Structural and functional characterization of novel F7 mutations identified in Chinese factor VII-deficient patients.

Hereditary factor VII (FVII) deficiency is a rare recessive bleeding disorder with an estimated prevalence of 1/500 000. We had investigated 50 unrelated Chinese patients with FVII deficiency and identified, in total, 25 mutations, including 18 missense mutations and 5 splicing mutations, on the F7 gene. The nucleotide transition c.1224T>G (p.His408Gln) in exon 9 constitutes a hotspot of mutation, with 19 patients harbouring this genetic variance. Few patients were homozygous or compound heterozygous for deleterious mutations, such as non-sense mutations, large insertion or deletions, indicating that complete deficiency of FVII may not be compatible with life. The eight novel mutations identified in the study, including one small deletion (p.Glu49GlyfsTer101), three type I missense mutations, p.Cys238Phe, p.Gly420Asp, p.Ala252Val and four type II missense mutations, p.Val336Met, p.Ser342Gly, p.Gly432Ser and p.Ile213Asn, were further analysed by in vitro expression and functional studies. The laboratory phenotype and structural analysis confirmed the functional consequence of p.Ile213Asn mutation involving cleavage and activation site. The molecular dynamic simulations and binding energy calculations along with functional probing of p.Gly432Ser mutation revealed the critical role of residue Gly432 in the binding between activated factor VII (factor VIIa) and tissue factor.

Inactivated SARS-CoV-2 vaccine does not influence the profile of prothrombotic antibody nor increase the risk of thrombosis in a prospective Chinese cohort.

The presence of antiphospholipid antibodies was shown to be associated with thrombosis in coronavirus disease 2019 (COVID-19) patients. Recently, according to reports from several studies, the vaccine-induced immune thrombotic thrombocytopenia is mediated by anti-platelet factor 4 (PF4)-polyanion complex in adenovirus-vectored COVID-19 vaccine recipients. It is impendent to explore whether inactivated COVID-19 vaccine widely used in China influences prothrombotic autoantibody production and induces thrombosis. In this prospective study, we recruited 406 healthcare workers who received two doses, 21 days apart, of inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine (BBIBP-CorV, Sinopharm). Paired blood samples taken before vaccination and four weeks after the second vaccination were used in detecting prothrombotic autoantibodies, including anticardiolipin (aCL), anti-ß2 glycoprotein I (aß2GP1), anti-phosphatidylserine/prothrombin (aPS/PT), and anti-PF4-heparin. The seroconversion rate of SARS-CoV-2 specific antibodies was 95.81% (389/406) four weeks after vaccination. None of the subjects had spontaneous thrombosis or thrombocytopenia over a minimum follow-up period of eight weeks. There was no significant difference in the presence of all ten autoantibodies between samples collected before and after vaccination: for aCL, IgG (7 vs. 8, P = 0.76), IgM (41 vs. 44, P = 0.73), IgA (4 vs. 4, P = 1.00); anti-ß2GP1, IgG (7 vs. 6, P = 0.78), IgM (6 vs. 5, P = 0.76), IgA (3 vs. 5, P = 0.72); aPS/PT IgG (0 vs. 0, P = 1.00), IgM (6 vs. 5, P = 0.76); aPF4-heparin (2 vs. 7, P = 0.18), and antinuclear antibody (ANA) (18 vs. 21, P = 0.62). Notably, seven cases presented with anti-PF4-heparin antibodies (range: 1.18-1.79 U/mL) after vaccination, and none of them exhibited any sign of thrombotic disorder. In conclusion, inactivated SARS-CoV-2 vaccine does not influence the profile of antiphospholipid antibody and anti-PF4-heparin antibody nor increase the risk of thrombosis.

Low factor V level ameliorates bleeding diathesis in patients with combined deficiency of factor V and factor VIII.

Combined factor V (FV) and FVIII deficiency (F5F8D) is a rare autosomal-recessive bleeding disorder caused by mutations in lectin mannose binding-1 (LMAN1) and multiple coagulation factor deficiency-2 (MCFD2). Six causative homozygous mutations (5 in LMAN1 and 1 in MCFD2) were identified in 6 patients with F5F8D. A thrombin-generation assay, triggered with tissue factor (1 pM) in F5F8D plasma, paradoxically exhibited enhanced thrombin generation compared with normal plasma. Significantly lower free tissue factor pathway inhibitor (fTFPI) was found in F5F8D patients compared with healthy controls (P < .01). Normalizing tissue factor pathway inhibitor α (TFPIα) in F5F8D plasma greatly delayed and reduced thrombin generation. Increasing FV concentrations by adding plasma FV to F5F8D plasma only caused a gradual decrease in thrombin generation, suggesting that low levels of TFPIα and FV cocontributed to the elevated thrombin generation by reducing anticoagulant effects. On the contrary, thrombin generation in F5F8D platelet-rich plasma (PRP) was significantly lower than in normal controls (P < .05); however, it was fully corrected by normalizing FVIII or after 1-deamino-8-d-arginine vasopressin (DDAVP) infusion, indicating that the hypocoagulable state of F5F8D patients is associated with low FVIII levels. In addition, plasma and platelet FV in F5F8D PRP were sufficient to support normal thrombin generation, and low TFPIα may have no effect on thrombin generation. DDAVP infusion induced a complete response in 5 F5F8D patients and a partial response in the remaining patient. Based on our findings, we suggest that DDAVP may be considered a potential substitute for FVIII concentrates, and fresh-frozen plasma (FFP) infusion may not be necessary for F5F8D patients with minor bleeding challenges.

Inhibitor eradication and bleeding management of acquired hemophilia A: a single center experience in China.

Objectives: Acquired hemophilia A (AHA) is a rare disease resulting from autoantibodies against coagulation factor VIII that leads to spontaneous bleeding. This study reports the clinical characteristics and treatment outcomes of a relatively sizable cohort of patients with AHA. Methods: We retrospectively analyzed the characteristics and outcomes of 42 patients with AHA diagnosed in our center from January 2014 through December 2018. Results: The FVIII activity (FVIII: C) was significantly suppressed (median 1.5%; interquartile range [IQR]: 0.9-3.5) by FVIII inhibitor (median 8 BU/mL; IQR: 4.0-16.0). Bypassing agents, PCC or FVIIa, were used in 14 patients for bleeding control without any adverse reaction; and most patients (90.5%, 38/42) were placed on immunosuppressive regimen, corticosteroid alone or in combination with cyclophosphamide. Patients treated with corticosteroids alone had a lower median inhibitor titer (8 BU/mL) than those treated with combination corticosteroids of cyclophosphamide (16 BU/mL) (p < 0.001). 97.4% (37/38) patients achieved complete remission (CR) after immunosuppression therapy, and the median time to CR in patients treated with corticosteroids alone was shorter than those with combination corticosteroids of cyclophosphamide (median 40 days; IQR: 31-65 vs. 51 days; IQR: 38-83, p = 0.301). 10 (26.3%) patients relapsed thereafter and were placed on combined corticosteroid and cyclophosphamide treatment, which yielded second remission in 8 patients (80%). Two patients died, one from uncontrolled post-surgical retroperitoneal hemorrhage and one from sepsis complicating corticosteroid therapy. Conclusion: The corticosteroid achieves a satisfactory outcome, particularly with low inhibitors titers; and combination of cyclophosphamide will facilitate remission in sever patients with high titers of inhibitors.

[Identification of genetic defects in a Chinese pedigree with factor XIII deficiency: case report and literature review].

OBJECTIVE: To perform phenotypic diagnosis, genetic diagnosis and prenatal diagnosis of inherited coagulation factor XIII (FXIII)deficiency in a Chinese family also provide a review of inherited coagulation F XIII deficiency. METHODS: The activity levels of F XIII (F XIII:C) of proband and family members were measured by clot solubility test and REA-chrom F XIII kit. The antigen levels of F XIII(FXIII:Ag) were measured by enzyme-linked immunosorbent assay. Thrombelastography (TEG) test was used to make a comprehensive evaluation of coagulation status in the proband. All 15 exons and exon-intron boundaries of the F13A1 gene were amplified by PCR, and DNA sequencing was performed then. The mutation identified in the proband was screened in the family members. Furthermore, the related literatures were reviewed to provide a profile of clinical manifestation, gene mutations, the relationship between the mutations and phenotype, and treatments of inherited coagulation F XIII deficient cases. RESULTS: The clot solubility test was positive in the proband. The FXIII:Ag level of the proband was less than 1% and the FXIII:C level was below the lower limit of detection (<3%). Two compound heterozygous missense mutations (p.Arg662* and p.Trp665*) were identified in the proband. Family study showed that the two mutations were both inherited from the parents. The fetus also carried two compound heterozygous mutations, the same as the proband, and was diagnosed with severe F XIII deficiency. As reported in the literatures, most mutations were missense mutations and nonsense mutations, and no hot spot was found. The clinical pattern of F XIII deficiency varied among patients, with potentially fatal consequences from severe bleeding complications. CONCLUSION: Better understanding of F XIII biochemical properties and function and developing of FXIII laboratory assays and genetic detection could prevent missed diagnosis, and patients moght benefit from better care.

Regional variations in the prevalence and misdiagnosis of air flow obstruction in China: baseline results from a prospective cohort of the China Kadoorie Biobank (CKB).

BACKGROUND: Despite the great burden of chronic respiratory diseases in China, few large multicentre, spirometry-based studies have examined its prevalence, rate of underdiagnosis regionally or the relevance of socioeconomic and lifestyle factors. METHODS: We analysed data from 512 891 adults in the China Kadoorie Biobank, recruited from 10 diverse regions of China during 2004-2008. Air flow obstruction (AFO) was defined by the lower limit of normal criteria based on spirometry-measured lung function. The prevalence of AFO was analysed by region, age, socioeconomic status, body mass index (BMI) and smoking history and compared with the prevalence of self-reported physician-diagnosed chronic bronchitis or emphysema (CB/E) and its symptoms. FINDINGS: The prevalence of AFO was 7.3% in men (range 2.5-18.2%) and 6.4% in women (1.5-18.5%). Higher prevalence of AFO was associated with older age (p<0.0001), lower income (p<0.0001), poor education (p<0.001), living in rural regions (p<0.001), those who started smoking before the age of 20 years (p<0.001) and low BMI (p<0.001). Compared with self-reported diagnosis of CB/E, 88.8% of AFO was underdiagnosed; underdiagnosis proportion was highest in 30-39-year olds (96.7%) compared with the 70+ age group (81.1%), in women (90.7%), in urban areas (89.4%), in people earning 5K-10 K ¥ monthly (90.3%) and in those with middle or high school education (92.6%). INTERPRETATION: In China, the burden of AFO based on spirometry was high and significantly greater than that estimated based on self-reported physician-diagnosed CB/E, especially in rural areas, reflecting major issues with diagnosis of AFO that will impact disease treatment and management.

Effectiveness of a television advertisement campaign on giving cigarettes in a chinese population.

BACKGROUND: Anti-tobacco television advertisement campaigns may convey messages on smoking-related health consequences and create norms against giving cigarettes. METHODS: Altogether, 156 and 112 slots of a television advertisement "Giving cigarettes is giving harm" were aired on Suzhou and Yizheng, respectively, over one month in 2010. Participants were recruited from 15 locations in Suzhou and 8 locations in Yizheng using a street intercept method. Overall 2306 residents aged 18-45 years completed questionnaires, including 1142 before the campaign and 1164 after, with respective response rates of 79.1% and 79.7%. Chi square tests were used to compare the difference between categorical variables. RESULTS: After the campaign, 36.0% of subjects recalled that they had seen the advertisement. Residents of Suzhou had a higher recall rate than those of Yizheng (47.6% vs. 20.6%, P < 0.001). The rate of not giving cigarettes dropped from 32.1% before the campaign to 28.5% after (P = 0.05). In the post-campaign evaluation, participants who reported seeing the advertisement were more likely not to give cigarettes in the future than those who reported not seeing the advertisement (38.7% vs. 27.5%, P < 0.001). CONCLUSIONS: Our study showed that an anti-tobacco television advertisements helped change societal norms and improve health behavior. Continuous and adequate funding of anti-tobacco media campaigns targeted at different levels of the general population is needed, in conjunction with a comprehensive tobacco control effort.

Prevalence and correlates of airflow obstruction in ∼317,000 never-smokers in China.

In China, the burden of chronic obstructive disease (COPD) is high in never-smokers but little is known about its causes in this group. We analysed data on 287 000 female and 30 000 male never-smokers aged 30-79 years from 10 regions in China, who participated in the China Kadoorie Biobank baseline survey (2004-2008). Prevalence of airflow obstruction (AFO) (pre-bronchodilator forced expiratory volume in 1 s (FEV1)/forced vital capacity (FVC) <0.7 and below the lower limit of normal (LLN)) was estimated, by age and region. Cross-sectional associations of AFO (FEV1/FVC <0.7), adjusted for confounding, were examined. AFO prevalence defined as FEV1/FVC <0.7 was 4.0% in females and 5.1% in males (mean ages 51 and 54 years, respectively). AFO prevalence defined as FEV1/FVC

Molecular basis and thrombotic manifestations of antithrombin deficiency in 15 unrelated Chinese patients.

INTRODUCTION: Antithrombin (AT) deficiency is associated with an increasing risk of thrombosis. MATERIALS AND METHODS: 15 unrelated patients with AT deficiency defined by thrombophilic assays were recruited and detailed clinical information about patients, focusing on the personal and family history of thromboembolism (TE), were recorded. Mutation analysis was performed by direct sequencing of an AT gene (SERPINC1) in the patients and their family members. RESULTS: A total of 15 heterozygous causative mutations, each being identified in one family, were identified. Five mutations (33.3%) were reported here for the first time, including three null mutations (Ser36X, Lys70X and Try307X) and two missense mutations (Phe123Cys and Leu340Phe) probably impairing the structural integrity and stability of protein based on the AT structural analysis. Of the 15 patients, 33.3% (5/15) had additional risk factors and only one patient presented with additional genetic alteration causing an early onset of thrombosis. Fourteen patients (93.9%) suffered from multisite recurrent thrombotic episodes after a first episode of thrombosis. 93.3% of the patients experienced deep vein thrombosis (DVT) and 40.0% presented with mesenteric venous thrombosis (MVT). In addition, both venous and arterial thrombosis was present in two unrelated patients. 51.0% subjects with AT deficiency in the 15 unrelated pedigrees experienced TE events. CONCLUSIONS: Prophylactic anticoagulation may be suggested in AT-deficient patients to avoid the recurrent and multisite thrombosis. The association of primary MVT and AT deficiency is highlighted.

[Functional study of abnormal fibrinogen caused by Arg275His mutation in fibrinogen γ chain].

OBJECTIVE: To investigate the function of abnormal fibrinogen in two inherited dysfibrinogenemia pedigrees. METHODS: Routine coagulation tests were conducted in the probands and related family members. The antigen and activity levels of fibrinogen were detected by immunoturbidimetry assay and clauss assay, respectively. All the exons and exon-intron boundaries of the three fibrinogen genes and antithrombin gene（AT3）were analyzed by PCR amplification and direct sequencing. Routine thrombelastography (TEG) test and functional fibrinogen TEG test were both used to make a comprehensive evaluation of coagulation status and functional fibrinogen level in patients. The molecular weights of the three peptides from fibrinogen were measured by Western blot. The function of abnormal fibrinogen was assessed by fibrinogen dynamic polymerization and fibrinolysis velocity. RESULTS: The coagulation routine tests were normal in two probands except for prolonged thrombin time (TT) and reptilase time (RT), as well as reduced activity levels of 0.5 g/L and 0.6 g/L fibrinogen, respectively. The antigen levels of fibrinogen were 2.32 g/L and 2.66 g/L in two probands, which were in the normal reference range. The genotype analysis showed that Arg275His in fibrinogen γ chain (γ Arg275His) existed in both probands and patients in these two pedigrees. Meanwhile, proband B's grandfather and aunt also carried heterozygote g.5876T>C (Ser116Pro) mutation in AT3. The results of routine TEG test demonstrated that the α values of proband B and his father were close to and lower than the lower limit of reference range, respectively, while the MA values were normal in both of them. However, functional fibrinogen TEG test revealed obviously reduced MA value. All the probands and patients demonstrated prolonged lag-off time and reduced peak value in fibrinogen dynamic polymerization tests. Meanwhile, most of fibrin formed from the patients' plasma could not be dissolved completely by plasminogen (PLG) and urokinase-typeplasminogenactivator (u-PA) at a certain time. CONCLUSION: We first reported cases of inherited dysgibrinogenemia associated with inherited AT deficiency. γArg275His mutation caused the abnormal fibrinogen in terms of fibrin mono polymerization and possibly in fibrinolysis. Combined use of routine TEG test and functional fibrinogen TEG test with comprehensive analyses of the parameters in both tests could better evaluate the level of functional fibrinogen and predict the risk of hemorrhage and thrombosis in patients with inherited dysfibrinogenemia.

[Phenotype and genotype analysis of two Chinese pedigrees with type 3 von Willebrand diseases].

OBJECTIVE: To analyze the phenotype and genotype of two Chinese pedigrees with von Willebrand diseases, and to investigate the molecular pathogenesis. METHODS: Bleeding time (BT), activated partial thromboplastin time (APTT), ristocetin-induced platelet aggregation (RIPA), von Willebrand factor-ristocetin cofactor (vWF:Rco), von Willebrand factor antigen (vWF:Ag), von Willebrand factor activity (vWF:A), von Willebrand factor collagen binding assay (vWF:CB) and multimer analysis were used for phenotype diagnosis. DNA was extracted. All of the 52 exons and exon-intron bounda ries of the VWF gene were amplified with polymerase chain reaction(PCR) and analyzed by direct sequencing. RESULTS: APTT and BT were prolonged. Plasma RIPA, vWF:Rco, vWF:Ag, vWF:A and vWF:CB was significantly decreased. No VWF multimer can be found by plasma VWF multimer analysis. Homozygous insertional mutation g.82888_82889insCATG in exon 17 was found in proband A. Compound heterozygous mutations g.94865 G to A (Trp856stop) in exon 20 and g.110698_110699delinsG in exon 28 were found in proband B. CONCLUSION: Homozygous insertional mutation g.82888_82889insCATG and compound heterozygous mutations g.94865G to A(Trp856X) and g.110698_110699delinsG probably have respectively induced type 3 von Willebrand diseases in the two probands.

[Genotype and function analyses of four inherited dysfibrinogenemia pedigree caused by Arg16 amino acid substitution in fibrinogen Aα chain].

OBJECTIVE: To analyze the phenotype, genotype and function in four Chinese pedigrees with inherited dysfibrinogenemia. METHODS: Routing tests including activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), reptilase time (RT), the activities of antithrombin (AT), protein C (PC) and protein S (PS) were detected in four pedigrees. The activity and antigen of plasma fibrinogen were analyzed by Clauss and immunoturbidimetry methods, respectively. The molecular weight of fibrinogen of four probands was assessed by Western blot. The function of abnormal fibrinogen was evaluated by fibrinogen clottability, fibrinogen dynamic polymerization and fibrinolysis velocity, respectively. The sequences of all the exons and exon-intron boundaries of the three fibrinogen genes were amplified by PCR and analyzed by direct sequencing. RESULTS: Four probands had prolonged TT and RT, reduced plasma fibrinogen activity levels and normal antigen levels. The assays of Western blot showed no abnormal molecular weight of fibrinogen. Function tests revealed reduced fibrinogen clottability, delayed and decreased fibrinogen dynamic polymerization and reduced fibrinolysis velocity. Aα chain Arg16His and Arg16Cys mutations were identified in the four probands, respectively. CONCLUSION: The four probands with dysfibrinogenemia were caused by the mutations of Aα chain Arg16His or Arg16Cys. Mutation of the fibrinogen induced dysfunction of plasma fibrinogen.

[The phenotypic and genotypic diagnosis of three Chinese patients with von Willebrand disease].

OBJECTIVE: To analyze the phenotype and genotype of three patients with von Willebrand disease (vWD), and to explore its molecular pathogenesis. METHODS: Bleeding time (BT), APTT, ristocetin induced platelet aggregation (RIPA), von Willebrand factor (vWF):ristocetin cofactor (Rco) (vWF:Rco), vWF antigen (vWF:Ag), vWF activity (vWF:A) test, vWF collagen binding assay (vWF:CB) and multimer analysis were detected for phenotype diagnosis. The dynamic process of blood coagulation was evaluated by using the thrombelastography. Genomic DNA was extracted from the peripheral blood. The vWF gene mutation was detected by sequencing. RESULTS: APTT, BT were prolonged in the three probands. Plasma vWF:Rco, vWF:Ag, vWF:A and vWF:CB were decreased in different degrees. RIPA was reduced in probands B and C. vWF multimer analysis found the lost of the large molecular weight multimers in proband B, while basically normal in probands A and C. The dynamic process of blood coagulation of proband C presented obvious hypocoagulability by using the thrombelastography. Heterozygous missense mutation g.106782G > T resulting in Cys1130Phe in exon 26, g.110988G > A resulting in Gly1579Arg in exon 28 and g.110373C > T resulting in Arg1374Cys in exon 28 were found in the probands A, B and C, respectively. CONCLUSION: Three probands were diagnosed as type 1, type 2A or type 2M vWD by phenotype detection. Heterozygous missense mutation Cys1130Phe, Gly1579Arg and Arg1374Cys induced vWD of three probands, respectively.

[Two novel mutations in one pedigree with hereditary Factor VII deficiency].

OBJECTIVE: To explore the mutations of coagulation factor VII (FVII) gene in one pedigree with hereditary FVII deficiency, and to investigate the molecular mechanisms of FVII deficiency. METHODS: FVII gene mutations were analysed in the pedigree by direct DNA sequencing. The mutated DNA fragments were cloned into pMD19-T simple TA vector, and sequenced to confirm their distribution on chromosome. The plasma activity of FVII of the probands and their family members was detected with coagulation assay. The antigen of FVII were identified with ELISA. RESULTS: Three gene mutations were detected in the pedigree: A/G to C at 15386 resulting in Arg353Pro/Gln353Pro, A to T at 15274 resulting in Lys316Stop, all three mutations were heterozygotes. Three kinds of polymorphisms were identified in his father: A to G transition at position 15386 resulting in Arg353Gln, heterozygotic deletion of 2050 - 2059 cctatatcct in promoter and G to A mutation in intron 1a, the same polymorphisms were found in his grandfather. The three polymorphisms were located in the same chromosome of his father. CONCLUSION: Two mutations were found in the pedigree with hereditary FVII deficiency. One is nonsense mutation (Lys316Stop), the other is missense one (Gln353Pro). Gln353Pro and Lys316Stop might be the molecular mechanisms of FVII deficiency. The two novel mutations were reported for the first time in the literature.

[Analysis of phenotype and genotype in three Chinese pedigrees with inherited dysfibrinogenemia].

OBJECTIVE: To analyze the phenotype and genotype in three Chinese pedigrees with inherited dysfibrinogenemia. METHODS: Laboratory tests including activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), reptilase time (RT), and the activities of antithrombin (AT:C), protein C (PC:C) and protein S(PS:C) were detected in three pedigrees. The activity and antigen of plasma fibrinogen (Fg) were analyzed by Clauss and immunoturbidimetry methods, respectively. The Fg of three probands was assessed by Western blot and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The sequences of all the exons and exon-intron boundaries of the three Fg genes FGA, GFB and FGG were amplified by PCR and analyzed by direct sequencing. RESULTS: Three probands had normal APTT, PT, PC:C, PS:C and AT:C, but prolonged TT and RT. The activity levels of the 3 probands's plasma Fg were reduced, but antigen levels were normal. Western blot and SDS-PAGE showed no abnormal molecular weight of Fg. The 3 heterozygous mutations of γ Arg275His, Aα Pro18Leu and Aα Arg16Cys were identified in the 3 probands, respectively. CONCLUSION: The three probands with dysfibrinogenemia were caused by the mutations of γ Arg275His, Aα Pro18Leu and Aα Arg16Cys, respectively. Both Aα Pro18Leu and Aα Arg16Cys were first reported in Chinese population.

[Phenotype and genotype analysis of three Chinese pedigrees with von Willebrand disease].

OBJECTIVE: To analyze phenotype and genotype of three Chinese pedigrees with von Willebrand disease (vWD), and explore the molecular mechanism. METHODS: Bleeding time (BT), activated partial thromboplastin time (APTT), ristocetin-induced platelet aggregation (RIPA), von Willebrand factor (vWF): ristocetin cofactor (RCof) (vWF:RCof), vWF antigen (vWF:Ag), vWF activity (vWF:A) test, vWF collagen binding assay (vWF:CB), vWF and Factor VIII (FVIII) binding assay (vWF:FVIII:B) and multimer analysis were used for phenotype diagnosis. Genomic DNA was extracted from the peripheral blood (PB). All the 52 exons and flanking sequences of the probands' vWF gene were amplified by PCR and analyzed by direct sequencing. RESULTS: APTT were prolonged in all three probands, while BT were normal excepting for proband 3. Plasma RIPA, vWF:RCo, vWF:Ag, vWF:A and vWF:CB were decreased in different extents. In multimer analysis, proband 3 lost the large and intermediate molecular weight multimers, while proband 1 and 2 were normal. Gene analysis in the three probands revealed three heterozygous missense mutations of 144067 G→A (R2287Q) in exon 39, 110374G→A (R1374H) and 110770C→T (S1506L) in exon 28 and heterozygous polymorphism 110667G→A (D1472H) in exon 28, respectively. CONCLUSION: The three heterozygous mutations (R2287Q, R1374H and S1506L) and an heterozygous polymorphism (D1472H) are genetic defects of the hereditary vWD of the three pedigrees respectively. R2287Q is a novel mutation reported for the first time in the literature.

[Molecular mechanisms of recurrent venous thrombosis in two pedigrees with type I antithrombin deficiency].

OBJECTIVE: To investigate the clinical phenotype, genotype and molecular mechanism of recurrent venous thrombosis in two Chinese pedigrees with type I antithrombin (AT) deficiency. METHODS: The routine coagulation screening tests were detected, thrombin generation tests was performed to evaluate the hypercoagulation. Anticardiolipin antibody (ACA) and lupus anticoagulant (LA) were detected with enzyme-linked immunosorbent assay (ELISA) and diluted viper venom time assay (DVVT), respectively. The activities of protein C, protein S and AT (PC:A, PS:A, AT:A) were tested with chromogenic substrate assay or clotting method. The antigen of AT (AT:Ag) was performed with immunoturbidimetry methods. Western blot was used to analyze the molecular weight (MW) and the plasma levels of AT:Ag. All 7 exons and the flanking sequences were amplified by PCR. The mutation of AT gene and thrombophilia associated gene polymorphisms were analyzed by direct DNA sequencing. The expression plasmid of Ala404Asp mutant was constructed with site-directed mutagenesis method based on the wild-type (WT) AT cDNA contained in pcDNA 3.1 vector, and transiently expression of AT WT and the Ala404Asp mutant was performed using HEK293T cells. Cultured supernatant and cell lysates were collected and measured for AT:Ag by ELISA and Western blot. RESULTS: The results of routine coagulation tests in two probands were normal, thrombin generation tests indicated that proband 1 presented hypercoagulable state with 2.8 and 1.5 times higher of the endogenous thrombin potential (ETP) and peak height compared with that of normal, respectively. The levels of PC:A, PS:A, ACA and LA were normal. AT:A in proband 1 and proband 2 were 45% and 32%, and AT:Ag were almost half of the normal (121 mg/L and 158 mg/L), respectively. The results of Western blot showed that both probands' plasma levels of AT:Ag were lower than the normal pooled plasma and MW was normal. Two heterozygous mutations of g.3291C→T(Thr98Ile), g.13863C > A(Ala404Asp) were identified in the probands, respectively. No proband had venous thrombosis associated gene polymorphisms. Expression in vitro showed that AT:Ag in culture media and lysates of Ala404Asp are 4.8% and 60.6% of that of WT, respectively. CONCLUSION: Thr98Ile and Ala404Asp mutation of AT gene significantly correlate with recurrent venous thrombosis in the two probands, respectively. Ala404Asp has not been described before. The mutant Ala404Asp protein can not be expressed due to impaired secretion and increased intracellular degradation, resulting in type I AT deficiency.

[Two new mutations of AT gene in type I inherited antithrombin deficiency.].

OBJECTIVE: To identify the clinical phenotype and gene mutation in two kindreds with type I inherited antithrombin (AT) deficiency. METHODS: The coagulation and anticoagulation testing and thrombophilia screening were used for phenotypic diagnosis and immunonephelometry and chromogenic assay for plasma level of AT antigen (AT:Ag) and AT activity (AT:A), respectively. All of the seven exons and intron-exon boundaries and untranslation regions of AT gene were amplified by PCR, and the PCR products analysis was by direct sequencing. The corresponding gene sites of the two family members and healthy individuals were detected according to the gene mutation sites. RESULTS: The plasma levels of AT:Ag of proband 1 and proband 2 were 126 mg/L and 117 mg/L, and AT:A was 49% and 48%, respectively. Heterozygotic deletion of 3239-3240delCT in proband 1 and nonsense mutation 3206A-->T (K70Stop) in proband 2 were rchaacterized in exon 2 of AT gene. And some of their family members were also detected with the heterozygotic gene mutation. CONCLUSION: Type I inherited antithrombin deficiency of the two probands were caused by AT gene mutation 3239-3240delCT and 3206A-->T (K70Stop).

[Analysis of phenotype and genotype in four Chinese pedigrees with inherited coagulation factor V deficiency.].

OBJECTIVE: To identify the phenotype and genotype in four Chinese pedigrees with inherited coagulation factor V (FV) deficiency. METHODS: The tests of activated partial thromboplastin time (APTT), prothrombin time (PT), FV activity (FV:C) and FV antigen (FV:Ag) were used for phenotype diagnosis. All the exons and exon-intron boundaries of F5 gene were amplified by PCR and analyzed by direct sequencing. RESULTS: The APTT and PT in each of the four probands were obviously prolonged, and both activity and antigen of FV in the four probands were extremely lower compared with that of normal mixed plasma. Sequencing of F5 gene in proband 1 identified a heterozygous mutation, G16088C (Asp68His), and four polymorphisms, T35788C (Met385Thr), A47295G (His1299Arg), A58668G (Met1736Val) and A74083G (Asp2194Gly), which were located in the same chromosome; proband 2 was homozygous for two mutations, C46253T (Arg952Cys) and C46724T(Gln1109stop); the F5 gene of proband 3 showed a homozygous missense mutation, C67793G(Pro2006Ala); and proband 4 was homozygous for one missense mutation, C74022T (Arg2174Cys). CONCLUSION: Five mutations (Asp68His, Arg952Cys, Gln1109stop, Pro2006Ala and Arg2174Cys) and four polymorphisms (Met385Thr, His1299Arg, Met1736Val and Asp2194Gly) may lead to type I inherited FV deficiency for these four probands, respectively. Gln1109stop, Pro2006Ala and Arg2174Cys haven't been identified before.

[The application of thrombin generation tests to warfarin anticoagulation monitoring].

OBJECTIVES: To explore the thrombin generation capacity in patients on warfarin therapy with different prothrombin time international normalized ratio (PT-INR), the capacity in relation to bleeding, and the application of thrombin generation tests to warfarin therapy monitoring. METHODS: Seventy eight blood samples were taken from patients on warfarin therapy for more than 3 months owing to valve replacement or atrial fibrillation. The patients' case history and PT-INR were collected and thrombin generation tests were performed in all samples. RESULTS: Patients were ranked into three groups according to different PT-INR. There were 23 patients in group I with PT-INR from 1.51 to 2.00, 39 patients in group II with PT-INR from 2.01 to 3.00, and 16 patients in group III with PT-INR from 3.01 to 4.26. There were significant differences between each two of the three groups in lag time, peak, and ttpeak (time to peak) (P <0.01). There was a significant difference between group I and group II in endogenous thrombin potential (ETP) (P = 0.0001), but not between group II and group III (P= 0.06). Five patients developed bleeding and their ETP was less than 15% of normal control. CONCLUSION: In patients on warfarin therapy, when the PT-INR was more than 3.0, increasing the dose of warfarin doesn' t decrease the thrombin generation, but increase bleeding risk. PT-INR combined with ETP may better reflect patient's coagulation status, therefore be of more significance in preventing bleeding.