Studies On The Association Of Polymorphisms In The VWF Gene With Thrombosis And Antithrombotic Recombinant Antibodies

von Willebrand factor (vWF) is a highly multimerized glycoprotein that promotes platelet adhesion and aggregation at a high shear rate,and also acts as a carrier of coagulation factor VIII.vWF has been identified as a risk factor for recurrent myocardial infarction in the general population.Two polymorphisms of vWF gene promoter and the Thr789Ala polymorphism in vWF gene,have been reported,are associated with arterial thrombosis.In order to develop a new assay for the Rsa I and Sma I polymorphisms (single nucleotide polymorphism,SNP) in the von Willebrand factor gene detection that utilizes oligonucleotide arrays (DNA chips) on glass supports and to explore the association of the Rsa I and Sma I polymorphisms in the vWF gene with thrombosis in Chinese,an allele-specific oligonucleotides are used which were covalently immobilized on glutaradeyd derivatized glass slides in arrays. Single strand PCR product of PCR- amplified genomic DNA is fluorescently labeled by assymetric PCR with fluorescently tagged dUTP and hybridized to the support-bound oligonucleotide array.The hybridization pattern is detected by fluorescence scenning.The effect of hybridization conditions were evaluated and optimized.The method was validated by the discrimination of blinded DNA samples identified by restriction enzymes.50 patients with thrombosis were examined by this method.The results show that the genotypes scored by oligonucleotide arrays assay were in 100% agreement with restriction enzymes results .No statistical difference in genotype distribution and allele frequencies was observed between patients with thrombosis and the control groups (all P>0.05).So a rapid and accurate method for the Rsa I and Sma I polymorphisms in the von Willebrand factor gene detection,or for theanalysis of single nucleotide polymorphism (SNP) has been developed using oligonucleotide arrays on glass supports. No association was observed between the Rsa I and Sma I polymorphisms in the vWF gene and thrombotic disease.However,the prevalence of the CC genotype of the Sma I polymorphism in 50 patients was 2.7 times higher than that of the normal controls.In order to investigate the true relationship between the Sma I polymorphism in the von Willebrand factor gene and arterial thrombosis,the Sma I polymorphism in vWF gene were analyzed in 107 case patients with acute ischemic stroke,49 case patients with acute myocardial infarction (AMI),and 113 health controls with PCR-RFLP. Plasma vWF:Ag was determined by ELISA on the frozen plasma of 122 subjects.The results show that the prevalence of the CC genotype of Sma I polymorphismin patients with acute ischemic stroke was significantly higher than that of the normal controls (Odds ratio=3.29, 95%CI=1.54-7.01,0.01>p>0.001).However,the prevalence of the CC genotype in AMI was not significantly different from that of the normal controls. The mean plasma vWF:Ag levels of the controls, patients with acute ischemic strokes and AMIs were 0.468,0.584 and 0.783U/ml respectively.The mean level of plasma vWF:Ag did not differ significantly between controls and patients with acute ischemic stroke (p=0.195),but had significantly different between controls and patients with AMIs (p=0.001).No association was found between the Sma I polymorphism and vWF plasma levels in controls, patients with acute ischemic stroke or AMI groups (ANOVA, p=0.323, p=0.315, p=0.96).So the Sma I polymorphism is strongly associated with increased risk pf acute ischemic stroke,however,no association was observed between this polymorphism and AMI.This polymorphism of vWF may represent newly identified risk factor for acute ischemic stroke in Chinese.A murine monoclonal antibody (MoAb) SZ-2 against GPIb a was previously generated and characterized.The antibody not only inhibited the ristocetin-dependent binding of vWF to platelets and ristocetin-induced platelet aggregation but also inhibited platelet aggregation induced by Type I collagen and platelet-activating factor (PAF),and produced a dose-dependent inhibition of platelet aggregation ind...