Ixonnexin from Tick Saliva Promotes Fibrinolysis by Interacting with Plasminogen and Tissue-Type Plasminogen Activator, and Prevents Arterial Thrombosis

Abstract

Tick saliva is a rich source of modulators of vascular biology. We have characterized Ixonnexin, a member of the “Basic-tail” family of salivary proteins from the tick Ixodes scapularis. Ixonnexin is a 104 residues (11.8 KDa), non-enzymatic basic protein which contains 3 disulfide bonds and a C-terminal rich in lysine. It is homologous to SALP14, a tick salivary FXa anticoagulant. Ixonnexin was produced by ligation of synthesized fragments (51–104) and (1–50) followed by folding. Ixonnexin, like SALP14, interacts with FXa. Notably, Ixonnexin also modulates fibrinolysis in vitro by a unique salivary mechanism. Accordingly, it accelerates plasminogen activation by tissue-type plasminogen activator (t-PA) with Km 100 nM; however, it does not affect urokinase-mediated fibrinolysis. Additionally, lysine analogue ε-aminocaproic acid inhibits Ixonnexin-mediated plasmin generation implying that lysine-binding sites of Kringle domain(s) of plasminogen or t-PA are involved in this process. Moreover, surface plasmon resonance experiments shows that Ixonnexin binds t-PA, and plasminogen (KD 10 nM), but not urokinase. These results imply that Ixonnexin promotes fibrinolysis by supporting the interaction of plasminogen with t-PA through formation of an enzymatically productive ternary complex. Finally, in vivo experiments demonstrates that Ixonnexin inhibits FeCl3-induced thrombosis in mice. Ixonnexin emerges as novel modulator of fibrinolysis which may also affect parasite-vector-host interactions.

Wonders of tick saliva

“…This multi-targeted approach of an ixodid tick to controlling the initiation of blood coagulation is remarkable and demonstrates the tick strategy of hitting the early steps of a cascade of host reactions. As if this was not enough, ixonnexin, a basic tail peptide from I. scapularis, promotes fibrinolysis through the host anticoagulation control system (Assumpção et al, 2018).…”

The Use of Tick Salivary Proteins as Novel Therapeutics

“…Salp14 and its smaller homolog Salp9Pac are anti-coagulants important for tick feeding, as evidenced by RNA interference studies in ticks (Narasimhan et al, 2002, 2004). Another BTSP family member, Ixonnexin, promoted fibrinolysis by accelerating plasminogen activation and inhibiting factor Xa (Assumpcao et al, 2018). An immunogen originally named P8 (Schuijt et al, 2011b) displayed lectin binding properties and acted as a complement inhibitor (Schuijt et al, 2011a), thus protecting Borrelia spirochetes from complement attack (Wagemakers et al, 2016).…”

Chemical Equilibrium at the Tick–Host Feeding Interface:A Critical Examination of Biological Relevance in Hematophagous Behavior

“…Plasminogen activators that result in degradation of the fibrin clot have already been discussed. Recently, a small peptide named ixonnexin that belongs to the basic tail family was described that act by promoting interaction of plasminogen and tissue plasminogen activator (tPA) by forming a enzymatically productive ternary complex that forms plasmin to promote fibrinolysis (Assumpção et al, 2018). Ixonnexin interacts with both plasminogen and tPA with similar K D values (4–20 nM).…”

“…Such an activator would need to be present at similar or higher concentrations than the target enzymes to be functionally relevant. In the case of ixonnexin, plasminogen and tPA occur at plasma concentrations of ~2 μM and 100 pM, respectively (MacGregor and Prowse, 1983; Assumpção et al, 2018). While no exact concentration has been established for ixonnexin, it has been estimated to occur at high concentrations in saliva (Assumpção et al, 2018).…”

“…In the case of ixonnexin, plasminogen and tPA occur at plasma concentrations of ~2 μM and 100 pM, respectively (MacGregor and Prowse, 1983; Assumpção et al, 2018). While no exact concentration has been established for ixonnexin, it has been estimated to occur at high concentrations in saliva (Assumpção et al, 2018). It has also been suggested that all basic tail proteins may perform this function, including the fXa inhibitor Salp14, since this family in which the C-terminal is rich in basic amino acids such as lysine and arginine mimics the C-terminal lysine present in fibrin that serves as recognition site for plasminogen and tPA lysine binding sites (Assumpção et al, 2018).…”