The leech product saratin is a potent inhibitor of platelet integrin α₂β₁ and von Willebrand factor binding to collagen

Abstract

Subendothelial collagen plays an important role, via both direct and indirect mechanisms, in the initiation of thrombus formation at sites of vascular injury. Collagen binds plasma von Willebrand factor, which mediates platelet recruitment to collagen under high shear. Subsequently, the direct binding of the platelet receptors glycoprotein VI and α2β1 to collagen is critical for platelet activation and stable adhesion. Leeches, have evolved a number of inhibitors directed towards platelet–collagen interactions so as to prevent hemostasis in the host during hematophagy. In this article, we describe the molecular mechanisms underlying the ability of the leech product saratin to inhibit platelet binding to collagen. In the presence of inhibitors of ADP and thromboxane A2, both saratin and 6F1, a blocking α2β1 mAb, abrogated platelet adhesion to fibrillar and soluble collagen. Additionally, saratin eliminated α2β1‐dependent platelet adhesion to soluble collagen in the presence of an Src kinase inhibitor. Moreover, saratin prevented platelet‐rich plasma adhesion to fibrillar collagen, a process dependent upon both α2β1 and von Willebrand factor binding to collagen. Furthermore, saratin specifically inhibited the binding of the α2 integrin subunit I domain to collagen, and prevented platelet adhesion to collagen under flow to the same extent as observed in the presence of a combination of mAbs to glycoprotein Ib and α2β1. These results demonstrate that saratin interferes with integrin α2β1 binding to collagen in addition to inhibiting von Willebrand factor–collagen binding, presumably by binding to an overlapping epitope on collagen. This has significant implications for the use of saratin as a tool to inhibit platelet–collagen interactions.

Structure of the Leech Protein Saratin and Characterization of Its Binding to Collagen

“…A recent study shows that Saratin interferes with the collagen interaction of the A3 domain of the vWF and of the I domain of integrin α2β1, indicating that overlapping binding epitopes on collagen may exist. 12 Structural information on the Saratin-collagen interaction is not available yet. As a prerequisite for the solution of these questions, the three-dimensional (3D) solution structure of Saratin has to be determined, a result we will present in this article.…”

“…It inhibits the first step of thrombocyte binding, that is, the interaction with the vWF by binding to collagen, presumably by binding to an overlapping epitope. 12 Therefore, Saratin affects the initial step of the blood coagulation, whereas the substances mentioned above affect coagulation later in the coagulation cascade. As a consequence, thrombocyte aggregation to vessel walls and thrombus formation is reduced.…”

Small-molecule inhibitors of integrin α 2 β 1 that prevent pathological thrombus formation via an allosteric mechanism

“…A variety of small molecules that weakly inhibit activation of integrin ␣ 2 ␤ 1 have been found, including naturally occurring collagen-mimetic peptides that bind the I-domain (18)(19)(20) and lipophilic compounds that stabilize a hydrophobic patch in the I-domain exposed in the inactive conformation (21). Here, we develop high-affinity small-molecule inhibitors of integrin ␣ 2 ␤ 1 that allosterically regulate the integrin through interaction with the I-like domain.…”

Chromoblastomycosis after a leech bite complicated by myiasis: a case report

“…The injection of platelet inhibitors during leech bites [18] might impair the local skin immunity increasing the risk for fungal infections. The coagulation system overlaps with the immune system and many acute-phase proteins of inflammation are also involved in the coagulation process.…”