Integrative structure and functional anatomy of a nuclear pore complex.

Nuclear pore complexes play central roles as gatekeepers of RNA and protein transport between the cytoplasm and nucleoplasm. However, their large size and dynamic nature have impeded a full structural and functional elucidation. Here we determined the structure of the entire 552-protein nuclear pore complex of the yeast Saccharomyces cerevisiae at sub-nanometre precision by satisfying a wide range of data relating to the molecular arrangement of its constituents. The nuclear pore complex incorporates sturdy diagonal columns and connector cables attached to these columns, imbuing the structure with strength and flexibility. These cables also tie together all other elements of the nuclear pore complex, including membrane-interacting regions, outer rings and RNA-processing platforms. Inwardly directed anchors create a high density of transport factor-docking Phe-Gly repeats in the central channel, organized into distinct functional units. This integrative structure enables us to rationalize the architecture, transport mechanism and evolutionary origins of the nuclear pore complex.

PFA-100 System: A New Method for Assessment of Platelet Dysfunction.

This is a celebratory reprint of a historical paper published in STH in 1998. The original Abstract follows.The PFA-100 system is a platelet function analyzer designed to measure platelet-related primary hemostasis. The instrument uses two disposable cartridges: a collagen/epinephrine (CEPI) and a collagen/ADP (CADP) cartridge. Previous experience has shown that CEPI cartridges detect qualitative platelet defects, including acetylsalicylic acid (ASA)-induced abnormalities, while CADP cartridges detect only thrombocytopathies and not ASA use. In this seven-center trial, 206 healthy subjects and 176 persons with various platelet-related defects, including 127 ASA users, were studied. The platelet function status was determined by a platelet function test panel. Comparisons were made as to how well the defects were identified by the PFA-100 system and by platelet aggregometry. The reference intervals for both cartridges, testing the 206 healthy subjects, were similar to values described in smaller studies in the literature (mean closure time [CT] of 132 seconds for CEPI and 93 seconds for CADP). The use of different lot numbers of cartridges or duplicate versus singleton testing revealed no differences. Compared with the platelet function status, the PFA-100 system had a clinical sensitivity of 94.9% and a specificity of 88.8%. For aggregometry, a sensitivity of 94.3% and a specificity of 88.3% were obtained. These values are based on all 382 specimens. A separate analysis of sensitivity by type of platelet defect, ASA use versus congenital thrombocytopathies, revealed for the PFA-100 system a 94.5% sensitivity in identifying ASA users and a 95.9% sensitivity in identifying the other defects. For aggregometry, the values were 100% for ASA users and 79.6% for congenital defects. Analysis of concordance between the PFA-100 system and aggregometry revealed no difference in clinical sensitivity and specificity between the systems (p > 0.9999). The overall agreement was 87.5%, with a Kappa index of 0.751. The two tests are thus equivalent in their ability to identify normal and abnormal platelet defects. Testing 126 subjects who took 325 mg ASA revealed that the PFA-100 system (CEPI) was able to detect 71.7% of ASA-induced defects with a positive predictive value of 97.8%. The overall clinical accuracy of the system, calculated from the area under the receiver operating characteristic curve, was 0.977. The data suggest that the PFA-100 system is highly accurate in discriminating normal from abnormal platelet function. The ease of operation of the instrument makes it a useful tool to use in screening patients for platelet-related hemostasis defects.

Architecture of the Human and Yeast General Transcription and DNA Repair Factor TFIIH.

TFIIH is essential for both RNA polymerase II transcription and DNA repair, and mutations in TFIIH can result in human disease. Here, we determine the molecular architecture of human and yeast TFIIH by an integrative approach using chemical crosslinking/mass spectrometry (CXMS) data, biochemical analyses, and previously published electron microscopy maps. We identified four new conserved "topological regions" that function as hubs for TFIIH assembly and more than 35 conserved topological features within TFIIH, illuminating a network of interactions involved in TFIIH assembly and regulation of its activities. We show that one of these conserved regions, the p62/Tfb1 Anchor region, directly interacts with the DNA helicase subunit XPD/Rad3 in native TFIIH and is required for the integrity and function of TFIIH. We also reveal the structural basis for defects in patients with xeroderma pigmentosum and trichothiodystrophy, with mutations found at the interface between the p62 Anchor region and the XPD subunit.

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.

Limited impact of clinician education on reducing inappropriate PF4 testing for heparin-induced thrombocytopenia.

A high frequency of PF4-ELISA testing in patients suspected to have heparin-induced thrombocytopenia (HIT) despite low 4T scores has been observed in multiple medical centers. Education of clinicians has been suggested to reduce inappropriate testing. We determined trends of PF4-ELISA testing in our institution after the introduction of a HIT education program for clinicians. A HIT Program was developed that included ongoing education, individual feedback, and continuous clinical audit of PF4-ELISA utilization. To assess the impact of education on PF4-ELISA testing trends, we conducted a prospective cohort review of all adult patients who had a PF4-ELISA ordered over a 3 month period (the last quarter of the academic year). 72 PF4-ELISA tests were ordered during the study period. Prospectively calculated 4T scores by investigators revealed 60 low-risk (83.3%), 9 intermediate-risk (12.5%), and 3 high-risk (4.16%). We observed divergent 4T scores with the ordering clinician calculating a higher 4T score compared to the Hematology Quality Improvement (QI) team. The majority of PF4-ELISA testing was ordered by the intensive care units (ICUs) (n = 32, 44.44%). Our study revealed that the frequency of calculation of 4T scores remains poor with the majority inappropriately performed in the ICU setting, with ordering clinicians calculating higher 4T scores than the Hematology QI team. This suggests that clinician education alone is insufficient. Introducing mandatory 4T score calculation prior to PF4-ELISA testing may not be helpful as ordering clinicians can bypass the restriction through inaccurate 4T score calculation.

Molecular Details Underlying Dynamic Structures and Regulation of the Human 26S Proteasome.

The 26S proteasome is the macromolecular machine responsible for ATP/ubiquitin dependent degradation. As aberration in proteasomal degradation has been implicated in many human diseases, structural analysis of the human 26S proteasome complex is essential to advance our understanding of its action and regulation mechanisms. In recent years, cross-linking mass spectrometry (XL-MS) has emerged as a powerful tool for elucidating structural topologies of large protein assemblies, with its unique capability of studying protein complexes in cells. To facilitate the identification of cross-linked peptides, we have previously developed a robust amine reactive sulfoxide-containing MS-cleavable cross-linker, disuccinimidyl sulfoxide (DSSO). To better understand the structure and regulation of the human 26S proteasome, we have established new DSSO-based in vivo and in vitro XL-MS workflows by coupling with HB-tag based affinity purification to comprehensively examine protein-protein interactions within the 26S proteasome. In total, we have identified 447 unique lysine-to-lysine linkages delineating 67 interprotein and 26 intraprotein interactions, representing the largest cross-link dataset for proteasome complexes. In combination with EM maps and computational modeling, the architecture of the 26S proteasome was determined to infer its structural dynamics. In particular, three proteasome subunits Rpn1, Rpn6, and Rpt6 displayed multiple conformations that have not been previously reported. Additionally, cross-links between proteasome subunits and 15 proteasome interacting proteins including 9 known and 6 novel ones have been determined to demonstrate their physical interactions at the amino acid level. Our results have provided new insights on the dynamics of the 26S human proteasome and the methodologies presented here can be applied to study other protein complexes.

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.

Structure of γ-tubulin small complex based on a cryo-EM map, chemical cross-links, and a remotely related structure.

Modeling protein complex structures based on distantly related homologues can be challenging due to poor sequence and structure conservation. Therefore, utilizing even low-resolution experimental data can significantly increase model precision and accuracy. Here, we present models of the two key functional states of the yeast γ-tubulin small complex (γTuSC): one for the low-activity "open" state and another for the higher-activity "closed" state. Both models were computed based on remotely related template structures and cryo-EM density maps at 6.9Šand 8.0Šresolution, respectively. For each state, extensive sampling of alignments and conformations was guided by the fit to the corresponding cryo-EM density map. The resulting good-scoring models formed a tightly clustered ensemble of conformations in most regions. We found significant structural differences between the two states, primarily in the γ-tubulin subunit regions where the microtubule binds. We also report a set of chemical cross-links that were found to be consistent with equilibrium between the open and closed states. The protocols developed here have been incorporated into our open-source Integrative Modeling Platform (IMP) software package (http://integrativemodeling.org), and can therefore be applied to many other systems.

Biology of Factor XIII and clinical manifestations of Factor XIII deficiency.

Factor XIII (FXIII) is activated by thrombin to form a transglutaminase (FXIIIa) that stabilizes clot formation by the cross-linking of fibrin monomers and antifibrinolytic proteins. Although rare, FXIII deficiency is characterized by variable bleeding manifestations depending on the magnitude of the deficiency. A congenital FXIII deficiency with levels less than 1% can be detected in children who present with prolonged bleeding from the umbilical stump as well as protracted bleeding after trauma. An acquired FXIII deficiency may occur in a number of diseases or clinical situations where FXIII levels and/or its activity are decreased. Patients may also develop a relative deficiency in FXIII as a result of hemorrhage or dilutional changes from transfusions during surgery or trauma and are at increased risk for postoperative bleeding. Genetic studies have identified a wide range of mutations that affect the activity of the FXIII protein but in lieu of molecular genetic analyses, FXIII deficiency can be identified by specific diagnostic assays that measure either the transglutaminase activity of the protein or the levels of the protein and its individual subunits. Replacement therapy has also been shown to increase FXIII levels and reduce bleeding symptoms in patients with congenital FXIII deficiency. This review presents recent findings on the biology of FXIII and the clinical manifestations observed among patients with congenital and acquired FXIII deficiencies.

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.

Review article: heparin sensitivity and resistance: management during cardiopulmonary bypass.

Heparin resistance during cardiac surgery is defined as the inability of an adequate heparin dose to increase the activated clotting time (ACT) to the desired level. Failure to attain the target ACT raises concerns that the patient is not fully anticoagulated and initiating cardiopulmonary bypass may result in excessive activation of the hemostatic system. Although antithrombin deficiency has generally been thought to be the primary mechanism of heparin resistance, the reasons for heparin resistance are both complex and multifactorial. Furthermore, the ACT is not specific to heparin's anticoagulant effect and is affected by multiple variables that are commonly present during cardiac surgery. Due to these many variables, it remains unclear whether decreased heparin responsiveness as measured by the ACT represents inadequate anticoagulation. Nevertheless, many clinicians choose a target ACT to assess anticoagulation, and interventions aimed at achieving the target ACT are routinely performed in the setting of heparin resistance. Treatments for heparin resistance/alterations in heparin responsiveness include additional heparin or antithrombin supplementation. In this review, we discuss the variability of heparin potency, heparin responsiveness as measured by the ACT, and the current management of heparin resistance.

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.