Improving normothermic machine perfusion and blood transfusion through biocompatible blood silicification.

The growing world population and increasing life expectancy are driving the need to improve the quality of blood transfusion, organ transplantation, and preservation. Here, to improve the ability of red blood cells (RBCs) for normothermic machine perfusion, a biocompatible blood silicification approach termed "shielding-augmenting RBC-in-nanoscale amorphous silica (SARNAS)" has been developed. The key to RBC surface engineering and structure augmentation is the precise control of the hydrolysis form of silicic acid to realize stabilization of RBC within conformal nanoscale silica-based exoskeletons. The formed silicified RBCs (Si-RBCs) maintain membrane/structural integrity, normal cellular functions (e.g., metabolism, oxygen-carrying capability), and enhance resistance to external stressors as well as tunable mechanical properties, resulting in nearly 100% RBC cryoprotection. In vivo experiments confirm their excellent biocompatibility. By shielding RBC surface antigens, the Si-RBCs provide universal blood compatibility, the ability for allogeneic mechanical perfusion, and more importantly, the possibility for cross-species transfusion. Being simple, reliable, and easily scalable, the SARNAS strategy holds great promise to revolutionize the use of engineered blood for future clinical applications.

[Neutrophil extracellular traps extrusion from neutrophils stably adhered to ICAM-1 by lipoteichoic acid stimulation].

The effect of neutrophil extracellular traps (NETs) on promoting intravascular microthrombi formation and exacerbating the severity of sepsis in patients has gained extensive attention. However, in sepsis, the mechanisms and key signaling molecules mediating NET formation during direct interactions of endothelial cells and neutrophils still need further explored. Herein, we utilized lipoteichoic acid (LTA), a component shared by Gram-positive bacteria, to induce NET extrusion from neutrophils firmly adhered to the glass slides coated with intercellular adhesion molecule-1(ICAM-1). We also used Sytox green to label NET-DNA and Flou-4 AM as the intracellular Ca 2+ signaling indicator to observe the NET formation and fluctuation of Ca 2+ signaling. Our results illustrated that LTA was able to induce NET release from neutrophils firmly attached to ICAM-1-coated glass slides, and the process was time-dependent. In addition, our study indicated that LTA-induced NET release by neutrophils stably adhered to ICAM-1 depended on Ca 2+ signaling but not intracellular reactive oxygen species (ROS). This study reveals NET formation mediated by direct interactions between endothelial ICAM-1 and neutrophils under LTA stimulation and key signaling molecules involved, providing the theoretical basis for medicine development and clinical treatment for related diseases.

Multivariate Silicification-Assisted Single Enzyme Structure Augmentation for Improved Enzymatic Activity-Stability Trade-Off.

The ability to finely tune/balance the structure and rigidity of enzymes to realize both high enzymatic activity and long-term stability is highly desired but highly challenging. Herein, we propose the concept of the "silicazyme", where solid inorganic silica undergoes controlled hybridization with the fragile enzyme under moderate conditions at the single-enzyme level, thus enabling simultaneous structure augmentation, long-term stability, and high enzymatic activity preservation. A multivariate silicification approach was utilized and occurred around individual enzymes to allow conformal coating. To realize a high activity-stability trade-off the structure flexibility/rigidity of the silicazyme was optimized by a component adjustment ternary (CAT) plot method. Moreover, the multivariate organosilica frameworks bring great advantages, including surface microenvironment adjustability, reversible modification capability, and functional extensibility through the rich chemistry of silica. Overall silicazymes represent a new class of enzymes with promise for catalysis, separations, and nanomedicine.

[The characteristics of neutrophil extracellular traps produced by all-trans retinoic acid-induced dHL-60 under PMA stimulation].

Extracellular traps released by neutrophils (neutrophil extracellular traps, NETs) are a double-edged sword, and understanding the mechanism of NET formation is of great significance for disease treatment. However, the short lifespan, the large individual differences, and the inability to perform gene editing render it difficult to decipher NET formation using neutrophils. It is necessary to find a model cell to replace neutrophils to study the mechanism of NET formation. In this study, we used different concentrations (0, 0.1, 1, and 10 µmol/L) of all-trans retinoic acid (ATRA) to differentiate HL-60 cells for different days (1, 3, 5, and 7 days). By detecting the cell viability and nuclear morphology of cells, we confirmed that HL-60 cells were differentiated to neutrophil-like cells (dHL-60) after treated with ATRA for at least 5 days. Using immunofluorescence staining to detect the formation of NETs, we demonstrated that dHL-60 cells differentiated for 5 days with 1 µmol/L ATRA could generate NETs comparable to those produced by neutrophils upon phorbol 12-myristate 13-acetate (PMA) stimulation, without histone H3 citrullination. Furthermore, the formation of NETs by dHL-60 cells were NADPH-dependent and PAD4-independent, consistent with neutrophils. Taken together, these observations suggest that dHL-60 cells differentiated with 1 µmol/L ATRA for 5 days can be used as a model cell for neutrophils to study the mechanism of NET formation.

Residues R1075, D1090, R1095, and C1130 Are Critical in ADAMTS13 TSP8-Spacer Interaction Predicted by Molecular Dynamics Simulation.

ADAMTS13 (A Disintegrin and Metalloprotease with Thrombospondin type 1 repeats, member 13) cleaves von Willebrand Factor (VWF) multimers to limit the prothrombotic function of VWF. The deficiency of ADAMTS13 causes a lethal thrombotic microvascular disease, thrombotic thrombocytopenic purpura (TTP). ADAMTS13 circulates in a "closed" conformation with the distal domain associating the Spacer domain to avoid off-target proteolysis or recognition by auto-antibodies. However, the interactions of the distal TSP8 domain and the Spacer domain remain elusive. Here, we constructed the TSP8-Spacer complex by a combination of homology modelling and flexible docking. Molecular dynamics simulation was applied to map the binding sites on the TSP8 or Spacer domain. The results predicted that R1075, D1090, R1095, and C1130 on the TSP8 domain were key residues that interacted with the Spacer domain. R1075 and R1095 bound exosite-4 tightly, D1090 formed multiple hydrogen bonds and salt bridges with exosite-3, and C1130 interacted with both exosite-3 and exosite-4. Specific mutations of exosite-3 (R568K/F592Y/R660K/Y661F/Y665F) or the four key residues (R1075A/D1090A/R1095A/C1130A) impaired the binding of the TSP8 domain to the Spacer domain. These results shed new light on the understanding of the auto-inhibition of ADAMTS13.

[LPS stimulating neutrophils firmly adhered to ICAM-1 to form extracellular traps depends on integrin Mac-1 and cytoskeletal proteins].

Neutrophil extracellular traps (NETs) play an important role in the formation of immunothrombosis. However, how vascular endothelial cells mediate the formation of NETs has not been fully understood. We stimulated neutrophils firmly attached on the endothelial cell surface intercellular adhesion molecule-1 (ICAM-1) with lipopolysaccharide (LPS) or phorbol-12-myristate-13-acetate (PMA) for 4 h, then labeled NETs-DNA with Sytox green dye and the formation of NETs was observed by fluorescent microscopy. The area and fluorescence intensity of NETs-DNA were analyzed to quantify the formation of NETs. The results showed that both PMA and LPS were able to induce firmly adhered neutrophils on ICAM-1 to produce NETs. NETs induced by PMA were independent of neither ß2 integrin lymphocyte function-associated antigen-1 (LFA-1) nor macrophage antigen complex-1 (Mac-1). In contrast, LPS-stimulated NETs were mediated by Mac-1 integrin, but not by LFA-1. After inhibition of actin filaments or Talin-1, the formation of NETs irrespective of the stimulus was significantly reduced. This study reveals the mechanism of the direct interaction between neutrophils and endothelial cells to produce NETs under inflammatory conditions, providing a new theoretical basis for the treatment of related diseases and the development of new drugs.

Characterization of adhesion properties of the cardiomyocyte integrins and extracellular matrix proteins using atomic force microscopy.

Integrins are transmembrane adhesion receptors that play important roles in the cardiovascular system by interacting with the extracellular matrix (ECM). However, direct quantitative measurements of the adhesion properties of the integrins on cardiomyocyte (CM) and their ECM ligands are lacking. In this study, we used atomic force microscopy (AFM) to quantify the adhesion force (peak force and mean force) and binding probability between CM integrins and three main heart tissue ECM proteins, ie, collagen (CN), fibronectin (FN), and laminin (LN). Functionalizing the AFM probes with ECM proteins, we found that the peak force (mean force) was 61.69 ± 5.5 pN (76.54 ± 4.0 pN), 39.26 ± 4.4 pN (59.84 ± 3.6 pN), and 108.31 ± 4.2 pN (129.63 ± 6.0 pN), respectively, for the bond of CN-integrin, FN-integrin, and LN-integrin. The binding specificity between CM integrins and ECM proteins was verified by using monoclonal antibodies, where α10 - and α11 -integrin bind to CN, α3 - and α5 -integrin bind to FN, and α3 - and α7 -integrin bind to LN. Furthermore, adhesion properties of CM integrins under physiologically high concentrations of extracellular Ca2+ and Mg2+ were tested. Additional Ca2+ reduced the adhesion mean force to 68.81 ± 4.0 pN, 49.84 ± 3.3 pN, and 119.21 ± 5.8 pN and binding probability to 0.31, 0.34, 0.40 for CN, FN, and LN, respectively, whereas Mg2+ caused very minor changes to adhesion properties of CM integrins. Thus, adhesion properties between adult murine CM integrins and its main ECM proteins were characterized, paving the way for an improved understanding of CM mechanobiology.

Cohesin SA2 is a sequence-independent DNA-binding protein that recognizes DNA replication and repair intermediates.

Proper chromosome alignment and segregation during mitosis depend on cohesion between sister chromatids, mediated by the cohesin protein complex, which also plays crucial roles in diverse genome maintenance pathways. Current models attribute DNA binding by cohesin to entrapment of dsDNA by the cohesin ring subunits (SMC1, SMC3, and RAD21 in humans). However, the biophysical properties and activities of the fourth core cohesin subunit SA2 (STAG2) are largely unknown. Here, using single-molecule atomic force and fluorescence microscopy imaging as well as fluorescence anisotropy measurements, we established that SA2 binds to both dsDNA and ssDNA, albeit with a higher binding affinity for ssDNA. We observed that SA2 can switch between the 1D diffusing (search) mode on dsDNA and stable binding (recognition) mode at ssDNA gaps. Although SA2 does not specifically bind to centromeric or telomeric sequences, it does recognize DNA structures often associated with DNA replication and double-strand break repair, such as a double-stranded end, single-stranded overhang, flap, fork, and ssDNA gap. SA2 loss leads to a defect in homologous recombination-mediated DNA double-strand break repair. These results suggest that SA2 functions at intermediate DNA structures during DNA transactions in genome maintenance pathways. These findings have important implications for understanding the function of cohesin in these pathways.

Functional interplay between SA1 and TRF1 in telomeric DNA binding and DNA-DNA pairing.

Proper chromosome alignment and segregation during mitosis depend on cohesion between sister chromatids. Cohesion is thought to occur through the entrapment of DNA within the tripartite ring (Smc1, Smc3 and Rad21) with enforcement from a fourth subunit (SA1/SA2). Surprisingly, cohesin rings do not play a major role in sister telomere cohesion. Instead, this role is replaced by SA1 and telomere binding proteins (TRF1 and TIN2). Neither the DNA binding property of SA1 nor this unique telomere cohesion mechanism is understood. Here, using single-molecule fluorescence imaging, we discover that SA1 displays two-state binding on DNA: searching by one-dimensional (1D) free diffusion versus recognition through subdiffusive sliding at telomeric regions. The AT-hook motif in SA1 plays dual roles in modulating non-specific DNA binding and subdiffusive dynamics over telomeric regions. TRF1 tethers SA1 within telomeric regions that SA1 transiently interacts with. SA1 and TRF1 together form longer DNA-DNA pairing tracts than with TRF1 alone, as revealed by atomic force microscopy imaging. These results suggest that at telomeres cohesion relies on the molecular interplay between TRF1 and SA1 to promote DNA-DNA pairing, while along chromosomal arms the core cohesin assembly might also depend on SA1 1D diffusion on DNA and sequence-specific DNA binding.

Flow-Enhanced Stability of Rolling Adhesion through E-Selectin.

Selectin-ligand interactions mediate tethering and rolling of circulating leukocytes on the vessel wall during inflammation. Extensive study has been devoted to elucidating the kinetic and mechanical constraints of receptor-ligand-interaction-mediated leukocyte adhesion, yet many questions remain unanswered. Here, we describe our design of an inverted flow chamber to compare adhesions of HL-60 cells to E-selectin in the upright and inverted orientations. This new, to our knowledge, design allowed us to evaluate the effect of gravity and to investigate the mechanisms of flow-enhanced adhesion. Cell rolling in the two orientations was qualitatively similar, and the quantitative differences can be explained by the effect of gravity, which promotes free-flowing cells to tether and detached cells to reattach to the surface in the upright orientation but prevents such attachment from happening in the inverted orientation. We characterized rolling stability by the lifetime of rolling adhesion and detachment of rolling cells, which could be easily measured in the inverted orientation, but not in the upright orientation because of the reattachment of transiently detached cells. Unlike the transient tether lifetime of E-selectin-ligand interaction, which exhibited triphasic slip-catch-slip bonds, the lifetime of rolling adhesion displayed a biphasic trend that first increased with the wall shear stress, reached a maximum at 0.4 dyn/cm(2), and then decreased gradually. We have developed a minimal mathematical model for the probability of rolling adhesion. Comparison of the theoretical predictions to data has provided model validation and allowed evaluation of the effective two-dimensional association on-rate, kon, and the binding affinity, Ka, of the E-selectin-ligand interaction. kon increased with the wall shear stress from 0.1 to 0.7 dyn/cm(2). Ka first increased with the wall shear stress, reached a maximum at 0.4 dyn/cm(2), and then decreased gradually. Our results provide insights into how the interplay between flow-dependent on-rate and off-rate of E-selectin-ligand bonds determine flow-enhanced cell rolling stability.

Mechanical regulation of calcium signaling of HL-60 on P-selectin under flow.

BACKGROUND: Binding of P-selectin to P-selectin glycoprotein ligand-1 (PSGL-1) makes neutrophils roll on and adhere to inflammatory site. Intracellular calcium bursting of adhered neutrophils is a key event for subsequent arresting firmly at and migrating into the injured tissue. But, it remains unclear how the cytoplasmic calcium signaling of the cells were modulated by the fluid shear stress. Here, we focus on mechanical regulation of P-selectin-induced calcium signaling of neutrophil-like HL-60 cells under flow. METHODS: HL-60 cells were loaded with Fluo-4 AM for fluorescent detection of intracellular calcium ion, and then perfused over P-selectin-coated bottom of parallel-plate flow chamber. The intracellular calcium concentration of firmly adhered cell under flow was observed in real time by fluorescence microscopy. RESULTS: Force triggered, enhanced and quickened cytoplasmic calcium bursting of HL-60 on P-selectin. This force-dependent calcium signaling was induced by the immobilized P-selectin coated on substrates in absence of chemokine. Increasing of both shear stress and P-selectin concentration made the calcium signaling intensive, through quickening the cytosolic calcium release and upregulating both probability and peak level of calcium signaling. CONCLUSIONS: Immobilized P-selectin-induced calcium signaling of HL-60 cells is P-selectin concentration- and mechanical force-dependent. The higher both the P-selectin concentration and the external force on cell, the more intensive the calcium signaling. It might provide a novel insight into the mechano-chemical regulation mechanism for intracellular signaling pathways induced by adhesion molecules.

TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel mechanism to search for protein partners at telomeres.

Human telomeres are maintained by the shelterin protein complex in which TRF1 and TRF2 bind directly to duplex telomeric DNA. How these proteins find telomeric sequences among a genome of billions of base pairs and how they find protein partners to form the shelterin complex remains uncertain. Using single-molecule fluorescence imaging of quantum dot-labeled TRF1 and TRF2, we study how these proteins locate TTAGGG repeats on DNA tightropes. By virtue of its basic domain TRF2 performs an extensive 1D search on nontelomeric DNA, whereas TRF1's 1D search is limited. Unlike the stable and static associations observed for other proteins at specific binding sites, TRF proteins possess reduced binding stability marked by transient binding (∼ 9-17 s) and slow 1D diffusion on specific telomeric regions. These slow diffusion constants yield activation energy barriers to sliding ∼ 2.8-3.6 κ(B)T greater than those for nontelomeric DNA. We propose that the TRF proteins use 1D sliding to find protein partners and assemble the shelterin complex, which in turn stabilizes the interaction with specific telomeric DNA. This 'tag-team proofreading' represents a more general mechanism to ensure a specific set of proteins interact with each other on long repetitive specific DNA sequences without requiring external energy sources.

Synthetic Biohybrids of Red Blood Cells and Cascaded-Enzymes@ Metal-Organic Frameworks for Hyperuricemia Treatment.

Hyperuricemia, caused by an imbalance between the rates of production and excretion of uric acid (UA), may greatly increase the mortality rates in patients with cardiovascular and cerebrovascular diseases. Herein, for fast-acting and long-lasting hyperuricemia treatment, armored red blood cell (RBC) biohybrids, integrated RBCs with proximal, cascaded-enzymes of urate oxidase (UOX) and catalase (CAT) encapsulated within ZIF-8 framework-based nanoparticles, have been fabricated based on a super-assembly approach. Each component is crucial for hyperuricemia treatment: 1) RBCs significantly increase the circulation time of nanoparticles; 2) ZIF-8 nanoparticles-based superstructure greatly enhances RBCs resistance against external stressors while preserving native RBC properties (such as oxygen carrying capability); 3) the ZIF-8 scaffold protects the encapsulated enzymes from enzymatic degradation; 4) no physical barrier exists for urate diffusion, and thus allow fast degradation of UA in blood and neutralizes the toxic by-product H2 O2 . In vivo results demonstrate that the biohybrids can effectively normalize the UA level of an acute hyperuricemia mouse model within 2 h and possess a longer elimination half-life (49.7 ± 4.9 h). They anticipate that their simple and general method that combines functional nanomaterials with living cell carriers will be a starting point for the development of innovative drug delivery systems.

Investigating bioconjugation by atomic force microscopy.

Nanotechnological applications increasingly exploit the selectivity and processivity of biological molecules. Integration of biomolecules such as proteins or DNA into nano-systems typically requires their conjugation to surfaces, for example of carbon-nanotubes or fluorescent quantum dots. The bioconjugated nanostructures exploit the unique strengths of both their biological and nanoparticle components and are used in diverse, future oriented research areas ranging from nanoelectronics to biosensing and nanomedicine. Atomic force microscopy imaging provides valuable, direct insight for the evaluation of different conjugation approaches at the level of the individual molecules. Recent technical advances have enabled high speed imaging by AFM supporting time resolutions sufficient to follow conformational changes of intricately assembled nanostructures in solution. In addition, integration of AFM with different spectroscopic and imaging approaches provides an enhanced level of information on the investigated sample. Furthermore, the AFM itself can serve as an active tool for the assembly of nanostructures based on bioconjugation. AFM is hence a major workhorse in nanotechnology; it is a powerful tool for the structural investigation of bioconjugation and bioconjugation-induced effects as well as the simultaneous active assembly and analysis of bioconjugation-based nanostructures.

Force-induced biphasic regulation of VWF cleavage by ADAMTS13.

It is crucial for hemostasis that platelets are rapidly recruited to the site of vascular injury by the adhesive ligand von Willebrand factor (VWF) multimers. The metalloproteinase ADAMTS13 regulates this hemostatic activity by proteolytically reducing the size of VWF and its proteolytic kinetics has been investigated by biochemical and single-molecule biophysical methods. However, how ADAMTS13 cleaves VWF in flowing blood remains poorly defined. To investigate the force-induced VWF cleavage, VWF A1A2A3 tridomains were immobilized and subjected to hydrodynamic forces in the presence of ADAMTS13. We demonstrated that the cleavage of VWF A1A2A3 by ADAMTS13 exhibited biphasic kinetics governed by shear stress, but not shear rate. By fitting data to the single-molecule Michaelis-Menten equation, the proteolytic constant kcat of ADAMTS13 had two distinct states. The mean proteolytic constant of the fast state (kcat-fast) was 0.005 ± 0.001 s-1, which is >10-fold faster than the slow state (kcat-slow = 0.0005 ± 0.0001 s-1). Furthermore, proteolytic constants of both states were regulated by shear stress in a biphasic manner, independent of the solution viscosity, indicating that the proteolytic activity of ADAMTS13 was regulated by hydrodynamic force. The findings provide new insights into the mechanism underlying ADAMTS13 cleaving VWF under flowing blood.

Force-induced cleavage of single VWFA1A2A3 tridomains by ADAMTS-13.

A disintegrin and metalloprotease with a thrombospondin type 1 motifs 13 (ADAMTS-13) regulates hemostasis by cleaving the folded A2 domain of von Willebrand factor (VWF). The cleavage is regulated by forces as it occurs in flowing blood. We tested the hypothesis that force-induced A2 domain unfolding facilitates cleavage using atomic force microscopy to pull single VWF A1A2A3 tridomain polypeptides by platelet glycoprotein Ibalpha or antibodies to measure time, distance, and force. Structural destabilization of A1A2A3 was induced by 5- to 80-pN forces, manifesting as an abrupt molecular length increase distributed around 20 and 50 nm, probably because of uncoupling A1A2A3 (or partially unfolding A2) and fully unfolding A2, respectively. Time required to destabilize A1A2A3 first increased (catch), reaching a maximum of 0.2 seconds at 20pN, then decreased (slip) with increasing force, independent of ADAMTS-13. The time required to rupture A1A2A3 exhibited a similar catch-slip behavior when pulled by glycoprotein Ibalpha but only slip behavior when pulled by antibody, which was progressively shortened by increasing concentration of ADAMTS-13 after (but not before) structural destabilization, indicating that cleavage of A2 requires the force-induced A2 unfolding. Analysis with a model for single-substrate trimolecular enzymatic kinetics estimated a cleavage rate k(cat) of 2.9 (+/- 59) seconds and a K(d) of 5.6 (+/- 3.4) nM for ADAMTS-13/A1A2A3 binding. These findings quantify the mechanical regulation of VWF cleavage by ADAMTS-13 at the level of single A1A2A3 tridomain.

Transcription Regulation of Tceal7 by the Triple Complex of Mef2c, Creb1 and Myod.

Tceal7 has been identified as a direct, downstream target gene of MRF in the skeletal muscle. The overexpression of Tceal7 represses myogenic proliferation and promotes cell differentiation. Previous studies have defined the 0.7 kb upstream fragment of the Tceal7 gene. In the present study, we have further determined two clusters of transcription factor-binding motifs in the 0.7 kb promoter: CRE#2-E#1-CRE#1 in the proximal region and Mef2#3-CRE#3-E#4 in the distal region. Utilizing transcription assays, we have also shown that the reporter containing the Mef2#3-CRE#3-E#4 motifs is synergistically transactivated by Mef2c and Creb1. Further studies have mapped out the protein-protein interaction between Mef2c and Creb1. In summary, our present studies support the notion that the triple complex of Mef2c, Creb1 and Myod interacts with the Mef2#3-CRE#3-E#4 motifs in the distal region of the Tceal7 promoter, thereby driving Tceal7 expression during skeletal muscle development and regeneration.

Spatiotemporal characteristics of P-selectin-induced ß2 integrin activation of human neutrophils under flow.

Activation of integrins is crucial for recruitment of flowing leukocytes to inflammatory or injured vascular sites, but their spatiotemporal characteristics are incompletely understood. We discovered that ß2-integrin activation over the entire surface of neutrophils on immobilized P-selectin occurred via mitogen-activated protein kinase (MAPK) or non-MAPK signaling with a minute-level timescale in a force-dependent manner. In flow, MAPK signaling required intracellular Ca2+ release to activate integrin within 2 min. Integrin activation via non-MAPK signaling occurred first locally in the vicinity of ligated P-selectin glycoprotein ligand-1 (PSGL-1) within sub-seconds, and then over the entire cell surface within 1 min in an extracellular Ca2+ influx-dependent manner. The transition from a local (but rapid) to global (but slow) activation mode was triggered by ligating the freshly activated integrin. Lipid rafts, moesin, actin, and talin were involved in non-MAPK signaling. Fluid loads had a slight effect on local integrin activation with a second-level timescale, but served as enhancers of global integrin activation.

MD Simulations on a Well-Built Docking Model Reveal Fine Mechanical Stability and Force-Dependent Dissociation of Mac-1/GPIbα Complex.

Interaction of leukocyte integrin macrophage-1 antigen (Mac-1) to platelet glycoprotein Ibα (GPIbα) is critical for platelet-leukocyte crosstalk in hemostasis and inflammatory responses to vessel injuries under hemodynamic environments. The mechano-regulation and its molecular basis for binding of Mac-1 to GPIbα remain unclear, mainly coming from the lack of crystal structure of the Mac-1/GPIbα complex. We herein built a Mac-1/GPIbα complex model through a novel computer strategy, which included a flexible molecular docking and system equilibrium followed by a "force-ramp + snapback" molecular dynamics (MD) simulation. With this model, a series of "ramp-clamp" steered molecular dynamics (SMD) simulations were performed to examine the GPIbα-Mac-1 interaction under various loads. The results demonstrated that the complex was mechano-stable for both the high rupture force (>250 pN) at a pulling velocity of 3 Å/ns and the conformational conservation under various constant tensile forces (≤75 pN); a catch-slip bond transition was predicted through the dissociation probability, examined with single molecular AFM measurements, reflected by the interaction energy and the interface H-bond number, and related to the force-induced allostery of the complex; besides the mutation-identified residues D222 and R218, the residues were also dominant in the binding of Mac-1 to GPIbα. This study recommended a valid computer strategy for building a likely wild-type docking model of a complex, provided a novel insight into the mechanical regulation mechanism and its molecular basis for the interaction of Mac-1 with GPIbα, and would be helpful for understanding the platelet-leukocyte interaction in hemostasis and inflammatory responses under mechano-microenvironments.

Characterization of the interactions of ADAMTS13 CUB1 domain to WT- and GOF-Spacer domain by molecular dynamics simulation.

Metalloprotease ADAMTS13 specifically cleaves VWF (von Willebrand Factor) to prevent excessive platelet aggregation and thrombus formation at the sites of vascular injury. To avoid non-specific cleavage, ADAMTS13 has the auto-inhibition effect in which the Spacer domain in N-terminal interacts with the CUB1 domain in C-terminal, resulting in decreased proteolytic activity. Previous studies reported that exosite-3 in the Spacer domain was a key binding site in the Spacer-CUB1 interaction. When exosite-3 was mutated (R660K/F592Y/R568K/Y661F/Y665F, GOF), the auto-inhibition of ADAMTS13 was disrupted and the enzymatic activity was markedly increased. However, the characteristics of the Spacer-CUB1 interaction is not fully understood. Here, we constructed the model of Spacer-CUB1 complex by homologous modeling and molecular docking to characterize the Spacer-CUB1 binding and predict key amino acid residues via molecular dynamics simulation. Our data showed that G607-S610 was a non-reported potential binding site in the Spacer domain; GOF mutation attenuated the formation of hydrogen bond between exosite-3 and the CUB1 domain; Residues E1231, R1251, L1258, D1259 and T1261 in the CUB1 domain might play an important role in the Spacer-CUB1 interaction. Our study advances the understanding of the structural basis of the auto-inhibition of ADAMTS13 and provides information about the key residues in the binding interface.