Abstract
Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) restricts platelet activation via platelet collagen receptor GPVI/FcRγ-chain. In this study, screening against collagen-induced platelet aggregation was performed to identify functional CEACAM1 extracellular domain fragments. CEACAM1 fragments, including Ala-substituted peptides, were synthesized. Platelet assays were conducted on healthy donor samples for aggregation, cytotoxicity, adhesion, spreading, and secretion. Mice were used for tail bleeding and FeCl3-induced thrombosis experiments. Clot retraction was assessed using platelet-rich plasma. Extracellular segments of CEACAM1 and A1 domain-derived peptide QDTT were identified, while N, A2, and B domains showed no involvement. QDTT inhibited platelet aggregation. Ala substitution for essential amino acids (Asp139, Thr141, Tyr142, Trp144, and Trp145) in the QDTT sequence abrogated collagen-induced aggregation inhibition. QDTT also suppressed platelet secretion and “inside-out” GP IIb/IIIa activation by convulxin, along with inhibiting PI3K/Akt pathways. QDTT curtailed FeCl3-induced mesenteric thrombosis without significantly prolonging bleeding time, implying the potential of CEACAM1 A1 domain against platelet activation without raising bleeding risk, thus paving the way for novel antiplatelet drugs.
Plain Language Summary
What is the context?
The study focuses on Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) and its role in platelet activation, particularly through the GPVI/FcRγ-chain pathway.The research aims to identify specific fragments of CEACAM1’s extracellular domain that could restrict platelet activation, without increasing bleeding risk.
What is new?
The researchers identified a peptide called QDTT derived from the A1 domain of CEACAM1’s extracellular segment. This peptide demonstrated the ability to inhibit platelet aggregation, secretion, and GP IIb/IIIa activation.
The study also revealed that specific amino acids within the QDTT sequence were essential for its inhibitory effects on collagen-induced aggregation.
What is the impact?
The findings suggest that the A1 domain-derived peptide QDTT from CEACAM1 could serve as a potential basis for developing novel antiplatelet drugs. This peptide effectively limits platelet activation and aggregation without significantly prolonging bleeding time, indicating a promising approach to managing thrombosis and related disorders while minimizing bleeding risks.
Introduction
Cardiovascular diseases are an important cause of morbidity and death worldwide.Citation1 Thrombosis is considered a major complication of cardiovascular disease, which leads to acute ischemic stroke, myocardial infarction, and venous thromboembolism.Citation2,Citation3 Platelets, which have a central role in hemostasis and thrombosis, have long been a primary target for therapeutic intervention for preventing occlusive thrombotic events.Citation1,Citation2 Over the years, various antiplatelet drugs, such as clopidogrel, aspirin, and tirofiban, have been developed. Yet, their side effects and drug resistance demand the introduction of novel, effective treatments.Citation4
Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a transmembrane glycoprotein expressed in platelets, endothelial cells, and immune cells.Citation5 Most epithelial, endothelial, and lymphocyte cells express CEACAM1-4 L and CEACAM1-4S among the 11 isoforms that make up the CEACAM1 family, all sharing the same extracellular domains,Citation6 comprising the N terminus of the immunoglobulin V (IgV)-like domain (N domain) and three immunoglobulin constant-region-type-2 (IgC2)-like domains (A1, B, and A2 domains). The differences in isoforms lie in the intracellular cytoplasmic tails of CEACAM1-4 L and CEACAM1-4S. The CEACAM1-4 L tail includes two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), whereas the CEACAM1-4S tail does not but has sequences for calmodulin and tropomyosin and may interact with the cytoskeleton.Citation7,Citation8 Previous studies using CEACAM1-deficient mice showed that CEACAM1 inhibits collagen-platelet interactions.Citation9 Furthermore, CEACAM1 is essential for platelet function through GP IIb/IIIa.Citation9,Citation10 The intracellular and extracellular domains of CEACAM1 has substantial role in regulating various cellular functions. It has been also demonstrated that the IgV-like N terminal domain of human CEACAM1 directly contributes to the implantation of cancer cells through its homotypic and heterotypic binding capabilities.Citation11–15 In addition, a study showed that the IgC2-like domain of CEACAM1 is crucial for regulating the cell-binding abilities of the protein.Citation16 Nevertheless, the regulatory mechanisms and critical domains involved in platelet activation have not been comprehensively explored.
Glycoprotein VI (GPVI) is a membrane protein expressed only in bone marrow-derived megakaryocytes and platelets. It is the primary platelet receptor for collagen.Citation17 A damaged vessel wall exposes matrix collagen, which binds to GPVI to stimulate platelet activation.Citation17 GPVI then mediates the activation of immunoreceptor tyrosine-based activation motif (ITAM) signaling pathways and promotes intracellular Ca2+ mobilization and platelet shape change.Citation17 GPVI induces platelet secretion of adenosine diphosphate (ADP), thromboxane, and soluble platelet agonists.Citation18,Citation19 These soluble factors can further activate and recruit circulation platelets adhering to the injured vessel wall surface.Citation20 Activating signaling molecules in platelet cytoplasm, known as “inside-out” signal transduction, ultimately makes integrin αIIbβ3 (GP IIb/IIIa) to adopt its high-affinity conformation.Citation21 The high-affinity conformations of GP IIb/IIIa bind to fibrinogen, facilitating the “outside-in” activation of platelets to form stable thrombi.Citation21 Antagonizing GPVI receptor function can effectively prevent thrombosis and slightly influence coagulation function.Citation17 In addition, antagonizing the function of the GPVI receptor is also conducive to reversing platelet participation in the inflammatory response.Citation17,Citation22–24 Therefore, GPVI has become a hot target for new antithrombotic therapies.
A study showed that matrix metallopeptidase-12 (MMP-12) could cleave recombinant human CEACAM1 (rhCEACAM1, aa A34-G252) into several peptide fragments in vitro.Citation14 One of these peptides, QLSNGNRTLT (QLSN), derived from an IgC2 domain (A1 domain), inhibits collagen-induced platelet activation.Citation25 Still, the rhCEACAM1 peptide fragments were only tested against platelet activation by a high dose of collagen (5 μg/mL), not by lower doses. Therefore, it is essential to determine whether other extracellular CEACAM1 domains can influence platelet activation under physiological conditions. Hence, the present study examined the effects of rhCEACAM1 protein and peptides on agonist-induced platelet aggregation. In addition, the effects of one specific fragment, QDTT, were examined on GPVI-mediated platelet activation and thrombus formation. Our findings provide novel insights into the mechanisms of thrombosis and identify potential new therapeutic targets.
Materials and methods
Antibodies and reagents
rhCEACAM1 was reconstituted in sterile phosphate-buffered saline (PBS, ST448) purchased from the Beyotime Institute of Biotechnology (Shanghai, China). GeneTex Inc. (Irvine, California, USA) provided the human FcγRIIa monoclonal antibody (IV.3, GTX14572). Convulxin (ALX-350-100-C050) was supplied by Enzo Life Sciences (Farmingdale, New York, USA). Chrono-Log Corp. (Havertown, PN, USA) provided collagen (P/N 385), ADP (P/N 384), thrombin (P/N 386), arachidonic acid (AA, P/N 390), and luciferin/luciferase (P/N 395). Prostaglandin E1 (PGE1, 900100P), fibrinogen (F3879), apyrase (A6410), and calcium ionophore (C9275) were supplied by Sigma-Aldrich (St. Louis, MO, USA). Fluo-3 AM (F880) and rhodamine (R7921) were purchased from Solarbio Life Science (Beijing, China). Thermo Fisher Scientific Inc. (Waltham, MA, US) provided Goat anti-Mouse (GAM) IgG F(ab’)2 (31166). Triton X-100 (A110694) was supplied by Sangon Biotech (Shanghai, China) Cell Signaling Technology Inc. (Danvers, Massachusetts, US) provided the antibodies for Src (2110), phospho-Src (Tyr 416, 6943), Akt (4691), phospho-Akt (Thr 308, 2965), Syk (12358), phospho-Syk (Tyr 525/526, 2710), and PLCγ2 (3872). Antibodies against phospho-PLCγ2 (Tyr 759, GTX133463) were from GeneTex Inc (Irvine, California, USA).
Synthesis, purification, and storage of the peptides
In order to explore the other active peptides, synthetic peptide sequences were designed according to the cleavage fragments of CEACAM1 (Table S2). As previously reported,Citation13 we also successfully synthesized 29 peptides of 14 amino acids in the length of extracellular CEACAM1 (Table S1). The peptides were produced using a peptide synthesizer, and their sequences were determined according to a standard method.
A Rainin Symphony Peptide Synthesizer (Mettler-Toledo, Columbus, OH, USA) was used to create the CEACAM1-derived peptides, Ala-substituted peptides, and scrambled peptides, as previously described.Citation25 Crude peptides were purified using a high-performance liquid chromatography (HPLC) column (Shimadzu LC-2010A) using a gradient elution of acetonitrile and triethylamine phosphate aqueous solution (flow rate of 1 ml/min). The peak samples were collected at A220 nm. The samples were tested for molecular weight and identification of peptide composition using MS (Shimadzu LCMS-2020). The peptides (purity ≥ 95%) were stored at −20°C. The peptides were dissolved in dimethyl sulfoxide (DMSO) and were diluted with 0.9% normal saline.
Platelet aggregation assay
All human-based experiments were performed in accordance with the Declaration of Helsinki. The study protocol was approved by the Institutional Review Board of the First Affiliated Hospital of Kunming Medical University (Kunming, China; approval #2020-L-17). All volunteers signed an informed consent form before donating blood samples.
For the determination of rhCEACAM1 concentration, previous studies determined that the serum CEACAM1 levels in healthy humans were about 7 ng/ml, while the serum CEACAM1 levels in patients with myocardial infarction were >11 ng/ml.Citation26,Citation27 On the other hand, Ergün et al.Citation28 used supraphysiological concentrations of rhCEACAM1 in the range of 100–600 ng/ml to treat human dermal microvascular endothelial cells (HDMECs) for 72 h before observing the promoter growth of HDMECs. In the present study, we used rhCEACAM concentrations ranging from 80 pM-50 nM (i.e., 3.6–2230 ng/ml) and observed that the incubation of rhCEACAM1 at 2 nM (i.e., 89 ng/ml, within the range of the physiological and pathological concentrations) for 10 min markedly inhibited collagen-induced platelet aggregation. For the determination of peptide concentration, the concentration of peptide that could inhibit collagen-induced platelet aggregation was screened using the peptides from Table S2, and the concentration was gradually increased until the concentration with the maximum collagen inhibition was reached.
Human apheresis platelets were obtained at the Yunnan Kunming Blood Center (Kunming, China) from healthy volunteers who had not taken any medication within 2 weeks.Citation14,Citation25 The methods are shown in the Supplementary Materials. ADP-induced platelet aggregation was performed using diluted human apheresis platelets, as previously described.Citation25 For FcγRIIa-mediated platelet aggregation, the washed platelets were first mixed with IV.3 (1.25 μg/mL), stirred at 1,200 rpm for 1 min, and then 20 μg/mL of GAM IgG F(ab’)2 antibody was added.Citation29
Platelet cytotoxicity assay
The measurement of lactate dehydrogenase (LDH) leakage was performed as previously reported.Citation30 The methods are shown in the Supplementary Materials.
Platelet adhesion and spreading assay
The methods are shown in the Supplementary Materials and performed as previously described.Citation31,Citation32
Platelet secretion assay
The release of ATP was detected during platelet aggregation using the Chrono-log Whole Blood/Optical Lumi-Aggregation System (Chrono-Log Corp.).Citation25 The methods are shown in the Supplementary Materials. The platelets were detected by flow cytometry, as described previously.Citation29
Western blotting
The suspended platelets were incubated with QDTT or DMSO for 10 min at 37°C. Eptifibatide (HY-B0686, MedChemExpress, Monmouth Junction, NJ, USA) prevents platelet aggregation.Citation33 The methods are shown in the Supplementary Materials.
Tail bleeding
Forty male C57BL/6 mice (4–6 weeks, 18–20 g) were purchased from SJA Laboratory Animal Company (Hunan, China). All animal procedures were carried out in accordance with institutional guidelines. The study was approved by the Experimental Animal Ethics Committee of Kunming Medical University (approval #kmmu2021737). All the animals were housed in an environment with a temperature of 18–26°C, a relative humidity of 40%–70%, and a light/dark cycle of 12/12 hr, and had free access to water and food.
The measurement of the bleeding time in mice was performed according to a previously reported method.Citation34 Male mice were randomly divided into three groups. The mice were anesthetized using an intraperitoneal injection of pentobarbital (45 mg/kg). After administration of QDTT (10 mg/kg), aspirin (100 mg/kg), or DMSO via the tail vein for 30 min, the tail was cut off 5 mm from the distal end of the mouse tail and soaked in saline at 37°C. Blood coagulation was assumed to occur when there was no blood flow for 60 s.
Mouse model of FeCl3-injured mesenteric arteriole thrombus
Injured mesenteric arterioles were observed and recorded, as previously described.Citation35 C57BL/6 mice aged 6–8 weeks were anesthetized by an intraperitoneal injection of pentobarbital (45 mg/kg). Injection of QDTT (10 mg/kg), aspirin (100 mg/kg), or DMSO and rhodamine (2.5 mg/kg) into the caudal vein was performed 30 min before thrombosis induction (10 mice/group). A filter paper (5 × 2 mm) saturated with 7.5% FeCl3 solution was placed on the mesenteric arterioles with a 60–200 μm diameter for 2 min to induce vascular injury. Within 2 min of removing the filter paper, a blood clot appeared in the vessel at the site of contact with the filter paper, indicating that the modeling was successful. During thrombosis, images were captured every 5 min using an MSHOT MD50 camera (Micro-Shot Technology Ltd.) and an Olympus i×73microscope (Olympus Corp., Tokyo, Japan).
Clot retraction
Clot retraction tests were carried out using a previously described technique with slight changes.Citation36 Platelet-rich plasma (PRP) was prepared at 500 × 109/L and incubated with QDTT at various doses (50, 250, and 500 μM) or DMSO for 10 min. Then, 200 μL of PRP, 1 mM CaCl2, 0.1 U/ml thrombin, and a siliconized clear glass tube were added, thoroughly mixed at 37°C, and images were taken every 10 min. Image J was used to determine the clot size.
Statistical analysis
GraphPad Prism 9 software (GraphPad Software Inc., San Diego, CA, USA) was used to analyze the experimental data. The results were expressed as means ± standard errors of the mean (SEM). The differences between the two experimental groups were analyzed using the t-test. One-way analysis of variance (ANOVA) and Tukey’s post hoc test were used to compare more than two groups. P-values < .05 were considered statistically significant.
Results
Screening of the extracellular domain of rhCEACAM1 and effects of the synthetic peptides on platelet aggregation
We attempted to determine whether rhCEACAM1 (aa Gln35-Gly428) contains the whole extracellular domain of amino acids of CEACAM1 and regulates platelet aggregation. We compared the aggregation of platelets in the presence or absence of 0 (vehicle: PBS), 0.08, 2, or 50 nM rhCEACAM1 over a range of acid-soluble type I collagen concentrations (0.625 or 1 μg/mL). Similar experiments were performed using GPCR agonists, including ADP (5 or 50 μM), thrombin (0.05 or 0.1 U/mL), and arachidonic acid (AA; 0.167 or 0.5 mM). As shown in , platelets displayed similar aggregation profiles in response to thrombin and AA in the presence of 0.08, 2, or 50 nM rhCEACAM1. Yet, 50 nM rhCEACAM1 treatment decreased platelet aggregation responses to collagen 0.625 µg/mL and ADP 5 µM but not to collagen 1 µg/mL and ADP 50 µM ().
We then screened the identified peptide fragments (Table S2) in low doses of collagen (1 μg/mL). Five out of the 10 peptides cleaved by MMP-12 were identified, and the remaining peptides were synthesized. The QNPV and CD66a-10 peptides were insoluble, and thus, they were not further investigated. As illustrated in , 50 μM of each peptide in Table S2 was screened for its effect on 1 μg/mL collagen-induced platelet aggregation. QDTTYLWW (QDTT) and QLSNGNRTLT (QLSN) significantly impaired collagen-induced platelet aggregation. Still, platelet aggregation in response to collagen stimulation was not substantially influenced by other peptides from the A1 domain of CEACAM1 ().
We also screened the data from Skubitz et al.Citation13 who reported 29 predicted peptide fragments (Table S1) of N, A1, B, and A2 ectodomains of CEACAM1 on collagen-induced platelet aggregation. Except for CD66a-12, which contains the QLSN amino acid sequence, the remaining 27 CEACAM1-derived peptides had no significant effect on collagen-induced platelet aggregation, even at the concentration of 200 μM (Figure S1).
Mapping the critical amino acids of QDTT
We attempted to identify the critical residues within QDTT by replacing each residue with alanine, one at a time. Our results showed that the replacement of the asparagine (D), the second threonine (T), tyrosine (Y) in the fifth position, and one or two tryptophan (W) residues in the N-terminus position resulted in the loss of the inhibitory effect of QDTT (). In addition, the scrambled peptide TLYWDWQT did not inhibit platelet aggregation (). The results show that QDTT exhibits a sequence-specific activity.
QDTT inhibits various agonist-induced platelet aggregation
rhCEACAM1 () and QDTT () significantly inhibited collagen-induced platelet aggregation. PGE1, which significantly hinders platelet activation,Citation37,Citation38 was used here as a positive control. Next, we examined the ability of QDTT to inhibit different doses of collagen-induced platelet responses. shows that 500 μM QDTT strongly inhibited platelet aggregation compared with the vehicle. Nevertheless, the efficacy of QDTT significantly decreased with the increase in collagen concentration (). QDTT also appeared to inhibit convulxin-induced platelet aggregation (). As displayed in , compared with the vehicle, 250–500 μM of QDTT reduced thrombin-, ADP-, and arachidonic acid-induced platelet aggregation while slightly influencing calcium ionophore-induced platelet aggregation and having no effect on immune-activated receptor FcγRIIa-induced platelet aggregation. Moreover, QDTT inhibited the static adhesion of platelets to a collagen surface (), and the half-maximal inhibitory concentration (IC50) was calculated as approximately 100 μM. Finally, the LDH release assay showed that 50–500 μM QDTT did not have toxicity effects on platelets under the conditions used (). The results of the present study indicate that QDTT substantially inhibits the aggregation and adhesion of platelets induced by collagen.
QDTT inhibits GPVI-mediated platelet secretion
GPVI reacts with collagen and activates platelets.Citation39 Since QDTT inhibits platelet aggregation induced by various agonists, we investigated how QDTT could affect platelet secretion induced by various agonists. As shown in , platelet P-selectin expression (representing α-granule release) and ATP release induced by convulxin were inhibited by QDTT. Next, we examined how QDTT could inhibit convulxin and other agonists-induced GP IIb/IIIa-mediated “inside-out” activation. It was revealed that both PAC-1 expression and Ca2+ release were increased in response to convulxin. PGE1 could decrease those effects, and QDTT significantly inhibited convulxin-induced platelet PAC-1 expression and Ca2+ release (). QDTT had little effect on ADP-, AA-, and thrombin-induced platelet PAC-1 expression (), and QDTT did not impair AA- and thrombin-induced platelet Ca2+ release (). The findings show that the inhibitory effects of QDTT on platelet activation are associated with GPVI-activated GP IIb/IIIa.
QDTT inhibits convulxin-induced platelet ITAM signaling
This study evaluated the hypothesis that QDTT might block convulxin-induced platelet ITAM signaling in human platelets, as QDTT was shown to reduce convulxin-induced platelet aggregation and secretion. The tyrosine phosphorylation of platelet ITAM signaling was examined under convulxin stimulation to examine this hypothesis. As shown in , the influence of QDTT on platelet ITAM signaling stimulated by convulxin was assessed. Convulxin stimulation activated Src, Syk, Akt, and PLCγ2 in platelets, and the addition of QDTT restrained the effect. Secondary mediators (such as ADP and thromboxane) are released during GPVI activation.Citation40 Therefore, to indicate whether QDTT could inhibit GPVI signaling, the signaling was investigated with inhibitors of secondary mediators by adding apyrase and indomethacin. Integrilin was added to both groups to prevent platelet aggregation during the experiment. As shown in , QDTT inhibited the activation of platelet ITAM signaling molecules induced by GPVI in the presence of apyrase and indomethacin. The evidence suggests that QDTT represses GPVI-mediated platelet activation via the Src-Syk-Akt-PLCγ2 signaling pathway.
Effects of QDTT on regulating phosphatidylinositol 3‑kinase (PI3K)/protein kinase B (Akt) signaling pathway
The PI3K pathway is crucial for GPVI-mediated platelet activation.Citation41,Citation42 The effects of QDTT on the PI3K signaling pathway were investigated using the PI3K-specific broad-spectrum antagonist Wort. and Akt activation because Akt is the main molecule downstream of the PI3K signaling pathway. shows that Wort. and QDTT reduced collagen-induced platelet aggregation and the addition of Wort. increased the inhibitory effects of QDTT. The phosphorylation levels of Akt were significantly different between the vehicle and Wort. groups. The Wort.+QDTT group had no difference in Akt activation compared with the Wort. group (). Hence, the PI3K/Akt signaling pathway could be associated with the effects of QDTT on collagen stimulation.
QDTT inhibits GP IIb/IIIa-mediated “outside-in” platelet activation
The “inside-out” activation of platelets induced by different activators causes conformational changes in GP IIb/IIIa and binding to its ligands, such as fibrinogen.Citation43 The ligand-bound integrins, in turn, cause clot retraction and spreading and “outside-in” mediated platelet activation. Therefore, we explored the effect of QDTT on platelet clot retraction and spreading. As shown in , QDTT significantly inhibited platelet spreading on fibrinogen-coated slides () and also significantly inhibited platelet clot retraction (). The above findings suggest that QDTT inhibits the function of platelet membrane protein GP IIb/IIIa.
QDTT inhibits FeCl3-induced mouse mesenteric arteriole thrombosis formation
Platelet activation suggested that QDTT could participate in platelet activation. Thus, the effects of QDTT on thrombosis in mice were explored using a FeCl3-induced mesenteric artery thrombosis model. As shown in , QDTT 10 mg/kg significantly prolonged FeCl3-induced stable mesenteric artery thrombosis. However, it was also found that QDTT was not as good as aspirin 100 mg/kg in inhibiting vessel occlusion; yet, different doses were applied.
QDTT did not prolong tail bleeding time in mice
The experimental results suggested that QDTT has an inhibitory effect on thrombosis. The present study explored the effects of QDTT on tail bleeding time in mice. As shown in , aspirin significantly prolonged the tail bleeding time in mice compared with DMSO, but QDTT did not significantly prolong tail bleeding time in mice compared with DMSO or aspirin. Also, no adverse events occurred or led to protocol modifications.
Discussion
The present study investigated the effects of the CEACAM1 extracellular domains on platelet function using rhCEACAM1 and synthetic peptides. By combining with earlier observations,Citation25 the results showed that the extracellular A1 domain of CEACAM1 contains at least two sites involved in collagen-induced platelet activation. A1 domain-derived peptide QDTT reduced platelet aggregation, and the release was mediated by convulxin in vitro and effectively inhibited thrombosis in vivo. This effect was attributed to the ITAM-dependent and PI3K/AKT signaling pathways. These results confirm that CEACAM1’s A1 domain is involved in the interaction between collagen and platelets, indicating that QDTT might be a valuable marker for developing antiplatelet drugs.
Studies using rhCEACAM1 and synthetic peptides were conducted to investigate the role of the CEACAM1 extracellular domain in collagen-induced platelet aggregation.Citation9,Citation25 Previous studies have shown that serum expression of soluble CEACAM1 is increased in various diseases closely related to thromboses, such as acute myocardial infarction and preeclampsia.Citation27,Citation44 Moreover, Skubitz et al.Citation12,Citation13 predicted and synthesized the extracellular peptide of CEACAM1 and screened seven peptides that could promote the activation and adhesion of neutrophils; these active peptides were all from the D1 domain, which mediates intercellular adhesion.Citation11–13,Citation15,Citation45–47 The present study revealed that rhCEACAM1 and A1 domain-derived polypeptides (QDTT and QLSN) were similar, inhibiting collagen-induced platelet aggregation. The findings confirmed that Asp139, Thr141, Tyr142, Trp144, and Trp145 are critical for the biological activities of QDTT on platelet aggregation. Also, the present study showed that a range of peptides generated from the N-terminal domain had no significant effect on collagen-induced platelet aggregation, and the same A2 and B domain-derived peptides did not have the functions mentioned above. This suggests that A1 domain-derived peptides (QLSN and QDTT) have a more profound role in the collagen-induced platelet response. Yet, the precise mechanism through which the A1 domain regulates platelet activity remains elusive. Multiple IgC2 domains and signals emerging from the IgC2 domain have an increased affinity for homophilic binding,Citation16 suggesting that the receptor’s capacity for intercellular binding is regulated by the number of existing IgC2 domains.Citation48
Next, we examined the regulatory mechanisms of QDTT involved in platelet activation. Our data suggested that the PI3K/Akt signaling and ITAM-dependent signaling pathways are associated with the fundamental molecular processes of the inhibitory effect of QDTT on platelet function. PI3K/Akt signaling pathway is pivotal in collagen-GPVI-induced platelet activation.Citation21,Citation39,Citation42 Human platelets express three ITAM-containing receptors: C-type lectin-like receptor 2 (CLEC-2), GPVI, and FcγRIIa,Citation49,Citation50 all having important roles in platelet function and thrombosis. The activation of these receptors involves Src kinases that promote the phosphorylation of two tyrosine residues within ITAM, and the phosphorylated receptors provide docking sites for signaling proteins containing the SH2 structural domain, such as the tyrosine kinase Syk, to initiate a signaling cascade based on intracellular tyrosine kinases, ultimately leading to the phosphorylation and activation of PLCγ2 and a cellular response.Citation51 Canobbio et al.Citation51 showed that the stimulation of platelets by agonists acting on the G protein-coupled seven transmembrane structural domain receptor (GPCR) induces the tyrosine phosphorylation of FcγRIIa, and a common feature of agonists acting on the GPCR is their ability to cause the activation of tyrosine kinases (particularly the members of the Src family) and promote tyrosine phosphorylation of a wide range of intracellular substrates. In this study, we found that QDTT represses GPVI-mediated platelet activation via the Src-Syk-Akt-PLCγ2 signaling pathway. Thus, we concluded that QDTT may indirectly inhibit platelet aggregation via the GPCR. Nevertheless, QDTT does not necessarily inhibit FcγRIIa activation during platelet activation, although the GPVI and FcγRIIa receptors share an ITAM signaling pathway.
The inside-out signaling of αIIbβ3 on platelets can be initiated by a variety of soluble agonists, e.g., ADP, thrombin, etc., which bind to the GPCR, where ligand binding induces integrin aggregation and subsequently promotes “outside-in” signaling, initiating and amplifying signaling cascade responses to drive essential platelet functions, e.g., spreading, aggregation, clot retraction, and thrombus coagulation, and integrin αIIbβ3 is the final common pathway for platelet activation.Citation21,Citation52 In this study, QDTT inhibited collagen- and convulxin-induced platelet aggregation at low concentrations, whereas it inhibited or did not inhibit platelet aggregation induced by other platelet agonists only at high concentrations. Therefore, it can be speculated that high concentrations of QDTT may inhibit platelet aggregation induced by other platelet agonists through integrin αIIbβ3, the final pathway of platelet activation. Hence, QDTT may inhibit platelet aggregation primarily through GPVI receptors and act by inhibiting integrin αIIbβ3. Still, more studies are needed to determine the exact signaling pathways involved in the effects of QDTT on platelets.
This study has a few limitations. First, the available experimental data cannot explain the polymerization state of QDTT. In future research, we plan to investigate whether QDTT may self-aggregate and clarify the effect of the aggregation state on platelet function. Second, various doses should be explored in vivo, and toxicity in vivo should be further investigated. Third, the exact mechanisms for the effects of QDTT should be explored and confirmed using knockdown, siRNAs, and overexpression of the identified genes. Multi-omics experiments could also be conducted.
In conclusion, the present study revealed that QDTT noticeably reduces convulxin-induced platelet activation. The PI3K/Akt and ITAM-dependent signaling pathways are associated with the fundamental molecular processes of QDTT’s inhibitory effects on platelet function. These results suggest that QDTT may be crucial against disorders caused by abnormal platelet activation and thrombosis.
Author contributions
Zhaohui Meng, Yujia Ye, Shengjie Chai, and Min Leng carried out the studies, participated in collecting data, and drafted the manuscript. Lihong Yang and Longcheng Ren performed the statistical analysis and participated in its design. Wen Wan, Huawei Wang, Longjun Li, and Chaozhong Li participated in the acquisition, analysis, or interpretation of data and drafted the manuscript. All authors read and approved the final manuscript.
Abbreviations
AA: | = | arachidonic acid |
BSA: | = | bovine serum albumin |
CEACAM1: | = | carcinoembryonic antigen-related cell adhesion molecule 1 |
DMSO: | = | dimethyl sulfoxide |
GPVI: | = | Glycoprotein VI |
GPIIb/IIIa: | = | Glycoprotein IIb/IIIa |
IgC: | = | immunoglobulin C |
IgG: | = | immunoglobulin G |
ITAM: | = | immunoreceptor tyrosine-based activation motif |
ITIMs: | = | immunoreceptor tyrosine-based inhibitory motifs |
LDH: | = | lactate dehydrogenase |
PBS: | = | phosphate-buffered saline |
QDTT: | = | QDTTYLWW |
QLSN: | = | QLSNGNRTLT |
rhCEACAM1: | = | recombinant human carcinoembryonic antigen-related cell adhesion molecule 1 |
Ethical statement
The study was approved by the Experimental Animal Ethics Committee of Kunming Medical University (approval #kmmu2021737) and by the Institutional Review Board of the First Affiliated Hospital of Kunming Medical University (Kunming, China; approval #2020-L-17). All methods were carried out in accordance with relevant guidelines and regulations.
Data declaration
All data generated or analyzed during this study are included in this published article [and its supplementary information files].
Supplemental Material
Download PDF (307.3 KB)Acknowledgments
We thank Dr. Peng Sang and Ph.D. Yirui Yin (College of Agriculture and Biological Science, Dali University, Dali, P. R. China.) for their insightful comments and suggestions in preparing this manuscript.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Supplementary material
Supplemental data for this article can be accessed online at https://doi.org/10.1080/09537104.2024.2308635.
Additional information
Funding
References
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