Free factor XIII activation peptide (fAP‐FXIII) is a regulator of factor XIII activity via factor XIII‐B

In a factor XIIIa (FXIIIa) generation assay with recombinant FXIII‐A₂ (rFXIII‐A₂) free FXIII activation peptide (fAP‐FXII) prolonged the time to peak (TTP) but did not affect the area under the curve (AUC) or concentration at peak (CP). Addition of recombinant factorXIII‐B₂ (rFXIII‐B₂) restored the characteristics of the FXIIIa generation parameters (AUC, TTP and CP) to those observed for plasma FXIII (FXIII‐A₂B₂). FXIII‐A₂B₂ reconstituted from rFXIII‐A₂ and rFXIII‐B₂ showed a similar effect on AUC, TTP and CP in the presence of fAP‐FXII as observed for plasma FXIII‐A₂B₂, indicating a role for FXIII‐B in this observation. An effect of fAP‐FXIII on thrombin, the proteolytic activator of FXIII, was excluded by thrombin generation assays and clotting experiments. In a purified system, fAP‐FXIII did not interfere with the FXIIIa activity development of thrombin‐cleaved rFXIII‐A₂ (rFXIII‐A₂′) also excluding direct inhibition of FXIIIa. However, FXIIIa activity development of FXIII‐A₂′B₂ was inhibited in a concentration‐dependent manner by fAP‐FXIII, indicating that an interaction between AP‐FXIII and FXIII‐B₂ contributes to the overall stability of FXIII‐A₂′B₂. In addition to its well‐known role, FXIII‐B also contributes to FXIII‐A₂B₂ stability or dissociation depending on fAP‐FXIII and calcium concentrations.     

Factor XIII deficiency

Summary. Inherited factor XIII (FXIII) deficiency is a rare bleeding disorder that can present with umbilical bleeding during the neonatal period, delayed soft tissue bruising, mucosal bleeding and life‐threatening intracranial haemorrhage. FXIII deficiency has also been associated with poor wound healing and recurrent miscarriages. FXIII plays an integral role in haemostasis by catalysing the cross‐linking of fibrin, platelet membrane and matrix proteins throughout thrombus formation, thus stabilizing the blood clot. The molecular basis of FXIII deficiency is characterized by a high degree of heterogeneity, which contributes to the different clinical manifestations of the disease. There have been more than 60 FXIII mutations identified in the current literature. In addition, single nucleotide polymorphisms have been described, some of which have been shown to affect FXIII activity, contributing further to the heterogeneity in patient presentation and severity of clinical symptoms. Although there is a lifelong risk of bleeding, the prognosis is excellent when current prophylactic treatment is available using cryoprecipitate or plasma‐derived FXIII concentrate.     

Pharmacokinetics and safety of plasma‐derived factor XIII concentrate (human) in patients with congenital factor XIII deficiency

Congenital factor XIII (FXIII) deficiency is a rare condition with substantial risk for life‐threatening bleeding. Replacement of deficient FXIII with plasma‐derived FXIII concentrate is a treatment option. The current 12‐week study evaluated the steady‐state pharmacokinetic (PK) and safety profile of prophylactic infusions of FXIII concentrate (human) in patients with congenital FXIII deficiency. Patients received FXIII concentrate (human) 40 IU kg^?1 on Days 0, 28, and 56. FXIII levels were assessed before and after each infusion; steady‐state PK parameters were assessed up to 28 days after the infusion on Day 56. Treatment effectiveness in maintaining trough FXIII activity levels ≥5% over 28 days and safety parameters were also assessed. Fourteen patients received FXIII concentrate (human) and 13 completed the study. Post‐infusion, FXIII activity levels increased to within the range found in patients without congenital FXIII deficiency without reaching supra‐therapeutic levels. Non‐baseline‐adjusted trough FXIII activity levels were maintained at or above 10% at all post‐baseline visits in all patients. Steady‐state PK parameters were baseline‐adjusted; maximum FXIII activity was 87.7% at 1.72 h post‐infusion, subsequently declining to a minimum of 5.0%. The half‐life was 6.6 days. FXIII concentrate (human) was generally well tolerated. Two patients had possibly treatment‐related adverse events. There were no reports of thromboembolism, viral transmission, bleeding events or treatment‐related hypersensitivity. These findings support use of FXIII concentrate (human) 40 IU kg^?1 every 28 days as an appropriate regimen for routine, long‐term prophylaxis in children and adults with congenital FXIII deficiency. Clinical trial registration: www.clinicaltrials.gov/ct2/show/NCT00883090 .     

An ELISA for factor × activation peptide: application to the investigation of thrombogenesis in cardiopulmonary bypass

Summary. An ELISA for measurement of factor × activation peptide (FXAP) in plasma has been developed. The capture antibody was generated by immunization with a carrier-coupled synthetic peptide based on the amino acid sequence of the C terminal region of native human FXAP: the tag antibody was a commercial polyclonal antibody to factor X. Because of limited specificity of the capture antibody to FXAP compared with factor X, a plasma processing step precipitated plasma factor × and also permitted a concentration step, enabling detection of FXAP below the lower limit of the normal range in plasma. The overall intra- and inter-assay coefficients of variation were ˜5% and ˜11%, respectively. 18 normal laboratory control subjects had FXAP levels of 2-12 – 0-82 ng/ml (mean –SEM). Eight patients under- going surgery and cardiopulmonary bypass progressively generated FXAP throughout the surgery with mean FXAP rising to 11-73 – 4'66ng/ml, and this resulted in increased generation of thrombin detected by measurement of plasma levels of Fl + 2. Levels of FXAP rose significantly ahead of those of factor IX activation peptide (FLXAP), supporting a suggestion that contact system activation can not be the primary stimulus to coagulation in bypass. The ELISA to FXAP will be useful in the study of mechanisms of thrombogenesis in clinical situations where the coagulation system is activated.     

Efficacy and safety of prophylactic treatment with plasma‐derived factor XIII concentrate (human) in patients with congenital factor XIII deficiency

Congenital factor XIII (FXIII) deficiency is an extremely rare, potentially life‐threatening bleeding disorder. Routine prophylactic management is recommended for individuals with clinically relevant FXIII deficiency. This prospective, multicentre, open‐label study evaluated the long‐term efficacy and safety of prophylactic infusions of FXIII concentrate (human) 40 IU kg^?1 in patients with congenital FXIII deficiency. FXIII concentrate (human) was administered every 4 weeks for 12 months. Dosing was adjusted to maintain trough FXIII activity levels of 5–20%. Logistical and ethical constraints precluded use of a placebo control group. Annualized incidence of spontaneous bleeding was compared with historical rates; safety was assessed as a secondary objective. Forty‐one patients were enrolled and completed the study. The annualized rate for spontaneous bleeding episodes requiring FXIII treatment was 0.000 episodes per patient‐year (95% CI: 0.000; 0.097). The study met its primary endpoint: the upper limit of the 95% CI was substantially below the historical rate of 2.5 bleeding episodes per patient‐year. Five spontaneous bleeding episodes (involving three patients; none requiring FXIII treatment) and eight trauma‐related bleeding episodes (two requiring FXIII treatment) occurred. Five patients had surgery during the study, only one of whom required FXIII treatment for post‐surgical bleeding. Most patients (≥85%) had trough FXIII activity levels ≥10%. No patient discontinued treatment due to an adverse event. No adverse events related to thromboembolism or viral transmission were reported. Prophylactic treatment with FXIII concentrate (human) was well tolerated and prevented spontaneous bleeding episodes that were serious enough to require treatment with FXIII‐containing product. Clinical trial registration: www.clinicaltrials.gov/ct2/show/NCT00885742 .     

Administration of factor XIII B subunit increased plasma factor XIII A subunit levels in factor XIII B subunit knock-out mice

Factor XIII (FXIII) is a proenzyme of plasma transglutaminase consisting of enzymatic A (FXIII-A) and noncatalytic B subunits (FXIII-B), and acts in hemostasis and wound healing. We freshly generated mice lacking either FXIII-A or FXIII-B to investigate the physiological functions of FXIII in vivo. Mice carrying the disrupted allele were born at the expected Mendelian ratios, and the homozygous mice were viable and fertile under specific pathogen-free conditions. Although all homozygous and heterozygous mice showed no marked difference from the wild-type animals in general appearance, homozygous mice of either FXIII-A- or FXIII-B-deficiency did have prolonged bleeding times. It was confirmed that thrombin-dependent amine incorporation and fibrin-crosslinking in plasma were undetectable in the FXIII-A-deficient mice and markedly reduced in the FXIII-B-deficient mice; however, the gene expression of each subunit was regulated independently. Recombinant human FXIII-B (rFXIII-B) was expressed in a baculovirus expression system. When rFXIII-B was injected into FXIII-B-deficient mice, FXIII-A levels, fibrin crosslinking, and amine-incorporation activities increased in their plasma, indicating that FXIII-B assisted the maintenance of FXIII-A levels in the circulation. These mouse strains will be useful in exploring the possible pathophysiological roles of each subunit in vivo.     

Platelet factor XIIIa release during platelet aggregation and plasma clot strength measured by thrombelastography in patients with coronary artery disease treated with clopidogrel

Abstract

It has been estimated that up to half of circulating factor XIIIa (FXIIIa) is stored in platelets. The release of FXIIIa from platelets upon stimulation with adenosine diphosphate (ADP) in patients with coronary artery disease treated with dual antiplatelet therapy has not been previously examined. Samples from 96 patients with established coronary artery disease treated with aspirin and clopidogrel were examined. Platelet aggregation was performed by light transmittance aggregometry in platelet-rich plasma (PRP), with platelet-poor plasma (PPP) as reference, and ADP 5?µM as agonist. Kaolin-activated thrombelastography (TEG) was performed in citrate PPP. PRP after aggregation was centrifuged and plasma supernatant (PSN) collected. FXIIIa was measured in PPP and PSN. Platelet aggregation after stimulation with ADP 5?µM resulted in 24% additional FXIIIa release in PSN as compared to PPP (99.3?±?27 vs. 80.3?±?24%, p?<?0.0001). FXIIIa concentration in PSN correlated with maximal plasma clot strength (TEG-G) (r?=?0.48, p?<?0.0001), but not in PPP (r?=?0.15, p?=?0.14). Increasing quartiles of platelet-derived FXIIIa were associated with incrementally higher TEG-G (p?=?0.012). FXIIIa release was similar between clopidogrel responders and non-responders (p?=?0.18). In summary, platelets treated with aspirin and clopidogrel release a significant amount of FXIIIa upon aggregation by ADP. Platelet-derived FXIIIa may contribute to differences in plasma TEG-G, and thus, in part, provide a mechanistic explanation for high clot strength observed as a consequence of platelet activation. Variability in clopidogrel response does not significantly influence FXIIIa release from platelets.     

Acquired factor XIII inhibitor: clinical features,treatment, fibrin structure and epitope determination

Summary. Acquired factor XIII (FXIII) deficiency, arising from an autoantibody against factor XIII, is a rare bleeding disorder. This autoimmune disorder most commonly occurs in the elderly. Patients who develop such acquired FXIII inhibitors may present with catastrophic bleeding events and are hard to be diagnosed with the normal general coagulation tests. Though the disease is relatively rare, it is known to cause significant mortality. In this article we briefly describe a patient who presented with extensive bleeding and a normal activated partial thromboplastin time and prothrombin time (PT), but had an acquired inhibitor to FXIII; her primary disease was systemic lupus erythematosus (SLE). Also, we will focus on the clinical features, treatment modalities, fibrin structure and epitope identification for acquired factor XIII inhibitor with a review of the literature.     

Incomplete Fibrin Formation and Highly Elevated Factor XIII Activity in Multiple Myeloma

Bleeding is a common complication in patients suffering from multiple myeloma. In some cases a defect in fibrin formation has been suggested as one possible cause of haemorrhagic tendency. As shown in this investigation the defect in fibrin formation, ascertained using PAGE, is due to a lack of α-chain polymerization of fibrin monomers in 5/11 patients with IgG myeloma and in 2/5 patients with IgM paraproteinaemia. No disturbed fibrin polymerization could be observed in IgA myeloma (n = 6). Factor XIII concentrations of subunit A and to a lesser extent of subunit S (Laurell technique) were highly elevated in all cases with regular fibrin formation. Comparable values were obtained by measuring the transamidase activity of factor XIII by incorporation of ¹⁴C-labelled purtrescin into casein. Levels up to 600% of normal could be recorded. In contrast, all patients with a lack of α-chain polymerization had a factor XIII activity within the normal range. Addition of factor XIII concentrate to plasma from patients with defective fibrin formation led in 5/8 cases to a partial cross-linking of α-monomers. We conclude that in some cases paraproteins can inhibit the factor XIII and prevent its action on fibrin.     

Refractory Duodenal Bleeding Ulcers Successfully Treated with Factor XIII Transfusion

A 67-year-old woman with a history of autoimmune hepatitis was admitted for fever, acute hepatic dysfunction, and acute kidney injury. She was diagnosed with multiple duodenal ulcers. Despite the administration of proton pump inhibitor and red blood cells, her black stool and anemia progressed, and she was therefore transferred to our hospital. Despite hemostatic treatments, she continued to bleed. On the 21st day of admission, an endoscopic examination showed the oozing of blood from the duodenal mucosa. A low factor XIII (FXIII) activity level was detected, and she was administered FXIII concentrate. The bleeding stopped and she was thereafter discharged.     

Molecular characterization of five Italian families with inherited severe factor XIII deficiency

Summary.  Factor XIII (FXIII) deficiency is a very rare (1:2 000 000) severe autosomal recessive bleeding disorder, mostly due to mutations in the coagulation FXIII A-subunit gene. We have studied the molecular basis of FXIII deficiency in five unrelated Italian families. The coding region, intron–exon boundaries and 5'- and 3'-untranslated regions of the FXIII gene encoding the A subunit were amplified and directly sequenced. Candidate mutations were identified in all the patients. Three novel mutations occurred in three patients. These include a novel homozygous deletion of two base pairs (bp) in exon 14 (c.2002–2003 DelCT). This deletion causes a frameshift from Leu667 and the formation of a stop codon at amino acid position 681. The second patient presents a novel homozygous (c.2126 G>A) transition in exon 15, predicting a Ser708Asn in Barrel 2 domain. The third patient is compound heterozygote for two missense mutations, a previously reported Arg260His substitution, and a novel transition in exon 4 (c.560 C>T) predicting a Pro186Leu in the core domain. The remaining two patients have two previously reported mutations: a 4-bp homozygous deletion in exon 11 (c.1392–1395 Del AATT), previously reported to occur in the Vicenza Area, and a homozygous nonsense mutation in exon 8 (c.979 C>T) predicting an Arg326X in the core domain. The novel mutations occurred at amino acid residues highly conserved among different species (pig, monkey, mouse and dog) and were not detected in 110 normal alleles. Structural analysis shows that Pro186Leu mutation leads to the replacement of the rigid proline pyrrolidine ring by the larger and more flexible leucine side chain and Ser708Asn may probably disrupt the hydrogen bond with Ala291.     

Double filtration plasmapheresis can decrease factor XIII Activity

Factor XIII (FXIII) produces cross-linkages among fibrin molecules within fibrin clots. Its deficiency is related with bleeding diathesis or retardation of wound healing. We report the possibility that intense double filtration plasmapheresis (DFPP) is associated with decreased FXIII activity. Five patients with various primary diagnoses were treated with DFPP and their FXIII activity was measured. Coagulation test results remained almost normal, but FXIII activities declined to less than 20% of their initial value before starting DFPP and 10% after DFPP in most cases. The cases that received intense DFPP therapy exhibited profoundly decreased FXIII activity. The clinical course demonstrated that DFPP caused the FXIII decrease. Fortunately, with careful observation, none of the patients experienced fatal bleeding. Only one case required fresh frozen plasma and an open hemostatic procedure because of prolonged postoperative bleeding. In general, DFPP most efficiently removes substances with the following characteristics: adequate molecular weight; long half-life; and small extravascular distribution volume. The FXIII properties meet all these characteristics. Consequently, we should devote much attention to FXIII activity during DFPP because it cannot be estimated from the usual coagulation tests. Patients who receive DFPP therapy, especially intensified therapy, should have FXIII measured during the course of therapy. Results show that DFPP can decrease FXIII activity. For this reason we recommend the measurement of FXIII when patients receive intense DFPP therapy with albumin replacement.     

A child with acquired factor XIII deficiency: case report and literature review

Factor XIII (FXIII) deficiency is a rare bleeding disorder, which can result in life threatening hemorrhage. Rarer still is acquired FXIII deficiency, in which the disorder is due to autoantibodies that inhibit the factor. To describe one of the youngest reported patients with this condition. To discuss the challenges we encountered in monitoring response with the available assays. To review the literature and provide a review of all acquired FXIII cases. We present the case of our patient, a 9‐year‐old girl with acquired FXIII deficiency. We present a comprehensive review of all acquired FXIII deficiency cases reported globally in English, with focus on clinical presentation, diagnostic assays, treatment and prognosis. There is no current standard for therapy and measuring response to therapy can be complicated by limitations of assays in the presence of inhibitors. Clinicians should be aware of acquired FXIII deficiency as a potentially life threatening bleeding disorder even in young children. The case presented illustrates a young patient with acquired FXIII deficiency with a good clinical response to cryoprecipitate and difficulty in hemostasis monitoring utilizing clinically available assays.     

Unusual presentation of factor XIII deficiency

Factor XIII deficiency is a rare inherited bleeding disorder that is often difficult to diagnose. The standard screening tests are normal in these patients and their bleeding phenotype may be variable. We report the case of a 3-year-old girl who presented with an intracranial haemorrhage. Several confounding factors, such as the suspicion of an arteriovenous malformation and the development of a deep venous thrombosis, led to a delay in the diagnosis of factor XIII deficiency. Subsequently, her brother was also found to have severe factor XIII deficiency. This case highlights the importance of a detailed history and of screening families in which index cases have been identified. It should also remind physicians that bleeding disorders may have unusual presentations and should be sought when investigating unexplained bleeding.     

Clinical pharmacokinetics of a placenta-derived factor XIII concentrate in type I and type II factor XIII deficiency

Limited data are available about the pharmacokinetics of placenta-derived factor XIII (FXIII) concentrate in patients with FXIII deficiency. This concentrate contains only the active subunit A but not the carrier subunit B of the factor, and perplexities have been raised about its clinical use. Moreover, no data are available on its use in the rare patients completely lacking both subunit A and subunit B. Therefore, we evaluated the pharmacokinetics of a commercial placenta concentrate in three patients with FXIII deficiency: two lacking subunit A (type II) and one lacking both subunits (type I). The elimination half-life of the infused placenta subunit A in the three patients was very similar (280, 283, and 272 hr) and was also consistent with the previously reported data for plasma-derived FXIII. No thrombin-independent activity was observed in our concentrate batches. The recovery was significantly lower in the type I patient, in whom infusion of subunit A was not able to elicit a monthly increment of subunit B, as usually observed in type II patients. Monthly infusions of placenta concentrate (at higher dosage in type I patient) have been administered to our patients for two to three years and no evidence of inhibitor against factor XIII activity has been observed. We conclude that placenta concentrates may be as effective as plasma derivatives in replacement therapy of factor XIII deficiency, even in patients who lack subunit B.     

Congenital deficiency of factor XIII caused by two missense mutations in a Dutch family

We present the clinical, biochemical and genomic findings of a family with congenital factor XIII (FXIII) deficiency. Congenital FXIII deficiency is a very rare autosomal recessive bleeding disorder, characterized by umbilical cord bleeding at birth and spontaneous intracranial haemorrhage. Routine clotting tests are normal, which may delay the diagnosis, leading to an increased chance of severe sequelae. The propositus and her brother, known with haemorrhagic diathesis, were found to be compound heterozygous with a known missense mutation (1050 G --> T transversion in exon 7, Val316Phe substitution) and a novel mutation 889 G --> A in exon 6, which predicts a Gly262Glu substitution. As these mutations were known in the family, DNA obtained from cord blood of the youngest sister was analysed for mutations in exons 6 and 7 only. We postulate that the diagnosis was facilitated by determining the two different mutations in the genotype of this family. The analysis showed that she was heterozygous for the exon 7 mutation. Hence, she was not at risk of experiencing haemorrhagic diathesis. This diagnosis avoided the administration of FXIII concentrate to the newborn.     

A complete deficiency of coagulation factor XIII A-subunit due to a novel compound heterozygote of Ser 413 Leu missense and an nt 389 (ins G) frameshift mutation

Coagulation factor XIII consists of two A- and two B-subunits, and either gene mutation can cause a complete deficiency. In a newborn patient with persistent bleeding from the umbilical cord stump, the plasma A-subunit protein was not detectable. Direct PCR sequencing revealed an nt 389 (ins G) frameshift mutation in exon 4 resulting in a new stop codon and a Ser 413 Leu missense mutation in exon 10 in either allele. His mother and father were heterozygous for the nt 389 (ins G) and the Ser 413 Leu, respectively, with about 50% reduction of the plasma A-subunit proteins. In all family members examined only those with either mutation showed the reduced subunit A protein levels. Thus, this complete deficiency of factor XIII was due to a novel compound heterozygous mutation in the A-subunit gene.     

Activated factor V is a cofactor for the activation of factor XI by thrombin in plasma

The mechanism by which the intrinsic pathway of coagulation contributes to physiological hemostasis is enigmatic. Thrombin activates factor XI, a key zymogen in this pathway, which leads to increased thrombin generation. As thrombin-dependent activation of factor XI in vitro is relatively inefficient, we hypothesized that a physiological cofactor supports this reaction in a plasma environment. We therefore investigated whether the cofactors of coagulation, activated factor V, activated factor VIII, high-molecular weight kininogen, or protein S, influenced activation of factor XI by thrombin. Only activated factor V stimulated activation of factor XI by thrombin in a purified system. Binding studies demonstrated that factor XI specifically interacts with both factor V and factor Va through multiple binding sites. We further investigated this cofactor function of activated factor V in plasma. Depletion of factor V, or the addition of activated protein C, decreased the activation of the intrinsic pathway by thrombin in plasma. However, activated protein C did not exert this effect in the plasma of a homozygous carrier of the prothrombotic factor V Leiden mutation. In conclusion, we propose a role for (activated) factor V as a cofactor in the activation of factor XI by thrombin. These findings offer insights into the coagulation system in both health and disease.     

Congenital factor XIII deficiency in Pakistan: characterization of seven families and identification of four novel mutations

Deficiency of coagulation factor XIII (FXIII) belongs to the rare bleeding disorders and its incidence is higher in populations with consanguineous marriages. The aims of this study were to characterize patients and relatives from seven families with suspected FXIII deficiency from Pakistan and to identify the underlying mutations. As a first indicator of FXIII deficiency, a 5M urea clot solubility test was used. Plasma FXIII A‐ and B‐subunit antigen levels were determined by ELISA. FXIII activity was measured with an incorporation assay. Sequencing of all exons and intron/exon boundaries of F13A was performed, and a novel splice site defect was confirmed by RT‐PCR analysis. Genetic analysis revealed six different mutations in the F13A gene. Two splice site mutations were detected, a novel c.1460+1G>A mutation in the first nucleotide of intron 11 and a previously reported c.2045G>A mutation in the last nucleotide of exon 14. Neither of them was expressed at protein level. A novel nonsense mutation in exon 4, c.567T>A, p.Cys188X, was identified, leading in homozygous form to severe FXIII deficiency. Two novel missense mutations were found in exons 8 and 9, c.1040C>A, p.Ala346Asp and c.1126T>C, p.Trp375Arg, and a previously reported missense mutation in exon 10, c.1241C>T, p.Ser413Leu. All patients homozygous for these missense mutations presented with severe FXIII deficiency. We have analysed a cohort of 27 individuals and reported four novel mutations leading to congenital FXIII deficiency.