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)

Anticoagulated whole blood from patients and control subjects was circulated through an annular perfusion chamber in which the fibrillar collagen of alpha chymotrypsin-digested subendothelium and intact subendothelium were exposed. The blood flow conditions corresponded to those in arteries (830 sec-1 wall shear rate). Platelet surface interaction was measured morphometrically. Decreased adhesion to fibrillar collagen associated with normal spreading and normal adhesion-induced formation of platelet thrombi was found with blood of patients with von Willebrand's disease and the Bernard Soulier Syndrome, indicating a defect in the initial attachment reaction of platelets with collagen. Platelets of patients with thrombasthenia did normally adhere to the collagen fibrils and also lost their subcellular organelles during this reaction, but they totally failed to adhere to each other. In storage pool disease platelet thrombus formation was consistently inhibited whereas adhesion and spreading was inhibited in some patients and normal in others. In contrast adhesion was always normal after ingestion of aspirin which consistently caused a marked inhibition of platelet thrombi. These findings correspond -- in essence -- to those previously described on intact subendothelium. However, the observed defects are more pronounced on the fibrillar collagen than on intact subendothelium.
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PMID:Platelet interaction with collagen fibrils in flowing blood. II. Impaired adhesion-aggregation in bleeding disorders. A comparison with subendothelium. 30 Jan 83

Integrins are membrane receptors which mediate cell-cell or cell-matrix adhesion. Integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) acts as a fibrinogen receptor of platelets and mediates platelet aggregation. Platelet activation is required for alpha IIb beta 3 to shift from noncompetent to competent for binding soluble fibrinogen. The steps involved in this transition are poorly understood. We have studied a variant of Glanzmann thrombasthenia, a congenital bleeding disorder characterized by absence of platelet aggregation and fibrinogen binding. The patient's platelets did not bind fibrinogen after platelet activation by ADP or thrombin, though his platelets contained alpha IIb beta 3. However, isolated alpha IIb beta 3 was able to bind to an Arg-Gly-Asp-Ser affinity column, and binding of soluble fibrinogen to the patient's platelets could be triggered by modulators of alpha IIb beta 3 conformation such as the Arg-Gly-Asp-Ser peptide and alpha-chymotrypsin. These data suggested that a functional Arg-Gly-Asp binding site was present within alpha IIb beta 3 and that the patient's defect was not secondary to a blockade of alpha IIb beta 3 in a noncompetent conformational state. This was evocative of a defect in the coupling between platelet activation and alpha IIb beta 3 up-regulation. We therefore sequenced the cytoplasmic domain of beta 3, following polymerase chain reaction (PCR) on platelet RNA, and found a T-->C mutation at nucleotide 2259, corresponding to a Ser-752-->Pro substitution. This mutation is likely to be responsible for the uncoupling of alpha IIb beta 3 from cellular activation because (i) it is not a polymorphism, (ii) it is the only mutation in the entire alpha IIb beta 3 sequence, and (iii) genetic analysis of the family showed that absence of the Pro-752 beta 3 allele was associated with the normal phenotype. Our data thus identify the C-terminal portion of the cytoplasmic domain of beta 3 as an intrinsic element in the coupling between alpha IIb beta 3 and platelet activation.
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PMID:Ser-752-->Pro mutation in the cytoplasmic domain of integrin beta 3 subunit and defective activation of platelet integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) in a variant of Glanzmann thrombasthenia. 143 6

Patient A.F. is a 28-year-old polytransfused woman with an inherited bleeding disorder, Glanzmann's thrombasthenia. An abnormal platelet function is linked to severe decreases in the platelet content of the integrins GP IIb and GP IIIa. In 1987 the patient gave birth to a child with severe anemia and thrombocytopenia. Serological tests revealed the presence of anti-platelet antibody together with an anti-Rhesus D. Western blotting identified a major antibody that reacted with a protein of 90-95 kDa present in platelets and endothelial cells. This was identified as the beta 3 integrin subunit (GP IIIa). Antibody-binding required intact disulfides, while controlled digestion with proteases showed the determinant(s) to be retained within chymotrypsin- (50, 63 kDa) and Staphylococcus aureus V8 protease-derived (25-38 kDa) fragments of GP IIIa. Direct binding assays performed in the presence of monoclonal antibodies specific for different epitopes on GP IIb-IIIa complexes confirmed that the epitope was exposed on intact platelets and revealed a specific inhibition of A.F. IgG binding by the monoclonal antibody, AP-3. Other tests confirmed that the antibody reacted independently of the PlA or Pen polymorphisms carried by GP IIIa. IgG purified from A.F. plasma by adsorption and elution from paraformaldehyde-fixed normal platelets or electrophoretically separated GP IIIa was an inhibitor of ADP-induced platelet aggregation. Unexpectedly, Western blotting showed trace amounts of abnormally migrating GP IIIa in A.F. platelets, which retained an ability to react with her antibody. This suggests that the patient has formed an autoantibody reactive with an active site of the beta 3 integrin subunit and linked to the development of neonatal thrombocytopenia.
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PMID:Characterization of an antibody to the integrin beta 3 subunit (GP IIIa) from a patient with neonatal thrombocytopenia and an inherited deficiency of GP IIb-IIIa complexes in platelets (Glanzmann's thrombasthenia). 163 70

Splenocytes from a patient with chronic, immune-mediated thrombocytopenic purpura (ITP) were transformed with Epstein-Barr virus. A stable lymphoblastoid cell line (LCL) derived from this transformation (2A3) produces IgM antibody reactive with platelet glycoprotein IIb. 2A3 was fused to the 6-thioguanine-resistant ouabain-resistant, murine-human heteromyeloma cell line, F6. The resultant heterohybridomas were selected by growth in medium containing hypoxanthine/aminopterin/thymidine and ouabain. One hybridoma line, 2E7, produces high levels of IgM antibody (2 to 4 micrograms IgM/ml/24 hr/10(5) cells) reactive with glycoprotein IIb. 2E7 has been repeatedly subcloned by limiting dilution and has been maintained in continuous culture for 26 months. 2E7 binds to human platelets but not endothelial cells, as determined by flow cytometry, and does not react with platelets of patients with Glanzmann's thrombasthenia that lack IIb-IIIa. The epitope recognized by 2E7 is likely to be a contiguous peptide sequence since the antibody binds to the IIb heavy chain in immunoblot assays of denatured, reduced platelet protein. Treatment of intact platelets or purified IIb-IIIa with papain or chymotrypsin, but not SV8 protease, destroys the epitope. Thus, the 2E7 epitope may be at or very close to a site on IIb that is cleaved by these proteases. The expression of the 2E7 epitope is significantly affected by the presence of divalent cations. Treatment of intact platelets with EDTA at 37 degrees C results in a three-to four-fold increase in the number of 2E7 molecules bound per platelet and an eight-fold increase in the affinity of the antibody. The binding of 2E7 to normal platelets does not inhibit any of the functions attributed to IIb-IIIa, such as fibrinogen-dependent platelet aggregation or clot retraction. 2E7 represents the first human monoclonal antibody reported to recognize an epitope on platelet glycoprotein IIb. The epitope is unique to IIb and not shared by other integrin alpha subunits.
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PMID:A human monoclonal autoantibody specific for human platelet glycoprotein IIb (integrin alpha IIb) heavy chain. 171 76

Platelets of a patient with Friedreich's ataxia have been investigated because of a codiagnosis of thrombasthenia. No aggregation occurred in response to adenosine diphosphate, platelet activating factor-acether, a stimulatory antiplatelet monoclonal antibody, or phorbol myristate acetate, although platelet aggregation could be induced with thrombin, the calcium ionophore A23187, or high concentrations of collagen. Shape change, adenosine triphosphate secretion, and the responses of the platelets' protein phosphorylation systems to all agonists were normal. Immunologic analysis of the patient's radiolabeled platelet surface proteins revealed normal levels of glycoproteins IIB and IIIa. However, no iodine 125-fibrinogen binding occurred after stimulation of the patient's platelets with adenosine diphosphate. In contrast, pretreatment of the patient's platelets with the proteolytic enzyme alpha-chymotrypsin resulted in the exposure of active 125I-fibrinogen binding sites. The patient's platelets exhibited normal aggregation to fibrinogen after their pretreatment with chymotrypsin and with elastases derived either from porcine pancreas or from human granulocytes. A murine monoclonal antibody directed against the human platelet membrane glycoproteins IIb and IIIa calcium-dependent epitope and rabbit polyclonal anti-human platelet membrane and human anti-P1A1 antibodies immunoprecipitated glycoproteins IIb and IIIa and a 66 kd cleavage product of glycoprotein IIIa from sodium dodecyl sulfate-Triton X-100 extracts of the patient's proteolytically treated platelets. The patient appears to exhibit a unique type of thrombopathy involving a defect in the exposure of fibrinogen receptors. The association between the neurologic disorder and the platelet defect is still unclear.
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PMID:Identification of a unique type of thrombopathy of human platelets: defect in the exposure of active fibrinogen receptors in a patient with Friedreich's ataxia. 283 36

Previous experiments demonstrated that chymotrypsin, but not adenosine diphosphate (ADP), exposed fibrinogen binding sites on platelets from patients with Glanzmann's thrombasthenia. Three of these patients have been reexamined, and previous observations were confirmed. The quantity of iodine 125-labeled glycoprotein IIb (GPIIb) and glycoprotein IIIa (GPIIIa) on the platelets of these patients was considerably less than normal but was detectable by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography. The amount of residual GPIIb and GPIIIa as measured by binding studies with radiolabeled monoclonal antibodies was between 3% and 12% of the normal value. Platelet suspensions from these patients did not aggregate with fibrinogen and did not bind 125I-fibrinogen on stimulation with ADP. However, incubation of these platelets with chymotrypsin or pronase resulted in fibrinogen binding and platelet aggregation. Monoclonal antibodies specific for the GPIIb-GPIIIa complex blocked both the fibrinogen binding and the aggregation of enzyme-treated platelets. The treatment of washed platelets of a fourth thrombasthenic patient with ADP or with chymotrypsin failed to result in fibrinogen binding and aggregation. However, the level of GPIIb and GPIIIa on these platelets as measured by a Western blot technique and by monoclonal antibody binding amounted to less than 0.35% to 0.5% of normal values. In conclusion, fibrinogen binding sites exposed on thrombasthenic platelets by chymotrypsin are derived from GPIIb-GPIIIa molecules. Aggregation of chymotrypsin-treated thrombasthenic platelets by fibrinogen appears to represent a sensitive test for detection of functionally active GPIIb-GPIIIa complex on the platelet surface.
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PMID:Aggregation of chymotrypsin-treated thrombasthenic platelets is mediated by fibrinogen binding to glycoproteins IIb and IIIa. 299 74

Sera from 28 of the 113 normal children and adults (25%) studied were found to contain an immunoglobulin capable of causing complement-dependent lysis of normal platelets treated with small quantities of papain. This factor reacts equally well at 4 degrees C and at 37 degrees C with a determinant induced on platelets from normal subjects by treatment with papain or bromelain, but not by trypsin, chymotrypsin, or neuraminidase. It does not bind to red cells treated with any of these enzymes. The site(s) for which the factor was specific could not be induced on platelets from six patients with type I Glanzmann's thrombasthenia (lacking glycoproteins IIb and IIIa), in contrast to platelets from each of 20 normal donors. Isolation and characterization of the factor has been difficult because of its intolerance to chemical and physical manipulation. In 11 of the 20 individuals studied, however, it was found to have the properties of an IgM immunoglobulin. The factor appears to be different from any previously described, naturally occurring human immunoglobulin. It has not yet been shown to be associated with any disease state, but in the presence of complement, it is capable of causing profound damage to platelets previously subjected to minimal proteolysis, and the possibility that it can provoke platelet destruction in some conditions deserves further study.
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PMID:A naturally occurring, warm-reactive macroglobulin specific for papain-treated human platelets: preliminary characterization. 394 32

Calcium is a cofactor of human platelet aggregation. Moreover a direct correlation between the ability of platelets to bind this divalent cation and to aggregate has been demonstrated. Since magnesium can substitute for calcium in supporting aggregation, especially in the presence of low calcium concentrations, and platelet aggregation is inhibited at low pH, the present study was designed to examine the effects of magnesium and low pH on 45calcium binding to human platelets, and to determine whether such effects might be associated with calcium binding to glycoproteins I (GPI) or IIb/IIIa (GPIIb/IIIa), the putative fibrinogen receptor. 45Calcium binding to aspirin-treated platelets that had been depleted of surface-associated calcium by brief exposure to EDTA was evaluated. Magnesium (5-10 mM) or a change in hydrogen ion concentration to decrease the pH from 7.5 to 6.0 was found to inhibit the binding of 45calcium to platelets from healthy donors by 34 +/- 6 and 32 +/- 8% (mean +/- SD, n = 13), respectively. Similar results were obtained with platelets incubated with chymotrypsin to selectively remove GPI or platelets from a patient with the Bernard Soulier Syndrome, congenitally deficient in GPI. In contrast, calcium binding to platelets from two patients with thrombasthenia, lacking GPIIb/IIIa, was reduced 49 +/- 6% and 42 +/- 8% (n = 4) by magnesium and hydrogen ions, respectively. This apparently increased inhibition was attributed to the combined effects of an overall decrease (approximately 50%) in calcium binding to thrombasthenic platelets compared with that in control platelets, and a similar absolute reduction in calcium binding in the presence of magnesium and/or hydrogen ions. No additional inhibition of 45calcium binding was noted in the presence of magnesium and at low pH, indicating that magnesium and hydrogen ions may affect the same platelet membrane binding sites. The data suggest that although modulation of platelet aggregation by magnesium and pH is accompanied by changes in platelet-associated calcium, calcium binding to the three major platelet membrane glycoproteins, GPI, IIb, and IIIa is unaffected.
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PMID:pH and magnesium alter 45calcium binding to platelets at sites other than glycoproteins I or IIb/IIIa. 399 8

We report the immunochemical characterization of a new platelet-specific alloantigen detected using an IgG antibody isolated from the serum of a patient with posttransfusion purpura (PTP). In indirect immunoprecipitation experiments, the antibody, termed anti-Leka, predominantly precipitated glycoprotein (GP) IIb from Triton X-100 lysates of normal human platelets. In an immunoblot procedure, which involved the transfer of platelet polypeptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to nitrocellulose membrane, anti-Leka bound exclusively to GP IIb. Under identical conditions, four anti-PlA1 antibodies each reacted with GP IIIa. No binding of anti-Leka IgG occurred to Leka (-) platelets or to their separated polypeptides although GP IIb was normally detected by Coomassie blue staining. After electrophoresis of reduced platelet proteins, the Leka determinant was localized to the IIb alpha chain. Thus, unlike the PlA1 antigen, the Leka determinant was not destroyed by disulfide reduction. Analysis of platelets from a patient with Glanzmann's thrombasthenia revealed little or no binding in the GP IIb position. Anti-Leka permitted the identification of 76,000 and 60,000 dalton fragments of GP IIb retained by the platelet following chymotrypsin treatment. Our results further highlight the immunogenicity of the GP IIb-IIIa complex. They also suggest that antibodies against GP IIb can cause the thrombocytopenia observed in PTP and that anti-PlA1 antibodies do not account exclusively for the pathophysiology of this immune disorder.
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PMID:Immunochemical characterization of the platelet-specific alloantigen Leka: a comparative study with the PlA1 alloantigen. 620 52

A monoclonal hybridoma antibody specific for platelet glycoprotein I complex is described. The nature od the antigen was determined by demonstration that it was chymotrypsin sensitive and gave a peak at 150 000 daltons on SDS-PAGE after immunoprecipitation. The expression of the antigen is restricted to platelets and megakaryocytes with at least 1.6 x 10(4) molecules of antigen per platelet. The antibody failed to bind to platelets from patients with Bernard Soulier syndrome, where there is known to be a deficiency of glycoprotein Ib/Is expression. Binding to platelets from patients with Glanzmann's thrombasthenia was normal.
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PMID:Monoclonal antibody to human platelet glycoprotein I. I. Immunological studies. 645 15


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