Plasma Factor XIII Research Articles

Reciprocal stabilization of coagulation factor XIII-A and -B subunits is a determinant of plasma FXIII concentration

AbstractTransglutaminase factor XIII (FXIII) is essential for hemostasis, wound healing, and pregnancy maintenance. Plasma FXIII is composed of A and B subunit dimers synthesized in cells of hematopoietic origin and hepatocytes, respectively. The subunits associate tightly in circulation as FXIII-A2B2. FXIII-B2 stabilizes the (pro)active site-containing FXIII-A subunits. Interestingly, people with genetic FXIII-A deficiency have decreased FXIII-B2, and therapeutic infusion of recombinant FXIII-A2 (rFXIII-A2) increases FXIII-B2, suggesting FXIII-A regulates FXIII-B secretion, production, and/or clearance. We analyzed humans and mice with genetic FXIII-A deficiency and developed a mouse model of rFXIII-A2 infusion to define mechanisms mediating plasma FXIII-B levels. Like humans with FXIII-A deficiency, mice with genetic FXIII-A deficiency had reduced circulating FXIII-B2, and infusion of FXIII-A2 increased FXIII-B2. FXIII-A-deficient mice had normal hepatic function and did not store FXIII-B in liver, indicating FXIII-A does not mediate FXIII-B secretion. Transcriptional analysis and polysome profiling indicated similar F13b levels and ribosome occupancy in FXIII-A-sufficient and -deficient mice and in FXIII-A-deficient mice infused with rFXIII-A2, indicating FXIII-A does not induce de novo FXIII-B synthesis. Unexpectedly, pharmacokinetic/pharmacodynamic modeling of FXIII-B antigen after rFXIII-A2 infusion in humans and mice suggested FXIII-A2 slows FXIII-B2 loss from plasma. Accordingly, comparison of free FXIII-B2 vs FXIII-A2-complexed FXIII-B2 (FXIII-A2B2) infused into mice revealed faster clearance of free FXIII-B2. These data show FXIII-A2 prevents FXIII-B2 loss from circulation and establish the mechanism underlying FXIII-B2 behavior in FXIII-A deficiency and during rFXIII-A2 therapy. Our findings reveal a unique, reciprocal relationship between independently synthesized subunits that mediate an essential hemostatic protein in circulation. This trial was registered at www.ClinicalTrials.com as #NCT00978380.

Distinguishing immune-mediated thrombotic thrombocytopenic purpura from septic disseminated intravascular coagulation using plasma levels of haptoglobin and factor XIII activity

BackgroundBoth immune-mediated thrombotic thrombocytopenic purpura (iTTP) and septic disseminated intravascular coagulation (DIC) are life-threatening disorders developed by platelet-consuming microvascular thrombi and necessitate immediate therapeutic interventions. Although severe deficiencies of plasma haptoglobin in iTTP and factor XIII (FXIII) activity in septic DIC have been reported, few studies have focused on the possibility of using these markers to distinguish between iTTP and septic DIC. ObjectivesWe investigated whether the plasma levels of haptoglobin and FXIII activity could be helpful for differential diagnosis. MethodsThirty-five patients with iTTP and 30 with septic DIC were enrolled in the study. Patient characteristics, coagulation, and fibrinolytic markers were collected from the clinical data. Plasma haptoglobin and FXIII activities were measured using chromogenic Enzyme-Linked Immuno Sorbent Assay and an automated instrument, respectively. ResultsThe median plasma haptoglobin level was 0.39 mg/dL and 54.20 mg/dL in the iTTP and septic DIC groups, respectively. The median plasma FXIII activities were 91.3% and 36.3% in the iTTP and septic DIC groups, respectively. In the receiver operating characteristic curve analysis, the cutoff level of plasma haptoglobin was 2.868 mg/dL and the area under the curve was 0.832. The cutoff level for plasma FXIII activity and the area under the curve were 76.0% and 0.931, respectively. The thrombotic thrombocytopenic purpura (TTP)/DIC index was defined by FXIII activity (percentage) and haptoglobin (milligrams per decilitre). Laboratory TTP was defined as an index ≥60 and laboratory DIC <60. The sensitivity and specificity of the TTP/DIC index were 94.3% and 86.7%, respectively. ConclusionThe TTP/DIC index, composed of plasma levels of haptoglobin and FXIII activity, is useful in differentiating iTTP from septic DIC.

The History of Factor XIII Deficiency.

Despite the early discovery of factor XIII (FXIII) in 1944, the diagnosis of FXIII deficiency was not made until 1960, after all the other coagulation factor deficiencies, most likely due to the normality of routine coagulation testing in FXIII deficiency. Although the first case was detected by the clot solubility test and this test has long since been used to detect FXIII deficiency, the test is no longer recommended by experts. Over the past 60 years, knowledge about FXIII deficiency has expanded considerably, between 1992, when the first variant was identified, and 2022, 197 mutations have been reported. Almost all missense mutations have a similar effect on FXIII, leading to instability and faster degradation of mutant FXIII protein. Therapeutic options have evolved from historical fresh frozen plasma (FFP), old plasma, whole blood, and cryoprecipitate, to plasma-derived and recombinant FXIII concentrates, respectively available since 1993 and 2012. These concentrate products were respectively approved by the Food and Drug Administration in 2011 and 2013. This historical review covers various aspects of FXIII related disorders, including the discovery of the FXIII, associated disorders, molecular basis, diagnosis, and treatment of FXIII deficiency.

Late Presentation of Factor XIII Deficiency with Recurrent Muscle Hematomas

Background: Plasma factor XIII is a proenzyme that gets activated in the final step of the coagulation cascade. Its physiological role is to stabilize clot formation by catalyzing the cross-linkage of fibrin. Factor XIII deficiency is a rare autosomal recessive disorder. Clinical Description: A 10-year-old boy presented with acute-onset swelling of the arm following a minor fall. There were no constitutional symptoms. There was a significant history of similar swellings 2 years earlier that had required surgery. The past and family history were otherwise noncontributory. The child was hemodynamically stable. Local signs suggested a hematoma or tumor. There was no pallor, icterus, petechiae, or ecchymosis. The remaining examination was normal. Provisional diagnoses included nonaccidental trauma or an underlying primary or secondary bleeding diatheses, tumor, or chronic osteomyelitis. Management: Soft-tissue ultrasonography showed a hematoma. The radiograph was normal. Hemograms, first-line tests for coagulopathy, infective biomarkers, and liver and kidney function tests were normal. There were no stigmata of neglect or physical/emotional abuse in the child. Since the clinical phenotype was of a bleeding disorder, but supportive investigations were inconclusive, we suspected factor XII deficiency. Specific levels showed zero activity. Fresh frozen plasma was administered due to the nonavailability of factor XIII. The hematoma was managed surgically. The child was discharged after genetic counseling and is receiving monthly cryoprecipitate. He is asymptomatic till now. Conclusion: Factor XIII deficiency should be suspected when there are clinical indicators of a bleeding diathesis, but supportive tests are not deranged.

Differential Diagnosis Distinguishing Immune TTP from Septic DIC Using Plasma Levels of Haptoglobin and Factor XIII Activity

Introduction Differential diagnosis of immune thrombotic thrombocytopenic purpura (iTTP) and septic disseminated intravascular coagulation (DIC) in daily clinical practice is difficult. iTTP is diagnosed by deficiency of ADAMTS13 activity and ADAMTS13 inhibitor positivity. However, it is difficult to rapidly measure ADAMTS13 activity in real-time in clinical practice. Prompt therapeutic interventions are directly dependent on iTTP prognosis. A method for differentiating between these two disorders using common laboratory tests is needed. Plasma haptoglobin levels can indicate hemolysis and are reported to be severely decreased in iTTP. Coagulation factor XIII (FXIII) activity is also severely decreased in septic DIC. To date, no reports have observed haptoglobin in septic DIC or FXIII activity in iTTP. We analyzed the plasma levels of haptoglobin and FXIII activity in patients with iTTP and septic DIC to investigate whether these markers can be used for differential diagnosis. Methods iTTP was defined as <10% normal ADAMTS13 activity, and >0.5 BU/mL ADAMTS13 inhibitor. Septic DIC was defined as meeting both Third International Consensus Definitions for Sepsis and Septic Shock, and ISTH DIC diagnostic criteria. Thirty-five patients with iTTP and 30 with septic DIC were enrolled in this study. Plasma haptoglobin levels were measured using Human Quantikine ELISA Kits (#DPHAGO, R&D Systems, Inc., Minneapolis, USA), which is a highly sensitive method compared to methods used in clinical practice. Values are expressed as medians (first quartile-third quartile). Results As shown in the table, iTTP patients were significantly younger than those with septic DIC. The iTTP group showed significantly lower ADAMTS13 activity, higher ADAMTS13 inhibitor titer, lower hemoglobin level, lower platelet counts, higher LDH level, and lower creatinine level than the septic DIC group. In a comparison of coagulation and fibrinolytic markers, the septic DIC group had significantly higher PT-INR, prolonged APTT, higher FDP and D-dimer levels, and lower AT activity than the iTTP group. The plasma haptoglobin level was 0.39 mg/dL (0.18-0.76 mg/dL) in the iTTP group, and 54.2 mg/dL (0.6-143.9 mg/dL) in the septic DIC group (P < 0.01). FXIII activity was 91.3% (80.4-115.0%) in the iTTP group, and 36.3% (14.7-61.9%) in the septic DIC group (p<0.01). The ROC curve of haptoglobin and FXIII activity in the differential diagnosis of TTP and septic DIC were investigated. The cut-off level of haptoglobin was 2.868 mg/dL, the area under the curve (AUC) was 0.832 (95% CI:0.727-0.938), specificity was 66.7%, and sensitivity was 100%. The cutoff level of FXIII activity was 76.0%, AUC was 0.931 (95%CI:0.873-0.989), specificity was 77.1%, and sensitivity was 93.3%. We developed a scoring system to differentiate iTTP from septic DIC using a combination of FXIII activity and haptoglobin; TTP/DIC index = plasma FXIII activity (%) - plasma haptoglobin (mg/dL). Laboratory TTP was defined when the index was ≥60, and laboratory DIC was defined when the index was <60. The AUC for the combination of FXIII activity and haptoglobin was 0.968 (95% CI: 0.9354-1). The accuracy of iTTP diagnosis in laboratory TTP was 89.2% (33/37), and the accuracy of septic DIC diagnosis in laboratory DIC was 92.9% (26/28). Conclusions A comparison of iTTP and septic DIC showed significant differences in haptoglobin and FXIII activity levels. A combination of haptoglobin and FXIII activity was considered helpful in differentiating iTTP from septic DIC. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A comparison of the ClotPro system with rotational thromboelastometry in cardiac surgery: a prospective observational study

Viscoelastic coagulation tests have been increasingly used for hemostasis management in cardiac surgery. The ClotPro system is a novel viscoelastic device based on principles of rotational thromboelastometry. We aimed to compare ClotPro with ROTEM and plasma coagulation assays in cardiopulmonary bypass (CPB) patients. Blood samples were collected from 25 CPB patients at (1) baseline, (2) start of CPB, (3) end of CPB, and (4) end of surgery. The EX-test, IN-test, HI-test, FIB-test parameters on ClotPro were compared with corresponding ROTEM assay (EXTEM, INTEM, HEPTEM, and FIBTEM). Standard plasma coagulation assays and endogenous thrombin generation (TG) were simultaneously evaluated. Pearson correlation analyses showed moderate correlations between clotting times (CTs) (r = 0.63–0.67; p < 0.001, respectively), and strong correlations with maximal clot firmness (MCF) (r = 0.93–0.98; p < 0.001, respectively) between ClotPro and ROTEM. EX-test and IN-test MCF parameters were interchangeable with acceptable percentage errors (EX-test MCF: 7.3%, IN-test MCF: 8.3%), but FIB-test MCF (27.0%) and CT results were not (EX-test CT: 44.7%, IN-test CT: 31.4%). The correlations of PT/INR or peak TG with EX-test CTs were higher than with EXTEM CTs (PT/INR: r = 0.80 and 0.41, peak TG: 0.43 and 0.18, respectively). FIB-test MCF has strong correlation with plasma fibrinogen and factor XIII level (r = 0.84 and 0.66, respectively). ROC analyses showed that ClotPro was capable of emulating well-established ROTEM thresholds (area under curves: 0.83–1.00). ClotPro demonstrated strong correlations in MCF parameters of ROTEM in CPB patients. It may be reasonable to modify ROTEM-based transfusion algorithm pertaining to MCF parameters to establish cut-off values for ClotPro device.

The Effect of Activated FXIII, a Transglutaminase, on Vascular Smooth Muscle Cells.

Plasma factor XIII (pFXIII) is a heterotetramer of FXIII-A and FXIII-B subunits. The cellular form (cFXIII), a dimer of FXIII-A, is present in a number of cell types. Activated FXIII (FXIIIa), a transglutaminase, plays an important role in clot stabilization, wound healing, angiogenesis and maintenance of pregnancy. It has a direct effect on vascular endothelial cells and fibroblasts, which have been implicated in the development of atherosclerotic plaques. Our aim was to explore the effect of FXIIIa on human aortic smooth muscle cells (HAoSMCs), another major cell type in the atherosclerotic plaque. Osteoblastic transformation induced by Pi and Ca2+ failed to elicit the expression of cFXIII in HAoSMCs. EZ4U, CCK-8 and CytoSelect Wound Healing assays were used to investigate cell proliferation and migration. The Sircol Collagen Assay Kit was used to monitor collagen secretion. Thrombospondin-1 (TSP-1) levels were measured by ELISA. Cell-associated TSP-1 was detected by the immunofluorescence technique. The TSP-1 mRNA level was estimated by RT-qPCR. Activated recombinant cFXIII (rFXIIIa) increased cell proliferation and collagen secretion. In parallel, a 67% decrease in TSP-1 concentration in the medium and a 2.5-fold increase in cells were observed. TSP-1 mRNA did not change significantly. These effects of FXIIIa might contribute to the pathogenesis of atherosclerotic plaques.

An international collaborative study to assign value for Total Factor XIII‐B Subunit Antigen to the WHO 1st International Standard for Factor XIII Plasma, (02/206): Communication from the ISTH SSC Subcommittee on Factor XIII and Fibrinogen

BackgroundFactor XIII (FXIII)‐B subunit measurements are required for the diagnosis and characterization of the type of FXIII deficiency. Furthermore, therapy for FXIII‐A deficiency with recombinant FXIII (rFXIII‐A) relies on available FXIII‐B. ObjectiveTo carry out a collaborative study to calibrate and assign value to the current WHO 1st International Standard (IS) FXIII Plasma for Total FXIII‐B subunit, relative to locally collected normal plasma pools. MethodsLaboratories were instructed to use a validated method (specific ELISA antibodies provided) for assessment of Total FXIII‐B subunit antigen potency. All laboratories used this method with one laboratory using an additional in‐house method. Nine data sets were received from seven laboratories (37 assays in total), which provided a total of 35 valid estimates for this new assignment. Total FXIII‐B subunit estimates were calculated relative to locally collected normal plasma pools, using an arbitrary value of 1.00 unit of Total FXIII‐B subunit per ml, for each pool. ResultsCombination of results produced an overall mean of 0.98 units/mL with an inter‐laboratory variability (geometric coefficients of variation – GCV%) of 18.3% [95% confidence interval: 0.86–1.11]. Real‐time and bench stability studies indicated good stability and preservation of the FXIII‐B subunit analyte in the WHO 1st IS FXIII Plasma (02/206). ConclusionFollowing agreement by study participants, ISTH/SSC Experts, WHO‐ISTH Liaison Group and the SSC Board, the WHO/ECBS established the current WHO 1st IS Factor XIII plasma (NIBSC code 02/206) by additionally assigning it with a Total FXIII‐B subunit antigen value of 0.98 IU/ampoule, in October 2019.

Effectiveness and failure factors of manual aspiration using a small needle for large pneumothorax in stable patients.

BackgroundManual aspiration as the initial management of a large pneumothorax in a clinically stable patient has been reported to be safe and effective. However, the effect with smaller needles, the number of aspiration, the indication other than spontaneous pneumothorax and failure factors are unknown. We assessed the effectiveness and failure risk factors of manual aspiration up to three using a 20- or 22-gauge (G) needle in patients with a large, clinically stable pneumothorax.MethodsWe included 107 clinically stable patients with large pneumothorax. Patients who were unstable, required a ventilator, underwent chest tube drainage or had an observed small pneumothorax, bilateral pneumothorax, hemopneumothorax, or postoperative pneumothorax were excluded. Up to three aspirations were performed using 20- or 22-G intravenous needles. When the aspiration volume was ≥2,500 mL or lung expansion did not occur, a chest tube was placed.ResultsThe first aspiration was successful in 57 patients (53.3%), the second in 16 patients (59.3%), and the third in eight patients (80.0%). No patient experienced any obvious complications or required emergent hospitalization or surgery after aspiration. Aspiration failure was correlated with an inter-pleural distance >20 mm at the level of the hilum [odds ratio (OR): 4.93; 95% confidence interval (CI): 1.49–22.71; P=0.0075], spontaneous secondary pneumothorax (OR: 3.11; 95% CI: 1.14–8.76; P=0.027), and ≤24 h from onset to presentation (OR: 2.95; 95% CI: 1.12–8.26; P=0.028). There were no significant differences in intrathoracic pressure after aspiration or plasma factor XIII levels between patients with resolved and persistent pneumothorax.ConclusionsManual aspiration up to three times using a small needle might be one of a treatment option in clinically stable patients with any large pneumothorax. Aspiration failure was correlated with an inter-pleural distance >20 mm at the level of the hilum, spontaneous secondary pneumothorax, and ≤24 h from onset to presentation.

Acquired Factor XIII Deficiency in a Patient with Metastatic Lung Cancer

Acquired factor XIII (FXIII) deficiency can result in life-long bleeding tendency and can be caused by enhanced consumption, impaired synthesis, or as an immune-mediated process. The latter can be related with solid neoplasms, through neutralizing or non-neutralizing antibodies. The relationship between FXIII activity and non-small cell lung cancer (NSCLC) is not well established. This case report is about a patient with NSCLC and acquired FXIII deficiency. Materials and Methods: Clinical records were obtained through the electronic process analysis, and the confidentiality of the patient was always assured. Results and Discussion: A 70-year-old male with no relevant past medical history and a recently diagnosed metastatic NSCLC was admitted for priapism. Five days later, a he developed a bleeding disorder, with slightly elevated coagulation times and normal fibrinogen levels and platelets count. FXIII level was found to be decreased (0.24 IU/mL) and FXIII plasma mixing studies did not confirm the presence of a neutralizing inhibitor. The FXIII level correction with standard plasma mixing studies was in favour of a non-neutralizing antibody. Despite treatment, haemorrhage control was not achieved and the patient died. Conclusion: This clinical report describes a rare case of a patient with metastatic NSCLC presenting a severe haemorrhagic event caused by FXIII deficiency immune-mediated by non-neutralizing antibodies and subsequent increased clearance.

Simultaneous Use of Factor XIII and Fibrin Degradation Products in Diagnosing Early Cases of NEC and Neonatal SEPSIS

Purpose: The study examined the use of factor XIII and fibrin degradation products in diagnosing early cases of NEC and neonatal sepsis. Methods: Sixty neonates were divided into two groups. 30 preterm neonates suspected with early NEC Diagnosis of NEC was confirmed by modified Bell’s score and 30 preterm neonates with symptoms of neonatal sepsis; where sepsis was confirmed by blood culture and CRP. Laboratory evaluation of FDPs and plasma factor XIII was done for all the patients. The study was carried out in a tertiary NICU of the pediatric department, Ain Shams University Hospital. All enrolled neonates had a matched mean birth weight and gestational age. They were either moderate preterms &gt;32 weeks, but &lt;34 weeks, and late preterms &gt;34 weeks, but &lt;37 weeks). Results: The results indicate a correlation between FDPs and the laboratory data of group B, and it was found out that FDPs were negatively correlated with TLC, Plate-lets, and CRP, reflecting FDPs increase with bone marrow suppression and progression of sepsis. Factor XIII was significantly lower in the group with NEC as compared to the group of sepsis (p&lt;0.001), while FDPs level was significantly higher in the group with sepsis (p&lt;. 0.001). The correlation between the clinical stages of NEC BELL's score and the level of Initial factor XIII level revealed that the factor level is negatively correlated with stage I of BELL's score. The follow-up revealed that there was no correlation between BELL's score and the level of follow-up factor XIII. On follow-up, the current study demonstrated that TLC, CRP, FDPS, PTT were significantly increased in the sepsis group with p values of 0.021,, 0.001, 0.001 and 0.01. The current study found significantly higher partial thromboplastin time (PTT) in the group with sepsis Conclusion: Factor XIII level can predict early cases of NEC and can differentiate it from neo-natal sepsis.

Predictive value of coagulation factor XIII on bleeding risk in ischemic stroke patients treated with intravenous thrombolysis.

Intravenous thrombolysis (IVT) therapy with recombinant tissue plasminogen activator (rtPA) in acute ischemic stroke (AIS) has a known risk of intracerebral hemorrhage (ICH). We aimed to identify the predictive value of coagulation factor XIII (FXIII) on post-thrombolytic ICH risk in AIS patients. The study cohort included 69 diagnosed AIS patients undergoing IVT treatment within 24 hours of symptom onset. Blood samples taken on admission were analyzed for FXIII antigen levels with an automated latex enhanced immunoassay. Conventional coagulation parameters including prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), and D-dimer (DD) were also tested. Of the 69 AIS patients, 23 (33.3%) developed post-thrombolytic ICH. Plasma FXIII levels showed a significant decrease, accompanied with elevated FIB and DD levels in AIS patients with post-thrombolytic ICH. Multivariable logistic regression (LR) revealed that FXIII and FIB were independently associated with post-thrombolytic ICH progression. Area under receiver operating characteristic curve of FXIII selected by the further forward logistic regression was 0.823 [95% confidence interval (CI): 0.712-0.904], and the cutoff value of 76.6% yielded good sensitivity at 91.3% and good negative predictive value (NPV) at 93.9%. Our findings indicated that plasma FXIII level may be an independent determinant for predicting post-thrombolytic bleeding risk in AIS patients.

A Large Case Series on Surgical Outcomes in Congenital FXIII Deficiency Patients in Iran

BackgroundFXIII requires low plasma FXIII levels for normal hemostasis. The lack of accepted standardized surgical guidelines necessitates dependence on the treating physicians' and centers' experiences.AimOur aim is to evaluate surgical outcomes on a large group of congenital FXIII deficiency (FXIIID) patients.MethodsA case series study was conducted on congenital FXIIID patients in two major referral centers located in Iran from 2010 to 2016. All patients were on prophylaxis using plasma factor XIII concentrate (10 U kg−1, every 28 days) except three patients prior to surgery. One single dose of 10 U kg−1 and 30 U kg−1 plasma factor XIII concentrate before a minor procedure and circumcision were given, respectively. Two doses of plasma factor XIII concentrate, one 30 U kg−1 prior to the procedure and the second dose of 30 U kg−1 on post-operative day 3, were given for major surgery. The plasma factor XIII concentrate dose was 50 U kg−1 both before and after the procedure for neurosurgery.ResultsOne hundred sixty-two FXIIID patients underwent minor, major and obstetrical and gynecological surgeries. The median age of the patients was 14 years (ages ranged 15 days to 47 years). The male-to-female ratio was 89/73. Five post-operative complications, two bleeding and three thrombosis, were recorded for two patients who were undiagnosed prior to surgery.ConclusionOur study showed excellent hemostasis in FXIIID patients undergoing surgeries. Of these surgeries, we observed only 1.8% post-operative complications. Surgery can be performed safely in FXIIID patients, and the proposed treatment regimens lead to adequate hemostatic coverage with minimal risk for both minor and major surgeries. DisclosuresPeyvandi:Freeline, Kedrion, LFB, Octapharma: Consultancy; Ablynx, Roche: Membership on an entity's Board of Directors or advisory committees; Ablynx, Bayer, Grifols, Novo Nordisk, Sobi: Speakers Bureau.

Factor XIII and Fibrin Clot Properties in Acute Venous Thromboembolism.

Coagulation factor XIII (FXIII) is converted by thrombin into its active form, FXIIIa, which crosslinks fibrin fibers, rendering clots more stable and resistant to degradation. FXIII affects fibrin clot structure and function leading to a more prothrombotic phenotype with denser networks, characterizing patients at risk of venous thromboembolism (VTE). Mechanisms regulating FXIII activation and its impact on fibrin structure in patients with acute VTE encompassing pulmonary embolism (PE) or deep vein thrombosis (DVT) are poorly elucidated. Reduced circulating FXIII levels in acute PE were reported over 20 years ago. Similar observations indicating decreased FXIII plasma activity and antigen levels have been made in acute PE and DVT with their subsequent increase after several weeks since the index event. Plasma fibrin clot proteome analysis confirms that clot-bound FXIII amounts associated with plasma FXIII activity are decreased in acute VTE. Reduced FXIII activity has been associated with impaired clot permeability and hypofibrinolysis in acute PE. The current review presents available studies on the role of FXIII in the modulation of fibrin clot properties during acute PE or DVT and following these events. Better understanding of FXIII’s involvement in the pathophysiology of acute VTE might help to improve current therapeutic strategies in patients with acute VTE.

Factor XIIIa Formation Promoted by Complexing of a-Thrombin, Fibrin, and Plasma Factor XIII

Fibrin polymers (des A,B fibrinogen) reduced the concentration of alpha-thrombin required for 50% activation of plasma factor XIII (a2b2 tetramer) by approximately 100-fold. In the presence of fibrin, the amount of gamma-thrombin required for activation was not affected. Catalytically inactive i-Pr2P- and D-Phe-Pro-Arg-CH2-alpha-thrombin were found to inhibit over 95% of the activation by alpha-thrombin in the presence of fibrin. Unlike plasma factor XIII, the concentration of alpha-thrombin required for 50% activation of platelet factor XIII (a2 dimer) was lower, and the activation was not enhanced by fibrin. However, when the a2 platelet factor XIII was incubated with purified b-chains, the alpha- and gamma-thrombin concentrations required for activation increased tenfold and reached levels similar to those required for activation of the plasma factor XIII. When fibrin was present, the alpha-thrombin concentrations needed for activation of the a2b2 complexes were reduced, and the presence of fibrin had no effect on gamma-thrombin cleavage of the a2b2 complexes. Therefore, the b-chains must inhibit a-chain cleavage by alpha-thrombin in the absence of fibrin. These results imply that the formation of a cocomplex involving alpha-thrombin, fibrin, and plasma factor XIII causes some conformational change in plasma factor XIII such that the b-chains no longer inhibit cleavage of the a-chains.

Transcription Factor RUNX1 Regulates Factor FXIIIA Subunit (F13A1) Expression in Megakaryocytic Cells and Platelet F13A1 Expression is Downregulated in RUNX1 Haplodeficiency

Plasma Factor XIII, a transglutaminase, is a complex of two A (gene F13A1) chains with catalytic activity and two B chains (gene F13B). FXIII induces clot stabilization and retraction via cross-linking of fibrin monomers and other proteins. Factor XIII deficiency in plasma causes a major bleeding disorder with impaired clot retraction. FXIIIA is synthesized in megakaryocytes, and is abundant in platelets. It is expressed on activated platelets and cross-links proteins on platelet surface. Little is known regarding the regulation of F13A1 in megakaryocytes and platelets. RUNX1 is a major hematopoietic transcription factor and regulates expression of numerous genes in megakaryocytes and platelets. Patients with RUNX1 haplodeficiency (RHD) with heterozygous mutations have thrombocytopenia, platelet dysfunction with impaired responses to activation, and predisposition to myeloid malignancies. Platelet expression profiling using Affymetrix microarrays of a patient (P1) with a heterozygous RUNX1 mutation (c.969-323G&amp;gt;T) revealed decreased F13A expression, which encodes for subunit A of FXIII: fold-change 0.30; p=0.006; patient profiled twice 10 months apart and compared to 6 healthy subjects. These findings were confirmed by qPCR With RT-PCR platelet F13A mRNA expression was decreased to ~ 20% of mean expression in 4 subjects. Platelet F13A protein expression was decreased by ~50% as assessed by corrected total cellular fluorescence imaging. Platelet F13A mRNA expression was also decreased (to less than 20% of expression in 5 controls) in two siblings (P2 and P3 ages 8 and 3 years) from an unrelated family with a heterozygous RUNX1 mutation 508+1G&amp;gt;A). Plasma FXIII was normal in the patient P1. We studied the regulation of F13A by RUNX1 in human erythroleukemia (HEL) cells treated with phorbol myristate acetate (PMA) to induce megakaryocytic transformation. In silico analyses of the 5' upstream region of F13A showed 7 RUNX1 consensus sites within 545bp from ATG. Chromatin immunoprecipitation studies performed using HEL cells and RUNX1 antibody showed that that RUNX1 binds in the region encompassing site 4 and sites 5-7. In parallel, electrophoretic mobility shift assay (EMSA) studies showed binding of nuclear protein to labeled DNA probes with sites 1, 4, 5, 6, and 7. RUNX1 antibody produced a "supershift" with addition suggesting RUNX1 binding. Site-directed mutagenesis of the RUNX1 binding sites in F13A1 promoter decreased promoter activity in luciferase reporter studies in HEL cells, providing evidence that the binding sites are functional. Further, overexpression of RUNX1 in HEL cells increased F13A1 promoter activity and protein while siRNA RUNX1 knockdown reduced F13A1 protein. We assessed clot retraction over 90 min at 37oC in the patient P1 and his daughter (also with RUNX1 mutation) in citrated whole blood following addition of tissue factor (1 pM) and CaCl2 (1 mM), and in washed platelets resuspended in platelet poor plasma from a healthy subject supplemented with added fibrinogen (0.5 mg/mL) and stimulated with 0.5 U/mL thrombin in the presence of CaCl2. These preliminary studies suggested that clot retraction was slower in the patient P1 compared to the controls, particularly in studies with platelet suspensions. Conclusions: Overall, these studies provide the first evidence that hematopoietic transcription factor RUNX1 regulates expression of F13A1 in megakaryocytic cells and that platelet expression of F13A1 is decreased in platelets from patients with RUNX1 haplodeficiency. The decreased expression of F13A1 in RHD may contribute to the platelet dysfunction in RHD. These findings are particularly interesting because they reflect regulation of a coagulation protein (FXIIIA) by hematopoietic transcription factor RUNX1. Disclosures Lambert: Dova: Consultancy, Membership on an entity's Board of Directors or advisory committees; Principia: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Shionogi: Consultancy; Educational Concepts in Medicine: Consultancy; Octapharma: Consultancy, Research Funding; Bayer: Consultancy; Platelet Disorder Support Association (PDSA): Consultancy; 22qSociety: Consultancy; ITP Australia: Consultancy; CdLS Foundation: Consultancy; RDMD ITP study: Consultancy; Sysmex: Research Funding; AstraZeneca: Research Funding; Argenix: Consultancy; ClinGen: Honoraria.

Activity and Substrate Specificity of Factor XIII Resulting from Different Activation Pathways

Factor XIII (FXIII) is a transglutaminase that catalyzes the formation of a calcium-dependent isopeptide bond between an acyl donor and a primary amine substrate. Plasma FXIII A2B2 is proteolytically activated by the concerted action of thrombin and low mM Ca2+ (FXIIIA∗) whereas cellular FXIII A2 is activated non-proteolytically by high mM Ca2+ concentration (FXIIIA°). Previous studies demonstrated that these activation pathways result in FXIIIA species that differ conformationally and functionally. The activities and substrate specificities of FXIIIA∗ and FXIIIA° were further examined using monodansyl cadaverine (MDC) incorporation and UV-spectrophotometric assays. In the MDC assay, reactive glutamines within fibrinogen αC (233-425) (Q237, Q328, Q366) were crosslinked by FXIII (A∗ versus A°) to MDC (amine substrate), and results were evaluated by SDS-PAGE. The assay revealed that with αC WT, Q328P (Seoul II), and Q237only, FXIIIA∗ had more transglutaminase activity than FXIIIA°. In contrast, αC E396A and αC 389stop (233-388) exhibited no substantial differences in activities between FXIIIA∗ and A°. This observation may be due to loss of αC E396 and surrounding residues from the putative FXIII anchoring site αC (389-403). From the UV-spectrophotometric assay, kinetic parameters for FXIII A∗ and FXIIIA° were determined using glutamine donor peptides (K9 (1-10), α2 AP (1-15) and α2 AP (1-15, Q4S)) crosslinked with the amine DMPDA. Km values for FXIIIA∗ were modestly lower than FXIIIA° for K9 (1-10) and α2 AP (1-15, Q4S) and higher for α2 AP (1-15). The smaller kinetic differences observed with peptides may be due to amino sequence or the need for larger FXIII substrates that better mimic physiological targets. Overall, FXIIIA activity can be a function of its activation pathway, in addition to substrate size and sequence.

Reduction of FXIII during myelosuppression in acute leukemia after chemotherapy and adverse relation with bleeding events

目的探讨急性白血病患者化疗后骨髓抑制期外周血凝血因子XIII（FXIII）浓度与出血事件的关系。方法纳入2017年8月至2018年3月期间55例PLT<50×109/L的化疗后骨髓抑制期急性白血病患者（非急性早幼粒细胞性急性髓系白血病35例，急性淋巴细胞白血病20例），以35例非血液疾病患者作为对照组。用ELISA方法检测外周血FXIII浓度，分析急性白血病患者化疗后骨髓抑制期外周血FXIII水平与出血事件的关系。结果急性白血病患者化疗后骨髓抑制期FXIII水平明显低于对照组（P<0.001），FXIII水平与出血评分呈负相关（Spearman相关系数为-0.761）。当FXIII浓度的临界值为103.9 µg/L时，诊断化疗后骨髓抑制期急性白血病患者出血的敏感性为0.939，特异性为0.909。结论化疗后骨髓抑制期急性白血病患者外周血FXIII水平减低，且与出血事件及严重程度呈负相关，是急性白血病化疗后骨髓抑制期患者出血的独立影响因素之一。

The Plasma Factor XIII Heterotetrameric Complex Structure: Unexpected Unequal Pairing within a Symmetric Complex.

Factor XIII (FXIII) is a predominant determinant of clot stability, strength, and composition. Plasma FXIII circulates as a pro-transglutaminase with two catalytic A subunits and two carrier-protective B subunits in a heterotetramer (FXIII-A2B2). FXIII-A2 and -B2 subunits are synthesized separately and then assembled in plasma. Following proteolytic activation by thrombin and calcium-mediated dissociation of the B subunits, activated FXIII (FXIIIa) covalently cross links fibrin, promoting clot stability. The zymogen and active states of the FXIII-A subunits have been structurally characterized; however, the structure of FXIII-B subunits and the FXIII-A2B2 complex have remained elusive. Using integrative hybrid approaches including atomic force microscopy, cross-linking mass spectrometry, and computational approaches, we have constructed the first all-atom model of the FXIII-A2B2 complex. We also used molecular dynamics simulations in combination with isothermal titration calorimetry to characterize FXIII-A2B2 assembly, activation, and dissociation. Our data reveal unequal pairing of individual subunit monomers in an otherwise symmetric complex, and suggest this unusual structure is critical for both assembly and activation of this complex. Our findings enhance understanding of mechanisms associating FXIII-A2B2 mutations with disease and have important implications for the rational design of molecules to alter FXIII assembly or activity to reduce bleeding and thrombotic complications.

Identification of Potential Novel Interacting Partners for Coagulation Factor XIII B (FXIII-B) Subunit, a Protein Associated with a Rare Bleeding Disorder.

Coagulation factor XIII (FXIII) is a plasma-circulating heterotetrameric pro-transglutaminase complex that is composed of two catalytic FXIII-A and two protective/regulatory FXIII-B subunits. FXIII acts by forming covalent cross-links within a preformed fibrin clots to prevent its premature fibrinolysis. The FXIII-A subunit is known to have pleiotropic roles outside coagulation, but the FXIII-B subunit is a relatively unexplored entity, both structurally as well as functionally. Its discovered roles so far are limited to that of the carrier/regulatory protein of its partner FXIII-A subunit. In the present study, we have explored the co-presence of protein excipients in commercial FXIII plasma concentrate FibrogamminP by combination of protein purification and mass spectrometry-based verification. Complement factor H was one of the co-excipients observed in this analysis. This was followed by performing pull down assays from plasma in order to detect the putative novel interacting partners for the FXIII-B subunit. Complement system proteins, like complement C3 and complement C1q, were amongst the proteins that were pulled down. The only protein that was observed in both experimental set ups was alpha-2-macroglobulin, which might therefore be a putative interacting partner of the FXIII/FXIII-B subunit. Future functional investigations will be needed to understand the physiological significance of this association.