Identification and characterization of a collagen‐induced platelet aggregation inhibitor, triplatin, from salivary glands of the assassin bug, Triatoma infestans

Abstract

Injury to the blood vessel wall exposes the subendothelial extracellular matrix, which is rich in collagens, providing a substrate for platelet adhesion and aggregation. This is the first step of hemostasis ending in formation of the thrombus. Several proteins on the platelet membrane participate in collagen-platelet interactions in a direct or indirect manner [1]. Glycoprotein (GP)VI [2-5] and a 2 b 1 integrin [6] bind to collagen directly, and GPIb-V-IX [7] and a IIb b 3 integrin [6,8] bind via von Willebrand factor (vWF). In the current 'two-site, two-step' model of platelet-collagen interaction [9][10][11][12], platelet aggregation proceeds as follows: first, GPIb-IX-V rapidly binds to vWF immobilized on collagen so that passing platelets are tethered to the latter. Following this event, GPVI, a surface signaling receptor, binds to collagen with low affinity, which triggers the signaling cascade for platelet activation. This leads to 'inside-out' activation of a 2 b 1 and a IIb b 3 integrins and secretion of platelet agonists such as ADP and thromboxane A 2 , accelerating platelet aggregation and thrombus formation. Therefore, GPVI has a central role in the initial phase of thrombus formation as the major signaling receptor for collagen. GPVI is composed of two extracellular immunoglobulin domains, a mucin-rich stalk, a single transmembrane domain, and a short cytoplasmic tail [2][3][4]. GPVI is coupled to the Fc receptor (FcR) c-chain homodimer in the transmembrane domain via a salt bridge [5,9,10,13,14]. The binding of collagen to GPVI leads to cross-linking of GPVI molecules [15], inducing tyrosine phosphorylation of the cytoplasmic tail of FcR c-chains. This phosphorylation leads to binding To facilitate feeding, certain hematophagous invertebrates possess inhibitors of collagen-induced platelet aggregation in their saliva. However, their mechanisms of action have not been fully elucidated. Here, we describe two major salivary proteins, triplatin-1 and -2, from the assassin bug, Triatoma infestans, which inhibited platelet aggregation induced by collagen but not by other agents including ADP, arachidonic acid, U46619 and thrombin. Furthermore, these triplatins also inhibited platelet aggregation induced by collagen-related peptide, a specific agonist of the major collagen-signaling receptor glycoprotein (GP)VI. Moreover, triplatin-1 inhibited Fc receptor c-chain phosphorylation induced by collagen, which is the first step of GPVI-mediated signaling. These results strongly suggest that triplatins target GPVI and inhibit signal transduction necessary for platelet activation by collagen. This is the first report on the mechanism of action of collagen-induced platelet aggregation inhibitors from hematophagus invertebrates.

Platelet aggregation inhibitors from hematophagous animals

“…Triplatin also inhibits platelet aggregation induced by collagen-related peptide (CRP), a GPVI agonist. While it has been suggested that triplatin targets GPVI, a direct demonstration has not been provided (Morita, et al, 2006). In fact, CRP-induced platelet aggregation is inhibitable by ADP receptor antagonists and cycloxygenase blockers (Watson, et al, 2005).…”

“…It is thus unclear whether the results observed were due to another activity such as ADP- or TXA2-binding properties. Additionally, triplatin displays high sequence similarity to pallidipin-1, works only at high concentrations, and at > 1 μM does not eliminate completely CRP- or collagen-induced shape change (Morita, et al, 2006). It is prudent to say that triplatin has not been confirmed as a GPVI antagonist.…”

A repertoire of the dominant transcripts from the salivary glands of the blood-sucking bug, Triatoma dimidiata, a vector of Chagas disease

“…Pallidipin 2 from T. pallidipennis and triplatin-1 and -2 from T. infestans saliva were characterized as inhibitors of collagen-induced platelet aggregation (Noeske-Jungblut et al, 1994; Haendler et al, 1995; Morita et al, 2006). Ten contigs from the T. dimidiata cDNA library (Td17, Td30, Td31, Td33, Td34, Td38, Td41, Td42, Td59 and Td153) had homology with pallidipin 2 and triplatin-1 and -2.…”

Comparison of fibrinolytic activity from Korean indigenous insects

“…2002a), 무당벌레의 항산화·염증효과 , Park et al 2005) 등이 보고되었고, 항혈 전 효과로는 선태(흑매미 우화허물), 자충(바퀴벌레), 상표 초(사마귀알집), 홍랑자(매미) 등의 중국 시판 곤충에서 활 성을 나타내었다 (Hahn et al 1999). 또한, 중국시판 곤충 을 포함하는 곤충시료 추출물 중 동충하초, 벼메뚜기, 전 갈의 물 추출물에서 항트롬빈 활성이 보고되었고 (Ahn et al 2002b), 침노린재과의 흡혈곤충들에서 혈전생성 억제 활성 (Morota et al 2006, Noeske-Junqblut et al 1994 Plasmin was used as a positive control. Fig.…”