Novel Insights into the Dual Anticoagulant and Antiplatelet Potency of Fructose - 1,6 - Diphosphate In Vitro: Precision Inhibition of Coagulation Initiation and Comprehensive Suppression of Platelet Aggregation

Novel Insights into the Dual Anticoagulant and Antiplatelet Potency of Fructose - 1,6 - Diphosphate In Vitro: Precision Inhibition of Coagulation Initiation and Comprehensive Suppression of Platelet Aggregation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Novel Insights into the Dual Anticoagulant and Antiplatelet Potency of Fructose - 1,6 - Diphosphate In Vitro: Precision Inhibition of Coagulation Initiation and Comprehensive Suppression of Platelet Aggregation Yalong Zhang, Xingguo Zhong, Lin Zhou, Yuan Fang, Tongqing Chen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7406959/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Jan, 2026 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Background Fructose-1,6-diphosphate (FDP), a metabolic intermediate widely used in clinical practice for managing ischemic conditions (e.g., myocardial infarction, hemorrhagic shock) due to its cytoprotective and ATP-enhancing properties, has poorly characterized effects on hemostasis. The current study aimed to systematically evaluate its in vitro impacts on coagulation and platelet function to address this knowledge gap. Methods Global hemostasis was assessed via thromboelastography (TEG) using three platforms (Maiketian, Lepu, Dingrun). Coagulation factor activities (II, V, VII, VIII, IX, X, XI, XII) were quantified with Sysmex CS5100. Platelet aggregation in platelet-rich plasma (PRP) from 5 healthy donors was tested with agonists (adenosine diphosphate [5 µmol/L], arachidonic acid [1 mmol/L], collagen [2.5 µg/mL], epinephrine [10 µmol/L]). Dose-response relationships (FDP: 0–6 mg/mL) were analyzed by linear regression. Results FDP prolonged TEG clot reaction time (R-time) concentration-dependently (P < 0.01). At therapeutic concentration (3.71 mg/mL), R-time increased by 21.8% (Maiketian), 48.3% (Lepu), and 34.1% (Dingrun) vs. controls (n=11). FDP inversely correlated with activities of factors V, VII, IX, XI, and XII (r = −0.989 to −0.997, all P < 0.001), but not factors II, VIII, X. FDP also inhibited platelet aggregation dose-dependently (maximal suppression: 94–95% at 6 mg/mL, P < 0.001), with epinephrine-induced aggregation most sensitive. Conclusions FDP exerts dual effects via selective inhibition of coagulation initiation factors and broad suppression of platelet aggregation. Caution is advised in high-risk populations (e.g., coagulation factor deficiencies, antithrombotic therapy). Further studies are needed to evaluate its clinical implications in anticoagulation therapy and bleeding risk. Health sciences/Cardiology Health sciences/Diseases Biological sciences/Drug discovery Health sciences/Medical research Fructose-1 6-diphosphate thromboelastography coagulation factors platelet aggregation anticoagulation R-time Figures Figure 1 Background Fructose-1,6-diphosphate (FDP), a key intermediate in the glycolytic pathway, has been clinically applied for several decades in the treatment of ischemic disorders, including myocardial infarction and hemorrhagic shock. Its therapeutic efficacy in these contexts is primarily attributed to its cytoprotective effects and ability to enhance adenosine triphosphate (ATP) production, which collectively mitigate tissue damage caused by hypoxia [ 1 , 2 ]. While the benefits of FDP in protecting hypoxic tissues have been well-documented in preclinical and clinical studies [ 3 , 4 , 5 ], its interactions with the hemostatic system—particularly its effects on coagulation dynamics and platelet function—remain incompletely understood. This knowledge gap holds significant clinical relevance. FDP is frequently administered systemically to critically ill patients, a population that often presents with underlying coagulopathies and platelet dysfunction [ 6 ]. Thus, clarifying FDP’s effects on hemostasis is crucial for ensuring its safe use in such high-risk groups. Emerging evidence has suggested that FDP may modulate hemostatic processes [ 6 , 7 ]. For instance, our preliminary work demonstrated that FDP can alter results of conventional coagulation assays (prothrombin time [PT], activated partial thromboplastin time [APTT], thrombin time [TT]) and inhibit platelet aggregation [ 8 ], indicating potential broad interference with components of the hemostatic system. However, prior studies have not systematically investigated FDP’s impact on the critical phase of clot initiation—a key determinant of bleeding or thrombotic risk—nor have they explored its combined effects on specific coagulation factors and platelet signaling pathways. To address these limitations, the present study employed thromboelastography (TEG), a viscoelastic assay that comprehensively evaluates clot formation kinetics [ 9 ], alongside quantitative analyses of coagulation factor activities and platelet aggregation. By using three distinct TEG platforms and assessing multiple coagulation factors and platelet agonists, this study aimed to characterize the in vitro effects of FDP on hemostasis, with a focus on its potential dual roles in coagulation and platelet function. Such insights are essential for refining clinical guidelines regarding FDP use and exploring its potential implications in anticoagulant therapy. Materials and Methods Reagents and Instruments Thromboelastography (TEG) systems: Maiketian Haema TX (Lot 20231101, Shenzhen Maiketian Biomedical Technology Co., Ltd., China); Lepu CFMS LEPU-8880 (with Thrombelastograph General Cup Test Kit, Lot 23SH0102, Lepu Medical Technology Co., Ltd., China); Dingrun DRNX-Ⅲ (with Activated Coagulation Reagent, Lot 20230504, Chongqing Dingrun Medical Equipment Co., Ltd., China). TEG parameters measured: clot reaction time (R-time, min), clot formation time (K-time, min), α-angle (°), maximum amplitude (MA, mm). Reagent mechanism: Kaolin activator (negatively charged particles) initiated intrinsic coagulation via factor XII contact activation; calcium chloride (CaCl₂) promoted fibrin polymerization. Coagulation factor assays: Sysmex CS5100 Coagulation Analyzer (Siemens, Germany) with screening reagents (APTT: Dade Actin Activated Cephaloplastin Reagent, Lot 562729A; PT: Thromborel S, Lot 568182), factor-deficient plasmas (factors II [Lot 503659], V [Lot 575712], VII [Lot 500776], VIII [Lot 560857A], IX [Lot 504172B], X [Lot 504029], XI [Lot 503358B], XII [Lot 503427]), and controls (Standard Human Plasma, Lot 563120; CONTROL N, Lot 507936; CONTROL P, Lot 556743). Platelet aggregation agonists (Sysmex, Japan): adenosine diphosphate (ADP, 5 μmol/L), arachidonic acid (AA, 1 mmol/L), collagen (Col, 2.5 μg/mL), epinephrine (Epi, 10 μmol/L). Test compound: Fructose-1,6-diphosphate (FDP, sodium salt; Anhui Weilman Pharmaceutical Co., Ltd., Lot 20231001, China). Study Participants and Laboratory Procedures Ethical Compliance: This study was approved by the Ethics Review Committee of Anhui No. 2 Provincial People's Hospital (Approval [R] 2024-037). All participants provided written informed consent. Quality Control: Before sample analysis, all assay systems were validated using manufacturer-provided controls (normal/abnormal ranges) to ensure reliability, alongside routine calibrations. Participants: Venous blood (14 mL) was collected from 11 healthy adult volunteers (6 males, 5 females; age 31–48 years) with no history of coagulopathies or anticoagulant use within 2 weeks. PRP was prepared from 5 additional healthy donors (3 males, 2 females; age 31–42 years; platelet counts of 150–450 × 10⁹/L) using standardized protocols (centrifugation at 150 × g for 10 min at 22°C). FDP Dosing Rationale: Based on clinical intravenous doses (5–10 g/70 kg; Anhui Weilman Pharmaceutical Co.), final plasma concentrations were calculated as 1.81–3.71 mg/mL. A pre-experiment at 3.71 mg/mL confirmed significant effects, prompting full concentration-response testing (0–6 mg/mL). Experimental Protocols: Thromboelastography (TEG): Whole blood supplemented with FDP (0, 1, 2, 3, 4, 5, 6 mg/mL) was incubated at 37°C for 1 h. R-time, K-time, α-angle, and MA were measured using Maiketian, Lepu, and Dingrun systems. Coagulation Factor Assays: Plasma spiked with FDP (0–6 mg/mL) was incubated at 37°C for 1 h. Activities of factors II, V, VII, VIII, IX, X, XI, and XII were quantified via Sysmex CS5100. Platelet Aggregation: PRP supplemented with FDP (0–6 mg/mL) was stimulated with agonists (ADP, AA, collagen, epinephrine). Maximum aggregation rate (MA%) was recorded within 4 h of PRP preparation using Sysmex CS5100 software. All samples were analyzed in triplicate within 2 h of preparation to minimize preanalytical variability. Statistical Analysis Samples with 0 mg/mL FDP served as baseline controls. Percentage changes relative to controls were computed to quantify FDP-induced alterations. Statistical analyses were performed using SPSS 20.0 (IBM, Armonk, NY, USA) and Microsoft Excel 2003 (Microsoft Corporation, Redmond, WA, USA). Pearson's correlation analysis evaluated associations between FDP concentrations (0–6 mg/mL) and TEG parameters (R-time, K-time, α-angle, MA) or coagulation factor activities. Paired Student's t-test compared maximum aggregation rates at each FDP concentration (1–6 mg/mL) vs. 0 mg/mL controls. Statistical significance was defined as P < 0.05 (two-tailed). Results FDP Concentration-Dependently Prolongs TEG Clot Reaction Time TEG analysis across three platforms (Maiketian, Lepu, Dingrun) showed that FDP prolonged R-time in a concentration-dependent manner (0–6 mg/mL). Strong positive correlations were observed between FDP concentrations and R-time (Table 1): Maiketian: r = 0.988 (P < 0.001), Y = 0.4507X + 5.8693 Lepu: r = 0.999 (P < 0.001), Y = 0.4939X + 6.8696 Dingrun: r = 0.996 (P 0.05). These findings indicate FDP selectively impairs the coagulation initiation phase (reflected by R-time) but not clot propagation, kinetics, or strength. FDP Dose-Dependently Inhibits Intrinsic and Extrinsic Pathway Coagulation Factors FDP selectively inhibited coagulation initiation factors in a concentration-dependent manner (0–6 mg/mL). Significant negative correlations were observed between FDP concentrations and activities of intrinsic/extrinsic pathway factors (Table 2): Factor V: r = −0.995 (P < 0.001) Factor VII: r = −0.990 (P < 0.001) Factor IX: r = −0.989 (P < 0.001) Factor XI: r = −0.997 (P < 0.001) Factor XII: r = −0.995 (P 0.05). At therapeutic concentrations (3.71 mg/mL), R-time increased by 21.8% (Maiketian), 48.3% (Lepu), and 34.1% (Dingrun) vs. controls (n=11 per platform) (Table 3). Despite modest reductions in factor activities (<10% at 3.71 mg/mL), R-time prolongation exceeded 20%, indicating functionally significant anticoagulant effects at clinically relevant doses. FDP Dose-Dependently Inhibits Platelet Aggregation FDP suppressed platelet aggregation across all agonists in a dose-dependent manner (Table 4, Figure 1). Maximum aggregation rates (MA%) decreased progressively with increasing FDP concentrations (0–6 mg/mL): Epinephrine (10 μmol/L): MA% declined to 5.86 ± 2.73% at 6 mg/mL (P < 0.001 vs. control), with near-complete inhibition (MA% < 10%) at ≥4 mg/mL. ADP (5 μmol/L): MA% was reduced to 6.02 ± 2.67% at 6 mg/mL (P < 0.001). Arachidonic acid (1 mmol/L) and collagen (2.5 μg/mL): Significant dose-dependent suppression was observed at all concentrations (P < 0.05). Notably, epinephrine-triggered aggregation was most sensitive to FDP, with significant suppression (P < 0.05) starting at 1–3 mg/mL for all agonists. Discussion This study systematically demonstrates that FDP exerts dual anticoagulant and antiplatelet effects in vitro, characterized by selective inhibition of coagulation initiation factors, prolonged clot initiation, and broad suppression of platelet aggregation. These findings provide novel insights into FDP’s uncharacterized hemostatic properties, with implications for clinical safety and potential therapeutic expansion. FDP Selectively Impairs Coagulation Initiation TEG revealed FDP concentration-dependently prolonged R-time across three platforms, while sparing other parameters. R-time, a critical marker of the coagulation initiation phase, reflects the time required for initial fibrin clot formation via intrinsic and extrinsic pathway activation [ 9 , 10 ]. Our observation of R-time prolongation (21.8–48.3% at therapeutic concentrations) aligns with selective reductions in activities of factors V, VII, IX, XI, and XII, highlighting FDP’s targeted interference with coagulation initiation. Factor VII, a key trigger of the extrinsic pathway (activated by tissue factor), and factors IX, XI, XII (intrinsic pathway components) are pivotal for amplifying coagulation cascades [ 11 , 12 ]. The robust negative correlations between FDP levels and these factors (r = − 0.989 to − 0.997) suggest a direct or indirect inhibitory interaction. Notably, factors II, VIII, and X—central to the common pathway—remained unaffected, indicating FDP does not disrupt late-stage fibrin formation or thrombin generation. This selectivity distinguishes FDP from conventional anticoagulants: heparin broadly inhibits thrombin and factor Xa via antithrombin III [ 13 , 14 , 15 ], while warfarin non-selectively reduces vitamin K-dependent factors (II, VII, IX, X) [ 16 ]. FDP’s mechanism may involve competitive binding to coagulation factors or modulation of their conformational activation, though further structural biology studies are needed to clarify this. The discrepancy in R-time prolongation across TEG platforms (Lepu > Dingrun > Maiketian) likely stems from differences in reagent compositions (e.g., kaolin activation efficiency) [ 9 ], emphasizing the need for platform-specific reference ranges when interpreting FDP’s effects. Potent Antiplatelet Effects: Broad Agonist Inhibition FDP dose-dependently suppressed platelet aggregation induced by ADP, AA, collagen, and epinephrine, with maximal inhibition (> 90%) at 6 mg/mL. Epinephrine-induced aggregation, which is particularly sensitive to adenylate cyclase-mediated cAMP elevation [ 17 ], was most sensitive to FDP, suggesting potential modulation of cyclic nucleotide signaling. These findings extend prior reports: Cavallini et al. first observed FDP-mediated inhibition of platelet activation in 1992 [ 6 ], and de Oliveira et al. demonstrated reduced ADP-induced aggregation in septic rats treated with FDP [ 7 ]. Our study expands this by showing broad efficacy across agonists, indicating FDP may target shared downstream pathways (e.g., glycoprotein IIb/IIIa activation or intracellular Ca²⁺mobilization) rather than agonist-specific receptors. For instance, AA-induced aggregation relies on cyclooxygenase-1 (COX-1) and thromboxane A₂ synthesis [ 18 ], while collagen activates glycoprotein VI [ 19 ]; FDP’s inhibition of both suggests a converging regulatory point, possibly related to energy metabolism. As a glycolytic intermediate, FDP enhances ATP production [ 1 , 5 ], and platelet function is highly ATP-dependent [ 20 , 21 ]; whether FDP disrupts ATP balance or signaling in platelets warrants investigation. Clinical Context: Balancing Cytoprotection and Hemostatic Risk FDP is clinically used for ischemic conditions due to its cytoprotective roles in ATP preservation and anti-inflammatory effects [ 2 , 3 , 4 ]. Our data raise concerns about its hemostatic safety in high-risk populations: patients with coagulation factor deficiencies (e.g., factor VII or IX deficiency) or concurrent antithrombotic therapy (e.g., aspirin, which inhibits AA-mediated aggregation) may experience exacerbated bleeding [ 22 ]. For example, in perioperative patients with acquired coagulopathy, FDP-induced R-time prolongation at therapeutic doses may amplify hemostatic dysfunction. Conversely, FDP’s dual profile may offer therapeutic potential. Unlike single-target agents (e.g., clopidogrel inhibits ADP receptors [ 23 ]; rivaroxaban targets factor Xa [ 24 ]), FDP simultaneously modulates coagulation initiation and platelet aggregation, which could be advantageous in thrombotic disorders (e.g., stroke, deep vein thrombosis) where multi-pathway activation occurs[ 25 ]. However, translating these in vitro findings requires in vivo validation, as plasma protein binding or metabolic conversion may alter FDP’s activity. Limitations and Future Directions This study has limitations: (1) The in vitro design cannot replicate in vivo complexities (e.g., endothelial interactions, blood flow dynamics). (2) Sample sizes for platelet aggregation (n = 5) and TEG (n = 11) are small, necessitating larger cohorts. (3) The mechanism underlying factor selectivity and platelet inhibition remains speculative; structural studies (e.g., FDP-coagulation factor binding assays) and proteomic analyses of FDP-treated platelets could provide clarity. Future research should focus on: (1) In vivo models (e.g., murine thrombosis/bleeding models) to confirm FDP’s hemostatic effects. (2) Clinical studies evaluating bleeding risk in FDP-treated patients with coagulopathies. (3) Exploration of FDP’s therapeutic window in thrombotic conditions, potentially in combination with existing antithrombotics. Conclusions In summary, this in vitro study demonstrates that fructose-1,6-diphosphate (FDP) exerts dual anticoagulant and antiplatelet effects, characterized by concentration-dependent prolongation of clot initiation (reflected by TEG R-time) through selective inhibition of coagulation initiation factors (V, VII, IX, XI, and XII), as well as broad suppression of platelet aggregation induced by multiple agonists (ADP, arachidonic acid, collagen, and epinephrine). These findings provide novel insights into the previously undercharacterized hemostatic properties of FDP, a metabolic intermediate widely used in clinical practice for ischemic conditions. Notably, the selective targeting of coagulation initiation phases and platelet aggregation distinguishes FDP from conventional antithrombotic agents, highlighting its unique profile that may hold potential for therapeutic exploration. However, the observed effects were limited to in vitro settings, and caution is warranted when extrapolating to clinical scenarios—particularly in high-risk populations with coagulation factor deficiencies or concurrent antithrombotic therapy, where FDP may exacerbate bleeding risks. Further studies are required to validate these findings in vivo, clarify the underlying molecular mechanisms (e.g., direct interactions with coagulation factors or modulation of platelet signaling pathways), and evaluate the clinical implications of FDP’s dual hemostatic effects in larger cohorts. Such investigations will be critical to balancing FDP’s established cytoprotective benefits with its newly identified impacts on hemostasis, ultimately optimizing its safe and effective use in clinical practice. Declarations Data Availability Statement The original contributions presented in this study are included in the article. The raw data supporting the conclusions will be made available by the corresponding author, Tongqing Chen, without restriction. Requests for raw data can be directed to Tongqing Chen via email: [email protected] . Ethics Statement All procedures involving human participants were in accordance with the ethical standards of the institutional research committee and the 1964 Helsinki Declaration and its later amendments. The study was approved by the Ethics Review Committee of Anhui No. 2 Provincial People's Hospital (Approval [R] 2024-037). Authorship Contribution Statement Yalong Zhang: Methodology, Writing—original draft,Provision of reagents. Xingguo Zhong: Supervision, Validation. Lin Zhou: Data analysis. Yuan Fang: Formal analysis, Data analysis, Provision of reagents. Tongqing Chen: Conceptualization, Experiment conduction, Provision of reagents/analytic tools. All authors reviewed the manuscript. Funding This work was supported by the Anhui Provincial Health Commission (Health Research Program of Anhui: AHWJ2023BAc20016 and AHWJ2023BAa20021). Conflict of Interest All authors declare no competing financial interests or personal relationships that could have influenced the work. Publisher's Note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or the publisher, editors, or reviewers. No product evaluation or manufacturer claim is guaranteed or endorsed by the publisher. Informed Consent Statement Informed consent was obtained from all subjects involved in the study. References Zhang CS, Hawley SA, Zong Y, et al. Fructose-1,6-bisphosphate and aldolase mediate glucose sensing by AMPK. Nature. 2017;548(7665):112-116. Li TT, Xie JZ, Wang L, et al. Rational application of fructose-1,6-diphosphate: From the perspective of pharmacokinetics. Acta Pharmacol Sin. 2015;65(2):147-157. Munger MA, Botti RE, Grinblatt MA, et al. Effect of intravenous fructose-1,6-diphosphate on myocardial contractility in patients with left ventricular dysfunction. Pharmacotherapy. 1994;14(5):522-528. Markov AK, Neely WA, Didlake RH, et al. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7406959","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":504024058,"identity":"669f099c-8056-4b02-b4cc-7dda12884a4a","order_by":0,"name":"Yalong Zhang","email":"","orcid":"","institution":"Guoyang Hospital of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yalong","middleName":"","lastName":"Zhang","suffix":""},{"id":504024059,"identity":"de094be5-b55f-4e0a-a093-59c794f5ba1f","order_by":1,"name":"Xingguo Zhong","email":"","orcid":"","institution":"Anhui No. 2 Provincial People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xingguo","middleName":"","lastName":"Zhong","suffix":""},{"id":504024060,"identity":"45f70036-2005-463c-8e01-a18f03c76f8e","order_by":2,"name":"Lin Zhou","email":"","orcid":"","institution":"Anhui No. 2 Provincial People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Lin","middleName":"","lastName":"Zhou","suffix":""},{"id":504024061,"identity":"424d839e-e7ee-4605-8293-a43472702809","order_by":3,"name":"Yuan Fang","email":"","orcid":"","institution":"Anhui No. 2 Provincial People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Fang","suffix":""},{"id":504024062,"identity":"ee052f37-aea2-48ed-bb47-896e768a9f91","order_by":4,"name":"Tongqing Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYBAC+wMg0gCIJYD4g4GNHEEtbAxIWhhnFKQZE6mFAaKFmefD4UTCWth7D794U3DHrn9287HHNgbMCQzsh49uwKuF51ya5RyDZ8kz7hxLN84xYMtj4ElLu4FXi0SOmTGPweFkAyBDOseAp5hBgseMWC3536QtDCQSG4jQYvwYqMUOaAubNIOBARFaeM6YMc4xOJwgcSPNTLLHIMGYjaBf2HuMP7z5c9ief0byM4kff/7L8bMfPoZXC9htPAwMiQ1wLgHlIMD8AajFngiFo2AUjIJRMFIBAL+OQ+nR+0MLAAAAAElFTkSuQmCC","orcid":"","institution":"Anhui No. 2 Provincial People's Hospital","correspondingAuthor":true,"prefix":"","firstName":"Tongqing","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2025-08-19 09:38:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7406959/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7406959/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-30815-7","type":"published","date":"2026-01-05T15:58:45+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89889334,"identity":"2de73a6e-6553-4922-beda-3f702825abbe","added_by":"auto","created_at":"2025-08-26 07:11:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":146725,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative platelet aggregation (%) curves in normal healthy volunteers following stimulation with 5 µmol/L ADP (adenosine diphosphate, 5 µmol/L), Ara (arachidonic acid, AA, 1 mmol/L),Col (collagen, 2.5 µg/mL), or Epi (epinephrine , 10 µmol/L).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7406959/v1/6df9b7d93df0d5fea6615f6e.png"},{"id":100070296,"identity":"8076afe2-b92d-41a4-b025-56a4bc490815","added_by":"auto","created_at":"2026-01-12 16:17:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":763054,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7406959/v1/51b63cb3-bfd4-4ec2-ae82-7d2eb8a515d3.pdf"},{"id":89889336,"identity":"3e298ad4-db31-44fa-ab83-fc4ead2e59d0","added_by":"auto","created_at":"2025-08-26 07:11:54","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":53941,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-7406959/v1/7b44266b482b91b46f754f7f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Novel Insights into the Dual Anticoagulant and Antiplatelet Potency of Fructose - 1,6 - Diphosphate In Vitro: Precision Inhibition of Coagulation Initiation and Comprehensive Suppression of Platelet Aggregation","fulltext":[{"header":"Background","content":"\u003cp\u003eFructose-1,6-diphosphate (FDP), a key intermediate in the glycolytic pathway, has been clinically applied for several decades in the treatment of ischemic disorders, including myocardial infarction and hemorrhagic shock. Its therapeutic efficacy in these contexts is primarily attributed to its cytoprotective effects and ability to enhance adenosine triphosphate (ATP) production, which collectively mitigate tissue damage caused by hypoxia [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. While the benefits of FDP in protecting hypoxic tissues have been well-documented in preclinical and clinical studies [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], its interactions with the hemostatic system\u0026mdash;particularly its effects on coagulation dynamics and platelet function\u0026mdash;remain incompletely understood.\u003c/p\u003e\u003cp\u003eThis knowledge gap holds significant clinical relevance. FDP is frequently administered systemically to critically ill patients, a population that often presents with underlying coagulopathies and platelet dysfunction [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Thus, clarifying FDP\u0026rsquo;s effects on hemostasis is crucial for ensuring its safe use in such high-risk groups. Emerging evidence has suggested that FDP may modulate hemostatic processes [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. For instance, our preliminary work demonstrated that FDP can alter results of conventional coagulation assays (prothrombin time [PT], activated partial thromboplastin time [APTT], thrombin time [TT]) and inhibit platelet aggregation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], indicating potential broad interference with components of the hemostatic system. However, prior studies have not systematically investigated FDP\u0026rsquo;s impact on the critical phase of clot initiation\u0026mdash;a key determinant of bleeding or thrombotic risk\u0026mdash;nor have they explored its combined effects on specific coagulation factors and platelet signaling pathways.\u003c/p\u003e\u003cp\u003eTo address these limitations, the present study employed thromboelastography (TEG), a viscoelastic assay that comprehensively evaluates clot formation kinetics [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], alongside quantitative analyses of coagulation factor activities and platelet aggregation. By using three distinct TEG platforms and assessing multiple coagulation factors and platelet agonists, this study aimed to characterize the in vitro effects of FDP on hemostasis, with a focus on its potential dual roles in coagulation and platelet function. Such insights are essential for refining clinical guidelines regarding FDP use and exploring its potential implications in anticoagulant therapy.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003ch4\u003eReagents and Instruments\u003c/h4\u003e\n\u003cp\u003eThromboelastography (TEG) systems: Maiketian Haema TX (Lot 20231101, Shenzhen Maiketian Biomedical Technology Co., Ltd., China); Lepu CFMS LEPU-8880 (with Thrombelastograph General Cup Test Kit, Lot 23SH0102, Lepu Medical Technology Co., Ltd., China); Dingrun DRNX-Ⅲ (with Activated Coagulation Reagent, Lot 20230504, Chongqing Dingrun Medical Equipment Co., Ltd., China). TEG parameters measured: clot reaction time (R-time, min), clot formation time (K-time, min), α-angle (°), maximum amplitude (MA, mm). Reagent mechanism: Kaolin activator (negatively charged particles) initiated intrinsic coagulation via factor XII contact activation; calcium chloride (CaCl₂) promoted fibrin polymerization.\u003c/p\u003e\n\u003cp\u003eCoagulation factor assays: Sysmex CS5100 Coagulation Analyzer (Siemens, Germany) with screening reagents (APTT: Dade Actin Activated Cephaloplastin Reagent, Lot 562729A; PT: Thromborel S, Lot 568182), factor-deficient plasmas (factors II [Lot 503659], V [Lot 575712], VII [Lot 500776], VIII [Lot 560857A], IX [Lot 504172B], X [Lot 504029], XI [Lot 503358B], XII [Lot 503427]), and controls (Standard Human Plasma, Lot 563120; CONTROL N, Lot 507936; CONTROL P, Lot 556743).\u003c/p\u003e\n\u003cp\u003ePlatelet aggregation agonists (Sysmex, Japan): adenosine diphosphate (ADP, 5 μmol/L), arachidonic acid (AA, 1 mmol/L), collagen (Col, 2.5 μg/mL), epinephrine (Epi, 10 μmol/L).\u003c/p\u003e\n\u003cp\u003eTest compound: Fructose-1,6-diphosphate (FDP, sodium salt; Anhui Weilman Pharmaceutical Co., Ltd., Lot 20231001, China).\u003c/p\u003e\n\u003ch3\u003eStudy Participants and Laboratory Procedures\u003c/h3\u003e\n\u003cp\u003eEthical Compliance: This study was approved by the Ethics Review Committee of Anhui No. 2 Provincial People's Hospital (Approval [R] 2024-037). All participants provided written informed consent.\u003c/p\u003e\n\u003cp\u003eQuality Control: Before sample analysis, all assay systems were validated using manufacturer-provided controls (normal/abnormal ranges) to ensure reliability, alongside routine calibrations.\u003c/p\u003e\n\u003cp\u003eParticipants: Venous blood (14 mL) was collected from 11 healthy adult volunteers (6 males, 5 females; age 31–48 years) with no history of coagulopathies or anticoagulant use within 2 weeks. PRP was prepared from 5 additional healthy donors (3 males, 2 females; age 31–42 years; platelet counts of 150–450 × 10⁹/L) using standardized protocols (centrifugation at 150 × g for 10 min at 22°C).\u003c/p\u003e\n\u003cp\u003eFDP Dosing Rationale: Based on clinical intravenous doses (5–10 g/70 kg; Anhui Weilman Pharmaceutical Co.), final plasma concentrations were calculated as 1.81–3.71 mg/mL. A pre-experiment at 3.71 mg/mL confirmed significant effects, prompting full concentration-response testing (0–6 mg/mL).\u003c/p\u003e\n\u003cp\u003eExperimental Protocols: Thromboelastography (TEG): Whole blood supplemented with FDP (0, 1, 2, 3, 4, 5, 6 mg/mL) was incubated at 37°C for 1 h. R-time, K-time, α-angle, and MA were measured using Maiketian, Lepu, and Dingrun systems.\u003c/p\u003e\n\u003cp\u003eCoagulation Factor Assays: Plasma spiked with FDP (0–6 mg/mL) was incubated at 37°C for 1 h. Activities of factors II, V, VII, VIII, IX, X, XI, and XII were quantified via Sysmex CS5100.\u003c/p\u003e\n\u003cp\u003ePlatelet Aggregation: PRP supplemented with FDP (0–6 mg/mL) was stimulated with agonists (ADP, AA, collagen, epinephrine). Maximum aggregation rate (MA%) was recorded within 4 h of PRP preparation using Sysmex CS5100 software.\u003c/p\u003e\n\u003cp\u003eAll samples were analyzed in triplicate within 2 h of preparation to minimize preanalytical variability.\u003c/p\u003e\n\u003ch3\u003eStatistical Analysis\u003c/h3\u003e\n\u003cp\u003eSamples with 0 mg/mL FDP served as baseline controls. Percentage changes relative to controls were computed to quantify FDP-induced alterations. Statistical analyses were performed using SPSS 20.0 (IBM, Armonk, NY, USA) and Microsoft Excel 2003 (Microsoft Corporation, Redmond, WA, USA).\u003c/p\u003e\n\u003cp\u003ePearson's correlation analysis evaluated associations between FDP concentrations (0–6 mg/mL) and TEG parameters (R-time, K-time, α-angle, MA) or coagulation factor activities.\u003c/p\u003e\n\u003cp\u003ePaired Student's t-test compared maximum aggregation rates at each FDP concentration (1–6 mg/mL) vs. 0 mg/mL controls.\u003c/p\u003e\n\u003cp\u003eStatistical significance was defined as P \u0026lt; 0.05 (two-tailed).\u003c/p\u003e"},{"header":"Results","content":"\u003ch4\u003eFDP Concentration-Dependently Prolongs TEG Clot Reaction Time\u003c/h4\u003e\n\u003cp\u003eTEG analysis across three platforms (Maiketian, Lepu, Dingrun) showed that FDP prolonged R-time in a concentration-dependent manner (0–6 mg/mL). Strong positive correlations were observed between FDP concentrations and R-time (Table 1):\u003c/p\u003e\n\u003cp\u003eMaiketian: r = 0.988 (P \u0026lt; 0.001), Y = 0.4507X + 5.8693\u003c/p\u003e\n\u003cp\u003eLepu: r = 0.999 (P \u0026lt; 0.001), Y = 0.4939X + 6.8696\u003c/p\u003e\n\u003cp\u003eDingrun: r = 0.996 (P \u0026lt; 0.001), Y = 0.5714X + 6.6486\u003c/p\u003e\n\u003cp\u003eIn contrast, no significant correlations were observed between FDP concentrations and other TEG parameters (K-time, α-angle, MA; all P \u0026gt; 0.05). These findings indicate FDP selectively impairs the coagulation initiation phase (reflected by R-time) but not clot propagation, kinetics, or strength.\u003c/p\u003e\n\u003ch4\u003eFDP Dose-Dependently Inhibits Intrinsic and Extrinsic Pathway Coagulation Factors\u003c/h4\u003e\n\u003cp\u003eFDP selectively inhibited coagulation initiation factors in a concentration-dependent manner (0–6 mg/mL). Significant negative correlations were observed between FDP concentrations and activities of intrinsic/extrinsic pathway factors (Table 2):\u003c/p\u003e\n\u003cp\u003eFactor V: r = −0.995 (P \u0026lt; 0.001)\u003c/p\u003e\n\u003cp\u003eFactor VII: r = −0.990 (P \u0026lt; 0.001)\u003c/p\u003e\n\u003cp\u003eFactor IX: r = −0.989 (P \u0026lt; 0.001)\u003c/p\u003e\n\u003cp\u003eFactor XI: r = −0.997 (P \u0026lt; 0.001)\u003c/p\u003e\n\u003cp\u003eFactor XII: r = −0.995 (P \u0026lt; 0.001)\u003c/p\u003e\n\u003cp\u003eIn contrast, common pathway factors (II, VIII, X) showed no significant alterations (P \u0026gt; 0.05). At therapeutic concentrations (3.71 mg/mL), R-time increased by 21.8% (Maiketian), 48.3% (Lepu), and 34.1% (Dingrun) vs. controls (n=11 per platform) (Table 3). Despite modest reductions in factor activities (\u0026lt;10% at 3.71 mg/mL), R-time prolongation exceeded 20%, indicating functionally significant anticoagulant effects at clinically relevant doses.\u003c/p\u003e\n\u003ch4\u003eFDP Dose-Dependently Inhibits Platelet Aggregation\u003c/h4\u003e\n\u003cp\u003eFDP suppressed platelet aggregation across all agonists in a dose-dependent manner (Table 4, Figure 1). Maximum aggregation rates (MA%) decreased progressively with increasing FDP concentrations (0–6 mg/mL):\u003c/p\u003e\n\u003cp\u003eEpinephrine (10 μmol/L): MA% declined to 5.86 ± 2.73% at 6 mg/mL (P \u0026lt; 0.001 vs. control), with near-complete inhibition (MA% \u0026lt; 10%) at ≥4 mg/mL.\u003c/p\u003e\n\u003cp\u003eADP (5 μmol/L): MA% was reduced to 6.02 ± 2.67% at 6 mg/mL (P \u0026lt; 0.001).\u003c/p\u003e\n\u003cp\u003eArachidonic acid (1 mmol/L) and collagen (2.5 μg/mL): Significant dose-dependent suppression was observed at all concentrations (P \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003eNotably, epinephrine-triggered aggregation was most sensitive to FDP, with significant suppression (P \u0026lt; 0.05) starting at 1–3 mg/mL for all agonists.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study systematically demonstrates that FDP exerts dual anticoagulant and antiplatelet effects in vitro, characterized by selective inhibition of coagulation initiation factors, prolonged clot initiation, and broad suppression of platelet aggregation. These findings provide novel insights into FDP\u0026rsquo;s uncharacterized hemostatic properties, with implications for clinical safety and potential therapeutic expansion.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eFDP Selectively Impairs Coagulation Initiation\u003c/h2\u003e\u003cp\u003eTEG revealed FDP concentration-dependently prolonged R-time across three platforms, while sparing other parameters. R-time, a critical marker of the coagulation initiation phase, reflects the time required for initial fibrin clot formation via intrinsic and extrinsic pathway activation [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Our observation of R-time prolongation (21.8\u0026ndash;48.3% at therapeutic concentrations) aligns with selective reductions in activities of factors V, VII, IX, XI, and XII, highlighting FDP\u0026rsquo;s targeted interference with coagulation initiation.\u003c/p\u003e\u003cp\u003eFactor VII, a key trigger of the extrinsic pathway (activated by tissue factor), and factors IX, XI, XII (intrinsic pathway components) are pivotal for amplifying coagulation cascades [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The robust negative correlations between FDP levels and these factors (r\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;0.989 to \u0026minus;\u0026thinsp;0.997) suggest a direct or indirect inhibitory interaction. Notably, factors II, VIII, and X\u0026mdash;central to the common pathway\u0026mdash;remained unaffected, indicating FDP does not disrupt late-stage fibrin formation or thrombin generation.\u003c/p\u003e\u003cp\u003eThis selectivity distinguishes FDP from conventional anticoagulants: heparin broadly inhibits thrombin and factor Xa via antithrombin III [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], while warfarin non-selectively reduces vitamin K-dependent factors (II, VII, IX, X) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. FDP\u0026rsquo;s mechanism may involve competitive binding to coagulation factors or modulation of their conformational activation, though further structural biology studies are needed to clarify this.\u003c/p\u003e\u003cp\u003eThe discrepancy in R-time prolongation across TEG platforms (Lepu\u0026thinsp;\u0026gt;\u0026thinsp;Dingrun\u0026thinsp;\u0026gt;\u0026thinsp;Maiketian) likely stems from differences in reagent compositions (e.g., kaolin activation efficiency) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], emphasizing the need for platform-specific reference ranges when interpreting FDP\u0026rsquo;s effects.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003ePotent Antiplatelet Effects: Broad Agonist Inhibition\u003c/h2\u003e\u003cp\u003eFDP dose-dependently suppressed platelet aggregation induced by ADP, AA, collagen, and epinephrine, with maximal inhibition (\u0026gt;\u0026thinsp;90%) at 6 mg/mL. Epinephrine-induced aggregation, which is particularly sensitive to adenylate cyclase-mediated cAMP elevation [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], was most sensitive to FDP, suggesting potential modulation of cyclic nucleotide signaling.\u003c/p\u003e\u003cp\u003eThese findings extend prior reports: Cavallini et al. first observed FDP-mediated inhibition of platelet activation in 1992 [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], and de Oliveira et al. demonstrated reduced ADP-induced aggregation in septic rats treated with FDP [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Our study expands this by showing broad efficacy across agonists, indicating FDP may target shared downstream pathways (e.g., glycoprotein IIb/IIIa activation or intracellular Ca\u0026sup2;⁺mobilization) rather than agonist-specific receptors. For instance, AA-induced aggregation relies on cyclooxygenase-1 (COX-1) and thromboxane A₂ synthesis [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], while collagen activates glycoprotein VI [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]; FDP\u0026rsquo;s inhibition of both suggests a converging regulatory point, possibly related to energy metabolism. As a glycolytic intermediate, FDP enhances ATP production [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], and platelet function is highly ATP-dependent [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]; whether FDP disrupts ATP balance or signaling in platelets warrants investigation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eClinical Context: Balancing Cytoprotection and Hemostatic Risk\u003c/h2\u003e\u003cp\u003eFDP is clinically used for ischemic conditions due to its cytoprotective roles in ATP preservation and anti-inflammatory effects [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Our data raise concerns about its hemostatic safety in high-risk populations: patients with coagulation factor deficiencies (e.g., factor VII or IX deficiency) or concurrent antithrombotic therapy (e.g., aspirin, which inhibits AA-mediated aggregation) may experience exacerbated bleeding [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. For example, in perioperative patients with acquired coagulopathy, FDP-induced R-time prolongation at therapeutic doses may amplify hemostatic dysfunction.\u003c/p\u003e\u003cp\u003eConversely, FDP\u0026rsquo;s dual profile may offer therapeutic potential. Unlike single-target agents (e.g., clopidogrel inhibits ADP receptors [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]; rivaroxaban targets factor Xa [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]), FDP simultaneously modulates coagulation initiation and platelet aggregation, which could be advantageous in thrombotic disorders (e.g., stroke, deep vein thrombosis) where multi-pathway activation occurs[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, translating these in vitro findings requires in vivo validation, as plasma protein binding or metabolic conversion may alter FDP\u0026rsquo;s activity.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eLimitations and Future Directions\u003c/h2\u003e\u003cp\u003eThis study has limitations: (1) The in vitro design cannot replicate in vivo complexities (e.g., endothelial interactions, blood flow dynamics). (2) Sample sizes for platelet aggregation (n\u0026thinsp;=\u0026thinsp;5) and TEG (n\u0026thinsp;=\u0026thinsp;11) are small, necessitating larger cohorts. (3) The mechanism underlying factor selectivity and platelet inhibition remains speculative; structural studies (e.g., FDP-coagulation factor binding assays) and proteomic analyses of FDP-treated platelets could provide clarity.\u003c/p\u003e\u003cp\u003eFuture research should focus on: (1) In vivo models (e.g., murine thrombosis/bleeding models) to confirm FDP\u0026rsquo;s hemostatic effects. (2) Clinical studies evaluating bleeding risk in FDP-treated patients with coagulopathies. (3) Exploration of FDP\u0026rsquo;s therapeutic window in thrombotic conditions, potentially in combination with existing antithrombotics.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, this in vitro study demonstrates that fructose-1,6-diphosphate (FDP) exerts dual anticoagulant and antiplatelet effects, characterized by concentration-dependent prolongation of clot initiation (reflected by TEG R-time) through selective inhibition of coagulation initiation factors (V, VII, IX, XI, and XII), as well as broad suppression of platelet aggregation induced by multiple agonists (ADP, arachidonic acid, collagen, and epinephrine). These findings provide novel insights into the previously undercharacterized hemostatic properties of FDP, a metabolic intermediate widely used in clinical practice for ischemic conditions.\u003c/p\u003e\n\u003cp\u003eNotably, the selective targeting of coagulation initiation phases and platelet aggregation distinguishes FDP from conventional antithrombotic agents, highlighting its unique profile that may hold potential for therapeutic exploration. However, the observed effects were limited to in vitro settings, and caution is warranted when extrapolating to clinical scenarios\u0026mdash;particularly in high-risk populations with coagulation factor deficiencies or concurrent antithrombotic therapy, where FDP may exacerbate bleeding risks.\u003c/p\u003e\n\u003cp\u003eFurther studies are required to validate these findings in vivo, clarify the underlying molecular mechanisms (e.g., direct interactions with coagulation factors or modulation of platelet signaling pathways), and evaluate the clinical implications of FDP\u0026rsquo;s dual hemostatic effects in larger cohorts. Such investigations will be critical to balancing FDP\u0026rsquo;s established cytoprotective benefits with its newly identified impacts on hemostasis, ultimately optimizing its safe and effective use in clinical practice.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch3\u003eData Availability Statement\u003c/h3\u003e\n\u003cp\u003eThe original contributions presented in this study are included in the article. The raw data supporting the conclusions will be made available by the corresponding author, Tongqing Chen, without restriction. Requests for raw data can be directed to Tongqing Chen via email: [email protected].\u003c/p\u003e\n\u003ch3\u003eEthics Statement\u003c/h3\u003e\n\u003cp\u003eAll procedures involving human participants were in accordance with the ethical standards of the institutional research committee and the 1964 Helsinki Declaration and its later amendments. The study was approved by the Ethics Review Committee of Anhui No. 2 Provincial People's Hospital (Approval [R] 2024-037).\u003c/p\u003e\n\u003ch3\u003eAuthorship Contribution Statement\u003c/h3\u003e\n\u003cp\u003eYalong Zhang: Methodology, Writing—original draft,Provision of reagents.\u003cbr\u003e\u0026nbsp;Xingguo Zhong: Supervision, Validation.\u003cbr\u003e\u0026nbsp;Lin Zhou: Data analysis.\u003cbr\u003e\u0026nbsp;Yuan Fang: Formal analysis, Data analysis, Provision of reagents.\u003cbr\u003e\u0026nbsp;Tongqing Chen: Conceptualization, Experiment conduction, Provision of reagents/analytic tools.\u003c/p\u003e\n\u003cp\u003eAll authors reviewed the manuscript.\u003c/p\u003e\n\u003ch3\u003eFunding\u003c/h3\u003e\n\u003cp\u003eThis work was supported by the Anhui Provincial Health Commission (Health Research Program of Anhui: AHWJ2023BAc20016 and AHWJ2023BAa20021).\u003c/p\u003e\n\u003ch3\u003eConflict of Interest\u003c/h3\u003e\n\u003cp\u003eAll authors declare no competing financial interests or personal relationships that could have influenced the work.\u003c/p\u003e\n\u003ch3\u003ePublisher's Note\u003c/h3\u003e\n\u003cp\u003eAll claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or the publisher, editors, or reviewers. No product evaluation or manufacturer claim is guaranteed or endorsed by the publisher.\u003c/p\u003e\n\u003ch3\u003eInformed Consent Statement\u003c/h3\u003e\n\u003cp\u003eInformed consent was obtained from all subjects involved in the study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eZhang CS, Hawley SA, Zong Y, et al. Fructose-1,6-bisphosphate and aldolase mediate glucose sensing by AMPK. Nature. 2017;548(7665):112-116.\u003c/li\u003e\n \u003cli\u003eLi TT, Xie JZ, Wang L, et al. Rational application of fructose-1,6-diphosphate: From the perspective of pharmacokinetics. Acta Pharmacol Sin. 2015;65(2):147-157.\u003c/li\u003e\n \u003cli\u003eMunger MA, Botti RE, Grinblatt MA, et al. Effect of intravenous fructose-1,6-diphosphate on myocardial contractility in patients with left ventricular dysfunction. Pharmacotherapy. 1994;14(5):522-528. \u0026nbsp;\u003c/li\u003e\n \u003cli\u003eMarkov AK, Neely WA, Didlake RH, et al. Metabolic responses to fructose-1,6-diphosphate in healthy subjects. Metabolism. 2000;49(6):698-703.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eRipari P, Pieralisi G. Effects of fructose-1,6-diphosphate on heart rate, ventilation, oxygen consumption and endurance performance. Pharmatherapeutica. 1988;5(4):249-255.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eCavallini L, Deana R, Francesconi MA, et al. Fructose-1,6-diphosphate inhibits platelet activation. Biochem Pharmacol. 1992;43(7):1539-1544.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003ede Oliveira LM, Sim\u0026otilde;es Pires MG, Magrisso AB, et al. Fructose-1,6-bisphosphate inhibits in vitro and ex vivo platelet aggregation induced by ADP and ameliorates coagulation alterations in experimental sepsis in rats. J Thromb Thrombolysis. 2010;29(4):387-394.\u003c/li\u003e\n \u003cli\u003eChen TQ, Chen D, Chen L,et al. The effects of fructose diphosphate on routine coagulation tests in vitro. Sci Rep.(2022)12(1):304.\u003c/li\u003e\n \u003cli\u003eWhiting D, DiNardo JA. TEG and ROTEM: Technology and clinical applications. Am J Hematol. 2014;89:228-232.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eDavie EW, Fujikawa K, Kisiel W. The coagulation cascade: initiation, maintenance, and regulation. Biochemistry. 1991;30:10363-10370.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eMann KG, Brummel-Ziedins K, Orfeo T, et al. Models of blood coagulation. Blood Cells Mol Dis. 2006;36:108-117.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eWinter WE, Flax SD, Harris NS. Coagulation Testing in the Core Laboratory. Lab Med. 2017 ;48(4):295-313.\u003c/li\u003e\n \u003cli\u003eHarenberg J, Fenyvesi T. Heparin, thrombin and Factor Xa inhibitors. Hamostaseologie.2004 ;24(4):261-78.\u003c/li\u003e\n \u003cli\u003eHao C, Xu H, Yu L, Zhang L. Heparin: An essential drug for modern medicine.Prog Mol Biol Transl Sci. 2019;163:1-19.\u003c/li\u003e\n \u003cli\u003eSingh P, Singh K, Jairajpuri MA. Energetics of hydrogen bond switch, residue burial and cavity analysis reveals molecular basis of improved heparin binding to antithrombin. J Biomol Struct Dyn. 2011 ;29(2):339-50.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eNakamura K, Toyohira H, Kariyazono H, et al. Anticoagulant effects of warfarin and kinetics of K vitamins in blood and feces. Artery. 1994;21(3):148-60.\u003c/li\u003e\n \u003cli\u003eSpalding A, Vaitkevicius H, Dill S,et al. Mechanism of epinephrine-induced platelet aggregation. Hypertension. 1998;31(2):603-607.\u003c/li\u003e\n \u003cli\u003eFloyd CN, Ferro A. Mechanisms of aspirin resistance. Pharmacol. Ther. 2014;141:69\u0026ndash;78.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eFeitsma LJ, Brondijk HC, Jarvis GE, et al. Structural insights into collagen binding by platelet receptor glycoprotein VI.Blood. 2022;139(20):3087-3098.\u003c/li\u003e\n \u003cli\u003eSpalding A,Vaitkevicius H, Dill S, et al. Mechanism of epinephrine-induced platelet aggregation.Hypertension. 1998;31(2):603-607.\u003c/li\u003e\n \u003cli\u003eLai KC, Flaumenhaft R. SNARE protein degradation upon platelet activation: calpain cleaves SNAP-23.J Cell Physiol. 2003;194(2):206-214. )\u003c/li\u003e\n \u003cli\u003eMousa SA, Forsythe MS, Bozarth JM, et al. Effect of single oral dose of aspirin on human platelet functions and plasma plasminogen activator inhibitor-1. Cardiology. 1993;83(5-6):367-73.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eCoukell AJ, Markham A. Clopidogrel. Drugs.1997;54(5):745-50.\u003c/li\u003e\n \u003cli\u003eIto Y, Maejima Y, Nakagama S,et al. Rivaroxaban, a Direct Oral Factor Xa Inhibitor, Attenuates Atherosclerosis by Alleviating Factor Xa-PAR2-Mediated Autophagy Suppression.JACC Basic Transl Sci. 2021;6(12):964-980.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eCushman M. Epidemiology and risk factors for venous thrombosis. Semin Hematol. 2007;44(2 ):62-69. \u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 4 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Fructose-1,6-diphosphate, thromboelastography, coagulation factors, platelet aggregation, anticoagulation, R-time","lastPublishedDoi":"10.21203/rs.3.rs-7406959/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7406959/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground \u003c/strong\u003eFructose-1,6-diphosphate (FDP), a metabolic intermediate widely used in clinical practice for managing ischemic conditions (e.g., myocardial infarction, hemorrhagic shock) due to its cytoprotective and ATP-enhancing properties, has poorly characterized effects on hemostasis. The current study aimed to systematically evaluate its in vitro impacts on coagulation and platelet function to address this knowledge gap.\u003cbr\u003e\n \u003cstrong\u003eMethods \u003c/strong\u003eGlobal hemostasis was assessed via thromboelastography (TEG) using three platforms (Maiketian, Lepu, Dingrun). Coagulation factor activities (II, V, VII, VIII, IX, X, XI, XII) were quantified with Sysmex CS5100. Platelet aggregation in platelet-rich plasma (PRP) from 5 healthy donors was tested with agonists (adenosine diphosphate [5 µmol/L], arachidonic acid [1 mmol/L], collagen [2.5 µg/mL], epinephrine [10 µmol/L]). Dose-response relationships (FDP: 0–6 mg/mL) were analyzed by linear regression.\u003cbr\u003e\n \u003cstrong\u003eResults \u003c/strong\u003eFDP prolonged TEG clot reaction time (R-time) concentration-dependently (P \u0026lt; 0.01). At therapeutic concentration (3.71 mg/mL), R-time increased by 21.8% (Maiketian), 48.3% (Lepu), and 34.1% (Dingrun) vs. controls (n=11). FDP inversely correlated with activities of factors V, VII, IX, XI, and XII (r = −0.989 to −0.997, all P \u0026lt; 0.001), but not factors II, VIII, X. FDP also inhibited platelet aggregation dose-dependently (maximal suppression: 94–95% at 6 mg/mL, P \u0026lt; 0.001), with epinephrine-induced aggregation most sensitive.\u003cbr\u003e\n \u003cstrong\u003eConclusions \u003c/strong\u003eFDP exerts dual effects via selective inhibition of coagulation initiation factors and broad suppression of platelet aggregation. Caution is advised in high-risk populations (e.g., coagulation factor deficiencies, antithrombotic therapy). Further studies are needed to evaluate its clinical implications in anticoagulation therapy and bleeding risk.\u003c/p\u003e","manuscriptTitle":"Novel Insights into the Dual Anticoagulant and Antiplatelet Potency of Fructose - 1,6 - Diphosphate In Vitro: Precision Inhibition of Coagulation Initiation and Comprehensive Suppression of Platelet Aggregation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-26 07:11:50","doi":"10.21203/rs.3.rs-7406959/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-26T14:12:51+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-26T06:36:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-24T18:19:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-22T13:41:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"335383489212649426002687827971989009958","date":"2025-09-19T12:26:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"238243202457494339200355221648576121950","date":"2025-09-17T12:51:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"211377813388578889124473028870697757107","date":"2025-09-10T17:43:53+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-05T19:50:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-05T15:07:41+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-08-28T06:12:15+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-21T11:15:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-08-21T09:31:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0e2cedbd-2bdc-4831-b2bc-df4e93315d62","owner":[],"postedDate":"August 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":53549116,"name":"Health sciences/Cardiology"},{"id":53549117,"name":"Health sciences/Diseases"},{"id":53549118,"name":"Biological sciences/Drug discovery"},{"id":53549119,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2026-01-12T16:13:27+00:00","versionOfRecord":{"articleIdentity":"rs-7406959","link":"https://doi.org/10.1038/s41598-025-30815-7","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-01-05 15:58:45","publishedOnDateReadable":"January 5th, 2026"},"versionCreatedAt":"2025-08-26 07:11:50","video":"","vorDoi":"10.1038/s41598-025-30815-7","vorDoiUrl":"https://doi.org/10.1038/s41598-025-30815-7","workflowStages":[]},"version":"v1","identity":"rs-7406959","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7406959","identity":"rs-7406959","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}