Low FXIII activity levels in intensive care unit hospitalized COVID-19 patients

Background COVID-19 infection is associated with a hypercoagulable state. Severe COVID-19 patients present with high plasma fibrinogen levels, continuous deposition of fibrin and the presence of microthrombi in their lungs, accompanied by significant fibrinolysis, resulting in high D-dimer levels. Due to the role of FXIII in fibrin crosslinking and clot stabilization, we analyzed its activity levels and dynamics in COVID-19 patients hospitalized in the intensive care unit (ICU). Methods FXIII levels were measured in thirty four COVID-19 patients hospitalized in the ICU and in fourteen non-severe COVID-19 patients. FVIII levels were measured for comparison. Laboratory data and clinical variables were recorded. Results The average FXIII activity level in 34 ICU hospitalized COVID-19 patients was 69.9±33 %, significantly lower compared to an average of 120±20.9 % FXIII activity in 14 non-severe COVID-19 patients. FXIII activity levels were below the low normal value (< 79 % FXIII activity) in 74 % of the ICU hospitalized COVID-19 patients. In contrast, high FVIII activity was measured among all severe COVID-19 patients. Consecutive measurements, performed in fourteen ICU hospitalized COVID-19 patients, pointed to a significant decrease in FXIII activity from the average of 85.7±28.2 %, (which is in the normal range), to an average of 68.0±20.4 %, below the low normal range, within 6.4±3.4 days of ICU hospitalization. Liver functions did not differentiate between patients with low and normal FXIII activity. No inhibitor to FXIII activity was found in the plasma of severe COVID-19 patients. Levels of FXIII-A antigen correlated with FXIII activity, and were low in severe COVID-19 patients. Conclusions Low FXIII activity levels were found in COVID-19 patients hospitalized in the ICU, with gradual decline during their hospitalization. A mechanism of consumption may account for the low FXIII activity in these patients.

Patient-centered approach to managing factor XIII deficiency

Factor XIII (FXIII) is a thrombin-activated protransglutaminase that plays a key role in blood clot formation. Congenital FXIII A-subunit deficiency represents a rare bleeding disorder that affects one in 2–3 million individuals worldwide and is treated with recombinant FXIII (rFXIII). However, due to the rarity of the disease, clinicians are often left to weigh individual variation in FXIII activity and/or symptoms to optimally guide dosing. Cases often become further complicated when patients experience refractory bleeding, which can be difficult to treat. This report describes an approach to rFXIII dosing in two patients who required deviation from standard protocols to maintain therapeutic FXIII troughs. We highlight limitations in our understanding of FXIII deficiency management, while also providing an example of the application of pharmacokinetic data to individualise therapy for improved outcomes. Finally, the case reminds us of the importance of patient-centered, cost-conscious care and multidisplinary teamwork in complex cases.

Acquired FXIII deficiency is associated with high morbidity

Background: A factor XIII (FXIII) level >30% is considered necessary to prevent spontaneous bleeding. Bleeding is also a risk in patients with acquired FXIII deficiency, but the hemostatic level of FXIII in this context remains to be determined. Material & Methods: We retrospectively analyzed all patients diagnosed with acquired FXIII deficiency at a large hospital over 3 years (study ID NCT04416594, http://www.clinicaltrials.gov) and assessed clinical data to identify the best cut-off point for FXIII activity to distinguish between low and high risk of major bleeding in a mixed medical and surgical population. Results: Of the 97 patients who experienced bleeding despite a normal coagulation test, 43.2% had FXIII activity <70%. FXIII activity was significantly lower in surgical patients and patients admitted to the intensive care unit (ICU). Low FXIII activity was significantly associated with long ICU stays and a high incidence of major bleeding. Conclusions: Acquired FXIII deficiency is associated with high morbidity. The hemostatic level of FXIII in the setting of acquired FXIII deficiency might be above 30%.

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.

Clinical Validation of an Automated Fluorogenic Factor XIII Activity Assay Based on Isopeptidase Activity

Hereditary factor XIII (FXIII) deficiency is a rare autosomal bleeding disorder which can cause life-threatening bleeding. Acquired deficiency can be immune-mediated or due to increased consumption or reduced synthesis. The most commonly used screening test is insensitive, and widely used quantitative assays have analytical limitations. The present study sought to validate Technofluor FXIII Activity, the first isopeptidase-based assay available on a routine coagulation analyser, the Ceveron s100. Linearity was evidenced throughout the measuring range, with correlation coefficients of >0.99, and coefficients of variation for repeatability and reproducibility were <5% and <10%, respectively. A normally distributed reference range of 47.0–135.5 IU/dL was derived from 154 normal donors. Clinical samples with Technofluor FXIII Activity results between 0 and 167.0 IU/dL were assayed with Berichrom® FXIII Activity, a functional ammonia release assay, and the HemosIL™ FXIII antigen assay, generating correlations of 0.950 and 0.980, respectively. Experiments with a transglutaminase inhibitor showed that Technofluor FXIII Activity can detect inhibition of enzymatic activity. No interference was exhibited by high levels of haemolysis and lipaemia, and interference by bilirubin was evident at 18 mg/dL, a level commensurate with severe liver disease. Technofluor FXIII Activity is a rapid, accurate and precise assay suitable for routine diagnostic use with fewer interferents than ammonia release FXIII activity assays.

Mild Acquired Factor XIII Deficiency and Clinical Relevance at the ICU—A Retrospective Analysis

Acquired FXIII deficiency is a relevant complication in the perioperative setting; however, we still have little evidence about the incidence and management of this rarely isolated coagulopathy. This study aims to help find the right value for the substitution of patients with an acquired mild FXIII deficiency. In this retrospective single-center cohort study, we enrolled critically ill patients with mild acquired FXIII deficiency (>5% and ≤70%) and compared clinical and laboratory parameters, as well as pro-coagulatory treatments. The results of the present analysis of 104 patients support the clinical relevance of FXIII activity out of the normal range. Patients with lower FXIII levels, beginning at <60%, had lower minimum and maximum hemoglobin values, corresponding to the finding that patients with a minimum FXIII activity of <50% needed significantly more packed red blood cells. FXIII activity correlated significantly with general coagulation markers such as prothrombin time, activated partial thromboplastin time, and fibrinogen. Nevertheless, comparing the groups with a cut-off of 50%, the amount of fresh frozen plasma, thrombocytes, PPSB, AT-III, and fibrinogen given did not differ. These results indicate that a mild FXIII deficiency occurring at any point of intensive care unit stay is also probably relevant for the total need of packed red blood cells, independent of pro-coagulatory management. In alignment with the ESAIC guidelines, the measurement of FXIII in critically ill patients with the risk of bleeding and early management, with the substitution of FXIII at levels <50%-60%, could be suggested.

A Novel Assay Using Enzyme Capture - ELISA for Accurate Determination of Factor XIII Activity

Background: The currently available, clinically approved factor XIII (fXIII) activity assays rely on indirect measurement of activated fXIII transglutaminase activity by detecting "ammonia-release" during an intermediate step of the reaction. Unfortunately, this assay is also sensitive to fXIII-independent ammonia-producing reactions that may take place physiologically in plasma, rendering it prone to overestimate fXIII activity, especially when levels are &lt;10% (0.1 IU/mL). High quality quantitative fXIII activity assays are required for the accurate and timely diagnosis of fXIII deficiency. fXIII circulates in complex with fibrinogen, and activated fXIII retards fibrinolysis by incorporating α2-antiplasmin (α2AP), a potent antifibrinolytic, into the forming clot via intermolecular α2AP:fibrin crosslinks. We have thus developed a new fXIII transglutaminase activity assay that directly measures α2-AP incorporation onto captured fibrin(ogen):fXIII complexes. We hypothesized that this novel assay has high sensitivity in detecting FXIII activity, especially at the low end of the dynamic range (&lt;10%; &lt;0.01 IU/mL). Methods: A standard curve was established by diluting known concentrations of recombinant factor XIII-A (rFXIII-A; Tretten) into (1) fXIII-A congenitally deficient and (2) fXIII immunodepleted plasma to determine if our assay is able to detect variable fXIII concentrations from 1 to 200% (0.01 - 2 IU/mL). Results: We demonstrated that α2-AP incorporation is dependent upon rfXIII-A concentration in the starting sample and was linear from 1-12% (0.01 - 0.12 IU/mL; Figure). Further expansion of the standard curve to include all concentrations within the normal physiologic range demonstrated that the assay remains strongly linear from 1-20% (0.01 - 0.2 IU/mL; R2 0.966). Moderate linearity (R2 0.908) extended to 50% (0.5 IU/mL). The curve plateaued between 50%-200% (0.5 IU/mL- 2 IU/mL). Thus, the dynamic range of the Enzyme Capture-ELISA is expected within 1-50% (0.01 - 0.5 IU/mL). Conclusion: We conclude that Enzyme Capture-ELISA is a promising novel method for accurate detection of FXIII activity and is expected to improve sensitivity at low levels (&lt;20%; &lt;0.02 IU/mL), which are most clinically relevant. Further experiments are needed to refine the upper limit of assay linearity and thereby refine the dynamic range of the assay. With further optimization and validation, the EC-ELISA method may provide an improved diagnostic assay. Disclosures No relevant conflicts of interest to declare.

The Need for Red Cell Support During Non-Cardiac Surgery Is Associated to Pre-Transfusion Levels of FXIII and the Platelet Count

Unexpected intraoperative bleeding is associated with a reduced availability of crosslinking capacity (provided through factor XIII (FXIII)) per unit of generated thrombin. Furthermore, FXIII deficiency and thrombocytopenia (but not fibrinogen deficiency) are the most prevalent modulators of clot firmness in the immediate postoperative setting. In this study, we therefore evaluated whether levels of FXIII, fibrinogen, or the platelet count influenced the probability of intraoperative red cell transfusions in patients in the operating theatre. This retrospective study was comprised of 1023 patients, which were in need of blood product support in the operating theatre and of which 443 received red cell transfusions. Due to standard operating procedures, FXIII activity, fibrinogen concentration, and platelet count were measured before transfusion took place, but without influencing the decision to transfuse. FXIII deficiency was frequent (50%), as was thrombocytopenia (49%), but not fibrinogen deficiency (9%). FXIII deficiency was associated with a significantly increased probability to receive red cell transfusions (OR 4.58, 95% CI 3.46–6.05) as was thrombocytopenia (OR 1.94, 95% CI 1.47–2.56), but not fibrinogen deficiency (OR 1.09, 95% CI 0.67–1.76). Similar results were seen for cut-off independent evaluations (receiver operating characteristics (ROC) curves, using continuously distributed variables), where the areas under the curves (AUC) of red cell transfusion for FXIII activity was 0.744 (95% CI 0.716–0.770)/0.632 (95% CI 0.601–0.661) for the platelet count, and 0.578 (95% CI 0.547–0.609) for fibrinogen concentration. All AUCs were significantly different from each other (p < 0.0001 and p = 0.0106, respectively), indicating that FXIII activity was a significantly better predictor of red blood cell (RBC) transfusion than platelet count and fibrinogen concentration. These results suggest that pre-transfusion FXIII activity and to a lesser extent the platelet count influence the probability of intraoperative red cell transfusions. Modifying FXIII activity and/or the platelet count might influence the need for downstream red cell transfusion, thus potentially reducing transfusion associated morbidity. This, however, needs confirmation in future studies.

Recurrent Hematomas following a Revision Total Hip Arthroplasty in Acquired Coagulation Factor XIII Deficiency

Coagulation factor XIII (FXIII) is the final enzyme in the coagulation cascade and plays an important role in catalyzing the intermolecular cross-linking of fibrin polymers. FXIII deficiency is a rare disorder that presents with recurrent soft tissue bleeding. In this case report, we describe a patient with recurrent hematomas, following a revision total hip arthroplasty (THA). A 50-year-old female patient with no past history of bleeding and with a normal perioperative coagulation profile presented with recurrent hip joint hematomas. Her plasma FXIII activity showed a slight decrease (69%). Therefore, the patient was diagnosed with an acquired deficiency and was administered FXIII to correct it. The bleeding did not recur once the FXIII activity had returned to a normal level (76%). At 2 months after the second evacuation procedure, the patient was discharged from the hospital in an ambulatory state. There has been no recurrence of a hematoma since. We managed a rare case of acquired FXIII deficiency, which highlighted that a patient can present with an acquired bleeding disorder despite having a normal coagulation profile. An acquired FXIII deficiency should be suspected in patients with inexplicable, sudden-onset bleeding, as early diagnosis and treatment are important to prevent life-threatening complications.

Mechanism Underlying a Role for Factor XIII (FXIII) Polymorphism in Sickle Cell Disease-Associated Priapism

Background: The pathophysiology of priapism in sickle cell disease (SCD) is poorly understood. While blood stasis is essential to tumescence, most research in SCD-associated priapism to date has focused on the potential role of abnormal signaling pathways. We have previously observed that a coding sequence single nucleotide polymorphism (rs5988) of the transglutaminase factor XIII gene (FXIII) is strongly associated with SCD priapism, with an odds ratio of 2.52 [C.I. 1.27 -5.03] for the risk genotype (G/G, expressing only FXIII E652) vs the most common non-risk genotype (G/C, expressing both FXIII E652 and FXIII Q652). We therefore explored the effect of the rs5988 polymorphism on various aspects of FXIII function. Methods: Recombinant FXIII (rFXIII) E652 and rFXIII Q652 were expressed by 293 kidney cells and isolated from serum-free tissue culture supernatant. Before use in assays, activation of rFXIII by thrombin was confirmed by generating activated rFXIII (rFXIIIa) with thrombin (10 U/ml), followed SDS-PAGE, western blotting for FXIII, and densitometry for quantitation. Whole blood samples and plasmas were obtained from previously genotyped subjects with SCD under an IRB-approved protocol. Plasma FXIII and rFXIII transglutaminase activity was measured by the ability to catalyze 5-(biotinamido)pentylamine incorporation into a suitable substrate. Plasma FXIII antigen was assayed by ELISA. Clot contraction was measured after tissue factor-initiated clotting of recalcified whole blood. Clot resistance to lysis was measured after exposure to tissue plasminogen activator (tPA). Results: Transglutaminase activity of each rFXIII was measured using fibrinogen and fibronectin as substrates. At 20 minutes, rFXIIIa E652 showed 1.44-fold more transglutaminase (crosslinking) activity toward fibrin(ogen) than rFXIIIa Q652 (p=0.027), and a nonsignificant trend toward more activity (1.32-fold, p=0.079) toward fibronectin. Kinetic assays also showed that rFXIII E652 had significantly greater activity toward both matrices (p=0.006 and 0.012, respectively), suggesting the risk genotype (homozygosity for the G allele) enhances fibrin(ogen) and/or fibronectin crosslinking. FXIII activity in the plasma of 18 genotyped SCD patients (3 CC, 7 GC, 8 GG) demonstrated a consistent, but not significant, trend toward increased FXIII activity with increasing presence of G alleles (80.01% CC, 98.90% CG, and 107.20% GG). Although results were not adjusted for genotype at other loci reported to affect FXIII expression, there was no significant difference in FXIII antigen among genotypes. Compared to contracted whole blood clots from patients with only one or no risk alleles, clots from SCD patients with two copies of the risk allele (GG, expressing only FXIII E652) did not differ in either RBC retention within clots or clot mass (weight). Moreover, inhibition of transglutaminase activity with T101 significantly increased RBC release and decreased clot weight to a similar degree in contracted clots from patients with either the GC or GG genotype. In clots formed from FXIII-deficient plasma supplemented with rFXIIIa E652 or rFXIIIa Q652 and ABO-compatible donor RBCs, clots containing rFXIIIa E652 were 14% more resistant to lysis than clots containing rFXIIIa Q652 (p=0.016). Parallel studies with SCD patient plasma samples also showed that clots containing only FXIII E652 were more resistant to lysis than clots containing both FXIII E652 and FXIII Q652(p=0.0001). Conclusions: These data suggest the FXIII rs5988 polymorphism does not alter protein expression or clot contraction but may regulate clot stability via slightly increased transglutaminase activity and enhanced resistance to lysis. These effects may predispose patients to formation of microclots during tumescence, thus impairing blood egress and increasing risk of priapism. Further studies should be conducted to determine if anticoagulation or fibrinolytic treatments are viable preventative or treatment strategies for SCD patients with the risk FXIIIGG genotype and recurrent priapism. Disclosures Telen: Pfizer, Inc.: Consultancy, Research Funding. Wolberg:GlaxoSmithKline: Employment; Novo Nordisk: Research Funding.