Factor XIII and Endothelial Dysfunction in Patients with Systemic Sclerosis.

Systemic sclerosis (SSc, scleroderma) is a severe autoimmune connective tissue disease which affects the skin and internal organs. There has been evidence that coagulation factor XIII (FXIII) has a positive impact on clinical results in patients with SSc. In a single-center cohort study, we investigated the relationship between coagulation FXIII, endothelial dysfunction, and skin infection in SSc. Fifty-six patients could be included and were divided into two groups (with and without scleroderma). Markers of inflammation, coagulation, and endothelial dysfunction like C-reactive protein, leucocytes, fibrinogen, FVIII, VWF-Ag (von Willebrand factor antigen), D-dimers, and vascular endothelial growth factor were analyzed as well as MRSS (modified Rodnan skin scores) data were evaluated. Reduced daily activities were evaluated by the Scleroderma Health Assessment Questionnaire (SHAQ). There were no significant correlations between FXIII activity, MRSS, and SHAQ score. There were correlations between FXIII activity and Raynaud's phenomenon-related symptoms and a weak but not significant positive correlation with the level of pain. A significant correlation between VWF-Ag and lung-associated complaints (n = 56; p = 0.41, p < 0.0001) was found. Moreover, the study showed a correlation between VWF-Ag and MRSS (r [N = 48] = 0.4, p = 0.01), which means that higher VWF-Ag levels come along with more severe skin involvement. A trend toward a negative correlation between FXIII activity and VWF-Ag as marker of endothelial dysfunction was found (r [N = 56] = - 0.20, p = 0.15). In our cohort, there is no FXIII deficiency in patients with SSc. FXIII might have a role in improving cutaneous manifestations indirectly by means of a moderating influence on endothelial dysfunction. Further clinical evaluation is needed.

Severe high-molecular-weight kininogen deficiency: clinical characteristics, deficiency-causing KNG1 variants, and estimated prevalence.

BACKGROUND: Severe high-molecular-weight kininogen (HK) deficiency is a poorly studied autosomal recessive contact system defect caused by pathogenic, biallelic KNG1 variants. AIM: We performed the first comprehensive analysis of diagnostic, clinical, genetic, and epidemiological aspects of HK deficiency. METHODS: We collected clinical information and blood samples from a newly detected HK-deficient individual and from published cases identified by a systematic literature review. Activity and antigen levels of coagulation factors were determined. Genetic analyses of KNG1 and KLKB1 were performed by Sanger sequencing. The frequency of HK deficiency was estimated considering truncating KNG1 variants from GnomAD. RESULTS: We identified 48 cases of severe HK deficiency (41 families), of these 47 have been previously published (n = 19 from gray literature). We genotyped 3 cases and critically appraised 10 studies with genetic data. Ten HK deficiency-causing variants (one new) were identified. All of them were truncating mutations, whereas the only known HK amino acid substitution with a relevant phenotype instead causes hereditary angioedema. Conservative estimates suggest an overall prevalence of severe HK deficiency of approximately one case per 8 million population, slightly higher in Africans. Individuals with HK deficiency appeared asymptomatic and had decreased levels of prekallikrein and factor XI, which could lead to misdiagnosis. CONCLUSION: HK deficiency is a rare condition with only few known pathogenic variants. It has an apparently good prognosis but is prone to misdiagnosis. Our understanding of its clinical implications is still limited, and an international prekallikrein and HK deficiency registry is being established to fill this knowledge gap.

Endothelial dysfunction and atherosclerosis related miRNA-expression in patients with haemophilia.

INTRODUCTION: Elevated markers of endothelial dysfunction and inflammation indicate worse endothelial function in the aging haemophilia population. MicroRNAs (miRNAs) regulate gene expression post-transcriptionally. Several miRNAs have been shown to be involved in the process of endothelial dysfunction and atherosclerosis. AIM: The aim of this study was to determine the underlying molecular pathways of endothelial dysfunction and inflammation in haemophilia patients. METHODS: A total of 25 patients with severe or moderate haemophilia A (20 patients) or B (5 patients), 14 controls and 18 patients with coronary artery disease (CAD) after myocardial infarction were included in this study. Expression of miRNA-126, -155, -222, -1, -let7a, -21 and -197 were analysed using a real time polymerase chain reaction. Network-based visualisation and analysis of the miRNA-target interactions were performed using the MicroRNA ENrichment TURned NETwork (MIENTURNET). RESULTS: Expression of miRNA-126 (p < .05) and miRNA-let7a (p < .05) were significantly higher in CAD patients compared to haemophilia patients and controls. MiRNA-21 (p < .05) was significantly elevated in CAD patients compared to controls. MiRNA-155 (p < .05), miRNA-1 (p < .05) and miRNA-197 (p < .05) were significantly higher expressed in CAD and haemophilia patients compared to controls and showed a strong correlation with increased levels of interleukin-6 (IL-6) and soluble intercellular adhesion molecule-1 (sICAM-1). The network analysis revealed interactions in the cytokine signalling, focal adhesion and VEGFA-VEGFR2 pathway (Vascular endothelial growth factor, -receptor). CONCLUSION: This study characterises miRNA expression in haemophilia patients in comparison to CAD patients and healthy controls. The results imply comparable biological processes in CAD and haemophilia patients.

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.

Endothelial Function in Patients With Von Willebrand Disease.

In patients with von Willebrand disease (vWD) the interest in age-related comorbidities has grown, because the life expectancy of these patients has increased. The research question of this study was whether patients with vWD show a different endothelial function compared to the general population. A total of 37 patients with type 1 (n = 23), type 2 (n = 10) and type 3 (n = 4) vWD, 14 controls and 38 patients with coronary artery disease (CAD) were included in this study. Five markers of endothelial dysfunction (MOED) were determined. Moreover, the endothelial function was examined using the Itamar Endo-PAT. The reactive hyperemia index (RHI) was calculated from the results. The markers soluble intercellular adhesion molecule-1 (p = 0.171), P-Selectin (p = 0.512), interleukin-6 (p = 0.734) and monocyte chemoattractant protein-1 (p = 0.761) showed higher levels in patients with vWD, but were not significantly different compared to the control group. RHI was impaired in CAD-patients (1.855), whereas vWD patients had mean results of 1.870 and controls 2.112 (p = 0.367). In this study, the endothelial function measurements of patients with von Willebrand disease were not significantly different compared to healthy controls.

Endothelial Function in Patients with Severe and Moderate Haemophilia A and B.

The life expectancy of patients with haemophilia has increased and therefore the interest in age-related comorbidities has grown. The aim of this study was to determine whether haemophilia patients have a different endothelial function compared with the general population. A total of 26 patients with severe or moderate haemophilia A or B, 14 controls and 36 patients with coronary artery disease (CAD) were included in this study. Five markers of endothelial dysfunction (MOEDs) were determined. Moreover, the endothelial function was examined using the Itamar Endo-PAT, and the reactive hyperemia index (RHI) was calculated from the results. The MOEDs soluble intercellular adhesion molecule-1 (p = 0.0095) and interleukin-6 (p = 0.010) were significantly higher for patients with haemophilia compared with the control group. The presence of increased adhesion molecule levels and low-grade inflammation is suggestive of a decreased endothelial function. RHI is impaired in CAD patients (1.862), whereas haemophilia patients have an RHI of 1.958 in comparison with 2.112 in controls (p = 0.127). Therefore, laboratory and functional measurements imply a possible higher risk for CAD in haemophilia patients.

Is gingival bleeding a symptom of patients with type 1 von Willebrand disease? A case-control study.

BACKGROUND: Von Willebrand disease (VWD) is the most common inherent bleeding disorder resulting in prolonged bleeding time. Gingival bleeding is a frequently reported symptom of VWD. However, gingival bleeding is also known as a leading symptom of plaque-induced gingivitis and untreated periodontal disease. Gingival bleeding in VWD patients (VWD) may be triggered by gingival inflammation and not a genuine symptom. Thus, this study evaluated whether type 1 VWD determines an increased susceptibility to gingival bleeding in response to the oral biofilm. METHODS: Fifty cases and 40 controls were examined haematologically (VWF antigen, VWF Ristocetin cofactor, factor VIII activity) and periodontally [Gingival Bleeding Index (GBI), bleeding on probing (BOP), Plaque Control Record (PCR), periodontal inflamed surface area (PISA), vertical probing attachment level]. RESULTS: GBI was significantly higher in controls (12.2%) than in VWD (10%). The study failed to find a significant difference regarding BOP between VWD (17%) and controls (17.2%). Multiple regressions identified PCR and PISA to be associated with GBI and BOP. VWD was negatively associated with GBI. Smoking and number of remaining teeth was negatively associated with BOP. CONCLUSION: VWD is not associated with a more pronounced inflammatory response to the oral biofilm in terms of GBI and BOP.

Impact of the type of SERPINC1 mutation and subtype of antithrombin deficiency on the thrombotic phenotype in hereditary antithrombin deficiency.

Mutations in the antithrombin (AT) gene can impair the capacity of AT to bind heparin (AT deficiency type IIHBS), its target proteases such as thrombin (type IIRS), or both (type IIPE). Type II AT deficiencies are almost exclusively caused by missense mutations, whereas type I AT deficiency can originate from missense or null mutations. In a retrospective cohort study, we investigated the impact of the type of mutation and type of AT deficiency on the manifestation of thromboembolic events in 377 patients with hereditary AT deficiencies (133 from our own cohort, 244 reported in the literature). Carriers of missense mutations showed a lower risk of venous thromboembolism (VTE) than those of null mutations (adjusted hazard ratio [HR] 0.39, 95% confidence interval [CI] 0.27-0.58, p<0.001), and the risk of VTE was significantly decreased among patients with type IIHBS AT deficiency compared to patients with other types of AT deficiency (HR 0.23, 95%CI 0.13-0.41, p<0.001). The risk of pulmonary embolism complicating deep-vein thrombosis was lower in all type II AT deficiencies compared to type I AT deficiency (relative risk 0.69, 95%CI 0.56-0.84). By contrast, the risk of arterial thromboembolism tended to be higher in carriers of missense mutations than in those with null mutations (HR 6.08-fold, 95%CI 0.74-49.81, p=0.093) and was 5.9-fold increased (95%CI 1.22-28.62, p=0.028) in type IIHBS versus other types of AT deficiency. Our data indicate that the type of inherited AT defect modulates not only the risk of thromboembolism but also the localisation and encourage further studies to unravel this phenomenon.

No detection of the retrovirus xenotropic murine leukemia virus-related virus in individuals with hemophilia.

BACKGROUND: Xenotropic murine leukemia virus-related virus (XMRV) is a retrovirus that has recently been related to prostate cancers and chronic fatigue syndrome. Since other human-pathogenic retroviruses, such as HIV, human T-lymphotropic virus type I (HTLV-I) and -II, are known blood-transmitted pathogens, XMRV might present another hazard associated with products derived from in vitro cultures of human or animal origin, or blood component-based therapeutics. Here, we investigated whether XMRV was transmitted to individuals with hemophilia and frequent exposure to plasma-derived or recombinant clotting factors. METHODS: We used highly sensitive real-time PCR to test plasma samples from 127 consecutive individuals with hemophilia who consulted our hemophilia center either for treatment or for a standard check-up. RESULTS: From the 127 hemophiliacs, 80 had prior contact to persons with either hepatitis B (n = 30), hepatitis C (n = 74) and/or HIV (n = 21), and 30 were currently being treated with plasma-derived and 97 with recombinant factor concentrates. None of the individuals tested positive for XMRV. CONCLUSIONS: Independent of the ongoing discussion on whether the positive XMRV testing in initial reports was a result of reagent, sample, or tissue contamination, and whether XMRV is a real threat or a testing artifact, our data suggest that XMRV might not play an important role for hemophiliacs.

Engineered factor IX variants bypass FVIII and correct hemophilia A phenotype in mice.

The complex of the serine protease factor IX (FIX) and its cofactor, factor VIII (FVIII), is crucial for propagation of the intrinsic coagulation cascade. Absence of either factor leads to hemophilia, a disabling disorder marked by excessive hemorrhage after minor trauma. FVIII is the more commonly affected protein, either by X-chromosomal gene mutations or in autoimmune-mediated acquired hemophilia. Whereas substitution of FVIII is the mainstay of hemophilia A therapy, treatment of patients with inhibitory Abs remains challenging. In the present study, we report the development of FIX variants that can propagate the intrinsic coagulation cascade in the absence of FVIII. FIX variants were expressed in FVIII-knockout (FVIII-KO) mice using a nonviral gene-transfer system. Expression of the variants shortened clotting times, reduced blood loss after tail-clip assay, and reinstalled clot formation, as tested by in vivo imaging of laser-induced vessel injury. In addition, we confirmed the therapeutic efficacy of FIX variants in mice with inhibitory Abs against FVIII. Further, mice tolerant to wild-type human FIX did not develop immune responses against the protein variants. Our results therefore indicate the feasibility of using variants of FIX to bypass FVIII as a novel treatment approach in hemophilia with and without neutralizing FVIII Abs.

Comprehensive treatment of periodontitis in patients with von Willebrand disease.

BACKGROUND: Hemophilia A and B and von Willebrand disease (VWD) belong to the most frequent congenital coagulation disorders and are a significant problem in patients who require periodontal therapy or tooth extraction. These patients need specialist management because even minor invasive procedures can precipitate a prolonged bleeding episode. However, although dental care presents major challenges in these patients, only a few studies are available. METHODS: In this case series, the comprehensive periodontal treatment of four patients with hemorrhagic disorders (VWD type I and mild hemophilia B) is described. There was a close collaboration between the periodontist and the hematologist: all patients were scheduled for premedication with desmopressin and other pharmaceuticals at the hematologist's office. After one session of scaling and root planing was performed in all patients, local agents such as tranexamic acid were used. In the course of periodontal therapy, access-flap surgery was performed in one of the four patients. RESULTS: Before treatment, the rates of probing depths (PDs) of 4 to 6 mm (20% to 57%) or ≥ 7 mm (2% to 20%) were high. Three months after treatment, the rates of PDs of 4 to 6 mm (5% to 42%) or ≥ 7 mm (0% to 2%) decreased significantly in all patients. Attachment gains were also observed. A secondary hemorrhage did not occur in any of the patients, and wound healing proceeded without any complications. CONCLUSION: Effective periodontal treatment can be provided to patients with hemorrhagic disorders with the combined efforts of the periodontist and hematologist.

Effect of freezing method and storage at -20 degrees C and -70 degrees C on prothrombin time, aPTT and plasma fibrinogen levels.

Clinical and epidemiological trials often involve central laboratory analyses of coagulation tests, including fibrinogen, which requires freezing of the plasma samples. Although rapid freezing by immersion of sample tubes in liquid nitrogen and storage at -70 degrees C is recommended, plasma samples are often transferred directly to the storage compartments, and stored at -20 degrees C. Prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen using a kinetic fibrinogen assay, PT-derived fibrinogen, and an immunoassay were measured in fresh plasma samples from 16 healthy blood donors. In addition, four sets of aliquots were prepared. Set A was transferred directly to a -20 degrees C storage compartment, set B was first snap-frozen in liquid nitrogen and then transferred to the -20 degrees C compartment. Set C was transferred directly to a -70 degrees C freezer, set D was first snap-frozen in liquid nitrogen and then stored at -70 degrees C. Aliquots were thawed after one, two, three and four months storage and laboratory assays repeated. PT and aPTT were strongly influenced by freezing and storage. In contrast, freezing had little effect on fibrinogen levels. Differences were below three percent for all variants. Changes were smaller for samples stored at -70 degrees C compared to -20 degrees C, and for snap-frozen compared to not snap-frozen samples. Frozen and thawed samples generated slightly higher fibrinogen levels compared to fresh samples. Prothrombin time and aPTT should be measured in fresh samples, since freezing has an inconstant and unpredictable effect on the results. In contrast, freezing and storage has little effect on results of fibrinogen assays. A limitation of the study is that only samples from healthy blood donors were used. Plasma samples with abnormal fibrinogen concentration, or with abnormal concentrations of coagulation factors might behave differently.