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Contains fulltext : 238007.pdf (Publisher’s version ) (Closed access) IMPORTANCE: Cases of cerebral venous sinus thrombosis in combination with thrombocytopenia have recently been reported within 4 to 28 days of vaccination with the ChAdOx1 nCov-19 (AstraZeneca/Oxford) and Ad.26.COV2.S (Janssen/Johnson & Johnson) COVID-19 vaccines. An immune-mediated response associated with platelet factor 4/heparin antibodies has been proposed as the underlying pathomechanism. OBJECTIVE: To determine the frequencies of admission thrombocytopenia, heparin-induced thrombocytopenia, and presence of platelet factor 4/heparin antibodies in patients diagnosed with cerebral venous sinus thrombosis prior to the COVID-19 pandemic. DESIGN, SETTING, AND PARTICIPANTS: This was a descriptive analysis of a retrospective sample of consecutive patients diagnosed with cerebral venous sinus thrombosis between January 1987 and March 2018 from 7 hospitals participating in the International Cerebral Venous Sinus Thrombosis Consortium from Finland, the Netherlands, Switzerland, Sweden, Mexico, Iran, and Costa Rica. Of 952 patients, 865 with available baseline platelet count were included. In a subset of 93 patients, frozen plasma samples collected during a previous study between September 2009 and February 2016 were analyzed for the presence of platelet factor 4/heparin antibodies. EXPOSURES: Diagnosis of cerebral venous sinus thrombosis. MAIN OUTCOMES AND MEASURES: Frequencies of admission thrombocytopenia (platelet count 0.4, in a subset of patients with previously collected plasma samples). RESULTS: Of 865 patients (median age, 40 years [interquartile range, 29-53 years], 70% women), 73 (8.4%; 95% CI, 6.8%-10.5%) had thrombocytopenia, which was mild (100-149 ×103/μL) in 52 (6.0%), moderate (50-99 ×103/μL) in 17 (2.0%), and severe (

Laboratory diagnosis of congenital and acquired deficiencies of coagulation factor XIII (FXIII) can be challenging. Determination of FXIII function requires specific and sensitive assays which are not always available. This brief review article summarizes currently used FXIII assay methods, their principles and difficulties, and discusses the recommended diagnostic workup in case of a suspected FXIII deficiency. The article also briefly touches on experimental methods used in FXIII research.

Bleeding and thrombosis are major clinical problems with high morbidity and mortality. Treatment modalities for these diseases have improved in recent years, but there are many clinical questions remaining and a need to advance diagnosis, management, and therapeutic options. Basic research plays a fundamental role in understanding normal and disease processes, yet this sector has observed a steady decline in funding prospects thereby hindering support for studies of mechanisms of disease and therapeutic development opportunities. With the financial constraints faced by basic scientists, the ISTH organized a basic science task force (BSTF), comprising Scientific and Standardization Committee subcommittee chairs and co-chairs, to identify research opportunities for basic science in hemostasis and thrombosis. The goal of the BSTF was to develop a set of recommended priorities to build support in the thrombosis and hemostasis community and to inform ISTH basic science programs and policy making. The BSTF identified three principal opportunity areas that were of significant overarching relevance: mechanisms causing bleeding, innate immunity and thrombosis, and venous thrombosis. Within these, five fundamental research areas were highlighted: blood rheology, platelet biogenesis, cellular contributions to thrombosis and hemostasis, structure-function protein analyses, and visualization of hemostasis. This position paper discusses the importance and relevance of these opportunities and research areas, and the rationale for their inclusion. These findings have implications for the future of fundamental research in thrombosis and hemostasis to make transformative scientific discoveries and tackle key clinical questions. This will permit better understanding, prevention, diagnosis, and treatment of hemostatic and thrombotic conditions. ispartof: JOURNAL OF THROMBOSIS AND HAEMOSTASIS vol:20 issue:6 pages:1496-1506 ispartof: location:England status: published

Background: A high prevalence of poor job satisfaction and high chronic stress is documented for general practitioners (GPs) and non-physician practice staff from various countries. The reasons are multifactorial and include deficits in leadership, communication and workflows. This publicly funded study evaluates the effectiveness of the newly developed participatory, interdisciplinary, and multimodal IMPROVEjob intervention on improving job satisfaction among GPs and practice personnel. Here, we report the baseline characteristics of the participating GPs and practice assistants, focusing on job satisfaction and perceived chronic stress. Methods: The IMPROVEjob study was performed as a cluster-randomised, controlled trial (cRCT) with German GP practices in the North Rhine Region. The IMPROVEjob intervention comprised two leadership workshops (one for practice leaders only; a second for leaders and practice assistants), a toolbox with supplemental printed and online material, and a nine-month implementation phase supported by IMPROVEjob facilitators. The intervention addressed issues of leadership, communication, and work processes. During study nurse visits, participants completed questionnaires at baseline and after nine months follow up. The primary outcome was the change in job satisfaction as measured by the respective scale of the validated German version of the Copenhagen Psychosocial Questionnaire (German COPSOQ, version 2018). Perceived chronic stress was measured using the Trier Inventory of Chronic Stress (TICS- SSCS). Results: Recruitment of 60 practices was successful: 21 were solo, 39 were group practices. At baseline, n = 84 practice owners, n = 28 employed physicians and n = 254 practice assistants were included. The mean age of all participants was 44.4 (SD = 12.8). At baseline, the job satisfaction score in the total sample was 74.19 of 100 (±14.45) and the perceived chronic stress score was 19.04 of 48 (±8.78). Practice assistants had a significantly lower job satisfaction than practice owners (p < 0.05) and employed physicians (p < 0.05). In the regression analysis, perceived chronic stress was negatively associated with job satisfaction (b= −0.606, SE b = 0.082, p < 0.001, ICC = 0.10). Discussion: The degree of job satisfaction was similar to those in other medical professionals published in studies, while perceived chronic stress was markedly higher compared to the general German population. These findings confirm the need for interventions to improve psychological wellbeing in GP practice personnel.

This year's Congress of the International Society of Thrombosis and Haemostasis (ISTH) was hosted virtually from Philadelphia July 17-21, 2021. The conference, now held annually, highlighted cutting-edge advances in basic, population and clinical sciences of relevance to the Society. Despite being held virtually, the 2021 congress was of the same scope and quality as an annual meeting held in person. An added feature of the program is that talks streamed at the designated times will then be available on-line for asynchronous viewing. The program included 77 State of the Art (SOA) talks, thematically grouped in 28 sessions, given by internationally recognized leaders in the field. The SOA speakers were invited to prepare brief illustrated reviews of their talks that were peer reviewed and are included in this article. The topics, across the main scientific themes of the congress, include Arterial Thromboembolism, Coagulation and Natural Anticoagulants, COVID-19 and Coagulation, Diagnostics and Omics, Fibrinogen, Fibrinolysis and Proteolysis, Hemophilia and Rare Bleeding Disorders, Hemostasis in Cancer, Inflammation and Immunity, Pediatrics, Platelet Disorders, von Willebrand Disease and Thrombotic Angiopathies, Platelets and Megakaryocytes, Vascular Biology, Venous Thromboembolism and Women's Health. These illustrated capsules highlight the major scientific advances with potential to impact clinical practice. Readers are invited to take advantage of the excellent educational resource provided by these illustrated capsules. They are also encouraged to use the image in social media to draw attention to the high quality and impact of the science presented at the congress. ispartof: RESEARCH AND PRACTICE IN THROMBOSIS AND HAEMOSTASIS vol:5 issue:5 ispartof: location:United States status: published

Background The lectin pathway serine protease mannan-binding lectin-associated serine protease 1 (MASP-1) has been demonstrated to be a major link between complement and coagulation, yet little is known about its interactions with the fibrinolytic system. The aim of this work was to assess the effects of MASP-1 on fibrin clot lysis in different experimental settings. Methods Rotational thrombelastometry was used to evaluate the effect of MASP-1 on the lysis of clots formed in whole blood under static conditions. Whole blood clots were also formed in the presence and absence of MASP-1 under flow conditions in the Chandler loop and their lysis was analysed separately by fluorescence release of incorporated labelled fibrin. Real-time observation by laser scanning confocal microscopy was used to investigate the lysis of plasma clots where MASP-1 was present either during clot formation or lysis. Cleavage of tPA or plasminogen by MASP-1 was analysed by gel electrophoresis. We performed a turbidimetric clot lysis assay in the presence and absence of the MASP-1 inhibitor SGMI-1 (Schistocerca gregaria protease inhibitor (SGPI)-based MASP inhibitor-1) to evaluate the effect of endogenous MASP-1 in normal plasma and plasma samples from sepsis patients. Results In the thrombelastometric experiments, where MASP-1 was present during the entire clotting and lysis process, MASP-1 had a significant profibrinolytic effect and accelerated clot lysis. When clots were formed in the presence of MASP-1 under flow in the Chandler loop, the effects on fibrinolysis were heterogenous with impaired fibrinolysis in some individuals (n = 5) and no (n = 3) or even the opposite effect (n = 2) in others. In plasma clot lysis observed by confocal microscopy, lysis was prolonged when MASP-1 was added to the lysis solution, yet there was no difference in lysis time when MASP-1 was present during clot formation. When MASP-1 was incubated with tPA or plasminogen, respectively, cleavage of single-chain tPA into two-chain tPA and a slight reduction of plasminogen were observed. SGMI-1 significantly prolonged clot lysis in the turbidimetric clot lysis assay suggesting that MASP-1 accelerated lysis in plasma samples. Conclusion MASP-1 is able to alter the susceptibility of blood clots to the fibrinolytic system. MASP-1 has complex, mostly promoting effects on fibrinolysis with high inter-individual variation. Interactions of MASP-1 with the fibrinolytic system may be relevant in the development and therapy of cardiovascular and thrombotic diseases.

Accumulating evidence suggests a role for the complement system in the pathogenesis of diabetes and the vascular complications that characterise this condition. Complement proteins contribute to the development of type 1 diabetes (T1D) by enhancing the underlying organ-specific autoimmune processes. Complement upregulation and activation is also an important feature of insulin resistance and the development of type 2 diabetes (T2D). Moreover, animal and human studies indicate that complement proteins are involved in the pathogenic mechanisms leading to diabetic microvascular and macrovascular complications. The adverse vascular effects of complement appear to be related to enhancement of the inflammatory process and the predisposition to a thrombotic environment, eventually leading to vascular occlusion. Complement proteins have been considered as therapeutic targets to prevent or treat vascular disease but studies have been mainly conducted in animal models, while human work has been both limited and inconclusive so far. Further studies are needed to understand the potential role of complement proteins as therapeutic targets for reversal of the pathological processes leading to T1D and T2D and for the prevention/treatment of diabetic vascular complications.

Diabetes is a proinflammatory and prothrombotic condition that increases the risk of vascular complications. The aim of this study was to develop a diabetic microvascular flow model that allows to study the complex interactions between endothelial cells, blood cells and plasma proteins and their effects on clot formation. Primary human cardiac microvascular endothelial cells from donors without diabetes or donors with diabetes (type 1 or type 2) were grown in a microfluidic chip, perfused with non-diabetic or diabetic whole blood, and clot formation was assessed by measuring fibrin deposition in real time by confocal microscopy. Clot formation in non-diabetic whole blood was significantly increased in the presence of endothelial cells from donors with type 2 diabetes compared with cells from donors without diabetes. There was no significant difference in clot formation between non-diabetic and diabetic whole blood. We present for the first time a diabetic microvascular flow model as a new tool to study clot formation as a result of the complex interactions between endothelial cells, blood cells and plasma proteins in a diabetes setting. We show that endothelial cells affect clot formation in whole blood, attributing an important role to the endothelium in the development of atherothrombotic complications.

ObjectiveTo investigate prediction of cerebral venous thrombosis (CVT) by clinical variables and D-dimer levels.MethodsThis prospective multicenter study included consecutive patients with clinically possible CVT. On admission, patients underwent clinical examination, blood sampling for D-dimers measuring (ELISA test), and magnetic resonance/CT venography. Predictive value of clinical variables and D-dimers for CVT was calculated. A clinical score to stratify patients into groups with low, moderate, or high CVT risk was established with multivariate logistic regression.ResultsCVT was confirmed in 26.2% (94 of 359) of patients by neuroimaging. The optimal estimate of clinical probability was based on 6 variables: seizure(s) at presentation (4 points), known thrombophilia (4 points), oral contraception (2 points), duration of symptoms >6 days (2 points), worst headache ever (1 point), and focal neurologic deficit at presentation (1 point) (area under the curve [AUC] 0.889). We defined 0 to 2 points as low CVT probability (negative predictive value [NPV] 94.1%). Of the 186 (51.8%) patients who had a low probability score, 11 (5.9%) had CVT. The frequency of CVT was 28.3% (34 of 120) in patients with a moderate (3–5 points) and 92.5% (49 of 53) in patients with a high (6–12 points) probability score. All low CVT probability patients with CVT had D-dimers >500 μg/L. Predictive value of D-dimers for CVT for >675 μg/L (best cutoff) vs >500 μg/L was as follows: sensitivity 77.7%, specificity, 77%, NPV 90.7%, and accuracy 77.2% vs sensitivity 89.4%, specificity 66.4%, NPV 94.6%, and accuracy 72.4%, respectively. Adding the clinical score to D-dimers >500 μg/L resulted in the best CVT prediction score explored (at the cutoff ≥6 points: sensitivity 83%/specificity 86.8%/NPV 93.5%/accuracy 84.4%/AUC 0.937).ConclusionThe proposed new clinical score in combination with D-dimers may be helpful for predicting CVT as a pretest score; none of the patients with CVT showed low clinical probability for CVT and D-dimers ClinicalTrials.gov identifierNCT00924859.

Cellular factor XIII (cFXIII, FXIII-A2), a transglutaminase, has been demonstrated in a few cell types. Its main function is to cross-link proteins by isopeptide bonds. Here, we investigated the presence of cFXIII in cells of human cornea. Tissue sections of the cornea were immunostained for FXIII-A in combination with staining for CD34 antigen or isopeptide cross-links. Isolated corneal keratocytes were also evaluated by immunofluorescent microscopy and flow cytometry. FXIII-A in the corneal stroma was quantified by Western blotting. FXIII-A mRNA was detected by RT-qPCR. The cornea of FXIII-A-deficient patients was evaluated by cornea topography. FXIII-A was detected in 68 ± 13% of CD34+ keratocytes. Their distribution in the corneal stroma was unequal; they were most abundant in the subepithelial tertile. cFXIII was of cytoplasmic localization. In the stroma, 3.64 ng cFXIII/mg protein was measured. The synthesis of cFXIII by keratocytes was confirmed by RT-qPCR. Isopeptide cross-links were detected above, but not within the corneal stroma. Slight abnormality of the cornea was detected in six out of nine FXIII-A-deficient patients. The presence of cFXIII in human keratocytes was established for the first time. cFXIII might be involved in maintaining the stability of the cornea and in the corneal wound healing process.

Coagulation factor XIII (FXIII) is the main stabilizer of the fibrin clot. It circulates in plasma as a tetramer of two A-subunits and two B-subunits. Under physiological conditions, FXIII-A exists as a dimer (FXIII-A2). The interactions between the FXIII-A-subunits that stabilize the FXIII-A2 dimer are not fully understood. We therefore designed a systematic approach to identify amino acid residues crucial for the expression and stability of FXIII-A2. Based on the available FXIII-A2 crystal structure, we identified 12 amino acid residues forming intersubunit salt bridges and 21 amino acid residues forming hydrogen bonds between the two A-subunits. We chose 10 amino acid residues that form 5 particularly strong interactions, performed site-directed mutagenesis, and expressed the mutants in CHO cells. Disruption of these interactions by single mutation of Lys257, Lys113, Asp343, Glu401, or Asp404 abolished the expression of properly folded, soluble, and functional FXIII-A in CHO cells. On the contrary, mutation of Glu111, Arg100, or Asn112 had no significant effect on FXIII-A expression. Our results suggest that 4 intersubunit interactions (Arg11-Asp343, Lys113-Asp367, Lys257-Glu401, and Arg260-Asp404) are essential for the stability of FXIII-A2. Our findings are supported by reported mutations at Lys257, Arg260, and Asp404 found in patients with congenital FXIII-A deficiency.

There is a need to improve the efficacy and safety of catheter-directed thrombolysis (CDT) for thrombo-occlusive diseases, and ultrasound-assisted CDT (USAT) is a promising approach. We tested if thrombolysis efficacy of USAT can be improved by adding gaseous microbubbles (MB). We developed an in vitro dynamic overflow model for iliofemoral deep vein thrombosis, and added MB to an USAT system with ultrasound energy and dose of tissue plasminogen activator according to clinical practice. A total of 64 clots (mean baseline weight of 8.23 ± 1.12 g, generated from citrated human whole blood from 7 healthy male volunteers) were randomly assigned to 1 of 4 study protocols of 30 minutes' duration: negative control, CDT, USAT, and USAT + MB.Thrombolysis efficacy was assessed by measuring the change in D-dimer levels in the overflow liquid and the percentage of clot weight reduction. Compared to negative control, change in D-dimer increased by 62% (p = 0.017), 128% (p = 0.002), and 177% (p

The activation peptide of blood coagulation factor XIII (AP-FXIII) has important functions in stabilizing the FXIII-A2 dimer and regulating FXIII activation. Contributions of many of its 37 amino acids to these functions have been described. However, the role of proline 36, which is adjacent to the thrombin cleavage site at Arg37, has not yet been studied in detail. We approached this question when we came across a patient with congenital FXIII deficiency in whom we detected a novel Pro36Ser mutation. We expressed the mutant FXIII-A Pro36Ser protein in Chinese hamster ovary cells and found that this mutation does not influence FXIII-A expression but significantly inhibits proteolytic activation by thrombin. The enzymatic transglutaminase activity is not affected as it can be induced in the presence of high Ca2+ concentrations. We performed nuclear magnetic resonance analysis to investigate AP-FXIII–thrombin interactions, which showed that the mutant Ser36 peptide binds less well to the thrombin surface than the native Pro36 peptide. The Arg37 at the P1 position still makes strong interactions with the active site cleft but the P4–P2 residues (34VVS36) appear to be less well positioned to contact the neighbouring thrombin active site region. In conclusion, we have characterized a novel mutation in AP-FXIII representing only the fourth case of the rare FXIII-A type II deficiency. This case served as a perfect in vivo model to shed light on the crucial role of Pro36 in the proteolytic activation of FXIII-A. Our results contribute to the understanding of structure–function relationship in FXIII.

Mannan-binding lectin-associated serine protease-1 (MASP-1), a protein of the complement lectin pathway, resembles thrombin in terms of structural features and substrate specificity, and it has been shown to activate coagulation factors. Here we studied the effects of MASP-1 on clot formation in whole blood (WB) and platelet-poor plasma (PPP) by thrombelastography and further elucidated the underlying mechanism. Cleavage of prothrombin by MASP-1 was investigated by SDS-PAGE and N-terminal sequencing of cleavage products. Addition of MASP-1 or thrombin to WB and PPP shortened the clotting time and clot formation time significantly compared to recalcified-only samples. The combination of MASP-1 and thrombin had additive effects. In a purified system, MASP-1 was able to induce clotting only in presence of prothrombin. Analysis of MASP-1-digested prothrombin confirmed that MASP-1 cleaves prothrombin at three cleavage sites. In conclusion, we have shown that MASP-1 is able to induce and promote clot formation measured in a global setting using the technique of thrombelastography. We further confirmed that MASP-1-induced clotting is dependent on prothrombin. Finally, we have demonstrated that MASP-1 cleaves prothrombin and identified its cleavage sites, suggesting that MASP-1 gives rise to an alternative active form of thrombin by cleaving at the cleavage site R393.

* University Clinic of Haematology, Haemostasis Research Laboratory, University Hospital of Bern, Bern, Switzerland † Department of Clinical Research, University of Bern, Bern, Switzerland; ‡ Department of Haematology, Haemostasis& Thrombosis, National Institute of Blood Disease and Bone Marrow Transplantation (NIBD), Karachi, Pakistan. ¶ Department of Internal Medicine, Spital Netz Bern, Bern, Switzerland

Summary Growing evidence suggests a prominent role of the complement system in the pathogenesis of cardio- and cerebrovascular diseases (CVD). Mannan-binding lectin-associated serine proteases (MASPs) MASP-1 and MASP-2 of the complement lectin pathway contribute to clot formation and may represent an important link between inflammation and thrombosis. MBL-associated protein MAp44 has shown cardioprotective effects in murine models. However, MAp44 has never been measured in patients with CVD and data on MASP levels in CVD are scarce. Our aim was to investigate for the first time plasma levels of MAp44 and MASP-1, -2, -3 concomitantly in patients with CVD. We performed a pilot study in 50 healthy volunteers, in stable coronary artery disease (CAD) patients with one-vessel (n = 51) or three-vessel disease (n = 53) and age-matched controls with normal coronary arteries (n = 53), 49 patients after myocardial infarction (MI) and 66 patients with acute ischaemic stroke. We measured MAp44 and MASP-1 levels by in-house time-resolved immunofluorometric assays. MASP-2 and MASP-3 levels were measured using commercial enzyme-linked immunosorbent assay kits. MASP-1 levels were highest in subacute MI patients and lowest in acute stroke patients. MASP-2 levels were lower in MI and stroke patients compared with controls and CAD patients. MASP-3 and MAp44 levels did not differ between groups. MASP or MAp44 levels were not associated with severity of disease. MASP and MAp44 levels were associated with cardiovascular risk factors including dyslipidaemia, obesity and hypertension. Our results suggest that MASP levels may be altered in vascular diseases. Larger studies are needed to confirm our results and elucidate the underlying mechanisms.

Over the last two decades, it became evident that factor XIII (FXIII) is not only a crucial determinant of clot characteristics but also has potentially important functions in many various fields such as bone biology, immunity, and adipogenesis. In this review, we aim to summarize the latest findings regarding structure and function of FXIII. In regard to FXIII structure, much progress has been made recently to understand how its subunits are held together. In the A subunit, the activation peptide has a crucial role in the formation of FXIII-A2 dimers. In the B subunit, Sushi domains that are involved in binding to the A subunit and in B2 dimer formation have been identified. In regard to FXIII function, interactions with immune cells and the complement system have been described. A novel function of FXIII-A in adipogenesis has been suggested. The role of FXIII-A in osteoblast differentiation has been further investigated; however, a novel double knockout mouse deficient in both FXIII-A and transglutaminase 2 showed normal bone formation. Thus, more research, in particular, into the cellular functions of FXIII-A is still required.

Background and method: Increased plasma clot density and prolonged lysis times are associated with cardiovascular disease. In this study, we employed a functional proteomics approach to identify novel clot components which may influence clot phenotypes. Results: Analysis of perfused, solubilised plasma clots identified inflammatory proteins, including complement C3, as novel clot components. Analysis of paired plasma and serum samples confirmed concentration-dependent incorporation of C3 into clots. Surface plasmon resonance indicated high-affinity binding interactions between C3 and fibrinogen and fibrin. Turbidimetric clotting and lysis assays indicated C3 impaired fibrinolysis in a concentration-dependent manner, both in vitro and ex vivo. Conclusion: These data indicate functional interactions between complement C3 and fibrin leading to prolonged fibrinolysis. These interactions are physiologically relevant in the context of protection following injury and suggest a mechanistic link between increased plasma C3 concentration and acute cardiovascular thrombotic events.

Coagulation factor XIII (FXIII) stabilizes fibrin fibers and is therefore a major player in the maintenance of hemostasis. FXIII is activated by thrombin resulting in cleavage and release of the FXIII activation peptide (AP-FXIII). The objective of this study was to characterize the released AP-FXIII and determine specific features that may be used for its specific detection. We analyzed the structure of bound AP-FXIII within the FXIII A-subunit and interactions of AP-FXIII by hydrogen bonds with both FXIII A-subunit monomers. We optimized our previously developed AP-FXIII ELISA by using 2 monoclonal antibodies. We determined high binding affinities between the antibodies and free AP-FXIII and demonstrated specific binding by epitope mapping analyses with surface plasmon resonance and enzyme-linked immunosorbent assay. Because the structure of free AP-FXIII had been characterized so far by molecular modeling only, we performed structural analysis by nuclear magnetic resonance. Recombinant AP-FXIII was largely flexible both in plasma and water, differing significantly from the rigid structure in the bound state. We suggest that the recognized epitope is either occluded in the noncleaved form or possesses a structure that does not allow binding to the antibodies. On the basis of our findings, we propose AP-FXIII as a possible new marker for acute thrombotic events.

Background Severe hereditary coagulation factor XIII deficiency is a rare homozygous bleeding disorder affecting one person in every two million individuals. In contrast, heterozygous factor XIII deficiency is more common, but usually not associated with severe hemorrhage such as intracranial bleeding or hemarthrosis. In most cases, the disease is caused by F13A gene mutations. Causative mutations associated with the F13B gene are rarer. Design and Methods We analyzed ten index patients and three relatives for factor XIII activity using a photometric assay and sequenced their F13A and F13B genes. Additionally, structural analysis of the wild-type protein structure from a previously reported X-ray crystallographic model identified potential structural and functional effects of the missense mutations. Results All individuals except one were heterozygous for factor XIIIA mutations (average factor XIII activity 51%), while the remaining homozygous individual was found to have severe factor XIII deficiency (

BACKGROUND: It has been suggested that changes in blood coagulation and fibrinolysis might explain the observed association between depression and coronary artery disease (CAD). So far, only a few coagulation factors have been investigated in this regard, and the results were not consistent. DESIGN: The aim of our study was to analyse a broad range of coagulation and fibrinolytic factors, with emphasis on factors directly involved in clot formation and degradation or reflecting coagulation activation, in patients with CAD and controls without CAD, as assessed by coronary angiography, who also underwent a diagnostic procedure for depression. METHODS: We screened 306 patients with CAD and controls without CAD for depression using the Hospital Anxiety and Depression Scale and Allgemeine Depressions Skala-L questionnaires. In participants with positive screening result, diagnosis of major depression was confirmed or excluded by a structured interview. We analysed the following coagulation and fibrinolytic factors: fibrinogen, prothrombin fragment F1+2, factor XIII A-subunit, factor XIII B-subunit, tissue plasminogen activator, plasminogen activator inhibitor-1, thrombin-activable fibrinolysis inhibitor, and D-dimer. RESULTS: We did not observe significant associations between depression and CAD, nor between depression and cardiovascular risk factors. Coagulation and fibrinolytic factors showed no differences between patients with CAD and controls, but they were associated with several cardiovascular risk factors. Depression was not associated with coagulation and fibrinolytic factors. No associations were found either when both CAD and depression were taken into account. CONCLUSION: Our study gives no evidence that there is a significant relation among depression, CAD, and blood coagulation and fibrinolysis.

Mannan-binding lectin-associated serine protease-1 (MASP-1), a protein of the complement lectin pathway, resembles thrombin in terms of structural features and substrate specificity. Due to its interplay with several coagulation factors, it has the ability to induce fibrin clot formation independent of the usual coagulation activation pathways. We have recently shown that MASP-1 activates prothrombin and identified arginine (R) 155, R271, and R393 as potential cleavage sites. FXa cleaves R320 instead of R393, and thrombin cleaves R155 and R284 in prothrombin. Here we have used three arginine-to-glutamine mutants of prothrombin, R271Q, R320Q, R393Q and the serine-to-alanine active site mutant S525A to investigate in detail the mechanism of MASP-1 mediated prothrombin activation. Prothrombin wildtype and mutants were digested with MASP-1 and the cleavage products were analysed by SDS-PAGE and N-terminal sequencing. A functional clotting assay was performed by thrombelastography. We have found that MASP-1 activates prothrombin via two simultaneous pathways, either cleaving at R271 or R393 first. Both pathways result in the formation of several active alternative thrombin species. Functional studies confirmed that both R393 and R320 are required for prothrombin activation by MASP-1, whereas R155 is not considered to be an important cleavage site in this process. In conclusion, we have described for the first time a detailed model of prothrombin activation by MASP-1.

Summary There is increasing evidence that the complement system plays an important role in diabetes and the development of diabetic vascular complications. In particular, mannan-binding lectin (MBL) levels are elevated in diabetes patients, and diabetes patients with diabetic nephropathy have higher MBL levels than diabetes patients with normal renal function. The MBL-associated serine proteases (MASPs) MASP-1, MASP-2 and MASP-3 and MBL-associated protein MAp44 have not yet been studied in diabetes patients. We therefore measured plasma levels of MASP-1, MASP-2, MASP-3 and MAp44 in 30 children with type 1 diabetes mellitus (T1DM) and 17 matched control subjects, and in 45 adults with T1DM and 31 matched control subjects. MASP-1 and MASP-2 levels were significantly higher in children and adults with T1DM than in their respective control groups, whereas MASP-3 and MAp44 levels did not differ between patients and controls. MASP-1 and MASP-2 levels correlated with HbA1c, and MASP levels decreased when glycaemic control improved. Because MASP-1 and MASP-2 have been shown to interact directly with blood coagulation, elevated levels of these proteins may play a role in the enhanced thrombotic environment and consequent vascular complications in diabetes.

Plasminogen activator inhibitor type-1 (PAI-1) is considered to be the main inhibitor of fibrinolysis in sepsis. However, the contribution of TAFI to the inhibition of fibrinolysis in sepsis is currently unknown. TAFI antigen and PAI-1 levels were measured in severe sepsis (n = 32) and septic shock (n = 8) patients. In addition, TAFI antigen levels had been determined in 151 controls. Septic patients had significantly (p

Nicorandil is a drug which has been developed as an anti-anginal medication. Its structure is characterized by a dual mechanism of action. The nicotinamide moiety acts as an opener of ATP-sensitive potassium channels, whereas the NO2 group explains its nitrate-like properties. The nitric oxide-like action leads to a dilatation of the large coronary arteries, whereas its potassium channel opening action is responsible for the dilatation of coronary resistance vessels. Nicorandil has also been found to dilate veins, enabling it to decrease both preload and afterload and to increase coronary blood flow. The ATP-sensitive potassium channel opening mimics preconditioning in the absence of ischemia and may therefore exert cytoprotective effects. These have been thought to be the reason for a reduction in major coronary events and all cardiovascular events of nicorandil in addition to a specific anti-anginal medication in the Impact Of Nicorandil in Angina study. This review summarizes the pharmacologic properties of nicorandil and assesses its actual place in different cardiovascular disease states.

BACKGROUND The mechanism of avascular osteonecrosis (AVN) is controversial. Besides an increased bone marrow pressure with reduced blood supply, an enhanced coagulation has been considered. We hypothesize that a genetic variant of the plasminogen activator inhibitor-1 (PAI-1) determines the risk of AVN in glucocorticoid-treated patients. METHODS Genotyping for the 4G/5G PAI-1 polymorphism was performed in 228 glucocorticoid-treated renal transplant patients. AVN of the hip was present in 26 patients. Magnetic resonance imaging (MRI) of the hips was obtained in 81 of the remaining renal transplant patients without clinical symptoms of AVN. RESULTS The presence of the homozygous 4G/4G PAI-1 genotype was higher in patients with AVN (60.3%) as compared with patients without either clinical (20.6%, P

SummaryDue to its role in the balance between coagulation and fibrinolysis, thrombin activatable fibrinolysis inhibitor (TAFI) may be involved in the development of cardiovascular diseases. We studied 362 patients with coronary artery disease (CAD) and 134 control subjects free of CAD, both groups investigated by angiography. TAFI antigen levels were determined in venous and intracoronary plasma samples and were related to metabolic and hemostatic risk factors and extent of coronary atherosclerosis. Venous TAFI levels tended to be higher in CAD patients compared to controls, whereas this difference was significant in intracoronary samples. A subgroup of patients who had not experienced acute myocardial infarction or undergone previous cardiac interventions showed significantly higher TAFI levels in both venous and intracoronary plasma samples. TAFI levels correlated with acute phase reactants indicating a role for TAFI in inflammation. However, TAFI levels did not correlate with extent of coronary atherosclerosis and among the classical cardiovascular risk factors TAFI levels only correlated with total cholesterol and fibrinogen concentration. Our results suggest that TAFI might be a risk factor for the development of CAD.

BACKGROUND The human activation peptide of factor XIII (AP-FXIII) comprises the first 37 amino acids of the N-terminus and holds the FXIII in an inactive state. FXIII is activated either proteolytically by cleavage of AP-FXIII by thrombin, or non-proteolytically by high calcium concentrations. OBJECTIVE To investigate the role of AP-FXIII in the expression and stability of FXIII. METHODS We cloned 13 FXIII variants with progressive truncations of AP-FXIII from the N-terminus (delN-FXIII-A), expressed them in mammalian cells, and measured their thermostability, activation, and transglutaminase activity. We also used in silico calculations to analyze the stability of hypothetical delN-FXIII dimers and to identify crucial motifs within AP-FXIII. RESULTS Variants with deletions longer than the first 10 amino acids and an R11Q point mutant were not expressed as proteins. In silico calculations indicated that the sequence (8) FGGR(12) R plays a substantial role in intersubunit interactions in FXIII-A2 homodimers. In agreement with this prediction, the temperature stability of delN-FXIII variants decreased with increasing length of deletion. These results may suggest a role of the N-terminus of AP-FXIII in dimer stability. Substantial sequence homology was found among activation peptides of vertebrate and even invertebrate (crustacean) FXIII-A orthologs, which further supports our conclusion. CONCLUSIONS We conclude that deletion of 11 or more N-terminal amino acids disrupts intersubunit interactions, which may prevent FXIII-A2 homodimer formation. Therefore, AP-FXIII plays an important role in the stability of the FXIII-A2 dimer.

AIMS/HYPOTHESIS Plasminogen activator inhibitor-1 (PAI-1) has been regarded as the main antifibrinolytic protein in diabetes, but recent work indicates that complement C3 (C3), an inflammatory protein, directly compromises fibrinolysis in type 1 diabetes. The aim of the current project was to investigate associations between C3 and fibrinolysis in a large cohort of individuals with type 2 diabetes. METHODS Plasma levels of C3, C-reactive protein (CRP), PAI-1 and fibrinogen were analysed by ELISA in 837 patients enrolled in the Edinburgh Type 2 Diabetes Study. Fibrin clot lysis was analysed using a validated turbidimetric assay. RESULTS Clot lysis time correlated with C3 and PAI-1 plasma levels (r = 0.24, p