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A molecularly imprinted polymer (MIP) was synthesized and evaluated for the selective extraction of cocaine (COC) and its main metabolite benzoylecgonine (BZE) in hair extracts. To this end, a screening of different conditions of synthesis was performed by changing the nature of the crosslinker, and the functional monomer and also by changing polymerization's initiation mode. The selectivity of the different MIPs was evaluated by comparing the retention of COC and BZE between the MIP supports and also compared to a non-imprinted polymer for each. All the supports were selective for one or both molecules, but, the best results in terms of selectivity and retention were obtained for a MIP using methacrylic acid as functional monomer, ethyleneglycol dimethacrylate as crosslinker, and a photochemical initiation. An optimized procedure in acetonitrile media was developed for the selective extraction of COC and BZE with a recovery close to 80% for both molecules from the MIP. The capacity of the MIP for COC retention was also evaluated, and MIP showed a specific capacity of 8.96 μmol g −1 . Finally, the potential of this material for sample clean-up was demonstrated by the selective extraction of both COC and BZE from acetonitrile hair extracts spiked at the cutoff value for COC in hair analysis. By the selective purification with the MIP, a limit of quantification inferior to 0.07 ng mg −1 of hair was reached for both molecules.

The thrombospondins are a family of extracellular calcium-binding proteins that modulate cellular phenotype. Thrombospondin-1 (TSP-1) reportedly regulates cellular attachment, proliferation, migration, and differentiation in vitro. To explore its function in vivo, we have disrupted the TSP-1 gene by homologous recombination in the mouse genome. Platelets from these mice are completely deficient in TSP-1 protein; however, thrombin-induced platelet aggregation is not diminished. TSP-1-deficient mice display a mild and variable lordotic curvature of the spine that is apparent from birth. These mice also display an increase in the number of circulating white blood cells, with monocytes and eosinophils having the largest percent increases. The brain, heart, kidney, spleen, stomach, intestines, aorta, and liver of TSP-1-deficient mice showed no major abnormalities. However, consistent with high levels of expression of TSP-1 in lung, we observe abnormalities in the lungs of mice that lack the protein. Although normal at birth, histopathological analysis of lungs from 4-wk-old TSP-1-deficient mice reveals extensive acute and organizing pneumonia, with neutrophils and macrophages. The macrophages stain for hemosiderin, indicating that diffuse alveolar hemorrhage is occurring. At later times, the number of neutrophils decreases and a striking increase in the number of hemosiderin-containing macrophages is observed associated with multiple-lineage epithelial hyperplasia and the deposition of collagen and elastin. A thickening and ruffling of the epithelium of the airways results from increasing cell proliferation in TSP-1-deficient mice. These results indicate that TSP-1 is involved in normal lung homeostasis.

Considering the important complexity of biological samples, a molecularly imprinted polymer (MIP) was applied to the selective extraction of cocaine and its two main metabolites, benzoylecgonine and ecgonine methyl ester from biological samples. The MIP was imprinted with cocaine and it was synthesized in acetonitrile with methacrylic acid as a functional monomer and ethylene glycol dimethacrylate as a crosslinker. The selectivity of the MIP was first assessed for the three target analytes in acetonitrile with recoveries higher than 80% on the MIP and lower than 30% on the non-imprinted polymer (NIP). The MIP was then evaluated for the selective extraction of these targets from real aqueous media, i.e. serum and urine samples. The pH adjustment of the sample as well as the optimization of the washing step led to a very selective extraction of cocaine from these media. A LOQ of 0.5ng/mL was obtained for cocaine in urine. Concerning cocaine metabolites, benzoylecgonine and ecgonine methyl ester, they were first extracted from urine by liquid-liquid extraction and the resulting extract was purified on the MIP. The results obtained with the MIP as compared to the LLE alone showed the great potential of the MIP extraction for the clean-up of the biological matrix. This procedure was tested for the extraction of the analytes from urine samples, leading to a very selective protocol with LOQs of 0.09ng/mL, 0.4ng/mL and 1.1ng/mL for cocaine, benzolecgonine and ecgonine methyl ester respectively in urine samples.

We have investigated the molecular requirements for thrombospondin (TSP) to bind to the platelet surface and to support the subsequent secretion-dependent platelet aggregation. For this, we used two distinct murine monoclonal antibodies (MoAbs), designated MAI and MAII, raised against human platelet TSP, and three polyclonal antibodies, designated R3, R6, and R5, directed against fusion proteins containing the type 1 (Gly 385-Ile 522), type 2 (Pro 559-Ile 669), and type 3 (Asp 784-Val 932) repeating sequences, respectively. Among them, R5 and R6, but not R3, inhibited thrombin-induced aggregation of washed platelets and the concomitant secretion of serotonin. These antibodies, however, did not inhibit the expression of TSP on thrombin-activated platelets, as measured by the binding of a radiolabeled MoAb to TSP, suggesting that they may inhibit platelet aggregation by interfering with a physiologic event subsequent to TSP binding. In contrast, MoAb MAII, which reacts with an epitope located within the heparin-binding domain of TSP, inhibited both TSP surface expression and platelet aggregation/secretion induced by thrombin. In addition, this MoAb inhibited in a dose-dependent manner (IC50 approximately 0.5 mumol/L) the interaction of 125I-TSP with immobilized fibrinogen and platelet glycoprotein IV, both potential physiologic receptors for TSP on thrombin-activated platelets. These results indicate that the interaction of TSP with the surface of activated platelets can be modulated at the level of a specific epitope located within the amino terminal heparin-binding domain of the molecule. Thus, selective inhibition of the platelet/TSP interaction may represent an alternative approach to the inhibition of platelet aggregation.

Summary. The distribution of the major platelet membrane glycoproteins (GP), Ib, IX, IIb-IIIa and IV (or CD36), which play important roles as receptors for adhesive molecules in haemostasis and thrombosis, was studied in 34 patients with myeloproliferative disorders (MPD): 13 had essential thrombocythaemia (ET), 12 had polycythaemia vera (PV) and nine had chronic myelogenous leukaemia (CML). Only occasionally were modifications of the numbers of GPIb or GPIIb-IIIa measured using the binding of specific radiolabeled antibodies to platelets. In contrast, 2-3-fold increases of the total CD36 content and the surface CD36 expression were measured in almost all patients studied, using a radioimmunoassay and the direct binding of the radiolabelled antibody, FA6-152, to the platelet surface, respectively. These results indicate that the abnormality affected both the external and internal CD36 pools. Therefore platelet CD36 may be a useful tool for the diagnosis and the follow-up of MPD patients. Surface CD36 has been proposed as a platelet receptor for thrombospondin, an adhesive glycoprotein that is released from platelets upon activation and promotes aggregate formation. Despite a 2-fold increase of CD36 molecules, resting and thrombin-activated platelets from ET patients expressed the same amount of thrombospondin as normal platelets, suggesting that there is not a direct correlation between the CD36 expression and thrombospondin binding either spontaneously or after activation.

SummaryGlycoprotein IV (GPIIIb, CD36) is a major platelet membrane glycoprotein which is thought to participate in a number of adhesive reactions and to mediate signal transduction. In order to measure the total content of GPIV in human platelets, we have developed a simple and sensitive solid-phase radioimmunoassay based on the immunocapture of GPIV from Triton X-100-solubilized platelets. FA6-152, a monoclonal antibody to GPIV was coated on microtiter plates and bound antigen was quantified with a radiolabeled polyclonal antibody to GPIV Using purified GPIV as a standard, the coefficients of variation of the assay were found to be less than 10% at concentrations of GPIV ranging from 0.15 to 0.75 µg/ml. The assay was validated by the parallelism obtained between purified GPIV dose-response curves and those obtained with platelet lysates, indicating a similar antigenic activity for GPIV in both samples. The level of GPIV in platelets from healthy donors was 0.23 ± 0.05 (mean ± SD, n = 15) µg per 100 µg of platelet proteins and a mean value of 27,440 ± 6,200 (SD) molecules per platelet was calculated. The radioimmunoassay could be used to discriminate between the high level of platelet GPIV in patients with essential thrombocythemia (mean ± SD = 81,850 ± 27,780 molecules/platelet; n = 8) and the normal GPIV level in patients with secondary thrombocytosis (mean ± SD = 26,810 ± 4,030 molecules/platelet; n = 5), thereby demonstrating the clinical usefulness of the assay. The specific increase in platelet GPIV in patients with essential thrombocythemia was confirmed by immunoblot analysis whereas no increase in platelet GPIb or GPIIb-IIIa was observed by this technique.