Factor XIII Activity Research Articles

Factor XIII Deficiency and Thrombocytopenia Are Frequent Modulators of Postoperative Clot Firmness in a Surgical Intensive Care Unit

Objective: Fibrinogen and factor XIII (FXIII) have been shown to critically influence clot firmness in the intraoperative setting and thus likely influence intraoperative bleeding. We were interested to identify potential modulators of postoperative clot firmness in a tertiary care hospital surgical intensive care unit setting, independent of their clinical course during surgery. Methods: 272 day-shift consecutive patients were evaluated for whole blood clot firmness evaluated by the ROTEM® EXTEM thrombelastometric assay and various potential modulators of clot firmness upon arrival at the surgical intensive care unit (SICU). Results: Maximum clot firmness on the SICU was found to be independently influenced by the amount of colloids given during surgery as well as by platelet count, fibrinogen concentration, and FXIII activity at the time of SICU admission. In patients with lowest clot firmness, FXIII activity was the most important independent modulator of clot firmness; in patients with the highest clot firmness, platelet count and fibrinogen concentration were the most important modulators of clot firmness. Deficiencies (i.e., results below normal range) of these modulators of clot firmness were most prevalent for FXIII (activity < 70%: 45% of cases), which was significantly more frequent than thrombocytopenia (<150 × 10⁹/l: 32%) or fibrinogen deficiency (<1.5 g/l: 6%). Conclusions: Postoperative clot firmness as evaluated by whole blood thrombelastometry (ROTEM EXTEM assay) is independently and frequently modulated though FXIII activity and the platelet count, while fibrinogen concentration is also an independent but much less frequent modulator. Different modulators show different influences, depending on the clot firmness being present. Colloids infused during surgery also independently modulate postoperative clot firmness. Based on our data, strategies can be developed to improving postoperative care of patients with bleedings or at risk for bleeding.

Coagulation factor XI improves host defence during murine pneumonia-derived sepsis independent of factor XII activation.

Bacterial pneumonia, the most common cause of sepsis, is associated with activation of coagulation. Factor XI (FXI), the key component of the intrinsic pathway, can be activated via factor XII (FXII), part of the contact system, or via thrombin. To determine whether intrinsic coagulation is involved in host defence during pneumonia and whether this is dependent on FXII activation, we infected in parallel wild-type (WT), FXI knockout (KO) and FXII KO mice with two different clinically relevant pathogens, the Gram-positive bacterium Streptococcus pneumoniae and the Gram-negative bacterium Klebsiella pneumoniae, via the airways. FXI deficiency worsened survival and enhanced bacterial outgrowth in both pneumonia models. This was accompanied with enhanced inflammatory responses in FXI KO mice. FXII KO mice were comparable with WT mice in Streptococcus pneumoniae pneumonia. On the contrary, FXII deficiency improved survival and reduced bacterial outgrowth following infection with Klebsiella pneumoniae. In both pneumonia models, local coagulation was not impaired in either FXI KO or FXII KO mice. The capacity to phagocytose bacteria was impaired in FXI KO neutrophils and in human neutrophils where activation of FXI was inhibited. Deficiency for FXII or blocking activation of FXI via FXIIa had no effect on phagocytosis. Taken together, these data suggest that FXI protects against sepsis derived from Streptococcus pneumoniae or Klebsiella pneumoniae pneumonia at least in part by enhancing the phagocytic capacity of neutrophils by a mechanism that is independent of activation via FXIIa.

Assessment of Factor XIII.

Blood coagulation factor XIII (FXIII) is essential for maintaining hemostasis. The absence of FXIII results in severe bleeding diathesis, which without prophylaxis frequently leads to fatal bleeding. As the usual hemostasis screening tests remain normal, the diagnosis of FXIII deficiency needs specific tests. Here, we describe FXIII activity determination by the ammonia release assay, which is the first-line test in the diagnostic algorithm for FXIII deficiency. The method for another activity test, the undeservedly rarely used fibrin cross-linking assay, is also presented. Further tests used for the classification of FXIII deficiencies, measurement of FXIII activity in platelets, ELISAs for the measurement of complex plasma FXIII (FXIII-A2B2) antigen and for FXIII-A2 in plasma and platelets are also included. Detailed description of the methods for the detection and measurement of neutralizing auto- and alloantibodies is also provided.

A novel missense mutation in the factor XII gene in a litter of cats with factor XII deficiency.

The feline F12 gene was examined to identify a mutation associated with coagulation factor XII (FXII) deficiency in a litter of 6 cats, including 2 cats with severely reduced FXII activity (7.1 and 9.3%, respectively) and 4 cats with moderately reduced FXII activity (range 36.0 to 46.3%). Cats with severely reduced FXII activity were homozygous for a G to C missense mutation in exon 13 of the F12 gene, resulting in an amino acid change (p.G544A). Cats with moderately reduced FXII activity were heterozygous for this mutation. Expression studies revealed reduced secretion of p.G544A mutant FXII protein from transfected HEK293 cells compared with wild type FXII. These results reveal a novel F12 mutation in FXII deficient cats and define the underlying mechanism for low FXII activity in homozygotes.

Impact of low coagulation factor XIII activity in patients with chronic subdural hematoma associated with cerebrospinal fluid hypovolemia: A retrospective study.

Background:Cerebrospinal fluid hypovolemia (CSFH) is sometimes associated with chronic subdural hematomas (CSHs). Affected patients often develop enlargement and recurrence of the CSH, even if appropriate treatments such as epidural blood patch (EBP) and/or burr-hole surgery for the CSH are performed. This situation may lead to subclinical coagulopathy, including low coagulation factor XIII (CFXIII) activity. We retrospectively analyzed whether CFXIII activity was involved in the development of CSHs and post-treatment exacerbation of CSHs in patients with CSFH.Methods:We diagnosed CSFH by radioisotope (RI), magnetic resonance imaging (MRI) and computed tomography (CT) findings, and CSH by CT and/or MRI findings. The plasma CFXIII activity was assessed on admission. All patients with CSFH initially received conservative treatments. When these treatments were ineffective, the patients underwent EBP and/or CSH surgery according to previously reported therapeutic strategies.Results:Among 206 patients with CSFH, 19 developed CSHs. Fourteen patients with a thin hematoma underwent EBP and three with a thick hematoma underwent CSH surgery immediately after EBP on the same day. We were unable to diagnose two patients with CSFH at the time of admission, and one of these two patients underwent repeated CSH surgery before obtaining the correct diagnosis. Seven patients (36.8%) developed CSH exacerbation after the treatment. The CFXIII activity was significantly lower in patients with than without a CSH (42.1% vs. 12.8%, respectively; P = 0.003). The CFXIII activity was significantly lower in patients with than without post-treatment CSH exacerbation (P = 0.046). All five patients with low CFXIII activity who developed CSH exacerbation received intravenous injection of CFXIII and had no recurrence of CSH after the additional treatment.Conclusion:In patients with CSFH, low CFXIII activity is one of the risk factors for both the development of a CSH and the post-treatment exacerbation CSH.

Intracardiac Hemostasis and Fibrinolysis Parameters in Patients with Atrial Fibrillation.

Aims To identify intracardiac hemostasis or fibrinolysis abnormalities, which are associated with atrial fibrillation (AF) and increase the risk of thromboembolism. Patients and Methods Patient group consisted of 24 patients with AF and control group included 14 individuals with other supraventricular tachycardia undergoing transcatheter radiofrequency ablation. Blood samples were drawn from the femoral vein (FV), left atrium (LA), and left atrial appendage (LAA) before the ablation procedure. Fibrinogen, factor VIII (FVIII) and factor XIII activity, von Willebrand factor (VWF) antigen, thrombin-antithrombin (TAT) complex, quantitative fibrin monomer (FM), plasminogen, α2-plasmin inhibitor, plasmin-α2-antiplasmin (PAP) complex, PAI-1 activity, and D-dimer were measured from all samples. Results Levels of FVIII and VWF were significantly elevated in the FV and LA of AF patients as compared to controls. TAT complex, FM, PAP complex, and D-dimer levels were significantly elevated in the LA as compared to FV samples in case of both groups, indicating a temporary thrombotic risk associated with the catheterization procedure. Conclusions None of the investigated hemostasis or fibrinolysis parameters showed significant intracardiac alterations in AF patients as compared to non-AF controls. AF patients have elevated FVIII and VWF levels, most likely due to endothelial damage, presenting at both intracardiac and systemic level.

Differential Roles for the Coagulation Factors XI and XII in Regulating the Physical Biology of Fibrin.

In the contact activation pathway of the coagulation, zymogen factor XII (FXII) is converted to FXIIa, which triggers activation of FXI leading to the activation of FIX and subsequent thrombin generation and fibrin formation. Feedback activation of FXI by thrombin has been shown to promote thrombin generation in a FXII-independent manner and FXIIa can bypass FXI to directly activate FX and prothrombin in the presence of highly negatively charged molecules, such as long-chain polyphosphates (LC polyP). We sought to determine whether activation of FXII or FXI differentially regulate the physical biology of fibrin formation. Fibrin formation was initiated with tissue factor, ellagic acid (EA), or LC polyP in the presence of inhibitors of FXI and FXII. Our data demonstrated that inhibition of FXI decreased the rate of fibrin formation and fiber network density, and increased the fibrin network strength and rate of fibrinolysis when gelation was initiated via the contact activation pathway with EA. FXII inhibition decreased the fibrin formation and fibrin density, and increased the fibrinolysis rate only when fibrin formation was initiated via the contact activation pathway with LC polyP. Overall, we demonstrate that inhibition of FXI and FXII distinctly alter the biophysical properties of fibrin.

Stimulated Platelets but Not Endothelium Generate Thrombin Via a Factor XIIa-Dependent Mechanism Requiring Phosphatidylserine Exposure

To inhibit pathological thrombus formation without impairing hemostasis is the holy grail of anticoagulant therapy. Recent data from animal models have indicated that factor XII (FXII) may be a promising new antithrombotic target that is particularly intriguing due to the longstanding clinical observation that severe congenital FXII deficiency is not associated with a bleeding diathesis in humans. FXII is thought to participate in thrombus formation after being activated in high shear arterial environments. FXIIa then initiates downstream activation of the contact pathway, culminating in thrombin generation. However, the relevant cell surface for FXII activation remains unclear. Here we compare the role of platelets versus endothelial cells in FXII activation and study the function of surface phospholipids in this process.To explore the effect of FXII inhibition on thrombus formation in vivo, we used antibody X210-C01, a novel human IgG1 developed using phage-display technology that blocks both mouse and human FXIIa. Using a mouse laser injury model of arterial thrombosis, we showed that X210-C01 inhibited both fibrin formation and platelet accumulation at sites of vascular injury. Plasma removed from animals after completion of these experiments was used to quantify the concentrations of X210-C01 achieved in vivo at a given dose. FXII inhibition was somewhat more potent in preventing platelet accumulation (IC50 dose = 27 mg/kg, R2=0.93) than fibrin formation (IC50 dose = 43 mg/kg, R2=0.95). Importantly, treatment with X210-C01 at 100 mg/kg did not prolong bleeding times or increase total blood loss in a tail bleeding assay.To evaluate the mechanism underlying our in vivo observations, we studied the differential role of FXII in thrombin generation by stimulated platelets and endothelium. X210-C01 did not globally inhibit SFLLRN-induced platelet aggregation or granule release. We next performed a fluorogenic thrombin generation assay (TGA) using human platelets treated with the peptide agonist SFLLRN. X210-C01 inhibited platelet-based thrombin generation in a dose-dependent fashion, whereas anti-tissue factor (TF) and anti-factor VIIa (FVIIa) antibodies did not. By contrast, in a similar TGA using SFLLRN-stimulated endothelial cells, X210-C01 had no effect, while anti-TF antibodies abrogated thrombin generation. These results indicate that stimulated endothelium generates thrombin by a mechanism distinct from that of platelets.FXII is known to be activated in vitro by anionic surfaces. Because phosphatidylserine (PS) is a negatively-charged phospholipid expressed on the surface of stimulated platelets, we reasoned that PS may serve as the platelet-based activator of FXII. To test this hypothesis, we used lactadherin, a potent and specific inhibitor of PS, in the platelet-based TGA and showed that PS blockade inhibited platelet-based thrombin generation at concentrations as low as 10 nM. We then used a chromogenic FXIIa activity assay to test the ability of PS-containing liposomes to activate FXII. Liposomes containing 80% phosphatidylcholine (PC) and 20% PS (PC-PS 80/20) failed to activate FXII at concentrations as high as 100 µM. In this assay, SFLLRN-stimulated platelets led to significantly greater FXII activation than either resting platelets or the PC-PS 80/20 liposomes tested.In summary, we have made the unexpected finding that thrombin generation on the surface of stimulated platelets proceeds by a FXIIa-dependent pathway and does not require FVIIa or TF. By contrast, thrombin generation on endothelium requires TF but not FXIIa. Additionally, surface PS is necessary but not sufficient for platelet-based FXII activation and thrombin generation, pointing to the involvement of a second platelet component. Further studies will be directed towards investigating the in vivo role of platelet-based FXII activation in arterial thrombus formation. DisclosuresMason:Shire Pharmaceuticals: Employment. Kenniston:Shire Pharmaceuticals: Employment.

Safety and Efficacy of Recombinant Factor XIII (FXIII) in Patients with Congenital FXIII A-Subunit Deficiency, Results from the Mentor™2 Trial

Safety and Efficacy of Recombinant Factor XIII (FXIII) in Patients with Congenital FXIII A-Subunit Deficiency, Results from the Mentor™2 Trial

Acquired Factor XIII Deficiency in the Inpatient Setting: Clinical Suspicion, Impact and Treatment: Retrospective Case Series

Introduction: Activated factor XIII (FXIII) stabilizes fibrin clots at the end of the coagulation cascade by bridging fibrin molecules. Disproportionate surgery related bleeding has been reported in association with FXIII deficiency, in patients with normal coagulopathies screening tests, and without a history of previous bleedings.Methods: Retrospective case series. We performed immunological factor XIIIA (FXIIIA) studies in the inpatient setting of the Hospital Italiano de Buenos Aires, between January 2014 and March 2016. FXIIIA below 50% were considered deficient. Patients with suspicion of congenital Factor XIII deficiency or 2 years old or less were excluded. Descriptive statistics were used. Populations were compared with chi-square, Fisher and T test, or Mann Whitney tests with Stata13 software.Results: FXIII was studied in 52 patients; 37 of these (26 females and 11 males) met inclusion and exclusion criteria. Median age was 43 years (range 9-81). Twenty two patients were studied because of disproportionate surgery related bleeding, 11 because of spontaneous bleeding; and 4 not specified. All patients presented normal coagulopathies screening tests, normal Von Willebrand factor and ristocetine cofactor activity and normal platelet function tests. Eleven patients (30%) had FXIIIA less than 50%. Statistically significant differences between groups were found for median drop in hematocrit points [3.5 (IQR 3-10) vs 1.5 (IQR 0-2, p=0.02)] and median number of red cell units transfused [4 (IQR 0-6) vs 0 (0-2), p=0.01]. Differences in rates of minor and major bleedings were not statistically significant. Only one patient of eleven presented FXIII deficiency associated spontaneous bleeding vs 8 out of 20 patients in the postsurgical setting. FXIII concentrate was used in two patients to treat persistent bleeding, resolving in less than 24 hours. Three patients died, none because of bleeding. Five of the 11 patients with FXIII deficiency returned to normal values when FXIII assay was repeated away from acute setting.Discussion: FXIII acquired deficiency is associated with disproportionate bleeding in postsurgical setting in patients without apparent coagulation defects. FXIII deficiency could be underdiagnosed. Patients with FXIII deficiency in our series had more hematocrit drop and more transfusion requirements. We found no association with spontaneous bleeding. Alltough deficiency could be transient, treatment with FXIII concentrate could be useful to manage serious, persistent or life threatening bleedings. DisclosuresNo relevant conflicts of interest to declare.

Effect of FXIII Polymorphism on Formation of Heterocellular Aggregates in SCD

▪ BACKGROUNDWe have recently identified a SNP (rs5988) in the gene encoding the factor XIII A chain (F13A) that is highly associated with risk for priapism in SCD. The P value for the comparison of G/G vs. C/G was 0.009, and the odds ratio for having priapism was 2.52 (CI 1.27-5.03) for men with the G/G F13A genotype (encoding FXIIIA 651E) compared to those with the C/G genotype (expressing FXIIIA 651Q/E). We hypothesized that this polymorphism leads to abnormal FXIII-mediated crosslinking and increased heterologous blood cell aggregates. The mechanism whereby heterologous blood cell aggregates form has only been partially elucidated to date. Brittain et al. (2008) showed that fibronectin is detectable within aggregates and plays a critical role in RBC-monocyte interactions, but it is unknown how fibronectin is recruited. We propose that aggregates form via interactions between fibrin(ogen), other plasma proteins (e.g., fibronectin, laminin), and blood cells during blood stasis in the penis during erection. These aggregates might then obstruct vessels, preventing blood egress, and result in SCD-associated priapism. METHODSWe quantitated circulating heterocellular aggregates using flow cytometry and directly labeled antibodies against CD45 (pan-leukocyte), CD235a (glycophorin A), CD41a (platelet GPIIb), and CD14 (monocytes and granulocytes). Heterocellular aggregates were defined as events simultaneously expressing CD45 and CD235a in whole anticoagulated blood. Events were analyzed by forward and side scatter as well as immunostaining characteristics (Canto II flow cytometer, Becton Dickinson, San Jose). Genotypes were ascertained by SNP genotyping using predesigned and custom Taqman SNP Genotyping Assays (ThermoFisher Scientific, Foster City, CA). Mean values for different genotypes were compared using two-tailed t-tests; FXIII activity was compared using 2-way ANOVA. RESULTSAs previously described, heterocellular aggregates occurred more frequently in blood samples from SCD subjects than from HBAA controls. Among SCD subjects, the % WBCs found in aggregates ranged from 14.9% to 88.5% (mean 58.85%). Specifically, lymphocytes and monocytes were found significantly more frequently in aggregates in HbSS compared to HbAA samples (p=0.042 and 0.025, respectively). When analyzed by F13A genotype, we also found that aggregates containing monocytes and lymphocytes were significantly more numerous in individuals with the GG (priapism risk) genotype than with the CC (low-risk) genotype (p = 0.008 and 0.015, respectively).We then tested recombinant (r) FXIIIA isoforms corresponding to the two alleles of F13A for their ability to bind to and crosslink both fibrinogen and fibronectin. Comparison of their ability to bind fibrinogen, fibrin and fibronectin showed no significant differences between the two rFXIII isoforms after activation. However, activated FXIIIA 651E (G allele) crosslinked both fibrinogen and fibronectin significantly more quickly than did activated FXIIIA 651Q (C allele) (p = 0.006 and p = 0.012, respectively), thus suggesting that the G allele might be associated with greater amounts of crosslinked fibrin(ogen) and fibronectin in the circulation to promote aggregate formation. CONCLUSIONSOur study has demonstrated that the F13A G allele at rs5988 carries a higher risk for SCD-related priapism and is associated with increased involvement of lymphocytes and monocytes in heterocellular aggregates. A possible mechanism is suggested by our observation that rFXIIIA protein encoded by the G allele more rapidly crosslinks fibrinogen and fibronectin than that encoded by the C allele. We theorize that the G genotype may be related to greater fibrin and fibronectin crosslinking, thus promoting the formation of circulating heterocellular aggregates. DisclosuresNo relevant conflicts of interest to declare.

Alteration of the Structure and Dynamics of Venous Clot Formation in Human and Murine Sickle Cell Disease

Sickle cell disease (SCD) is associated with chronic activation of coagulation and an increased risk of venous thromboembolism. Traditionally, it is believed that during venous thrombosis, red blood cells (RBC) are simply trapped within fibrin-rich thrombi and do not actively affect thrombosis. However, a study from our group showed that factor XIII (FXIII) activity is critical for the retention of RBC within clots and directly affects thrombus size. Others reported that during clot contraction polyhedral shaped RBC formed a densely packed core and that SCD alters the formation of polyhedrocytes which may affect clot stability (Strauss et al, 2015, ASH abstract).We further investigated if SCD affects the structure and the dynamics of clot formation. Ex vivo clot retraction was performed using blood from sickle cell patients and Townes mice (a model of SCD). Citrated blood was added to siliconized wells of 96-well plates containing CaCl2 (10mM) and tissue factor (1pM) and incubated for 2 hours at 37°C. The number of RBC extruded from the clot was counted in serum by Hemavet™ and expressed as a percentage of initial RBC number in the anticoagulated blood. Morphology of the clots was evaluated using scanning and transmission electron microscopy (EM). Thrombosis in Townes SS (sickle) and AA (control) mice was studied using femoral vein thrombosis induced by electrolytic injury and inferior vena cava stenosis models.The number of mouse SS RBC in the serum extruded during clot contraction was dramatically reduced compared to the number of AA RBC (0.8±0.8% vs. 19.4±0.8%, n=3, p<0.0001, Fig. A). A similar result was observed for human RBC. Since SS mice and sickle patients have a lower hematocrit (HCT), we investigated if the number of RBC affects the extrusion of these cells during clot retraction. Indeed, lowering HCT in AA mouse blood reduced RBC extrusion from the clots. However, increasing HCT in SS mouse blood to that of AA blood did not increase the number of SS RBC extruded from the clot. Furthermore, inhibition of FXIIIa activity with T101 (10µM) increased the release of AA (by 64%, n=6, p<0.05) but not SS RBC from mouse clots. These data indicate that the entrapment of SS RBCs within the clot is not simply caused by lower RBC number, and is FXIII-independent. Mixing the platelet poor plasma (PPP) and cellular fraction of AA and SS mouse blood revealed that the entrapment of SS RBC is not mediated by PPP (AA blood recombined = 6.9±3.6%; SS blood recombined = 0.4±0.4%; AA cells/SS PPP = 6.7±6.4%; SS cells/AA PPP = 0.1±0.2%, n=3 per group).Clots formed ex vivo from AA blood had a gel-like, soft structure, whereas SS clots were more firm and stiff. EM demonstrated that RBC within AA clots had polyhedral shapes and were tightly packed in the central part of the clot. In contrast, most of the mouse SS RBC did not have polyhedral shapes, underwent sickling and were not compacted within the clot. They also formed long "spicule-like" processes that intertwined with fibrin fibers (Fig. C). Similar results were observed in blood of sickle patients; however, the sickled RBC phenotype was less prominent. Importantly, sickling of RBC was observed in clots formed in the inferior vena cava of SS mice, 2 hours after vessel stenosis (Fig. D). We also performed a tPA challenge assay on clots formed ex vivo from human blood and showed that SS clots challenged with low tPA concentration (0.6nM) were more resistant to fibrinolysis compared to AA clots (clot lysis time, 714±6 vs. 388.3±120.7 minutes, n=6, p=0.08).The electrolytic injury model of venous thrombosis was used to investigate the dynamics of clot formation in SS mice in vivo. Mice were infused with fluorescently labeled antibodies for fibrin (green) and platelets (red). Electrolytic injury was applied to the femoral vein; a relative intensity of fibrin and platelet accumulation was assessed by fluorescence microscopy for one hour at 10-minute intervals. SS mice have increased platelet and fibrin accumulation compared to AA mice (~ 2 fold, n=5-7, p<0.05 for 40, 50 and 60 minute time points). Interestingly, in the AA clots, platelets were mostly localized on the surface, in contrast to their widespread distribution throughout the clot in SS mice (Fig. E, yellow color).Our data demonstrated that SCD alters the structure and dynamics of venous clot formation. Experiments investigating the consequence of these observations in mouse models of stroke and pulmonary embolism are currently ongoing. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Factor XIII deficiency enhances thrombin generation due to impaired fibrin polymerization — An effect corrected by Factor XIII replacement

IntroductionFactor XIII (FXIII) cross-links fibrin, completing blood coagulation. Congenital FXIII deficiency is managed with plasma-derived FXIII (pdFXIII) or recombinant FXIII (rFXIII) concentrates. AimAs the mechanisms protecting patients with low FXIII levels (<5IU/dL) from spontaneous bleeds remain unknown we assessed the interplay between thrombin generation (TG), fibrin formation and clot kinetics before and after FXIII administration in three patients with FXIII deficiency. MethodsPatients received initially rFXIII (35IU/kg, A-subunit) following with pdFXIII at 1250IU or 2500IU (12–30IU/kg) monthly. TG (CAT), thromboelastometry (ROTEM), prothrombin fragments F1+2, fibrinogen and FXIII activity (FXIII:C) were measured at baseline and one-hour recovery. ResultsFXIII was at the target level of 20±6IU/dL at the 4-week trough. rFXIII corrected FXIII to 98±15 and high-dose pdFXIII to a level of 90±6, whereas low-dose/half dose pdFXIII reached 45±4IU/dL. Although fibrinogen (Clauss Method) was normal, coagulation in FIBTEM was impaired, which FXIII administration tended to correct. CAT implied 1.6- to 1.9-fold enhanced TG, which FXIII administration normalized. Inhibition of fibrin polymerization by Gly-Pro-Arg-Pro peptide mimicked FXIII deficiency in CAT by enhancing TG both in control and FXIII recovery plasma. Antithrombin, α2-macroblobulin–thrombin complex and prothrombin were normal, whereas F1+2 were elevated compatible with in vivo TG. DiscussionFXIII deficiency impairs fibrinogen function and fibrin formation simultaneously enhancing TG on the poorly polymerizing fibrin strands, when fibrin’s antithrombin I -like function is absent. Our study suggests an inverse link between low FXIII levels and enhanced TG modifying structure-function relationship of fibrin to support hemostasis.

Regulation of plasma factor XIII levels in healthy individuals; a major impact by subunit B intron K c.1952 + 144 C > G polymorphism

BackgroundThe regulation of plasma factor XIII (FXIII) levels in healthy individuals has been only partially explored. The identification of major non-genetic and genetic regulatory factors might provide important information on the contribution of FXIII to the risk of cardio/cerebrovascular diseases. ObjectivesTo determine the effect of age, smoking, BMI, fibrinogen concentration on plasma FXIII activity, complex FXIII antigen (FXIII-A2B2) and total FXIII-B subunit (tFXIII-B) level, to correlate FXIII-B level with the other two FXIII parameters and to assess the variation of FXIII levels in carriers of major FXIII subunit polymorphisms. Methods268 healthy individuals were enrolled in the study. FXIII activity was measured by the ammonia release assay; FXIII-A2B2 and tFXIII-B were determined by ELISAs. FXIII-A p.Val34Leu, FXIII-B p.His95Arg and FXIII-B intron K c.1952+144 C>G polymorphisms were identified by RT-PCR using melting point analysis with fluorescence resonance energy transfer detection. ResultsAll investigated FXIII parameters showed significant positive correlation with age and fibrinogen level; gender and BMI influenced only tFXIII-B. A highly significant positive correlation was demonstrated between tFXIII-B and the other FXIII parameters. FXIII-A p.Val34Leu polymorphism had only slight, if any effect on FXIII levels. The FXIII-B Arg95 allele moderately increased all three FXIII parameters, but the effect became statistically significant only after adjustment. The FXIII-B intron K G allele drastically decreased FXIII levels, and it seemed to be in synergism with the FXIII-A Leu34 allele. ConclusionsPlasma FXIII levels are subjected to multifactorial regulation, in which age, fibrinogen level and FXIII-B intron K polymorphism are major determinants.

Regulation of Complement and Contact System Activation via C1 Inhibitor Potentiation and Factor XIIa Activity Modulation by Sulfated Glycans - Structure-Activity Relationships.

The serpin C1 inhibitor (C1-INH) is the only regulator of classical complement activation as well as the major regulator of the contact system. Its importance is demonstrated by hereditary angioedema (HAE), a severe disease with potentially life-threatening attacks due to deficiency or dysfunction of C1-INH. C1-INH replacement is the therapy of choice in HAE. In addition, C1-INH showed to have beneficial effects in other diseases characterized by inappropriate complement and contact system activation. Due to some limitations of its clinical application, there is a need for improving the efficacy of therapeutically applied C1-INH or to enhance the activity of endogenous C1-INH. Given the known potentiating effect of heparin on C1-INH, sulfated glycans (SG) may be such candidates. The aim of this study was to characterize suitable SG by evaluating structure-activity relationships. For this, more than 40 structurally distinct SG were examined for their effects on C1-INH, C1s and FXIIa. The SG turned out to potentiate the C1s inhibition by C1-INH without any direct influence on C1s. Their potentiating activity proved to depend on their degree of sulfation, molecular mass as well as glycan structure. In contrast, the SG had no effect on the FXIIa inhibition by C1-INH, but structure-dependently modulated the activity of FXIIa. Among the tested SG, β-1,3-glucan sulfates with a Mr ≤ 10 000 were identified as most promising lead candidates for the development of a glycan-based C1-INH amplifier. In conclusion, the obtained information on structural characteristics of SG favoring C1-INH potentiation represent an useful elementary basis for the development of compounds improving the potency of C1-INH in diseases and clinical situations characterized by inappropriate activation of complement and contact system.

Investigation of the anticoagulant and antithrombotic effects of chlorogenic acid.

Thrombosis is a leading cause of morbidity and mortality throughout the world. Thrombolytic agents are important for both the prevention and treatment of thrombosis. Fibrin clot and turbidity assays revealed that it was able to inhibit the formation of fibrin clot. Chlorogenic acid degraded blood clot and inhibited the enzymatic activity of procoagulant proteases, thrombin, activated factor X (FXa), and activated factor XIII (FXIIIa). Chlorogenic acid was found to delay activated partial thromboplastin time, prothrombin time, and thrombin time. PFA-100 assays showed that it prolonged the closure time of citrated whole human blood. It demonstrated the antithrombotic effect in collagen and epinephrine-induced acute thromboembolism mice model. These antithrombotic profiles together with its anticoagulant and platelet disaggregation properties, and lack of toxicity to NIH-3T3 and 3T3-L1 cells, make it a potential agent for thrombotic treatment and prevention.

Leptospira Immunoglobulin-Like Protein B (LigB) Binds to Both the C-Terminal 23 Amino Acids of Fibrinogen αC Domain and Factor XIII: Insight into the Mechanism of LigB-Mediated Blockage of Fibrinogen α Chain Cross-Linking.

The coagulation system provides a primitive but effective defense against hemorrhage. Soluble fibrinogen (Fg) monomers, composed of α, β and γ chains, are recruited to provide structural support for the formation of a hemostatic plug. Fg binds to platelets and is processed into a cross-linked fibrin polymer by the enzymatic clotting factors, thrombin and Factor XIII (FXIII). The newly formed fibrin-platelet clot can act as barrier to protect against pathogens from entering the bloodstream. Further, injuries caused by bacterial infections can be confined to the initial wound site. Many pathogenic bacteria have Fg-binding adhesins that can circumvent the coagulation pathway and allow the bacteria to sidestep containment. Fg expression is upregulated during lung infection providing an attachment surface for bacteria with the ability to produce Fg-binding adhesins. Fg binding by leptospira might play a crucial factor in Leptospira-associated pulmonary hemorrhage, the main factor contributing to lethality in severe cases of leptospirosis. The 12th domain of Leptospira immunoglobulin-like protein B (LigB12), a leptospiral adhesin, interacts with the C-terminus of FgαC (FgαCC). In this study, the binding site for LigB12 was mapped to the final 23 amino acids at the C-terminal end of FgαCC (FgαCC8). The association of FgαCC8 with LigB12 (ELISA, KD = 0.76 μM; SPR, KD = 0.96 μM) was reduced by mutations of both charged residues (R608, R611 and H614 from FgαCC8; D1061 from LigB12) and hydrophobic residues (I613 from FgαCC8; F1054 and A1065 from LigB12). Additionally, LigB12 bound strongly to FXIII and also inhibited fibrin formation, suggesting that LigB can disrupt coagulation by suppressing FXIII activity. Here, the detailed binding mechanism of a leptospiral adhesin to a host hemostatic factor is characterized for the first time and should provide better insight into the pathogenesis of leptospirosis.

Association between the C46T polymorphism of coagulation factor Ⅻ gene and the involvement of factor Ⅻ activity in patients with unexplained recurrent spontaneous abortion

To explore the association between the C46T polymorphism of coagulation factor Ⅻ (FⅫ) gene and the involvement of FⅫ activity (FⅫ:C) in patients with unexplained recurrent spontaneous abortion (URSA), and to elucidate its role in the pathogenesis of URSA. This study included 203 patients with URSA (URSA group) and 171 healthy women with at least one child and no history of infertility or miscarriage (control group) in the southern area of Zhejiang Province. The C46T polymorphism of the FⅫ gene was analyzed with matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS) in all subjects. The values of prothrombin time, activated partial thromboplastin time (APTT), fibrinogen, FⅫ:C and other coagulant parameters were determined. The frequency distribution of the wild-type (CC), heterozygote (CT), homozygote (TT) genotypes and C and T alleles were compared between the patients and controls. A comprehensive analysis of association was conducted between C46T genotypes and the FⅫ:C levels in URSA patients. The CC, CT, TT genotypes of the FⅫ gene were observed in 7 (3.4%, 7/203), 83 (40.9%, 83/203) and 113 (55.7%, 113/203) patients with URSA versus 7 (4.1%, 7/171), 46 (26.9%, 46/171) and 118 (69.0%, 118/171) controls. The frequency of CT in the patients with URSA was significantly higher than that in controls, but the frequency of TT in the patients was lower than that in controls (χ(2)=7.939, OR=1.884, 95%CI: 1.210-2.935, P<0.05). The frequencies of allele C and allele T were observed in 97 (23.9%, 97/406) and 309 (76.1%, 309/406) patients with URSA versus 60 (17.5%, 60/342) and 282 (82.5%, 282/342) controls. The distribution frequency of allele T in URSA group was lower than that in control group (χ(2)=4.510, OR=1.475, 95%CI: 1.029-2.115, P<0.05). The FⅫ: C levels in the patients were (102±13)% in CC genotype, (78±11)% in CT genotype and (59±9)% in TT genotype, respectively. The differences of the FⅫ: C levels between the CC and CT, CT and TT, CC and TT genotypes in the patients were significant (all P<0.05). The low level of FⅫ:C maybe result from the T allele of the FⅫ gene in URSA patients. The CT genotype might be relative to the pathogenesis of URSA in a Chinese Han female population from the southern area of Zhejiang province.

Coagulation during elective neurosurgery with hydroxyethyl starch fluid therapy: an observational study with thromboelastometry, fibrinogen and factor XIII

BackgroundSeveral studies have described hypercoagulability in neurosurgery with craniotomy for brain tumor resection. In this study, hydroxyethyl starch (HES) 130/0.42 was used for hemodynamic stabilization and initial blood loss replacement. HES can induce coagulopathy with thromboelastographic signs of decreased clot strength. The aim of this study was to prospectively describe perioperative changes in coagulation during elective craniotomy for brain tumor resection with the present fluid regimen.MethodsForty patients were included. Perioperative whole-blood samples were collected for EXTEM and FIBTEM assays on rotational thromboelastometry (ROTEM) and plasma fibrinogen analysis immediately before surgery, after 1 L of HES infusion, at the end of surgery and in the morning after surgery. Factor (F)XIII activity, thrombin-antithrombin complex (TAT) and plasmin-α2-antiplasmin complex (PAP) were analysed in the 25 patients receiving ≥1 L of HES.ResultsMost patients (37 of 40) received HES infusion (0.5–2 L) during surgery. Preoperative ROTEM clot formation/structure, plasma fibrinogen and FXIII levels were generally within normal range but approached a hypocoagulant state during and at end of surgery. ROTEM variables and fibrinogen levels, but not FXIII, returned to baseline levels in the morning after surgery. Low perioperative fibrinogen levels were common. TAT levels were increased during and after surgery. PAP levels mostly remained within the reference ranges, not indicating excessive fibrinolysis. There were no differences in ROTEM results and fibrinogen levels in patients receiving <1 L HES and ≥1 L HES.ConclusionsOnly the increased TAT levels indicated an intra- and postoperative activation of coagulation. On the contrary, all other variables deteriorated towards hypocoagulation but were mainly normalized in the morning after surgery. Although this might be an effect of colloid-induced coagulopathy, we found no dose-dependent effect of HES. The unactivated fibrinolysis indicates that prophylactic use of tranexamic acid does not seem warranted under normal circumstances in elective neurosurgery. Individualized fluid therapy and coagulation factor substitution is of interest for future studies.

Revisiting the mechanism of coagulation factor XIII activation and regulation from a structure/functional perspective.

The activation and regulation of coagulation Factor XIII (FXIII) protein has been the subject of active research for the past three decades. Although discrete evidence exists on various aspects of FXIII activation and regulation a combinatorial structure/functional view in this regard is lacking. In this study, we present results of a structure/function study of the functional chain of events for FXIII. Our study shows how subtle chronological submolecular changes within calcium binding sites can bring about the detailed transformation of the zymogenic FXIII to its activated form especially in the context of FXIIIA and FXIIIB subunit interactions. We demonstrate what aspects of FXIII are important for the stabilization (first calcium binding site) of its zymogenic form and the possible modes of deactivation (thrombin mediated secondary cleavage) of the activated form. Our study for the first time provides a structural outlook of the FXIIIA2B2 heterotetramer assembly, its association and dissociation. The FXIIIB subunits regulatory role in the overall process has also been elaborated upon. In summary, this study provides detailed structural insight into the mechanisms of FXIII activation and regulation that can be used as a template for the development of future highly specific therapeutic inhibitors targeting FXIII in pathological conditions like thrombosis.