Activated platelets shed surface proteins, potentially modifying platelet function as well as providing a source of bioactive fragments. Previous studies have identified several constituents of the platelet sheddome, but the full extent of shedding is unknown. Here we have taken a global approach, analyzing protein fragments in the supernate of activated platelets using mass spectroscopy and looking for proteins originating from platelet membranes. After removing plasma proteins and microparticles, 1048 proteins were identified, including 69 membrane proteins. Nearly all of the membrane proteins had been detected previously, but only 10 had been shown to be shed in platelets. The remaining 59 are candidates subject to confirmation. Based on spectral counts, protein representation in the sheddome varies considerably. As proof of principle, we validated one of the less frequently detected proteins, semaphorin 7A, which had not previously been identified in platelets. Surface expression, cleavage, and shedding of semaphorin 7A were demonstrated, as was its association with ␣-granules. Finally, cleavage of semaphorin 7A and 12 other proteins was substantially reduced by an inhibitor of ADAM17, a known sheddase. These results define a subset of membrane proteins as sheddome candidates, forming the basis for further studies examining the impact of ectodomain shedding on platelet function. (Blood. 2011;117(1): e15-e26)

A novel approach to pancreatic cancer biomarker discovery has been developed, which employs a stable isotope labeled proteome (SILAP) standard coupled with extensive multidimensional separation coupled with tandem mass spectrometry (MS/MS). Secreted proteins from CAPAN-2 human pancreatic cancer derived cells were collected after conducting stable isotope labeling by amino acids in cell culture (SILAC). The resulting SILAP standard contained <0.5% of individual unlabeled proteins. Pooled sera from patients with early stage pancreatic cancer or controls were prepared, and an equal amount of the SILAP standard was added to each sample. Proteins were separated by isoelectric focusing (IEF) prior to two-dimensional liquid chromatography (2D-LC)–MS/MS analysis. A total of 1065 proteins were identified of which 121 proteins were present at 1.5-fold or greater concentrations in the sera of patients with pancreatic cancer. ELISA validation of these findings was successfully performed for two proteins, ICAM-1 and BCAM. Results of these studies have provided proof of principle that a SILAP standard derived from the CAPAN-2 secreted proteome can be used in combination with extensive multidimensional LC-MS/MS for the identification and relative quantitation of potential biomarkers of pancreatic cancer. This technique allows for the detection of low-abundance proteins, and focuses only on biologically relevant proteins derived from pancreatic cancer cells.

We have investigated the reactions of [PtCl(en)(ACRAMTU-S)](NO(3))(2) (2) (en = ethane-1,2-diamine; ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea, acridinium cation, 1), the prototype of a new class of cytotoxic DNA-targeted agents, with 2'-deoxyguanosine (dGuo) and random-sequence native DNA by in-line liquid chromatography/mass spectrometry (LC/MS) and NMR spectroscopy ((1)H, (195)Pt) to identify the covalent adducts formed by platinum. In the mononucleoside model system, two adducts are observed, [Pt(en)(ACRAMTU)(dGuo)](3+) (P1, major) and [Pt(en)(dGuo)(2)](2+) (P2, minor). The reaction, which proceeds significantly slower (half-life 11-12 h at 37 degrees C, pH 6.5) than analogous reactions with cisplatin and reactions of 2 with double-stranded DNA, results in the unexpected displacement of the sulfur-bound acridine ligand in approximately 15% of the adducts. This reactivity is not observed in double-stranded DNA, rendering 1 a typical nonleaving group in reactions with this potential biological target. In enzymatic digests of calf thymus DNA treated with 2, three adducts were identified: [Pt(en)(ACRAMTU)(dGuo)](3+) (A1, approximately 80%), [Pt(en)(ACRAMTU)[d(GpA)]](2+) (A2, approximately 12%), and [Pt(en)(ACRAMTU)[d(TpA)]](2+) (A3, approximately 8%). A1 and P1 proved to be identical species. In the dinucleotide adducts A2 and A3, complex 2 covalently modifies adenine at GA and TA base steps, which are high-affinity intercalation sites of the acridine derivative 1. A2 and A3, which may be formed in the minor groove of DNA, are the first examples of monofunctional adenine adducts of divalent platinum formed in double-stranded DNA. The analysis of the adduct profile indicates that the sequence specificity of 1 plays an important role in the molecular recognition between DNA and the corresponding conjugate, 2. Possible biological consequences of the unusual adduct profile are discussed.

The interactions of [Pt(en)Cl(ACRAMTU-S)](NO3)2 (PT-ACRAMTU, en = ethane-1,2-diamine, ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea) with adenine in DNA have been studied using a combination of analytical and high-resolution structural methods. For the first time, a cytotoxic platinum(II) complex has been demonstrated to form adducts in the minor groove of DNA through platination of the adenine-N3 endocyclic nitrogen. An acidic depurination assay was developed that allowed the controlled and selective (pH 2, 60 degrees C, 12 h) release of platinum-modified adenine from drug-treated nucleic acid samples. From the digested mixtures, three adducts were isolated by semipreparative reverse phase high-performance liquid chromatography and studied by electrospray ionization mass spectrometry (in-line LC-MS), variable-pH 1H NMR spectroscopy, and, where applicable, X-ray crystallography. The three species were identified as the N7 (A-I), N3 (A-II), and N1 (A-III) linkage isomers of [Pt(en)(ACRAMTU-S)(adenine)]3+ (A). Incubations carried out with the single- and double-stranded model sequences, d(TA)5 and d(TA)15, as well as native DNA indicate that the adduct profiles (A-I:A-II:A-IIIratios) are sensitive to the nature of the nucleic acid template. A-II was found to be a double-strand specific adduct. The crystal structure of this adduct has been determined, providing ultimate evidence for the N3 connectivity of platinum. A-II crystallizes in the triclinic space group P in the form of centrosymmetric dimers, {[Pt(en)(ACRAMTU-S)(adenine-N3)]2}6+. The cations are stabilized by a combination of adenine-adenine base pairing (N6...N1 2.945(5) A) and mutual acridine-adenine base stacking. Tandem mass spectra and 1H chemical shift anomalies indicate that this type of self-association is not merely a crystal packing effect but persists in solution. The monofunctional platination of adenine at its N7, N3, and N1 positions in a significant fraction of adducts breaks a longstanding paradigm in platinum-DNA chemistry, the requirement for nucleophilic attack of guanine-N7 as the principal step in cross-link formation. The biological consequences and potential therapeutic applications of the unique base and groove recognition of PT-ACRAMTU are discussed.

The synthesis, cytotoxicity, and nucleoside binding of some platinum-acridinylthiourea conjugates derived from the prototypical compound [PtCl(en)(ACRAMTU)](NO3)2 ("PT-ACRAMTU"; en=ethane-1,2-diamine, ACRAMTU=1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea, protonated form) are reported. To establish structure-activity relationships within this class of compounds, systematic changes were made to the thiourea nonleaving group, which links the intercalator to platinum. Three new derivatives of ACRAMTU, one di-, one tri-, and one tetraalkylated, were generated, where the degree of alkylation indicates the number of alkyl groups attached to the SCN2 framework. Subsequent reaction of the tri- and tetraalkylated derivatives with activated [PtCl2(en)] yielded the corresponding platinum conjugates. The dialkylated thiourea gave an unstable complex, which was not included in the studies. The crystal structure of PT-ACRAMTU x MeOH has been determined. In the solid state, one axial position of the square-planar platinum coordination sphere is partially shielded by the bulky thiourea group, providing a strong rationale for the kinetic inertness of the compound. The cytotoxicity of the prototype, the two new conjugates, and cisplatin was assessed in ovarian (A2780, A2780/CP), lung (NCI-H460), and colon (RKO) cancer cell lines using clonogenic survival assays. The derivatives containing trialkylated thiourea groups showed activity similar or superior to cisplatin, with IC50 values in the low micromolar concentration range. The complex modified with the tetraalkylated (bulkiest) thiourea was significantly less active, possibly due to the greatly decreased rate of binding to nucleobase nitrogen (1H NMR spectroscopy), but was most efficient at overcoming cross resistance to cisplatin in A2780/CP. Possible consequences of the reported structural modifications for the mechanism of action of these agents are discussed.