Study On The Influential Factors Of Von Willebrand Factor Proteolysis By ADAMTS13

Von Willebrand factor (VWF), the largest human plasma protein, is synthesized and secreted by platelets and endothelial cells. VWF plays an important role both in primary hemostasis and in secondary hemostasis. VWF is present in the form of multimeric glycoprotein in plasma and the molecular weight varies between 500 and 20,000 kDa. The adhesive activity of VWF is positively correlated with the size of the multimers in plasma. VWF multimer size is regulated by metalloproteinase ADAMTS13, which cleaves the central A2 domain of VWF at the Tyr1605-Met1606 bond. This physiological proteolysis of VWF is modulated and influenced by multiple factors including VWF conformation, fluid shear stress, ABO bood group, pH value, divalent metal ion, and so on. At the same time, a number of pathological factors or states can also affect the cleavage of VWF by ADAMTS13, such as anti-ADAMTS13 autoantibody, ADAMTS13 gene polymorphisms or mutations, VWF gene polymorphisms or mutations. The importance of VWF proteolysis by ADAMTS13 is demonstrated in two syndromes, i.e., thrombotic thrombocytopenic purpura and von Willebrand disease type 2A and type 2B. The former is associated with the deficiency of plasma ADAMTS13 activity, either due to congenital mutations or acquired autoantibodies. The latter is mostly caused by mutations in the A2 domain or A1 domain of VWF that lead to the increased proteolysis of VWF multimers by ADAMTS13.The theme of my doctor graduate study is focused on the related factors influencing ADAMTS13 cleavage of VWF. My dissertation consists of three parts. Part I describes the effects of eight monoclonal antibodies (mAbs) against various domains of human VWF on the proteolytic cleavage of VWF by ADAMTS13 under shear stress and static conditions. Additionally, the epitope of anti-VWF mAb (SZ34) was identified. Part II shows the increased susceptibility of recombinant type 2A VWF mutant A1500E to proteolysis by ADAMTS13. Part III illustrates the effect of R673C mutation, a novel mutation of ADAMTS13, on ADAMTS13 activity. Furthermore, the distribution and secretion of the mutant protein in cell has also been studied.1. Study on the effects of monoclonal antibodies against von Willebrand factor on its proteolysis by ADAMTS13Many factors modulate ADAMTS13-mediated VWF proteolysis by interaction with VWF. These ligands include platelet, which binds to the A1 domain of VWF, and factor VIII, which binds to the D’D3 domain of VWF. They both significantly enhance the cleavage of VWF multimers by ADAMTS13 under fluid shear stress. On the contrary, thrombospondin-1(TSP-1), an extracellular matrix adhesion protein, may compete with ADAMTS13 for binding to the A3 domain of VWF, which reduces the rate of VWF proteolysis by ADAMTS13.In this section, to explore whether mAbs against human VWF affect the susceptibility of VWF to proteolysis by ADAMTS13 by means of their interactions with VWF in vitro, eight murine mAbs to different domains of human VWF were used in this study. These mAbs include 1C1E7 and 75H4B12 (anti-VWF D’D3 domain), SZ129 and SZ130 (anti-VWF A1 domain), SZ29 and SZ34 (anti-VWF A2 domain) and SZ123 and SZ125 (anti-VWF A3 domain). The effects of various mAbs to VWF on its proteolysis by ADAMTS13 were investigated under shear stress and static conditions, respectively. In addition to this, the epitope of SZ34, an anti-VWF mAb decreasing the proteolysis of VWF by ADAMTS13 under shear stress, was mapped.Firstly, purified plasma-derived human VWF (pVWF) was incubated separately with 8 anti-VWF mAbs followed by adding into rADAMTS13 that had been activated by 5 mM CaCl2. The mixture was then subjected to a mini vortexer with constant vortexing at 2,500 rpm for 5 min and the reaction was terminated by EDTA. At last the cleavage reactions were measured by SDS-PAGE under non-reducing condition or by VWF multimeric analysis, and then analyzed by Western blotting. The results showed that the proteolysis of VWF by ADAMTS13 was sharply inhibited by SZ34 under shear stress. And the inhibition was in a concentration-dependent manner. In contrast, other anti-VWF mAbs had no effects on the proteolytic cleavage of VWF by ADAMTS13.Secondly, pVWF was pre-denatured with guanidine-HCl. After dilution, the denatured VWF was incubated with anti-VWF mAbs and then treated with rADAMTS13 that had been activated by CaCl2. Subsequently, the reaction was stopped by EDTA. At last, the cleavage reactions were measured by SDS-PAGE under non-reducing conditions or by VWF multimeric analysis, and then analyzed by Western blotting. The results showed that SZ34 had no effect on the digestion of unfolded VWF by ADAMTS13. In the meantime, other seven anti-VWF mAbs exhibited no effects on ADAMTS13-mediated proteolysis of the unfolded VWF under static conditions either. In addition to this, under no shear stress and no denaturants, VWF-R1597W mutant, the most common mutation of type 2A VWD, was cleaved by rADAMTS13 in the absence or in the presence of SZ34. It is shown that SZ34 had no effect on the proteolysis of VWF-R1597W by ADAMTS13 under satic/nondenaturing conditions.At last, the epitope of SZ34, an anti-VWF mAb inhibiting VWF proteolysis by ADAMTS13 under shear stress, was identified. Except for pVWF purified from human factor VIII concentration, ten recombinant protein containing different domains or structures of mature VWF subunit were used. These recombinant proteins related to VWF include D’D3(S764P1247-H), A1A2A3(Q1238G1874-H), A1(H-E1260P1467), A2(H-G1481R1668), A3(S1681R1877-H), A2-12(GST-D1459G1595-H), A2-23 (VWF114,GST-A1555R1668-H), A2-1(GST-D1459E1554-H), A2-2 (GST-A1555G1595-H) and A2-3(VWF73, GST-D1596R1668-H). The epitope of SZ34 was mapped using pVWF and these recombinant proteins by enzyme-linked immunosorbent assay (ELISA) in combination with Western blot analysis. The results indicate that the binding epitope of SZ34 mAb is located between A1555 and G1595 in the central A2 domain of VWF and SZ34 is a conformation-sensitive mAb against VWF. These data show that it is possible that part or all of A1555-G1595 region (i.e. theα2-helix andα3-helix structure) of A2 domain is constitutively exposed on the native VWF multimers and is involved in the regulation of VWF proteolysis by ADAMTS13.2. Increased susceptibility of recombinant type 2A von Willebrand factor mutant A1500E to proteolysis by ADAMTS13Some VWF mutations responsible for VWD type 2A and 2B can lead to the increases in the susceptibility of VWF to proteolysis by ADAMTS13. The pathogenesis of VWD type 2A includes two mechanisms. Group I mutations are characterized by defects in intracellular transport and decreased VWF secretion due to improperly folded protein, whereas the VWF synthesized by patients with Group II, type 2A mutations is processed and secreted normally. The absence of the large and intermediate molecular weight multimers observed in these patients’plasma is attributed to increased proteolysis by ADAMTS13 between Y1605-M1606 of the mature VWF subunit.To investigate the susceptibility of 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 VWD type 2A, recombinant wild-type VWF (WT-VWF) and A1500E mutant VWF were expressed transiently in transfected HeLa cell lines, respectively. Expression supernatant were collected and concentrated, then were cleaved directly by recombinant ADAMTS13. Compared with WT-VWF, the susceptibility of A1500E mutant VWF to proteolysis by ADAMTS13 was analyzed using SDS-agarose gel VWF multimers analysis. Results showed that A1500E mutant VWF can 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 and low molecular weight multimers obviously increased. Conversely, WT-VWF could not be cleaved by ADAMTS13 under this condition. So it is concluded that the A1500E mutation results in increased susceptibility of mutant VWF to proteolysis by ADAMTS13 and belongs to group 2 mutation of VWD type 2A.3. Study on the effects of ADAMTS13 R498C mutation on its activity in VWF proteolysisThrombotic thrombocytopenic purpura (TTP) can be classified into at least three distinct types according to its pathogenesis: congenital TTP, idiopathic TTP, and secondary (nonidiopathic) TTP. There is a decreased ADAMTS13 activity in both congenital and idiopathic TTP, in which, the former is caused by a constitutive deficiency resulting from mutations in the ADAMTS13 gene. There are no hot spots in ADAMTS13 mutations. More than half of these mutations are missense mutations. Others include frameshift mutation, nonsense mutation, aberrant splicing and base deletion.In this section the pathogenisis of a female patient with TTP has been investigated. The attack occurred in her first pregnancy. Firstly, her plasma ADAMTS13 activity under denaturing conditions was detected by means of VWF multimers analysis using SDS-agarose gel electrophoresis. Results showed that the ADAMTS13 activity was absent and the inhibitor of ADAMTS13 was under detectable level. Genomic DNA was extracted from the peripheral leukocytes of this patient. All 29 exons and its intron-exon boundaries of ADAMTS13 gene were amplified by polymerase chain reaction (PCR). All PCR products were directly sequenced in both directions and aligned with the ADAMTS13 gene and cDNA. We found a novel ADAMTS13 gene mutation responsible for this disease: a missense mutation in exon 13 [1492 C→T (R498C)]. ADAMTS13 mutant containing R498C was constructed by site-directed mutagenesis and expressed in Hela cell. It was shown that the protein amounts in mutant-ADAMTS13 cell lysates were same as those of WT-ADAMTS13 cell lysates using western blot analysis, whereas the protein was absent in the culture medium of mutant ADAMTS13. Investigation on immunofluorescence location showed that the distribution and amount of R498C-ADAMTS13 were normal in endoplasmic reticulum and Golgi apparatus in cells, suggesting that the R498C mutation in ADAMTS13 do not impact the synthesis and transport of ADAMTS13 in cell but lead to its markedly impaired extracellular secretion. So both ADAMTS13 protein and its activity are absent in plasma, which results in the onset of TTP.