BackgroundNumerous interactions between the coagulation and complement systems have been shown. Recently, links between coagulation and mannan-binding lectin-associated serine protease-1 (MASP-1) of the complement lectin pathway have been proposed. Our aim was to investigate MASP-1 activation of factor XIII (FXIII), fibrinogen, prothrombin, and thrombin-activatable fibrinolysis inhibitor (TAFI) in plasma-based systems, and to analyse effects of MASP-1 on plasma clot formation, structure and lysis.Methodology/Principal FindingsWe used a FXIII incorporation assay and specific assays to measure the activation products prothrombin fragment F1+2, fibrinopeptide A (FPA), and activated TAFI (TAFIa). Clot formation and lysis were assessed by turbidimetric assay. Clot structure was studied by scanning electron microscopy. MASP-1 activated FXIII and, contrary to thrombin, induced FXIII activity faster in the Val34 than the Leu34 variant. MASP-1-dependent generation of F1+2, FPA and TAFIa showed a dose-dependent response in normal citrated plasma (NCP), albeit MASP-1 was much less efficient than FXa or thrombin. MASP-1 activation of prothrombin and TAFI cleavage were confirmed in purified systems. No FPA generation was observed in prothrombin-depleted plasma. MASP-1 induced clot formation in NCP, affected clot structure, and prolonged clot lysis.Conclusions/SignificanceWe show that MASP-1 interacts with plasma clot formation on different levels and influences fibrin structure. Although MASP-1-induced fibrin formation is thrombin-dependent, MASP-1 directly activates prothrombin, FXIII and TAFI. We suggest that MASP-1, in concerted action with other complement and coagulation proteins, may play a role in fibrin clot formation.

Aims/hypothesis Impaired fibrin clot lysis is a key abnormality in diabetes and complement C3 is one protein identified in blood clots. This work investigates the mechanistic pathways linking C3 and hypofibrinolysis in diabetes using ex vivo/in vitro studies. Methods Fibrinolysis and C3 plasma levels were determined in type 1 diabetic patients and healthy controls, and the effects of glycaemia investigated. C3 incorporation into fibrin clots and modulation of fibrinolysis were analysed by ELISA, immunoblotting, turbidimetric assays and electron and confocal microscopy. Results Clot lysis time was longer in diabetic children than in controls (599±18 and 516±12 s respectively; p<0.01), C3 levels were higher in diabetic children (0.55±0.02 and 0.43± 0.02 g/l respectively; p<0.01) and both were affected by improving glycaemia. An interaction between C3 and fibrin was confirmed by the presence of lower protein levels in sera compared with corresponding plasma and C3 detection in plasma clots by immunoblot. In a purified system, C3 was associated with thinner fibrin fibres and more prolongation of lysis time of clots made from fibrinogen from diabetic participants compared with controls (244±64 and 92±23 s respectively; p<0.05). Confocal microscopy showed higher C3 incorporation into diabetic clots compared with controls, and fully formed clot lysis was prolonged by 764±76 and 428±105 s respectively (p<0.05). Differences in lysis, comparing diabetes and controls, were not related to altered plasmin generation or C3-fibrinogen binding assessed by plasmon resonance. Conclusions/interpretation C3 incorporation into clots from diabetic fibrinogen is enhanced and adversely affects fibrinolysis. This may be one novel mechanism for compromised clot lysis in diabetes, potentially offering a new therapeutic target.

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MASP-1 is a versatile serine protease that cleaves a number of substrates in human blood. In recent years it became evident that besides playing a crucial role in complement activation MASP-1 also triggers other cascade systems and even cells to mount a more powerful innate immune response. In this review we summarize the latest discoveries about the diverse functions of this multi-faceted protease. Recent studies revealed that among MBLassociated serine proteases, MASP-1 is the one responsible for triggering the lectin pathway via its ability to rapidly autoactivate then cleave MASP-2, and possibly MASP-3. The crystal structure of MASP-1 explains its more relaxed substrate specificity compared to the related complement enzymes. Due to the relaxed specificity, MASP-1 interacts with the coagulation cascade and the kinin generating system, and it can also activate endothelial cells eliciting pro-inflammatory signaling.

To cite this article: Schroeder V, Kohler HP. New developments in the area of factor XIII. J Thromb Haemost 2013; 11: 234-44.Summary. Coagulation factor (F)XIII is best known for its role in fibrin stabilization and cross-linking of antifibrinolytic proteins to the fibrin clot. From patients with congenital FXIII deficiency, it is known that FXIII also has important functions in wound healing and maintaining pregnancy. Over the last decade more and more research groups with different backgrounds have studied FXIII and have unveiled putative novel functions for FXIII. FXIII, with its unique role as a transglutaminase among the other serine protease coagulation factors, is now recognized as a multifunctional protein involved in regulatory mechanisms and construction and repair processes beyond hemostasis with possible implications in many areas of medicine. The aim of this review was to give an overview of exciting novel findings and to highlight the remarkable diversity of functions attributed to FXIII. Of course, more research into the underlying mechanisms and (patho-)physiological relevance of the many described functions of FXIII is needed. It will be exciting to observe future developments in this area and to see if and how these interesting findings may be translated into clinical practice in the future.

BackgroundSevere 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 MethodsWe 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 wildtype protein structure from a previously reported X-ray crystallographic model identified potential structural and functional effects of the missense mutations. ResultsAll 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 (<5% of normal factor XIII activity). Eight of the 12 heterozygous patients exhibited a bleeding tendency upon provocation. ConclusionsThe identified missense (Pro289Arg, Arg611His, Asp668Gly) and nonsense (Gly390X, Trp664X) mutations are causative for factor XIII deficiency. A Gly592Ser variant identified in three unrelated index patients, as well as in 200 healthy controls (minor allele frequency 0.005), and two further Tyr167Cys and Arg540Gln variants, represent possible candidates for rare F13A gene polymorphisms since they apparently do not have a significant influence on the structure of the factor XIIIA protein. Future in vitro expression studies of the factor XIII mutations are required to confirm their pathological mechanisms.Key words: factor XIII deficiency, FXIII-A, FXIII-B, structural analysis. Citation: Ivaskevicius V, Biswas

Confirmation of suspected congenital factor XIII (FXIII) deficiency still represents a diagnostic challenge in the field of rare bleeding disorders. Because of the lack of awareness and difficulties associated with timing of blood sampling, FXIII laboratory assays, and interpretation of laboratory results, diagnoses of FXIII deficiency are still missed all over the world with potentially fatal consequences from severe bleeding complications. Better knowledge of FXIII biochemical properties and function and understanding of the principles and limitations of FXIII laboratory assays can prevent missed diagnoses, and patients will benefit from better care. This review gives a detailed overview and update about congenital FXIII deficiency, its epidemiology, and molecular genetics. It highlights the importance of newer specific FXIII assays and their principles to avoid any missed diagnosis of FXIII deficiency. This review also gives an update on the therapeutic options for patients suffering from this rare but life-threatening disease.

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.