Envenomation and death resulting from snakebites represent a significant public health problem worldwide, particularly in tropical and subtropical regions. The WHO has defined snakebite as a neglected tropical health concern. Bites from Macrovipera lebetina obtusa usually cause life-threatening systemic hemodynamic disturbances, reduced functionality of the kidneys, and other serious symptoms, including hypotension shock, edema, and tissue necrosis, at the bite site. Herein, we highlight five cases of M. l. obtusa envenomation that presented with wide-ranging manifestations. Many recovered cases were left with long-term musculoskeletal disabilities. In a particular case, a 15-year-old male patient was envenomed in his palm by an 80-cm M. l. obtusa. Within 12 hours, swelling extended to near the shoulder. Fasciotomy was performed on the forearm and part of the upper arm of this patient. Symptoms of severe localized pain and swelling, dizziness, weakness, low blood pressure, and itching around the bite area were documented. The patient remained in the hospital for 13 days.

Snake venoms contain components selected to immobilize prey. The venoms from Elapidae mainly contain neurotoxins, which are critical for rapid prey paralysis, while the venoms from Viperidae and Colubridae may contain fewer neurotoxins but are likely to induce circulatory disorders. Here, we show that the venoms from Protobothrops mucrosquamatus and Trimeresurus stejnegeri are comparable to those of Naja atra in prey immobilization. Further studies indicate that snake C-type lectin-like proteins (snaclecs), which are one of the main nonenzymatic components in viper venoms, are responsible for rapid prey immobilization. Snaclecs (mucetin and stejnulxin) from the venoms of P. mucrosquamatus and T. stejnegeri induce the aggregation of both mammalian platelets and avian thrombocytes, leading to acute cerebral ischemia, and reduced animal locomotor activity and exploration in the open field test. Viper venoms in the absence of snaclecs fail to aggregate platelets and thrombocytes, and thus show an attenuated ability to cause cerebral ischemia and immobilization of their prey. This work provides novel insights into the prey immobilization mechanism of Viperidae snakes and the understanding of viper envenomation-induced cerebral infarction.

Protease inhibitors have been reported rarely from the leech Hirudinaria manillensis. In this study, we purified a novel protease inhibitor (bdellin-HM-2) with anticoagulant properties from H. manillensis. With a molecular weight of 1.4x104, bdellin-HM-2 was also characterized with three intra-molecular disulfide bridges at the N-terminus and multiple HHXDD and HXDDmotifs at the C-terminus. cDNAcloning revealed that the putative nucleotide-encoding protein of bdellin-HM-2 contained 132 amino acids and was encoded by a 399 bp open reading frame (ORF). Sequence alignment showed that bdellin-HM-2 shared similarity with the"non-classical" Kazal-type serine protease inhibitors, but had no inhibitory effect on trypsin, elastase, chymotrypsin, kallikrein, factor XIIa (FXIIa), factor XIa (FXIa), factor Xa (FXa), thrombin, or plasmin. Bdellin-HM-2 showed anticoagulant effects by prolonging the activated partial thromboplastin time (aPTT), indicating a role in enabling H. manillensis to obtain a blood meal from its host. Our results suggest that bdellin-HM-2 may play a crucial role in blood-sucking in this leech species and may be a potential candidate for the development of clinical anti-thrombotic drugs.

Envenomation by viperid snakes may lead to severe bleeding, consumption coagulopathy, and thrombotic microangiopathy symptoms. The exact etiology or toxins responsible for thrombotic microangiopathy symptoms after snake envenomation remain obscure. Snake C-type lectin-like proteins (snaclecs) are one of the main non-enzymatic protein constituents in viper venoms, of which a majority are considered as modulators of thrombosis and hemostasis. In this study, we demonstrated that two snaclecs (mucetin and stejnulxin), isolated and identified from Protobothrops mucrosquamatus and Trimeresurus stejnegeri venoms, directly induced platelet degranulation and clot-retraction in vitro, and microvascular thrombosis has been confirmed in various organs in vivo. These snaclecs reduced cerebral blood flow and impaired motor balance and spatial memories in mice, which partially represent the thrombotic microangiopathy symptoms in some snakebite patients. The functional blocking of these snaclecs with antibodies alleviated the viper venom induced platelet activation and thrombotic microangiopathy-like symptoms. Understanding the pathophysiology of thrombotic microangiopathy associated with snake envenoming may lead to emerging therapeutic strategies.

From a survival perspective, it is hypothesized that leech saliva exhibits certain physiological effects to ensure fast blood-feeding, including analgesia, anesthesia, and anti-inflammation to stay undetected by the host and vasodilatation and anti-hemostasis to ensure a steady, rapid, and sustained blood flow to the feeding site. Many anti-hemostatic compounds have been identified in leech saliva, such as hirudin, calin, and bdellin A. However, no specific substance with direct vasodilatory and anti-inflammatory function has been reported from forest leech saliva. Herein, using activity-guided analysis, prostaglandin E1 (PGE1) was identified for the first time as an efficient molecular tool for forest leech blood sucking. The structure of PGE1 was analyzed by nuclear magnetic resonance spectroscopy and high-resolution electrospray ionization mass spectroscopy. PGE1 was found to be primarily distributed in the leech salivary gland (1228.36 ng/g body weight). We also analyzed how forest leech PGE1 affects platelet aggregation, skin vascular permeability, bleeding time, and pain. Results indicated that PGE1 efficiently inhibited platelet aggregation induced by adenosine diphosphate (ADP) (5 μM) with an IC₅₀of 21.81 ± 2.24 nM. At doses of 10, 100 nM, and 1 μM, PGE1 increased vascular permeability by 1.18, 5.8, and 9.2 times. It also prolonged bleeding time in a concentration-independent manner. In the formalin-induced mouse paw pain model, PGE1 suppressed acute pain. To the best of our knowledge, this is the first report on PGE1 in invertebrates. The functions of PGE1, such as vasodilation, platelet aggregation inhibition, anti-inflammation, and pain alleviation, may facilitate the ingestion of host blood by leeches.