Identification of amino acid residues that are crucial for FXIII-A intersubunit interactions and stability.

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.

Factor XIII: Structure and Function.

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.

Interaction between FXIII and fibrinogen.

In this issue of Blood, Smith and colleagues report on the functional role of the interaction between these 2 proteins by studying the involved binding sites responsible for clot stabilization.(1)

Factor XIII deficiency: an update.

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.

Sensitive and selective detection of free FXIII activation peptide: a potential marker of acute 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.

Prediction of cerebral venous thrombosis with a new clinical score and D-dimer levels.

OBJECTIVE: To investigate prediction of cerebral venous thrombosis (CVT) by clinical variables and D-dimer levels. METHODS: This 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. RESULTS: CVT 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). CONCLUSION: The 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 <500 µg/L. CLINICALTRIALSGOV IDENTIFIER: NCT00924859.

Proline 36 of the Factor XIII Activation Peptide Plays a Crucial Role in Substrate Recognition and Zymogen Activation.

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.

Factor XIII activation peptide is released into plasma upon cleavage by thrombin and shows a different structure compared to its bound form.

The first step of coagulation factor XIII (FXIII) activation involves cleavage of the FXIII activation peptide (FXIII-AP) by thrombin. However, it is not known whether the FXIII-AP is released into plasma upon cleavage or remains attached to activated FXIII. The aim of the present work was to study the structure of free FXIII-AP, develop an assay for FXIII-AP determination in human plasma, and to answer the question whether FXIII-AP is released into plasma. We used ab-initio modeling and molecular dynamics simulations to study the structure of free FXIII-AP. We raised monoclonal and polyclonal antibodies against FXIII-AP and developed a highly sensitive and specific ELISA method for direct detection of FXIII-AP in human plasma. Structural analysis showed a putative different conformation of the free FXIII-AP compared to FXIII-AP bound to the FXIII protein. We concluded that it might be feasible to develop specific antibodies against the free FXIII-AP. Using our new FXIII-AP ELISA, we found high levels of FXIII-AP in in-vitro activated plasma samples and serum. We showed for the first time that FXIIIAP is detached from activated FXIII and is released into plasma, where it can be directly measured. Our findings may be of major clinical interest in regard to a possible new marker in thrombotic disease.

International registry on factor XIII deficiency: a basis formed mostly on European data.

FXIII deficiency is known as one of the rarest blood coagulation disorders. In this study, the phenotypic and in part genotypic data of 104 FXIII-deficient patients recorded from 1993 - 2005 are presented. The most common bleeding symptoms were subcutaneous bleeding (57%) followed by delayed umbilical cord bleeding (56%), muscle hematoma (49%), hemorrhage after surgery (40%), hemarthrosis (36%), and intracerebral bleeding (34%). Prophylactic treatment was initiated in about 70% of all patients. FXIII-B subunit-deficient patients had a milder phenotype than patients with FXIII-A subunit deficiency. The most frequent mutation affecting the F13A gene was a splice site mutation in intron 5 (IVS5-1G>A). This mutation was found in eight (17%) of 46 analyzed families. The haplotype analysis of patients carrying the IVS5-1A allele was consistent with a founder effect. The international registry (http://www.f13-database.de) will provide clinicians and scientists working on FXIII deficiency with a helpful tool to improve patient care and direct future studies towards better understanding and treatment of the disease.

Characterisation of six novel A-subunit mutations leading to congenital factor XIII deficiency and molecular analysis of the first diagnosed patient with this rare bleeding disorder.

In 1960, the first case report on factor XIII deficiency was published describing a seven-year-old Swiss boy with a so far unknown bleeding disorder. Today, more than 60 mutations in the factor XIIIA- and B-subunit genes are known leading to congenital factor XIII deficiency. In the present study, we describe six novel mutations in the factor XIII A-subunit gene. Additionally, we present the molecular characterisation of the first described patient with congenital factor XIII deficiency. The six novel mutations include a small deletion, Glu202 delG, leading to a premature stop codon and truncation of the protein, and a splice site mutation at the exon 10/intron 10 boundary, +1G/A, giving rise to an incorrect spliced mRNA lacking exons 10 and 11. The remaining four mutations are characterised by the single amino acid changes Met159Arg, Gly215Arg, Trp375Cys, and His716Arg, and were expressed in COS-1 cells. Antigen levels and activity of the mutants were significantly reduced compared to the wild-type. The patient described in 1960 also shows a single amino acid change, Arg77Cys. Structural analysis of all mutant enzymes suggests several mechanisms leading to destabilisation of the protein.

Association of plasminogen activator inhibitor-1 genotype with avascular osteonecrosis in steroid-treated renal allograft recipients.

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<0.007) or radiological signs of AVN (17.3%, P<0.002). The prevalence of AVN by genotype was 1.8% with the 5G/5G, 7.7% with the 5G/4G, and 30.3% with the 4G/4G alleles (P<0.001 vs. 5G/4G and 5G/5G). The prevalence of AVN increased with increasing body mass index (BMI) (P=0.04). The prevalence of AVN by genotype in subjects with persistent hyperparathyroidism was 4.2% with the 5G/5G, 15.2% with the 5G/4G, and 55.5% with the 4G/4G alleles (P<0.003 vs. 5G/4G and P<0.001 vs. 5G/5G). CONCLUSIONS: Hypofibrinolysis conferred by the 4G/4G PAI-1 gene variant is a major predisposing factor for AVN in renal transplant patients. The risk is particularly high in obese subjects or patients with persistent hyperparathyroidism. A prospective intervention study of early anticoagulation after renal transplantation is needed to assess whether glucocorticoid-associated AVN can be prevented.

Role of blood coagulation factor XIII in patients with acute pulmonary embolism. Correlation of factor XIII antigen levels with pulmonary occlusion rate, fibrinogen, D-dimer, and clot firmness.

In patients with acute pulmonary embolism (PE), pulmonary occlusion rate is directly related to D-dimer and inversely related to fibrinogen levels. The role of coagulation factor XIII (FXIII) levels in acute venous thromboembolism is not known. A total of 120 consecutive patients with suspected PE and VIDAS D-dimer levels >500 micro g/L were investigated by helical computed tomography (CT). Pulmonary occlusion rate was assessed by CT using the modified Miller index. D-dimer, fibrinogen, and FXIII A- and B-subunit antigen levels were taken on admission. Thrombelastography (TEG) was performed in a subset of patients (n = 12). The 71 patients with PE had lower FXIII A-subunit levels than the 49 patients with excluded PE (78.6 +/- 24.5% vs. 91.3+/-28.8%, p=0.01). In both groups, FXIII A-subunit was inversely related to D-dimer levels. FXIII A-subunit correlated with fibrinogen levels in patients with PE but not in patients without PE. FXIII A-subunit decreased with increasing pulmonary occlusion rate. The risk of PE was increased in the presence of A-subunit levels < 60% (OR 7.0 [95% CI 1.4-35.3], p = 0.019). Clot firmness determined by TEG was lower in patients with PE than in patients without PE. In patients with PE, circulating FXIII A-subunit is decreased compared to patients with suspected but excluded PE. The higher the clot burden within the pulmonary arteries the lower the FXIII antigen. In these patients, direct relation of FXIII A-subunit to fibrinogen levels argues for significant consumption of these coagulation factors in PE. This consumption of FXIII can also be detected by a global coagulation test like TEG.

Thrombin activatable fibrinolysis inhibitor (TAFI) levels in patients with coronary artery disease investigated by angiography.

Due 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.