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ABSTRACT

BackgroundThis study was designed to test a new approach to drug treatment of autism spectrum disorders (ASDs) in the Fragile X (Fmr1) knockout mouse model.MethodsWe used behavioral analysis, mass spectrometry, metabolomics, electron microscopy, and western analysis to test the hypothesis that the disturbances in social behavior, novelty preference, metabolism, and synapse structure are treatable with antipurinergic therapy (APT).ResultsWeekly treatment with the purinergic antagonist suramin (20 mg/kg intraperitoneally), started at 9 weeks of age, restored normal social behavior, and improved metabolism, and brain synaptosomal structure. Abnormalities in synaptosomal glutamate, endocannabinoid, purinergic, and IP3 receptor expression, complement C1q, TDP43, and amyloid β precursor protein (APP) were corrected. Comprehensive metabolomic analysis identified 20 biochemical pathways associated with symptom improvements. Seventeen pathways were shared with human ASD, and 11 were shared with the maternal immune activation (MIA) model of ASD. These metabolic pathways were previously identified as functionally related mediators of the evolutionarily conserved cell danger response (CDR).ConclusionsThe data show that antipurinergic therapy improves the multisystem, ASD-like features of both the environmental MIA, and the genetic Fragile X models. These abnormalities appeared to be traceable to mitochondria and regulated by purinergic signaling.Electronic supplementary materialThe online version of this article (doi:10.1186/2040-2392-6-1) contains supplementary material, which is available to authorized users.

Tissue factor is a membrane-bound procoagulant protein that activates the extrinsic pathway of blood coagulation in the presence of factor VII and calcium. X Phage containing the tissue factor gene were isolated from a human placental cDNA library. The amino acid sequence deduced from the nucleotide sequence of the cDNAs indicates that tissue factor is synthesized as a higher molecular weight precursor with a leader sequence of 32 amino acids, while the mature protein is a single polypeptide chain composed of 263 residues. The derived primary structure of tissue factor has been confirmed by comparison to protein and peptide sequence data. The sequence of the mature protein suggests that there are three distinct domains: extracellular, residues 1-219; hydrophobic, residues 220-242; and cytoplasmic, residues 243-263. Three potential N-linked carbohydrate attachment sites occur in the extracellular domain. The amino acid sequence of tissue factor shows no significant homology with the vitamin Kdependent serine proteases, coagulation cofactors, or any other protein in the National Biomedical Research Foundation sequence data bank (Washington, DC).Blood coagulation can be initiated by a complex of tissue factor (TF), a membrane-bound glycoprotein, and factor VII, a plasma coagulation factor (for reviews, see refs. 1 and 2). The physiological significance of this extrinsic pathway can be judged by the severe bleeding frequently observed in individuals who are markedly deficient in factor VII (3, 4). In contrast, individuals who have deficiencies or abnormalities in proteins that are involved in the early steps of the intrinsic pathway of coagulation-i.e., high molecular weight kininogen, prekallikrein, and factor XII-are asymptomatic (5). The TF-factor VII complex activates factor IX, a component of the intrinsic pathway, as well as factor X (6). Thus, it is reasonable that the association of TF and factor VII may be the crucial event triggering the initiation of clotting in vivo.The cDNAs for all of the proteins involved in TF-initiated coagulation, with the exception of TF itself, have already been cloned and sequenced (7-13). The TF apoprotein has been purified from both bovine and human sources (14-16). Approximately 50-70% of the amino acid sequence of both species has now been determined, and this has permitted us to select suitable amino acid sequences to serve as a basis for constructing oligonucleotide probes. This in turn has enabled the isolation and characterization of two human placental TF cDNA clones that contain the entire coding region of the mature protein. The nucleotide sequence of these clones, together with amino acid sequence data, has allowed us to formulate a primary structure for the human TF apoprotein §. MATERIALS AND METHODSTF Purification and Sequencing. A monoclonal antibody (17) prepared against human TF that had been purified by using factor VII affinity columns (15) was used for immunoaffinity isolation of TF. Briefly, TF was extracted from human brain or placental tissue acet...