Severe fletcher factor (plasma prekallikrein) deficiency with partial deficiency of hageman factor (factor xii): Report of a case with observation on in vivo and in vitro leukocyte chemotaxis

A case of cross-reacting material-negative Fletcher trait with additional partial deficiency of Hageman factor (HF, Factor XII) is described. Although the patient presented with a recent history of frequent epistaxis, he had no other personal or family history of a tendency toward bleeding or infection. Similar to other cases of Fletcher trait, his plasma showed a markedly prolonged partial thromboplastin time which could be corrected by prolonged incubation with the surface-activator kaolin. Surface-induced fibrinolysis, amidolysis of α-N-benzoyl-proline-L -phenylalanine-L -arginine-p-nitro- anilide, and cold-promoted enhancement of factor VII activity, reactions requiring the presence in the plasma of Fletcher factor (pre-kallikrein), in addition to Hageman factor and Fitzgerald factor (high-molecular weight kininogen), were also defective. In vivo chemotaxis of polymorphonuclear leukocytes and monocytes (Rebuck's skin window technique) in response to skin abrasions was defective, but was normal when diphtheria-tetanus toxoid was also applied. In vitro leukocyte chemotaxis (Boyden chamber technique) in response to normal or patient's own serum activated with zymosan was normal. Together with previous observations that kallikrein generated chemotactic activity, possibly via activation of C5, the present observations suggest that prekallikrein activation may be important for in vivo leukocyte chemotactic response to skin abrasion. The inheritance of Fletcher trait in this patient is unclear. Although the father was an apparent heterozygote, the mother was completely normal for Fletcher factor procoagulant activity and antigen. The mild Hageman factor deficiency in the patient did not contribute significantly to the plasma defects described and was likely inherited from the father who had a low HF procoagulant activity.     

Factor XII: a novel target for safe prevention of thrombosis and inflammation

Plasma protein factor XII (FXII) activates the procoagulant and proinflammatory contact system that drives both the kallikrein–kinin system and the intrinsic pathway of coagulation. When zymogen FXII comes into contact with negatively charged surfaces, it auto‐activates to the serine proteaseactivated FXII (FXIIa). Recently, various in vivo activators of FXII have been identified including heparin, misfolded protein aggregates, polyphosphate and nucleic acids. Murine models have established a central role of FXII in arterial and venous thrombosis. Despite its central function in thrombosis, deficiency in FXII does not impair haemostasis in animals and humans. In a preclinical cardiopulmonary bypass system in large animals, the FXIIa‐blocking antibody 3F7 prevented thrombosis; however, in contrast to traditional anticoagulants, bleeding was not increased. In addition to its function in thrombosis, FXIIa initiates formation of the inflammatory mediator bradykinin. This mediator increases vascular leak, causes vasodilation, and induces chemotaxis with implications for septic, anaphylactic and allergic disease states. Therefore, targeting FXIIa appears to be a promising strategy for thromboprotection without associated bleeding risks but with anti‐inflammatory properties.     

Genetic analysis of a hereditary factor XII deficiency pedigree of a consanguineous marriage due to a homozygous F12 gene mutation: Gly341Arg

Objective and Importance: To study the gene mutations of factor XII (FXII) in a Chinese family of consanguineous marriage with FXII deficiency and illuminate the possible molecular pathogenic mechanism. It will contribute to our comprehension of the pathogenesis of the disease.

Clinical presentation: The proband was a 26-year-old Chinese pregnant woman who was discovered, in a pregnancy test, with a prolonged activated partial thromboplastin time (APTT) at 61.6s (reference range, 29.0–43.0s).

Techniques: The FXII activity (FXII:C) and FXII antigen (FXII:Ag) were tested with clotting assay and ELISA, respectively. The FXII gene (F12) was ampli?ed by PCR with direct sequencing. A ClustalX-2.1-win and four online bioinformatics software (PolyPhen-2, PROVEAN, SIFT, and Mutation Taster) were used to study the conservatism and harm of the mutation. The reference range of each test indicator in our laboratory was established with 150 healthy subjects.

Conclusion headings: The FXII:C and FXII:Ag of the proband were 12% and 10% (normal range, 72–113%), respectively. Gene sequencing detected a homozygous c.1078G?>?A point mutation in exon 10 resulting in a substitution of glycine 341 by arginine (Gly341Arg) in the proline-rich domain of FXII. Family study showed that her elder brother had the same phenotype and genotype with her. In addition, there were another six heterozygous members in her family. Both conservatism and bioinformatics results indicated the mutation probably had affected the function of the protein. We thought the Gly341Arg mutation was responsible for the decreased activity of FXII:C and FXII:Ag. And in vitro expression experiment is performed to elucidate the precise pathological mechanism of the mutation.     

Genetic Variant CFH rs6677604 Might Play a Protective Role in lupus Nephritis

BackgroundThis study aimed to explore the associations between the complement factor H (CFH) rs6677604 and clinico-pathological characteristics of lupus nephritis.Materials and methodsA total of 188 patients with lupus nephritis with complete clinico-pathological data were enrolled and genotyping of CFH rs6677604 was conducted by TaqMan SNP genotyping assays. Patients were divided into two groups by rs6677604-AA/AG or -GG, and the clinico-pathological features between the two groups were further compared.ResultsWe found that patients with rs6677604-AA/AG presented with lower prevalence of anti-dsDNA antibody (12/24 [50.0%] vs 121/164 [73.8%], P = 0.028), higher level of plasma C3a (2642.96 ± 1575.05 vs 1640.01 ± 1209.40, ng/ml, P = 0.024), and a tendency for higher level of plasma CFH (505.76 ± 169.28 vs 397.67 ± 179.11, μg/ml, P = 0.087). Patients with rs6677604-AA/AG had milder renal histopathological features, including total activity indices score (4.5[0, 13] vs 8[0, 19], P = 0.013), endocapillary hypercellularity (1.5[0, 3] vs 3[0, 3], P = 0.013), sub-endothelial hyaline deposits (0.5[0, 3] vs 1[0,3], P = 0.021), glomerular leukocyte infiltration (0.5[0, 1] vs 1[0, 12], P = 0.023) and tubular atrophy (1[0, 1] vs 1[0, 3], P = 0.027) than those with rs6677604-GG, which was further confirmed by the stratified analysis. The rs6677604-A was not a risk factor for patients’ renal outcomes (hazard ratio=0.898; 95% CI: 0.264–3.059, P = 0.863).ConclusionsThe rs6677604-A genotype in CFH was associated with milder renal pathological features in lupus nephritis, and its protective effect on the pathogenesis of the disease remained to be elucidated.     

Prediction of severe illness due to COVID-19 based on an analysis of initial Fibrinogen to Albumin Ratio and Platelet count

Abstract

Concomitant coagulation disorder can occur in severe patients withCOVID-19, but in-depth studies are limited. This study aimed to describe the parameters of coagulation function of patients with COVID-19 and reveal the risk factors of developing severe disease. This study retrospectively analyzed 113patients with SARS-CoV-2 infection in Taizhou Public Health Center. Clinical characteristics and indexes of coagulation function were collected. A multivariate Cox analysis was performed to identify potential biomarkers for predicting disease progression. Based on the results of multivariate Cox analysis, a Nomogram was built and the predictive accuracy was evaluated through the calibration curve, decision curve, clinical impact curve, and Kaplan–Meier analysis. Sensitivity, specificity, predictive values were calculated to assess the clinical value. The data showed that Fibrinogen, FAR, and D-dimer were higher in the severe patients, while PLTcount, Alb were much lower. Multivariate Cox analysis revealed that FAR and PLT count were independent risk factors for disease progression. The optimal cutoff values for FAR and PLT count were 0.0883 and 135*10⁹/L, respectively. The C-index [0.712 (95% CI = 0.610–0.814)], decision curve, clinical impact curve showed that Nomogram could be used to predict the disease progression. In addition, the Kaplan–Meier analysis revealed that potential risk decreased in patients with FAR<0.0883 and PLT count>135*10⁹/L.The model showed a good negative predictive value [(0.9474 (95%CI = 0.845–0.986)].This study revealed that FAR and PLT count were independent risk factors for severe illness and the severity of COVID-19 might be excluded when FAR<0.0883 and PLT count>135*10⁹/L.     

Activation of Coagulation and Fibrinolysis During Coronary Artery Bypass Grafting: A Comparison Between On-Pump and Off-Pump Techniques

Coronary artery bypass grafting (CABG) with cardiopulmonary bypass (CPB) is associated with intense activation of hemostatic mechanisms. But the precise knowledge of the effects of eliminating CPB in patients undergoing off-pump coronary artery bypass grafting (CABG) are not well established. The present study was carried out to compare and document the changes in selected coagulation and fibrinolysis variables in patients undergoing on-pump and off-pump CABG (OPCAB). A total of 42 patients of on-pump and 31 patients of off-pump CABG were selected for the study. Platelet count, prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), Fibrinogen and D-dimer levels were measured immediately, 24 h and 7 days after operation and compared with the baseline preoperative values. Statistical analysis was done by mixed ANOVA for repeated measures and Post-hoc tests using the Bonferroni correction, Chi square and unpaired t test. All the parameters were significantly changed (P < 0.05) with the time. Platelet counts, fibrinogen and D-dimer levels were significantly different between on-pump and off-pump CABG patients on immediate and 24 h postoperative period and attained almost same level after 7 days of operation. Fibrinogen level and platelet counts were increased after a sharp fall in the immediate post-operative period whereas D-dimer levels were persistently increased with a sharp peak of rise in the immediate post-operative period in on-pump group. On-pump surgery was associated with excessive fibrinolytic activity immediately after operation. The off-pump group demonstrated less activation of coagulation and fibrinolysis and delayed postoperative response that became almost equal to the on-pump group in the later postoperative period.     

反式载脂蛋白A1模拟肽R-D4F对内皮祖细胞黏附及迁移功能的影响

目的探讨反式载脂蛋白A1模拟肽R-D4F对人内皮祖细胞黏附及迁移功能的影响。方法取新鲜人脐静脉血,采用密度梯法从中分离、诱导培养人内皮祖细胞,并使用Dil-ac-LDL和FITC-UEA-1染料进行双荧光染色以及流式细胞术进行鉴定。分别给于牛血清白蛋白、顺式D-4F(50μg/ml)及R-D4F(5~50μg/ml)处理后,采用黏附能力测定实验及Transwell小室观察内皮祖细胞黏附及迁移能力的变化。结果与牛血清白蛋白对照组相比,D-4F显著增强内皮祖细胞的黏附及迁移功能。相似的是,R-D4F呈剂量依赖性地增强内皮祖细胞的黏附与迁移。结论 R-D4F具有与D-4F相似的生物学作用,可促进人内皮祖细胞的黏附及迁移。     

Enhancing Effect of Hydroxyurea on Hb F in Sickle Cell Disease: Ten-Year Egyptian Experience

Patients with sickle cell disease experience hemolytic anemia and vaso-occlusions that result in pain, organ injury, and premature mortality. Several prospective studies have verified the efficacy and tolerability of hydroxyurea (HU), and demonstrated its efficacy in reducing painful vaso-occlusive crises (VOCs) in addition to its ability to increase Hb F levels. We aimed to evaluate the long-term effects of HU therapy on Hb F and assess its long term efficacy and safety in sickle cell disease patients. A retrospective study on 60 sickle cell disease patients was conducted. We studied the laboratory changes, frequency of VOCs per year, frequency of hospital admisions per year and number of transfusions per year, both before and after HU therapy. The follow-up period was 4 to 120 months. Hb F levels after HU therapy positively correlated with the duration of HU therapy, baseline Hb F levels and baseline total hemoglobin (Hb) (r?=?0.4, p?=?0.04; r?=?0.45, p?=?0.001; r?=?0.5, p?=?0.019, respectively) and inversely correlated with baseline total leucocyte count (r?=?–0.33, p?=?0.034). Hydroxyurea therapy was associated with an increase in the total Hb and mean corpuscular volume (MCV) (p?=?0.009, p?=?0.000; respectively) and with a decrease in total leucocyte count, platelet count and reticulocyte count (p?=?0.00, p?=?0.03, p?=?0.02, respectively). Moreover, a significant reduction in the frequency of VOCs, transfusion frequency and hospital admissions per year after HU therapy was shown in the studied subjects. Hydroxyurea induced an increase in Hb F level, which was maintained over time and was associated with clinical efficacy and acceptable safety.     

抑制性消减杂交技术克隆和筛选丙型肝炎病毒F蛋白的反式调节基因

目的 构建丙型肝炎病毒（HCV）F蛋白反式激活相关基因差异表达的差异cDNA，克隆HCV-F蛋白反式激活相关基因。方法 以HCV-F表达质粒pcDNA3．1（-）-F转染HepG2细胞，以空载体pcDNA3．1（-）为对照；制备转染后的细胞裂解液，从中提取mRNA并合成cDNA，经RsaI酶切后将实验组cDNA分成两组，分别与两种不同的接头衔接，再与对照组cDNA进行两次消减杂交及两次抑制性聚合酶链反应，将产物与T／A载体连接，构建cDNA消减文库，并转染大肠杆菌进行文库扩增，随机挑选克隆聚合酶链反应后进行测序及同源性分析。结果 成功构建人HcVF蛋白反式激活相关基因差异表达的cDNA。扩增后得到56个200～1000bP插入片段的克隆，随机挑选其中28个插入片段测序，并通过生物信息学分析获得其全长基因序列，结果共获得19种编码基因，其中2个为未知功能的新基因。结论 筛选到的cDNA全长序列，包括一些与细胞生长调节、物质代谢和细胞凋亡密切相关的蛋白编码基因。     

Resistance to activated protein C is a risk factor for pregnancy-related venous thrombosis in the absence of the F5 rs6025 (factor V Leiden) polymorphism

Resistance to activated protein C (aPC) is most commonly due to the F5 rs6025 (factor V Leiden) polymorphism, which increases the risk of venous thrombosis. In the present population-based study of 313 cases and 353 controls, we investigated whether reduced sensitivity to aPC was associated with a history of pregnancy-related venous thrombosis. Calibrated automated thrombography was used to determine the sensitivity to aPC, and normalized aPC sensitivity ratio (n-aPC-sr) was calculated. Pregnant women and women using oral contraceptives and/or anticoagulants were excluded due to the effect on the n-aPC-sr. In women without the F5 rs6025 polymorphism, free tissue factor pathway inhibitor (TFPI), free protein S and protein C activity were associated with n-aPC-sr. Unadjusted odds ratio for venous thrombosis for women with n-aPC-sr in the 4th quartile as compared with n-aPC-sr below the 4th quartile was 2·4 (95% confidence interval 1·7-3·6). Adjusting for free protein S, free TFPI and age did not influence the odds ratios. Also in carriers of the F5 rs6025 polymorphism the risk for venous thrombosis was increased for women with higher n-aPC-sr. Our findings substantiate the importance of the aPC resistant phenotype as a risk factor for pregnancy-related venous thrombosis.     

胶体金免疫层析法与双单克隆抗体夹心ELISA检测鼠疫F1抗原的比较

目的比较胶体金免疫层析法（GICA）和双单克隆抗体夹心酶联免疫吸附试验（DMcAbS-ELISA）检测鼠疫F1抗原的敏感性和特异性。方法用GICA试纸条和DMcAbS-ELISA平行检测308份强毒鼠疫耶尔森菌感染鼠脏器标本和327份对照鼠标本,用RIHA微量法同时检测作为参照。结果327份对照鼠鼠疫细菌学检验及GICA,DMcAbS-ELISA和RIHA的F1抗原检测均为阴性,GICA,DMcAbS-ELISA和RIHA在108cfu/mL水平上未发现与近缘假结核耶尔森菌有交叉反应;308只感染鼠样本细菌培养阳性284只,24只样本未分离到鼠疫菌;F1抗原检测GICA阳性287份,DM-cAbS-ELISA阳性280份,RIHA阳性276份,GICA阳性检出率最高93.19%,与DMcAbS-ELISA和RIHA比较,差异均有统计学意义（χ^2=5.14,9.09;P=0.016,0.001）;GICA与DMcAbS-ELISA符合率97.73%（Kappa=0.845）;GICA和DMcAbS-ELISA与RIHA符合率分别是96.43%和98.70%（Kappa=0.774,0.926）;284份细菌培养阳性鼠标本F1抗原检测GICA阳性率是98.59%,高于DMcAbS-ELISA和RIHA,差异有统计学意义（χ^2=4.17,5.14;P=0.031,0.016）;24份细菌培养阴性实验鼠标本F1抗原检测：GICA检出7份阳性,阳性率29.17%,DMcAbS-ELISA检出6份阳性,阳性率25%,RIHA检出3份阳性,阳性率12.5%,GICA高于DMcAbS-ELISA和RIHA,但差异无统计学意义（χ^2=2.25,0;P=0.125,1.000）。结论GICA检测鼠疫F1抗原敏感特异,快速简便,优于DMcAbS-ELISA和RIHA,是鼠疫快速诊断中有应用价值的检测技术。     

Congenital Defects in the Expression of the Glycosylphosphatidylinositol-Anchored Complement Regulatory Proteins CD59 and Decay-Accelerating Factor

CD59 and decay-accelerating factor (DAF) are glycosylphosphatidylinositol (GPI)-anchored complement regulatory proteins critical for regulating complement activation on the host cell surface. Defective expressions of CD59 or DAF caused by mutations in the genes coding for these proteins or genes involved in the biosynthesis of GPI, such as PIGA, PIT, and PIGM, are associated with various clinical symptoms that are mediated by dysregulated activation of complement, especially the C5 component. Eculizumab, an anti-C5 antibody, is effective in relieving the symptoms seen in patients with complement dysregulation.     

"Off-license" use of recombinant activated factor VII

Recombinant factor VIIa (rFVIIa) has been widely used in the treatment of bleeding episodes in haemophiliac patients with inhibitors. In haemostatic circles it has also been assessed in reversing oral anticoagulant therapy. Over the last few years, it has been used "off-label" in patients with uncontrolled bleeding due to haemostatic abnormalities due to trauma and/or massive blood loss, thrombocytopenia, platelet dysfunction or liver dysfunction. This review examines the proposed mechanism of action of rFVIIa in the context of current concepts of haemostasis and its pharmacological properties. The "off-license" use of rFVIIa is reviewed. The latter are reported mainly as case reports, case series. There is an overwhelming need for randomized controlled trials to assess rFVIIa's efficacy, dosing and safety in current "off-license" use.