Echicetin, a GPIb-binding snake C-type lectin from Echis carinatus, also contains a binding site for IgMκ responsible for platelet agglutination in plasma and inducing signal transduction

Section: Methodsmentioning

confidence: 99%

The Collagen-binding Integrin α2β1 Is a Novel Interaction Partner of the Trimeresurus flavoviridis Venom Protein Flavocetin-A

Arlinghaus

Eble

2013

“…PolyScreen PVDF membranes were from PerkinElmer Life Sciences (Zaventem, Belgium). Echicetinspecific IgM was prepared as previously described (15). Polyvinyl alcohol (30 -70 kDa, Sigma)-based microscope slide mounting medium was prepared by adding 2.4 g to 6 ml of water and stirring for a few hours.…”

Section: Methodsmentioning

confidence: 99%

“…We recently showed that cross-linking GPIb by the snake C-type lectin echicetin using IgM is a practical approach to the specific activation of platelets via GPIb (15). The use of echicetin as ligand avoids two complications of VWF, the requirement for shear to induce (VWF)/GPIb binding as well as VWF/␣ IIb ␤ 3 interactions following platelet activation.…”

mentioning

confidence: 99%

Glycoprotein Ib Cross-linking/Ligation on Echicetin-coated Surfaces or Echicetin-IgMκ in Stirred Suspension Activates Platelets by Cytoskeleton Modulated Calcium Release

Navdaev

Clemetson

2002

Self Cite

Cross-linking platelet GPIb with the snake C-type lectin echicetin provides a specific technique for activation via this receptor. This allows GPIb-dependent mechanisms to be studied without the necessity for shear stress-induced binding of von Willebrand factor or primary ␣ IIb ␤ 3 involvement. We already showed that platelets are activated, including tyrosine phosphorylation, by echicetin-IgM -induced GPIb cross-linking. We now investigate the mechanism further and demonstrate that platelets, without modulator reagents, spread directly on an echicetin-coated surface, by a GPIb-specific mechanism, causing exocytosis of ␣-granule markers (Pselectin) and activation of ␣ IIb ␤ 3 . This spreading requires actin polymerization and release of internal calcium stores but is not dependent on external calcium nor on src family tyrosine kinases. Cross-linking of GPIb complex molecules on platelets, either in suspension or via specific surface attachment, is sufficient to induce platelet activation.Binding of platelets to damaged vessel wall is the first step in sealing a wound to prevent bleeding. Under the high shear conditions encountered in smaller vessels, this involves glycoprotein (GP) 1 Ib on platelets binding to von Willebrand factor (VWF) on the subendothelium, to slow the platelets down via a rolling interaction, so that other platelet receptors can be activated and engaged to give stable adhesion. This is a complex process and many aspects are still not understood. In particular, the mechanism regulating the shear-dependent binding between GPIb and VWF is still unknown, as well as the way in which this binding activates platelets. Several recent studies showed that the cytoskeleton has a critical role in this process. Although about 75% of GPIb is bound to membrane cytoskeleton through filamin, another 25% appears to be free (1). Increasing the amount of free GPIb by preventing actin polymerization with cytochalasin D also enhances platelet aggregation responses to VWF. Similar results were also obtained in model systems where GPIb/IX was transfected into CHO cells (2, 3). CHO cells expressing a truncated form of GPIb, where the filamin binding domain was deleted, also show a higher agglutination response to VWF than those expressing wild-type GPIb/IX. However, Dong et al. (4) found a decrease in ristocetin-induced VWF binding to CHO cells expressing a filamin binding domain deleted form of GPIb, compared with cells expressing the wild type.

“…It was already known that Dami cells express amounts of GPVI adequate to respond to convulxin (19). Ophioluxin gave a strong calcium signal compared with convulxin when the same amounts were used.…”

Section: Fig 3 Ophioluxin Binds Mainly To Gpvimentioning

confidence: 90%

“…Some inhibit the function of platelets by preventing ligand binding, others activate platelets either directly via receptors or indirectly using plasma proteins. Examples of these are echicetin, which uses IgM (19), or botrocetin and bitiscetin that bind von Willebrand factor (20,21). Earlier, these proteins were thought to activate or inhibit platelets via only one receptor.…”

mentioning

confidence: 99%

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

Clemetson

Navdaev

et al. 2002

Self Cite

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