ADAMTS13 cleaves VWF multimer

Von Willebrand factor (VWF) is synthesized by endothelial cells and megakaryocytes, and released as a multimeric glycoprotein into the peripheral blood stream. Ultra large VWF multimers are formed in the Golgi apparatus. In circulation, VWF senses a vessel injury and induces platelet adhesion to vascular injury sites (Reininger AJ 2008; Mojzisch A & Brehm MA 2021). VWF also functions as a carrier protein for factor VIII (FVIII), stabilizing FVIII, which otherwise has a very short half-life in the bloodstream (Kaufman RJ et al., 1997). VWF activity is dependent on its extent of multimerization, as larger VWF structures are more thrombogenic and display higher platelet tethering capacity at sites of a vascular injury. Under normal physiological conditions, ultra large VWF multimers are cleaved into smaller units by a disintegrin and metalloproteinase with thrombospondin type 1 repeats 13 (ADAMTS13) in a shear-dependent manner (Shim K et al., 2008; Zhang X et al., 2009). ADAMTS13 downregulates VWF procoagulant activity by cleaving the peptide bond between Tyr1605 and Met1606 within the A2 domain of VWF (Furlan M et al., 1996; Tsai HM 1996; Crawley JTB et al., 2011). ADAMTS13 is primarily expressed by hepatic stellate cells in the liver and is secreted into the bloodstream as an active enzyme (Zhou W et al., 2005) which circulates in its inactive (closed) conformation (South K et al., 2014; Petri A et al., 2019; Geist N et al., 2022). The closed conformation of ADAMTS13 is maintained by the interaction between the C-terminal CUB1-2 domains and the spacer domain of ADAMTS13 (South K et al., 2014; Kim HJ et al., 2021; reviewed in Ercig B et al., 2021). Structural and biochemical studies have revealed that ADAMTS13 becomes proteolytically active upon binding to its substrate, VWF (Crawley JTB et al., 2011; South K et al., 2014; Petri A et al., 2019; Geist N et al., 2022). The cleavage of VWF by ADAMTS13 is thought to occur on the surface of endothelial cells during secretion of VWF or at sites of vascular damage where VWF binds to exposed collagen and forms VWF-platelet strings (Dong JF et al., 2003; Shim K et al., 2008; Turner N et al., 2008). Cleavage of VWF multimer has also been detected in circulating blood (Majerus EM et al., 2005). Deficiency or dysfunction of ADAMTS13 has been linked to various bleeding disorders such as thrombotic thrombocytopenic purpura (TTP) (Zheng XL 2015; Sukumar S et al. 2021). <p>ADAMTS13 binding to VWF is controlled by the conformational changes in the mechanosensitive VWF multimer, which undergoes shear stress-induced transition from a folded, inactive conformation to an unfolded, elongated VWF multimers. In the inactive state, VWF is stabilized by autoinhibitory interdomain interactions that mask binding sites for platelets and ADAMTS13 within the A1 and A2 domain of VWF, respectively (Aponte-Santamaría C et al., 2015; Arce NA et al., 2021; Bonazza K et al., 2022; Zhao YC et al., 2022). In addition, the stability of the VWF A2 domain is maintained by the Ca2+ ion-binding site (CBS) and the vicinal disulfide bond formed by Cys1669-Cys1670 within the A2 domain (Xu AJ & Springer TA 2012; Lynch CJ et al., 2014). These structural features prevent ADAMTST13-mediated cleavage of VWF (Xu AJ & Springer TA 2012; Lynch CJ et al., 2014; Aponte-Santamaría C et al., 2015; Arce NA et al., 2021; Bonazza K et al., 2022; Zhao YC et al., 2022). Shear-induced destabilization of the A2 domain of VWF results in exposing Tyr1605-Met1606 to ADAMTS13 (Zhang X et al., 2009; Baldauf C et al., 2009; Crawley JTB et al., 2011; Petri A et al., 2019). The ADAMTS13:VWF interaction involves multiple contact sites (Gao W et al., 2008; de Groot R et al., 2015; South K et al., 2017; Kretz CA et al., 2018; Petri A et al., 2019; Geist N et al., 2022; reviewed by Crawley JTB et al., 2011; DeYoung V et al., 2022). Surface plasmon resonance and equilibrium binding assays showed binding between CUB1-2 domains of ADAMTS13 and the D4-CK domain of VWR suggesting a release of the spacer domain (South K et al., 2017). Kinetic analyses of substrate proteolysis revealed that the unfolded A2 domain of VWF is recognized by exosites within the cysteine-rich and spacer domains of ADAMTS13, which conjugate VWF and ADAMTS13 in close proximity to each other (Petri A et al., 2019). Then, interaction of the disintegrin-like domain of ADAMTS13 with VWF allosterically activates the adjacent metalloprotease domain of ADAMTS13 (Petri A et al., 2019). Structural insights further confirm the allosteric activation of ADAMTS13 induced by the multi-step VWF binding (Petri A et al., 2019; Geist N et al., 2022). <p>This Reactome event shows ADAMTS13-mediated cleavage of VWF between Tyr1605-Met1606.

因子VIII相关蛋白（VWF）由内皮细胞和巨核细胞合成，并以多聚糖蛋白的形式释放至外周血液中。在高尔基体中，VWF形成超大型多聚体。在血液循环中，VWF可感知血管损伤并诱导血小板在血管损伤部位粘附（Reininger AJ，2008；Mojzisch A & Brehm MA，2021）。VWF亦作为载体蛋白，稳定因子VIII（FVIII），否则FVIII在血液中的半衰期极短（Kaufman RJ 等，1997）。VWF的活性取决于其多聚化程度，较大的VWF结构具有更高的血栓形成性和在血管损伤部位的血小板结合能力（Shim K 等，2008；Zhang X 等，2009）。在正常生理条件下，超大型VWF多聚体在剪切依赖性的作用下，由金属蛋白酶与凝血素样蛋白13（ADAMTS13）裂解成较小的单位（Shim K 等，2008；Zhang X 等，2009）。ADAMTS13通过裂解VWF A2结构域内的酪氨酸1605和甲硫氨酸1606之间的肽键，下调VWF的促凝活性（Furlan M 等，1996；Tsai HM，1996；Crawley JTB 等，2011）。ADAMTS13主要由肝脏星状细胞表达，并以活性酶的形式分泌至血液中，循环于其非活性（闭合）构象中（South K 等，2014；Petri A 等，2019；Geist N 等，2022）。ADAMTS13的闭合构象由其C端CUB1-2结构域与ADAMTS13的间隔结构域之间的相互作用维持（South K 等，2014；Kim HJ 等，2021；Ercig B 等，2021年综述）。结构学和生化研究表明，ADAMTS13在与其底物VWF结合后，会变得蛋白水解活性化（Crawley JTB 等，2011；South K 等，2014；Petri A 等，2019；Geist N 等，2022）。ADAMTS13通过裂解VWF被认为是在内皮细胞分泌VWF时或在VWF与暴露的胶原蛋白结合形成VWF-血小板串的血管损伤部位发生（Dong JF 等，2003；Shim K 等，2008；Turner N 等，2008）。VWF多聚体的裂解也已在循环血液中被检测到（Majerus EM 等，2005）。ADAMTS13的缺乏或功能障碍与各种出血性疾病相关，如血栓性血小板减少性紫癜（TTP）（Zheng XL，2015；Sukumar S 等，2021）。ADAMTS13与VWF的结合受机械敏感型VWF多聚体构象变化的控制，该多聚体在剪切应力诱导下从折叠的非活性构象转变为展开的延长VWF多聚体。在非活性状态下，VWF通过域间自抑制性相互作用得到稳定，这些相互作用掩盖了VWF A1和A2结构域内的血小板和ADAMTS13的结合位点（Aponte-Santamaría C 等，2015；Arce NA 等，2021；Bonazza K 等，2022；Zhao YC 等，2022）。此外，VWF A2结构域的稳定性由钙离子结合位点（CBS）和A2结构域内的半胱氨酸1669-1670之间形成的邻近二硫键维持（Xu AJ & Springer TA，2012；Lynch CJ 等，2014）。这些结构特征防止了ADAMTST13介导的VWF裂解（Xu AJ & Springer TA，2012；Lynch CJ 等，2014；Aponte-Santamaría C 等，2015；Arce NA 等，2021；Bonazza K 等，2022；Zhao YC 等，2022）。VWF A2结构域的剪切应力诱导的不稳定导致酪氨酸1605-甲硫氨酸1606暴露于ADAMTS13（Zhang X 等，2009；Baldauf C 等，2009；Crawley JTB 等，2011；Petri A 等，2019）。ADAMTS13:VWF相互作用涉及多个接触位点（Gao W 等，2008；de Groot R 等，2015；South K 等，2017；Kretz CA 等，2018；Petri A 等，2019；Geist N 等，2022；Crawley JTB 等，2011年综述；DeYoung V 等，2022年综述）。表面等离子体共振和平衡结合实验表明，ADAMTS13的CUB1-2结构域与VWR的D4-CK结构域之间的结合释放了间隔结构域（South K 等，2017）。底物蛋白水解的动力学分析揭示了展开的VWF A2结构域被ADAMTS13的半胱氨酸丰富和间隔结构域内的外位点识别，这使得VWF和ADAMTS13在彼此邻近处共轭（Petri A 等，2019）。然后，ADAMTS13的解聚酶样结构域与VWF的别构激活相邻的金属蛋白酶结构域（Petri A 等，2019）。结构上的见解进一步证实了ADAMTS13通过多步骤VWF结合引起的别构激活（Petri A 等，2019；Geist N 等，2022）。此Reactome事件展示了ADAMTS13介导的VWF在酪氨酸1605-甲硫氨酸1606之间的裂解。