Glycoprotein (GP) Ib is the functionally dominant subunit of the platelet GPIb-IX-V receptor com... more Glycoprotein (GP) Ib is the functionally dominant subunit of the platelet GPIb-IX-V receptor complex, with the von Willebrand factor (vWF) binding site residing on the amino-terminus. A threonine for methionine-145 replacement of GPIb is associated with the human platelet antigen (HPA)-2 system. To study the structural and functional consequences of this mutation, both forms of GPIb were expressed as calmodulin fusion proteins in insect cells. Both recombinant proteins were recognized by their respective alloantibodies, independent of glycosylation or intactness of disulfide bonds, and gave similar results to platelet-derived GPIb in antibody detection assays. Resonant mirror studies showed that vWF binding was not affected by the HPA-2 mutation; however, vWF binding was partially inhibited by IgG HPA-2 antibodies. Our data are compatible with an involvement of the leucine-rich repeat domain of GPIb in vWF binding and indicate that recombinant GPIb may be used to detect HPA-2 ...
Serpin polymerization is the underlying cause of several diseases, including thromboembolism, emp... more Serpin polymerization is the underlying cause of several diseases, including thromboembolism, emphysema, liver cirrhosis, and angioedema. Understanding the structure of the polymers and the mechanism of polymerization is necessary to support rational design of therapeutic agents. Here we show that polymerization of antithrombin is sensitive to the addition of synthetic peptides that interact with the structure. A 12‐mer peptide (homologous to P14‐P3 of antithrombin reactive loop), representing the entire length of s4A, prevented polymerization totally. A 6‐mer peptide (homologous to P14‐P9 of antithrombin) not only allowed polymerization to occur, but induced it. This effect could be blocked by the addition of a 5‐mer peptide with the s1C sequence of antithrombin or by an unrelated peptide representing residues 26–31 of cholecystokinin. The s1C or cholecystokinin peptide alone was unable to form a complex with native antithrombin. Moreover, an active antitrypsin double mutant, Pro 3...
Antithrombin, the main inhibitor of thrombosis in blood, is bound and activated by the heparin-li... more Antithrombin, the main inhibitor of thrombosis in blood, is bound and activated by the heparin-like side-chains that line the small vasculature. We now have good depictions of the heparin-binding site on antithrombin, and of the way in which mutations at this site cause thrombotic disease. The interaction of heparin with antithrombin is, however, a kinetic one, with binding being followed by formation of a complex with thrombin and then release from the heparin. Our understanding of the processes involved is currently based on crystallographic models but, for a mobile mechanism, these merely provide snapshots - what is needed is a movie.
Biochemical and Biophysical Research Communications, 2002
The high resolution structure of hemalbumin was determined by single crystal X-ray diffraction to... more The high resolution structure of hemalbumin was determined by single crystal X-ray diffraction to a resolution of 1.9 A. The structure revealed the protoporphyrin IX bound to a single site within a hydrophobic cavity in subdomain IB, one of the principal binding sites for long chain fatty acid. The iron is penta coordinated with the fifth ligand comprised of the hydroxyl oxygen of Tyr-161 (phenolic oxygen to heme plane distance: 2.73 A) in an otherwise completely hydrophobic pocket. The heme propionic acid residues form salt bridges with His-142 and Lys-190, which together with a series of hydrophobic interactions, enclose and secure the heme within the IB helical motif. A detailed discussion of the structure together with its implications for the development of potential blood substitutes is presented.
Glycoprotein (GP) Ib is the functionally dominant subunit of the platelet GPIb-IX-V receptor com... more Glycoprotein (GP) Ib is the functionally dominant subunit of the platelet GPIb-IX-V receptor complex, with the von Willebrand factor (vWF) binding site residing on the amino-terminus. A threonine for methionine-145 replacement of GPIb is associated with the human platelet antigen (HPA)-2 system. To study the structural and functional consequences of this mutation, both forms of GPIb were expressed as calmodulin fusion proteins in insect cells. Both recombinant proteins were recognized by their respective alloantibodies, independent of glycosylation or intactness of disulfide bonds, and gave similar results to platelet-derived GPIb in antibody detection assays. Resonant mirror studies showed that vWF binding was not affected by the HPA-2 mutation; however, vWF binding was partially inhibited by IgG HPA-2 antibodies. Our data are compatible with an involvement of the leucine-rich repeat domain of GPIb in vWF binding and indicate that recombinant GPIb may be used to detect HPA-2 ...
Serpin polymerization is the underlying cause of several diseases, including thromboembolism, emp... more Serpin polymerization is the underlying cause of several diseases, including thromboembolism, emphysema, liver cirrhosis, and angioedema. Understanding the structure of the polymers and the mechanism of polymerization is necessary to support rational design of therapeutic agents. Here we show that polymerization of antithrombin is sensitive to the addition of synthetic peptides that interact with the structure. A 12‐mer peptide (homologous to P14‐P3 of antithrombin reactive loop), representing the entire length of s4A, prevented polymerization totally. A 6‐mer peptide (homologous to P14‐P9 of antithrombin) not only allowed polymerization to occur, but induced it. This effect could be blocked by the addition of a 5‐mer peptide with the s1C sequence of antithrombin or by an unrelated peptide representing residues 26–31 of cholecystokinin. The s1C or cholecystokinin peptide alone was unable to form a complex with native antithrombin. Moreover, an active antitrypsin double mutant, Pro 3...
Antithrombin, the main inhibitor of thrombosis in blood, is bound and activated by the heparin-li... more Antithrombin, the main inhibitor of thrombosis in blood, is bound and activated by the heparin-like side-chains that line the small vasculature. We now have good depictions of the heparin-binding site on antithrombin, and of the way in which mutations at this site cause thrombotic disease. The interaction of heparin with antithrombin is, however, a kinetic one, with binding being followed by formation of a complex with thrombin and then release from the heparin. Our understanding of the processes involved is currently based on crystallographic models but, for a mobile mechanism, these merely provide snapshots - what is needed is a movie.
Biochemical and Biophysical Research Communications, 2002
The high resolution structure of hemalbumin was determined by single crystal X-ray diffraction to... more The high resolution structure of hemalbumin was determined by single crystal X-ray diffraction to a resolution of 1.9 A. The structure revealed the protoporphyrin IX bound to a single site within a hydrophobic cavity in subdomain IB, one of the principal binding sites for long chain fatty acid. The iron is penta coordinated with the fifth ligand comprised of the hydroxyl oxygen of Tyr-161 (phenolic oxygen to heme plane distance: 2.73 A) in an otherwise completely hydrophobic pocket. The heme propionic acid residues form salt bridges with His-142 and Lys-190, which together with a series of hydrophobic interactions, enclose and secure the heme within the IB helical motif. A detailed discussion of the structure together with its implications for the development of potential blood substitutes is presented.
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