Effects of the ITGA2B Nonsense Mutation (c.2659C > T, p.Q887X) on Platelet Function in a Mouse Model of Glanzmann's Thrombasthenia Generated with CRISPR/Cas9 Technology / 中国实验血液学杂志

OBJECTIVE@#To construct a mouse model of Glanzmann's thrombasthenia (GT) with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation by CRISPR/Cas9 technology, and then further explore the expression and function of glycoprotein αIIbβ3 on the surface of platelet membrane.@*METHODS@#The donor oligonucleotide and gRNA vector were designed and synthesized according to the ITGA2B gene sequence. The gRNA and Cas9 mRNA were injected into fertilized eggs with donor oligonucleotide and then sent back to the oviduct of surrogate mouse. Positive F0 mice were confirmed by PCR genotyping and sequence analysis after birth. The F1 generation of heterozygous GT mice were obtained by PCR and sequencing from F0 bred with WT mice, and then homozygous GT mice and WT mice were obtained by mating with each other. The phenotype of the model was then further verified by detecting tail hemorrhage time, saphenous vein bleeding time, platelet aggregation, expression and function of αIIbβ3 on the surface of platelet.@*RESULTS@#The bleeding time of GT mice was significantly longer than that of WT mice (P<0.01). Induced by collagen, thrombin, and adenosine diphosphate (ADP), platelet aggregation in GT mice was significantly inhibited (P<0.01, P<0.01, P<0.05). Flow cytometry analysis showed that the expression of αIIbβ3 on the platelet surface of GT mice decreased significantly compared with WT mice (P<0.01), and binding amounts of activated platelets to fibrinogen were significantly reduced after thrombin stimulation (P<0.01). The spreading area of platelet on fibrinogen in GT mice was significantly smaller than that in WT mice (P<0.05).@*CONCLUSION@#A GT mouse model with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation has been established successfully by CRISPR/Cas9 technology. The aggregation function of platelet in this model is defective, which is consistent with GT performance.

Effect of Recombinant Protein in the Spacer Domain on ADAMTS13 Activity / 中国实验血液学杂志

OBJECTIVE@#To obtain the recombinant protein of spacer domain in von Willebrand factor cleaving protease (ADAMTS13), and further study its biological function in ADAMTS13.@*METHODS@#The prokaryotic expression vector was constructed by using the template of plasmid with full-length ADAMTS13, and then transfected into E coli., following the induction of IPTG with the low temperature (30 ℃). The recombinant protein was purified with Ni-NTA agarose column by gradient imidazole. The purity and immune activity of purified products were identified with SDS-PAGE and Western blot respectively. By Adding the recombinant protein to the plasma of immune-mediated thrombotic thrombocytopenic purpura (iTTP) patients, the activity of ADAMTS13 was tested.@*RESULTS@#The prokaryotic expression vector was successfully constructed and the protein of spacer domain with the high purity was obtained. Western blot showed that the recombinant fragment could both react with monoclonal antibody against 6×His and polyclonal sheep IgG against ADAMTS13 (Gln34-Trp688). The protein formed a main lane at the position of 15 kDa with SDS-PAGE. It was demonstrated that the recombinant protein could efficiently elevate the ADAMTS13 activity in plasma of iTTP patients to reach normal level by functional experiment.@*CONCLUSION@#The recombinant protein has high purity and immune activity, which provides the experimental basis for further research on mechanism of iTTP involved in spacer domain.

Development of Hybridoma Cell Lines Secreting Monoclonal Antibodies against ADAMTS13 / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To establish the hybridoma cell lines secreting anti-ADAMTS13-T7 monoclonal antibodies (MA6) and to construct the MAb directed against different domains of ADAMTS13-T7.</p><p><b>METHODS</b>The trucated type protein ADAMTS13 of eukaryote-expressed recombinant ADAMTS13-T7 was constructed and was purified by using Ni-NTA agarose. Then the purified ADAMTS13 was used to immunize the BALB/c mice; the antiserum titer of ADAMTS13 protein of immunized mice was deteceted by ELISA. The spleen was taken from mice for construcing the single cell suspension, then the single cell suspension was mixed with myeloma cells SP2/0 at 10:1 ratio for cell fusion, and the elution culture of hybridoma cells was carried out; after 2 weeks, the wells in which clones were well grown were selected for detection, then the positive clonal wells were expansively cultured and the detection again was performed.</p><p><b>RESULTS</b>The purified ADAMTS13-T7 protein was gained. The ELISA showed that the antiserum titer in mice immumized by ADAMTS13-T7 protein was 1:20000. The results of fusion culture by hybridoma techmique showed that 30 hyridoma cell lines secreting MAb against recombinant ADAMTS13 were established.</p><p><b>CONCLUSION</b>The hybridoma cell lines secreting MAb against recombinant ADAMTS13 have been successfully established by fusion culture, which provide more powerful tools for further screening the MAb with certain functional activity and studying the structure and function of ADAMTS13.</p>

Identification of Monoclonal Antibodies against von Willebrand Factor Cleaving Protease (ADAMTS13) and Their Function / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To construct and identify the monoclonal antibodies against von willebrand factor cleaving protease(ADAMTS13), and to study their biological function.</p><p><b>METHODS</b>BALB/c mice were immunized by purified recombinant ADAMTS13 truncated eukaryotic protein (ADAMTS13-T7). Murine anti-human ADAMTS13 monoclonal antibodies (McAbs) were constructed by standard hybridoma technology and identified by ELISA. The recognition of McAbs with full-length recombinant ADAMTS13 protein was identified by Western blot. In function assay, the influence of McAbs on the proteolysis of vWF by ADAMTS13 was observed.</p><p><b>RESULTS</b>A group of 6 murine anti-ADAMTS13 McAbs was obtained with the clone number 1G11, 2F11, 6G3, 9E1, 10A8 and 10B4. In ELISA, the highest titers of 1G11 and 2F11 were observed, both of which showed a higher affinity to ADAMTS13-T7 than full-length ADAMTS13. The Western blot demonstrated that the 6 McAbs all could recognize ADAMTS13, among which 1G11 and 2F11 showed stronger reaction with ADAMTS13. In addition, under the denatured conditions, 1G11 and 2F11 could inhibit hydrolysis of vWF by ADAMTS13, and that was stronger with the increasing of McAbs concentration.</p><p><b>CONCLUSION</b>McAbs against ADAMTS13 have been gained, two of which are inhibitory antibodies.</p>

Changes of ADAMTS13 activity and vWF antigen level in patients with acute myelogenous leukemia and their significance / 中国实验血液学杂志

This study was purposed to investigate the changes of von Willebrand factor cleaving protease (ADAMTS13) activity and vWF antigen level in patients with acute myelogenous leukemia (AML) before and after treatment and evaluate their clinical significance. Seventy-three AML patients were enrolled in this study, the sodium citrate anticoagulated plasma was collected before and after their induction chemotherapy. Fluorescence resonance energy transfer substrate vWF73 (FRETS-vWF73) assay was established to detect the plasma ADAMTS13 activity while vWF antigen level was measured by ELISA. The results showed that the ADAMTS13 activity in newly diagnosed patients with AML before induction therapy was obviously lower than that in normal controls (63.3 ± 25.5)% vs (105.1 ± 37.7)(P < 0.01), while the vWF antigen level was higher than that in normal controls (226.6 ± 127.0)% vs (111.4 ± 39.7)% (P < 0.01). After standard induction chemotherapy, the ADAMTS13 activity of AML patients in complete remission period was higher than that in AML patients before therapy (P < 0.01), and was not significant difference with that in normal controls; the vWF antigen was significantly lower than that in AML patients before therapy (P < 0.01), but it still was higher than that in controls (P < 0.05). The ADAMTS13 activity in newly diagnosed AML patients complicated with infection before therapy was obviously lower than that in AML patients without infection (52.2 ± 20.6)% vs (73.9 ± 24.7)% (P < 0.01), while the vWF antigen level was significantly higher than that in AML patients without infection (262.2 ± 135.7)% vs (193.8 ± 110.2)% (P < 0.05). The ADAMTS13 activity in AML patients with disseminated intravascular coagulation (DIC) was significantly lower than that in AML patients without DIC (42.0 ± 14.5)% vs (73.4 ± 22.7)% (P < 0.01), while the vWF antigen level was obviously higher that in AML patients without DIC (274.2 ± 140.0)% vs (204.7 ± 115.5)% (P < 0.01). It is concluded that the ADAMTS13 activity in newly diagnosed AML patients befor induction therapy has been confiremed to be lower and the vWF antigen level to be higher, especially in AML patients with infection or DIC. The ADAMTS13 and vWF antigen may play a role in the pathogenesis of AML and the formation of infection and DIC.

Isolation, culture and characterization of outgrowth endothelial cells from the human peripheral blood / 中国实验血液学杂志

Compared with endothelial progenitor cells, outgrowth endothelial cells (BOECs) from peripheral blood are rich in protein for blood angiogenesis and cell adhesion, similar to mature endothelial cells in biological characteristics. Moreover, they are now replacing human umbilical vein endothelial cells for the latter's limited life span and drift of phenotype, and might become a new tool for exploring the vascular abnormalities. This study was aimed to establish the protocol of producing BOECs, and then analyze the cell phenotype and function of BOECs. Mononuclear cells were collected from peripheral blood by gradient centrifugation and then seeded on plates and cultivated in EGM-2 medium for 4 weeks. The morphological changes of cells were observed and cell phenotype was examined by flow cytometry. VWF multimers were used to analyse the distribution of vWF multimers in superment of BOECs and the storage of vWF in BOECs, and the secretion of vWF in BOECs under stimulation was detected by confocal fluorescence microscopy. The results showed that after 4-week-culture in vitro, the cell colonies and characteristic cobblestone-like morphology of BOECs were found in plates. For another three weeks of expansion, BOECs expressed CD31, CD34, and EPCR, without the expression of CD14, CD45 and CD133. The vWF from BOECs cell supernatant shared the same multimer pattern as that in normal plasma. By confocal fluorescence microscopy, vWFs were observed in BOECs. The amount of vWF increased in cells, and vWF strings were formed on cell surface by the stimulation of phorbol-12-myristate-13-acetate(PMA). It is concluded that the BOECs are first successfully established, and the phenotype and function of BOECs are analyzed. They are the native cell models for the pathogenesis of von Willebrand diseases (vWD), and may be used as new gene therapy tools for vWD.

Congenital afibrinogenemia caused by a novel insertion mutation in the FGB gene / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the genetic defect and its mechanism in a patient with congenital afibrinogenemia.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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].</p><p><b>CONCLUSION</b>Congenital afibrinogenemia was caused by this frameshift mutation in exon 2 of FGB. This novel mutation impaired fibrinogen assembly and secretion.</p>

Construction of ADAMTS13-pEGFP-N1 vector and its expression in HeLa cells / 中国实验血液学杂志

This study was aimed to construct a pEGFP-N1 vector of von Willebrand factor cleaving protease (ADAMTS13, a disintegrin and metalloprotease with a thrombospondin type 1 motifs 13) so as to pave the way for further studying its synthesis and secretion. Human full-length cDNA sequence of ADAMTS13 was acquired by polymerase chain reaction (PCR) with Phusion(®) High-Fidelity (NEB), then the PCR product was double digested with EcoRI and XhoI. After digestion, the ADAMTS13 cDNA sequence was purified and recombined with the pEGFP-N1 vector. The DNA sequence analysis showed that ADAMTS13 was ligated to the pEGFP-N1 vector correctly. After transient expression in HeLa cells, the expression of EGFP could be detected by fluorescent microscopy, and the expression of ADAMTS13 protein could be detected by SDS-PAGE and Western blot. It is concluded that the ADAMTS13-pEGFP-N1 vector is successfully constructed, and it can be widely used in further research on the mechanism of the synthesis and secretion of ADAMTS13.

Increased susceptibility of recombinant type 2A von Willebrand factor mutant A1500E to proteolysis by ADAMTS13 / 中华血液学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>The A1500E mutation resulted in VWF more susceptible to ADAMTS13-dependent proteolysis, which belonged to VWD type 2A group 2 mutation.</p>

Expression of vWF73 and VWF114 fragments of von Willebrand factor A2 domain and their utilization in detecting ADAMTS13 activity / 中华血液学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>

Evaluation and clinical application of a new method for detecting ADAMTS13 activity / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>A severe deficiency of ADAMTS13 activity contributes to the pathogenesis of thrombotic thrombocytopenic purpura (TTP). Measuring the activity of ADAMTS13 is helpful for the diagnosis of TTP and the prognostic monitor in TTP patients. Most available assays are cumbersome and costly, so not easily adapted to routine laboratories. ADAMTS13 cleaves von Willebrand factor (VWF) within the domain A2, located between domains A1 and A3. Therefore, specific assays for ADAMTS13 activity could be based on the different structures of VWF before and after the cleavage. Using this hypothesis we try to establish a new and simple method to determine ADAMTS13 activity.</p><p><b>METHODS</b>First, plasma samples were exposed in denaturing condition to allow cleavage of VWF by ADAMTS13. Then, the ADAMTS13 activity was measured with two novel monoclonal antibodies, SZ-129 and SZ-125, which specifically recognize the VWF A1 and A3 domains by using a two-site sandwich ELISA. Compared with a residual-collagen binding assay (R-CBA), plasma ADAMTS13 activities in 161 samples were assessed, and the inhibitory activities of ADAMTS13 autoantibody in 24 TTP patients were determined. The relationship of these two assays was analyzed by linear correlation, and the sensitivity and specificity of the new assay was also evaluated.</p><p><b>RESULTS</b>Plasma ADAMTS13 activities in normal people and TTP, acute myocardial infarction (AMI), and idiopathic thrombocytopenic purpura (ITP) patients determined by the new assay were (89.75 +/- 7.93)%, (17.63 +/- 18.71)%, (68.55 +/- 18.08)%, (85.83 +/- 9.84)%, respectively. Results were consistent with those of R-CBA, the squared correlation factor was 0.9183 of the two assays. The new assay can easily discriminate a TTP plasma sample from a non-TTP plasma sample (P < 0.01), and the coefficient of variation for the new assay was 6.17%. In 23 idiopathic TTP patients, the inhibitor activity of ADAMTS13 autoantibody ranged from 12% to 100%, while no inhibitory activity was detected in one hereditary TTP patient.</p><p><b>CONCLUSION</b>This new and simple assay for ADAMTS13 activity could be used routinely in the clinic to determine the activity of ADAMTS13.</p>

Inherited dysfibrinogenemia caused by Arg16His mutation in alpha chain of fibrinogen / 中华血液学杂志

<p><b>OBJECTIVE</b>To analyze the phenotype and genotype of a family with inherited dysfibrinogenemia.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>Inherited dysfibrinogenemia was caused by Arg16His mutation in exon 2 of FGA, and this is the first case reported in a Chinese family.</p>

Research on the C-terminal domain of ADAMTS13 regulates its cleaving activity / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the influence of C-terminal domain of ADAMTS13 on its cleaving activity.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Effects of Annexin II gene silencing by siRNA on proliferation and invasive potential of Jurkat lymphoma cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of Annexin II (AnxA2) gene silencing by siRNA on proliferation and invasive potential of lymphoma cell line Jurkat cells.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>AnxA2 gene silenced by siRNA can inhibit the proliferation and the invasive potential of Jurkat cells remarkably.</p>