缺血性心脑血管疾病（如冠心病与缺血性卒中）因高发病率和高致死率已成为全球公共卫生的重大挑战。中药水蛭及水蛭素多肽因其高效的抗凝活性在传统医学中广泛应用。尽管宽体金线蛭（Whitmania pigra）作为中药水蛭药材基原物种应用历史悠久，但其抗凝活性的物质基础仍存在科学争议。传统观点认为水蛭素是抗凝核心成分，但宽体金线蛭中既未分离出水蛭素类物质，也未发现相关基因；此外，其非吸血食性与多数吸血性药用水蛭差异显著，有学者认为将其作为抗凝药材可能源于历史认知局限，需借助现代技术重新评估其药效物质基础。此外，酪氨酸磺酸化修饰作为增强天然水蛭素多肽抗凝活性的关键天然修饰，目前缺乏有效的人工磺酸化修饰体系，严重限制了重组水蛭素的临床应用。本研究旨在从宽体金线蛭基因组中序列鉴定具有明确抗凝活性的水蛭素新变体，解析其抗凝作用的分子作用基础并基于此对天然序列进行理性改造优化，提高抗凝活性；构建基于宽体金线蛭来源的酪氨酸蛋白磺基转移酶（TPST）的体外酶催化体系，实现重组水蛭素的天然结构修饰与增强抗凝活性。本研究为开发新型抗凝多肽提供研究基础，对缺血性心脑血管疾病的治疗具有积极意义。主要研究内容及结果如下：

1. 宽体金线蛭水蛭素新变体的基因组序列鉴定与功能表征：本研究利用基因组序列鉴定与分子克隆技术，从宽体金线蛭中鉴定水蛭素新变体WP_HV1。其基因全长667 bp，编码88 aa多肽，N端含三对二硫键，C端富含酸性氨基酸。cDNA文库的表达模式分析显示，该基因存在三种可变剪切转录本，但只有全长编码序列能翻译功能性蛋白。重组蛋白的体外抗凝活性检测结果表明，0.05 mg/mL重组WP_HV1将凝血酶时间（TT）由17.40 s延长至30.20 s，而等质量浓度仅含C端结构域的重组蛋白WPHV_C使TT延长至38.83 s。分子对接模拟表明，WP_HV1通过其N端插入凝血酶的催化口袋，同时C端酸性氨基酸簇与凝血酶exosite I结合，形成类似经典水蛭素的“双位点”抗凝作用机制。

2. 水蛭素新变体C端结构抗凝分子基础的解析与验证：本研究表明，WPHV_C通过其酸性氨基酸簇与凝血酶exosite I的正电荷区域产生强静电相互作用，竞争性抑制纤维蛋白原结合，从而发挥显著抗凝活性。同时，芳香性氨基酸的突变可通过π-π堆叠及π-阳离子作用增强结合稳定性。关键位点突变实验表明，将13位谷氨酸突变为丙氨酸，WPHV_C的解离ΔG由19.27 kcal/mol降至10.93 kcal/mol，TT从42.00 s降至30.94 s（0.1 mg/mL）；而26位赖氨酸突变为色氨酸后，ΔG提升至24.70 kcal/mol，TT延长至51.92 s。此外，20位酪氨酸残基的侧链芳香效应及潜在磺酸化修饰通过静电与π效应相互作用协同增效，增强与凝血酶的结合活性。

3. 后生动物TPST家族的功能结构域保守性分析与HMM鉴定工具开发：TPST是体内催化蛋白酪氨酸磺酸化的关键酶，具有对重组水蛭素进行磺酸化修饰的潜力。本研究构建了基于隐马尔可夫模型（HMM）的TPST特异性鉴定工具（TPST-HMM），并利用该工具从宽体金线蛭中鉴定并表达了同源TPST酶（WP_TPST）。进一步对后生动物TPST家族进行多序列比对和系统发育分析，发现其催化结构域、磺酸盐供体及底物酪氨酸识别与结合位点高度保守，主要通过静电相互作用实现酶-底物识别。基于此，建立了TPST数据库，提供序列储存、快速鉴定、序列比对、基序识别和活性位点注释的云服务，为酶介导的酪氨酸磺酸化修饰提供分析平台。

4. 宽体金线蛭中同源TPST介导的水蛭素酪氨酸磺酸化修饰与分子结合方式初探：利用分子对接和分子动力学模拟初步发现WP_TPST与水蛭素底物通过酸碱氨基酸间的静电作用实现特异性识别，且底物-酶复合物显示出高度构象稳定性，表明该特异性识别可能是进化适应性的结果。体外酶促反应及ELISA检测进一步确认，重组WP_TPST能催化WPHV_C的酪氨酸磺酸化修饰，并显著增强其抗凝活性，使TT延长约10 s，证明TPST在体外完成重组水蛭素磺酸化修饰的可行性。

5. 水蛭素新变体衍生抗凝先导化合物的理性设计与体内外抗凝活性验证：基于WPHV_C与凝血酶结合的分子基础，采用多点芳香性氨基酸突变策略设计合成了抗凝先导化合物WPHVC_V1。分子动力学模拟结果表明WPHVC_V1与凝血酶exosite I形成更强π-π堆叠及π-阳离子相互作用，使解离ΔG提升至37.24 kcal/mol（天然WPHV_C为19.27 kcal/mol）。体内外检测显示，WPHVC_V1在TT、APTT和PT检测中抑制凝血酶活性上均优于天然WPHV_C，其抗凝效果接近肝素钠。在小鼠尾部血栓模型中，WPHVC_V1组与CK相比抑制形成的平均血栓长度从3.562 cm减少至1.853 cm，与肝素钠组的1.729 cm接近，显著低于WPHV_C组的2.530 cm；而在小鼠颈动脉模型中，WPHVC_V1组在研究时间内能够完全抑制血栓形成，保持正常的血流通过。该结果为新型直接凝血酶抑制剂的开发提供了候选分子。

综上所述，本研究为新一代水蛭素衍生直接凝血酶抑制剂的设计、优化与修饰提供了序列来源、研究基础与催化方法参考，推动水蛭类抗凝多肽的抗凝研究。具体而言，本研究利用基因组手段在非吸血性药用水蛭中发现并鉴定了具有明确抗凝活性的水蛭素新变体，为宽体金线蛭抗凝活性物质基础研究提供了水蛭素类多肽的支持；揭示了水蛭素新变体C端结构域的抗凝作用的分子基础，初步解析酸性氨基酸与芳香性氨基酸协同作用方式；开发后生动物TPST家族专属鉴定隐马尔可夫模型，利用宽体金线蛭的重组TPST实现重组水蛭素的体外酪氨酸磺酸化修饰，为人工获得具有天然结构的重组水蛭素药物提供了生物酶参考；基于水蛭素新变体C端天然序列通过理性设计抗凝先导化合物WPHVC_V1，通过体内外药效实验证实其抗凝活性得到了优化，为直接凝血酶抑制剂药物研发提供设计策略和序列参考。

Ischemic cardiovascular and cerebrovascular diseases (such as coronary heart disease and ischemic stroke) had become major global public health challenges due to their high morbidity and mortality. Traditional Chinese medicinal leeches and hirudin peptides had been widely used in traditional medicine because of their potent anticoagulant activity; however, all the active hirudin peptides discovered thus far had been derived from hematophagous species. Whitmania pigra, an important primitive species of medicinal leeches and a non-hematophagous leech, had an unclear material basis for its anticoagulant activity. In addition, tyrosine sulfation modification, which served as a key natural modification to enhance the anticoagulant activity of natural hirudin peptides, had lacked an effective artificial sulfation modification system, thereby severely limiting the clinical application of recombinant hirudin. This study was designed to mine a new variant of hirudin with defined anticoagulant activity from the genome of W. pigra, to elucidate the molecular basis of their anticoagulant effects, and to rationally modify and optimize the natural sequences to enhance anticoagulant activity. An in vitro enzyme catalytic system based on tyrosylprotein sulfotransferase (TPST) derived from W. pigra was established to achieve natural structural modification and enhanced anticoagulant activity of recombinant hirudin. This work provided a research foundation for the development of novel anticoagulant peptides and was expected to have positive significance for the treatment of ischemic cardiovascular and cerebrovascular diseases. The main research contents and results were as follows:

1. Genomic Mining and Functional Characterization of a New Variant of Hirudin from W. pigra: Genomic mining and molecular cloning techniques were employed to identify a new hirudin variant, designated WP_HV1, from non-hematophagous W. pigra. The full-length gene was 667 bp in size and encoded an 88 aa peptide, with its N-terminus containing three pairs of disulfide bonds and its C-terminus being rich in acidic amino acids. Expression pattern analysis of the cDNA library revealed that three alternatively spliced transcripts were present, but only the full-length coding sequence was translated into a functional protein. In vitro anticoagulant activity assays demonstrated that 0.05 mg/mL of recombinant WP_HV1 prolonged thrombin time (TT) from 17.40 s to 30.20 s, whereas the recombinant protein containing only the C-terminal domain (WPHV_C) prolonged TT to 38.83 s. Molecular docking simulations revealed that WP_HV1 interacted with thrombin by inserting its N-terminus into the thrombin catalytic pocket and engaging its C-terminal acidic amino acid cluster with thrombin exosite I, thereby forming a “dual binding” anticoagulant mechanism similar to the classical hirudin anticoagulant mechanism.

2. Analysis and Validation of the Anticoagulant Molecular Basis of the C-terminal Structure of the New Variant of Hirudin: Based on the significant anticoagulant activity of WPHV_C, analyses revealed that its anticoagulant mechanism primarily derived from the strong electrostatic interactions between its acidic amino acid cluster and the positively charged region of thrombin exosite I, which competitively blocked fibrinogen binding. Aromatic amino acid mutations were found to enhance binding stability through π-π stacking and π-cation interactions. Key site mutation experiments indicated that mutating glutamic acid at position 13 to alanine reduced the dissociation ΔG from 19.27 kcal/mol to 10.93 kcal/mol and decreased TT from 42.00 s to 30.94 s (at 0.1 mg/mL); conversely, mutating lysine at position 26 to tryptophan increased the ΔG to 24.70 kcal/mol and prolonged TT to 51.92 s. Moreover, the aromatic effect of the tyrosine residue at position 20, together with its potential sulfation modification, was found to synergistically enhance binding activity through both electrostatic and π effects.

3. Conservation Analysis of the Metazoan TPST Family and Development of an HMM-Based TPST Identification Tool: TPST, a key enzyme that catalyzed tyrosine sulfation in vivo, was considered to have the potential for the sulfation modification of recombinant hirudin. An HMM-based TPST identification tool (TPST-HMM) was constructed and used to identify and express a homologous TPST enzyme (WP_TPST) from W. pigra. Multiple sequence alignment and phylogenetic analyses of the metazoan TPST family revealed that the catalytic domain, the sulfate donor, and the substrate tyrosine recognition and binding sites were highly conserved, with enzyme-substrate recognition mainly mediated by electrostatic interactions. Based on these analyses, a TPST database was established to provide cloud services for sequence storage, rapid identification, sequence alignment, motif recognition, and active site annotation, thereby offering an analytical platform for enzyme-mediated tyrosine sulfation modification.

4. Preliminary Exploration of Hirudin Tyrosine Sulfation Modification Mediated by Homologous TPST from W. pigra: Molecular docking and molecular dynamics simulations were conducted to preliminarily elucidate that WP_TPST recognized the hirudin substrate through electrostatic interactions between acidic and basic amino acids, and that the substrate-enzyme complex exhibited high conformational stability, suggesting that this specific recognition might have arisen as an evolutionary adaptation. In vitro enzymatic reactions and ELISA detection further confirmed that recombinant WP_TPST catalyzed the tyrosine sulfation modification of WPHV_C and significantly enhanced its anticoagulant activity, as evidenced by an approximate 10 s prolongation of TT, thereby proving the feasibility of using TPST for in vitro modification of recombinant hirudin.

5. Rational Design and In Vitro/In Vivo Validation of a New Anticoagulant Lead Compound Derived from the New Variant of Hirudin: Based on the molecular basis of the interaction between WPHV_C and thrombin, a multi-site aromatic amino acid mutation strategy was employed to design and synthesize the lead compound WPHVC_V1. Molecular dynamics simulations indicated that WPHVC_V1 formed stronger π-π stacking and π-cation interactions with thrombin exosite I, which increased the dissociation ΔG to 37.24 kcal/mol (compared to 19.27 kcal/mol for WPHV_C). In vitro and in vivo assays demonstrated that WPHVC_V1 exhibited superior thrombin inhibition in TT, APTT, and PT assays compared to natural WPHV_C, with its anticoagulant effect being comparable to that of heparin sodium. In the mouse tail thrombosis model, the average thrombus length in the WPHVC_V1 group was reduced from 3.562 cm in the control group to 1.853 cm, which was close to the 1.729 cm observed in the heparin sodium group and significantly lower than the 2.530 cm observed in the WPHV_C group. In the mouse carotid artery model, WPHVC_V1 was found to completely inhibit thrombus formation during the observation period, thereby maintaining normal blood flow. These findings provided candidate molecules for the development of novel direct thrombin inhibitors.

In summary, this study provided sequence resources, a research foundation, and catalytic methodologies for the design, optimization, and modification of a new generation of hirudin-derived direct thrombin inhibitors, thereby advancing the research on anticoagulant peptides derived from leeches. Specifically, the study utilized genomic approaches to identify and characterize a new hirudin variant with defined anticoagulant activity in non-hematophagous medicinal leeches, supporting investigations into the material basis of anticoagulant activity in W. pigra; it elucidated the molecular basis of the anticoagulant effect of the C-terminal domain of the new hirudin variant and provided a preliminary analysis of the synergistic interactions between acidic and aromatic amino acids; it achieved in vitro tyrosine sulfation modification of recombinant hirudin using recombinant TPST from W. pigra, thereby offering a biocatalytic reference for obtaining recombinant hirudin with a natural structure; and it rationally designed the anticoagulant lead compound WPHVC_V1 based on the natural sequence of the C-terminal domain, with in vitro and in vivo pharmacodynamic assays confirming its optimized anticoagulant activity. These efforts were anticipated to provide design strategies and sequence references for the development of direct thrombin inhibitor drugs.