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A 28-year-old woman was found to have coagulation factor Ⅷ activity (FⅧ∶C) <1% and von Willebrand factor antigen (VWF∶Ag) <1% during routine prenatal examinations. No pathogenic variation was found in the exon region of the VWF gene using next-generation sequencing. The clinical presentation of this patient does not match the clinical characteristics of type Ⅲ hemophilia [von Willebrand disease (VWD) ]; therefore, third-generation sequencing technology was used to perform whole-genome sequencing on the patient and her family members. Multiple members of the patient's paternal family carried a heterozygous variant of VPS33B, c.869G>C. The family members carrying this variant all had varying degrees of reduced VWF levels (39% -56% ). Moreover, the proband was detected with the heterozygous variant c.1474dupA in GP1BA. The ACMG and Clinvar databases determined that this variation was associated with platelet-type pseudo VWD. The decrease in VWF levels caused by heterozygous variations in VPS33B in families is the first international report, and no previous studies have reported cases of severe decrease in plasma VWF levels caused by double heterozygous variations in VPS33B and GP1BA.

Objective: To assess the significance of DDAVP use in the diagnosis and treatment of VWD. Methods: An analysis of 15 VWD cases who referred to Hematology Division of First affiliated Hospital of Soochow University and treated with DDAVP from March 2016 to August 2018 was conducted. Efficacy and treatment response of DDAVP were monitored by observations of changes in factor Ⅷ procoagulant (FⅧ∶C) and von Willebrand Factor (VWF) related indicators before and 2 h after DDAVP injection. Results: Of 15 cases with VWD, 7 males and 8 females with a median age of 23 (6-46) years, 7 of 9 type I VWD patients achieved complete response (CR) , 1 type 2A VWD case CR, 5 type 3 VWD ones no response (NR) . The VWF multimer analysis in 5 patients combined with other plasma VWF values were in accordance with the known diagnosis. Conclusions: DDAVP was effective in most type 1 patients, and ineffective in some type 2 and almost all type 3 cases. It was helpful for diagnosis and subsequent treatment planning.

Objective: To deepen the understanding of acquired von Willebrand syndrome (AVWS)., Methods: The clinical data of 3 patients were analyzed and related literature were reviewed., Results: ① Case 1, a 70- year- old male, diagnosed as Waldenstrom macroglobulinemia and AVWS, was presented with spontaneous epitaxis and bruising. The VWF∶Ag level was 16%. Treatment was initiated with VWF concentrates. Two cycles of chemotherapy with Bortezomib, thalidomide and Dexamethasone were followed. Partial remission was achieved. Half- year' follow- up showed no sign of spontaneous hemorrhage. ② Case 2, a 48- year- old female, diagnosed as monoclonal gammopathy of undetermined significance and AVWS, was presented with repeated epitaxis. The VWF∶Ag level was 7%. Because the bleeding was slight and self-relieved, no specific treatment was addressed. She was followed up for one and a half year. ③ Case 3, a 50- year- old man, diagnosed as monoclonal gammopathy of undetermined significance and AVWS, was referred to our hospital for presentation with significant hematomas. VWF∶ Ag was reduced to 12%. VWF- containing cryoprecipitate, plasma, intravenous immunoglobulin and rituximab were used to control his bleeding symptom. During the follow-up, spontaneous hemorrhage still occurred occasionally., Conclusions: Acquired von Willebrand syndrome presented with heterogeneous symptoms. The level of VWF∶Ag and VWF∶Rco for patients with bleeding disorder should be performed. Abnormal bleeding symptoms in elderly patients without personal or family history of bleeding should prompt consideration of the underlying disorders. Treatment included controlling acute bleeding, curing the underlying diseases and preventing bleeding in high- risk situations. The prognosis of acquired von Willebrand syndrome is mainly related to the underlying diseases.

Objective: To investigate the genetic defect and its mechanism in a patient with congenital afibrinogenemia., Methods: The plasma fibrinogen activity and antigen of the patient was determined using the Clauss method and immuno-nephelometric assay, respectively. Genomic DNA was isolated from peripheral blood of the proband and his related family members. All exons and exon-intron boundaries of the three fibrinogen genes (FGA, FGB, FGG) were amplified by PCR followed by direct sequencing. Thrombin fibrin aggregation curve were detected in the plasma of the patient. Wild-type and mutation type fibrinogen vectors were constructed, and then transfected into COS-7 cells. The wild-type and mutant proteins from the culture media and cell lysates were tested by Western blot and ELISA., Results: APTT, PT, TT were significantly longer in the proband. Plasma fibrinogen activity and antigen of the patient could not be detected using the Clauss method and immuno-nephelometry, respectively. Gene analysis revealed that a novel homozygous GTTT insertion between nucleotides 2833 and 2834 in FGB exon 2 in the proband. The proband's father, mother, brother and son were heterozygous. The polymerization curves of the patient did not show a lag phase or final turbidity, compared with the normal controls. Western blot analysis showed the lack of complete half-molecules of the fibrinogen molecule and fibrinogen in patient's plasma under non-reducing conditions. It also could not detect the truncated Bβ chain under reducing conditions. Abnormal fibrinogen molecule (molecule weight>340 000) were found in transfected COS-7 cells by Western blot, which indicated that the mutation caused the abnormal intracellular fibrinogen molecule assembly. The fibrinogen band was absent in culture media transfected by the mutation. Fibrinogen levels of mutant fibrinogen were no significant different from those of wild-type fibrinogen in cell lysates by ELISA analysis [(2.47 ± 0.30) μg/ml vs (2.65±0.60) μg/ml, P=0.0889]; However, the levels of the mutant fibrinogen were statistically significant lower than those of wild type fibrinogen in culture media [(0.01 ± 0.01) μg/ml vs (3.80±0.80) μg/ml, P=0.0001]., Conclusion: Congenital afibrinogenemia was caused by this frameshift mutation in exon 2 of FGB. This novel mutation impaired fibrinogen assembly and secretion.

Objective: To investigate the susceptibility of von Willebrand factor (VWF) type 2A mutant A1500E to proteolysis by metalloprotease ADAMTS13 and to provide the direct supports for the pathogenesis of VWF mutation A1500E responsible for von Willebrand disease (VWD) type 2A., Methods: Recombinant wild-type VWF (WT-VWF) and A1500E mutant VWF transiently expressed on transfected HeLa cell lines. Expression media were collected and concentrated, then cleaved directly by recombinant ADAMTS13 (rADAMTS13). Compared with WT-VWF, the susceptibility of A1500E mutant VWF to proteolysis by ADAMTS13 was analyzed using SDS-agarose gel VWF multimers analysis., Results: In vitro the expression of VWF:Ag in the supernatants of WT-VWF and A1500E mutant VWF were 1.10 U/ml and 0.78 U/ml, respectively, while VWF:Ag in cells lysates of A1500E mutant VWF was 90.6% of that of WT-VWF. The SDS-agarose gel VWF multimers analysis showed that there were no differences between WT-VWF and A1500E mutant VWF. The A1500E mutant VWF could be efficiently cleaved by ADAMTS13 under static condition without denaturants such as urea and guanidine HCl. VWF multimeric analysis showed that high and intermediate molecular weight multimers dramatically decreased while low molecular weight multimers obviously increased. Conversely, WT-VWF could not be cleaved by ADAMTS13 under the same condition., Conclusion: The A1500E mutation resulted in VWF more susceptible to ADAMTS13-dependent proteolysis, which belonged to VWD type 2A group 2 mutation.

Objective: To construct the expression vectors of vWF73 and vWF114 fragments of von Willebrand factor (vWF) A2 domain, and to express glutathione S-transferase (GST) fusion proteins in E. coli, and to explore their values in measuring ADAMTS13 activity as substrates., Methods: The DNA fragments encoding vWF73 and vWF114 were generated using PCR and separately cloned into pGEX-6P-1, a Schistosoma japonicum GST fusion expression vector. The expression of GST-vWF73-H and GST-vWF114-H was induced in liquid culture, followed by purification with Ni-NTA agarose column. The cleavage of two GST fusion proteins by recombinant ADAMTS13 (rADAMTS13) or plasma from normal individuals and thrombotic thrombocytopenic purpura (TTP) patients were identified by Western blot. Based on an enzyme-linked immunosorbent assay (ELISA) with anti-GST and anti-His monoclonal antibodies, GST-vWF73-H and GST-vWF114-H were used to measure plasma ADAMTS13 activity as substrates., Results: Two small molecular substrates of ADAMTS13, GST-vWF73-H and GST-vWF114-H, are expressed and purified, which could be specifically cleaved by rADAMTS13 or plasma from healthy individuals, but not by plasma from congenital or idiopathic TTP patients. An ELISA assay was established to detect plasma ADAMTS13 activity using GST-vWF73-H and GST-vWF114-H as substrates., Conclusions: Two GST fusion proteins in vWF A2 domain, vWF73 and vWF114, were expressed effectively using a prokaryotic expression system and could be used to detect ADAMTS13 activity as substrates.

Objective: To study the influence of C-terminal domain of ADAMTS13 on its cleaving activity., Methods: The full-length wild-type (WT) and C-terminal domain truncated type (TT, TSP8 + CUB domains were deleted) of human ADAMTS13 recombinant protein were transfected into and permanent expressed on Hela cells. Western blot and R-CBA were used to directly detect the activities of the two recombinant proteins under the static and stressed condition respectively. ELISA was used to compare the binding abilities of the two proteins by coating with vWF., Results: The recombinant proteins were identified by Western blot with anti-his-tag or anti-ADAMTS13 antibodies. With pretreatment of 1.5 M urea, the enzyme activity of TT was significantly higher than that of WT, and so did in binding ability with vWF While, only WT could cleave vWF under high stress., Conclusion: The distal carboxyl-terminal TSP8 together with CUB domains of ADAMTS13 may affect the enzyme activity by regulating the binding of ADAMTS13 to vWF in different conditions, and they are very important for the enzyme activity under high stress force condition.

Objective: To analyze the phenotype and genotype of a family with inherited dysfibrinogenemia., Methods: Assays of coagulation, including activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT), were carried out with Stago Compact in the proband and his family members. The activity and antigen of fibrinogen in plasma were determined by Clauss and immunoturbidimetry, respectively. Fibrinogen and its constituent were analyzed by Western blot with nonreducing 4%-20% SDS-polyacrylamide gel electrophoresis (PAGE). All exons and exon-intron boundaries of fibringen genes FGA, FGB and FGG were analyzed by PCR and then direct sequencing., Results: The proband had normal APTT and PT, but prolonged TT. The activity of fibrinogen in plasma was decreased while its antigen level was normal. These abnormalities were also found in his mother and a sister. Genetic analysis revealed heterozygous G1233A in the exon 2 of FGA originating from his mother, which resulted in Arg16His missense mutation., Conclusion: Inherited dysfibrinogenemia was caused by Arg16His mutation in exon 2 of FGA, and this is the first case reported in a Chinese family.

Objective: To investigate the effects of Annexin II (AnxA2) gene silencing by siRNA on proliferation and invasive potential of lymphoma cell line Jurkat cells., Methods: A synthesized siRNA duplex targeting to AnxA2 was transfected into Jurkat cells. Transfection efficiency was analyzed by real-time PCR and flow cytometry. MTT assay for cell proliferation and transwell plates for invasive potential were performed., Results: Compared with the negative controls, the cell proliferation inhibitory rate of the AnxA2 siRNA transfected Jurkat cells was significantly increased at 24 h, 48 h and 72 h [(17.4 +/- 2.3)%, (22.4 +/- 3.8)%, (37.6 +/- 1.5)% vs (-1.3 +/- 5.1)%, (-5.5 +/- 4.4)%, (-10.8 +/- 5.5)%, respectively, P<0.05]. The cell invasive potential of the transfected Jurkat cells was inhibited remarkably at 48 h (11.3 +/- 4.2 vs 54.3 +/- 8.7, P<0.01)., Conclusion: AnxA2 gene silenced by siRNA can inhibit the proliferation and the invasive potential of Jurkat cells remarkably.