ABSTRACT
OBJECTIVE: To investigate mechanisms responsible for increased thrombotic activity in systemic lupus erythematosus (SLE) associated with the antiphospholipid syndrome (APS). We had reported that anticardiolipin/beta2-glycoprotein I (aCL/beta2-GPI) complexes induce platelet overactivity resulting in excessive production of thromboxane A2 (TXA2). Presumably this occurs by decreased platelet cyclic AMP (cAMP) activity and results in increased platelet aggregation. METHODS: We stimulated platelet intracellular cAMP generation with known cAMP agonists (dibutyryl cAMP, theophylline, and prostaglandin E1) and measured aCL/beta2-GPI induced platelet TXB2 production in vitro. Isolated human platelets were prelabeled with 14C-arachidonic acid and then challenged with aCL/beta2-GPI in the presence or absence of cAMP-activating substances. The resulting 14C labeled TXB2 was quantified by thin layer chromatography and radioactive scanning. RESULTS: We found a marked decrease in aCL/beta2-GPI induced platelet TXB2 production by the cAMP agonists in a dose dependent manner. CONCLUSION: Our findings suggest the usefulness of cAMP agonists in the control of thrombosis in some patients with SLE and APS.
Subject(s)
Antiphospholipid Syndrome/metabolism , Blood Platelets/metabolism , Cyclic AMP/agonists , Glycoproteins/pharmacology , Thromboxane A2/biosynthesis , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Antibodies, Anticardiolipin/pharmacology , Antiphospholipid Syndrome/immunology , Arachidonic Acid/pharmacokinetics , Blood Platelets/drug effects , Carbon Radioisotopes , Cyclic AMP/metabolism , Hemostatics/pharmacology , Humans , In Vitro Techniques , Indomethacin/pharmacology , Lupus Erythematosus, Systemic/immunology , Lupus Erythematosus, Systemic/metabolism , Thrombin/pharmacology , beta 2-Glycoprotein IABSTRACT
Sp17 was initially thought to be a sperm specific protein involved in the interaction of the spermatozoon with the oocyte's surrounding extracellular glycoprotein matrix. Recent reports, however, indicate that Sp17 expression is neither testis-specific nor is it exclusively used for binding to the zona pellucida of the oocyte. In this study, we provide comprehensive characterization of the genomic structure of Sp17. We identified an intron-containing gene (Sp17-1) containing five exonic and four intronic sequences. Analysis of Sp17 transcripts using rapid amplification of DNA complementary to RNA (cDNA) ends (RACE) and polymerase chain reaction (PCR) techniques showed the presence of alternative polyadenylation resulting in the production of varying lengths of mRNAs as well as the usage of different transcriptional start sites. Moreover, an earlier description of the human Sp17 mRNA describing a splice variant could not be confirmed. Comparison to mouse Sp17 gene organization demonstrated a high degree of conservation, suggesting selective evolutionary pressure for this protein to retain a conserved gene architecture. Additionally, we identified a second gene (Sp17-2), whose most striking characteristic was the complete absence of introns. This Sp17-2 gene has likely arisen by reverse transcription (RT) of a spliced Sp17-1 mRNA with subsequent integration into the human genome. Its open reading frame (ORF) is interrupted by stop codons, giving rise to a pseudogene. Furthermore, Southern blot analysis of human genomic DNA indicated the possibility of additional Sp17 species within the human genome.
Subject(s)
Carrier Proteins/genetics , Introns , Pseudogenes/genetics , Antigens, Surface , Base Sequence , Blood Cells/metabolism , Blotting, Southern , Calmodulin-Binding Proteins , Chromosomes, Human, 6-12 and X , DNA, Complementary/chemistry , DNA, Complementary/isolation & purification , Humans , Male , Membrane Proteins , Molecular Sequence Data , Promoter Regions, Genetic , RNA, Messenger/analysis , Restriction Mapping , Reverse Transcriptase Polymerase Chain Reaction , Testis/metabolism , Transcription Initiation SiteABSTRACT
Rheumatoid arthritis (RA) is an autoimmune disease characterized by proliferative synovial tissue. We used mRNA differential display and library subtraction to compare mRNA expression in RA and osteoarthritis (OA) synoviocytes. We initially compared the mRNA expression patterns in 1 female RA and 1 OA synovia and found a differentially expressed 350 bp transcript in the RA synoviocytes which was, by sequence analysis, 100% homologous to sperm protein 17 (Sp17). Moreover, the Sp17 transcript was found differentially expressed in a RA synovial library that was subtracted with an OA synovial library. Using specific primers for full length Sp17, a 1.1 kb transcript was amplified from the synoviocytes of 7 additional female RA patients, sequenced and found to 100% homologous to Sp17. Thus, we found the unexpected expression of Sp17, a thought to be gamete-specific protein, in the synoviocytes of 8/8 female RA patients in contrast to control OA synoviocytes. Interestingly, Sp17's structural relationship with cell-binding and recognition proteins, suggests that Sp17 may function in cell-cell recognition and signaling in the RA synoviocyte. Further, Sp17 could have a significant regulatory role in RA synoviocyte gene transcription and/or signal transduction. Thus, Sp17 could have an important role in RA synoviocyte proliferation or defective apoptosis. Finally, the presence of Sp17 in synoviocytes has interesting developmental considerations.
