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Vascular endothelial growth factor (VEGF), a potent and specific activator of endothelial cells, is expressed as multiple homodimeric forms resulting from alternative RNA splicing. VEGF121 does not bind heparin while the other three isoforms do, and it has been documented that the binding of VEGF165 to its receptor is dependent upon cell surface heparin sulfate proteoglycans. Little is known about the biochemical mechanism that allows for heparin regulation of growth factor binding. For example, it is not clear whether heparin interactions with growth factor or with cell surface receptors or both are essential for VEGF binding to its receptor. In this manuscript we provide results which are consistent with the hypothesis that an interaction between heparin and a site on the KDR receptor subtype is essential for VEGF165 binding. First, we demonstrate that expression of KDR into a CHO cell line deficient in heparan sulfate biosynthesis does not allow VEGF165 binding unless heparin is exogenously added during the binding assay. Secondly, we show that a ten amino acid synthetic peptide, corresponding to a sequence from the extracellular domain of the KDR, both inhibits VEGF165 binding to the receptor and also binds heparin with high avidity. Third, affinity purification of heparin molecules on a KDR-derived peptide affinity column, together with capillary electrophoresis and polyacrylamide electrophoresis analysis, was used to show that the KDR-derived peptide interacts with a specific subset of polysaccharide chains contained in the unfractionated heparin. Taken together, these results are consistent with the hypothesis that interactions between cell surface heparan sulfate proteoglycans and the VEGF receptor contribute to allowing maximal VEGF binding.

A series of aryl phosphoglyceride (3, 19-61) and bis-aryl phosphate (67-135) antagonists of platelet activating factor (PAF) were prepared. A group of four bifunctional phosphorus reagents (5a-c and 7) were developed that allowed the preparation of these aryl phosphates in which the position of aromatic substitution can be varied. These compounds were examined for their ability to inhibit PAF-induced platelet aggregation of rabbit platelets. Selected compounds were also evaluated for their ability to displace [3H]PAF from its receptor on rabbit platelets. These in vitro data were compared to similar data obtained for a number of known PAF antagonists. The compounds were evaluated in vivo, in both the mouse and rabbit, for their ability to prevent death induced by a lethal challenge of PAF. The relationships between the biological activity and the nature, lipophilicity, and position of substituents of the aromatic rings were studied. Compound 105 (CL 184005) has been selected to undergo further development as a potential therapeutic agent for the treatment of septic shock in man.

By using inhibition of histamine release from antigen-challenged, sensitized human basophils as a means of identifying a potentially prophylactic drug for the treatment of asthma, a series of substituted imidazo[1,5-d][1,2,4]triazines were found, which were active. These compounds were prepared by treating imidazolecarboxaldehydes with excess Grignard agent and then oxidizing the resulting alcohols to ketones with Jones reagent. Pyrolysis of a mixture of ketone and methyl carbazate at 200 degrees C in diphenyl ether produced the desired imidazo[1,5-d][1,2,4]triazines. Those compounds with the greatest basophil activity were tested for in vivo activity in the mouse passive cutaneous anaphylaxis (PCA) and the guinea pig passive anaphylaxis tests. The best compounds, 1-ethyl-8-methyl-6-propylimidazo[1,5-d][1,2,4]triazin-4(3H)- one (4-17) and 1,8-dimethyl-6-propylimidazo[1,5-d][1,2,4]triazin-4-(3H)-one (4-16) were chosen for further study.

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