Textilinin-1, an alternative anti-bleeding agent to aprotinin: Importance of plasmin inhibition in controlling blood loss.

Aprotinin has been used widely in surgery as an anti-bleeding agent but is associated with a number of side effects. We report that textilinin-1, a serine protease inhibitor from Pseudonaja textilis venom with sequence relatedness to aprotinin, is a potent but reversible plasmin inhibitor and has a narrower range of protease inhibition compared to aprotinin. Like aprotinin, textilinin-1 at 5 micromol/l gave almost complete inhibition of tissue plasminogen activator-induced fibrinolysis of whole blood clots. The activated partial thromboplastin time for plasma was markedly increased by aprotinin but unaffected by textilinin-1. In a mouse tail-vein bleeding model, intravenous textilinin-1 and aprotinin caused similar decreases in blood loss but time to haemostasis in the textilinin-treated animals was significantly shorter than in aprotinin-treated mice. Based on these data, textilinin-1 merits further investigation as a therapeutic alternative to aprotinin.

Altered clot kinetics in patients with non-alcoholic fatty liver disease.

BACKGROUND: Emerging evidence has linked the presence of non-alcoholic fatty liver disease (NAFLD) with an increased risk for cardiovascular events. We hypothesised that altered clot kinetics and platelet function may contribute to this increased risk. This study compared whole blood clotting kinetics in patients with 1) non-cirrhotic NAFLD (n = 28) and 2) healthy control subjects (n = 22). METHODS: Clotting kinetics were assessed in whole blood using thromboelastography (TEG) and assessed for correlations with cardiovascular risk factors. RESULTS: Clot kinetics in patients with NAFLD showed significantly stronger clot development (maximum amplitude (MA); 58.3 +/- 6.3 mm vs. 52.0 +/- 10.1 mm, p = 0.01) and reduced clot lysis in the presence of thrombin (35 +/- 30% vs. 51 +/- 26% clot lysis 30 minutes after MA, p = 0.03) compared to control subjects. Clot strength was independently positively associated with body mass index in NAFLD, but not in control subjects. There was a greater platelet contribution to clot strength in patients with NAFLD compared to controls despite similar platelet counts. There was no association between clot kinetics and features of the metabolic syndrome or presence of type 2 diabetes. CONCLUSION: Patients with NAFLD have disturbances in ex-vivo clot kinetics including increased clot strength and clots that are more resistant to thrombin-stimulated lysis.

Cloning and characterisation of natriuretic peptides from the venom glands of Australian elapids.

The venom from Australian elapid snakes contains a complex mixture of polypeptide toxins that adversely affect multiple homeostatic systems within their prey in a highly specific and targeted manner. Included in these toxin families are the recently described venom natriuretic peptides, which display similar structure and vasoactive functions to mammalian natriuretic peptides. This paper describes the identification and detailed comparative analysis of the cDNA transcripts coding for the mature natriuretic peptide from a total of nine Australian elapid snake species. Multiple isoforms were identified in a number of species and represent the first description of a natriuretic peptide from the venom gland for most of these snakes. Two distinct natriuretic peptide isoforms were selected from the common brown snake (Pseudonaja textilis), PtNP-a, and the mulga (Pseudechis australis), PaNP-c, for recombinant protein expression and functional analysis. Only one of these peptides, PtNP-a, displayed cGMP stimulation indicative of normal natriuretic peptide activity. Interestingly, both recombinant peptides demonstrated a dose-dependent inhibition of angiotensin converting enzyme (ACE) activity, which is predictive of the vasoactive effects of the toxin. The natriuretic peptides, however, did not possess any coagulopathic activity, nor did they inhibit or potentiate thrombin, adenosine diphosphate or arachidonic acid induced platelet aggregation. The data presented in this study represent a significant resource for understanding the role of various natriuretic peptides isoforms during the envenomation process by Australian elapid snakes.

Resistance of porcine blood clots to lysis relates to poor activation of porcine plasminogen by tissue plasminogen activator.

In-vitro experimentation was performed on porcine and human blood to determine their comparative responsiveness to a novel fibrinolytic inhibitor and thereby assess whether the pig is a suitable animal model for subsequent in-vivo testing of this inhibitor. Thromboelastography showed the clots formed from porcine whole blood to be highly resistant to tissue plasminogen activator (t-PA)-catalyzed lysis, and this communication offers the resistance of porcine plasminogen to activation by t-PA as an explanation. Porcine blood containing 100 and 1500 IU/ml added t-PA lysed very slowly, having LY30 values of 1.9 +/- 1.4 and 2.9 +/- 1.9%, respectively. In contrast, the LY30 values for the human clots containing 100 and 1500 IU/ml t-PA were 77.1 +/- 6.3 and 93.3 +/- 1.3%, respectively. Moreover, purified porcine plasminogen was activated very slowly by added t-PA in the presence of both human and porcine fibrin. Activation of plasminogen by the endogenous activators, as measured by the euglobulin clot lysis time, was greatly prolonged for the pig (22 +/- 3 h) compared with the human (3.5 +/- 1.5 h). These results suggest caution in using the pig as an experimental model when studying the effects of various agents on fibrinolysis.

Identification and characterisation of Kunitz-type plasma kallikrein inhibitors unique to Oxyuranus sp. snake venoms.

As part of a wider study on Australian snake venom components, we have identified and characterised Kunitz-type protease inhibitors from the venoms of Oxyuranus scutellatus and Oxyuranus microlepidotus (Australian taipans) with plasma kallikrein inhibitory activity. Each inhibitor had a mass of 7 kDa and was purified from the venom as part of a protein complex. Mass spectrometry and N-terminal sequencing was employed to obtain amino acid sequence information for each inhibitor and a recombinant form of the O. scutellatus inhibitor, termed TSPI, was subsequently expressed and purified. TSPI was investigated for inhibition against a panel of 12 enzymes involved in haemostasis and estimates of the K(i) value determined for each enzyme. TSPI was found to be a broad spectrum inhibitor with most potent inhibitory activity observed against plasma kallikrein that corresponded to a K(i) of 0.057 ± 0.019 nM. TSPI also inhibited fibrinolysis in whole blood and prolonged the intrinsic clotting time. These inhibitors are also unique in that they appear to be found only in Oxyuranus sp. venoms.

Comparison of active venom components between Eastern brown snakes collected from South Australia and Queensland.

The abundance and activity of the prothrombin activator (pseutarin C) within the venom of the Eastern brown snake (Pseudonaja textilis textilis) is the primary determinant of its coagulation potency. Textilinin-1, also in this venom, is a plasmin inhibitor which is thought to exert its toxic effects through the slowing of fibrinolysis. The aim of this report is to determine if there are differences in the potency of the venom from Eastern brown snakes collected from South Australia (SA) compared to those from Queensland (QLD). A concentration of 0.4 microg/ml venom protein from six QLD specimens clotted citrated plasma in an average time of 21.4+/-3.3 s compared to 68.7+/-2.4 s for the same amount of SA venom (averaged for six individuals). The more potent procoagulant activity of the QLD venom was measured between 0.4 and 94 microg/ml venom protein in plasma. The anti-plasmin activity of textilinin was also greater in the venom of the snakes collected from QLD, causing full inhibition of plasmin at approximately 1.88 microg/ml of venom protein compared to approximately 7.5 microg/ml for the SA venoms. It is concluded that geographic differentiation of the Eastern brown snakes results in significant differences venom potency.

Comparison of textilinin-1 with aprotinin as serine protease inhibitors and as antifibrinolytic agents.

Textilinin-1 (Q8008) was isolated from the venom of the Pseudonaja textilis and has a 47% sequence identity to the antihaemorrhagic therapeutic agent aprotinin. When equimolar concentrations of enzyme and aprotinin were pre-incubated, plasmin was inhibited 100%, plasma kallikrein 58%, and tissue kallikrein 99%. Under the same conditions, textilinin-1 inhibited plasmin 98%, plasma kallikrein 16% and tissue kallikrein 17%. Whole blood clot lysis was inhibited strongly by both aprotinin and textilinin-1, as shown by thrombelastography. At 2 microM inhibitor lysis initiated by t-PA was greater than 99% inhibited by aprotinin (LY60 = 0.4 +/- 0.1) whereas textilinin-1, inhibited lysis by 91% (LY60 = 8.9 +/- 0.7). The same trend was found with the lysis of euglobulin fractions. From these data textilinin-1 appears to be a more specific plasmin inhibitor than aprotinin but aprotinin inhibits clot lysis to a greater extent.

Cloning and functional expression of venom prothrombin activator protease from Pseudonaja textilis with whole blood procoagulant activity.

The snake venom group C prothrombin activators contain a number of components that enhance the rate of prothrombin activation. The cloning and expression of full-length cDNA for one of these components, an activated factor X (factor Xa)-like protease from Pseudonaja textilis as well as the generation of functional chimeric constructs with procoagulant activity were described. The complete cDNA codes for a propeptide, light chain, activation peptide (AP) and heavy chain related in sequence to mammalian factor X. Efficient expression of the protease was achieved with constructs where the AP was deleted and the cleavage sites between the heavy and light chains modified, or where the AP was replaced with a peptide involved in insulin receptor processing. In human kidney cells (H293F) transfected with these constructs, up to 80% of the pro-form was processed to heavy and light chains. Binding of the protease to barium citrate and use of specific antibodies demonstrated that gamma-carboxylation of glutamic acid residues had occurred on the light chain in both cases, as observed in human factor Xa and the native P. textilis protease. The recombinant protease caused efficient coagulation of whole citrated blood and citrated plasma that was enhanced by the presence of Ca2+. This study identified the complete cDNA sequence of a factor Xa-like protease from P. textilis and demonstrated for the first time the expression of a recombinant form of P. textilis protease capable of blood coagulation.

Use of defined estrone glucuronide-hen egg white lysozyme conjugates as signal generators in homogeneous enzyme immunoassays for urinary estrone glucuronide.

Three structurally characterized estrone glucuronide-lysozyme conjugates, E1 (a 60:40 mixture acylated at K3 and K97), E3 (acylated at K33), and E5 (acylated at both K33 and K97) were isolated and purified using a combination of cation-exchange chromatography on S-sepaharose in 7M urea and hydrophobic interaction chromatography on butyl sepharose. Urea was essential to separate the conjugates into six chromatographically homogeneous fractions. In the absence of urea, complex mixtures of lysozyme and the six conjugate fractions were always encountered. The E1, E3, and E5 conjugates were highly inhibited by a sheep polyclonal anti-estrone glucuronide antibody only after the hydrophobic interaction chromatography step. The high level of inhibition enabled all three conjugates to be utilized as signal generators in homogenous enzyme immunoassays for urinary estrone glucuronide. Despite the apparently higher affinity of E3 for the antibody, both E1 and E3 gave standard curves that were indistinguishable provided that 1.7-fold more antiserum was used for E1. Both E1 and E3 yielded menstrual cycle urinary data that agreed with that provided by the Ovarian Monitor pre-coated assay tubes. Although, the menstrual cycle pattern was similar for the three signal generators, the E1G excretion rates yielded by E5 as the signal generator were only 60% of the reference values. Despite structural differences, there was no advantage gained in separating E1 and E3, but higher substituted conjugates such as E5 need removal for best assay performance.