Effect of oxidative stress occurring in diabetes mellitus on VWF structure and function : its relevance for thrombotic complications in this clinical setting

Abstract

The Von Willebrand Factor (VWF) is a plasmatic glycoprotein with a large multimeric structure, produced in endothelial cells and megakaryocytes and involved in primary haemostasis. VWF induces adhesion and aggregation of platelets at sites of vascular injury, playing an important role in both haemorrhagic and thrombotic processes. The turnover of VWF depends on the cleavage specifically operated by ADAMTS13 in the A2 domain of VWF and in particular, at the Tyr1605–Met1606 peptide bond. Methionine and tyrosine represent substrates particularly sensible to oxidative modifications, that may easily be transformed in methionine-sulfoxide and 3-nitrotyrosine, respectively. Previous studies demonstrated that notable levels of oxidative stress contribute to pro-thrombotic effects, by inhibiting VWF cleavage by ADAMTS13. This kind of resistance to the hydrolysis operated by the metalloprotease leads to accumulation of high molecular weight VWF multimers (Ultra Large-VWF, UL-VWF), that have the highest ability to bind and activate platelets. Basing on these considerations, I focused the researches performed during the course of my PhD on discovering the differences in VWF hydrolysis when performed by leukocyte serine proteases (LSPs) or ADAMTS13, in different pathological settings characterized by conditions of high oxidative stress. Recent studies showed in fact that not only ADAMTS-13, but even the three polymorphonuclear (PMN) proteases, human neutrophil elastase (HNE), proteinase 3 (PR3) and cathepsin G (CG), are able to cleave VWF. Beginning from such evidence, with our experiments we discovered that PR3 and CG can cleave VWF (as a peptide mimicking its A2 domain or as full length protein) near or at the same cleavage site as ADAMTS13, either under conditions of normal or increased oxidation. The same results were even obtained with stimulated PMN secreting both reactive oxygen species (ROS) and LSPs, in the presence or in absence of ROS scavengers or LSPs inhibitors, from where we observed that not only VWF hydrolysis by LSPs is not affected by PMN-generated ROS, but it is even accelerated. In the light of the obtained data, we could hence consider the LSPs-dependent VWF hydrolysis as a limiting step for the adhesion of those bacteria (i.e., S.aureus) that use VWF for adhering to vessels and migrate into tissues, particularly in a high oxidant environment, where VWF hydrolysis by ADAMTS13 results inefficient. On the other hand, the reduced hydrolysis of VWF by ADAMTS13 in the presence of high oxidative stress remains a point still requiring investigations, particularly in those pathological settings characterized by both high ROS levels and the onset of thrombotic events. My researches have been therefore focused on Type 2 Diabetes Mellitus (T2DM) and Chronic Kidney Disease (CKD), two pathologies that, in addition to the above characteristics, even showed a strong accumulation of circulating UL-VWF. In particular, concerning DM, it was highlighted the role of oxidative stress in acquired thrombotic micro- and macro-angiopathy related to the interaction between VWF and ADAMTS13. When compared to a matched group of healthy subjects, T2DM patients showed increased plasma proteins and VWF carbonyl levels (used as a marker of oxidative stress), increased VWF antigen and activity levels and ADAMTS13 antigen and activity values within the normal range, meaning that the reduced cleavage of VWF in T2 diabetics did not depend on impaired ADAMTS13 quantity or functionality. The level of VWF-bound carbonyls was even found correlated with the presence of either UL-VWF forms and several degrees of micro- or macro-angiopathic complications in T2DM. Moreover, samples characterized by the highest carbonyl content even resulted more resistant to the cleavage by ADAMTS13 when compared to a control group, after 60 minutes of incubation with the enzyme. Altogether, the obtained evidences led us to conclude that in T2DM, the ROS-mediated induction of UL-VWF release from endothelial cells causes the observed elevation of VWF levels, which is associated with the increase of major adverse cardiovascular diseases proper of this pathological setting. As last, we investigated the role of VWF in CKD, another pathology characterized by conditions of high oxidative stress as well as a persistent inflammatory state. Patients suffering from CKD have increased risk of developing cardiovascular and septic complications. In our research, we purified VWF from CKD patients and characterized its oxidative state, noting even in this case increased levels of both UL-VWF and carbonyl content and a relevant resistance to the cleavage by ADAMTS13. These results led us to conclude that VWF oxidation, impairing ADAMTS13 cleavage, determines UL-VWF polymers accumulation, inducing in turn platelets’ recruitment and activation and enhancing, at the same time, UL-VWF binding to bacterial adhesins, resulting in the development of thrombotic and septic complications in CKD.