Platelet receptor recognition domains on the .alpha. chain of human fibrinogen: structure-function analysis

Biochemistry ◽  
1989 ◽  
Vol 28 (7) ◽  
pp. 2909-2914 ◽  
Author(s):  
Jacek Hawiger ◽  
Marek Kloczewiak ◽  
Maria A. Bednarek ◽  
Sheila Timmons
Keyword(s):  
Structure Function ◽  
Function Analysis ◽  
Human Fibrinogen ◽  
Alpha Chain ◽  
Platelet Receptor ◽  
Receptor Recognition

Platelet receptor recognition domain on the .gamma. chain of human fibrinogen and its synthetic peptide analogues

Biochemistry ◽  
1989 ◽  
Vol 28 (7) ◽  
pp. 2915-2919 ◽  
Author(s):  
Marek Kloczewiak ◽  
Sheila Timmons ◽  
Maria A. Bednarek ◽  
Masato Sakon ◽  
Jacek Hawiger
Keyword(s):  
Synthetic Peptide ◽  
Gamma Chain ◽  
Human Fibrinogen ◽  
Platelet Receptor ◽  
Receptor Recognition ◽  
Peptide Analogues

scholarly journals Expression of a fibrinogen fusion peptide in Escherichia coli: a model thrombin substrate for structure/function analysis

Blood ◽  
1989 ◽  
Vol 73 (1) ◽  
pp. 166-171
Author(s):  
ST Lord ◽  
DM Fowlkes
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Function Analysis ◽  
Fibrin Clot ◽  
Human Fibrinogen ◽  
Alpha Chain ◽  
Cell Lysates ◽  
Amino Terminal ◽  
Thrombin Cleavage ◽  
Initial Event

The initial event in fibrin clot formation is the thrombin-catalyzed cleavage of the A alpha chain of human fibrinogen. Most of the information required for thrombin recognition and cleavage of the A alpha chain lies in the amino terminal 51 residue CNBr fragment. By selective modification of residues in this region, we probed the features that participate in thrombin interactions. We constructed a vector which expressed a tripartite protein (tribrid) consisting of amino acids 1 to 50 of the A alpha chain followed by 60 amino acids of chicken collagen and the beta-galactosidase protein from Escherichia coli. Cell lysates run on NaDodSO4-polyacrylamide gels contained the predicted band of molecular weight (mol wt) 125,000. The tribrid reacted with a monoclonal antibody, Mab-Y18, which recognizes the amino terminus of the A alpha chain. When cell lysates were incubated with thrombin, FPA was released. By including one heterogeneous oligonucleotide in the construction, we generated plasmids that encoded three specific amino acid substitutions. Surprisingly, changing Gly14 to Val did not alter thrombin cleavage, although recognition by Mab-Y18 was lost. Substitution of lie for Arg23 did not alter either thrombin cleavage or monoclonal recognition. Substitution of Leu for Arg 16 altered thrombin cleavage; unexpectedly, recognition by Mab-Y18 was not changed.

Platelet receptor recognition domain on the .gamma. chain of human fibrinogen and its synthetic peptide analogs [Erratum to document cited in CA110(17):151810E]

Biochemistry ◽  
1989 ◽  
Vol 28 (19) ◽  
pp. 7974-7974
Author(s):  
Marek Kloczewiak ◽  
Sheila Timmons ◽  
Maria A. Bednarek ◽  
Masato Sakon ◽  
Jacek Hawiger
Keyword(s):  
Synthetic Peptide ◽  
Gamma Chain ◽  
Human Fibrinogen ◽  
Peptide Analogs ◽  
Platelet Receptor ◽  
Receptor Recognition

scholarly journals Expression of a fibrinogen fusion peptide in Escherichia coli: a model thrombin substrate for structure/function analysis

Blood ◽  
1989 ◽  
Vol 73 (1) ◽  
pp. 166-171 ◽  
Author(s):  
ST Lord ◽  
DM Fowlkes
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Function Analysis ◽  
Fibrin Clot ◽  
Human Fibrinogen ◽  
Alpha Chain ◽  
Cell Lysates ◽  
Amino Terminal ◽  
Thrombin Cleavage ◽  
Initial Event

Abstract The initial event in fibrin clot formation is the thrombin-catalyzed cleavage of the A alpha chain of human fibrinogen. Most of the information required for thrombin recognition and cleavage of the A alpha chain lies in the amino terminal 51 residue CNBr fragment. By selective modification of residues in this region, we probed the features that participate in thrombin interactions. We constructed a vector which expressed a tripartite protein (tribrid) consisting of amino acids 1 to 50 of the A alpha chain followed by 60 amino acids of chicken collagen and the beta-galactosidase protein from Escherichia coli. Cell lysates run on NaDodSO4-polyacrylamide gels contained the predicted band of molecular weight (mol wt) 125,000. The tribrid reacted with a monoclonal antibody, Mab-Y18, which recognizes the amino terminus of the A alpha chain. When cell lysates were incubated with thrombin, FPA was released. By including one heterogeneous oligonucleotide in the construction, we generated plasmids that encoded three specific amino acid substitutions. Surprisingly, changing Gly14 to Val did not alter thrombin cleavage, although recognition by Mab-Y18 was lost. Substitution of lie for Arg23 did not alter either thrombin cleavage or monoclonal recognition. Substitution of Leu for Arg 16 altered thrombin cleavage; unexpectedly, recognition by Mab-Y18 was not changed.

Clues for Understanding the Structure and Function of a Prototypic Human Integrin: The Platelet Glycoprotein IIb/IIIa Complex

1994 ◽  
Vol 72 (01) ◽  
pp. 001-015 ◽  
Author(s):  
Juan J Calvete
Keyword(s):  
Structure Function ◽  
Thrombus Formation ◽  
Platelet Glycoprotein ◽  
Primary Role ◽  
Clot Retraction ◽  
Inhibitory Peptide ◽  
Rgd Sequence ◽  
Platelet Receptor ◽  
Adhesive Proteins ◽  
And Function

SummaryThe glycoprotein (GP) IIb/IIIa, a Ca2+-dependent heterodimer, is the major integrin on the platelet plasma membrane. On resting platelets GPIIb/IIIa is maintained in an inactive conformation and serves as a low affinity adhesion receptor for surface-coated fibrinogen, whereas upon platelet activation signals within the cytoplasma alter the receptor function of GPIIb/IIIa (inside-out signalling), which undergoes a measurable conformational change within its exoplasmic domains, and becomes a competent receptor for soluble fibrinogen and some other RGD sequence-containing plasma adhesive proteins. Upon ligand binding, further structural alterations trigger the association of receptor-occupied GPIIb/IIIa complexes with themselves within the plane of the membrane. The simultaneous binding of dimeric fibrinogen molecules to GPIIb/IIIa clusters on adjacent platelets leads to platelet aggregation, which promotes attachment of fibrinogen-GPIIb/IIIa clusters to the cytoskeleton (outside-in signalling). This, in turn, provides the necessary physical link for clot retraction to occur, and generates a cascade of intracellular biochemical reactions which result in the formation of a multiprotein signalling complex at the cytoplasmic domains of GPIIb/IIIa. Glycoprotein IMIIa, also called αIIbβ3 in the integrin nomenclature, plays thus a primary role in both platelet adhesion and thrombus formation at the site of vascular injury. In addition, the human glycoprotein Ilb/IIIa complex is the most thoroughly studied integrin receptor, its molecular biology and major features of its primary structure having been elucidated mainly during the last six years. Furthermore, localization of functionally relevant monoclonal antibody epitopes, determination of the cross-linking sites of inhibitory peptide ligands, proteolytic dissection of the isolated integrin, and analysis of natural and artificial GPIIb/IIIa mutants have recently provided a wealth of information regarding structure-function relationships of human GPIIb/IIIa. The aim of this review is to summarize these many structural and functional data in the perspective of an emerging model. Although most of the interpretations based on structural elements of this initial biochemical model require independent confirmation, they may help us to understand the structure-function relationship of this major platelet receptor, and of other members of the integrin superfamily, as well as to perform further investigations in order to test current hypotheses.

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