Bioevaluation of Pheretima vulgaris Antithrombotic Extract, PvQ, and Isolation, Identification of Six Novel PvQ-Derived Fibrinolytic Proteases

Abstract

Thrombosis is a disease that seriously endangers human health, with a high rate of mortality and disability. However, current treatments with thrombolytic drugs (such as recombinant tissue-plasminogen activator) and the oral anticoagulants (such as dabigatran and rivaroxaban) are reported to have a tendency of major or life-threatening bleeding, such as intracranial hemorrhage or massive gastrointestinal bleed with non-specific antidotes. In contrast, lumbrokinase is very specific to fibrin as a substrate and does not cause excessive bleeding. It can dissolve the fibrin by itself or convert plasminogen to plasmin by inducing endogenous t-PA activity to dissolve fibrin clots. Therefore, searching for potentially new therapeutic molecules from earthworms is significant. In this study, we first collected a strong fibrinolytic extract (PvQ) from the total protein of the Pheretima vulgaris with AKTA pure protein purification systems; its fibrinolytic bioactivity was verified by the fibrin plate assay and zebrafish thrombotic model of vascular damage. Furthermore, according to the cell culture model of human umbilical vein endothelial cells (HUVECs), the PvQ was proven to exhibit the ability to promote the secretion of tissue-type plasminogen activator (t-PA), which further illustrated that it has an indirect thrombolytic effect. Subsequently, extensive chromatographic techniques were applied to reveal the material basis of the extract. Fortunately, six novel earthworm fibrinolytic enzymes were obtained from the PvQ, and the primary sequences of those functional proteins were determined by LC-MS/MStranscriptome cross-identification and the Edman degradation assay. The secondary structures of these six fibrinolytic enzymes were determined by circular dichroism spectroscopy and the three-dimensional structures of these proteases were predicted by MODELLER 9.23 based on multi-template modelling. In addition, those six genes encoding blood clot-dissolving proteins were cloned from P. vulgaris by RT-PCR amplification, which further determined the accuracy of proteins primary sequences identifications and laid the foundation for subsequent heterologous expression.

Traditional Chinese medicines treat ischemic stroke and their main bioactive constituents and mechanisms

“…Lumbrukinase has become a commonly used anti-thrombotic drug, which can effectively thrombolyze and improve cerebrovascular microcirculation, and exerts neuroprotective effects probably by regulating JNK1 and PI3K/Akt signaling pathways (Yang et al, 2021).Lumbrukinase has a special affinity for fibrin and can hydrolyse both fibrin-rich and fibrin-free fibrin; in addition, it can directly hydrolyse fibrinogen to make it soluble, thereby lowering fibrin and dissolving blood clots (Qiuping et al, 2021). Pheretima regulates ERK1/2, p38, t-PA, and MMP signaling pathways that may play an important role in Schwann cell migration and neural regeneration (Chang et al, 2011).…”