Paweena Pradniwat

Snake venom thrombin-like enzymes

Abstract Thrombin-like enzymes (TLEs) are serine proteases, found mainly in pit viper venoms that induce coagulopathy. Hypofibrinogenemia is a usual result of fibrinogen degradation by TLEs. Viper-derived TLEs are promising as diagnostic or therapeutic agents and research tools to reveal the fibrinopeptide release mechanisms from fibrinogen. The TLEs have highly conserved structures, such as catalytic triads and six disulfide bridges, yet very diverse activities through accelerated evolution mechanisms. Glycosylation of the molecules may provide protein stability and contribute to some activities of the enzymes. In contrast to thrombin, which contains well-defined anion-binding exosites 1 and 2, the presence of substrate recognition sites on TLEs is unclear. Further protein structure-function analysis is still required.

The structure-function relationship of thrombin-like enzymes from the green pit viper (Trimeresurus albolabris).

Pit viper venoms can decrease fibrinogen levels in snakebite patients. Studies have shown that the hypofibrinogenemia is a consequence of snake venom thrombin-like enzymes (TLEs), the serine proteases that have the potential to be both diagnostic and therapeutic agents. Exosites of thrombin are the molecular regions that determine the substrate specificities, but its presence and significance in TLEs are unclear. Therefore, the putative exosites of recombinant TLEs derived from Green pit viper (Trimeresurus albolabris), GPV-TL1 and GPV-TL2, were mutated in a Pichia pastoris system. In a previous report, GPV-TL1 showed a strong fibrinogenolytic activity on the Aα and Bβ chains of fibrinogen, as well as a plasma clotting activity. Compared with GPV-TL1, the GPV-TL1m mutated in the putative exosite (TRN to RRR at residues 60-62) showed a weaker fibrinogenolytic activity with a similar clotting activity of 207.1 thrombin units/mg. GPV-TL2 contained two-residue differences from GPV-TL1 in the putative exosite (N73M and V74Y). GPV-TL2 selectively cleaved only the Aα chain of fibrinogen without detectable clotting activity. The mutated GPV-TL2 (GPV-TL2m) showed a weaker fibrinogenolytic activity compared with that of the wild type. These results support the important roles of the putative exosite in snake venom TLE activities. This information is helpful for future protein engineering.

Analysis of green pit viper (Trimeresurus alborabris) venom protein by LC/MS-MS.

Green pit viper venom has major effect on the hematological system having a thrombin‐like effect. Thus, this study is designed to analyze the composition of Trimeresurus albolabris venom by performing gel filtration and LC/MS‐MS. The purified protein was then digested by trypsin, and the tryptic fragments were analyzed by iontrap spectrophotometry. This study found four types of proteins, namely jerdonitin, stejaggregin‐A β chain‐1, stejnobin, and stejnihagin‐A, as the components of T. albolabris venom. All of these toxins played a greater or lesser role in clot formation or otherwise contributed to cross‐reactions in antivenom production.

Platelet Count and Platelet Parameters in Hemoglobin E Carriers

Hemoglobinopathies are an important inherited disorder with a high prevalence in Thailand. Of several hemoglobinopathies, hemoglobin E (Hb) disorder (beta 26, GAG-AAG, Glu-Lys) is the most common. Coagulation disorders in these patients have also been proposed. Even though thrombotic risks in the patients with hemoglobin disorders from standpoints of platelet dysfunction and coagulation factors are controversial, they are in favor of thrombosis due to thrombocytosis. A study was performed in 57 healthy subjects to evaluate platelet count and platelet parameters in hemoglobin E carriers compared to values in healthy subjects. Classified by standard hemoglobin electrophoresis, there were 46 healthy subjects and 11 hemoglobin E carriers. There are no significant differences in platelet count and platelet parameters between the two groups (p > 0.05). Although there are reports that indicate the change in the quality of platelet in hemoglobin E disorders, no quantitative disorder was detected. There was no trend toward increased platelet count in the HbE carriers.

Flavonoid Component Determination and Apoptotic Induction Evaluation of Houttuynia cordata Thunb Extract on Human Acute Leukemic Cells

Development of resistance to currently used drugs and side effects of several allopathic drugs have led to increased emphasis on plant materials uses as a source of medicines for a wide variety of human illnesses including leukemia. Houttuynia cordata Thunb (H.cordata), a Northern Thailand local plant and commonly known as Plucao, has various biological activities such as anti-inflammatory, anti-cancer and anti-leukemic activities. We aimed to determine active flavonoid components of H.cordata and to investigate the effect of H.cordata ethanolic extract on apoptotic induction on human acute lymphoblastic leukemia cells. In this study, we found that H.cordata ethanolic extract had total flavonoid of 231.21 ± 4.19 mg QE/g dried H.cordata. The extract analyzed by quantitative LC-MS consists of several flavonoid components including hyperin 6.35 ± 0.41, quercetin 0.34 ± 0.02, isoquercetin 1.10 ± 0.03, and rutin 0.88 ± 0.04 (%w/w). The cytotoxicity results showed dose dependent decrease in growth of Jurkat leukemic cells. Blebbing pattern of cell apoptosis was found in cells treated with H.cordata ethanolic extract for 24 and 48 hrs. Moreover, we found that the extract could substantially induce Jurkat cell death through apoptosis at both 12 and 24 hrs. In conclusion, these results indicated that H.cordata ethanolic extract which is composed of several flavonoids, possesses anti-leukemic activity through apoptotic induction in Jurkat leukemia cells.