Triplatin, a platelet aggregation inhibitor from the salivary gland of the triatomine vector of Chagas disease, binds to TXA(2) but does not interact with glycoprotein PVI

Abstract

Salivary glands from haematophagous animals express a notable diversity of negative modulators of platelet function. Triplatin is an inhibitor of collagen-induced platelet aggregation which has been described as an antagonist of glycoprotein VI (GPVI). Because triplatin displays sequence homology to members of the lipocalin family of proteins, we investigated whether triplatin mechanism of action could be explained by interaction with pro-haemostatic prostaglandins. Our results demonstrate that triplatin inhibits platelet aggregation induced by low doses of collagen, thromboxane A2 (TXA(2)) mimetic (U46619), and arachidonic acid (AA). On the other hand, it does not inhibit platelet aggregation by convulxin, PMA, or low-dose ADP. Isothermal titration calorimetry (ITC) revealed that triplatin binds AA, cTXA(2), TXB(2), U46619 or prostaglandin (PG)H(2) mimetic (U51605). Consistent with its ligand specificity, triplatin induces relaxation of rat aorta contracted with U46619. Triplatin also interacts with PGF(2α) and PGJ(2), but not with leukotrienes, AA or biogenic amines. Surface plasmon resonance experiments failed to demonstrate interaction of triplatin with GPVI; it also did to inhibit platelet adhesion to fibrillar or soluble collagen. Because triplatin displays sequence similarity to apolipoprotein D (ApoD) - a lipocalin associated with high-density lipoprotein, ApoD was tested as a putative TXA(2)-binding molecule. ITC failed to demonstrate binding of ApoD to all prostanoids described above, or to AA. Furthermore, ApoD was devoid of inhibitory properties towards platelets activation by AA, collagen, or U46619. In conclusion, triplatin mechanism of action has been elucidated without ambiguity as a novel TXA(2)- and PGF(2α)- binding protein. It conceivably blocks platelet aggregation and vasoconstriction, thus contributing to successful blood feeding at the vector-host interface.

Fig. 1. Characterization of triplatin

(A) Clustal alignment of triplatin (gi109240370) and DPTL (gi344190590), pallidipin-2 (gi388359), RPAI-1 (gi1572725), triabin (gi1122389) and several other triatomine putative TXA2-binding proteins. (B) Phylogenetic tree of triplatin and related salivary lipocalins. Tree was generated using the neighbor-joining method after 10,000 bootstraps. The numbers in the phylogram nodes indicates percent bootstrap support for the phylogeny. The bar at the bottom indicates 20% amino acid divergence in sequences. (C) Triplatin was loaded in a Sephadex G75 gel-filtration column and eluted at 1 ml/min in TBS, pH 7.4. Inset, gel electrophoresis of triplatin (reducing [+] and non-reducing [−] conditions) was carried out in a 4–12% NuPAGE gel. N-terminus was performed by Edman degradation. (D) Mass spectrometry of triplatin reveals a mass of 18,799 Da, which is in agreement with a theoretical mass of 18,672.7 Da with an extra methionine and 6xhis.

Fig. 2. Triplatin inhibits platelet aggregation

Triplatin was incubated for 1 min with platelet-rich plasma (2 × 10⁵/μl) followed by addition of collagen, U46619, and arachidonic acid, PMA, convulxin and ADP. Triplatin and agonist concentrations are indicated. In some experiments, platelets were treated with indometacin (50 μM) for 3 minuets before addition of the agonist. Typical experiments are shown (n = 3).

Fig. 3. Isothermal titration calorimetry

Upper panels: base line-adjusted heats per injection of different ligands (40 μM) into triplatin (4.0 μM). The lower panels indicate the molar enthalpies per injection for ligand interaction with triplatin. Filled squares measured enthalpies; solid line, fit of experimental data to a single site binding model. Thermodynamic parameters: ΔH in kcal/mol, TΔS in kcal/mol, and K_D are indicated in the inset for each ligand. Experiments were repeated at least 2 times for each ligand.

Fig. 4. Inhibition of vasoconstriction by triplatin

Rat aorta was placed in a chamber and contraction induced by U-46619 (0.1 μM). After stabilization, triplatin was added and relaxation recorded isometrically. Triplatin or buffer was injected at 30 min. Representative experiment is shown.

Fig. 5. Triplatin does not interact with GPVI and its activity is loss in the presence of indometacin

(A) Convulxin (10 nM), triplatin (1 μM), or DPTL (1 μM) was injected for 180 sec over immobilized GPVI using HBSP as a buffer. Dissociation was carried out for 5 min followed by regeneration of CM5 chip with HCl (10 mM). As a control, Cvx was injected again without loss of interaction with GPVI. (B) Calcein-labeled human platelets (2 × 10⁵/μl) were incubated with fibrillar or soluble collagen for 1 h at indicated concentrations of triplatin. EDTA was used to discriminate GPVI- or integrin α2β1-mediated adhesion. Absolute fluorescence values are reported. Platelet adhesion BSA-blocked wells was negligible (n=6). (C) platelet-rich plasma (2 × 10⁵/μl) was incubated with 50 μM indometacin, or 0.2 μM SQ29548 for 3 min alone, or with and without triplatin (1 μM) followed by addition of collagen (6.6 μg/ml).

Fig. 6. Apolipoprotein D (ApoD) does not inhibit platelet aggregation

(A) Alignment of ApoD and triplatin. (B) Purification of ApoD, PAGE and N-terminus identification were carried out as described in Methods. (C) ApoD was incubated for 1 min with platelets (2 × 10⁵/μl) followed by addition of collagen, U46619, and arachidonic acid. (D) Isothermal titration calorimetry for ApoD interaction with AA, cTXA_2α and PGF2 is shown.