Novel 6-substituted uracil analogs as inhibitors of the angiogenic actions of thymidine phosphorylase.

Thymidine phosphorylase (TP) catalyzes the reversible phosphorolysis of thymidine and other pyrimidine 2'-deoxyribonucleosides. In addition, TP has been shown to possess angiogenic activity in a number of in vitro and in vivo assays, and its angiogenic activity has been linked to its catalytic activity. A series of 5- and 6-substituted uracil derivatives were synthesized and evaluated for their abilities to inhibit TP activity. Among the most active compounds was a 6-amino-substituted uracil analog, 6-(2-aminoethyl)amino-5-chlorouracil (AEAC), which was a competitive inhibitor with a K(i) of 165 nM. The inhibitory activity of AEAC was selective for TP, as it did not inhibit purine nucleoside phosphorylase or uridine phosphorylase at concentrations up to 1 mM. Human recombinant TP induced human umbilical vein endothelial cell (HUVEC) migration in a modified Boyden chamber assay in vitro, and this action could be abrogated by the TP inhibitors. The actions of the inhibitors were specific for TP, as they had no effect on the chemotactic actions of vascular endothelial growth factor (VEGF). HUVEC migration was also induced when TP-transfected human colon and breast carcinoma cells were co-cultured in the Boyden chamber assay in place of the purified angiogenic factors, and a TP inhibitor blocked the tumor cell-mediated migration almost completely. These studies suggest that inhibitors of TP may be useful in pathological conditions that are dependent upon TP-driven angiogenesis.

Physical proximity and functional association of glycoprotein 1balpha and protein-disulfide isomerase on the platelet plasma membrane.

Platelet function is influenced by the platelet thiol-disulfide balance. Platelet activation resulted in 440% increase in surface protein thiol groups. Two proteins that presented free thiol(s) on the activated platelet surface were protein-disulfide isomerase (PDI) and glycoprotein 1balpha (GP1balpha). PDI contains two active site dithiols/disulfides. The active sites of 26% of the PDI on resting platelets was in the dithiol form, compared with 81% in the dithiol form on activated platelets. Similarly, GP1balpha presented one or more free thiols on the activated platelet surface but not on resting platelets. Anti-PDI antibodies increased the dissociation constant for binding of vWF to platelets by approximately 50% and PDI and GP1balpha were sufficiently close on the platelet surface to allow fluorescence resonance energy transfer between chromophores attached to PDI and GP1balpha. Incubation of resting platelets with anti-PDI antibodies followed by activation with thrombin enhanced labeling and binding of monoclonal antibodies to the N-terminal region of GP1balpha on the activated platelet surface. These observations indicated that platelet activation triggered reduction of the active site disulfides of PDI and a conformational change in GP1balpha that resulted in exposure of a free thiol(s).

Exposure of the cryptic Arg-Gly-Asp sequence in thrombospondin-1 by protein disulfide isomerase.

Thrombospondin-1 is a matrix protein that inhibits proliferation, motility and sprouting of endothelial cells in vitro and angiogenesis in vivo. One mechanism by which thrombospondin-1 may influence endothelial cell biology is through interaction with the endothelial cell alphav beta3 integrin receptor. This interaction is mediated via a cryptic Arg-Gly-Asp sequence in the C-terminal Ca2+-binding region of thrombospondin-1. Exposure of the Arg-Gly-Asp sequence is controlled by disulfide interchange events in the Ca2+-binding loops and C-globular domain. Limited reduction of thrombospondin-1 by dithiothreitol exposes the Arg-Gly-Asp sequence which can bind to the alphav beta3 integrin receptor and support endothelial cell spreading (X. Sun, K. Skorstengaard, D.F. Mosher, J. Cell Biol. 118 (1992) 693-701). Our aim was to identify possible physiological reductants that can mediate Arg-Gly-Asp exposure. We now report that protein disulfide isomerase, which is known to catalyze disulfide interchange in thrombospondin-1 and change its enzyme inhibitory properties and its binding to monoclonal antibodies, was secreted by bovine aortic endothelial cells and deposited on the cell surface. There was an average of approximately 2.2 fg of protein disulfide isomerase on the surface of a bovine aortic endothelial cell. Treatment of thrombospondin-1 with purified protein disulfide isomerase enhanced adhesion of endothelial cells to thrombospondin-1 in an Arg-Gly-Asp-dependent manner through the alphav beta3 integrin receptor and supported cell spreading. Both Ca2+-depleted and Ca2+-replete thrombospondin-1 were substrates for protein disulfide isomerase. These results suggest that endothelial cell derived protein disulfide isomerase may regulate Arg-Gly-Asp-dependent binding of thrombospondin-1.

Interaction of platelet-derived growth factor with thrombospondin 1.

Key factors that mediate vascular smooth muscle cell proliferation and migration are platelet-derived growth factor (PDGF) and thrombospondin 1 (TSP1). We now report that PDGFBB bound tightly and specifically to TSP1, that this interaction was markedly dependent on the disulphide bond arrangement in TSP1, and that binding of PDGFBB to TSP1 did not preclude PDGFBB from binding to its receptor on rat aortic vascular smooth-muscle cells. At physiologic ionic strength and pH, PDGFBB bound to Ca2+-depleted TSP1 with a dissociation constant of 11 +/- 2 nM and to Ca2+-replete TSP1 with a dissociation constant of 32 +/- 5 nM. Binding was specific, as both soluble TSP1 and unlabelled PDGFBB competed for binding of iodinated PDGFBB to immobilized TSP1, whereas other platelet alpha-granule proteins did not compete. The tertiary structure of TSP1 is regulated by intramolecular disulphide interchange; we found that catalysis of disulphide interchange in TSP1 by protein disulphide isomerase ablated the binding of PDGFBB. The interaction of PDGFBB with TSP1 was weakened by increasing salt concentration and essentially ablated at 0.65 ionic strength; it was inhibited by heparin with a half-maximal effect at 20 i.u./ml, implying that the binding was mediated largely by ionic interactions. An anti TSP1 monoclonal antibody decreased the binding of iodinated PDGFBB to PDGF receptor on rat aortic vascular smooth-muscle cells by 37 +/- 2%, whereas platelet TSP1 non-competitively inhibited binding of iodinated PDGFBB. Uncomplexed PDGFBB bound to PDGF receptor with an affinity 5 +/- 2 times that of PDGFBB-TSP1 complexes. These results suggest that TSP1 might assist in the targeting of PDGF to its receptor on vascular smooth-muscle cells.

Catalysis of disulfide isomerization in thrombospondin 1 by protein disulfide isomerase.

Thrombospondin 1 is a multidomain glycoprotein from platelets and most cells that participates in diverse biological processes. The structure and some functional properties of thrombospondin 1 are regulated by disulfide interchange in the Ca(2+)-binding repeats and C-globular domain. The recent identification of the enzyme, protein disulfide isomerase, on the platelet surface suggested that protein disulfide isomerase may catalyze disulfide isomerization in platelet thrombospondin 1. Protein disulfide isomerase was found to form disulfide-linked complexes with thrombospondin 1, which is consistent with protein disulfide isomerase-mediated rearrangement of disulfide bonds in thrombospondin 1. To quantitate disulfide interchange in thrombospondin 1, perturbation of the enzyme inhibitory properties of platelet thrombospondin 1 were measured, specifically changes in the apparent dissociation constant for inhibition of neutrophil cathepsin G by thrombospondin 1. The inhibition constant increased > or = 10-14-fold following incubation of either Ca(2+)-replete or Ca(2+)-depleted thrombospondin 1 with protein disulfide isomerase and reduced glutathione. The rate of protein disulfide isomerase-catalyzed disulfide interchange in thrombospondin 1 increased linearly with protein disulfide isomerase concentration and the K(m) for reduced glutathione was 0.4 +/- 0.2 mM. Disulfide isomerization in both platelet and fibroblast thrombospondin 1 was probed by measuring perturbation in epitopes for two anti-thrombospondin 1 monoclonal antibodies. Antibody D4.6 binds to the C-terminal Ca(2+)-binding domains which are involved in disulfide interchange, whereas antibody HB8432 binds toward the N-terminus of the thrombospondin 1 subunit. In accordance with the location of these epitopes, incubation of platelet thrombospondin 1 or fibroblast thrombospondin 1 with protein disulfide isomerase and reduced glutathione resulted in 2-fold enhancement of binding of D4.6, whereas binding of HB8432 did not significantly change. In summary, protein disulfide isomerase catalyzes disulfide interchange in thrombospondin 1 which alters binding of neutrophil cathepsin G and antibody D4.6 to thrombospondin 1.

Inhibition of heparin activity in plasma by soluble fibrin: evidence for ternary thrombin-fibrin-heparin complex formation.

The ability of heparin to dramatically enhance the inactivation of thrombin (IIa) by antithrombin III (ATIII) in buffer is negated through formation of a IIa-fibrin-heparin ternary complex (Hogg and Jackson, Proc Natl Acad Sci USA 86:3619, 1989; Hogg and Jackson, J Biol Chem 265:241, 1990). IIa, in this ternary complex, is protected from inactivation by ATIII. Our aim was to determine whether fibrin also compromises heparin efficacy in plasma. We found that soluble fibrin ablated the heparin-mediated prolongation of the thrombin time with half-maximal effect at 60 nmol/L fibrin. The heparin-mediated prolongation of the activated partial thromboplastin time (APTT) was also reduced by fibrin with half-maximal effects at 140 nmol/L fibrin using 0.12 U/mL heparin and 500 nmol/L fibrin using 0.25 U/mL heparin. The mechanism of inhibition of heparin activity by fibrin in plasma was determined by measuring IIa-ATIII complexes by enzyme-linked immunosorbent assay (ELISA). Fibrin was found to inhibit the heparin-catalyzed inactivation of IIa by ATIII with half-maximal effect at 97 +/- 19 nmol/L fibrin. Fibrin had no effect on the heparin-catalyzed inactivation of factor Xa by ATIII in plasma, using either standard heparin, a heparinoid preparation (Orgaran; Organon, Lane Cove, Sydney, Australia), or low-molecular weight heparin. These findings imply that fibrin is a potent modulator of heparin activity in vivo by inhibiting heparin-catalyzed IIa-ATIII complex formation through formation of ternary IIa-fibrin-heparin complexes.