Probing the determinants of disulfide stability in native pancreatic trypsin inhibitor.

The effects of single amino acid replacements on the stability of the 14-38 disulfide bond in the native form of bovine pancreatic trypsin inhibitor (BPTI) were measured. A total of 17 mutant proteins, with substitutions at one of 7 residues located 5-15 A from the disulfide in the native wild-type protein, were examined. The replacements were found to decrease the thermodynamic stability of the disulfide, as measured by exchange with thiol-disulfide reagents, by 0.6-5 kcal/mol, corresponding to a range of nearly 100 mV in redox potentials. The effects of the substitutions on disulfide stability were roughly correlated with the changes in side-chain volume, suggesting that optimal packing is a major factor in determining the stability of the disulfide in the wild-type protein. With only one exception, the substitutions also led to increases, as large as 50-fold, in the rates of disulfide reduction by dithiothreitol. The increased rates of reduction suggest that at least a fraction of the mutational destabilization of the disulfide is due to strain in the native protein that is relieved in the transition state for reduction. The stability of the disulfide in a peptide corresponding to the segments that are linked together by the 14-38 disulfide in native BPTI was found to be about 5 kcal/mol less than that of the disulfide in the intact wild-type protein. Together, the results with the mutant proteins and the peptide indicate that the stability of the disulfide in the native protein depends on both the local environment of the disulfide and on the ability of the rest of the protein to favor a conformation that promotes disulfide formation.

Platelet glycoprotein IIb-IIIa-like proteins mediate endothelial cell attachment to adhesive proteins and the extracellular matrix.

The adherence of human umbilical vein endothelial (HUVE) cells to adhesive matrix proteins was examined to determine if cell attachment and spreading were mediated by the glycoprotein (GP) IIb-IIIa complex on endothelial cells. The HUVE cells adhered well to glass slides that had been coated with fibronectin, vitronectin, fibrinogen, or von Willebrand factor but failed to adhere to albumin-coated or to uncoated slides. The HUVE cell attachment and spreading on vitronectin, fibrinogen, and von Willebrand factor were greatly inhibited by a GP IIb-IIIa monoclonal antibody (7E3). In contrast, HUVE cell attachment to fibronectin was not inhibited by 7E3 but was inhibited by a fibronectin-receptor antibody (alpha GP140), which had no effect on cell attachment to the other adhesive proteins. The 7E3 antibody, but not alpha GP140, disrupted HUVE cell monolayers by detaching cells from their naturally occurring extracellular matrix. These data indicate that platelet GP IIb-IIIa-like proteins mediate the adherence of HUVE cells to specific adhesive proteins and to the extracellular matrix.

Platelet glycoproteins IIb and IIIa: evidence for a family of immunologically and structurally related glycoproteins in mammalian cells.

Human and bovine cultured cell lines and circulating leukocytes were examined for the presence of surface proteins similar to platelet glycoproteins IIb (GPIIb) and IIIa (GPIIIa). Human endothelial cells, smooth muscle cells, and MG-63 fibroblast-like cells were found to have surface proteins that cross-reacted with platelet GPIIb and GPIIIa antibodies, existed as complexes, and had molecular weights similar to those of the corresponding platelet glycoproteins. Bovine endothelial cells and smooth muscle cells also expressed GPIIb- and GPIIIa-like surface proteins. Metabolic labeling studies with [35S]methionine demonstrated that the cultured cells synthesized these glycoproteins. The GPIIIa-like protein in human endothelial and smooth muscle cells had the same isoelectric point as platelet GPIIIa, whereas their GPIIb alpha-like protein was slightly more acidic than platelet GPIIb alpha (pI = 5.2-5.3 versus 5.5). Platelet and endothelial cell GPIIb alpha (but not GPIIIa) showed an increased electrophoretic mobility in Ca2+ -containing versus EDTA-containing gels, implying a Ca2+ -GPIIb alpha interaction. The amino acid sequence of the amino termini of platelet GPIIb alpha and GPIIb beta and of the alpha chains of the leukocyte LFA-1 and Mac-1 glycoprotein complexes had significant sequence homology. These data indicate that glycoproteins that have either immunological cross-reactivity or amino-terminal sequence homology with the platelet GPIIb-IIIa complex are widely distributed in human and non-human adherent cells and circulating leukocytes and suggest that these proteins may be the products of a large gene family whose expression is cell specific.