Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.1 (chymotrypsin)
 10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

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

To determine a thrombin-binding site on GPIb alpha on platelet membrane, we have examined the binding activities of tryptic or chymotryptic fragments of purified GPIb alpha to a monoclonal antibody against GPIb (TM60) and thrombin using (immuno)affinity chromatography. When purified GPIb alpha was digested with trypsin, two fragments (94-kDa, and 43-kDa) were obtained. The 43-kDa fragment was shown to bind to both affinity columns of TM60- and thrombin-Affi-Gel, while the 94-kDa fragment did not bind to either Affi-Gel columns. When trypsin fragments were incubated with TM60 and then applied to the column of thrombin-Affi-Gel, neither fragments were bound to the column. When the same experiment was performed using chymotrypsin, three fragments (94-kDa, 45-kDa and 39-kDa) were observed. On TM60- and thrombin-Affi-Gel columns, the smaller fragments (45-kDa and 39-kDa) were bound to the column. After incubation of these fragments with TM60, neither bound to the thrombin column. These results indicate (i) that the epitope for TM60 is located near, or on the thrombin-binding site of GPIb alpha, and (ii) that the thrombin-binding site is located on the tail portion of GPIb alpha, especially on a chymotrypsin cleavage site.
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PMID:Localization of a thrombin-binding site on human platelet membrane glycoprotein Ib determined by a monoclonal antibody. 242 16

Tertiary amine local anesthetics modify a variety of platelet membrane-related functions. The present study explored dibucaine (DB)-induced inhibition of platelet cohesion by examining structural and functional alterations of the human platelet membrane glycoprotein IIb-IIIa complex (GPIIb-IIIa) and platelet Ca2+ homeostasis. Complete inhibition of ADP-induced aggregation was achieved five minutes after platelet exposure to 0.10 to 0.25 mmol/L of DB when fibrinogen binding was reduced by 50%. At higher concentrations of DB (approximately 1 mmol/L), ADP-induced fibrinogen binding was completely blocked. Scatchard analysis revealed loss of high-affinity binding sites in addition to reduction in Bmax. In contrast, chymotrypsin-treated platelets sustained 50% inhibition of fibrinogen binding when incubated with 0.4 to 0.5 mmol/L DB, and kinetic analysis showed that the high-affinity platelet-fibrinogen interactions were reduced but not absent. Fibrinogen binding to chymotrypsin-treated platelets could not be completely inhibited even at high DB concentrations (1 mmol/L). The inhibition of fibrinogen binding to chymotrypsin-treated platelets correlated with changes in binding of a monoclonal antibody (10E5) specific for an epitope on the GPIIb-IIIa complex. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and radioelectroimmunoassay of DB-treated platelets, however, showed no evidence of a reduction or degradation of GP IIb or IIIa. Platelet incubation with DB (five minutes, 0.1 to 1.0 mmol/L) was also accompanied by: increased platelet membrane-associated Ca2+ involving low-affinity binding sites [Kd = 5 X 10(-5) mol/L-]; increased 45Ca2+ uptake which correlated with degradation of actin-binding protein (ABP) and digestion of GPIb as visualized on periodic-acid Schiff (PAS)-stained SDS gels and as inferred from decreased binding of a monoclonal antibody (6D1) directed against this glycoprotein; and enhanced Ca2+ exchange. Thus, exposure of platelets to DB results in membrane-related alterations that may contribute to inhibition of platelet cohesion: Decreased fibrinogen receptor exposure by traditional agonists and diminished accessibility of the GPIIb-IIIa complex to extracellular ligands correlate with DB-induced inhibition of platelet aggregation; and increased calcium uptake and exchange across the platelet membrane likely leads to activation of the calcium-dependent protease(s) which was previously shown to correlate with DB-induced inhibition of ristocetin-induced platelet agglutination.
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PMID:Platelet membrane alterations induced by the local anesthetic dibucaine. 301 84

Human platelets were surface-labeled by the periodate/NaB3H4 method or by lactoperoxidase-catalysed iodination with 125I. The labeled platelets were treated with chymotrypsin under conditions known to give platelets which aggregate with fibrinogen without stimulation with ADP. Platelets and supernatant were then analysed by various gel electrophoretic techniques including isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing or non-reducing conditions and two-dimensional non-reduced/reduced sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by fluorography or indirect autoradiography. Chymotrypsin-treatment of surface-labeled platelets degraded the major glycoproteins Ib, IIb and IIIa but also GP120(4.9-5.4), GPIc and GPV. The membrane-bound fragments of GPIb, IIb and IIIa could be identified and also the supernatant fragments of GPIb and GPV. GPIIIa was also cleaved within a loop structure formed by disulfide bond(s). The fact that remnants of both GPIIb and IIIa are left on chymotrypsin-treated platelets which aggregate spontaneously with fibrinogen may indicate that a complex formed by these remnants constitutes the fibrinogen-binding site on platelets.
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PMID:Identification and characterization of fragments of major glycoproteins from platelet membrane after chymotrypsin treatment. 397 99

Treatment of intact human platelets with chymotrypsin released a glycopolypeptide that was shown to be derived from the major membrane glycoprotein, GPIb. The glycopolypeptide contained 59% carbohydrate on a molar basis and was rich in serine, threonine and proline. Almost all the carbohydrate could be released from the glycopolypeptide by treatment with alkali in the presence of NaBH4. The major component (comprising 80% of the released sugar) was purified and shown to be a hexasaccharide containing sialic acid, galactose, N-acetylglucosamine and N-acetylgalactosaminitol in the molar ratios 2:2:1:1. Two possible structures for this hexasaccharide are proposed on the basis of the known biosynthetic pathways of mucus-type glycoproteins. Our data is consistent with the occurrence of an O-glycosidically linked oligosaccharide on one amino acid in four of the glycopolypeptide. These results suggest that glycoprotein Ib can best be described as a membrane-bound mucus-type glycoproteins. Our data are consistent with the occurrence of an O- in the process by which platelets adhere to the exposed subendothelium of damaged blood-vessel walls. The possible role of the glycopolypeptide portion of GPIb in this process was investigated. Neither the major oligosaccharide nor the glycopolypeptide itself inhibited ristocetin-induced platelet agglutination at the concentrations tested. It is suggested that the carbohydrate moieties of GPIb molecules at the cell surface interact to form a barrier to macromolecules. Such a barrier could play a major role in modulating platelet function.
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PMID:Isolation and characterization of the major oligosaccharide of human platelet membrane glycoprotein GPIb. 621 34

Present knowledge of the structure and function of platelet glycocalicin is reviewed. Glycocalicin (M 150,000) is a glycoprotein component of the outer surface of intact platelets which is released in soluble form following platelet homogenization. Glycocalicin has been purified and shown to inhibit platelet aggregation induced by thrombin or by ristocetin. Thrombin binding activity is associated with the peptide "tail" of the molecule (Mr 45,000), the macroglycopeptide portion (Mr 120,000) being without effect. Glycocalicin and membrane-bound glycoprotein I have been shown to be functionally and immunologically identical. Studies with platelets modified by chymotrypsin, and with platelets from patients with Bernard-Soulier disease and an ill-defined bleeding abnormality show that the amount of thrombin bound is proportional to the total amount of glycocalicin and glycoprotein I present. These results support the concept of a single class of binding site for thrombin in platelets.
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PMID:Structure and function of platelet glycocalicin. 736 63

The platelet-membrane glycoprotein IIb-IIIa (GPIIb-IIIa) complex is essential for platelet aggregation and is involved in the attachment of platelets to thrombogenic surfaces. This study shows the retention of GPIIb and GPIIIa on immobilized fibrinogen after Triton X-100 (Sigma Chemical Co, St Louis, MO) lysis of adherent platelets. Glycoproteins were detected using subunit specific monoclonal antibodies in a modified enzyme-linked immunosorbent assay procedure. GPIIb-IIIa retention was judged to be specific relative to GPIb recovery, and was modulated by platelet activation. Platelet exposure to adenosine diphosphate or thrombin, but not A23187 or chymotrypsin, markedly enhanced GPIIb and GPIIIa recovery relative to that observed with unstimulated platelets, or prostaglandin E1-treated platelets. Moreover, lysis of adherent platelets in the presence of 10 mmol/L EDTA, under conditions promoting GPIIb-IIIa complex dissociation (pH 8.1, 60 minutes, 37 degrees C), had no effect on GPIIb or GPIIIa subunit recovery. Platelet activation with Zn+2 also enhanced GPIIb and GPIIIa recovery on fibrinogen-coated surfaces over that observed with unstimulated platelets, but GPIIb and IIIa retention was EDTA sensitive. This correlated with the EDTA-reversible nature of Zn+2-activated platelet adhesion to fibrinogen-coated surfaces. The data (1) show that platelet adhesion to fibrinogen is accompanied by the induction of high-affinity interactions between GPIIb-IIIa and immobilized fibrinogen that are EDTA-resistant and enhanced by platelet activation with some but not all agonists, and (2) implicate these interactions in stabilizing platelet contacts with fibrinogen-coated surfaces.
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PMID:Glycoprotein IIb and IIIa retention on fibrinogen-coated surfaces after lysis of adherent platelets. 824 6

Present knowledge of the structure and function of platelet glycocalicin is reviewed. Glycocalicin (M,. 150,000) is a glycoprotein component of the outer surface of intact platelets which is released in soluble form following platelet homogenization. Glycocalicin has been purified and shown to inhibit platelet aggregation induced by thrombin or by ristocetin. Thrombin binding activity is associated with the peptide "tail" of the molecule (Mr 45,000), the macroglycopeptide portion (Mr 120,000) being without effect. Glycocalicin and membrane-bound glycoprotein I have been shown to be functionally and immunologically identical. Studies with platelets modified by chymotrypsin, and with platelets from patients with Bemard-Soulier disease and an ill-defined bleeding abnormality show that the amount of thrombin bound is proportional to the total amount of glycocalicin and glycoprotein I present. These results support the concept of a single class of binding site for thrombin in platelets.
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PMID:Structure and Function of Platelet Glycocalicin. 3078 29