Robyn Summerhayes

Early evolution of the venom system in lizards and snakes

Among extant reptiles only two lineages are known to have evolved venom delivery systems, the advanced snakes and helodermatid lizards (Gila Monster and Beaded Lizard). Evolution of the venom system is thought to underlie the impressive radiation of the advanced snakes (2,500 of 3,000 snake species). In contrast, the lizard venom system is thought to be restricted to just two species and to have evolved independently from the snake venom system. Here we report the presence of venom toxins in two additional lizard lineages (Monitor Lizards and Iguania) and show that all lineages possessing toxin-secreting oral glands form a clade, demonstrating a single early origin of the venom system in lizards and snakes. Construction of gland complementary-DNA libraries and phylogenetic analysis of transcripts revealed that nine toxin types are shared between lizards and snakes. Toxinological analyses of venom components from the Lace Monitor Varanus varius showed potent effects on blood pressure and clotting ability, bioactivities associated with a rapid loss of consciousness and extensive bleeding in prey. The iguanian lizard Pogona barbata retains characteristics of the ancestral venom system, namely serial, lobular non-compound venom-secreting glands on both the upper and lower jaws, whereas the advanced snakes and anguimorph lizards (including Monitor Lizards, Gila Monster and Beaded Lizard) have more derived venom systems characterized by the loss of the mandibular (lower) or maxillary (upper) glands. Demonstration that the snakes, iguanians and anguimorphs form a single clade provides overwhelming support for a single, early origin of the venom system in lizards and snakes. These results provide new insights into the evolution of the venom system in squamate reptiles and open new avenues for biomedical research and drug design using hitherto unexplored venom proteins.

Reference values for kaolin-activated thromboelastography in healthy children.

BACKGROUND:The hemostatic system of children changes with age and differs significantly from the hemostatic system of adults. Age-specific reference values are therefore required for most hemostatic variables. Thromboelastography (TEG®) is a point-of-care coagulation test that may provide superior evaluation and management of coagulopathies after cardiac surgery, when large-dose unfractionated heparin is administered for cardiopulmonary bypass. In this study, we established reference values for kaolin-activated TEG in healthy children, to facilitate accurate interpretation of pediatric TEG results. METHODS:Kaolin-activated TEG was performed on 100 healthy children undergoing elective day surgery and 25 healthy adult volunteers. The following TEG variables were recorded: reaction time, coagulation time, &agr; angle, maximum amplitude, percentage lysis 30 min after maximum amplitude was reached, and the coagulation index. Differences between age-groups were evaluated using analysis of variance. RESULTS:Age-specific reference values for kaolin-activated TEG in healthy children between 1 mo and 16 yr of age are presented. No significant differences between children and adults were observed. CONCLUSIONS:TEG results, from a particular clinical setting, must be compared to age-specific, as well as analyzer- and activator-specific, reference values to allow for correct interpretation of the results. Reference values provided here will be of use in acute clinical situations where a practical monitor of hemostasis is required.

Therapeutic range for unfractionated heparin therapy: age- related differences in response in children

1 Xiao T, Takagi J, Coller BS, Wang JH, Springer TA. Structural basisforallosteryinintegrinsandbindingtofibrinogen-mimetictherapeutics.Nature 2004; 432: 59–67.2 LuoBH,TakagiJ,SpringerTA.Lockingtheb3 integrin I-like domaininto high and low affinity conformations with disulfides. JBiolChem2004; 279: 10215–21.3 Yamanouchi J, Hato T, Tamura T, Fu jita S. Identification of criticalresidues for ligand binding in the integrin b3 I-domain by site-directedmutagenesis. Thromb Haemost 2002; 87: 756–62.4 Tozer EC, Liddington RC, Sutcliffe MJ, Smeeton AH, Loftus JC.Ligand binding to integrin a

In vivo age dependency of unfractionated heparin in infants and children

INTRODUCTION Unfractionated Heparin (UFH) is used widely in paediatrics. Paediatric specific recommendations for UFH therapy are few, with the majority of recommendations being extrapolated from adult practice. In vitro studies have shown that this practice may be suboptimal. This study aimed to improve the understanding of the impact of age upon UFH response in vivo. MATERIALS AND METHODS This prospective, observational study, conducted in the Paediatric Intensive Care Unit (PICU), included: patients 16 years or younger; treated with UFH of at least 10 U/Kg/hr. Laboratory analysis included: Antithrombin, APTT, Anti-Xa, Anti-IIa and thrombin generation expressed as the Endogenous Thrombin Potential. Results were grouped according to patient age (i.e. <1, 1-5, 6-10 and 11-16 years). RESULTS 85 patients received an equivalent mean UFH dose with a median duration of 3 days. Antithrombin levels were decreased compared to age-related norms in children up to 11 years of age. APTT results were comparable across the age-groups. The Anti-Xa results using two different assays showed a trend for lower values in younger children. All children less than one year old recorded Anti-Xa values outside the therapeutic range for heparin therapy, for both assays. There was a trend for decreased Anti-IIa activity in younger children. Endogenous Thrombin Potential showed a significant trend for increased inhibition in older children. In vitro Antithrombin supplementation did not change the Anti-Xa or thrombin generation. CONCLUSIONS This study confirms that, in vivo, for the same dose of UFH, the anti Xa and anti IIa effect, as well as the inhibition of endogenous thrombin potential is age dependent and that these differences are not purely AT dependent. The implication is that the anticoagulant and antithrombotic effect of a given dose of UFH differs with age. Clinical outcome studies to determine the optimal dosing for each age group are warranted.

Age-related differences in plasma proteins: how plasma proteins change from neonates to adults.

The incidence of major diseases such as cardiovascular disease, thrombosis and cancer increases with age and is the major cause of mortality world-wide, with neonates and children somehow protected from such diseases of ageing. We hypothesized that there are major developmental differences in plasma proteins and that these contribute to age-related changes in the incidence of major diseases. We evaluated the human plasma proteome in healthy neonates, children and adults using the 2D-DIGE approach. We demonstrate significant changes in number and abundance of up to 100 protein spots that have marked differences in during the transition of the plasma proteome from neonate and child through to adult. These proteins are known to be involved in numerous physiological processes such as iron transport and homeostasis, immune response, haemostasis and apoptosis, amongst others. Importantly, we determined that the proteins that are differentially expressed with age are not the same proteins that are differentially expressed with gender and that the degree of phosphorylation of plasma proteins also changes with age. Given the multi-functionality of these proteins in human physiology, understanding the differences in the plasma proteome in neonates and children compared to adults will make a major contribution to our understanding of developmental biology in humans.

Dosing and monitoring of enoxaparin (Low molecular weight heparin) therapy in children.

Low molecular weight heparins (LMWHs) are commonly used in paediatric tertiary institutions for primary prophylaxis and treatment of thromboembolic events. Despite this widespread use, the therapeutic and prophylactic guidelines for LMWH therapy in children are extrapolated from adult guidelines. In fact, there is very little information regarding the pharmacokinetics, clinical effectiveness, adverse event profile and optimal dose schedule for LMWH therapy in children. This study was designed to determine whether paediatric‐specific dosage requirements for LMWH are justified, by investigating the doses required to achieve target therapeutic ranges. Patients who were treated with enoxaparin between October 2003 and July 2007 were identified for inclusion in this study. One hundred forty patients had an anti‐activated factor X assay result with a total of 55 (39%) patients achieving therapeutic levels 4–6 h post dose. Children younger than 1 year required the highest dose of enoxaparin/kg and highest number of dose changes to achieve the target therapeutic range. Major bleeding occurred in one patient, equating to 0·7%, with complete clot resolution recorded in 16 (11%) patients. This study demonstrated a 2–3‐fold variation in individual dose requirements for LMWH in children ≤5 years of age, and further mandates the need for age‐specific dosage requirements in children receiving enoxaparin.

Age is a determinant factor for measures of concentration and effect in children requiring unfractionated heparin

Previous studies investigating continuous unfractionated heparin (UFH) therapy report age-related differences in UFH response in children, as measured by APTT and anti-Xa assay. This study determined the age-related response following administration of a single UFH bolus of 75-100 IU/kg in children. Venous blood samples were collected from children (n=56) at 15, 30, 45 and 120 minutes post-UFH. Anti-Xa, anti-IIa, APTT, TCT and protamine titration were performed on all samples. Age-dependent differences in the effect and concentration of UFH were identified for the anti-Xa, anti-IIa and protamine titration assays, respectively. In addition, a trend suggesting a proportional increase in anti-Xa and anti-IIa-mediated UFH effect with age was evident. Logistic regression demonstrated an increase in protamine titration of 0.6 IU/ml for every year of age in samples collected 15 minutes post-UFH. UFH-mediated anti-IIa activity was reduced compared to anti-Xa activity across childhood, with a two-fold increase in anti-Xa to anti-IIa ratio in infants less than one year of age compared to teenagers in the setting of high UFH concentrations. This study demonstrates that the previously reported age-dependent response to UFH occurs in the context of an age-dependent serum concentration of UFH. The trend toward increased UFH serum concentration and anticoagulant activity with age may be related to short-term differences in UFH binding to coagulant and competitive plasma proteins in vivo.

Point-of-care monitoring of oral anticoagulation therapy in children. Comparison of the CoaguChek XS system with venous INR and venous INR using an International Reference Thromboplastin preparation (rTF/95).

Point-of-care (POC) monitoring of oral anticoagulation has been widely adopted in both paediatric and adult patients. A new POC system, the CoaguChek XS has recently been developed to measure the international normalised ratio (INR) and may offer significant advantages. The CoaguChek XS utilises a new method of electrochemical clot detection based on thrombin generation. This system has not been previously evaluated in children with reference to the laboratory gold standard, the prothrombin time using reference thromboplastin. It was the objective to compare values obtained by the CoaguChek XS system with both the venous INR and the gold standard for anticoagulant monitoring, prothrombin time with reference thromboplastin (rTF/95). To evaluate the impact of testing using the CoaguChek XS on clinical anticoagulant dosing decisions. Fifty paired venous INR and capillary CoaguChek XS results were obtained from 31 children (aged up to 16 years). The laboratory gold standard, a manual prothrombin time with reference thromboplastin (rTF/95) was additionally performed on 26 samples. Correlation between the CoaguChek XS result and the venous INR was r = 0.810. Agreement between the CoaguChek XS result and the reference INR was shown to be higher (r=0.95), in the subset analysed by this method. Correlation between the venous INR and reference INR was r=0.90. Despite changes to the methodology of testing with the CoaguChek XS POC monitoring system, the accuracy of this method when compared with both the venous INR and gold standard reference INR was satisfactory. This resulted in infrequent changes to clinical decision making regarding anticoagulation.

Age-specific differences in binding of heparin to plasma proteins

Summary.  Background: Clinically significant age‐related differences in the anticoagulation effect of heparin have previously been established in vitro as well as in different clinical settings in vivo. These differences were hypothesized to be due to the age‐specific differences in binding of heparin to plasma proteins. Objectives: The aim of this project was to investigate global age‐related differences in heparin binding to plasma proteins. Patients/Methods: Heparin‐binding proteins were identified by incubating heparin‐coated magnetic beads with plasma samples from neonates, children and adults, and purifying the proteins that were bound to the beads in this reaction system. Results: These results provide the first preliminary evidence of age‐related differences in the total number and concentration of proteins bound to heparin. The results also suggest, for the first time, that there are age‐related differences of heparin binding to antithrombin and thrombin. Conclusions: The results of this study, although preliminary, support and contribute to the explanation of the mechanism of age‐related differences in the effect of heparin observed previously in vitro and in vivo.

Thrombin generation: the functional role of alpha‐2‐macroglobulin and influence of developmental haemostasis

Currently available chromogenic and fluorogenic substrates for endogenous thrombin potential (ETP) measurement are cleaved by both free (active) and alpha‐2‐macroglobulin‐bound (inactive) thrombin, leading to an overestimation of ETP. Commercial methods for ETP measurement determine this using a mathematical algorithm, which assumes the contribution of alpha‐2‐macroglobulin to the ETP. This limits application of such methods to populations where variation in alpha‐2‐macroglobulin concentrations is observed, primarily children. This study examined the contribution of alpha‐2‐macroglobulin‐bound thrombin to the ETP measurement in neonates, children and adults, to determine whether automated methods are appropriate for use in neonates and children.