To avoid pathological platelet aggregation by von Willebrand factor (VWF), VWF multimers are regulated in size and reactivity for adhesion by ADAMTS13-mediated proteolysis in a shear flow dependent manner. We examined if tensile stress in VWF under shear flow activates the VWF A2 domain for cleavage by ADAMTS13 using molecular dynamics simulations. We indeed observed stepwise unfolding of A2 and exposure of its deeply buried ADAMTS13 cleavage site. Interestingly, disulfide bonds in the adjacent and highly homologous VWF A1 and A3 domains obstruct their mechanical unfolding. We generated a full length mutant VWF featuring a homologous disulfide bond in A2 (N1493C and C1670S), in an attempt to lock A2 against unfolding. We find this mutant to feature ADAMTS13-resistant behavior in vitro. Our results yield molecular-detail evidence for the force-sensoring function of VWF A2, by revealing how tension in VWF due to shear flow selectively exposes the A2 proteolysis site to ADAMTS13 for cleavage while keeping the folded remainder of A2 intact and functional. We find the unconventional knotted Rossman fold of A2 to be the key to this mechanical response, tailored for regulating VWF size and activity. Based on our model we can explain the pathomechanism of some natural mutations in the VWF A2 domain that significantly increase the cleavage by ADAMTS13 without shearing or chemical denaturation, and provide with the cleavage-activated A2 conformation a structural basis for the design of inhibitors for VWF type 2 diseases.
Deep Dive into Shear-Induced Unfolding Activates von Willebrand Factor A2 Domain for Proteolysis.
To avoid pathological platelet aggregation by von Willebrand factor (VWF), VWF multimers are regulated in size and reactivity for adhesion by ADAMTS13-mediated proteolysis in a shear flow dependent manner. We examined if tensile stress in VWF under shear flow activates the VWF A2 domain for cleavage by ADAMTS13 using molecular dynamics simulations. We indeed observed stepwise unfolding of A2 and exposure of its deeply buried ADAMTS13 cleavage site. Interestingly, disulfide bonds in the adjacent and highly homologous VWF A1 and A3 domains obstruct their mechanical unfolding. We generated a full length mutant VWF featuring a homologous disulfide bond in A2 (N1493C and C1670S), in an attempt to lock A2 against unfolding. We find this mutant to feature ADAMTS13-resistant behavior in vitro. Our results yield molecular-detail evidence for the force-sensoring function of VWF A2, by revealing how tension in VWF due to shear flow selectively exposes the A2 proteolysis site to ADAMTS13 for cleav
Von Willebrand factor (VWF) is a huge multimeric protein found in blood plasma. VWF mediates the adhesion of platelets to the sub-endothelial connective tissue and is the key protein in primary hemostasis in arterial vessels and the microcirculation (1,2). Monomeric VWF is synthesized solely in megakaryocytes and endothelial cells. After transfer from the cytosol to the endoplasmatic reticulum, dimers form by C terminal disulfide bonds between CK domains (cf. Fig. 1A). Multimers consisting of up to 100 VWF monomers are then formed in the Golgi and post-Golgi compartment by cystin formation between the N terminal D3 domains. VWF is highly glycosylated, oligosaccharides make up about 20% of the mass of VWF (3). The multimers are either constitutively secreted or stored in endothelial Weibel-Palade bodies and platelet α-granules and released from these storage organelles by adequate stimuli. The VWF multimers released from storage are particularly rich in ultra-large VWF (ULVWF). These highly active forms get rapidly yet only partially cleaved by the protease ADAMTS13 at the cleavage site Tyr1605-Met1606 within the A2 domain (4,5).
ADAMTS13 is a zinc containing metallo-protease from the ADAMS/ADAMTS family. Shear stress in blood vessels has been shown to drive VWF multimers into an elongated conformation with increased activity for adsorption to the blood vessel surface, a mechanism to stop bleeding after mechanical injury (6,7). Mechanical forces due to shear flow regulate selective cleavage of ULVWF and thereby their size distribution (8,9). If this size regulation fails, ULVWF accumulates and results in phenotypic manifestation of thrombotic thrombocytopenic purpura (TTP) (10). In contrast, reduced VWF concentration or complete absence of VWF results in the different types of von Willebrand disease (VWD) (11), the most common inherited bleeding disorder in humans. While the shear stress induced adhesion and cleavage have been demonstrated in detail in vitro, the underlying molecular mechanism of shear induced activation of VWF for ADAMTS13 cleavage is currently unknown.
Structural information at atomic detail on the VWF is scarce. A single VWF is a multidomain protein featuring a multitude of functionalities (Fig. 1A). Dimerisation and multimerisation are mediated by domains CK and D3, respectively. The central A domain triplet is pivotal for adhesion and clotting, featuring binding sites for collagen (A1, A3) and glycoprotein Ib (GPIb, A3), and the ADAMTS13 cleavage site (A2). A1 and A3 have been shown by X-ray crystallography (12,13) and A2 by homology modeling to adopt a Rossman α/β-fold (14). The ADAMTS13 cleavage site in A2 appears to be buried, suggesting that forces in stretched VWF multimers induce unfolding and exposure (15).
We here reveal the unfolding and activation mechanism of A2 for ADAMTS13 cleavage under force by molecular simulations. By applying force distribution analysis, a method previously introduced by our group, (16) we reveal how the atypical Rossman fold topology of the VWF A2 domain senses mechanical force by selectively exposing and activating the ADAMTS13 cleavage site. Furthermore, we predict and analyze the impact of mutations stabilizing the A2 domain by introducing a disulfide bond into VWF A2, in analogy to A1 and A3. We demonstrate this mutant VWF to be resistant against ADAMTS13 in vitro. Our results clearly show VWF A2 domain unfolding as a response to shear stress to be the essential event in VWF size regulation.
To reveal the molecular process of enforced unfolding and activation of A2 at atomic detail, a homology model including residues 1488 to 1676 of human VWF was created (Fig. 1B). The model fully includes the very terminal sequences of A2, and thereby the site of mutagenesis for introducing a disulfide bond (see below). It is therefore more comprehensive but otherwise highly similar to a previous homology model that covers only the VWF residues 1496 to 1669 (14). The multiple sequence alignment and ProSA 2003 (17) results are shown in the Supporting Information. The model was subjected to equilibrium Molecular Dynamics (MD) simulations. Within the 30 ns simulation time for each of the three independent trajectories the structures converged fast to a backbone root mean square deviation (rmsd) between 0.2 and 0.25 nm (Supporting Information Fig. S3). The agreement with the previous model and the overall high stability indicate the quality of this A2 model and its appropriateness for the subsequent studies.
The secondary structure elements of VWF A2 are organized in the typical Rossman fold as follows (Fig. 1C): β1 L1497 to E1504), α1 (E1511 to Q1526), β2 (I1535 to Y1542), β3 (V1546 to P1551), α2 (D1560 to R1566), α3 (T1578 to D1587), β4 (P1601 to T1608), α4 (R1618 to G1621), β5 (Q1624 to V1630), α5 (Q1635 to R1641), β6 (P1648 to I1651), α6 (F1654 to C1670). The ADAMTS13 cleavage site Y1605-M1606 is located on strand β4 in the protein core, buried on all sides
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