Heterozygosity in factor XIII genes and the manifestation of mild inherited factor XIII deficiency.

BACKGROUND: The characterization of inherited mild factor XIII deficiency is more imprecise than its rare, inherited severe forms. It is known that heterozygosity at FXIII genetic loci results in mild FXIII deficiency, characterized by circulating FXIII activity levels ranging from 20% to 60%. There exists a gap in information on 1) how genetic heterozygosity renders clinical bleeding manifestations among these individuals and 2) the reversal of unexplained bleeding upon FXIII administration in mild FXIII-deficient individuals. OBJECTIVES: To assess the prevalence and burden of mild FXIII deficiency among the apparently healthy German-Caucasian population and correlate it with genetic heterozygosity at FXIII and fibrinogen gene loci. METHODS: Peripheral blood was collected from 752 donors selected from the general population with essentially no bleeding complications to ensure asymptomatic predisposition. These were assessed for FXIII and fibrinogen activity, and FXIII and fibrinogen genes were resequenced using next-generation sequencing. For comparison, a retrospective analysis was performed on a cohort of mild inherited FXIII deficiency patients referred to us. RESULTS: The prevalence of mild FXIII deficiency was high (∼0.8%) among the screened German-Caucasian population compared with its rare-severe forms. Although no new heterozygous missense variants were found, certain combinations were relatively dominant/prevalent among the mild FXIII-deficient individuals. CONCLUSION: This extensive, population-based quasi-experimental approach revealed that the burden of heterozygosity in FXIII and fibrinogen gene loci causes the clinical manifestation of inherited mild FXIII deficiency, resulting in ''unexplained bleeding'' upon provocation.

Glycated albumin modulates the contact system with implications for the kallikrein-kinin and intrinsic coagulation systems.

BACKGROUND: Human serum albumin (HSA) is the most abundant plasma protein and is sensitive to glycation in vivo. The chronic hyperglycemic conditions in patients with diabetes mellitus (DM) induce a nonenzymatic Maillard reaction that denatures plasma proteins and forms advanced glycation end products (AGEs). HSA-AGE is a prevalent misfolded protein in patients with DM and is associated with factor XII activation and downstream proinflammatory kallikrein-kinin system activity without any associated procoagulant activity of the intrinsic pathway. OBJECTIVES: This study aimed to determine the relevance of HSA-AGE toward diabetic pathophysiology. METHODS: The plasma obtained from patients with DM and euglycemic volunteers was probed for activation of FXII, prekallikrein (PK), and cleaved high-molecular-weight kininogen by immunoblotting. Constitutive plasma kallikrein activity was determined via chromogenic assay. Activation and kinetic modulation of FXII, PK, FXI, FIX, and FX via in vitro-generated HSA-AGE were explored using chromogenic assays, plasma-clotting assays, and an in vitro flow model using whole blood. RESULTS: Plasma obtained from patients with DM contained increased plasma AGEs, activated FXIIa, and resultant cleaved cleaved high-molecular-weight kininogen. Elevated constitutive plasma kallikrein enzymatic activity was identified, which positively correlated with glycated hemoglobin levels, representing the first evidence of this phenomenon. HSA-AGE, generated in vitro, triggered FXIIa-dependent PK activation but limited the intrinsic coagulation pathway activation by inhibiting FXIa and FIXa-dependent FX activation in plasma. CONCLUSION: These data indicate a proinflammatory role of HSA-AGEs in the pathophysiology of DM via FXII and kallikrein-kinin system activation. A procoagulant effect of FXII activation was lost through the inhibition of FXIa and FIXa-dependent FX activation by HSA-AGEs.

Kallikrein directly interacts with and activates Factor IX, resulting in thrombin generation and fibrin formation independent of Factor XI.

Kallikrein (PKa), generated by activation of its precursor prekallikrein (PK), plays a role in the contact activation phase of coagulation and functions in the kallikrein-kinin system to generate bradykinin. The general dogma has been that the contribution of PKa to the coagulation cascade is dependent on its action on FXII. Recently this dogma has been challenged by studies in human plasma showing thrombin generation due to PKa activity on FIX and also by murine studies showing formation of FIXa-antithrombin complexes in FXI deficient mice. In this study, we demonstrate high-affinity binding interactions between PK(a) and FIX(a) using surface plasmon resonance and show that these interactions are likely to occur under physiological conditions. Furthermore, we directly demonstrate dose- and time-dependent cleavage of FIX by PKa in a purified system by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and chromogenic assays. By using normal pooled plasma and a range of coagulation factor-deficient plasmas, we show that this action of PKa on FIX not only results in thrombin generation, but also promotes fibrin formation in the absence of FXII or FXI. Comparison of the kinetics of either FXIa- or PKa-induced activation of FIX suggest that PKa could be a significant physiological activator of FIX. Our data indicate that the coagulation cascade needs to be redefined to indicate that PKa can directly activate FIX. The circumstances that drive PKa substrate specificity remain to be determined.

Structure functional insights into calcium binding during the activation of coagulation factor XIII A.

The dimeric FXIII-A2, a pro-transglutaminase is the catalytic part of the heterotetrameric coagulation FXIII-A2B2 complex that upon activation by calcium binding/thrombin cleavage covalently cross-links preformed fibrin clots protecting them from premature fibrinolysis. Our study characterizes the recently disclosed three calcium binding sites of FXIII-A concerning evolution, mutual crosstalk, thermodynamic activation profile, substrate binding, and interaction with other similarly charged ions. We demonstrate unique structural aspects within FXIII-A calcium binding sites that give rise to functional differences making FXIII unique from other transglutaminases. The first calcium binding site showed an antagonistic relationship towards the other two. The thermodynamic profile of calcium/thrombin-induced FXIII-A activation explains the role of bulk solvent in transitioning its zymogenic dimeric form to an activated monomeric form. We also explain the indirect effect of solvent ion concentration on FXIII-A activation. Our study suggests FXIII-A calcium binding sites could be putative pharmacologically targetable regions.

Evaluation of the Total Thrombus-Formation System (T-TAS): application to human and mouse blood analysis.

The Total Thrombus-formation Analyser System (T-TAS) is a whole blood flow chamber system for the measurement of in vitro thrombus formation under variable shear stress conditions. Our current study sought to evaluate the potential utility of the T-TAS for the measurement of thrombus formation within human and mouse whole blood. T-TAS microchips (collagen, PL chip; collagen/tissue thromboplastin, AR chip) were used to analyze platelet (PL) or fibrin-rich thrombus formation, respectively. Blood samples from humans (healthy and patients with mild bleeding disorders) and wild-type (WT), mice were tested. Light transmission lumi-aggregometer (lumi-LTA) was performed in PRP using several concentrations of ADP, adrenaline, arachidonic acid, collagen, PAR-1 peptide and ristocetin. Thrombus growth (N = 22) increased with shear within PL (4:40 ± 1.11, 3:25 ± 0.43 and 3:12 ± 0.48 mins [1000, 1500 and 2000s-1]) and AR chips (3:55 ± 0.42 and 1:49 ± 0.19 [240s-1 and 600s-1]). The area under the curve (AUC) on the PL chip was also reduced at 1000s-1 compared to 1500/2000s-1 (260 ± 51.7, 317 ± 55.4 and 301 ± 66.2, respectively). In contrast, no differences in the AUC between 240s-1 and 600s-1 were observed in the AR chip (1593 ± 122 and 1591 ± 158). The intra-assay coefficient of variation (CV) (n = 10) in the PL chip (1000s-1) and AR chip (240s-1) were T1014.1%, T6016.7%, T10-6022.8% and AUC1024.4% or T10 9.03%, T808.64%, T10-8023.8% and AUC305.1%. AR chip thrombus formation was inhibited by rivaroxaban (1 µM), but not with ticagrelor (10 µM). In contrast, PL chip thrombus formation was totally inhibited by ticagrelor. T-TAS shows an overall agreement with lumi-LTA in 87% of patients (n = 30) with normal PL counts recruited into the genotyping and phenotyping of platelet (GAPP) study and suspected to have a PL function defect. The onset (T10) of thrombus formation in WT mice (N = 4) was shorter when compared to humans e.g. PL chip (1000s-1) T10 were 02:02 ± 00:23 and 03:30 ± 0:45, respectively). T-TAS measures in vitro thrombus formation and can be used for monitoring antithrombotic therapy, investigating patients with suspected PL function defects and monitoring PL function within mice.

Coagulation Factor XIIIA Subunit Missense Mutations Affect Structure and Function at the Various Steps of Factor XIII Action.

Inherited defects of coagulation Factor XIII (FXIII) can be categorized into severe and mild forms based on their genotype and phenotype. Heterozygous mutations occurring in F13A1 and F13B genes causing mild FXIII deficiency have been reported only in the last few years primarily because the mild FXIII deficiency patients are often asymptomatic unless exposed to some kind of a physical trauma. However, unlike mutations causing severe FXIII deficiency, many of these mutations have not been comprehensively characterized based on expression studies. In our current article, we have transiently expressed 16 previously reported missense mutations detected in the F13A1 gene of patients with mild FXIII deficiency and analyzed their respective expression phenotype. Complimentary to expression analysis, we have used in silico analysis to understand and explain some of the in vitro findings. The expression phenotype has been evaluated with a number of expression phenotype determining assays. We observe that the mutations influence different aspects of FXIII function and can be functionally categorized on the basis of their expression phenotype. We identified mutations which even in heterozygous form would have strong impact on the functional status of the protein (namely mutations p.Arg716Gly, p.Arg704Gln, p.Gln602Lys, p.Leu530Pro, p.His343Tyr, p.Pro290Arg, and p.Arg172Gln).

The role of activated coagulation factor XII in overall clot stability and fibrinolysis.

Activated coagulation factor XII (α-FXIIa) is able to bind to fibrin(ogen) and increases the density and stiffness of the fibrin clot. Conversely, proteins of the contact system and the fibrinolytic system show a high degree of homology and α-FXIIa can convert plasminogen into plasmin resulting in fibrin degradation. Therefore, we studied the contribution of α-FXIIa to overall clot stability and plasmin driven fibrinolysis in the absence and presence of tissue plasminogen activator (tPA). We observed that α-FXIIa directly converted plasminogen into plasmin and reduced clot lysis time at all tPA concentrations tested (15-1500 pM). Simultaneous assessment of plasmin generation (chromogenic substrate S-2251) and fibrin formation and degradation (absorbance at 405nm), showed an earlier onset of fibrinolysis and plasmin formation in the presence of α-FXIIa. Fibrinolysis of clots formed under flow conditions, revealed that incorporation of α-FXIIa accelerated clot breakdown (fluorescence release of labeled fibrin) by additional plasmin generation on top of formation by tPA. Scanning electron microscopy (SEM) revealed that the surface area pore size increased in the presence compared with the absence of α-FXIIa when fibrinolysis was initiated by the conversion of plasminogen with tPA during clot formation. α-FXIIa enhances fibrinolysis in the presence of plasminogen, irrespective of whether tPA was present during clot formation or was added afterwards to initiate fibrinolysis. We postulate that FXIIa first strengthens the clot structure during clot formation and thereafter contributes towards fibrinolysis.