Abstract

Von Willebrand factor (vWF) is a large multimeric protein produced by endothelial cells and megakaryocytes. vWF mediates adhesion of plateletes at sites of vascular injury, and transports factor VIII. After secretion, vWF multimers are cleaved by ADAMTS-13 (A disintegrin –like and metalloprotease with thrombospondin type 1 motif 13) enzyme to smaller multimers. Decreased activity of ADAMTS-13 by autoantibodies, mutations and other etiologies causes thrombotic microangiopathy (intraluminal platelet thrombosis in the microvasculature, thrombocytopenia, and red blood cell fragmentation). Antiphospholipid syndrome (APS) is an autoimmune disorder characterized by recurrent arterial and venous thrombosis and fetal losses associated with elevated levels of antiphospholipid antibodies. The pathology of small vessel thrombosis in APS patients has been shown to be related with thrombotic microangiopathy. Recently, Austin et al reported that ADAMTS-13 activity was reduced in patients with APS. They also showed an increase of large vWF multimers in plasma of those patients. In this study, we aimed to investigate ADAMTS-13 mutations which decrease the activity of enzyme and the quantitative gene transcrition rates in patients with primary APS and healthy controls. We evaluated 70 primary APS patients (male to female ratio was 25/45), and age and sex matched healthy controls. Exons 10+11,12,13+14 of ADAMTS-13 gene were amplified with the PCR assay for 365del, Q449stop C508Y mutations and P475S polymorphism analysis. PCR products were cleaved with restriction digestion enzymes and genotyped. C508Y mutation was screened by a DNA sequencer system. Total RNA samples were extracted from peripheral blood and cDNA samples were synthesized with reverse transcriptase. Original primers and probes were designed for the target gene ADAMTS-13 and the reference gene HPRT- 1. cDNA amounts of target and reference genes in patient and healthy control groups were determined by real-time PCR. We did not find C365del, Q449stop mutations and P475S polymorphism in the control group and the patients with APS (Table1.) We used Pfaffl method for transcription analysis and found that mRNA amounts of ADAMTS-13 was significantly decreased in APS group compared to helathy controls (Table 1). Our results may suggest that ADAMTS-13 gene is down regulated at the transcription stage in patients with APS. Transcription factors or existence of other mutation(s) at promotor region may explain this effect. This study demonstrated that ADAMTS 13- vWF related mechanisms may have important role in the development of thrombosis in APS patients.

Table 1. Data of transcription analysis and mutations which affect the activity of ADAMTS13.

 C365del Q449stop P475S C508Y ADAMTS13 CP* HPRT1 CP* ADAMTS13 mRNA ratio (PAPS/ Control) 
* Cross Point 
APS n=70 33,71 30,43 0,5 
Control n=100 32,23 30,05  
 C365del Q449stop P475S C508Y ADAMTS13 CP* HPRT1 CP* ADAMTS13 mRNA ratio (PAPS/ Control) 
* Cross Point 
APS n=70 33,71 30,43 0,5 
Control n=100 32,23 30,05  

Disclosures: No relevant conflicts of interest to declare.

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