Mechanochemistry of von Willebrand factor

Von Willebrand factor (VWF), a blood multimeric protein with a very high molecular weight, plays a crucial role in the primary haemostasis, the physiological process characterized by the adhesion of blood platelets to the injured vessel wall. Hydrodynamic forces are responsible for extensive conformational transitions in the VWF multimers that change their structure from a globular form to a stretched linear conformation. This feature makes this protein particularly prone to be investigated by mechanochemistry, the branch of the biophysical chemistry devoted to investigating the effects of shear forces on protein conformation. This review describes the structural elements of the VWF molecule involved in the biochemical response to shear forces. The stretched VWF conformation favors the interaction with the platelet GpIb and at the same time with ADAMTS-13, the zinc-protease that cleaves VWF in the A2 domain, limiting its prothrombotic capacity. The shear-induced conformational transitions favor also a process of self-aggregation, responsible for the formation of a spider-web like network, particularly efficient in the trapping process of flowing platelets. The investigation of the biophysical effects of shear forces on VWF conformation contributes to unraveling the molecular mechanisms of many types of thrombotic and haemorrhagic syndromes.

Angiotensin-I converting enzyme (ACE): structure, biological roles, and molecular basis for chloride ion dependence

Somatic angiotensin-I converting enzyme (sACE) has an essential role in the regulation of blood pressure and electrolyte fluid homeostasis. It is a zinc protease that cleaves angiotensin-I (AngI), bradykinin, and a broad range of other signalling peptides. The enzyme activity is provided by two homologous domains (N- and C-), which display clear differences in substrate specificities and chloride activation. The presence of chloride ions in sACE and its unusual role in activity was identified early on in the characterisation of the enzyme. The molecular mechanisms of chloride activation have been investigated thoroughly through mutagenesis studies and shown to be substrate-dependent. Recent results from X-ray crystallography structural analysis have provided the basis for the intricate interactions between ACE, its substrate and chloride ions. Here we describe the role of chloride ions in human ACE and its physiological consequences. Insights into the chloride activation of the N- and C-domains could impact the design of improved domain-specific ACE inhibitors.

PROTEOLYTIC PROCESSING OF VON WILLEBRAND FACTOR BY ADAMTS13 AND LEUKOCYTE PROTEASES

ADAMTS13 is a 190 kDa zinc protease encoded by a gene located on chromosome 9q34. This protease specifically hydrolyzes von Willebrand factor (VWF) multimers, thus causing VWF size reduction. ADAMTS13 belongs to the A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats (ADAMTS) family, involved in proteolytic processing of many matrix proteins. ADAMTS13 consists of numerous domains including a metalloprotease domain, a disintegrin domain, several thrombospondin type 1 (TSP1) repeats, a cysteine-rich domain, a spacer domain and 2 CUB (Complement c1r/c1s, sea Urchin epidermal growth factor, and Bone morphogenetic protein) domains. ADAMTS13 cleaves a single peptide bond (Tyr1605-Met1606) in the central A2 domain of the VWF molecule. This proteolytic cleavage is essential to reduce the size of ultra-large VWF polymers, which, when exposed to high shear stress in the microcirculation, are prone to form with platelets clumps, which cause severe syndromes called thrombotic microangiopathies (TMAs). In this review, we a) discuss the current knowledge of structure-function aspects of ADAMTS13 and its involvement in the pathogenesis of TMAs, b) address the recent findings concerning proteolytic processing of VWF multimers by different proteases, such as the leukocyte-derived serine and metallo-proteases and c) indicate the direction of future investigations

A disintegrin and metalloprotease 10 activity sheds the ectodomain of the amyloid precursor-like protein 2 and regulates protein expression in proximal tubule cells

A disintegrin and metalloprotease 10 (ADAM10) is a zinc protease that mediates ectodomain shedding of numerous receptors including Notch and members of the amyloid precursor protein family (APP, APLP1, and APLP2). Ectodomain shedding frequently activates a process called regulated intramembrane proteolysis (RIP) that links cellular events with gene regulation. To characterize ADAM10 in kidney and in opossum kidney proximal tubule (OKP) cells, we performed indirect immunofluorescence microscopy and immunoblotting of renal membrane fractions using specific antibodies. These studies show that ADAM10 and APLP2 are coexpressed in the proximal tubule and in OKP cells. To study the role of ADAM10 activity in the proximal tubule, we stably overexpressed wild-type ADAM10 or an inactive mutant ADAM10 in OKP cells. We found a direct correlation between the amount of active ADAM10 expressed and 1) the amount of APLP2 ectodomain shed into the culture supernatant and 2) the amount of Na+/H+ exchanger 3 (NHE3) and megalin mRNA and protein expressed compared with control proteins. To establish a link between ADAM10-mediated shedding of APLP2 and the effect on NHE3 and megalin mRNA expression we performed RNA interference experiments using APLP2-specific short hairpin RNA (shRNA) in OKP cells. Cells expressing the APLP2 shRNA showed >80% knock down of APLP2 protein and mRNA as well as 60–70% reduction in NHE3 protein and mRNA. Levels of megalin and Na-K-ATPase protein and mRNA were not changed. These studies show 1) ADAM10 and APLP2 are expressed in proximal tubule cells and, 2) ADAM10 activity has a pronounced effect on expression of specific brush-border proteins. We postulate that ADAM10 and APLP2 may represent elements of a here-to-fore unknown signaling pathway in proximal tubule that link events at the brush border with control of gene expression.