The Study On Relationship Between Von Willebrand Factor And Cerebral Infarction

Background:Cerebrovascular disease(CVD) is the leading cause of disability and the third mortality worldwide and is the major factor of cognitive impairment and affective disorder in elderly. China Health Statistical data which was released by Chinese Center for Disease Control and Prevention in 2012 indicated that C hinese have a higher morbidity of CVD, the incidence of CVD increased from 6.6‰ in 2003 to 9.7‰ in 2008 and the CVD mortality of urban dweller was 138.68 in one hundred thousand in 2011. CVD has jumped recently to the second place as a cause of death in the total population of China. Stroke refers to cerebrovascular clinical events of acute onset, rapid localized or diffuse cerebral function deficits. Cerebral infarction(CI) is also called cerebral ischemic stroke(CIS), is due to brain blood supply disturbance, local brain tissue ischemia and hypoxia caused ischemic necrosis or softening of the brain, which accounts for about 80% of all stroke.Platelet and inflammatory cells were leaded to adhesion and aggregation in the endothelial injury department, and ultimately thrombosis, which is the main reason of cerebral infarction. Von Willebrand factor(VWF) is a kind of high molecular weight multimeric glycoprotein with adhesion function, which plays an important role in the process of hemostasis and thrombosis. Upon vessel walldamage, VWF as a bridge connect the platelet membrane glycoprotein(GP) Ib and collagen in the exposed subendothelial matrix, can mediate platelet adhesion to the vessel wall. In recent years, more and more studies showed that high levels of VWF and low levels of von Willebrand factor cleaving protease(ADAMTS13) are related to the occurrence and development of ischemic stroke. The novel antagonist of platelet adhesion and activation which was mediated by VWF, as a more powerful, secure anti-thrombosis drug, also acquired more and more attention.We have reported an ELISA methods with simple operation, high specificity and sensitivity for detecting VWF antigen levels(VWF:Ag), VWF ristocetin cofactor activity(VWF:Rcof) and VWF collagen binding activity(VWF:C B). In this study, we used these assays to measure plasma VWF:Ag, VWF:Rcof and VWF:C B and combined with the activity of ADAMTS13(ADAMTS13) detection, VWF polymer gel electrophoresis, providing more reliable basis for the molecular pathogenesis study of cerebral infarction in the Chinese population. Objective:The aim of this study is to prepare recombinant platelet glycoprotein Ibα-Fc fusion protein(rf GPIbα-Fc), to measure plasma levels of VWF:Ag, VWF:Rcof, VWF:CB, ADAMTS13 and VWF polymer gel electrophoresis in cerebral infarction patients and controls and to analyze the ratios of VWF:Rcof/VWF:Ag, VWF:C B/VWF:Ag, ADAMTS13/VWF:Ag, ADAMTS13/VWF:Rcof and ADAMTS13/VWF:C B. We will use this information to assess their relationship to cerebral infarction. Methods:(1). The CHO cells expressing rf GP Ibα-Fc were cultured. It was purified with affinity chromatography column and its immune activity were identified by ELISA, 10% SDS-PAGE and Western blot methods.(2). The subject group comprised 94 patients who were diagnosed with cerebral infarction in the department of neurology of the first affiliated hospital of Soochow University, and 103 control subjects who were health volunteers and matched by sex and index year, and have no history, signs or symptoms of cerebral infarction.(3). Plasma levels of VWF:Ag, VWF:Rcof and VWF:C B were measured by ELISA. ADAMTS13 was measured with FRETS-VWF73.(4). The relationship between plasma levels and ratios of VWF and ADAMTS13 and the occurrence of cerebral infarction were analyzed with software SPSS version 20.0 and Med Calc version 13.2.2.0.(5). The VWF multimeric patterns were analyzed by VWF polymer gel electrophoresis in patients and controls.Results:(1). The CHO cell supernatants with rf GPIbα-Fc were prepared successfully and the purified product of rf GPIbα-Fc was obtained with highly activity.(2). Cerebral infarction patients displayed higher VWF:Ag(median [IQR], 176% [114%-293%]) and VWF:Rcof(197% [105%-381%]) levels, and lower ADAMTS13(109% [80%-132%]), VWF:CB/VWF:Ag(0.43 [0.28-0.90]), ADAMTS13/VWF:Ag(0.54 [0.32-0.99]) and ADAMTS13/VWF:Rcof(0.57 [0.29-0.91]) levels compared to controls(105% [61%-145%], 100% [60%-175%],117% [103%-146%], 1.13 [0.72-1.81], 1.23 [0.79-2.16] and 1.18 [0.71-1.79], P < 0.01, respectively). The highest quartiles of VWF:Ag(OR = 5.11, 95% CI, 1.49-17.50), VWF:Rcof(OR = 5.04, 95% CI, 1.62-15.66), and the lowest quartiles of VWF:C B/VWF:Ag(O R = 5.91, 95% CI, 1.95-17.93), ADAMTS13/VWF:Ag(OR = 9.11, 95% CI, 2.49-33.33) and ADAMTS13/VWF:Rcof(OR = 3.73, 95% CI, 1.39-10.03) are associated with cerebral infarction.(3). The VWF polymer gel electrophoresis showed that there is no significantly difference between patients and controls.Conclusion:(1). The rf GPIbα-Fc protein have high immune activity.(2). An association was found between increased levels of VWF:Ag, VWF:Rcof, reduced levels of VWF:CB/VWF:Ag, ADAMTS13/VWF:Ag and ADAMTS13/VWF:Rcof ratios and cerebral infarction.(3). There are the abnormal amount o f VWF and the normal VWF multimeric patterns in cerebral infarction.(4). The combination of VWF:Ag, VWF:Rcof, VWF:CB/VWF:Ag, ADAMTS13/VWF:Ag and ADAMTS13/VWF:Rcof may present a clear relationship between VWF and cerebral infarction, and provide references for the clinical application of VWF target antithrombotics in the future.