Association between vitamin D and cardiovascular health in Chinese children and adolescents: Basing on Life's Essential 8.

BACKGROUND AND AIMS: The relationship between vitamin D and cardiovascular health (CVH) in children remains unclear. We aimed to explore the association between vitamin D and CVH metrics using the latest Life's Essential 8 (LE8) among Chinese children and adolescents. METHODS AND RESULTS: A cross-sectional study containing 2680 participants aged 7-18 years (1340 boys and 1340 girls) was performed in South China in 2013. Vitamin D levels were categorized as follows: ≥20 ng/mL (sufficiency), 12 - < 20 ng/mL (inadequacy), and <12 ng/mL (deficiency). The CVH metrics of LE8 was assessed by overall CVH score, health behavior score, health factor score and high CVH, among which the health behaviors included diet, physical activity, nicotine exposure, and sleep health, as well as the health factors contained body mass index, blood lipids, blood glucose, and blood pressure. Different regression models were used to assess the associations between vitamin D levels and CVH metrics of LE8. Results showed that the prevalence of vitamin D deficiency and inadequacy was 7.5% and 44.4%, respectively. Boys had lower levels in overall CVH score, health behavior score, and health factor score than girls. After adjusting for potential confounds, upward trends in diet score, health behavior score, and high CVH were observed with increasing vitamin D levels. CONCLUSION: Vitamin D levels were positively associated with high CVH based on LE8, and more attention should be paid on boys due to whose lower levels in CVH metrics.

Molecular genetic analysis of two novel a allele to cause Ax phenotype in Chinese.

BACKGROUND: Mutations of ABO gene may cause the dysfunction of ABO glycosyltransferase (GT) that can result in weak ABO phenotypes. Here, we identified two novel weak ABO subgroup alleles and explored the mechanism that caused Ax phenotype. MATERIALS AND METHODS: The ABO phenotyping and genotyping were performed by serological studies and direct DNA sequencing of ABO gene. The role of the mutations was evaluated by 3D model, predicting protein structure changes, and in vitro expression assay. The total glycosyltransferase transfer capacity in supernatant of transfected cells was examined. RESULTS: The results of serological showed the subject RJ23 and RJ52 both were Ax phenotypes. The novel A alleles, Avar-1 and Avar-2 were identified according to the gene analysis. Both Avar-1 and Avar-2 harbored recombinant heterozygous alleles, specifically A2.05 and O.01.02. These alleles showcased substitutions at positions c.106G > T, c.189C > T, c.220C > T, and c.1009A > G in their respective exons. It is worth noting that the crossing-over regions of these two alleles differed from each other. In vitro expression study showed that GTA mutant impaired H to A antigen conversion, and the mutant did not affect the production of GTA though the Western bolt. In silico analysis showed that GTA mutant may change the local conformation and the stability of GT. CONCLUSIONS: The Avar-1 and Avar-2 alleles were identified, which could cause the Ax phenotype through changing the local conformation and reducing stability of the GTA.

Unraveling the molecular basis underlying nine putative splice site variants of von Willebrand factor.

Approximately 10% of von Willebrand factor (VWF) gene variants are suspected to disrupt messenger RNA (mRNA) processing, the number of which might be underestimated due to the lack of transcript assays. In the present study, we provided a detailed strategy to evaluate the effects of nine putative splice site variants (PSSVs) of VWF on mRNA processing as well as protein properties and establish their genotype-phenotype relationships. Eight of nine PSSVs affected VWF splicing: c.322A>T, c.1534-13_1551delinsCA, and c.8116-2del caused exon skipping; c.221-2A>C, c.323+1G>T, and c.2547-13T>A resulted in the activation of cryptic splice sites; c.2684A>G led to exon skipping and activation of a cryptic splice site; c.2968-14A>G created a new splice site. The remaining c.5171-9del was likely benign. The efficiency of nonsense-mediated mRNA decay (NMD) was much higher in platelets compared to leukocytes, impairing the identification of aberrant transcripts in 4 of 8 PSSVs. The nonsense variant c.322A>T partially impaired mRNA processing, leaking a small amount of correct transcripts with c.322T (p.Arg108*), while the missense variant c.2684A>G totally disrupted normal splicing of VWF, rather than produced mutant protein with the substitution of Gln895Arg. The results of this study would certainly add novel insights into the molecular events behind von Willebrand disease.

Prothrombin Arg541Trp Mutation Leads to Defective PC (Protein C) Pathway Activation and Constitutes a Novel Genetic Risk Factor for Venous Thrombosis.

OBJECTIVE: Defective PC (protein C) pathway predisposes patients to venous thromboembolism (VTE) and is mostly, but not exclusively, attributed to hereditary PC or PS (protein S) deficiencies and activated PC resistance caused by factor V Leiden mutation. Approach and Results: In a patient with acute mesenteric venous thrombosis and positive family history of VTE associated with the impaired PC pathway function determined by thrombin generation test, we identified a novel heterozygous prothrombin mutation p.Arg541Trp. Two more patients with positive family history of VTE carrying the same mutation were identified in a cohort of another 373 unrelated patients, making an overall prevalence of 0.8%. Family investigation revealed 11 individuals in the 3 pedigrees harboring the heterozygous prothrombin p.Arg541Trp mutation, and 8 of them (72%) had experienced episodes of VTE. Functional studies indicated the mutation moderately decreased procoagulant activity of prothrombin and had mild impact on the inactivation of thrombin by its inhibitor antithrombin. However, the amino acid residue substitution significantly compromised PC activation by thrombin, both in the absence and presence of soluble thrombomodulin, and thus rendered prothrombin function procoagulant biased. CONCLUSIONS: In summary, the prothrombin p.Arg541Trp mutation constitutes a new genetic risk factor of VTE by impairing function of PC pathway and tilting thrombin's procoagulant activity over anticoagulant function.

Ile73Asn mutation in protein C introduces a new N-linked glycosylation site on the first EGF-domain of protein C and causes thrombosis.

Activated protein C exerts its anticoagulant activity by protein S-dependent inactivation of factors Va and VIIIa by limited proteolysis. We identified a venous thrombosis patient who has plasma protein C antigen level of 63% and activity levels of 44% and 23%, as monitored by chromogenic and clotting assays. Genetic analysis revealed the proband carries compound heterozygous mutations (c.344T>A, p.I73N and c.1181G>A, p.R352Q) in PROC We individually expressed protein C mutations and discovered that thrombin-thrombomodulin activates both variants normally and the resulting activated protein C mutants exhibit normal amidolytic and proteolytic activities. However, while protein S-dependent catalytic activity of activated protein C-R352Q toward factor Va was normal, it was significantly impaired for activated protein C-I73N. These results suggest that the Ile to Asn substitution impairs interaction of activated protein C-I73N with protein S. This conclusion was supported by a normal anticoagulant activity for activated protein C-I73N in protein S-deficient but not in normal plasma. Further analysis revealed Ile to Asn substitution introduces a new glycosylation site on first EGF-like domain of protein C, thereby adversely affecting interaction of activated protein C with protein S. Activated protein C-R352Q only exhibited reduced activity in sub-physiological concentrations of Na+ and Ca2+, suggesting that this residue contributes to metal ion-binding affinity of the protease, with no apparent adverse effect on its function in the presence of physiological levels of metal ions. These results provide insight into the mechanism by which I73N/R352Q mutations in activated protein C cause thrombosis in proband carrying this compound heterozygous mutation.

The characteristics and spectrum of F9 mutations in Chinese sporadic haemophilia B pedigrees.

INTRODUCTION: Sporadic haemophilia B (HB) without obvious familial history poses challenges for genetic diagnosis and counselling. AIM: To identify the F9 variants in sporadic HB patients and probe the origin of these de novo mutations. METHOD: A total of 294 unrelated HB pedigrees sought genetic diagnosis were analysed in this single-centre study. The F9 gene was analysed by direct sequencing, and AccuCopy technique was adopted to screen for gene copy number variations. Six short tandem repeats approximal or within F9 gene were applied for linkage analysis. Mosaicism of sequence variant was determined by ddNTP Primer Extension method. RESULTS: Sporadic HB patients constituted 36% (61/294) of cases enrolled in current study. The sporadic and familial HB patients shared similar spectrum of F9 variants, with single nucleotide substitution as predominant form of disease-causing mutation and no mutation prone hotspot sites, including CpG dinucleotide sequences, had been identified. Majority of the mothers of sporadic HB patients were F9 mutation carriers (70%, 43/61), and most of them (95%, 41/43) had the inherited bleeding trait traced back to maternal grandfathers. Although most de novo mutations occur in germ cells, 2 maternal grandfathers, who had somatic mosaic mutations of F9, were also revealed to be the source of genetic variations identified in patients. In our cohort, FIX inhibitor incidence was 1%, developed only in patients carrying null mutations. CONCLUSION: The diversity of F9 genetic variants and possible mosaicism of de novo mutation demand extensive study and more cautious in genetic counselling of sporadic HB.

Vps33b regulates Vwf-positive vesicular trafficking in megakaryocytes.

Mutations of vacuolar protein sorting-associated protein 33b (VPS33B) cause arthrogryposis, renal dysfunction, and cholestasis syndrome, and a lack of platelet α-granules in the affected patients. Conditional Vps33b knockout mice were developed to investigate the function(s) of Vps33b in platelet α-granule formation. We found that early embryonic deletion of Vps33b was lethal. PF4-Cre-driven megakaryocyte-targeted Vps33b gene deletion greatly diminished Vps33b expression in platelets, but had no effect on platelet α-granule formation and protein content. Tamoxifen-induced, haematopoietic stem cell (HSC)-specific Vps33b deletion completely depleted Vps33b in platelets, caused the absence of α-granules, and increased the number of vacuoles in platelets and megakaryocytes. VPS33B association with VIPAS39, α-tubulin, and SEC22B was identified by co-immunoprecipitation, mass spectra, and immunoblotting in human embryonic kidney 293T (HEK293T) cells. Also, pull-down experiments revealed that VIPAS39 bound to intact VPS33B; in contrast, α-tubulin and SEC22B separately interacted with the sec1-like domains of VPS33B. Vps33b deficiency in megakaryocytes disturbs the redistribution of Vipas39 and Sec22b to proplatelets, and interrupted the co-localization of Sec22b with Vwf-positive vesicles. The data presented in this study suggest that Vps33b is involved in α-granule formation possibly by facilitating the Vwf-positive vesicular trafficking to α-granule-related vacuoles in megakaryocytes. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Clinical manifestations and mutation spectrum of 57 subjects with congenital factor XI deficiency in China.

Congenital factor XI (FXI) deficiency is a rare bleeding disorder with unpredictable bleeding tendency. Few studies in a large cohort have been reported regarding associations between FXI activity (FXI:C) or genotypes and bleeding symptoms currently. This study characterized clinical manifestations and mutation spectrum of 57 subjects with FXI deficiency in China. Clinical data were collected and mutations were identified by direct sequencing and determined by mRNA analysis. The result revealed bleeding symptoms were only found in 12 patients (12/57, 21.1%) with severely reduced FXI:C, and prolonged bleeding post injury/surgery as well as easy bruising were the commonest bleeding manifestations presented in respective 5 cases (5/12, 41.7%). A total number of 37 mutations were identified including 19 missense mutations, 9 nonsense mutations, 6 splice site mutations and 3 small deletions. Among them, 4 missense mutations, 5 splice mutations, 3 small deletions and a nonsense mutation were newly detected. W228*, G400V, Q263* and c.1136-4delGTTG with a total frequency of 48.3% were the most four common mutations in Chinese patients. RT-PCR analysis was carried out and confirmed that both c.596-8T>A and c.1136-4delGTTG were pathogenic due to frameshift resulting in respective truncated proteins. Our findings suggested clinical manifestations had little to do with FXI:C or genotypes, which required further study. This study, the largest investigation of FXI deficiency in China revealed that the F11 mutation spectrum of Chinese population was distinct from those of other populations earlier established.

Paired immunoglobulin-like receptor B regulates platelet activation.

Murine paired immunoglobulin-like receptors B (PIRB), as the ortholog of human leukocyte immunoglobulin-like receptor B2 (LILRB2), is involved in a variety of biological functions. Here, we found that PIRB and LILRB2 were expressed in mouse and human platelets, respectively. PIRB intracellular domain deletion (PIRB-TM) mice had thrombocythemia and significantly higher proportions of megakaryocytes in bone marrow. Agonist-induced aggregation and spreading on immobilized fibrinogen were facilitated in PIRB-TM platelets. The rate of clot retraction in platelet-rich plasma containing PIRB-TM platelets was also increased. Characterization of signaling confirmed that PIRB associated with phosphatases Shp1/2 in platelets. The phosphorylation of Shp1/2 was significantly downregulated in PIRB-TM platelets stimulated with collagen-related peptide (CRP) or on spreading. The results further revealed that the phosphorylation levels of the linker for activation of T cells, SH2 domain-containing leukocyte protein of 76kDa, and phospholipase C were enhanced in PIRB-TM platelets stimulated with CRP. The phosphorylation levels of FAK Y397 and integrin ß3 Y759 were also enhanced in PIRB-TM platelet spread on fibrinogen. The PIRB/LILRB2 ligand angiopoietin-like-protein 2 (ANGPTL2) was expressed and stored in platelet α-granules. ANGPTL2 inhibited agonist-induced platelet aggregation and spreading on fibrinogen. The data presented here reveal that PIRB and its ligand ANGPTL2 possess an antithrombotic function by suppressing collagen receptor glycoprotein VI and integrin αIIbß3-mediated signaling.

Thromboelastography predicts risks of obstetric complication occurrence in (hypo)dysfibrinogenemia patients under non-pregnant state.

Congenital (hypo)dysfibrinogenemia patients may have obstetric complications during their pregnancies. This study aimed to evaluate thromboelastography (TEG) as a potential tool for assessing the tendency for obstetric complications in those patients in a non-pregnant state. A total of 22 female subjects with congenital (hypo)dysfibrinogenemia were recruited. Nine subjects had histories of obstetric complications and the other 13 subjects had at least one uneventful pregnancy without obstetric complications as yet. Detailed clinical investigation and phenotype/genotype detection were carried out, and both kaolin-activated TEG and functional fibrinogen TEG (FF-TEG) were applied in all subjects. Significant differences were identified in all TEG parameters except for R and angle between these two groups (P < 0.05) by covariance analysis. Receiver operating characteristic (ROC) analysis of discrimination between these two groups of patients was performed for TEG parameters. Significantly high odds ratio (OR) of obstetric complications occurrence were demonstrated in K ≥ 3.8 min, maximum amplitude (MA) ≤ 54.2 mm, comprehensive index (CI) ≤ -3 (11.67, 95% CI 1.527-89.121, P < 0.05 in all), and MA-CFF ≤ 12.1 mm (20.00, 95% confidence interval (95% CI) 1.967-203.322, P = 0.002). Moreover, MA-CFF had better prognostic performance, with a corresponding area under the receiver operating curve of 0.923 (range 0.815-1.031, P = 0.001). This study suggests that (hypo)dysfibrinogenemia patients with values outside of the cut-off values of TEG assays under non-pregnant state may have a higher risk of obstetric complications occurring when they are pregnant. No parameters under non-pregnant state in clinical laboratory have ever been reported to be risk factors for obstetric complication occurrence in (hypo)dysfibrinogenemia patients. This study explored such parameters in TEG assays and found that parameters of TEG assays under non-pregnant status might predict the occurrence of obstetric complications, which could provide physicians with important information about whether fibrinogen replacement therapy is required, so as to prevent the occurrence of obstetric complications, especially for patients who are asymptomatic in daily life.

Clinical features and molecular basis of 102 Chinese patients with congenital dysfibrinogenemia.

INTRODUCTION: Congenital dysfibrinogenemia (CD) is a rare qualitative disorder of fibrinogen (Fg) with heterogeneous clinical manifestations. We aimed to analyze clinical phenotype and molecular basis of 102 Chinese CD patients and to evaluate the application of thromboelastography (TEG). MATERIALS AND METHODS: Clinical manifestations were recorded and quantified using the consensus ISTH bleeding assessment tool. Kaolin activated TEG and functional Fg TEG were applied in 30 patients. Genetic analysis of Fg genes were performed by direct sequencing. RESULTS: 27.5% patients experienced bleeding, 3.9% had thrombosis and 68.6% were asymptomatic. Females were more prone to experience bleeding (P=0.01). Significant difference (P<0.05) in TEG results were found between patients with hot-spot mutations at AαArg35(16) and γArg301(275), but were not identified between patients with and without bleeding. Normal TEG results were found in patients with mutations at AαArg35(16), AαPro37(18) or AαArg38(19). Six novel mutations were identified, including AαGly33(14)del, AαAsp57(38)_Trp60(41)delIVS2+1_+2GTdel, AαPhe742(723)Tyr, γAsn334(308)Thr, γGly335(309)Cys and γTrp395(369)Leu. CONCLUSIONS: CD patients have similar clinical manifestations and hot-spot mutations worldwide with no ethnic difference. TEG results could not indicate the bleeding risk in patients, but priority of mutation screening at thrombin cleavage site or polymerization site on Aа chain may be given if TEG results are normal.

Molecular mechanisms and clinical manifestations of hereditary hemorrhagic telangiectasia.

INTRODUCTION: Hereditary Hemorrhagic Telangiectasia (HHT) is charactered by telangiectasia and arteriovenous malformations (AVMs). Recurrent visceral and mucocutaneous bleeding is frequently reported among HHT patients, while data on the prevalence of thrombosis remains limited. This study aims to describe the clinical manifestations and molecular biological characteristics of HHT patients. METHODS: We conducted a retrospective study at Ruijin Hospital, affiliated with Shanghai Jiao Tong University School of Medicine. A total of 24 HHT patients, observed between January 2019 and December 2023, were included. We recorded the biological, clinical, and therapeutic events, with particular attention to bleeding and thrombotic events. Gene mutation analysis and blood constituent measurements were performed. RESULTS: The prevalence of bleeding among all HHT patients was 100 %, while thrombotic events were noted in 41.70 % of cases. Hepatic arteriovenous malformations (HAVMs) were identified in six patients, pulmonary arteriovenous malformations (PAVMs) in five patients, and cerebral arteriovenous malformations (CAVMs) in one patient. For patients with thrombosis, the discontinuation rates were 23.08 % for antiplatelet therapy and 33.33 % for anticoagulant therapy due to the increased risk of bleeding. Genetic mutations related to HHT were present in 16 patients, with ACVRL1 (activin A receptor-like type 1) mutations being the most frequent at 41.67 %, followed by ENG (endoglin) mutations at 20.83 %, and GDF2 (growth differentiation factor 2) mutations at 4.17 %. The incidence of PAVMs was 75.00 % in HHT1 patients with ENG mutations and 20 % in HHT2 patients with ACVRL1 mutations, while HAVMs occurred in 0 % and 40.00 % of these groups, respectively. Patients were divided into non-AVMs and AVMs groups. Compared to normal controls, von Willebrand factor (vWF) activity was significantly increased in all HHT patients (149.10 % vs. 90.65 %, P < 0.001). In the non-AVMs group, the median level of stromal cell-derived factor-1 (SDF-1) was significantly elevated (124.31 pg/mL vs. 2413.57 pg/mL, P < 0.05), while vWF antigen levels were markedly higher in the AVMs group (165.30 % vs. 130.60 %, P = 0.021). Further grouping of HHT patients based on bleeding and thrombosis phenotypes revealed that those with thrombosis had significantly higher median percentages of schistocytes (3.50 % vs. 0 %, P = 0.002), ferritin concentrations (318.50 µg/L vs. 115.50 µg/L, P = 0.001), and lactate dehydrogenase (LDH) levels (437 U/L vs. 105 U/L, P < 0.001). There were no significant differences in the activity of vWF, protein C (PC), protein S (PS), and factor VIII (FVIII) between the two groups. CONCLUSION: This study highlighted the complex relationship between arteriovenous malformations and genetic mutations in HHT patients. A comprehensive assessment of bleeding and thrombosis risks should be conducted for each HHT patient, additionally, further clinical studies are needed to explore the risk factors for thrombosis and anticoagulant-related bleeding in HHT.

Clinical and Molecular Characteristics of Megakaryocytes in Myelodysplastic Syndrome.

Objective Myelodysplastic syndrome (MDS) is a malignant clonal disorder of hematopoietic stem cells which is characterized by morphologic dysplasia. However, the pathological characteristics of megakaryocytes (MKs) in MDS patients with gene mutation are not well established. Methods Bone marrow MK specimens from 104 patients with primary MDS were evaluated, and all patients were distributed into two groups according to gene mutation associated with functional MKs. The morphologic and cellular characteristics of MKs and platelets were recorded and compared. Results The more frequently mutated genes in MDS patients were TUBB1 (11.54%), VWF (8.65%), NBEAL2 (5.77%), and the most common point mutation was TUBB1 p.(R307H) and p.(Q43P). Patients with MK mutation showed a decrease in adenosine diphosphate-induced platelet aggregation, high proportion of CD34 + CD61 + MKs (10.00 vs. 4.00%, p = 0.012), and short overall survival (33.15 vs. 40.50 months, p = 0.013). Further, patients with a higher percent of CD34 + CD61 + MKs (≧20.00%) had lower platelet counts (36.00 × 10 9 /L vs. 88.50 × 10 9 /L, p = 0.015) and more profound emperipolesis ( p = 0.001). By analyzing RNA-sequencing of MKs, differentially expressed mRNA was involved in physiological processes including platelet function and platelet activation, especially for MDS patients with high percent of CD34 + CD61 + MKs. The high levels of expression of CD62P, CXCL10, and S100A9 mRNA, shown by RNA sequencing, were validated by PCR assay. Conclusion High proportion of CD34 + CD61 + MKs was a poor prognostic factor in MDS patients with MK mutation. CD62P, CXCL10, and S100A9 may be the potential targets to evaluate the molecular link between gene defects and platelet function.

Mutation Ter462GlnextTer17 introduces a tail to C-terminus of protein C and causes venous thrombosis.

Protein C (PC), a vitamin K-dependent serine protease zymogen in plasma, can be activated by thrombin-thrombomodulin(TM) complex, resulting in the formation of activated protein C (APC). APC functions to downregulate thrombin generation by inactivating active coagulation factors V(FVa) and VIII(FVIIIa). Deficiency in PC increases the risk of venous thromboembolism (VTE). We have identified two unrelated VTE patients with the same heterozygous mutation (c.1384 T > C, p.Ter462GlnextTer17) in PROC. To comprehend the role of this mutation in VTE development, we expressed recombinant PC-Ter462GlnextTer17 in mammalian cells and evaluated its characteristics using established coagulation assay systems. Functional studies revealed a significant impairment in the activation of the mutant by thrombin or thrombin-TM complex. Furthermore, APC-Ter462GlnextTer17 demonstrated diminished hydrolytic activity towards the chromogenic substrate S2366. APTT and FVa degradation assays showed that both the anticoagulant activity of the mutant protein was markedly impaired, regardless of whether protein S was present or absent. These results were further supported by a thrombin generation assay conducted using purified and plasma-based systems. In conclusion, the Ter462GlnextTer17 mutation introduces a novel tail at the C-terminus of PC, leading to impaired activity in both PC zymogen activation and APC's anticoagulant function. This impairment contributes to thrombosis in individuals carrying this heterozygous mutation and represents a genetic risk factor for VTE.

Met343Val mutation disrupts the shuttling of Trp380 leading to a low-activity conformer of activated protein C and causes thrombosis.

BACKGROUND: Protein C (PC) pathway serves as a major defense mechanism against thrombosis by the activation of PC through the thrombin-thrombomodulin complex and subsequent inactivation of the activated factor (F)V (FVa) and FVIII (FVIIIa) with the assistance of protein S, thereby contributing to hemostatic balance. We identified 2 unrelated patients who suffered from recurrent thrombosis and carried the same heterozygous mutation c.1153A>G, p.Met343Val (M343V), in PROC gene. This mutation had not been previously reported. OBJECTIVES: To explore the molecular basis underlying the anticoagulant defect in patients carrying the M343V mutation in PROC. METHODS: We expressed PC-M343V variant in mammalian cells and characterized its properties through coagulation assays. RESULTS: Our findings demonstrated that while activation of mutant zymogen by thrombin-thrombomodulin complex was slightly affected, cleavage of chromogenic substrate by APC-M343V was significantly impaired. However, Ca2+ increased the cleavage efficiency by approximately 50%. Additionally, there was a severe reduction in affinity between APC-M343V and Na+. Furthermore, the inhibitory ability of APC-M343V toward FVa was markedly impaired. Structural and simulation analyses suggested that Val343 might disrupt the potential hydrogen bonds with Trp380 and cause Trp380 to orient closer to His211, potentially interfering with substrate binding and destabilizing the catalytic triad of APC. CONCLUSION: The M343V mutation in patients adversely affects the reactivity and/or folding of the active site as well as the binding of the physiological substrate to the protease, resulting in impaired protein C anticoagulant activity and ultimately leading to thrombosis.

Heterozygous Prothrombin Mutation-Associated Thrombophilia.

BACKGROUND: Venous thromboembolism (VTE) is predisposed by thrombotic mutations in patients with hereditary thrombophilia. Although prothrombin deficiencies caused by homozygous or compound heterozygous mutations are associated with bleeding diathesis, rare cases have shown a correlation between heterozygous prothrombin mutations and thrombosis. MATERIALS AND METHODS: We surveyed genetic variants involved in thrombosis and hemostasis in 347 patients with unprovoked VTE or having a positive family history of thrombosis. For patients identified with heterozygous prothrombin mutations, we conducted family investigations and performed a thrombin generation test (TGT) to elucidate the thrombotic risk. Novel mutants were expressed and subjected to functional assays to clarify the underlying thrombotic mechanisms. RESULTS: Heterozygous prothrombin mutations were identified in 3.5% of patients (12/347), including three novel mutations Phe382Ser, Phe382Leu, and Asp597Tyr found in one patient each, as well as previously reported Arg541Trp mutation in four patients and Arg596Gln mutation in five patients. A total of 42 mutation carriers were identified within the 12 pedigrees, among whom 64.3% (27/42) had experienced thrombotic events. TGT results demonstrated hypercoagulability for carriers of the five mutations, with Arg596Gln showing the highest thrombin generation potential followed by Arg541Trp. The Phe382-associated mutations severely impaired thrombomodulin-binding ability of thrombin, resulting in obviously reduced protein C (PC) activation. The Asp597Tyr mutation exhibited a mild reduction in both inactivation by antithrombin and PC activation reactions. CONCLUSION: The presence of heterozygous prothrombin mutations represents a potential genetic predisposition for VTE. All thrombosis-associated mutations potentiate coagulation activity by either conferring antithrombin resistance and/or impairing PC pathway activity.

Ser252Asn Mutation Introduces a New N-Linked Glycosylation Site and Causes Type IIb Protein C Deficiency.

BACKGROUND: Protein C (PC) is a vitamin K-dependent anticoagulant serine protease zymogen which upon activation by the thrombin-thrombomodulin (TM) complex downregulates the coagulation cascade by degrading cofactors Va and VIIIa by limited proteolysis. We identified a thrombosis patient who carried a heterozygous mutation c.881G > A, p.Ser252Asn (S252N) in PROC. This mutation was originally described in a report of novel mutations in patients presenting with defective PC anticoagulant activity in Paris. The research identified PC-S252N (the "Paris" mutation) in a propositus and her family members and highlighted the critical role of Ser252 in the anticoagulation process of activated PC (APC). MATERIAL AND METHODS: We expressed the PC-S252N mutant in mammalian cells and characterized the properties in coagulation assays to decipher the molecular basis of anticoagulant defect of this mutation. RESULTS: We demonstrated that PC-S252N had a diminished ability to TM binding, which resulted in its impaired activation by the thrombin-TM complex. However, APC-S252N exhibited a slightly stronger cleavage capacity for the chromogenic substrate. Meanwhile, the catalytic activity of APC-S252N toward FVa was significantly reduced. Sequence analysis revealed that Ser252 to Asn substitution introduced a new potential N-linked glycosylation site (252NTT254) in the catalytic domain of PC, which adversely affected both the activation process of PC and anticoagulant activity of APC. CONCLUSION: The new N-glycosylation site (252NTT254) resulting from the mutation of Ser252 to Asn252 in PROC affects the overall structure of the protease, thereby adversely affecting the anticoagulant function of protein C. This modification has a negative impact on both TM-promoted activation of protein C and APC cleavage of FVa, ultimately leading to thrombosis in the patient.

Molecular basis of type I antithrombin deficiency in two women with recurrent venous thromboembolism in the first trimester of pregnancy.

Inherited antithrombin (AT) deficiency carries a 50% risk of venous thromboembolism (VTE) during pregnancy. Here, we investigated the molecular basis of type I AT deficiency in two women with recurrent VTE in the first trimester of pregnancy. Phenotype analysis showed both probands had almost 50% of normal AT levels. Two novel heterozygous AT mutations were identified: g.7920C>T resulting in a Trp225Cys mutation in case 1 and g.13863C>A causing an Ala404Asp mutation in case 2. Transient expression of either wild-type (WT) or mutant AT expression vectors in HEK293T and CHO cells showed impaired secretion of both AT mutant proteins. Immunofluorescence analysis revealed that the staining of AT-Trp225Cys in both endoplasmic reticulum (ER) and Golgi apparatus was similar to that of AT-WT, and the staining of AT-Ala404Asp was mainly present in ER but was weaker than that of AT-WT. These results revealed that the type I AT deficiency in two patients was caused by impaired secretion of the AT-Trp225Cys and AT-Ala404Asp mutant proteins, respectively. The two mutations are associated with a high risk of thrombotic onset and women with these AT mutations are prone to VTE in early pregnancy.

[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.

[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.