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Abstract
Dipetalodipin (DPTL) is an 18 kDa protein cloned from salivary glands of the triatomine Dipetalogaster maxima. DPTL belongs to the lipocalin superfamily and has strong sequence similarity to pallidipin, a salivary inhibitor of collagen-induced platelet aggregation. DPTL expressed in Escherichia coli was found to inhibit platelet aggregation by collagen, U-46619, or arachidonic acid without affecting aggregation induced by ADP, convulxin, PMA, and ristocetin. An assay based on incubation of DPTL with small molecules (e.g. prostanoids, leukotrienes, lipids, biogenic amines) followed by chromatography, mass spectrometry, and isothermal titration calorimetry showed that DPTL binds with high affinity to carbocyclic TXA(2), TXA(2) mimetic (U-46619), TXB(2), PGH(2) mimetic (U-51605), PGD(2,) PGJ(2), and PGF(2α). It also interacts with 15(S)-HETE, being the first lipocalin described to date to bind to a derivative of 15-lipoxygenase. Binding was not observed to other prostaglandins (e.g. PGE(1), PGE(2), 8-iso-PGF(2α), prostacyclin), leukotrienes (e.g. LTB(4), LTC(4), LTD(4), LTE(4)), HETEs (e.g. 5(S)-HETE, 12(S)-HETE, 20-HETE), lipids (e.g. arachidonic acid, PAF), and biogenic amines (e.g. ADP, serotonin, epinephrine, norepinephrine, histamine). Consistent with its binding specificity, DPTL prevents contraction of rat uterus stimulated by PGF(2α) and induces relaxation of aorta previously contracted with U-46619. Moreover, it inhibits angiogenesis mediated by 15(S)-HETE and did not enhance inhibition of collagen-induced platelet aggregation by SQ29548 (TXA(2) antagonist) and indomethacin. A 3-D model for DPTL and pallidipin is presented that indicates the presence of a conserved Arg(39) and Gln(135) in the binding pocket of both lipocalins. Results suggest that DPTL blocks platelet aggregation, vasoconstriction, and angiogenesis through binding to distinct eicosanoids involved in inflammation.
Highlights
The hemostatic process, a host defense mechanism to preserve the integrity of the circulatory system, remains inactive until vascular injury occurs, leading to activation of hemo
In an attempt to verify the inhibitory profile of DPTL toward other agonists that activate platelets independently of secondary mediators, it was tested as an inhibitor for U-46619 (TXA2 mimetic) and AA-induced platelet aggregation
We have identified DPTL as a novel salivary lipocalin that binds to distinct prostanoids
Summary
Materials—Horse tendon insoluble Horm fibrillar collagen (quaternary, polymeric structure) composed of collagen types I (95%) and III (5%), and Chrono-Lume were from Chrono-Log. Selected fractions (20 l) were mixed with 1 l of methanol containing 1 M HCl, centrifuged at 14,000 ϫ g for 10 min, and the supernatant injected into a 0.3 ϫ 150-mm C18 reverse phase column (Magic C18 200 Å; Michrom BioResources, Inc, Auburn CA) equilibrated with 10% methanol/water containing 0.1% acetic acid at a flow rate of 3 l/min maintained by an ABI 140D pump (Applied Biosystems). Calorimetric assays for measuring DPTL binding to a number of ligands were performed using a VP-ITC microcalorimeter (Microcal, Northampton, MA) at 35 °C. Where Q is total heat content of the solution contained in the cell volume (Vo), at fractional saturation , ⌬H is the molar heat of ligand binding, n is the number of sites, and Mt is the bulk concentration of macromolecules in Vo. The binding constant, Ka, is described as Equation 2, Ka ϭ ͞(1Ϫ)[X]. Statistical Analysis—Results are expressed as mean Ϯ S.E. (GraphPad Software, Inc., San Diego, CA)
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