The Insect Salivary Protein, Prolixin-S, Inhibits Factor IXa Generation and Xase Complex Formation in the Blood Coagulation Pathway

Abstract

Prolixin-S is a salivary anticoagulant of the bloodsucking insect, Rhodnius prolixus, and known as an inhibitor of the intrinsic Xase. We report here its inhibitory mechanisms with additional important anticoagulation activities. We found prolixin-S specifically bound to factor IX/IXa in the presence of Ca 2؉ ions. Light scattering and surface plasmon resonance studies showed that prolixin-S interfered with factor IX/IXa binding to the phospholipid membrane, indicating that prolixin-S inhibit Xase activity of factor IXa by interference with its Xase complex formation. Furthermore, reconstitution experiments showed that prolixin-S binding to factor IX strongly inhibited factor IXa generation by factor XIa. We also found that prolixin-S inhibited factor IXa generation by factor VIIa-tissue factor complex and factor IX␣ generation by factor Xa. These results suggest that prolixin-S inhibits both intrinsic and extrinsic coagulations by sequential inhibition of all coagulation pathways in which factor IX participates. It was also suggested that prolixin-S may bind to factor IX/IXa by recognizing conformational change of the Gla domain induced by Ca 2؉ binding.

Exploring the sialome of the blood-sucking bug Rhodnius prolixus

“…Several distinct nitrophorins (NP), which are hemecontaining lipocalins, carry nitric oxide (NO) from the salivary glands to the injured tissue, causing vasodilation and further inhibiting platelet aggregation Champagne et al, 1995). These NP also bind histamine with high affinity (Ribeiro and Walker, 1994;Andersen et al, 1998), and one, NP2, also inhibits the Xase complex of the intrinsic pathway of the blood coagulation cascade Zhang et al, 1998;Isawa et al, 2000). An additional lipocalin, biogenic amine binding protein (BABP), removes serotonin and adrenergic mediators of vasoconstriction (Andersen et al, 2003).…”

A Mosquito Salivary Protein Inhibits Activation of the Plasma Contact System by Binding to Factor XII and High Molecular Weight Kininogen

“…Dextran sulfate of molecular weight 500,000 (DS 500) and soybean trypsin inhibitor (SBTI) were purchased from Wako Pure Chemical Industries Ltd. Chromogenic substrates (S-2302, H-D-Pro-L-Phe-L-Arg-p-nitroanilide; S-2366, pyro-Glu-Pro-Argp-nitroanilide) were purchased from Chromogenix AB (Mö lndal, Sweden). Phosphatidylcholine (PC) and phosphatidylinositol phosphate (PtdInsP) were purchased from Sigma Chemical Co. Small unilamellar phospholipid vesicle suspension (PC and PtdInsP in a 6:4 (w/w) ratio) was prepared by sonication as described previously (23).…”

Nitrophorins and nitrobindins: structure and function

“…The saliva of Rp has long been known to act as an anticoagulant, this activity possibly residing in Rp-NP2 and Rp-NP7 (55,56,60,65). Rp-NP2 (also called prolixin-S) acts not only as a NO carrier but also in the blood coagulation cascade preventing the conversion of factor X to Xa (55,56).…”

“…The saliva of Rp contains several proteins including: (i) three platelet aggregation inhibitors (named from RPAI1 to RPAI3) (52); (ii) an anti-thrombin protein that modulates the blood-pumping frequency, facilitating the feeding efficiency of Triatominae (53,54); (iii) six RpNPs (designated from Rp-NP1 to Rp-NP6) that induce the vasodilation releasing NO and impair the activation of the host inflammation and immune responses by trapping histamine; (iv) Rp-NP2, also called prolixin-S, which blocks both the intrinsic and extrinsic coagulation pathways by inhibiting the factor IX (55,56); and (v) Rp-NP7 that is devoted to impair platelet aggregation (26,27,37). Although Rp-NPs have been extensively studied and are structurally well characterized, it remains a matter of debate why Rp uses a set of NPs.…”

“…Rp-NP2 (also called prolixin-S) acts not only as a NO carrier but also in the blood coagulation cascade preventing the conversion of factor X to Xa (55,56). Indeed, Rp-NP2 is a good therapeutic molecule in the care of arterial thrombosis: 20-40 μg/ml of Rp-NP2 can reduce thrombin formation by 50%-80%, and 400 μg/kg of Rp-NP2 displays a better effect than heparin; furthermore, 1 mg/kg of Rp-NP2 can even increase the time taken for carotid artery occlusion (56).…”