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A novel role for protein disulfide isomerase ERp18 in venous thrombosis

Thrombosis Journal volume 22, Article number: 110 (2024) Cite this article

Abstract

Background

Previous studies using genetically modified mouse models and inhibitors have shown that protein disulfide isomerase (PDI) family plays a significant role in arterial thrombosis. However, their role in venous thrombosis remains unknown. In this study, using gene-modified mouse models, we determined whether PDI family members contribute to venous thrombosis.

Methods

Mice deficient of the PDI family members, including PDI, PDIp, ERp57, PDIr, ERp5, ERp27, ERp29, TMX4, ERdj5, and ERp18, were generated. The venous thrombosis phenotype of these deficient strains was evaluated using an inferior vena cava (IVC) stenosis model. Moreover, the recombinant human ERp18 (rhERp18) protein was generated and its reductase activity was assessed using a Di-E-GSSG method. The effect of ERp18 in venous thrombosis was tested in the IVC stenosis model. The levels of von Willebrand factor (vWF) at the site of venous thrombi were measured.

Results

The mice deficient in PDI, PDIp, ERp57, PDIr, ERp5, ERp27, ERp29, TMX4, and ERdj5 had no effects on venous thrombosis in the IVC stenosis model. However, the mice lacking ERp18 developed significantly less venous thrombosis compared with the WT mice. ERp18 contains one CGAC active motif. When WT or ERp18-KO mice received injection of rhERp18-WT or inactive rhERp18-mutant (Mut) protein whose CGAC was mutated to SGAS, rhERp18-Mut protein inhibited venous thrombosis in the IVC stenosis model, suggesting that the role of ERp18 is dependent on its enzymatic activity. As determined by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence staining, the levels of vWF in the plasma at the site of venous thrombus in ERp18-KO mice were significantly lower than those in WT mice.

Conclusion

ERp18 enhances the development of venous thrombosis, and its function and its enzymatic activity and regulation of the vWF release are involved.

Introduction

Venous thromboembolism (VTE), mainly caused by deep vein thrombosis (DVT) and pulmonary embolism (PE) [1], has emerged as a significant global health problem, affecting approximately 10 million patients annually [2, 3]. Surprisingly, despite advancements in diagnostic measures, the prevalence and mortality rates of VTE have continuously remained high over the past 30 years [4]. Moreover, some anticoagulant strategies appear to be effective in combating venous thrombosis, but they often have the risk of bleeding [5, 6, 7]. Therefore, a comprehensive understanding of the molecular mechanisms underlying venous thrombosis is critical for the development of efficient and safe treatment preventing and managing DVT development.

The family of protein disulfide isomerases (PDIs) is a group of oxidoreductases with 21 members in mammals [8]. Their primary role is catalyzing the correct disulfide bond formation during the protein folding process in the endoplasmic reticulum (ER) [9]. Using genetically-modified mouse models and inhibitors, we and others have found that some members of the PDI family, such as ERp57, PDI, TMX4, and ERp5 containing the Cys-X-X-Cys (CXXC) active sites, enhance platelet and coagulation activation supporting arterial thrombosis [10, 11, 12, 13, 14, 15, 16, 17]. However, the mechanism for arterial and venous thrombosis are distinct in many aspects [18], whether the PDI family enzymes play a role in venous thrombosis remains unknown.

In this study, by screening the phenotype of the mice deficient in the PDI family members in the inferior vena cava (IVC) stenosis model, we found that ERp18, which is a member of the PDI family with a CGAC motif [19, 20], is critical for venous thrombosis. ERp18 regulates the structure and function of substrates by catalyzing the reduction and oxidation of allosteric disulfide bonds [19, 21]. We found that the role of ERp18 in venous thrombosis is associated with its enzymatic activity. Moreover, ERp18 deficiency decreased the level of vWF at the site of IVC stenosis. Thus, this study provides the first genetic evidence demonstrating that ERp18 is critical for venous thrombosis.

Material and methods

Reagents

PCR Green Taq Mix (P131-01), GelRed (GR501-01), DNA marker (MD104-02), Trizol (R401-01), and HiScript II Q Select RT SuperMix (R233-01) were purchased from Vazyme Inc. The protein marker (26616) was from the Thermo Fisher Scientific Inc. The vWF ELISA kit (E-EL-1247c) was purchased from Elabscience Inc. The following antibodies used in this study were obtained from the commercial companies: Anti-ERp18 antibody (Abcam Inc, ab134938), rabbit anti-vWF antibody (Dako Inc, A0082), anti-β-actin antibody (GeneTex Inc., GTX109639), IRDye 800CW goat anti-rabbit lgG (LI-COR Bioscience Inc., 926–32,211).

Mice

To generated the knockout (KO) mice, the embryonic stem cells for PDIp-KO mice (clone number: EPD0753_5_D11), PDIr-KO mice (clone number: DEPD00576_3_G10), ERp5-floxed mice (clone number: HEPD0530-2_F02), ERp27-KO mice (clone number: EPD0688_4_C05), ERp29-KO mice (clone number: EPD0667_5_E05), TMX4-KO mice (clone number: EPD0684_1_D07), and ERdj5-KO mice (clone number: HEPD0507_5_B06) were obtained from the International Knockout Mouse Consortium (IKMC) at the Cambridge-Suda Genomic Resource Center. After passing production quality control, the ES cells were injected into murine blastocysts and transferred to pseudopregnant female mice to generate the target KO mice [22]. Because whole body ERp5 deficiency was embryonically lethal, platelets specific knockout mice deficient of ERp5 were generated by mating ERp5-floxed mice with PF4-Cre mice. Generation of the knockout mice lacking ERp57 and PDI were described in our previous study [14, 23]. ERp18-floxed mice were generated by CRISPR/Cas9 technique (Cyagen). ERp18-floxed mice were mated with CAG-Cre mice (Gempharmatech company) to produce ERp18-knockout mice. CAG serves as a robust promoter that is widely expressed and not interfered with by silencing mechanisms. Using the strong promoter ability of CAG, researchers have developed CAG-cre mice, which express cre recombinase throughout the body. When mated with floxed mice, whole-body knockout mice can be generated more efficiently. Offspring were identified by genotyping tail DNA using the following primers: CAG-cre-F 5’-CCTGCTGTCCATTCCTTATTCCATA-3’, CAG-cre-R 5’-ATATCCCCTTGTTCCCTTTCTGC-3’, ERp18-F 5’-CCCAGAAGTTCTAGTGACTGTAGG-3’, ERp18-R 5’-CCACAGATTTGGGCTTTAAAAGTAGG-3’. PCR generated 271 bp for wild type allele and 344 bp for floxed allele. The mice were fed standard rodent chow and water ad libitum, and were maintained under climate-controlled conditions in a 12-h light/dark cycle in a pathogen-free facility. The health status of the animals was monitored in accordance with the guidelines of the Institutional Animal Care and Use Committee (Soochow University) with the approved animal protocol.

RT-PCR and immunoblotting

To detect the levels of ERp18 mRNA, total RNA was extracted from major organs of the mouse to verify the knockout efficiency. In the RT-PCR reactions, cDNA was obtained through reverse transcription and amplified by PCR using specific primers: qERp18-F 5’-TCTTGGTGTGGAGCCTGCAAAG-3’, qERp18-R.

5’-CATCAGGGCTGAAGTCTTCATCC-3’. β-actin (forward primer, 5’-GTGCTATGTTGCTCTAGACTTCG-3’, reverse primer, 5’-ATGCCACAGGATTCCATACC-3’).

Major organ homogenates from mice were separated by 12% SDS-PAGE and then transferred onto a PVDF membrane. 5% skim milk in Tris-buffered saline (TBS) was used to block the membrane for 1 h at room temperature. After thorough washing with TBS, the membrane was incubated with the primary antibodies overnight at 4℃. Antibody binding was detected using IRDye 800-conjugated goat anti-rabbit IgG and visualized with an ODYSSEY infrared imaging system (LI-COR).

IVC stenosis model

The murine model of IVC was performed as described [24, 25]. In brief, mice at 8- to 12-week-old were fasted for 8 h before surgery and had free access to water. The mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital and secured on an operating table. The intestines were exteriorized, and the IVC was separated. A suture was placed on the IVC below the renal veins over a spacer, and then the spacer was removed. After 200 µL of ampicillin (10 mg/mL) was injected into the peritoneum, the peritoneum and skin were closed. After 48 h, the mice were sacrificed and thrombi formed in the IVC were harvested [24].

Preparation of recombinant ERp18 protein

Human ERp18 plasmid was a gift from Dr. Lloyd W. Ruddock (University of Oulu, Finland). Human ERp18 (UniProtKB: O95881, CGAC, rhERp18-WT) was expressed in Escherichia coli strain BL21 (DE3) pLysS and purified on an Ni Sepharose High-Performance column (GE Healthcare). The mutant recombinant ERp18 was created by mutating cysteine residues of the active site of human ERp18 to serine residues (SGAS, rhERp18-Mut). DNA sequencing confirmed the correct base substitutions.

Di-E-GSSG assay

As we previously described [26], Di-E-GSSG assay was performed in an assay buffer (0.1 M potassium phosphate buffer, 2 mM EDTA, pH 7.0). rhERp18-WT and rhERp18-Mut (2 µM) were added to the buffer containing Di-E-GSSG (150 nM) and DTT (5 µM) in a quartz cuvette. The increase in fluorescence was monitored at 545 nm with excitation at 525 nm.

Measurement of vWF level in plasma

Six hours after IVC blood flow restriction, blood samples were collected from the mice by retroorbital bleeding and vWF concentration in the diluted plasma was measured using an ELISA kit following the manufacturer’s instructions.

Confocal fluorescence microscopy analysis

Freshly isolated mouse aortas from mice were placed into a 12-well plate and fixed with 4% paraformaldehyde 2 h at room temperature. The fixed tissues were washed and dehydrated in 20% sucrose solution at 4 °C for 24 h. Each tissue was then immersed in a Tissue-Tek Optimal cutting temperature compound (O.C.T.) at room temperature for 30 min and transferred to a tinfoil cryomold, filled with O.C.T., and frozen at −20 °C until solidified. Tissue Sects. (8 μm) were prepared on microscopy slides (Citotest Scientific). The sections were fixed with cold acetone for 20 min and washed three times with 1 × PBS. After blocking with 1% BSA at room temperature for 1 h, the sections were stained with antibodies against CD31 and ERp18, as well as DAPI (SouthernBiotech). The sections were imaged using a laser confocal microscope (Olympus).

Detection of ERp18 in platelet releasate

Washed human platelets (1.5 × 109/mL) in Tyrode buffer were activated with 1 U/mL Thrombin for 10 min at room temperature in stirring. The platelets were centrifuged at 16,000 g for 20 min at 4℃ and the supernatant was collected. Triton X-100 (final 1%) and SDS (final 2.5%) were added to dissolve the micro-particles. The supernatant samples were pre-cleared with protein G for 2 h, followed by incubation with anti-ERp18 and then with protein G for 60 min. The samples were washed × 3 using platelet lysis buffer, boiled in 2 × SDS-Laemmli buffer with β-mercapoethanol for 10 min at 100℃, and electrophoresed using 12% PAGE. The band density was measured using Image J [16].

Statistical analysis

The data were expressed as the Mean ± SEM. Data were analyzed using the statistical software GraphPad Prism 9. P values < 0.05 were considered statistically significant.

Results

Generation and characterization of ERp18-KO mice

To investigate the role of ERp18 in venous thrombosis, ERp18-floxed mice were first generated by introducing loxP sites flanking exon 3 of the ERp18 gene, followed by crossing ERp18-floxed mice with CAG-Cre mice (Fig. 1, A). CAG-Cre/ERp18fl/fl (ERp18-KO) mice exhibited a homozygous floxed allele (344 bp) and the presence of the CAG-Cre gene (377 bp) (Fig. 2A). In major organs such as the liver, spleen, kidney, lung, and heart, ERp18-KO mice showed no expression of ERp18 mRNA and protein (Fig. 2B and C). ERp18 protein was also not present in ERp18-KO platelets (Fig. 2D). Confocal fluorescence microscopy revealed the expression of ERp18 in endothelial cells in arterial sections of WT mice, while it was absent in ERp18-KO mice (Fig. 2E). These data confirm the successful deletion of the ERp18 gene and ERp18 deficiency did not cause developmental problem.

Fig. 1
figure 1

The schematic diagram for generation of ERp18-KO mice. A schematic diagram of mouse ERp18 gene deletion. ERp18-floxed mice were generated by CRISPR/Cas9 technology, and ERp18-KO mice were obtained by mating ERp18-floxed mice with CAG-Cre mice

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Fig. 2
figure 2

Characterization of ERp18-KO mice. A Genotyping of WT (ERp18fl/fl) mice and ERp18-KO (CAG-Cre/ERp18fl/fl) mice using tail DNA. The bands represent PCR products of the floxed allele (the upper panel, 344 bp) and the CAG-Cre gene (the lower panel, 337 bp), respectively. B ERp18 mRNA expression in different organs from WT and KO mice was measured by RT-PCR with β-actin as the loading control. (C) ERp18 protein expression in different organs from WT and KO mice was detected by western blotting, with β-actin as the loading control. D Expression of ERp18 in platelets from WT and KO mice was evaluated by immunoblotting, with GAPDH as the loading control. E Absence of ERp18 expression (green) in the mouse aorta between WT and KO mice indicated by anti-ERp18 labeling in confocal fluorescence microscopy images, anti-CD31 (red), and DAPI (blue) were used as the marker of endothelial cells and nuclei. The scale bar was 50 μm

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ERp18 enhances venous thrombosis

To investigate the role of the PDI family members in venous thrombosis, the mice deficient of PDIs were assessed in the IVC stenosis-induced venous thrombosis model. Whole-body deficiency of PDI, ERp57, and ERp5 were embryonically lethal and the cells involved in venous thrombosis primarily include endothelial cells, leukocytes, and platelets [27]. MX1-cre/PDIfl/fl, Tie2-cre/ERp57fl/fl mice lack PDI or ERp57 in endothelial and blood cells, so these two mice can be used to evaluate the roles of PDI and ERp57 in venous thrombosis. Currently, PF4-Cre/ERp5fl/fl mice were only available because even Tie2-cre/ERp5fl/fl mice could not survive. PF4-Cre/ERp5fl/fl can be utilized to assess the role of platelet-derived ERp5 in venous thrombosis. From the results of the IVC stenosis model, we found that the knockout mice lacking PDI, ERp57, ERp5, PDIp, PDIr, ERdj5, and TMX4, which have 1 to 4 CXXC active sites, showed no significant difference in the weight of the thrombus compared to control mice (Fig. 3A-G). ERp27 and ERp29 are two PDI family members lacking the CXXC active site. Compared to the control mice, ERp27-KO or ERp29-KO mice did not exhibit a significant difference in IVC stenosis thrombosis (Fig. 3H and I). Interestingly, ERp18-KO mice had significant decrease in the thrombus weight and length development compared to the WT mice. Meanwhile, only half of the ERp18-KO mice developed thrombus (Fig. 4A-D). These results suggest that ERp18 is selectively required for venous thrombosis.

Fig. 3
figure 3

PDI, ERp57, ERp5, PDIp, PDIr, ERdj5, TMX4, ERp27 and ERp29 have a limited role in venous thrombosis. Mice were subjected to IVC ligation-induced stenosis. Forty-eight hours following the IVC ligation, mice were euthanized, and thrombus formation in the ligated vein was evaluated. A Weight of thrombus from PDI-WT and MX1-Cre/PDIfl/fl mice was evaluated. Mean ± SEM, ns, not significant, n = 7, t-test. B Weight of thrombus from ERp57-WT and Tie2-Cre/ERp57fl/fl mice was evaluated. Mean ± SEM, ns, not significant, n = 7, t-test. C Weight of thrombus from ERp5-WT and PF4-Cre/ERp5fl/fl mice was evaluated. Mean ± SEM, ns, not significant, n = 7, t-test. D Weight of thrombus from PDIp-WT and KO mice was evaluated. Mean ± SEM, ns, not significant, n = 7, t-test. E Weight of thrombus from PDIr-WT and KO mice was evaluated. Mean ± SEM, ns, not significant, n = 7, t-test. (F) Weight of thrombus from ERdj5-WT and KO mice was evaluated. Mean ± SEM, ns, not significant, n = 7, t-test. G Weight of thrombus from TMX4-WT and KO mice was evaluated. Mean ± SEM, ns, not significant, n = 7, t-test. (H) Weight of thrombus from ERp27-WT and KO mice was evaluated. Mean ± SEM, ns, not significant, n = 7, t-test. (I) Weight of thrombus from ERp29-WT and KO mice was evaluated. Mean ± SEM, ns, not significant, n = 7, t-test

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Fig. 4
figure 4

ERp18 deficiency decreases venous thrombosis. ERp18-KO and WT mice underwent IVC stenosis. After 48 h, the mice were euthanized and thrombi were harvested. A A typical thrombus formed within 48 h was shown. Additionally, B thrombus weight was assessed. Mean ± SEM, *P < 0.05, n = 6-8, t-test. C Thrombus length was assessed. Mean ± SEM, *P < 0.05, n = 6-8, t-test. D Thrombus incidence was assessed. Mean ± SEM, ***P < 0.001, n = 6-8, t-test

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Recombinant ERp18 protein plays an important role in venous thrombosis in mice and is related to its CGAC activity site

ERp18 has one CGAC active site. To determine whether the role of ERp18 in venous thrombosis is dependent on enzymatic activity, recombinant human ERp18 (rhERp18-WT, CGAC) and ERp18-inactive mutant (rhERp18-Mut, SGAS) proteins were generated in Escherichia coli (Fig. 5A). A Di-E-GSSG assay was used to verify their disulfide reductase activity. The rhERp18-WT protein, but not the rhERp18-Mut protein, exhibited a strong reductase activity (Fig. 5B). When C57BL/6 mice received the injection of the rhERp18-Mut protein, they developed significantly fewer and smaller thrombi than those treated with the PBS (Fig. 5C-F). In mice injected with rhERp18-WT was comparable to that in mice injected with PBS (data not known), suggesting that endogenous ERp18 is sufficient to support thrombosis. Additionally, ERp18-KO mice were injected with rhERp18-WT or rhERp18-Mut protein. ERp18-KO mice injected with the rhERp18-WT protein showed significantly higher thrombus weight and length compared to ERp18-KO mice injected with the rhERp18-Mut protein (Fig. 5G-J). These results suggest that the catalytic activity of ERp18 is necessary for venous thrombosis.

Fig. 5
figure 5

Enzymatice activity of ERp18 is needed for venous thrombosis in mice. A Recombinant ERp18-WT (CGAC, rhERp18-WT) and ERp18-Mut (SGAS, rhERp18-Mut) proteins were verified by coomassie blue staining (left) and western blotting (right). B A Di-E-GSSG substrate cleavage assay was used to determine the reductase activity of rhERp18-WT and rhERp18-Mut proteins. C57BL/6 mice treated with PBS or rhERp18-Mut protein underwent IVC stenosis to evaluate differences in venous thrombosis. C A typical thrombus formed in 48 h was presented. D Thrombus weight was evaluated. Mean ± SEM, *P < 0.05, n = 7-10, t-test. E Thrombus length was evaluated. Mean ± SEM, **P < 0.01, n = 7-10, t-test. F Thrombus incidence was evaluated. Mean ± SEM, ***P < 0.001, n = 7-10, t-test. ERp18-KO mice treated with rhERp18-WT or rhERp18-Mut were performed IVC stenosis to evaluate difference in venous thrombosis. G A typical thrombus formed in 48 h was presented. H Thrombus weight was evaluated. Mean ± SEM, *P < 0.05, n = 7, t-test. I Thrombus length was evaluated. Mean ± SEM, *P < 0.05, n = 7, t-test. (J) Thrombus incidence was evaluated. Mean ± SEM, ***P < 0.001, n = 7, t-test

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The level of plasma vWF at the site of venous thrombi is decreased in ERp18-KO mice

vWF plays a crucial role in platelet adhesion during venous thrombosis [28, 29]. To determine whether ERp18 affects the levels of vWF in circulation, plasma vWF level in ERp18-KO and WT mice was measured using ELISA. Under physiological conditions, the deficiency of ERp18 did not affect the level of vWF in the IVC. However, the plasma vWF level of ERp18-KO mice at the site of stenosis was significantly lower at 6 h compared to WT mice (Fig. 6A). Additionally, the vWF content in paraformaldehyde-fixed OCT-embedded thrombi was evaluated using immunofluorescence analysis. The results showed that thrombi from ERp18-KO mice contained significantly less vWF than those from WT mice (Fig. 6B-C). In wild-type mice, treatment with rhERp18-WT or rhERp18-Mut did not affect the plasma vWF levels (Supplemental Fig. 2). These results indicate that ERp18 contributes to vWF release in the model of IVC flow-restriction venous thrombosis via an intracellular mechanism.

Fig. 6
figure 6

Decreased level of vWF in venous thrombi in ERp18-KO mice. A ELISA was used to detect the concentration of vWF in the ERp18-KO and WT mice plasma after IVC stenosis. Mean ± SEM, *P < 0.05, ns, not significant, n = 5, t-test. B Mice were subjected to IVC ligation-induced stenosis. Forty-eight hours after the IVC ligation, mice were euthanized, and collected thrombi were processed for tissue sectioning. The sections were than stained with antibodies against vWF. C Thrombus constituents were quantified using integrated density. The scale bar was 500 μm. Mean ± SEM, *P < 0.05, n = 9, t-test

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Discussion

Venous thrombosis is an abnormal coagulation of blood in the venous lumen, resulting in the occlusion of the vein. In a rodent IVC stenosis model, PDI and tissue factor (TF) were found to be co-localized at the site of venous thrombi [30]. However, in this study, we found only the deficiency of ERp18 inhibited venous thrombosis, and no effects were caused by the deficiency of PDI or other members. Thus, ERp18 plays a critical role in venous thrombosis. Additionally, the role of ERp18 in venous thrombosis involves its enzymatic activity and vWF release.

ERp18 is a newly discovered member of the PDI family. Although ERp18 contributes to activation of ATF6α during the unfolded protein response in cells [31], and its role has never been characterized using genetically-modified modes. The cells involved in venous thrombosis primarily include endothelial cells, leukocytes, and platelets. In this study, we found that ERp18 is expressed in endothelial cells, leukocytes, and platelets using the western blotting (Supplemental Fig. 1, A). As detected by ERp18 antibody, ERp18 was found on the surface of nonactivated platelets and the platelet activation enhanced its expression on the surface and its release into the supernatant (Supplemental Fig. 1, B-D). On the other hand, we successfully generated a new mouse strain lacking ERp18 (Fig. 1 and 2), by which we found that the deficiency of ERp18 inhibited venous thrombosis (Fig. 4A-D). Additionally, using the rhERp18-WT and rhERp18-Mut in the IVC stenosis model, we confirmed the phenotype of ERp18-KO mice. Collectively, these data demonstrate that ERp18, like other PDIs, is secreted from either activated platelets or vascular endothelial cells and plays a critical biological role of ERp18 in facilitating venous thrombosis and its role in venous thrombosis depends on its enzymatic activity (Fig. 5 and Supplemental Fig. 1).

Several studies have documented the role of vWF in venous thrombosis [29, 32]. In our study, we found that the level of vWF was decreased in plasma and thrombus in venous thrombi in ERp18-KO mice compared with WT mice (Fig. 6A-C). However, ERp18-KO mice have similar basal vWF levels as WT mice, suggesting that ERp18 is not required for vWF biosynthesis. The decrease in plasma vWF levels of ERp18-KO mice suggests that ERp18 contributes to endothelial cell activation or exocytosis of endothelial Weibel-Palade bodies. The detailed mechanism and the substrates of ERp18 in endothelial cell activation and vWF release await further investigations.

It has been known that the hemostatic proteins important in venous thrombosis are controlled by the redox regulation of disulfide bonds. For example, the disulfide bond Cys1669-Cys1670 of vWF is necessary to maintain high affinity for glycoprotein GPIb [33]. Oxidation of the disulfide Cys186-Cys209 of TF promotes its activation [34]. The disulfide Cys362-Cys482 regulates the activation of coagulation factor FXI [35]. The binding of coagulation factor VIII to cell surface phospholipids depends on the regulation of thiol-disulfide exchange [36]. Considering that the enzymatic activity of ERp18 is necessary for venous thrombosis, ERp18 probably catalyzes the thiol-disulfide exchange in some molecules that are important in venous thrombosis. Additionally, it is also possible that ERp18 contributes to the correct folding of substrates that participate in venous thrombosis. Therefore, in our future studies, we plan to determine the substrates of ERp18 and its target cysteines using trap mutants that capture ERp18 substrates and mass spectrometry analysis.

In summary, the current study demonstrates that ERp18 plays a critical role in venous thrombosis, which is related to its enzymatic activity and vWF release. ERp18 may serve as a new therapeutic target for the treatment of venous thrombosis.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

CXXC:

Cys-X-X-Cys

DVT:

Deep Vein Thrombosis

ELISA:

Enzyme-Linked ImmunoSorbent Assay

ERp18:

Endoplasmic Reticulum protein 18

IVC:

Inferior Vena Cava

KO:

Knock Out

O.C.T.:

Optimal Cutting Temperature compound

PDI:

Protein Disulfide Isomerase

PE:

Pulmonary Embolism

TF:

Tissue Factor

VTE:

Venous Thromboembolism

vWF:

von Willebrand factor

WT:

Wild Type

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Funding

This work was supported by grants from the National Natural Science Foundation of China (82270136, 82170129, 82470132, 81970128, 81770138, 31970890, 82200147), the Suzhou Science and Technology Development Project (SKJY2021043), the Translational Research Grant of NCRCH (2020ZKPA02, 2020WSA04), the Jiangsu Provincial Medical Innovation Center (CXZX202201), the collaboration fund from State Key Laboratory of Radiation Medicine and Protection (GZN1201802), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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Author notes

  1. Chao He and Aizhen Yang contributed equally to this work.

Authors and Affiliations

  1. Collaborative Innovation Center of Hematology, State Key Laboratory of Radiation Medicine and Prevention, Cyrus Tang Medical Institute, Soochow University, Suzhou, 215123, China

    Chao He, Aizhen Yang, Zhenzhen Zhao & Yi Wu

  2. Departemnt of Hematology, The Second Hospital of Hebei Medical University, Shijiazhuang, 050000, China

    Yuxin Zhang & Jingyu Zhang

  3. Hunan Sinozex Biosciences Co, Ltd, Changsha, China

    Yi Lu

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  1. Chao He
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Contributions

Conceptualization, Y.W. and A.Y.; Methodology, C.H., A.Y., Y.Z., Z.Z.; Investigation, C.H., A.Y., Z.Z.; Formal Analysis, C.H., A.Y.; Resources, Y.L.; Validation, A.Y., Y.W.; Data curation, C.H; Writing, C.H., A.Y., J.Z., Y.W.; Supervision, A.Y., J.Z., Y.W.; Project administration, J.Z., Y.W.; Funding Acquisition, A.Y., Z.Z., J.Z., Y.W. All authors reviewed the manuscript.

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Correspondence to Aizhen Yang or Yi Wu.

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Supplementary Information

12959_2024_678_MOESM1_ESM.pptx

Supplemental Fig. 1. Detection of ERp18 expression and secretion. (A) Expression of ERp18 was detected in human endothelial, platelets, and white blood cell using western blotting. (B) Expression of ERp18 on the surface of non-activated platelets and (C) Thrombin (1 U/mL)-activated platelets. Normal rabbit IgG (nI IgG, 50 μg/ml, black) or anti-ERp18 antibody (50 μg/ml, green) were added and goat anti-rabbit (Alexa Fluor® 488) (ab150077, Abcam) was used as secondary antibody to detect. MFI ± SEM, **P < 0.01, ***P < 0.001, ns, not significant, n = 3, t-test. (D) Thrombin (1 U/mL)-induced platelet activation/aggregation releases ERp18 into the supernatant. Shown was the fold increase of ERp18 protein in the supernatant with platelet activation. ERp18 was immunoprecipitated from the supernatant and analyzed by immunoblotting. Mean ± SEM, ***P < 0.001, n = 3, t-test. Supplemental Fig. 2. Measurement of vWF levels in mice pretreated with rhERp18-WT and rhERp18-Mut proteins. As described in the legend of Fig. 5, C57BL/6 mice were treated with control buffer, rhERp18-WT, or rhERp18-Mut proteins, followed by IVC blood flow restriction. Six hours after the surgery,blood samples were collected and vWF concentration was measured using an ELISA kit. Mean ± SEM, n = 6.

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He, C., Yang, A., Zhang, Y. et al. A novel role for protein disulfide isomerase ERp18 in venous thrombosis. Thrombosis J 22, 110 (2024). https://doi.org/10.1186/s12959-024-00678-5

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Keywords

Thrombosis Journal

ISSN: 1477-9560

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