Method for detecting von willebrand factor micro shear force response sensitivity based on FRET biosensor and application thereof

Abstract

The invention discloses a method for detecting response sensitivity of von Willebrand Factors (von Willebrand Factors, von Willebrand Factors, von Willebrand Factors, von Willebrand Factors, von Willebrand Factors, von Willebrand Factors, von Willebrand Factors, von Willebrand The method comprises the following steps: firstly, inserting an FRET pair at two sides of an A2 structural domain of a full-length vWF to construct an FRET-based vWF biosensor; transfecting the vWF biosensor into cells, so that the vWF biosensor is anchored on the surface of a cytoplasmic membrane; the cells are inoculated in a fluid experiment shearing device, and an FRET microscopic imaging system collects image data of ECFP and FRET imaging channels of the cells under the action of fluid shearing force; performing data processing and analysis on the image data; and analyzing the response sensitivity of the vWF under the action of the micro shear force. The constructed biosensor and the analysis method thereof can be used for screening research of drugs for treating hemophilia.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescence resonance energy transfer (FRET) detection, and particularly relates to a method for detecting the micro-shear force response sensitivity of von Willebrand Factor (vWF) based on a FRET biosensor and its application, and a FRET probe for detecting fluid shear stress developed based on the vWF molecule and its application in the study of vWF mechanical response. Background Art

[0002] Hemophilia is a disease in which blood cannot properly clot at the site of vascular injury and can be serious for health, especially during internal bleeding. Von Willebrand Factor (vWF), as a large glycoprotein, is a crucial coagulation factor in the circulatory system. It can sense the hydrodynamic force at the site of vascular injury and then recruit platelets to form a thrombus. Insufficient quantity or abnormal function of it can lead to von Willebrand diseases (vWD), a type of hemophilia.

[0003] vWF is a macromolecular protein with a molecular weight of 220 kDa, synthesized by endothelial cells / megakaryocytes, stored in Weibel-Palade bodies or floating in the circulatory system. vWF can sense the hydrodynamic force at the site of vascular injury, thereby helping the protein to unfold, recruit platelets to form a thrombus and stop bleeding. The A2 domain in vWF is considered a mechanical sensing element during the thrombus formation process. A large number of studies on in vitro models have shown that under the action of high shear flow (such as several hundred dynes per square centimeter), the vWF molecule will elongate.

[0004] As a large polymer, vWF can sense changes in shear stress in the blood flow and block the rupture of the blood vessel wall by binding to platelets. The molecular mechanism behind this dynamic process is very complex and difficult to study using traditional methods. To gain in-depth understanding of this process, researchers used single-molecule fluorescence imaging and high-pressure fast-switching microfluidics technology to reveal the key role of electrostatic guidance in accelerating the binding between flow-activated vWF and GPIbα and in rapidly immobilizing platelets under flow conditions. It was found that during the elongation and tension-dependent activation of vWF multimers, electrostatic guidance significantly increases the binding rate with changes in ionic strength and pH value. In high-concentration physiological saline, strong electrostatic shielding greatly reduces the binding of platelets to vWF in flow, indicating that electrostatic attraction plays a key role in the binding of vWF to platelets during bleeding. Notably, the force on vWF at the site of vascular injury increases, and at these sites, the hemostatic potential of vWF is important for mediating platelet aggregation and recruiting platelets to the subendothelial layer. However, increased force in narrow blood vessels increases the risk of vWF-mediated thrombosis. Therefore, understanding the structure and function of vWF is of great significance for the diagnosis and treatment of related diseases.

[0005] How von Willebrand factor (vWF) achieves molecular unfolding by mechanically sensing shear flow is an important research topic. Currently, there is a lack of research means to study the molecular mechanism of how vWF generates shear stress. Fluorescence resonance energy transfer (FRET) technology and genetically encoded FRET biosensors provide powerful tools for visualizing signaling molecules in living cells with high spatiotemporal resolution. The present invention designs a Förster resonance energy transfer (FRET) biosensor to visually detect the mechanical response of vWF. Using FRET technology to visually study the structural and functional changes of vWF and explore its sensitivity to force is expected to provide a new approach for treating blood diseases using this means. Summary of the Invention

[0006] The present invention aims to provide a method for detecting the response sensitivity of von Willebrand Factor (vWF) under micro-shear force based on a FRET biosensor and its application.

[0007] The present invention first provides a method for detecting the response sensitivity of vWF under micro-shear force based on a FRET biosensor, comprising the following steps: (1) Inserting a FRET pair on both sides of the A2 domain of vWF to construct a FRET-based biosensor; (2) Transfecting the vWF biosensor obtained in step (1) into cells to anchor the vWF biosensor on the surface of the cytoplasmic membrane; (3) Seed the cells obtained in step (2) into a fluid experimental shear device, and the FRET microscopy system collects the image data of the ECFP and FRET imaging channels of the cells under the action of fluid shear force; (4) Perform data processing and analysis on the image data obtained in step (3); analyze the response sensitivity of vWF under micro-shear force.

[0008] In the above technical solution, the FRET pair described in step (1) consists of the fluorescent proteins ECFP and YPet, and ECFP and YPet are located on both sides of the target protein respectively.

[0009] Furthermore, the specific method for constructing the FRET-based biosensor is as follows: construct a fusion protein containing the A2 domain and ECFP and YPet, labeled as ECFP-A2-YPet; connect the ECFP-A2-YPet fragment with vWF without the A2 domain to obtain the vWF-ECFP-A2-YPet fusion protein; subclone the plasmid containing vWF-ECFP-A2-YPet into a vector to construct the vWF-ECFP-A2-YPet biosensor.

[0010] Among them, the vector is the pDisplay vector. The pDisplay vector can direct the protein to the secretory pathway and anchor the sensor on the cell membrane surface through the transmembrane domain of the platelet-derived growth factor receptor (PDGFR). This localization method helps to simulate the sensing of hydrodynamic force when vWF adheres to the blood vessel wall.

[0011] Furthermore, the present invention also mutates asparagine 1493 at the N-terminus and cysteine 1669 at the C-terminus of the A2 domain into cysteine and glycine respectively, generating a pDisplay-vWF-ECFP-LOCK-A2-YPet version of the biosensor. The mutated A2 domain forms a disulfide bond between its N-terminus and C-terminus, thereby preventing proper conformational changes, and is named LOCK-A2. Specifically, the A2 sequences of CCC AAG AGG AAC TCC at the N-terminus and AGTTGT TGT ACC GGT at the C-terminus are changed to CCC AAG AGG TGC TCC and AGT GGT TGT ACC GGT.

[0012] Furthermore, the present invention also constructs short - version FRET biosensors of ECFP - A2 - YPet only and ECFP - LOCK - A2 - YPet only without the full - length vWF structure in the pDisplay vector based on two full - length vWF FRET biosensors provided by BGI (BGI).

[0013] In a specific embodiment of the present invention, the cell transfection method in step (2) is as follows: according to the transfection method of Lipofectamine 3000 liposome, the vWF biosensor is transfected into HEK293T cells.

[0014] In the above - mentioned technical solution, in step (3), a fluid shear experiment device is used to apply shear force to the cells; the fluid shear experiment device includes a single - channel flow chamber slide and a peristaltic pump. A Luer connector is installed on the single - channel flow chamber slide, and a suitable hose is assembled on the Luer connector and connected to the peristaltic pump. During use, the air inlet and outlet are inserted into a centrifuge tube filled with Hank's balanced salt solution (HBSS). Before starting the fluid experiment, the HBSS solution at the liquid inlet is pumped into the Luer connector by the peristaltic pump to ensure that the single - channel flow chamber slide is filled with liquid and seamlessly connected, so as to prevent the generation of bubbles during the liquid flow and affect the stability of the experiment.

[0015] In the above - mentioned technical solution, for the image acquisition in step (3), in the FRET microscopy imaging system, the filter parameters of the ECFP imaging channel are excitation (436 ± 10) nm, dichroic mirror 455 nm, and emission (475 ± 20) nm; the filter parameters of the FRET (YPet) channel are excitation (436 ± 10) nm, dichroic mirror 455 nm, and emission (535 ± 15) nm.

[0016] In a specific embodiment of the present invention, in the imaging experiment, a ×100 Oil objective lens is selected to obtain FRET images. The Zeiss software system controls the rapid switching of the ECFP and FRET imaging channels to ensure the simultaneous acquisition of image data of the two channels. During the experimental flow process, fluorescence images are collected every 2 minutes. The peristaltic pump is turned on 10 minutes after FRET imaging according to the experimental parameters set in advance, and the total flow management time is 30 minutes.

[0017] In the above technical solution, the data processing and analysis in step (4) are to quantitatively analyze the experimental image data using the FRET image analysis software FluoCell 6.0.0. Specifically, the fluorescence intensity ratio between the ECFP and FRET channels is calibrated pixel by pixel after subtracting the background influence, and a ratio image and FRET / ECFP ratio data are obtained. The GraphPad Prism 6.0 software is used to statistically analyze the data. The time-course curve (mean ± S.E.M.) and scatter plot (mean ± S.E.M.) of the FRET ratio, as well as the differences between the two sets of data, are analyzed.

[0018] Fluid shear stress is closely related to biological growth, development, and related physiological activities, and may have an important impact on various cardiovascular and blood system diseases. How shear stress participates in regulating the function and activity of vWF protein has always been a hot topic in biological research. The present invention uses FRET technology to observe the activity of vWF probes in cells in real time and attempts to study the responses of cells transfected with vWF FRET probes under different shear stresses. By using vWF FRET technology, it is confirmed that vWF is particularly sensitive to shear stress, which is different from the situation without applying shear stress. When applying shear stresses of 0 dyn / cm 2 ~ 20 dyn / cm 2 at different levels, the probes are significantly activated, and the activity increases with time; while when applying a very small shear stress of less than 1 dyn / cm 2 , the probes of the present invention can still make sensitive responses, and the degree of response varies with the magnitude of the applied shear stress.

[0019] The present invention has developed a Förster resonance energy transfer (FRET) biosensor for monitoring the conformational changes of von Willebrand factor (vWF) under hydrodynamic forces. The biosensor based on full-length von Willebrand factor (vWF) is anchored on the cell surface, with FRET pairs linked on both sides of the A2 domain. 293T cells were seeded into the microfluidic channel, and a shear force of 2.8 dynes / cm² induced a significant FRET change (about 60%) within 30 minutes. Experiments on gradient micro-shear forces below 2.8 dynes / cm² showed that the FRET response was positively correlated with the fluid intensity, and a shear force of 0.14 dynes / cm² could trigger an obvious change (about 16%). The increase in FRET indicates that the two terminal positions of the A2 domain in von Willebrand factor (vWF) are closer, which is supported by the high FRET value of the biosensor based only on the A2 domain. This is probably due to the dissociation of the A2 domain from the intramolecular binding of von Willebrand factor (vWF) induced by the fluid. Interestingly, when the fluid shear force gradually increased from 2.8 dynes / cm² to 28 dynes / cm², the FRET change decreased instead, indicating a secondary unfolding of the A2 domain. The LOCK-vWF biosensor with the A2 domain bridged at both ends or the biosensor based only on the A2 domain could not sense the shear force, indicating that the A2 domain with a flexible structure and the larger von Willebrand factor (vWF) molecule play important roles in mechanical sensing.

[0020] Secondly, the present invention provides the above-mentioned FRET-based biosensor and its use in drug screening for detecting the response sensitivity of vWF under micro-shear forces. The detection and analysis method of the present invention can be used for the screening research of candidate new drugs acting on the A2 domain target.

[0021] Compared with the prior art research on the elongation of von Willebrand factor (vWF) molecules under high shear forces such as several hundred dynes / cm², the present invention applies Förster (fluorescence) resonance energy transfer (FRET) technology to study the conformational changes of von Willebrand factor (vWF) under shear forces. Fluorescence resonance energy transfer (FRET) technology has high sensitivity in measuring subtle intramolecular spatial changes, which provides a suitable method for visualizing the mechanical sensing of von Willebrand factor (vWF) under physiological shear conditions.

[0022] By anchoring a fluorescence resonance energy transfer (FRET) biosensor based on von Willebrand factor (vWF) on the surface of the cytoplasmic membrane, the present invention reveals that von Willebrand factor (vWF) has a mechanical sensing property of two-level horizontal unfolding in response to shear force: First, under the action of micro-shear force, the A2 domain dissociates from the intramolecular binding of von Willebrand factor (vWF); then, under the action of higher shear force, the A2 domain in von Willebrand factor (vWF) unfolds. The flexible A2 domain structure and the large molecular size of von Willebrand factor (vWF) are important contributing factors, providing a new perspective for in-depth understanding of the mechanical sensing property of von Willebrand factor (vWF) and its role in physiological functions and related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1. Design of vWF-based biosensor and flow experimental device. (A) Schematic diagram of FRET biosensor based on vWF or LOCK-vWF. The A2 domain is flanked by ECFP and YPet, and the biosensor protein is anchored on the surface of the cytoplasmic membrane. (B) Schematic diagram of FRET biosensor based on vWF or LOCK-vWF. (C) Setup of flow experiment on the microscope. (D) Working principle diagram of vWF-based FRET probe.

[0024] Figure 2 FRET changes of vWF-based biosensor in HEK293T cells under gradient flow shear. (A) FRET ratio plots of cells expressing vWF-based sensor at 0, 10, and 30 minutes under fluid shear of 0, 1, 2, 5, 10, 20 dyn / cm2 with 0 dyn as the control. (B) Time-course curves (mean ± S.E.M.) of the FRET ratio (FRET / ECFP) quantification of the cells in each group in (A) under the specified shear force. (C) Scatter plot of the quantified change in FRET ratio of vWF-based sensor at 30 minutes under the specified shear force (mean ± S.E.M.).

[0025] Figure 3Response of vWF-based FRET under a microshear force of 0.1 - 1.0 dyn / cm2. (A) Representative ratio metric FRET images of HEK293T cells expressing the vWF-based biosensor after applying microshear forces of 0, 0.1, 0.2, 0.4, 0.8, 1.0 dyn / cm2 for 0 minutes and 30 minutes. (B) Time course curves (mean ± S.E.M.) of normalized FRET changes before and after microshear flow (0.1 - 1.0 dyn / cm2) with 0 dyn / cm2 as the control. (C) Statistical comparison of the percentage changes in FRET after applying different microshear flows (0.1 - 1.0 dyn / cm2) at 6, 10, 20, 30 minutes (mean ± S.E.M.).

[0026] Figure 4 Comparison of flow-induced FRET responses of vWF- and LOCK-vWF-based biosensors and small-sized MT1-MMP biosensors. (A) Representative ratio metric FRET images of HEK293T cells expressing the vWF or LOCK-vWF-based biosensor or the MT1-MMP biosensor under a shear force of 2 dyn / cm2. (B) Time course curves (left) and scatter plots (right at 30 minutes) of the quantified FRET changes in each group of cells. (C, D) FRET responses and time course curves of the vWF-based biosensor induced by a shear force of 2 dyn / cm2, followed by stopping the flow.

[0027] Figure 5 Response of the A2-based FRET biosensor to hydrodynamic forces. (A) Design of the FRET biosensor based only on A2 and LOCK-A2 and anchored on the cytoplasmic membrane surface. (B) Representative ratio metric FRET images of HEK293T cells expressing the vWF FRET, A2FRET, or LOCK-A2 FRET biosensor under a shear force of 2 dyn / cm2. (C, D) Time course curves (C) and scatter plots (D at 30 minutes) of the quantified FRET changes in each group of cells under the condition of 2 dyn / cm2 in (B). (E, F) Time course curves (E) and scatter plots (F at 30 minutes) of the quantified FRET changes in each group of cells under the action of 10 dyn / cm2. Detailed implementation mode

[0028] The present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0029] Cell types and sources used in the following specific embodiments of the present invention: Human embryonic kidney cells (HEK-293T) are epithelial cells extracted from human embryonic kidneys and purchased from Bena Biology (BNCC). These cells grow relatively fast and contain SV40 antigen, which can express certain types of vectors and genes more effectively and are widely used in transfection experiments with high transfection efficiency. The HEK-293T cells used in this experiment have undergone ten subcultures and passages.

[0030] The main reagents and instruments used in the following specific embodiments of the present invention are as follows: DMEM high-glucose medium, fetal bovine serum (FBS), Opti-MEM medium, 0.25% trypsin-EDTA, cell dissociation and digestion reagent Accutase, and transfection kit Lipofectamine 3000 are purchased from Thermo Fisher Scientific. Hank's balanced salt solution (HBSS) is purchased from Procell Company in Wuhan, China; single-channel flow chamber slides are purchased from ibidi Company in Germany; flow rate peristaltic pumps are purchased from Figwit Company in Shanghai. The FRET microscopy system and inverted microscope PrimoVert are purchased from Carl Zeiss in Germany.

[0031] The present invention applies the FRET reaction to study the sensing of vWF to hydrodynamic forces. This research tool has high sensitivity in monitoring molecular transformation. The FRET pair of fluorescent proteins ECFP and YPet is inserted into full-length vWF because the FRET pair is located on both sides of the A2 domain (as Figure 1 shown in A). The vWF-based FRET sensor is subcloned into the pDisplay vector, which can direct proteins to the secretory pathway and anchor the sensor on the cell membrane surface through the transmembrane domain of platelet-derived growth factor receptor (PDGFR). This localization method helps to simulate the sensing of hydrodynamic forces when vWF adheres to the blood vessel wall. The present invention also prepares a negative control version of the sensor by forming a disulfide bond (LOCK-A2) between the N-terminus and C-terminus of the A2 domain, named the LOCK-vWF FRET sensor (Figure 1B). LOCK-A2 can resist conformational changes caused by flow shear.

[0032] In the fluid and FRET microscopy setup (Figure 1C), 24 hours after transfection of the sensor, HEK293T cells were seeded into single-channel fluid coverslips and cultured in a culture medium containing 1% FBS (low serum to reduce the possibility of sensor shedding from the cell surface). Flow was maintained using HBSS solution, and the cells could be maintained for several hours. A peristaltic pump could regulate the flow rate through the chamber coverslip (µ-Slide I0.4 Luer) from 0.0027 to 178.1 mL / min with an accuracy within 0.5%. FRET imaging was performed at room temperature, and the imaging process was generally completed within 40 minutes.

[0033] The fluid shear experiment setup and FRET microscopy imaging analysis involved in the following specific embodiments of the present invention are as follows: Before the experiment began, a suitable hose (Size-14#) was connected to the single-channel flow chamber coverslip (µ-Slide I 0.4 Luer) through a Luer connector, and then the hose was connected to the peristaltic pump. The inlet and outlet were inserted into a centrifuge tube containing 20 ml of Hank's balanced salt solution (HBSS), and then the centrifuge tube was placed in a centrifuge tube rack and fixed. It should be noted that before starting the fluid experiment, the HBSS solution at the inlet should be pumped into the Luer connector using the peristaltic pump to ensure that the single-channel flow chamber coverslip is filled with liquid and seamlessly connected, thus preventing the generation of bubbles during liquid flow and not affecting the stability of the experiment. According to the protocol provided by the manufacturer ibidi, the shear stress of μ-Slide I0.4 Luer was calculated as follows: τ = η ∙ 131.6 ∙ Φ (Φ: flow rate ml / min, τ: shear stress dyn / cm², η: dynamic viscosity dyn∙s / cm²).

[0034] In the FRET microscopy imaging system, the filter parameters for the ECFP imaging channel were excitation (436 ± 10) nm, dichroic mirror 455 nm, emission (475 ± 20) nm; the filter parameters for the FRET (YPet) channel were excitation (436 ± 10) nm, dichroic mirror 455 nm, emission (535 ± 15) nm. In the imaging experiment, a ×100 Oil objective was selected to acquire FRET images. The Zeiss software system controlled the rapid switching of the ECFP and FRET imaging channels to ensure simultaneous acquisition of image data from both channels. During the experimental flow, fluorescence images were acquired every 2 minutes. The peristaltic pump was turned on 10 minutes after FRET imaging according to the experimental parameters set in advance, and the total flow management time was 30 minutes. Since there was no suitable temperature control device for the fluid system in the laboratory, the images were acquired at room temperature.

[0035] The experimental image data were quantitatively analyzed using the FRET image analysis software FluoCell 6.0.0. The fluorescence intensity ratio between the ECFP and FRET channels was calibrated pixel by pixel after subtracting the background influence, and the ratio image and FRET / ECFP ratio data were obtained. The data were statistically analyzed using GraphPad Prism 6.0 software. The time-course curve (mean ± S.E.M.) and scatter plot (mean ± S.E.M.) of the FRET ratio, as well as the differences between the two groups of data, were analyzed.

[0036] The differences between the data sets were analyzed by one-way ANOVA between the control group and the experimental group, and the differences between a pair of data sets were analyzed by Student's t-test. *, **, ***, **** indicate that the p-values are less than 0.05, 0.01, 0.001, and 0.0001, respectively, indicating significant differences, and "ns" indicates no significant difference.

[0037] Example 1, Construction of a vWF-based FRET biosensor and related mutant plasmids

[0038] In the first step, using pCS-CG-WT-Cer-vWF as the amplification template (Addgene.com), the target fragment of the A2 domain of human full-length vWF was amplified by PCR; using the pcDNA3.1-Ca 2+ -YPet biosensor as the amplification template ( Mingxing Ouyang , Jie Sun, Shu Chien* & Yingxiao Wang* (2008) Determinationof hierarchical relationship of Src and Rac at subcellular locations withnovel FRET biosensors. Proc Natl Acad Sci USA105(38): 14353-8.), the linear vector fragment of pcDNA3.1-ECFP-YPet without the Camodulin-M13 part was obtained by PCR. Two linear fragments were ligated using the Gibson Assembly Kit (NEB) to obtain the plasmid pcDNA3.1-ECFP-A2-YPet. The primers used in this step were designed according to the gene sequences of the ligated genes. The PCR primers used for the amplification of the A2 domain were the forward primer FP 1: ttcgtg acc gcc gcc CCC AAG AGG AAC TCC, and the reverse primer RP 1: ttc acc ttt aga cat ACC GGTACA ACA ACT; the primers used for the amplification of the linear pcDNA3.1-ECFP-YPet fragment were FP 2: AGT TGT TGT ACC GGTatg tct aaa ggt gaa gaa tta ttc act ggt; RP 2: GGA GTT CCT CTT GGG ggc ggc ggtcac gaa ctc cag (the capital letters refer to the annealing parts on the template DNA).

[0039] In the second step, using pcDNA3.1-ECFP-A2-YPet as a template, the ECFP-A2-YPet fragment was amplified by PCR; meanwhile, using pcDNA3.1-WT-vWF as an amplification master plate, PCR amplification was carried out to obtain the linear fragment pcDNA3.1-vWF-ΔA2 (without the A2 domain). These two fragments were ligated using the Gibson assembly kit, and then the A2 domain in vWF was replaced with ECFP-A2-YPet to obtain the plasmid pcDNA3.1-vWF-ECFP-A2-YPet. The primers used in the second step were as follows: The primers used for ECFP-A2-YPet amplification were FP3: TTG GGG GTT TCG ACC CTG GGG atg gtg agcggc gag gag, RP3: GGT GGG GAT CTG CAG CCC CTC ttt gta caa ttc att cat acc caattc att cat acc ctc ggt aat acc; for the amplification of pcDNA3.1-vWF-ΔA2, FP4: ggt att acc gaggggt atg aat gaa ttg tac aaa GAG GGG CTG CAG ATC CCC ACC, RP4: CTC CTC GCC CTTGCT CAC CAT ccc cag ggt cga aac ccc caa.

[0040] In the third step, the obtained plasmid pcDNA3.1-vWF-ECFP-A2-YPet was further subcloned into the vector pDisplay, which can present the expressed biosensor protein on the cell membrane surface. This subcloning step was completed by the commercial company UW Genomics (BGI) to obtain the plasmid pDisplay-vWF-ECFP-A2-YPet, and sequencing confirmation was performed. Then, the company generated the pDisplay-vWF-ECFP-LOCK-A2-YPet version by mutating asparagine 1493 at the N-terminus and cysteine 1669 at the C-terminus of the A2 domain to cysteine and glycine, respectively. Thus, the mutated A2 domain formed a disulfide bond between its N-terminus and C-terminus, preventing proper conformational changes, and was named LOCK-A2. Specifically, the A2 sequences of CCC AAG AGG AAC TCC at the N-terminus and AGT TGT TGT ACCGGT at the C-terminus were changed to CCC AAG AGG TGC TCC and AGT GGT TGT ACC GGT. Based on the two full-length vWF FRET biosensors provided by BGI, short versions of FRET biosensors, ECFP-A2-YPetonly and ECFP-LOCK-A2-YPet only, were constructed in the pDisplay vector.

[0041] The plasmids used in this study were all ampicillin (Amp)-resistant and amplified using a Midi Plasmid Extraction Kit (QIAGEN). The DNA concentration was determined by NanoDrop (TECAN), and the plasmids were available for subsequent series of cell transfection experiments.

[0042] Example 2, 293T cell culture and transfection of FRET probes

[0043] 293T cells were cultured in high-glucose DMEM medium containing 10% FBS, placed in a constant temperature incubator at 37 °C, with 5% CO2 and saturated humidity. The cells used in the experiment were generally passaged no more than 10 times. According to the transfection method of Lipofectamine 3000 liposome, the cells were seeded in a 24-well plate containing 10% FBS and antibiotic-free culture medium one day before transfection, and the cell density at the time of transfection was about 60% - 80%. 1 μg of plasmid DNA was transfected into each well; 8 - 12 h after transfection, the cells were rinsed once with 1×PBS and replaced with fresh medium without antibiotics; 24 h after the start of transfection, the cells were digested with Accutase solution and transferred into a new unsealed or completely sterilized single-channel flow chamber glass slide, and replaced with high-glucose DMEM medium containing 1% FBS. The cell flow cytometry microscopy experiment was carried out two days after the start of transfection.

[0044] Example 3, Changes in vWF-based FRET under different shear forces

[0045] To detect whether the vWF-based sensor can detect hydrodynamic forces, we applied different shear stresses of 0 to 20 dyn / cm 2 to the cells in the microfluidic channel.

[0046] Figure 2 shows the FRET changes of the vWF-based biosensor in HEK293T cells under gradient flow shear. (A) Using 0 dyn as a control, at 0, 1, 2, 5, 10, 20 dyn / cm 2FRET ratio plots of cells expressing vWF-based sensors under fluid shear for 0, 10, and 30 minutes. (B) Time-course curves (mean ± S.E.M.) of the FRET ratios (FRET / ECFP) of the cells in each group in (A) under the specified shear forces. (C) Scatter plot of the quantified changes in FRET ratios of the vWF-based sensors at 30 minutes under the specified shear forces (mean ± S.E.M.). The experiments were independently repeated 3 times. Specific values (mean ± S.E.M.) in (C): 0 dyn, -0.32 ± 0.90, N = 40; 1 dyn, 34.41 ± 2.80, N = 38; 2 dyn, 60.49 ± 2.55, N = 29; 5 dyn, 42.77 ± 1.81, N = 24; 10 dyn, 34.14 ± 2.20, N = 19; 20 dyn, 38.04 ± 3.89, N = 20. N refers to the sample size. *, **, ***, **** indicate P < 0.05, 0.01, 0.001, and 0.0001 by one-way ANOVA test; "ns" indicates no significant difference, and so on.

[0047] HEK293T cells expressing vWF-based sensors on the cell surface showed a significant FRET response to shear flow, as shown by the FRET ratio plots in Figure 2A. Specifically, for cells implanted on a single-channel flow chamber slide, without applying shear force to the cells, there was no significant change in FRET values (0 dyn); when the peristaltic pump was turned on to apply shear force, the sensor showed a significant FRET change, and the ratio (FRET / ECFP) increased over time. The FRET changes at the cell boundary were generally greater, corresponding to a greater shear force on the cell surface.

[0048] The time-course curves of the quantification of the FRET / ECFP ratio of the cells showed that there was no change in FRET before flow, and FRET gradually increased after flow (Figure 2B). Comparing the FRET changes at different flow rates, within 30 minutes, 2 dyn / cm 2 caused a greater change (60%) than 1 dyn / cm 2 (38%) (Figure 2C). At higher flow rates from 5 to 20 dyn / cm 2 , the change in FRET did not continue to increase (Figure 2C), possibly due in part to the unstable adhesion of 293T cells and cell sample loss at higher flow rates. Another possible reason is that the vWF-based sensor is highly sensitive to flow rate, and at 2 dyn / cm 2Reached the maximum change. The root cause still needs further study.

[0049] Interestingly, according to the calibration of the FRET / ECFP ratio in cells, flow induced an increase in FRET efficiency (Figure 2A&B). This indicates that under the action of hydrodynamic forces, the two ends of the A2 domain in vWF are getting closer in space. This result seems inconsistent with the previous prediction that shear force would induce the unfolding of the A2 domain, thereby exposing the cleavage site of ADAMTS 13, although theoretically the unfolding of the A2 domain and the proximity of its N-terminus and C-terminus can coexist. Compared with previous visualization studies of vWF elongation under high flow rates (hundreds of dynes / cm²), this FRET-based study was able to measure conformational changes in vWF under low shear forces (a few dynes / cm²).

[0050] Example 4, Sensitive FRET response of a vWF-based biosensor to micro-shear flow

[0051] The above results show that the FRET response of the vWF-based biosensor had no obvious difference in the flow rate range of 2 - 20 dyn / cm 2 . This example further studied the sensitivity of vWF molecules under micro-shear forces below 1.0 dyn / cm 2 . We selected a gradual increase in micro-shear forces from 0.1 to 1.0 dyn / cm 2 to study the sensitivity of the FRET response based on vWF.

[0052] Figure 3Response of vWF-based FRET under micro-shear forces of 0.1 - 1.0 dyn / cm2. (A) Representative ratio-metric FRET images of HEK293T cells expressing vWF-based biosensors after applying micro-shear forces of 0, 0.1, 0.2, 0.4, 0.8, 1.0 dyn / cm2 for 0 minutes and 30 minutes. (B) Time-course curves (mean ± S.E.M.) of normalized FRET changes before and after micro-shear flow (0.1 - 1.0 dyn / cm2) with 0 dyn / cm2 as the control. (C) Statistical comparison of percentage changes in FRET after applying different micro-shear flows (0.1 - 1.0 dyn / cm2) at 6, 10, 20, 30 minutes respectively (mean ± S.E.M.). The percentage values (mean ± S.E.M.) at 30 minutes are as follows: 0.1 dyne, 16.16 ± 2.06, N = 36; 0.2 dyne, 21.71 ± 2.04, N = 35; 0.4 dyne, 19.68 ± 1.65, N = 37; 0.8 dyne, 16.90 ± 1.95, N = 25; 1 dyne, 35.48 ± 2.86, N = 38. N refers to the sample size. The experiment was independently repeated three times.

[0053] The results showed that these micro-shear forces were sufficient to induce conformational changes in the vWF-based biosensor to generate FRET responses ( Figure 3 A). The FRET changes were positively correlated with the magnitude of the micro-shear forces (0.1 - 1.0 dyn / cm2) ( Figure 3 B), indicating that the cell membrane-anchored vWF biosensor could sense minute hydrodynamic forces. By quantitatively comparing the responses of FRET to gradient micro-shear forces, differences in FRET changes were observed as early as 6 minutes after flow, with the lowest response at 0.1 dyn / cm 2 and the response at 1.0 dyn / cm 2 being relatively higher than other smaller shear forces during the 30-minute flow (Figure 3C). These data indicate that membrane-anchored vWF molecules are highly sensitive to hydrodynamic forces.

[0054] Example 5, Shear Force-induced vWF FRET Response of Conformational Change A2

[0055] To further investigate the molecular mechanism of shear stress-induced vWF FRET response, we constructed an A2-LOCK version of the vWF FRET biosensor, in which the N-terminus and C-terminus of the A2 domain were linked by a disulfide bond (Figure 1B). Previous studies have shown that the elongation of vWF mutants with LOCK-A2 is inhibited under shear flow. To detect whether flow-induced FRET changes are dependent on vWF, we also added the MT1-MMP biosensor as a control.

[0056] Figure 4 Comparison of flow-induced FRET responses of vWF- and LOCK-vWF-based biosensors and small-sized MT1-MMP biosensors. (A) Representative ratio-measured FRET images of HEK293T cells expressing vWF or LOCK-vWF-based biosensors or MT1-MMP biosensors under a shear stress of 2 dyn / cm2. (B) Time-course curves (left) and scatter plots (right at 30 minutes) of the quantified FRET changes in the cells of each group in (A). The scatter plot values (mean ± S.E.M.) are as follows: vWF-FRET, 60.49 ± 2.56, N = 29; LOCK-vWF-FRET, 7.36 ± 1.81, N = 27; MT1-MMP FRET, 8.08 ± 1.73, N = 27. N refers to the sample size. (C, D) FRET response and time-course curve of the vWF-based biosensor under shear stress, followed by flow cessation. 2 FRET response and time-course curve under shear stress induction, followed by flow cessation.

[0057] At a flow rate of 2 dyn / cm 2 , the vWF-based biosensor underwent approximately 60% FRET change within 30 minutes, while the FRET changes of the LOCK-vWF or MT1-MMP biosensors were less than 10% (Figure 4A&B). This indicates that the hydrodynamic-induced FRET response depends on the specific characteristics of vWF molecules. The LOCK-A2 domain can resist flow-induced conformational changes, suggesting that the unfolding of A2 is crucial for vWF to sense hydrodynamic forces. The small FRET response of the MT1-MMP biosensor also indicates that the large molecular size or spring-like characteristics of vWF are essential in flow mechanosensation.

[0058] Given the plasticity of vWF molecules, we examined whether the flow-induced conformational changes of vWF in this biosensor were reversible. Surprisingly, 30 minutes after flow cessation, the change in FRET continued to increase from 60% to 78% within the subsequent 20 minutes (Figure 4C & D).

[0059] Example 6, Response of the FRET Biosensor with Only the A2 Domain to Hydrodynamics

[0060] The A2 domain is the mechanically sensitive element in vWF that responds to hydrodynamic forces. Therefore, we constructed an A2-based FRET biosensor to investigate whether only the A2 domain can sense low-amplitude flow or depends on the large-sized vWF molecule.

[0061] Figure 5 Response of the A2-based FRET biosensor to hydrodynamics. (A) Design of the FRET biosensor based only on A2 and LOCK-A2 and anchored on the cytoplasmic membrane surface. (B) Representative ratio assay FRET images of HEK293T cells expressing vWF FRET, A2FRET, or LOCK-A2 FRET biosensors under a shear force of 2 dyn / cm2. (C, D) Time-course curves (C) and scatter plots (at 30 minutes, D) of the quantification of FRET changes in each group of cells under the condition of 2 dyn / cm2 in (B). The specific values (mean ± S.E.M) of the scatter plots are as follows: vWF, 60.49 ± 2.55, N = 29; only A2, 3.13 ± 2.76, N = 23; only LOCK-A2, 5.92 ± 2.09, N = 23. (E, F) Time-course curves (E) and scatter plots (at 30 minutes, F) of the quantification of FRET changes in each group of cells under the action of 10 dyn / cm2. The scatter plot values (mean ± S.E.M) in (F): vWF, 34.14 ± 2.20, N = 19; only A2, -2.77 ± 2.66, N = 24; only LOCK-A2, 3.01 ± 2.19, N = 22. N refers to the sample size. The experiment was independently repeated 3 times.

[0062] The expressed A2 biosensor and LOCK-A2 biosensor were immobilized on the cytoplasmic membrane surface (Figure 5A). Under a shear force of 2 dyn / cm2, the seeded cells in the fluid channel showed an obvious FRET response within 30 minutes, with a change rate of approximately 60%, while the A2 FRET or LOCK-A2 FRET biosensor did not show an obvious FRET response ( Figure 5B-D). We noted that the basal FRET level of the A2-only biosensor (FRET / ECFP ratio: ~2.0) was much higher than that of the vWF-based biosensor (ratio: ~1.3) (Figure 5C), indicating that the N- and C-terminal positions of A2-only are closer, or that vWF exerts a pulling force on the A2 domain in the full-length molecule. We further applied a higher shear force (10 dyn / cm2) to the three biosensors, and the A2 biosensor was not activated either (Figure 5E&F). Thus, under low flow conditions (below 10 dyn / cm2), the spring-like vWF with a large molecular size seems to be the key to sensing shear force and inducing A2 folding.

[0063] The work of the present invention can be summarized as follows: (1) The vWF-based biosensor is anchored on the cell surface to simulate the physiological shear scenario, and shows 35% and 60% FRET responses to hydrodynamic forces of 1.4 and 2.8 dyn / cm 2 in the microfluidic channel respectively; (2) The FRET response is positively correlated with the flow rate below 2.8 dyn / cm 2 , and 0.14 dyn / cm 2 can cause a significant change (~16%), but under the experimental conditions, starting from 7-28 dyn / cm 2 , the FRET change no longer increases; (3) The flow-induced increase in FRET indicates that the two ends of A2 are closer in vWF, which is further supported by the high FRET efficiency of the A2-only biosensor, probably due to the flow-induced dissociation of A2 from the intramolecular binding of vWF; (4) The LOCK vWF biosensor with A2 ends bridged by disulfide bonds cannot sense shear force, nor can the A2-only biosensor, indicating that the structurally flexible A2 and the large vWF molecule are important in the shear mechanical response.

[0064] Considering that our probe design is mainly based on the important role of the A2 domain of the vWF protein, we confirmed that the point mutation at the relevant site of this domain almost makes the protein lose its response to shear force. When using the probe with only the A2 domain in the experiment, the cells did not show an obvious response to the shear force stimulus. The important function of the A2 domain of the vWF protein and the integrity of the whole protein jointly establish a series of roles of it in life activities, and our experimental results also confirm this theory. However, the biochemical activities of shear stress binding to other receptors involved in the blood coagulation process still need further research.

[0065] Based on the speculation from the current experimental results, shear stress can activate the activity of vWF protein, and even a very small shear force (1 dyne / cm²) can cause varying degrees of sensitive responses in cells. This indicates that cells are very sensitive to shear stress and can adapt to different hydrodynamic environments by changing the activity of vWF protein. And the A2 domain of vWF plays an important role in its function. By comparing the experimental data of the probe containing only the A2 domain and the mutant LOCK probe under the same experimental conditions, it can be concluded that the A2 domain is crucial for maintaining the overall structure and function of vWF protein. This further confirms that the A2 domain is a key functional region of vWF protein. These results also provide new insights for the related fields.

[0066] Previous studies have shown that the main conformational changes of vWF are closely related to its A2 domain, and some causes of von Willebrand disease are also due to the inability of the A2 domain of vWF protein in patients to undergo conformational changes compared with normal people. By studying the relevant research and literature in this field, we designed and constructed a LOCK-vWF FRET probe with point mutations at relevant positions in the A2 domain of vWF protein, thus obtaining the mutant A2 domain.

[0067] As a macromolecular polysaccharide protein involved in the in vivo blood coagulation process, the structural integrity of vWF plays an important role in its function. Based on the previous experimental proof that inhibiting the deformation of the vWF A2 domain may hinder its related functions, we designed and constructed fluorescent probes containing only the vWF A2 domain and its corresponding LOCK version. Under the same experimental conditions, 293T cells were transfected with these three probes for fluid shear cell experiments. In the cell state diagrams before and after applying shear force, neither A2 FRET nor LOCK-A2 FRET was activated (Figure 5 A). Combining the corresponding graphs and scatter plots, compared with the complete vWF FRET probe, in the case of only its A2 domain, the related physiological functions could not be activated (Figure 5B and C). Subsequently, under the same conditions, the shear force was increased to 10 dyn / cm 2 The activation of the three probes was also observed. Analyzing the processed graphs and scatter plots, the results were no different from the former (Figure 5 D and E).

[0068] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for detecting the response sensitivity of vWF under micro-shear force using a FRET-based biosensor, characterized in that: The steps include: (1) Inserting FRET pairs on both sides of the A2 domain of vWF to construct a FRET-based biosensor; (2) transfecting cells with the biosensor of step (1) so that the biosensor is anchored on the surface of the cell plasma membrane; (3) The cells obtained in step (2) are inoculated in a fluid experimental shear device, and the FRET microscopic imaging system collects image data of the ECFP and FRET imaging channels of the cells under the action of fluid shear force; (4) Processing and analyzing the image data obtained in step (3); analyzing the response sensitivity of the A2 domain of vWF under microshear force.

2. The method for detecting the response sensitivity of vWF under micro-shear force by a FRET-based biosensor according to claim 1, characterized in that: The FRET pair described in step (1) is composed of fluorescent proteins ECFP and YPet, and ECFP and YPet are located on both sides of the target protein respectively.

3. The method for detecting the response sensitivity of vWF under micro-shear force by a FRET-based biosensor according to claim 1, characterized in that: The specific method for constructing the FRET-based biosensor described in step (1) is: constructing a fusion protein containing the A2 domain and ECFP and YPet, labeled as ECFP-A2-YPet; connecting the ECFP-A2-YPet fragment with the vWF fragment without the A2 domain to obtain the vWF-ECFP-A2-YPet fusion protein; subcloning the plasmid containing vWF-ECFP-A2-YPet into the vector to construct the FRET-based biosensor.

4. The method for detecting the response sensitivity of vWF under micro-shear force by a FRET-based biosensor according to claim 3, characterized in that: The carrier is a pDisplay carrier.

5. The method for detecting the response sensitivity of vWF under micro-shear force using a FRET-based biosensor according to claim 1, characterized in that: The FRET-based biosensor described in step (1) also includes a vWF-ECFP-LOCK-A2-YPet biosensor with A2 domain mutation constructed by mutating the asparagine at position 1493 of the N-terminus of the A2 domain to cysteine ​​and the cysteine ​​at position 1669 of the C-terminus to glycine, and connecting the vWF fragment, as well as an ECFP-A2-YPet only biosensor not connected to vWF and an ECFP-LOCK-A2-YPet only biosensor with A2 domain mutation not connected to vWF.

6. The method for detecting the response sensitivity of vWF under micro-shear force by a FRET-based biosensor according to claim 1, characterized in that: The fluid shear experimental device described in step (3) includes a single-channel flow chamber slide and a peristaltic pump. The single-channel flow chamber slide is equipped with a Luer connector, and a hose is assembled on the Luer connector to connect with the peristaltic pump.

7. The method for detecting the response sensitivity of vWF under micro-shear force by a FRET-based biosensor according to claim 1, characterized in that: In step (3), in the FRET microscopy imaging system, the filter parameters of the ECFP imaging channel are excitation wavelength 426~446 nanometers, dichroic mirror 455 nanometers, and emission wavelength 455~495 nanometers; the filter parameters of the FRET (YPet) channel are excitation 426~446 nanometers, dichroic mirror 455 nanometers, and emission wavelength 520~550 nanometers; the Zeiss software system controls the rapid switching of ECFP and FRET imaging channels.

8. The method for detecting the response sensitivity of vWF under micro-shear force using a FRET-based biosensor according to claim 1, characterized in that: The fluid shear force in step (3) is 0 dyn / cm 2 ~ 20dyn / cm 2 .

9. A biosensor, characterized in that: A biosensor constructed by the method of claim 1 or claim 3 or claim 5.

10. Use of the method according to any one of claims 1 to 8 or the biosensor according to claim 9 in drug screening.

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