Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Platelet membrane glycoprotein (GP)IIb-IIIa exists as a divalent cation-dependent heterodimer which recognizes the Arg-Gly-Asp (RGD) sequence of adhesive proteins. To isolate the RGD binding domain of GPIIb-IIIa we performed proteolysis of GPIIb-IIIa with alpha-chymotrypsin. GPIIb-IIIa was bound to an affinity matrix of GRGDSPK-coupled Sepharose 4B and was then treated with chymotrypsin. After washing the unbound fragments, two discrete polypeptides of 55 and 85 kDa remained bound to the RGD affinity matrix and were specifically eluted by soluble HHLGGAKQAGDV (H12) or by GRGDSP, but not by GRGESP. Immunoblotting with subunit-specific polyclonal antibodies showed that the 55- and 85-kDa fragments were derived from GPIIb and GPIIIa, respectively. Amino-terminal sequencing and immunoblotting using site-specific antibodies indicated that these fragments contained the amino termini of their parent molecules. In the presence of 1 mM Ca2+ and 1 mM Mg2+, these two fragments were maintained as a heterodimer inasmuch as both fragments were immunoprecipitated by the polyclonal anti-GPIIIa antibodies. In contrast, chelating the divalent cations with 5 mM EDTA resulted in the lack of co-immunoprecipitation of the 55-kDa GPIIb fragment. After removal of the H12 peptide, the 55/85-kDa heterodimer bound to immobilized fibrinogen in an enzyme-linked immunosorbent assay by an RGD-dependent mechanism. These findings suggest that the RGD binding domain and structures required for heterodimer maintenance are present within the 55/85-kDa chymotryptic fragment of GPIIb-IIIa.
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PMID:Isolation and characterization of a chymotryptic fragment of platelet glycoprotein IIb-IIIa retaining Arg-Gly-Asp binding activity. 154 38

Recent studies have shown that antibodies characteristic of quinine- and quinidine-induced thrombocytopenia sometimes recognize the platelet membrane glycoprotein (GP) complex IIb/IIIa in addition to their well known target, GPIb/IX. We have investigated the frequency with which drug-induced antibodies bind to GPIIb/IIIa and the nature of their target epitopes. In studies of sera from 13 patients sensitive to quinidine or quinine, we found that 10 contained IgG antibodies specific for both GPIb/IX and GPIIb/IIIa, two reacted with GPIb/IX alone, and one reacted with GPIIb/IIIa alone. In all cases, the presence of drug was required for binding of IgG to target GPs. By immunoabsorption, we found that each of five polyspecific sera contained at least two different antibodies, one reactive with GPb/IX and the other with GPIIb/IIIa. Further studies with eight drug-dependent antibodies (DDAb) specific for GPIIb/IIIa showed that three recognized the GPIIb/IIIa complex only, one recognized GPIIb alone, and three recognized GPIIIa alone. The eighth serum appeared to bind to both GPIIIa alone and to an epitope determined by the GPIIb/IIIa complex. The three antibodies specific for GPIIIa alone also reacted with GPIIIa deglycosylated with endo-H, and with the major (61 Kd) fragment obtained by chymotryptic digestion of GPIIIa but failed to react with reduced GPIIIa. These findings demonstrate that, in drug-induced, immunologic thrombocytopenia, the anti-platelet immune response is typically directed against epitopes on both GPIb/IX and GPIIb/IIIa. The three DDAb we studied that were specific for GPIIIa alone recognize epitopes resistant to chymotrypsin and endo-H treatment that are dependent on intrachain disulfide bonding.
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PMID:Characteristics of quinine- and quinidine-induced antibodies specific for platelet glycoproteins IIb and IIIa. 171 May 17

Glycoprotein (GP) IIb-IIIa serves as the platelet fibrinogen receptor. Studies of the tertiary structure of GPIIIa have shown that the protein has a large loop structure of at least 325 amino acids in length. To further characterize this loop structure, intact platelets were digested with alpha-chymotrypsin. Digestion products were examined using the anti-GPIIIa monoclonal antibodies (MoAbs) AP3, D3GP3, and C5GP3, as well as the human alloantibody, anti-PLA1. AP3 recognized GPIIIa digestion products of 109, 95, and 68 Kd. D3GP3 and C5GP3 recognized an additional band of 51 Kd. Time course digestions demonstrated that the 51-Kd fragment was generated by proteolysis of the 68-Kd peptide. Sequence analysis of the reduced 51-Kd peptide showed that this fragment began at amino acid 422. The nonreduced 51-Kd peptide was reactive with antibodies directed against the first 13 amino acids of GPIIIa, demonstrating the presence of a covalently attached N-terminal peptide. These data suggest that: (1) the minimum length of the loop structure is at least 384 amino acids; (2) the AP3 epitope is formed at least in part by a determinant contained within residues 348 to 421; and (3) the D3GP3 and C5GP3 epitopes are contained within amino acids 422 to 692 of GPIIIa, a region that may be flexible and involved in conformational changes that occur after ligand binding.
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PMID:Further characterization of the loop structure of platelet glycoprotein IIIa: partial mapping of functionally significant glycoprotein IIIa epitopes. 172 Jun 99

Quinine-dependent (Q) IgG antibodies (Q.Ab) in drug-induced immune thrombocytopenia are heterogeneous and bind to different platelet surface glycoproteins (GP), namely GPIb, IX, IIb, IIIa and an unidentified 57-kDa membrane proteins. Although both the Q-dependent epitope on GPIIIa and the P1A1 antigen require intact disulphide bonds for their expression, they are distinct because Q.Ab bind to GPIIIa lacking P1A1. Epitopes for both antigens were examined on Western blots of either intact washed human platelets or purified GPIIIa. When intact platelets were digested with trypsin and washed and solubilised prior to electrophoresis, membrane-associated fragments of GPIIIa of 78 kDa were found to be reactive with both antibodies. In addition, 60- and 68-kDa fragments bound anti-P1A1 but not Q.Ab. Similar digestion with chymotrypsin produced only 60-kDa fragments containing both epitopes. Digestion of purified GPIIIa with chymotrypsin produced 60-kDa peptides reactive with Q.Ab and anti-P1A1 in immunoblotting studies. Similar digestion with elastase produced 58-kDa fragments also containing the epitopes for both antibodies. Longer digestion times or sequential digestion with different enzymes did not reveal extra fragments. However, immunoprecipitation of trypsin-digested 125I-labelled GPIIIa with affinity-purified Q.Ab produced a 17-kDa fragment containing the Q-dependent epitope.
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PMID:Location of the quinine-dependent epitope on platelet glycoprotein IIIa. 172 84

The serine proteinase alpha chymotrypsin from bovine pancreas (CT) is known to expose fibrinogen binding sites on the surface of human platelets in the absence of cell activation and granular secretion. This is accompanied by the appearance of membrane-bound chymotryptic fragments of both glycoprotein (GP) IIb and GPIIIa, the two subunits of the platelet fibrinogen receptor, the GPIIb-IIIa complex. However, no clear relationship between discrete proteolytic event(s) within GPIIb-IIIa and fibrinogen-binding-site expression has yet been established. We have now evaluated the proteolysis of GPIIb-IIIa by CT by Western blot analyses using a panel of polyclonal and monoclonal antibodies against GPIIb or GPIIIa. The different proteolytic events were then correlated with the kinetics of the expression of active fibrinogen binding sites on platelets, as measured through the binding of 125I-labelled purified fibrinogen and to the capacity of CT-treated platelets to aggregate. Treatment of platelets with CT at 22 degrees C resulted in the expression of fibrinogen binding sites prior to cleavage of GPIIIa (Mr approximately 90,000) into a previously described, major membrane-bound fragment with Mr 60,000. In contrast, fibrinogen receptor expression closely paralleled a proteolytic cleavage at the carboxy terminus of the GPIIb heavy chain (Mr approximately 120,000), which was converted into a faster migrating species with Mr approximately 115,000). This proteolysis resulted in the release of a soluble peptide with an expected molecular mass of less than 3.7 kDa. Quantitation of this peptide using a competitive immunoenzymatic assay, confirmed that its release from the platelet surface correlated with the expression of fibrinogen binding sites and aggregability. When platelets were exposed to CT at 37 degrees C, a prompt increase in fibrinogen binding sites and platelet aggregability was observed, whereas the GPIIb heavy chain was rapidly converted into the carboxy-terminal-cleaved form. However, incubation at 37 degrees C for longer than 10 min resulted in extensive and simultaneous degradation of both the GPIIb heavy and light chains and of GPIIIa, with the latter being converted into the 60-kDa fragment. These later events were associated with a sharp decline of platelet aggregability and a reduction in the number of fibrinogen binding sites. These data allow us to propose that an early and limited proteolytic processing of the GPIIb component of the platelet fibrinogen receptor is associated with a shift of this receptor complex into a state which expresses specific binding sites for fibrinogen. Further cleavage of GPIIIa to generate the 60-kDa fragment results in loss of receptor activity.
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PMID:Activation of the fibrinogen receptor on human platelets exposed to alpha chymotrypsin. Relationship with a major proteolytic cleavage at the carboxyterminus of the membrane glycoprotein IIb heavy chain. 188 10

The proteolytic digestion of GPIIIa on intact platelets by chymotrypsin, thrombin, plasmin, trypsin, and staphylococcal V8 protease was monitored in immunoblot studies employing three different antibodies to GPIIIa, one of which was made against a 13-residue synthetic peptide containing the amino terminus of GPIIIa. Chymotrypsin, plasmin, and trypsin gave similar patterns, from which it could be inferred that the major products after extensive digestion were two-chain molecules composed of amino-terminal fragments of Mr approximately 17,000-18,000 disulfide bonded to carboxyl-terminal remnants of Mr approximately 58,000-70,000. These patterns suggest that GPIIIa contains a large disulfide-bonded loop of at least 325 amino acids that is susceptible to proteolytic cleavage, and that the 4 cysteine residues between residues 177 and 273 bond with each other. Such a structure can also account for the presence of the PIA1 epitope, which has recently been localized to a polymorphism at position 33 on these late digestion products. Thrombin did not proteolyze GPIIIa even at 2.5 units/ml. Still to be resolved is whether the minor immunoreactive GPIIIa bands found on normal platelets are related to in vivo or in vitro proteolysis and whether GPIIIa proteolysis plays a role in chymotrypsin-induced exposure of the GPIIb/IIIa receptor.
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PMID:Evidence that platelet glycoprotein IIIa has a large disulfide-bonded loop that is susceptible to proteolytic cleavage. 252 61

In vitro recirculation of fresh human heparinized blood in an extracorporeal circuit with a membrane oxygenator decreased fibrinogen-induced platelet aggregation and diminished the number of fibrinogen receptors and glycoprotein IIb/IIIa (GPIIb/GPIIIa) antigenic sites on the platelet surface. In seven experiments, the mean +/- SD Km value for fibrinogen (i.e., molar concentration of fibrinogen required to cause 50% of the maximal rate of aggregation) was 1.58 X 10(-7) mol/L +/- 0.68 X 10(-7) mol/L. After recirculation, this value increased to 3.8 X 10(-7) mol/L +/- 1.94 X 10(-7) mol/L (P less than or equal to 0.025). The maximal aggregation rate of chymotrypsin-treated platelets decreased by 40% after 2 hours of recirculation (P less than or equal to 0.025). The number of fibrinogen receptors on platelets, which were treated with chymotrypsin after a recirculation, decreased from 41,370 +/- 24,000 to 13,230 +/- 10,230/platelet under the same conditions (P less than or equal to 0.025). The number of antigenic sites for monoclonal antibody reacting with GPIIb/GPIIIa complex of adenosine diphosphate-stimulated platelets decreased from 34,200 +/- 5,940 to 19,500 +/- 9,680/platelet after recirculation (P less than or equal to 0.025). Prostaglandin E1 (0.3 mumol/L) in the perfusion circuit preserved the ability of platelets to react with fibrinogen. In conclusion, the loss of fibrinogen receptors from the surface of platelet membranes results from the interaction of platelets with the surfaces of perfusion circuits.
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PMID:Loss of fibrinogen receptors from the platelet surface during simulated extracorporeal circulation. 298

Considerable evidence indicates that the glycoprotein (GP) IIb/IIIa complex on human platelets functions as a receptor for fibrinogen, but little is known about the mechanism of receptor "exposure." To investigate this mechanism, our previously described murine monoclonal antibody (10E5) and a new monoclonal antibody (7E3), both of which block the binding of fibrinogen to platelets and bind to GPIIb and/or GPIIIa, were radiolabeled and their rates of binding to native and ADP-activated platelets were studied. At low concentrations, 125I-10E5 bound nearly equally rapidly to both native and activated platelets, whereas 125I-7E3 bound slowly to native platelets and much more rapidly to activated platelets. This increased rate of 7E3 binding is unlikely to be due to an increase in the number of GPIIb/IIIa sites on the surface of activated platelets because: (a) the rate of 10E5 binding was unchanged; (b) the total number of surface GPIIb/IIIa sites increased by only 2-10% with activation as judged by equilibrium binding of near-saturating concentrations of 10E5 and 7E3, and (c) there was less than 1% release of platelet factor 4 with activation, indicating minimal fusion of alpha-granule membranes (a potential source of GPIIb/IIIa) with the plasma membrane. Other activators (epinephrine, thrombin, and ionophore A 23187) also increased the rate of 7E3 binding, as did digestion of platelets with chymotrypsin. Aspirin did not affect the rate of binding of 7E3, whereas apyrase, prostaglandin E1, and dibucaine all inhibited the enhancement of the 7E3-binding rate produced by ADP. These data provide evidence for an activation-dependent change in the conformation and/or microenvironment of the GPIIb/IIIa complex, and offer a method of studying the receptor exposure mechanism that does not rely on the binding of fibrinogen itself.
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PMID:A new murine monoclonal antibody reports an activation-dependent change in the conformation and/or microenvironment of the platelet glycoprotein IIb/IIIa complex. 299 35

The following lines of evidence suggest that MP100, a putative ADP receptor, and GPIIIa are distinct proteins. [3H]FSBA incorporated equally into normal and thrombasthenic platelets (less than 5% GPIIIa), quantitatively as well as qualitatively. The dose-dependent inhibition of ADP-induced platelet shape change by FSBA is identical for normal and thrombasthenic platelets. Polyclonal rabbit antibodies precipitate MP100 and GPIIIa, but monoclonal antibodies directed against GPIIb/GPIIIa complex GPIIIa and P1A1 fail to precipitate the ADP receptor protein. A monoclonal antibody which inhibits ADP-induced platelet aggregation and fibrinogen binding fails to inhibit ADP-induced shape change. Thus, both functional and immunochemical evidence clearly indicates the distinct character of the ADP and fibrinogen receptors. We hypothesize that conformational changes in an ADP receptor on binding ADP or proteolytic changes as with chymotrypsin digestion may be responsible for exposure of the normally latent fibrinogen receptor (GPIIb/GPIIIa complex).
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PMID:Separation of the 100-kDa membrane protein mediating ADP-induced platelet shape change and activation from glycoprotein IIIa. 360 28

Trigramin, a highly specific inhibitor of fibrinogen binding to platelet receptors, was purified to homogeneity from Trimeresurus gramineus snake venom. Trigramin is a single chain (approximately 9 kDa) cysteine-rich peptide with the Glu-Ala-Gly-Glu-Asp-Cys-Asp-Cys-Gly-Ser-Pro-Ala NH2-terminal sequence. Chymotryptic fragmentation showed the Arg-Gly-Asp sequence in trigramin. Trigramin inhibited fibrinogen-induced aggregation of platelets stimulated by ADP (IC50 = 1.3 X 10(-7)M) and aggregation of chymotrypsin-treated platelets. It did not affect the platelet secretion. Trigramin was a competitive inhibitor of the 125I-fibrinogen binding to ADP-stimulated platelets (Ki = 2 X 10(-8) M). 125I-Trigramin bound to resting platelets (Kd = 1.7 X 10(-7) M; n = 16,500), to ADP-stimulated platelets (Kd = 2.1 X 10(-8) M; n = 17,600), and to chymotrypsin-treated platelets (Kd = 8.8 X 10(-8) M; n = 13,800) in a saturable manner. The number of 125I-trigramin binding sites on thrombasthenic platelets amounted to 2.7-5.4% of control values obtained for normal platelets and correlated with the reduced number of GPIIb-GPIIIa molecules on the platelet surface. EDTA, monoclonal antibodies directed against the GPIIb-GPIIIa complex, and synthetic peptides (Arg-Gly-Asp-Ser and Tyr-Gly-Gln-Gln-His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val) blocked both 125I-fibrinogen binding and 125I-trigramin binding to platelets. Fibrinogen binding was more readily inhibited by these compounds than was trigramin binding. Monoclonal antibodies directed either against GPIIb or GPIIIa molecules did not block the interaction of either ligand with platelets. Reduced, S-pyridylethyl, trigramin did not inhibit platelet aggregation and fibrinogen binding to platelets and it did not bind to platelets, suggesting that the secondary structure of this molecule is critical for expression of its biological activity.
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PMID:Trigramin. A low molecular weight peptide inhibiting fibrinogen interaction with platelet receptors expressed on glycoprotein IIb-IIIa complex. 368 Feb 47


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