increase portal pressure and carry a risk of transfusion-associated circulatory overload, transfusion-related acute lung injury, infection transmission, alloimmunization, and/or transfusion reactions. BEST PRACTICE ADVICE 4: The following transfusion thresholds for management of active bleeding or high-risk procedures may optimize clot formation in advanced liver disease: hematocrit ≥25%, platelet count >50,000, and fibrinogen >120 mg/dL. Commonly utilized thresholds for international normalized ratio correction are not supported by evidence. BEST PRACTICE ADVICE 5: Thrombopoietin agonists are a good alternative to platelet transfusion, but require time (about 10 days) to elevate platelet levels. BEST PRACTICE ADVICE 6: The large volume of fresh frozen plasma required to reach an arbitrary international normalized ratio target, limitations of the usual target, minimal effect on thrombin generation, and adverse effects on portal pressure limit the utility of this agent significantly. BEST PRACTICE ADVICE 7: The 4-factor prothrombin complex concentrate contains both pro- and anticoagulant factors that offer an attractive low-volume therapeutic to rebalance a disturbed hemostatic system. However, dosage is, in part, based on international normalized ratio, which is problematic in cirrhosis, and published experience in liver disease is limited. BEST PRACTICE ADVICE 8: Anti-fibrinolytic therapy may be considered in patients with persistent bleeding from mucosal oozing or puncture wound bleeding consistent with impaired clot integrity. Both ε-aminocaproic acid and tranexamic acid inhibit clot dissolution. Neither is believed to generate a hypercoagulable state, although both may exacerbate pre-existing thrombi. BEST PRACTICE ADVICE 9: Desmopressin releases von Willebrand factor as its primary hemostatic mechanism. As this factor is usually elevated in cirrhosis, the agent lacks a sound evidence-based foundation, but may be useful in patients with concomitant renal failure. BEST PRACTICE ADVICE 10: Systemic heparin infusion is recommended for symptomatic deep vein thrombosis and portal and mesenteric vein thrombosis, but there are unresolved issues regarding monitoring with both the anti-Xa assay and the partial thromboplastin time due to cirrhosis-related antithrombin deficiency (heparin cofactor). BEST PRACTICE ADVICE 11: Treatment of incidental portal and mesenteric vein thrombosis depends on estimated impact on transplantation surgical complexity vs risks of bleeding and falls. Therapy with low-molecular-weight heparin, vitamin K antagonists, and direct-acting anticoagulants improve portal vein repermeation vs observation alone. BEST PRACTICE ADVICE 12: Direct-acting anticoagulants, such as the factor Xa and thrombin inhibitors, are relatively safe and effective in stable cirrhotic patients, but are in need of further study in patients with more advanced liver disease.

Multicenter Evaluation of the Quantra QPlus System in Adult Patients Undergoing Major Surgical Procedures.

BACKGROUND: The management of perioperative bleeding and the optimization of the available therapies are subjects of significant clinical interest. Clinical guidelines recommend the use of whole blood viscoelastic testing devices to target the utilization of blood products during major surgical procedures. The Quantra QPlus System is a new cartridge-based viscoelastic testing device based on an innovative ultrasound technology. The aim of this study was to evaluate this new system in a surgical population. METHODS: Two hundred seventy-seven adult subjects were enrolled in a multicenter, prospective observational study consisting primarily of patients undergoing cardiac and major orthopedic surgeries. Samples were obtained at multiple time points for testing on the Quantra QPlus System, the rotational thromboelastometry (ROTEM) delta, and standard coagulation tests. Quantra measurements included Clot Time (CT), Heparinase Clot Time (CTH), Clot Time Ratio (CTR), Clot Stiffness (CS), Fibrinogen (FCS), and Platelet (PCS) Contributions to CS. Data analyses included assessment of the concordance of Quantra parameters with a series of clinical composite indexes formed on the basis of standard coagulation tests in 3 domains representing increased, decreased, and normal/subclinical coagulation function. Linear regression and receiver operator characteristic (ROC) analyses of Quantra parameters with corresponding parameters from ROTEM assays were also performed. RESULTS: The accuracy (overall percent agreement or ratio of true positives and true negatives over the entire population) between the Quantra and the composite indexes was between 72% and 98% depending on the specific parameter. Linear regression analysis indicated that the correlation between ROTEM delta and Quantra was very strong with r values ranging between 0.84 and 0.89. Results from ROC analysis demonstrated sensitivities and specificities in the 80%-90% range when QPlus parameters were used to discriminate ROTEM threshold values currently used in goal-directed treatment algorithms. CONCLUSIONS: This study demonstrated that the Quantra QPlus System is strongly correlated with a well-established viscoelastic testing device and its parameters effectively represent the results from multiple standard laboratory assays. The Quantra has been designed to operate at the point of care with the potential to provide rapid and comprehensive results to aid in the management of coagulopathic patients.

Impact of Benign Hematology Didactic Lectures on In-service Exam in a Hematology-Oncology Fellowship Program: a Cross-sectional Longitudinal Study.

It remains unclear on whether the traditional formal didactic lecture sessions improve knowledge acquisition with conflicting data in the literature. This study evaluates the impact of an additional benign hematology didactic curriculum on the American Society of Hematology In-Service Exam (ASHISE). During the first 5 years of the study (2012-2016), formal didactic lectures consisted of medical oncology and malignant hematology topics only. Formal benign hematology didactic lectures were added during the last 2 years of the study (2017-2018). All fellows are required to take the ASHISE annually. All fellows' ASHISE scores from 2012 to 2018 were collected. The mean total and Coagulation scale score were calculated by year of fellowship training. Pre-intervention (2012-2016) and post-intervention (2017-2018) scores were analyzed using a Student's t test. Over a 7-year period, 34 hematology-oncology fellows took the ASHISE. There was no statistical difference in the mean total and Coagulation scale score for the ASHISE in the pre-intervention and post-intervention group. The addition of a benign hematology curriculum did not improve fellows' performance on the ASHISE.

Analyzing the impact of systems-based hematologist in the healthcare system at an academic medical center.

Over the next decade, there is a predicted shortage of nonmalignant hematologist to maintain the workforce in the United States. To address this, the American Society of Hematology described the creation of the healthcare systems-based hematologist (SBH). The role of SBH has the potential to provide high-value, cost-conscious care to the healthcare system. In 2011, an Anticoagulation and Bleeding Management Medical Directorship position for a SBH was created at our healthcare system. We described our 6-year experience as SBH at a 750-bed tertiary academic medical center to improve clinical outcomes while reducing costs. Via four different initiatives, we were able to provide high-value, cost-conscious care as SBH by reducing cost of heparin-induced thrombocytopenia management, optimizing blood product utilization using goal-directed algorithms, reducing inappropriate thrombophilia testing and improving inferior vena cava filter retrieval rates. To ensure continuing success as a SBH, business plans need to include education, enforcement, monitoring, feedback, validation of safety and outcomes and a shared vision among leadership.

Initial and long term impact of a multi-disciplinary task force in the diagnosis and management of heparin-induced thrombocytopenia.

Many medical centers are faced with a major challenge in making an accurate diagnosis of heparin-induced thrombocytopenia (HIT) and ensuring appropriate changes in management strategy in line with guideline recommendations. We report the initial and long-term impact and challenges of institution-wide changes in the diagnosis and management of HIT in the inpatient setting at an academic medical center. We established a HIT Task Force, consisting of a multidisciplinary team of non-malignant hematologists, nursing, pharmacist, pathology, blood bank and clinical lab informatics. Changes were implemented from 2011 to 2012. In 2013, testing for PF4 and SRA decreased by 37.5 and 85%, respectively. 100% of positive PF4 received an automatic hematology consult to guide management, leading to a 78% reduction in the use of direct thrombin inhibitors. Annual audits in the subsequent years demonstrated increasing testing for HIT due to changes in the electronic ordering system. Through continuous monitoring, these shortfalls were detected and intervene early on with continued success. The implementation of a centralized hospital-wide protocol via a multidisciplinary task force that coordinates testing and treatment of patients suspected of having HIT led to a substantial reduction in PF4 and SRA testing, as well as use of DTIs, resulting in a safe and cost-effective approach for the diagnosis and treatment of HIT. Our study highlights the important of continuous monitoring to maintain the improvements made. Despite our initial success, annual re-auditing allowed for early detection of challenges, which then allowed appropriate early intervention.

Role of tissue transglutaminase-2 (TG2)-mediated aminylation in biological processes.

Post-translational modification (PTM) is an important mechanism in modulating a protein's structure and can lead to substantial diversity in biological function. Compared to other forms of PTMs such as phosphorylation, acetylation and glycosylation, the physiological significance of aminylation is limited. Aminylation refers to the covalent incorporation of biogenic/polyamines into target protein by calcium-dependent transglutaminases (TGs). The development of novel and more sensitive techniques has led to more proteins identified as tissue transglutaminase (TG2) substrates and potential targets for aminylation. Many of these substrate proteins play a role in cell signaling, cytoskeleton organization, muscle contraction, and inflammation. TG2 is well studied and widely expressed in a variety of tissues and will be the primary focus of this review on recent advance in transglutaminase-mediated aminylation.

Histaminylation of fibrinogen by tissue transglutaminase-2 (TGM-2): potential role in modulating inflammation.

Plasma fibrinogen plays an important role in hemostasis and inflammation. Fibrinogen is converted to fibrin to impede blood loss and serves as the provisional matrix that aids wound healing. Fibrinogen also binds to cytokine activated endothelial cells and promotes the binding and migration of leukocytes into tissues during inflammation. Tissue transglutaminase (TGM-2) released from injured cells could cross-link fibrinogen to form multivalent complexes that could promote adhesion of platelets and vascular cells to endothelium. Histamine released by mast cells is a potent biogenic amine that promotes inflammation. The covalent attachment of histamine to proteins (histaminylation) by TGM-2 could modify local inflammatory reactions. We investigated TGM-2 crosslinking of several biogenic amines (serotonin, histamine, dopamine and noradrenaline) to fibrinogen. We identified histaminylation of fibrinogen by TGM-2 as a preferred reaction in solid and solution phase transglutaminase assays. Histamine caused a concentration-dependent inhibition of fibrinogen cross-linking by TGM-2. Fibrinogen that was not TGM-2 crosslinked bound to unactivated endothelial cells with low affinity. However, the binding was increased by sevenfold when fibrinogen was cross-linked by TGM-2. Histaminylation of fibrinogen also inhibited TGM-2 crosslinking of fibrinogen and the binding to un-activated HUVEC cells by 75­90 %. In summary, the histaminylation of fibrinogen by TGM-2 could play a role in modifying inflammation by sequestering free histamine and by inhibiting TGM-2 crosslinking of fibrinogen.

D-dimer antigen: current concepts and future prospects.

The D-dimer antigen is a unique marker of fibrin degradation that is formed by the sequential action of 3 enzymes: thrombin, factor XIIIa, and plasmin. First, thrombin cleaves fibrinogen producing fibrin monomers, which polymerize and serve as a template for factor XIIIa and plasmin formation. Second, thrombin activates plasma factor XIII bound to fibrin polymers to produce the active transglutaminase, factor XIIIa. Factor XIIIa catalyzes the formation of covalent bonds between D-domains in the polymerized fibrin. Finally, plasmin degrades the crosslinked fibrin to release fibrin degradation products and expose the D-dimer antigen. D-dimer antigen can exist on fibrin degradation products derived from soluble fibrin before its incorporation into a fibrin gel, or after the fibrin clot has been degraded by plasmin. The clinical utility of D-dimer measurement has been established in some scenarios, most notably for the exclusion of VTE. This article consists of 2 sections: in the first, the dynamics of D-dimer antigen formation is discussed and an overview of commercially available D-dimer assays is provided. The second section reviews available evidence for the clinical utilization of D-dimer antigen measurement in VTE, as well as emerging areas of D-dimer utilization as a marker of coagulation activation in other clinical settings.

The use of activated recombinant factor VII in a patient with fulminant hepatic failure requiring placement of an intracranial pressure monitor.

OBJECTIVE: To evaluate the use of recombinant activated factor VII (rFVIIa) in a patient with fulminant hepatic failure (FHF) requiring placement of an intracranial pressure monitor. CASE SUMMARY: A 21-year-old female with no significant medical history was admitted to an outside hospital with elevated results of liver function tests. Subsequently, the patient was diagnosed with autoimmune hepatitis. Systemic corticosteroids were started, but her condition continued to decompensate. She was transferred to our tertiary care facility 5 days after initial presentation. The liver function test results remained elevated (eg, total bilirubin 27 mg/dL), and international normalized ratio (INR) was 3.57. The medical team decided to place an intracranial pressure monitor, with the neurosurgery team's goal being an INR less than 1.5 before placement of the monitor. After multiple units of fresh frozen plasma (FFP) failed to lower the patient's INR, rFVIIa 40 µg/kg was administered. A rapid decrease of the INR allowed the neurosurgery team to perform the procedure without complications. DISCUSSION: The use of rFVIIa allowed for decrease of this patient's INR after multiple units of FFP had failed to correct it. The utility of INR as a marker of coagulopathy in fulminant hepatic failure has been debated, but it is currently used as the standard laboratory test prior to invasive procedures, as in the case presented here. CONCLUSIONS: The use of rFVIIa for rapid decrease of INR in a patient with FHF prior to an invasive procedure was safe and efficacious. When considering the use of rFVIIa, clinicians should be aware of the risk of thrombosis. In our experience, and in the limited literature on the matter, rFVIIa 40 µg/kg appears to be an appropriate dose for decrease of the INR. Further studies are needed to confirm this finding.

Clinical usefulness of recombinant activated factor VII in patients with liver failure undergoing invasive procedures.

OBJECTIVE: To evaluate the use of recombinant activated factor VII (rFVIIa) in patients with liver failure undergoing invasive procedures. METHODS: An OVID/MEDLINE and PubMed search (1997-June 2011) was performed to identify literature on the use of rFVIIa to reduce bleeding risk in patients with liver failure undergoing invasive procedures. STUDY SELECTION AND DATA EXTRACTION: English-language data evaluating the efficacy of rFVIIa to reverse coagulopathies prior to invasive procedures in patients with liver disease were included. DATA SYNTHESIS: Following administration of rFVIIa, prothrombin time (PT) and international normalized ratio (INR) response is within 30 minutes. Doses ranging from 20 to 120 µg/kg have been studied, with a reduction in PT seen in a dose-dependent manner. One study in patients with no bleeding administered 5, 20, and 80 µg/kg sequentially during a 24-day period. All doses provided reversal of prolonged PT within 10 minutes, and the duration was dose-dependent. In a study of 15 patients with fulminant liver failure, requiring intracranial pressure monitor placement, a rFVIIa dose of 40 µg/kg was compared to fresh frozen plasma. In patients who received rFVIIa, the PT and INR normalized, compared to none of the patients in the fresh frozen plasma group. CONCLUSIONS: Retrospective and prospective data demonstrate that rFVIIa effectively reverses elevated PT and INR, reducing the risk of bleeding and safely facilitating invasive procedures. Based on available data, a dose of 20-40 µg/kg 30 minutes prior to an invasive procedure should be considered in patients with acute or chronic liver failure at risk for bleeding complications. A major limitation of rFVIIa use is the high cost of therapy. A prospective, randomized trial could help determine the appropriate dose of rFVIIa, timing of dose in relationship to procedure, and usefulness of subsequent doses.

Association of Sickle Cell Trait With Incidence of Coronary Heart Disease Among African American Individuals.

Importance: The incidence of and mortality from coronary heart disease (CHD) are substantially higher among African American individuals compared with non-Hispanic White individuals, even after adjusting for traditional factors associated with CHD. The unexplained excess risk might be due to genetic factors related to African ancestry that are associated with a higher risk of CHD, such as the heterozygous state for the sickle cell variant or sickle cell trait (SCT). Objective: To evaluate whether there is an association between SCT and the incidence of myocardial infarction (MI) or composite CHD outcomes in African American individuals. Design, Setting, and Participants: This cohort study included 5 large, prospective, population-based cohorts of African American individuals in the Women's Health Initiative (WHI) study, the Reasons for Geographic and Racial Differences in Stroke (REGARDS) study, the Multi-Ethnic Study of Atherosclerosis (MESA), the Jackson Heart Study (JHS), and the Atherosclerosis Risk in Communities (ARIC) study. The follow-up periods included in this study were 1993 and 1998 to 2014 for the WHI study, 2003 to 2014 for the REGARDS study, 2002 to 2016 for the MESA, 2002 to 2015 for the JHS, and 1987 to 2016 for the ARIC study. Data analysis began in October 2013 and was completed in October 2020. Exposures: Sickle cell trait status was evaluated by either direct genotyping or high-quality imputation of rs334 (the sickle cell variant). Participants with sickle cell disease and those with a history of CHD were excluded from the analyses. Main Outcomes and Measures: Incident MI, defined as adjudicated nonfatal or fatal MI, and incident CHD, defined as adjudicated nonfatal MI, fatal MI, coronary revascularization procedures, or death due to CHD. Cox proportional hazards regression models were used to estimate the hazard ratio for incident MI or CHD comparing SCT carriers with noncarriers. Models were adjusted for age, sex (except for the WHI study), study site or region of residence, hypertension status or systolic blood pressure, type 1 or 2 diabetes, serum high-density lipoprotein level, total cholesterol level, and global ancestry (estimated from principal components analysis). Results: A total of 23 197 African American men (29.8%) and women (70.2%) were included in the combined sample, of whom 1781 had SCT (7.7% prevalence). Mean (SD) ages at baseline were 61.2 (6.9) years in the WHI study (n = 5904), 64.0 (9.3) years in the REGARDS study (n = 10 714), 62.0 (10.0) years in the MESA (n = 1556), 50.3 (12.0) years in the JHS (n = 2175), and 53.2 (5.8) years in the ARIC study (n = 2848). There were no significant differences in the distribution of traditional factors associated with cardiovascular disease by SCT status within cohorts. A combined total of 1034 participants (76 with SCT) had incident MI, and 1714 (137 with SCT) had the composite CHD outcome. The meta-analyzed crude incidence rate of MI did not differ by SCT status and was 3.8 per 1000 person-years (95% CI, 3.3-4.5 per 1000 person-years) among those with SCT and 3.6 per 1000 person-years (95% CI, 2.7-5.1 per 1000 person-years) among those without SCT. For the composite CHD outcome, these rates were 7.3 per 1000 person-years (95% CI, 5.5-9.7 per 1000 person-years) among those with SCT and 6.0 per 1000 person-years (95% CI, 4.9-7.4 per 1000 person-years) among those without SCT. Meta-analysis of the 5 study results showed that SCT status was not significantly associated with MI (hazard ratio, 1.03; 95% CI, 0.81-1.32) or the composite CHD outcome (hazard ratio, 1.16; 95% CI, 0.92-1.47). Conclusions and Relevance: In this cohort study, there was not an association between SCT and increased risk of MI or CHD in African American individuals. These disorders may not be associated with sickle cell trait-related sudden death in this population.

Successful Management of Thrombotic Thrombocytopenic Purpura in a Jehovah's Witness: An Individualized Approach With Joint Decision-Making.

The management of thrombotic thrombocytopenic purpura (TTP) presents a unique challenge in individuals who are unable to accept plasma due to religious beliefs, given that therapeutic plasma exchange (TPE) is the standard of care. A 61-year-old Jehovah's Witness woman presented to our hospital with neurological symptoms and laboratory findings suggestive of TTP. On admission, she refused transfusion of blood products, specifically red blood cells, platelets, and plasma but accepted albumin and intravenous immunoglobulin (IVIG); fractions of plasma. She was started on steroids, IVIG, and TPE with albumin as replacement therapy with minimal improvement. After a detailed discussion with the patient and family, they agreed to accept cryosupernatant. The patient started TPE with cryosupernatant for replacement therapy, which resulted in clinical improvement. This case highlights the importance of an individualized approach with joint decision-making given the significant heterogeneity that exists in Jehovah's Witnesses' attitude toward the receipt of blood products.

Identification of chemical inhibitors to human tissue transglutaminase by screening existing drug libraries.

Human tissue transglutaminase (TGM2) is a calcium-dependent crosslinking enzyme involved in the posttranslational modification of intra- and extracellular proteins and implicated in several neurodegenerative diseases. To find specific inhibitors to TGM2, two structurally diverse chemical libraries (LOPAC and Prestwick) were screened. We found that ZM39923, a Janus kinase inhibitor, and its metabolite ZM449829 were the most potent inhibitors with IC(50) of 10 and 5 nM, respectively. In addition, two other inhibitors, including tyrphostin 47 and vitamin K(3), were found to have an IC(50) in the micromolar range. These agents used in part a thiol-dependent mechanism to inhibit TGM2, consistent with the activation of TGM2 by reduction of an intramolecular disulfide bond. These inhibitors were tested in a polyglutamine-expressing Drosophila model of neurodegeneration and found to improve survival. The TGM2 inhibitors we discovered may serve as valuable lead compounds for the development of orally active TGM2 inhibitors to treat human diseases.

Acute kidney injury is associated with low factor XIII in decompensated cirrhosis.

BACKGROUND/AIMS: The coagulation system is known to be rebalanced but fragile in stable cirrhosis. Acute kidney injury (AKI) is common in these patients and associated with an increased bleeding risk. We aimed to assess coagulation parameters in this population. METHODS: We prospectively enrolled 43 hospitalized patients with decompensated cirrhosis with (n = 22) or without (n = 21) AKI. Coagulation factor levels, viscoelastic coagulation assay, and thrombin generation assay were performed and compared between these groups and a healthy reference group. RESULTS: Conventional markers of coagulation were not statistically different between patients with and without AKI. Factor XIII was significantly reduced in all patients with cirrhosis compared to healthy controls (p = <0.0001). In patients with AKI, factor XIII was significantly lower compared to patients without AKI (AKI 38% vs. non-AKI 60% p = 0.002). In patients with cirrhosis, factor XIII had a significantly positive correlation with EXTEM maximal clot firmness (r = 0.5440, p = 0.0002) and FIBTEM maximal clot firmness (r = 0.7397, p = <0.0001) and a negative correlation with EXTEM clot formation time (-0.413, p = 0.0065). CONCLUSIONS: Factor XIII was significantly reduced in decompensated cirrhosis patients with AKI compared to decompensated patients without AKI. These findings suggest that exacerbation of factor XIII deficiency in AKI in decompensated cirrhosis may affect bleeding risk and warrants further study.

Identification of two GTP-independent alternatively spliced forms of tissue transglutaminase in human leukocytes, vascular smooth muscle, and endothelial cells.

Tissue transglutaminase (tTG) is a multifunctional enzyme with transglutaminase crosslinking (TGase), GTP binding, and hydrolysis activities that play a role in many different disorders. We identified, characterized, and investigated the function and stability of two alternatively spliced forms of tTG using biochemical, cellular, and molecular biological approaches. Using a human aortic vascular smooth muscle cells (VSMC) cDNA library, we identified two cDNAs encoding C-terminal truncated forms, tTG(V1) and tTG(V2). tTG(V1,2) mRNAs were synthesized by a rare splicing event using alternate splice sites within exons 12 and 13 of the tTG gene, respectively. Quantitative PCR and immunoblotting demonstrated that there was unique expression and localization of tTG(V1,2) compared with tTG in human umbilical vein endothelial cells (HUVECs), VSMC, and leukocytes. The loss of C-terminal 52 amino acid residues (AAs) in tTG(V1,2) altered GTP binding, enhanced GTP hydrolysis, rendered the variants insensitive to GTP inhibition, and resulted in <10% residual Ca(+2)-dependent TGase activity. Transfection in HEK293 demonstrated a 28- and 5-fold reduction in the expression of tTG(V1) and tTG(V2), respectively, demonstrating that the C-terminal GTP-binding domain is important in stabilizing and promoting the half-life of tTG. The altered affinity for GTP allowed tTG(V1,2) to exhibit enhanced TGase activity when there is a transient increase in Ca(+2) levels. The abundance of tTG(V1,2) and its distinct intracellular expression patterns in human vascular cells and leukocytes indicate these isoforms likely have unique physiological functions.