scholarly journals Proteolytic dissection of the isolated platelet fibrinogen receptor, integrin GPIIb/IIIa. Localization of GPIIb and GPIIIa sequences putatively involved in the subunit interface and in intrasubunit and intrachain contacts

1992 ◽  
Vol 282 (2) ◽  
pp. 523-532 ◽  
Author(s):  
J J Calvete ◽  
K Mann ◽  
M V Alvarez ◽  
M M López ◽  
J González-Rodríguez
Keyword(s):  
Limited Proteolysis ◽  
Peptide Fragments ◽  
Fibrinogen Receptor ◽  
Subunit Interface ◽  
Covalent Structure ◽  
Quaternary Structures ◽  
Adhesive Proteins ◽  
Proteolytic Product ◽  
Sequence Stretch ◽  
Integrin Family

Human platelet glycoproteins IIb (GPIIb) and IIIa (GPIIIa) form the subunits of the Ca(2+)-dependent heterodimer GPIIb/IIIa, which belongs to the integrin family of phylogenetically related receptors mediating a wide variety of cell-cell and cell-substratum interactions. GPIIb/IIIa plays a central role in haemostasis as a receptor for fibrinogen and other adhesive proteins at the surface of activated platelets. The covalent structure of the subunits is largely known; however, the tertiary and quaternary structures of the heterodimer remain to be determined. To this end, our approach consisted of limited proteolysis of the isolated heterodimer with proteinases of different specificities, followed by protein-chemical and immunochemical analyses of the peptide fragments within each isolated proteolytic product. From the information obtained, we have drawn a rudimentary map which outlines the demarcation of compact domains and the subunit peptide stretches carrying the sequences putatively involved in intrachain, intrasubunit and intersubunit non-covalent connectivity in the heterodimer. Three compact domains have been well defined: one in the heavy (H) chain of GPIIb [GPIIbH-(600-700)], and two in GPIIIa, the N-terminal [GPIIIa-(1-52)] and the core [GPIIIa-(423-622)] domains. Between the latter two domains there is a proteolysis-susceptible region, which is partly involved in ligand binding [GPIIIa-(100-220)] and partly implicated as being in teh subunit interface of the heterodimer. Contrary to GPIIIa, GPIIbH is highly susceptible to proteolysis all along its sequence. Equally susceptible are the extracellular end of the transmembrane segment of both GPIIIa and the light (L) chain of GPIIb (GPIIbL), and the N-terminal end of GPIIbL. Three sequence stretches along the C-terminal half of GPIIbH, one sequence stretch in GPIIbL and three sequence stretches within the GPIIIa-(217-421) region were putatively involved in the subunit interface of the heterodimer. Most likely, the N-terminal end of GPIIbL is folded over the N- and C-terminal regions of GPIIbH, and the N-terminal end of GPIIbH is folded against the GPIIbH-(600-700) domain. This map of GPIIb/IIIa does not fit the current accommodation of the amino acid sequence of GPIIb and GPIIIa in the head/two-tails image of the heterodimer obtained by metal-rotary-shadowing electron microscopy.

scholarly journals Proteolytic degradation of the RGD-binding and non-RGD-binding conformers of human platelet integrin glycoprotein IIb/IIIa: clues for identification of regions involved in the receptor's activation

1994 ◽  
Vol 298 (1) ◽  
pp. 1-7 ◽  
Author(s):  
J J Calvete ◽  
K Mann ◽  
W Schäfer ◽  
R Fernandez-Lafuente ◽  
J M Guisán
Keyword(s):  
Conformational Changes ◽  
Quaternary Structure ◽  
Conformational Transition ◽  
Limited Proteolysis ◽  
Membrane Expression ◽  
Fibrinogen Receptor ◽  
Subunit Interface ◽  
Plasma Membrane Expression ◽  
Fragmentation Patterns ◽  
Adhesive Proteins

The human integrin glycoprotein (GP)IIb/IIIa plays a central role in haemostasis as an inducible receptor for fibrinogen and other RGD-containing adhesive proteins at the platelet plasma membrane. Expression of the fibrinogen receptor on platelet activation involves conformational changes in the quaternary structure of GPIIb/IIIa. Little is known, however, about the nature of this conformational transition. Given that isolated GPIIb/IIIa contains a mixture of RGD-binding and non-RGD-binding heterodimers, we used limited proteolysis as a tool for investigating the structural differences between the two conformers. Comparison of their fragmentation patterns shows that, whereas in the non-RGD-binding form of GPIIb/IIIa the N-terminal half of the heavy chain of GPIIb (GPIIbH) and the central region of GPIIIa are cleaved by endoproteinase Arg-C, these domains associate tightly with one another in the RGD-binding GPIIb/IIIa and are thus protected from proteolysis. In addition, the C-terminal half of GPIIb becomes more susceptible to degradation in the non-RGD-binding GPIIb/IIIa conformer. Our interpretation, in the context of available structural and functional data, is that a major relative reorientation of the GPIIbH and GPIIIa extracellular domains takes place along the subunit interface during the conformational transition of the platelet integrin.

Exposure of Human Platelets to Plasmin Results in the Expression of Irreversibly Active Fibrinogen Receptors

1995 ◽  
Vol 73 (04) ◽  
pp. 693-701 ◽  
Author(s):  
Samia Rabhi-Sabile ◽  
Dominique Pidard
Keyword(s):  
Shape Change ◽  
Limited Proteolysis ◽  
Metabolic Activation ◽  
Human Platelets ◽  
Plasmin Activity ◽  
Low Velocity ◽  
Fibrinogen Receptor ◽  
Fibrinogen Binding ◽  
Adhesive Proteins

SummaryAlthough plasmin can trigger strong platelet responses such as shape change and exocytosis of internal granules, limited platelet aggregation is induced by this proteinase, owing to its capacity to rapidly proteolyse secreted adhesive proteins. In this context, we have investigated the state of activation of the fibrinogen receptor, the integrin αIIbβ3, on platelets exposed to plasmin. Following incubation with plasmin at 37 °C, washing, and resuspension, platelets exhibit a moderate, low-velocity aggregation when stirred in the presence of fibrinogen. Optimum aggregability is observed when platelets have been exposed to plasmin activity of ≈0.5 CU/ml for 20 min, and aggregation is insensitive to the presence of antagonists such as prostaglandin (PG) E1 and apyrase. Plasmin-induced platelet aggregability is associated with the expression of active fibrinogen receptors on the cell surface, which, using a l25I-fibrinogen binding assay, can be quantified to ≈2,300 molecules per platelet. Exposure of active αIIbβ3 receptors appears to depend partially, but not totally on a metabolic activation and granule exocytosis at the time of incubation with plasmin. In contrast with a-thrombin, plasmin-induced activation of αIIbβ3 is sustained and cannot be reversed by exposure of platelets to PGE1. Immunoblotting analysis of the receptor subunits shows no extensive proteolytic modification of αIIbβ3 by plasmin, and only reveals a limited proteolysis of the aminoterminal domain of the αIIb subunit. In addition to their capacity to aggregate in the presence of fibrinogen alone, plasmin-treated platelets also show a potentiated aggregability in response to low doses of ADP. Thus, plasmin has the potential to activate the platelet fibrinogen receptor in such a way that it remains irreversibly available to fibrinogen on the surface of nonaggregated cells, a feature that may participate to pathological states of in vivo platelet hyperaggregability.

STRUCTURAL DIVERSITY AMONG CELLULAR ADHESION RECEPTORS: FIBRINOGEN BINDING IS A NOVEL BIOLOGICAL PROPERTY OF THE MONOCYTE DIFFERENTIATION ANTIGEN OKM1

1987 ◽  
Author(s):  
Dario C Altieri ◽  
Rossella Bader ◽  
Pier M Mannucci
Keyword(s):  
Molecular Size ◽  
Structural Diversity ◽  
Cellular Adhesion ◽  
Differentiation Antigen ◽  
Cell Surface Antigens ◽  
Monocyte Differentiation ◽  
Fibrinogen Receptor ◽  
Biological Adhesion ◽  
Fibrinogen Binding ◽  
Adhesive Proteins

A family of related glycoproteins (GP) mediate the interaction between the circulating adhesive proteins and a variety of cells (cytyoadhesins). In this study we have compared two cell-surface antigens which share the property to bind fibrinogen: the platelet GP IIb/IIIa, prototype of the cytoadhesins, and the receptor for fibrinogen costitutively synthesized by monocytes. Two anti-GP IIb/IIIa monoclonal antibodies (Mabs) (LJP9, LJP5), recognizing functionally distinct epitopes of the GP IIb/IIIa did not react with monocytes nor inhibited 125I-fibrinogen binding to monocytes. Similarly, an Arg-Gly-Asp containing peptide which completely abolished platelet-fibrinogen interaction, had no effect on monocytes. Structurally, the monocyte fibrinogen receptor was dimeric and composed of two subunits with molecular weight (Mr) of 155,000 and 95,000. This structural organization was different from that of the GP IIb/IIIa (Mr= 116,000), but in close analogy with the family of leukocyte differentiation antigens OKM1, LFA-1. Therefore, this possible relationship was investigated. A Mab to OKM1 antigen (10 μg/ml) completely suppressed fibrinogen binding to monocytes while it was ineffective on plateles. Iodinated monocyte lysate subjected to immunoprecipitation with OKM1 Mab (60 μg/ml) showed a dimeric antigen with the same molecular size of the monocyte fibrinogen receptor. Moreover, preclearing of the monocyte lysate with OKM1 Mab removed the immunoprecipitate corresponding to the monocyte fibrinogen receptor. These data indicate that the immunologic differentiation antigen OKM1, in addition to function as a complement receptor, displays also the novel biological adhesion property to mediate the binding of fibrinogen to monocytes.

A mutation in the integrin αIIb subunit that selectively inhibits αIIbβ3 receptor function

2003 ◽  
Vol 90 (11) ◽  
pp. 853-862
Author(s):  
Kim Perkins ◽  
Joseph Loftus
Keyword(s):  
Ligand Binding ◽  
Single Amino Acid ◽  
Β Subunit ◽  
Receptor Function ◽  
Central Cavity ◽  
Fibrinogen Receptor ◽  
Binding Function ◽  
Subunit Interface ◽  
Active Variant

SummaryThe αIIband αvintegrins have been shown to play a significant role in a variety of disease processes. αIIbβ3is a platelet-specific fibrinogen receptor that is critical for thrombosis and hemo-stasis. Determination of the basis of ligand recognition by αIIbβ3is essential for modulation of platelet function. To identify αIIbresidues involved in αIIbβ3ligand binding function, cells expressing a constitutively active variant of αIIbβ3were randomly mutagenized and selected for loss of αIIbβ3ligand binding function. One mutant isolated in this manner contained a single amino acid substitution at position 96 in αIIb(Ser96→Leu). Cells expressing this αIIbmutant did not bind the ligand mimetic antibody PAC1 or adhere to fibrinogen. In addition, the mutant receptor did not bind to an RGD affinity matrix. Substitution of conserved serine residues at position 1 in β strand A of all seven repeats of αIIbsimilarly inhibited ligand binding to αIIbβ3. αIIbS96 maps to the central cavity of the β–propeller fold of the αIIbsubunit immediately adjacent to a structurally important sequence at the center of the α and β subunit interface. In contrast, substitution of the analogous residues in αvor α4did not disrupt the ligand binding function of αvβ3or α4β1. These data support a potential unique structural or mechanistic role for this residue in αIIbβ3receptor function.

SR 121787, a New Orally Active Fibrinogen Receptor Antagonist

1998 ◽  
Vol 80 (09) ◽  
pp. 469-476 ◽  
Author(s):  
Pierre Savi ◽  
Alain Badorc ◽  
Alain Lalé ◽  
Marie-Françoise Bordes ◽  
Josette Bornia ◽  
...  
Keyword(s):  
Human Platelets ◽  
Thrombosis Model ◽  
Fibrinogen Receptor ◽  
Shunt Thrombosis ◽  
Venous Shunt ◽  
Adhesive Proteins ◽  
Induced Aggregation ◽  
Orally Active

SummaryThe aim of this study was to describe the pharmacological properties of SR 121787, a new antiaggregating drug which is metabolized in vivo into SR 121566, a potent non-peptide antagonist of Gp IIb/IIIa. In vitro, SR 121566 antagonized the binding of [125I]-fibrinogen (IC50 = 19.8 ± 6.3 nM) and of [125I]-L-692,884, an RGD-containing peptide (IC50 = 291 ± 96 nM) to activated human platelets. SR 121566 inhibited the aggregation of human platelets induced by ADP, collagen, thrombin, arachidonic acid and PAF at concentrations lower than 0.1 μM. Adhesion of human platelets to adhesive proteins was inhibited by SR 121566 (IC50 = 40.3 ± 2.5 nM) only when Gp IIb/IIIa and fibrinogen were involved. No effect was found with regard to other adhesive proteins and/or other integrins. SR 121787 demonstrated a potent and sustained antiaggregating effect when administered intravenously to baboons at a dose 50 μg/kg, and eight hours after the administration of 100 μg/kg, ADP-induced aggregation was still strongly inhibited (more than 80%). A single oral administration of 2 mg/kg of SR 121787 produced a nearly complete inhibition of platelet aggregation for up to 8 h (ED50 at 8 h = 193 ± 20 μg/kg), a significant residual antiaggregating activity being still observed 24h after the administration. When administered orally to rabbits, SR 121787 exhibited a potent antiaggregating (ED50 = 2.3 ± 0.3 mg/kg) and antithrombotic activity in an arterio-venous shunt thrombosis model (ED50 = 10.4 ± 0.8 mg/kg). After oral and IV administration, SR 121787 was well tolerated suggesting that SR 121787, the most potent and long lasting orally active Gp IIb/IIIa antagonist described to date, is a promising antithrombotic compound.

scholarly journals PROTEOLYTIC MICRODISSECTION OF SMOOTH-SURFACED VESICLES OF LIVER MICROSOMES

1969 ◽  
Vol 40 (1) ◽  
pp. 179-189 ◽  
Author(s):  
Akio Ito ◽  
Ryo Sato
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Cytochrome B5 ◽  
Limited Proteolysis ◽  
Liver Microsomes ◽  
Sucrose Density Gradient ◽  
Peptide Fragments ◽  
Sucrose Density Gradient Centrifugation ◽  
Bacillus Subtilis Protease

Digestion of rabbit liver microsomal smooth vesicles with Bacillus subtilis protease released proteins and peptide fragments from the vesicles, without solubilizing phospholipids and cholesterol. The proteolysis was, however, limited when about 30% of the protein had been solubilized. The same limitation was observed when the vesicles were treated with trypsin, chymotrypsin, or their combinations with the bacterial protease. The limited proteolysis was accompanied by selective solubilization of cytochrome b5 and microsomal NADPH-specific flavoprotein, leaving the CO-binding hemoprotein and some other enzymes still attached to the vesicular membranes. Sucrose density gradient centrifugation of protease-treated vesicles indicated that all the vesicles had been attacked by the protease to similar extents. The behavior of intact and digested vesicles in dextran density gradient centrifugation suggested that the vesicles, even after proteolytic digestion, existed in the form of closed sacs which were impermeable to macromolecules such as dextran and proteases. It was concluded that only the outside surface of the vesicles is susceptible to the proteolytic action and that cytochrome b5 and the NADPH-specific flavoprotein are located in the susceptible area.

NMR studies of the R2 repeat and related peptide fragments of the DNA binding domain of c-Myb. New light on the structure and folding of R2.

1999 ◽  
Vol 96 (9/10) ◽  
pp. 1580-1584 ◽  
Author(s):  
I. Ségalas ◽  
S. Desjardins ◽  
H. Oulyadi ◽  
Y. Prigent ◽  
S. Tribouillard ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Peptide Fragments ◽  
Nmr Studies ◽  
Related Peptide

Conformational Changes in Adhesive Proteins Modulate Their Adhesive Function

1995 ◽  
Vol 74 (01) ◽  
pp. 253-257 ◽  
Author(s):  
Tatiana Ugarova ◽  
Francisca R Agbanyo ◽  
Edward F Plow
Keyword(s):  
Conformational Changes ◽  
Adhesive Proteins

Limited Proteolysis of Vitronectin by Plasmin Destroys Heparin Binding Activity

1991 ◽  
Vol 66 (03) ◽  
pp. 310-314 ◽  
Author(s):  
David C Sane ◽  
Tammy L Moser ◽  
Charles S Greenberg
Keyword(s):  
Limited Proteolysis ◽  
Plasminogen Activator Inhibitor ◽  
Binding Activity ◽  
Binding Domain ◽  
Heparin Binding ◽  
Inhibitor Type ◽  
Activator Inhibitor Type ◽  
Activator Inhibitor ◽  
Pai 1

SummaryVitronectin (VN) stabilizes plasminogen activator inhibitor type 1 (PAI-1) activity and prevents the fibrin(ogen)-induced acceleration of plasminogen activation by t-PA. These antifibrinolytic activities as well as other functions are mediated by the glycosaminoglycan (GAG) binding domain of VN. Since the GAG binding region is rich in arginyl and lysyl residues, it is a potential target for enzymes such as plasmin. In this paper, the dose and time-dependent proteolysis of VN by plasmin is demonstrated. The addition of urokinase or streptokinase (200 units/ml) to plasma also produced proteolysis of VN. With minimal proteolysis, the 75 kDa band was degraded to a 62-65 kDa form of VN. This minimal proteolysis destroyed the binding of [3H]-heparin to VN and reversed the neutralization of heparin by VN.Thus, the plasmin-mediated proteolysis of the GAG binding activity of VN could destroy the antifibrinolytic activity of VN during physiologic conditions and during thrombolytic therapy. Furthermore, other functions of VN in complement and coagulation systems that are mediated by the GAG binding domain may be destroyed by plasmin proteolysis.

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