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These results allow us to consider the use of LAAOs as anticancer agents, as tools in biochemical studies to investigate cellular processes, and to obtain a better understanding of the envenomation mechanism.

Aggretin, a potent platelet activator, was isolated from Calloselasma rhodostoma venom, and 30-amino acid N-terminal sequences of both subunits were determined. Aggretin belongs to the heterodimeric snake Ctype lectin family and is thought to activate platelets by binding to platelet glycoprotein ␣ 2 ␤ 1 . We now show that binding to glycoprotein (GP) Ib is also required. Aggretin-induced platelet activation was inhibited by a monoclonal antibody to GPIb as well as by antibodies to ␣ 2 ␤ 1 . Binding of both of these platelet receptors to aggretin was confirmed by affinity chromatography. No binding of other major platelet membrane glycoproteins, in particular GPVI, to aggretin was detected. Aggretin also activates platelets from Fc receptor ␥ chain (Fc␥)-deficient mice to a greater extent than those from normal control mice, showing that it does not use the GPVI/Fc␥ pathway. Platelets from Fc␥-deficient mice expressed fibrinogen receptors normally in response to collagen, although they did not aggregate, indicating that these platelets may partly compensate via other receptors including ␣ 2 ␤ 1 or GPIb for the lack of the Fc␥ pathway. Signaling by aggretin involves a dose-dependent lag phase followed by rapid tyrosine phosphorylation of a number of proteins. Among these are p72 SYK , p125 FAK , and PLC␥2, whereas, in comparison with collagen and convulxin, the Fc␥ subunit neither is phosphorylated nor coprecipitates with p72 SYK . This supports an independent, GPIb-and integrin-based pathway for activation of p72 SYK not involving the Fc␥ receptor.Platelet-collagen interactions are integral to primary hemostasis (1, 2). Resting platelets using several receptors adhering to subendothelium of damaged blood vessels are activated and spread to provide finally a new nonthrombogenic surface until vasculature regeneration occurs. Reversible binding between GPIb-V-IX 1 and von Willebrand factor, associated with collagen, is crucial to slow down the platelet (especially under high shear) so that it can bind more firmly via other receptors (3, 4). This mechanism strongly parallels that of the selectins in leukocyte adhesion (5). Another important receptor is the ␣ 2 ␤ 1 integrin, which is essential for anchoring the platelet to collagen in the subendothelium (6) and for linking to the platelet cytoskeleton to prevent the receptor being torn from the membrane by the forces that it has to withstand. Activation induces the release of storage granules and the expression of new receptors on the platelet surface (7) as well as changes in other receptors such as the fibrinogen receptor, ␣ IIb ␤ 3 , which is critical for spreading. Although GPIb-V-IX and ␣ 2 ␤ 1 also participate in signaling to the platelet interior (8, 9), recent studies, particularly in patients with platelet receptor deficiencies, have implicated GPVI/Fc␥ as a major collagen receptor for platelet activation (10 -12). Patients with platelets lacking any one of these receptors (GPIb-V-IX, ␣ 2 ␤ 1 , or GPVI/Fc␥) have increased bleeding times, and platelet adhesio...

The snake venom C-type lectin alboaggregin A (or 50-kd alboaggregin) from Trimeresurus albolabris was previously shown to be a platelet glycoprotein (GP) Ib agonist. However, investigations of the signal transduction induced in platelets showed patterns of tyrosine phosphorylation that were different from those of other GPIb agonists and suggested the presence of an additional receptor. In this study, the binding of biotinylated alboaggregin A to platelet lysates, as well as affinity chromatography evaluations of platelet lysates on an alboaggregin A-coated column, indicated that this other receptor is GPVI. Additional experiments with reagents that inhibit either GPIb or GPVI specifically supported this finding. These experiments also showed that both GPIb and GPVI have a role in the combined signaling and that the overall direction this takes can be influenced by inhibitors of one or the other receptor pathway.

Echicetin, a heterodimeric snake C-type lectin from Echis carinatus, is known to bind specifically to platelet glycoprotein (GP)Ib. We now show that, in addition, it agglutinates platelets in plasma and induces platelet signal transduction.

Snake venoms contain a wide range of components, many of which affect haemostasis by activation or inhibition of platelets or coagulation factors. They can be classified into groups based on structure and mode of action. One group is the snake C-type lectins, so called because of the typical folding which closely resembles that found in classical C-type lectins, such as selectins and mannose-binding proteins. Unlike the classic C-type lectins, those from snakes are generally heterodimeric with two subunits, α and β. Some are multimeric heterodimers. The subunits have homologous sequences and are generally linked by a disulphide bond as well as by swapping loops. One of the first C-type lectins with a defined function was echicetin which was demonstrated to bind to platelet GPIb and block several functions of this receptor. Since then, many proteins with similar structure have been reported to act on platelet receptors or coagulation factors and several have been crystallized. These proteins were thought to be specific for a single platelet receptor or coagulation factor, i.e. they had only one receptor per heterodimer. Recent studies show that most of these C-type lectins have binding sites for more than one ligand and have complex mechanisms of action.

Rationale: Decades of research have examined immune modulatory strategies to protect the heart after an acute myocardial infarction and prevent progression to heart failure, but have failed to translate to clinical benefit. Objective: To determine anti-inflammatory actions of apoA-I nanoparticles (n-apoA-I) that contribute to cardiac tissue recovery after myocardial infarction. Methods and Results: Using a preclinical mouse model of myocardial infarction, we demonstrate that a single intravenous bolus of n-apoA-I (CSL111, 80mg/kg) delivered immediately after reperfusion, reduced the systemic and cardiac inflammatory response. N-apoA-I treatment lowered the number of circulating leukocytes by 30{plus minus}7% and their recruitment into the ischemic heart by 25{plus minus}10% (all p<5.0E-2). This was associated with a reduction in plasma levels of the clinical biomarker of cardiac injury, cardiac troponin-I by 52{plus minus}17% (p=1.01E-2). N-apoA-I reduced the cardiac expression of chemokines that attract neutrophils and monocytes by 60-80%, and lowered surface expression of integrin CD11b on monocytes by 20{plus minus}5% (all p<5.0E-2). Fluorescently labeled n-apoA-I entered the infarct and peri-infarct regions and co-localized with cardiomyocytes undergoing apoptosis and with leukocytes. We further demonstrate that n-apoA-I binds to neutrophils and monocytes, with preferential binding to the pro-inflammatory monocyte subtype and partially via scavenger receptor BI (SR-BI). In patients with type 2 diabetes mellitus, we also observed that intravenous infusion of the same n-apoA-I (CSL111, 80mg/kg) similarly reduced the level of circulating leukocytes by 12{plus minus}5% (all p<5.0E-2). Conclusions: A single intravenous bolus of n-apoA-I delivered immediately post-myocardial infarction reduced the systemic and cardiac inflammatory response through direct actions on both the ischemic myocardium and leukocytes. These data highlight the anti-inflammatory effects of n-apoA-I and provide preclinical support for investigation of its use for management of acute coronary syndromes in the setting of primary percutaneous coronary interventions.

Aggretin is a C-type lectin purified from Calloselasma rhodostoma snake venom. It is a potent activator of platelets, resulting in a collagen-like response by binding and clustering platelet receptor CLEC-2. We present here the crystal structure of aggretin at 1.7 A which reveals a unique tetrameric quaternary structure. The two alphabeta heterodimers are arranged through 2-fold rotational symmetry, resulting in an antiparallel side-by-side arrangement. Aggretin thus presents two ligand binding sites on one surface and can therefore cluster ligands in a manner reminiscent of convulxin and flavocetin. To examine the molecular basis of the interaction with CLEC-2, we used a molecular modeling approach of docking the aggretin alphabeta structure with the CLEC-2 N-terminal domain (CLEC-2N). This model positions the CLEC-2N structure face down in the "saddle"-shaped binding site which lies between the aggretin alpha and beta lectin-like domains. A 2-fold rotation of this complex to generate the aggretin tetramer reveals dimer contacts for CLEC-2N which bring the N- and C-termini into the proximity of each other, and a series of contacts involving two interlocking beta-strands close to the N-terminus are described. A comparison with homologous lectin-like domains from the immunoreceptor family reveals a similar but not identical dimerization mode, suggesting this structure may represent the clustered form of CLEC-2 capable of signaling across the platelet membrane.