Department of Animal Physiology, Zoological Institute and Museum, University of Greifswald, Greifswald, Germany.
J Thromb Haemost. 2022 Aug;20(8):1808-1817. doi: 10.1111/jth.15762. Epub 2022 May 30.
Several leech species of the genera Hirudo, Hirudinaria, and Whitmania are widely used in traditional Chinese medicine (TCM) for the oral treatment of disorders associated with blood stasis. Among them, the non-hematophagous leech Whitmania pigra expresses a variety of components that have the potential to act on the vertebrate blood coagulation system.
Whether the thrombin inhibitor hirudin, probably the most prominent leech-derived anticoagulant, is actually present in Whitmania pigra, is still a matter of debate. To answer that open question was the aim of the study.
We identified several putative hirudin-encoding sequences in transcriptome data of Whitmania pigra. Upon gene synthesis and molecular cloning the respective recombinant proteins were expressed in Escherichia coli, purified, processed, and eventually functionally characterized for thrombin-inhibitory potencies in coagulation assays.
We were successful in the identification and functional characterization of several putative hirudins in Whitmania pigra. Some, but not all, of these factors are indeed thrombin inhibitors. Whitmania pigra hence expresses both hirudins (factors that inhibit thrombin) and hirudin-like factors (that do not or only very weakly inhibit thrombin). Furthermore, we revealed the exon/intron structures of the corresponding genes. Coding sequences of some putative hirudins of Whitmania pigra were present also in transcriptome datasets of Hirudo nipponia, a hematophagous leech that is likewise used in TCM.
Based on both structural and functional data we provide very strong evidence for the expression of hirudins in Whitmania pigra. This is the first description of hirudins in a non-hematophagous leech.
几种医用水蛭属（ Hirudo ）、舌蛭属（ Hirudinaria ）和白线蛭属（ Whitmania ）的水蛭物种被广泛应用于传统中医药中，用于口服治疗与血瘀相关的疾病。其中，非吸血性水蛭白线蛭（ Whitmania pigra ）表达了多种可能作用于脊椎动物凝血系统的成分。
作为水蛭来源的最主要抗凝剂之一，抗凝血酶水蛭素是否实际存在于白线蛭（ Whitmania pigra ）中，仍然存在争议。本研究旨在回答这个悬而未决的问题。
我们在白线蛭转录组数据中鉴定了几个可能编码水蛭素的序列。通过基因合成和分子克隆，我们在大肠杆菌中表达了相应的重组蛋白，进行了纯化、加工，并最终在凝血测定中对其抗凝血酶活性进行了功能表征。
我们成功鉴定并功能表征了白线蛭中的几个可能的水蛭素。其中一些因子确实是凝血酶抑制剂，但并非全部。因此，白线蛭既表达水蛭素（抑制凝血酶的因子），也表达水蛭素样因子（不抑制或仅弱抑制凝血酶）。此外，我们揭示了相应基因的外显子/内含子结构。某些白线蛭可能的水蛭素编码序列也存在于同样用于中医药的吸血性水蛭宽体金线蛭（ Hirudo nipponia ）的转录组数据集。
基于结构和功能数据，我们提供了非常有力的证据，证明了水蛭素在白线蛭中的表达。这是首次在非吸血性水蛭中描述水蛭素。
