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

Abstract

Ophioluxin, a potent platelet agonist, was purified from the venom of Ophiophagus hannah (King cobra). Under nonreducing conditions it has a mass of 85 kDa, similar to convulxin, and on reduction gives two subunits with masses of 16 and 17 kDa, slightly larger than those of convulxin. The N-terminal sequences of both subunits are very similar to those of convulxin and other C-type lectins. Ophioluxin induces a pattern of tyrosine-phosphorylated proteins in platelets like that caused by convulxin, when using appropriate concentrations based on aggregation response, because it is about 2-4 times more powerful as agonist than the latter. Ophioluxin and convulxin induce [Ca 2؉ ] i elevation both in platelets and in Dami megakaryocytic cells, and each of these C-type lectins desensitizes responses to the other. Convulxin agglutinates fixed platelets at 2 g/ml, whereas ophioluxin does not, even at 80 g/ml. Ophioluxin resembles convulxin more than echicetin or alboaggregin B because polyclonal anti-ophioluxin antibodies recognize both ophioluxin and convulxin, but not echicetin, and platelets adhere to and spread on ophioluxin-or convulxin-precoated surfaces in the same way that is clearly different from their behavior on an alboaggregin B surface. Immobilized ophioluxin was used to isolate the glycoprotein VI-Fc␥ complex from resting platelets, which also contained Fyn, Lyn, Syk, LAT, and SLP76. Ophioluxin is the first multiheterodimeric, convulxin-like snake C-type lectin, as well as the first platelet agonist, to be described from the Elapidae snake family.Collagens in the vascular subendothelium and vessel wall are important platelet activators that have a critical role in hemostasis. A large number of different receptors have been proposed for collagen on platelets, including CD36 (glycoprotein (GP) 1 IV, GPIIIb) (1), GPIb-V-IX complex as an indirect collagen receptor acting via von Willebrand factor (2), a 65-kDa collagen type I specific receptor (3), and a 68 -72-kDa doublet collagen type III-specific receptor (4), but the major ones have been identified as the integrin ␣ 2 ␤ 1 and Ig superfamily member GPVI. Integrin ␣ 2 ␤ 1 belongs to the class containing an I-domain, which has been well characterized and is considered to be responsible for platelet adhesion to collagen rather than platelet activation (5, 6) for which GPVI is largely responsible. The evidence that GPVI is the other major, established receptor essential for signaling and platelet activation was based on studies on patients with mild bleeding problems, whose platelets had an impaired response to collagen but not to other agonists and were 8). This was substantiated by studies with GPVI-specific agonists including collagen-related peptide (9

Regulation of Chemerin Bioactivity by Plasma Carboxypeptidase N, Carboxypeptidase B (Activated Thrombin-activable Fibrinolysis Inhibitor), and Platelets

“…Platelet-poor plasma was prepared by spinning the PRP at 1200 ϫ g for 10 min at room temperature. The platelets were washed with PIPES buffer (25 mM PIPES, 137 mM NaCl, 4 mM KCl, and 0.1% glucose) at pH 6.4 as previously described (25). Platelet lysates were obtained by lysing washed platelets with radioimmune precipitation assay lysis buffer (Upstate, NY) with protease inhibitors.…”

“…Preparation of Platelet-rich Plasma, Platelet-poor Plasma, Washed Platelets, and Platelet Lysates-Blood was drawn into tubes containing 3.8% sodium citrate (9:1 v/v) and platelet-rich plasma (PRP) prepared following standard procedure (25). Platelet-poor plasma was prepared by spinning the PRP at 1200 ϫ g for 10 min at room temperature.…”

Snake C-Type Lectin-Like Proteins and Platelet Receptors

“…[10] . Convulxin, stejnulxin and ophioluxin activate platelets via GPVI [11][12][13] . They are multimeric proteins composed of heterodimers.…”

“…Convulxin was the fi rst identifi ed member of this group and was instrumental in the cloning of GPVI [11,19] . Additional C-type lectin-like proteins from snake venoms shown to activate GPVI include alboaggregin A [17] , stejnulxin [12] , ophioluxin [13] and alboluxin. All are multimeric and induce platelet activation by clustering the receptor.…”

Análise transcriptômica das glândulas de veneno de Micrurus corallinus (cobra-coral) e identificação de candidatos antigênicos para um anti-soro alternativo

“…As lectinas tipo C têm sido bastante utilizadas como ferramenta para investigar e estudar receptores plaquetários (DU et al, 2002). Alguns inibem a função plaquetária e previnem a interação com o ligante, outros ativam as plaquetas também diretamente via receptores ou indiretamente por meio de proteínas plasmáticas.…”