Activated platelets bind numerous adhesive and procoagulant proteins by receptor-mediated processes. Although there is little evidence to suggest that these processes are heterogeneous in platelets, we previously found that platelets co-stimulated with collagen and thrombin express functional alpha-granule factor V only on a subpopulation of cells. Here we show that these cells, referred to as 'COAT-platelets', bind additional alpha-granule proteins, including fibrinogen, von Willebrand factor, thrombospondin, fibronectin and alpha2-antiplasmin. These proteins are all transglutaminase substrates, and inhibitors of transglutaminase prevent the production of COAT-platelets. A synthetic transglutaminase substrate (CP15) also binds to COAT-platelets, and analysis by high performance liquid chromatography/mass spectrometry shows that a product is formed with a relative molecular mass (Mr) equal to CP15 plus 176. Serotonin, an abundant component of platelet-dense granules, has an Mr of 176, and fibrinogen isolated from COAT-platelets contains covalently linked serotonin. Synthetic bovine serum albumin-(serotonin)6 binds selectively to COAT-platelets and also inhibits the retention of procoagulant proteins on COAT-platelets. These data indicate that COAT-platelets use serotonin conjugation to augment the retention of procoagulant proteins on their cell surface through an as yet unidentified serotonin receptor.

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Leukemia, the malignancy of the hematopoietic system accounts for 10% of cancer cases with poor overall survival rate in adults; therefore, there is a high unmet medical need for the development of novel therapeutics. Eight imidazo[1,2-b]pyrazole-7-carboxamides have been tested for cytotoxic activity against five leukemia cell lines: Acute promyelocytic leukemia (HL-60), acute monocytic leukemia (THP-1), acute T-lymphoblastic leukemia (MOLT-4), biphenotypic B myelomonocytic leukemia (MV-4-11), and erythroleukemia (K-562) cells in vitro. Imidazo[1,2-b]pyrazole-7-carboxamides hampered the viability of all five leukemia cell lines with different potential. Optimization through structure activity relationship resulted in the following IC50 values for the most effective lead compound DU385: 16.54 nM, 27.24 nM, and 32.25 nM on HL-60, MOLT-4, MV-4-11 cells, respectively. Human primary fibroblasts were much less sensitive in the applied concentration range. Both monolayer or spheroid cultures of murine 4T1 and human MCF7 breast cancer cells were less sensitive to treatment with 1.5–10.8 μM IC50 values. Flow cytometry confirmed the absence of necrosis and revealed 60% late apoptotic population for MV-4-11, and 50% early apoptotic population for HL-60. MOLT-4 cells showed only about 30% of total apoptotic population. Toxicogenomic study of DU385 on the most sensitive MV-4-11 cells revealed altered expression of sixteen genes as early (6 h), midterm (12 h), and late response (24 h) genes upon treatment. Changes in ALOX5AP, TXN, and SOD1 expression suggested that DU385 causes oxidative stress, which was confirmed by depletion of cellular glutathione and mitochondrial membrane depolarization induction. Imidazo[1,2-b]pyrazole-7-carboxamides reported herein induced apoptosis in human leukemia cells at nanomolar concentrations.

Combination therapy of bortezomib with other chemotherapeutics is an emerging treatment strategy. Since both curcumin and bortezomib inhibit NF-κB, we tested the effects of their combination on leukemia cells. To improve potency, a novel Mannich-type curcumin derivative, C-150, was synthesized. Curcumin and its analogue showed potent antiproliferative and apoptotic effects on the human leukemia cell line, HL60, with different potency but similar additive properties with bortezomib. Additive antiproliferative effects were correlated well with LPS-induced NF-κB inhibition results. Gene expression data on cell cycle and apoptosis related genes, obtained by high-throughput QPCR, showed that curcumin and its analogue act through similar signaling pathways. In correlation with in vitro results similar additive effect could be obsereved in SCID mice inoculated systemically with HL60 cells. C-150 in a liposomal formulation given intravenously in combination with bortezomib was more efficient than either of the drugs alone. As our novel curcumin analogue exerted anticancer effects in leukemic cells at submicromolar concentration in vitro and at 3 mg/kg dose in vivo, which was potentiated by bortezomib, it holds a great promise as a future therapeutic agent in the treatment of leukemia alone or in combination.

Since the introduction of tyrosine kinase inhibitors, the overall survival of patients with chronic myeloid leukemia has markedly improved. However long term use of these drugs results in various adverse events. Treatment with second generation dasatinib is often complicated by hemorrhagic events. Previous lumi-aggregometry studies have shown impaired platelet function in patients on dasatinib therapy. Dual agonist activated platelets (coated-platelets) are also sensitive indicators of platelet function. We hypothesized that dual activation with convulxin and thrombin of platelets in a flow cytometric assay could be a more sensitive method for detecting platelet dysfunction as compared to single agonist studies used in lumi-aggregometer. Platelets of healthy volunteers incubated with dasatinib as well as platelets from patients on dasatinib therapy were investigated. Low therapeutic plasma level dasatinib concentrations at which a considerable reduction in coated-platelet generation was observed in vitro, did not cause detectable change in platelet aggregation response. Coated-platelet assay and lumi-aggregometry were also investigated at 0, 1 and 4 hours after drug administration in dasatinib treated CML patients. Significant decrease was observed at 1 hour in maximal aggregation by collagen. Although the aggregation curves became normalized by 4 hours, coated-platelet generation was still inhibited in dasatinib treated patients. Nilotinib, another second generation tyrosine kinase inhibitor, had no effect on aggregation and on coated-platelet formation neither in vitro nor in ex vivo samples. At therapeutic plasma levels coated-platelet assay is more sensitive than lumi-aggregometry studies for the demonstration of the inhibitory effect of dasatinib on platelet function.