Development Of A Novel Flow Cytometric Immunobead Array To Quantify VWF:Ag And VWF:GPIbR And Its Clinical Application

Background:Von Willebrand factor(VWF)is a large multimeric plasma glycoprotein,which is synthesized by vascular endothelial cells and megakaryocytes.It has a series of oligomers ranging from 500 kDa up to more than 10,000 kDa.Generally,VWF is stored in?-granules of platelets and Weibel-Palade bodies of endotheliocytes,where it is in the form of large multimeric species(ultra-large VWF,UL-VWF).Once there is blood vessel damage,VWF will be released into the circulation to participate in hemostasis process.VWF can be as a bridge between endothelial matrix(via its A3 domain bind collage)and platelet(via its A1domain bind platelet glycoprotein Ib?).VWF can also be a carrier protein for F VIII to help the latter escape enzymatic hydrolysis of thrombin,which can extend the half-life of F VIII greatly.The large molecular weight of VWF is the major functional molecular,and ADAMTS13(a disintegrin and metalloprotease with thrombospondin type 1 repeats,member 13)can bind the A2 domain of VWF to cleave VWF into an inactive small fragment.ADAMTS13 plays an important role in maintaining the balance between different molecular weights of VWF.The quantitative deficiencies and/or qualitative defects in VWF will lead to von Willebrand disease(VWD),the most common hereditary hemorrhagic disease with 1%.Generally,von Willebrand factor antigen(VWF:Ag)and VWF ristocetin-triggered GPIb binding assay(VWF:GPIbR)will be first chosen for initial diagnosis of VWD,other assays are then used for further diagnosis and classification of VWD.Acute myocardial infarction(AMI)results from the rupture or erosion of an atherosclerotic plaque,which can induce thrombosis,and thus coronary artery occlusion and myocardial ischemia.The latest data shows that cardiovascular death has surpassed the tumor,ranking the first cause of death in urban and rural areas:the urban and rural AMI mortality rate has reached 55.32/100 thousand and 68.60/100 thousand,respectively.It's reported that there are also quantitative deficiencies and/or qualitative defects of VWF in AMI.Hence,VWF is a novel potential anti-thrombosis target drug as a risk factor for bleeding and thrombosis.Objective:The aim of this study is to establish a high-specific,accurate and rapid detection of VWF:Ag,VWF:GPIbR by flow cytometric immunobread array(FCIA)comparing the traditional method(time-consuming,poor reproducibility),then apply it in AMI,then evaluate the role of VWF:GPIbR and VWF multimer in the development of AMI.Methods:(1)21 patients with VWD from the Department of Hematology,the First Affiliated Hospital of Soochow University,146 patients with AMI in the First Affiliated Hospital of Henan University of Science and Technology,and the First Affiliated Hospital of Soochow University were collected,and 105 normal volunteers were used as control group(control,CTL).(2)The establish of VWF:Ag-FCIA:anti-human VWF monoclonal antibody SZ29IgG was coated microspheres overnight,added diluted plasma after blocked,finally incubated with fluorescein isothiocyanate(FITC)conjugated sheep-anti-human VWF IgG polyclonal antibody(FITC-GAH VWF IgG),and detected by flow cytometry.(3)The establish of VWF:GPIbR-FCIA:anti-human platelet GPIb?monoclonal antibody SZ151 IgG was coated microspheres overnight,added diluted plasma and ristocetin after blocked,finally incubated with FITC-GAH VWF IgG,and detected by flow cytometry.(4)Plasma VWF:Ag and VWF:GPIbR in CTL and VWD patients were tested by FCIA and enzyme-linked immunosorbent assay(ELISA)in parallel,and the feasibility and merits of FCIA method were evaluated.(5)Plasma VWF:Ag and VWF:GPIbR in AMI patients were tested by FCIA;plasma VWF:CB,ADAMTS13 activity in CTL and AMI patients were tested by ELISA;and plasma in CTL and AMI was also analyzed by VWF multimer analysis.(6)SPSS 19.0 and GraphPad Prism 5.0 software were used to analyse the data,and evaluate the clinical value in AMI.Results:(1)The VWF:Ag-FCIA method was successfully established.The VWF:Ag-FCIA showed a good linear regression(R~2=0.9941).The intra-assay and inter-assay coefficient variations(CVs)were 9.2%and 12.6%,respectively.The sensitivity,specificity and accuracy were 95.2%,89.5%and 90.5%.Linear regression analysis was used to compare the correlation between VWF:Ag-FCIA and ELISA,and the Pearson correlation could reach 0.855(P<0.0001).The Bland-Altman test showed that the quantification of VWF:Ag by FCIA agreed well with ELISA.Bland-Altman bias was 1.123%with 95%limits of agreement spanning from-45.06%to 47.30%.In addition,VWF:Ag-FCIA was superior to VWF:Ag-ELISA in detecting low level of VWF:Ag(1.22%vs 6.41%),so it has better sensitivity to the diagnosis of type 3 VWD.(2)The VWF:GPIb R-FCIA method was successfully established.The VWF:GPIbR-FCIA showed a good linear regression(R~2=0.9876).The intra-assay and inter-assay coefficient variations(CVs)were 7.7%and 13.5%,respectively.The sensitivity,specificity and accuracy were 95.2%,98.1%and 97.6%.Linear regression analysis was used to compare the correlation between VWF:GPIbR-FCIA and VWF:GPIbR-ELISA,and the Pearson correlation could reach 0.813(P<0.0001).And VWF:GPIbR-FCIA had higher specificity and sensitivity comparing with ELISA.The Bland-Altman test showed that the quantification of VWF:GPIbR using FCIA agreed well with ELISA.Bland-Altman bias was 9.947%with 95%limits of agreement spanning from-41.00%to 60.90%.(3)The application of VWF:Ag-FCIA and VWF:GPIbR-FCIA:the mean levels of VWF:Ag,VWF:GPIbR in AMI patients were significantly higher in comparison with those in CTL(299.4%[37.3%-966.7%]vs 73.5%[7.8%-193.3%],242.0%[17.1%-1136.0%]vs65.5%[8.6%-189.0%],all P<0.0001).The mean levels of ADAMTS13/VWF:Ag,ADAMTS13/VWF:GPIbR,ADAMTS13/VWF:CB in AMI patients were significantly lower in comparison with those in CTL(0.25[0.03-1.30]vs 1.16[0.24-4.82],0.34[0.03-2.53]vs 1.50[0.18-11.78],0.29[0.04-1.66]vs 2.43[0.51-14.23],all P<0.0001).There was no significant difference in the ratios(VWF:GPIbR/VWF:Ag,VWF:CB/VWF:Ag,VWF:GPIbR/VWF:CB)between AMI patients and CTL patients(P>0.05).There was also no significant difference in VWF:Ag,VWF:GPIb R,VWF:CB and ratios of VWF:GPIbR/VWF:Ag,and VWF:CB/VWF:Ag for Pre-PCI and Post-PCI AMI patients(P>0.05).(4)The mean levels of VWF:CB in AMI patients were significantly higher in comparison with those in CTL(309.2%[21.2%-987.3%]vs 75.6%[14.5%-342.1%],P<0.0001),while the mean levels of ADAMTS13 activity in AMI patients were lower in comparison with those in CTL(44.8%[28.3%-79.2%]vs 48.8%[30.6%-86.4%],P<0.01).VWF:CB level in male patients was lower than that in female patients(284.3%[29.6%-981.2%]vs 397.9%[21.2%-987.3%],P=0.0174),and VWF:CB level in below50 years old patients was lower than that in over 50 years old patients(238.0%[29.6%-878.5%]vs 336.1%[21.2%-987.3%],P=0.0271).(5)The VWF multimer analysis:AMI patients not only had more VWF:Ag,but also had significantly increased UL-VWF multimer contrast to that in CTL.Conclusion:(1)The VWF:Ag-FCIA method was successfully established.The method was consistent with the traditional ELISA method and had faster detection speed,and it also has better sensitivity to the diagnosis of type 3 VWD;(2)The VWF:GPIbR-FCIA method was successfully established.The method was consistent with the traditional ELISA method and had better specificity and sensitivity;(3)The VWF:Ag-FCIA and VWF:GPIbR-FCIA assays can not only be used for both hemorrhagic diseases such as VWD,but also has application in valuing the thrombotic risk of AMI;(4)The level of VWF in AMI patients is not only increased significantly,but also has more UL-VWF,which may be a signifcant factor leading to dangerous myocardial infarction;the elevated plasma levels of VWF:Ag,VWF:GPIbR,VWF:CB,low level of ADAMTS13 activity contribute to the pathogenesis of AMI.These findings provide a theoretical basis for the development of new VWF antagonists and further enriched and perfect the mechanism of cardiovascular and cerebrovascular diseases in China,thus has important clinical value.