Aggretin, a Heterodimeric C-type Lectin from Calloselasma rhodostoma (Malayan Pit Viper), Stimulates Platelets by Binding to α2β1 Integrin and Glycoprotein Ib, Activating Syk and Phospholipase Cγ2, but Does Not Involve the Glycoprotein VI/Fc Receptor γ Chain Collagen Receptor

Abstract

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...

Ophioluxin, a Convulxin-like C-type Lectin from Ophiophagus hannah (King Cobra) Is a Powerful Platelet Activator via Glycoprotein VI

“…Many snake C-type lectins that activate platelets to various extents have been reported that either have different receptors or have different affinities for the same receptors, even though they share a high degree of sequence similarities. For example, bilinexin only agglutinates platelets using GPIb and ␣ 2 ␤ 1 as receptors (36), whereas these same glycoproteins are also employed by aggretin to stimulate full platelet activation (25). This may indicate that they have different binding sites for/on each receptor and/or that aggretin may bind to (an) additional receptor(s).…”

“…Echicetin, aggretin, rhodocetin, and convulxin were purified from Echis carinatus sochureki venom (Latoxan, Valence, France), Calloselasma rhodostoma venom (CV Herpafauna, Indonesia), and Crotalus durissus terrificus venom (Sigma), respectively, as described previously (10,25,28,29). Alboaggregin B and alboaggregin A were isolated from Trimeresurus albolabris venom from Huangshang, China, and from Latoxan (Valence, France), respectively (22,30).…”

“…Alboaggregin A was previously reported to target GPIb but more recently has been shown to act principally by GPVI (22)(23)(24). Aggretin has been shown to interact with both GPIb and the integrin ␣ 2 ␤ 1 (25,26). Obviously, these proteins have evolved in snakes as powerful agonists by interacting with several platelet receptors simultaneously.…”

Convulxin Binds to Native, Human Glycoprotein Ibα

“…A second C-type lectin with dual affinity for GPIb␣ and GPVI is alboluxin, identified in the venom of Trimeresurus albolabris (7). Another example of the promiscuity of C-type lectins might be the behavior of aggretin, derived from Calloselasma rhodostoma (18). Aggretin (rhodocytin) was initially reported to bind to the integrin collagen receptor ␣ 2␤1 (19) and later reported to also bind to GPIb (18).…”

“…Another example of the promiscuity of C-type lectins might be the behavior of aggretin, derived from Calloselasma rhodostoma (18). Aggretin (rhodocytin) was initially reported to bind to the integrin collagen receptor ␣ 2␤1 (19) and later reported to also bind to GPIb (18). However, more recent studies with genetically engineered murine platelets that lack ␣ 2␤1 and GPVI and were stripped of GPIb by proteolysis challenge these conclusions, showing that none of these receptors is required for platelet activation by rhodocytin (20).…”