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:3.4.21.7 (plasmin)
9,023 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We demonstrate that unstimulated platelets attach to immobilized fibrinogen in a selective process mediated by the membrane glycoprotein (GP) complex IIb-IIIa (alpha IIb beta 3). The initial attachment, independent of platelet activation, is followed by spreading and irreversible adhesion even in the presence of activation inhibitors. Using fibrinogen fragments derived from plasmin digestion, we found that unstimulated platelets do not attach to immobilized fragment E, which contains an Arg-Gly-Asp sequence at A alpha 95-97, and adhere to fragments X and D, both containing the gamma 400-411 dodecapeptide adhesion sequence, less efficiently than to intact fibrinogen. Thus, the carboxyl terminus of the A alpha chain, missing in the "early" fragment X used in these studies, appears to be involved in the interaction of fibrinogen with unstimulated platelets. In contrast, activated platelets adhere to immobilized fibrinogen and fragments X, D, and E in a time-dependent and equivalent manner. Although activated platelets adhere to immobilized vitronectin, fibronectin, and von Willebrand factor through GP IIb-IIIa, unstimulated platelets fail to adhere to vitronectin and have only a limited capacity to adhere to fibronectin and von Willebrand factor. These results demonstrate that GP IIb-IIIa on unstimulated platelets displays a recognition specificity for attachment to immobilized adhesive proteins that is distinct from that seen following platelet activation. Thus, unstimulated platelets selectively interact with fibrinogen, and the initial attachment is followed by spreading and irreversible adhesion in the absence of exogenous agonists. This process may be regulated by plasmin cleavage of the fibrinogen A alpha chain and may play an important role during normal hemostasis and during the pathological development of thrombotic vascular occlusions.
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PMID:Selective recognition of adhesive sites in surface-bound fibrinogen by glycoprotein IIb-IIIa on nonactivated platelets. 204 Jun 30

Platelet membrane glycoprotein Ib (GPIb), a receptor for von Willebrand factor and thrombin, is present on the platelet surface membrane, in intraplatelet stores, and in plasma (as the proteolytic fragment glycocalicin). We examined the hypothesis that after plasmin-mediated cleavage of platelet surface GPIb, platelets can replenish their surface GPIb pool. Incubation of washed platelets with plasmin (1 hour, 22 degrees C) resulted in loss of platelet surface GPIb, but further incubation (3 hours, 37 degrees C) in autologous plasma resulted in restoration of platelet surface GPIb, as determined by ristocetin-induced platelet agglutination and a flow cytometric assay of platelet binding of three GPIb-specific monoclonal antibodies. Despite the restoration of platelet surface GPIb after the 3-hour incubation of plasmin-treated platelets in autologous plasma, the whole platelet GPIb content (measured by enzyme-linked immunosorbent assay [ELISA], sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and flow cytometry) remained reduced, quantitatively corresponding to an increase in plasma glycocalicin concentration (measured by ELISA). The loss and restoration of platelet surface GPIb occurred on all platelets and, as evidenced by lack of inhibition by prostaglandin E1, EDTA, and cytochalasins, was not mediated by cyclic AMP, extracellular Ca2+, or the platelet microfilament system. In summary, this study shows that after plasmin-mediated cleavage of platelet surface GPIb, platelets can replenish their surface GPIb pool by recruitment of GPIb molecules from the intraplatelet pool (or from a sequestered surface site).
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PMID:Plasmin-induced redistribution of platelet glycoprotein Ib. 214 79

The presence and localization of the plasmin system components urokinase (UPA), tissue type plasminogen activator (TPA), plasminogen (PG), a neoantigen expressed by the plasmin-alpha 2-antiplasmin complex, and plasmin inhibitors alpha 2-antiplasmin (AP) and alpha 2-macroglobulin (MG) have been tested by immunofluorescence on sections of 11 benign and 40 malignant lesions of the breast in an attempt to apply a morphological approach to the problem of tumor invasion in vivo. In benign lesions, TPA was seen in secretions of mammary glands and MG was seen in edematous zones. In one involuting lactating adenoma, UPA, TPA, PG, PAP, and AP were associated with glandular cells. UPA was detected in 11 carcinomas, TPA in 22, PG in 31, PAP in 12, AP in 23, and MG in all 40. All these components were essentially present in invasive territories, with a cellular labeling for UPA and TPA and a fluorescent staining frequently at the periphery of tumoral foci for PG and PAP. AP was more closely associated with cancer cells than MG, which was present in the stroma. Intraductal proliferations were rarely positive and there was no correlation between the localization of PG and the distribution of a basement membrane glycoprotein laminin. These data argue strongly for the involvement of the plasmin system in the infiltrating process of the stroma. This system seems to play a limited role in the breakdown of basement membrane in breast carcinomas in vivo.
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PMID:Detection of the plasmin system in human mammary pathology using immunofluorescence. 242 83

A remarkable reduction of postoperative blood loss after cardiopulmonary bypass (CPB) has been achieved by prophylactic treatment with the proteinase inhibitor aprotinin. To reveal the mode of action of aprotinin, 23 CPB patients were randomised for aprotinin (2 x 10(6) KIU in the pump prime) or placebo treatment during CPB. Blood samples were collected before and during operation. Blood loss and blood requirements were 50% lower in the aprotinin treated patients than in the untreated patients. The adhesive capacity of platelets assessed by the amount of platelet membrane glycoprotein Ib (GP Ib) decreased by 50% in the untreated patients within 5 min of CPB and remained low during CPB, whereas GP Ib did not decrease in the aprotinin treated patients. Fibrinogen degradation products indicating plasmin activity could only be measured after 30 min of CPB in the untreated, but not in the aprotinin treated patients. The kallikrein inhibiting capacity was 34% decreased in the untreated patients within 5 min of CPB, while it increased by 84% and remained high during CPB in the aprotinin treated patients. Our results demonstrate that the improved haemostasis during and after CPB in patients treated with aprotinin can be attributed to the preserved adhesive capacity of platelets. It remains to be found whether aprotinin has a primary effect on platelets or a secondary effect by plasmin or kallikrein inhibition.
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PMID:Platelet preservation during cardiopulmonary bypass with aprotinin. 248 79

Gelfiltered unstimulated human platelets neither bound 125-I-fibrinogen nor 125-I-fibrin. Fibrin-binding was, however, stimulated by N-terminal fibronectin 30 kD-and 70 kD-fragments while fibronectin was ineffective. The 30 kD-fragment also stimulated some platelet preparations to bind fibrinogen which, however, was suppressed by minute amounts of the thrombin inhibitor PPACK. PPACK hardly influenced fibrin-binding. Fragment-promoted fibrinogen-binding was also inhibited by a monoclonal antibody recognizing the membrane glycoprotein IIb/IIIa complex known to act as fibrinogen receptor. This antibody failed to influence fragment-stimulated fibrin-binding giving evidence that fibrinogen and fibrin were retained by different receptors. In contrast to 125-I-fibrin its plasmin-derived and 125-I-labelled fragment X was not recognized by the platelets in presence of the fibronectin 30 kD-fragment. Fragment-stimulated binding of 125-I-fibrin showed a lag phase and was completely inhibited by 0.25 mM putrescine as well as by 1 mM EDTA or 0.1 mM N-ethylmaleinimide. Evidently, a cell-attached transamidase was involved in fibrin-binding possibly by forming a ternary complex with fibrin and the fibronectin fragment.
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PMID:Free N-terminal fibronectin 30-kD-domain mediates binding of soluble fibrin to gelfiltered unstimulated thrombocytes. 317 84

Platelet membrane glycoprotein (GP) Ib contains receptor for von Willebrand factor and thrombin. Its proteolytic fragment, glycocalicin, circulates in normal plasma. In this study, storage of platelet concentrates for 5 d resulted in a 221% increase in plasma glycocalicin (1.3 times the total amount of glycocalicin present on the surface of all platelets), an 8% overall increase in platelet surface GPIb, and the appearance of a surface GPIb-negative subpopulation of platelets. Total platelet GPIb content of fresh washed platelets, determined by gel electrophoresis and immunoassay of Triton X-100 lysates, averaged 159,740 molecules per platelet. There were 36,360 surface GPIb molecules per platelet, determined by immunoassay of the supernatant of fresh washed platelets whose surface GPIb had been completely plasmin-cleaved. In summary, these studies provide evidence for (a) a redistribution of GPIb molecules with platelet storage, and (b) a large intraplatelet pool of GPIb (approximately threefold larger than the platelet surface pool of GPIb).
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PMID:Platelet storage results in a redistribution of glycoprotein Ib molecules. Evidence for a large intraplatelet pool of glycoprotein Ib. 338 48

Glycoprotein IIb (GPIIb) and glycoprotein IIIa (GPIIIa) form a macromolecular complex on the activated platelet surface which contains the fibrinogen-binding site necessary for normal platelet aggregation. To identify the specific region of the fibrinogen molecule responsible for its interaction with the GPIIb-GPIIIa complex, purified fragment D1 (Mr = 100,000) and fragment E (Mr = 50,000) were prepared from plasmin digests of purified human fibrinogen. In addition, the polypeptide chain subunits A alpha, B beta, and gamma of fibrinogen were prepared. Using an enzyme-linked immunosorbent assay we have demonstrated that isolated fragment D1 in a solid phase system forms a complex with a mixture of GPIIb and GPIIIa. The binding of the GPIIb-GPIIIa mixture to fragment D1-coated plates reached saturation at 8 nM and to fibrinogen-coated plates at 24 nM. Isolated A alpha, B beta, and gamma chains were not reactive with added glycoproteins. Fragment E coated directly on plastic plates or immobilized on antibody-coated plastic plates did not form a complex with GPIIb-GPIIIa. Only fluid phase fibrinogen and fragment D1 but not fragment E were inhibitory toward formation of a complex between solid phase fibrinogen and GPIIb-GPIIIa. Isolated A alpha, B beta, and gamma chains at concentrations equivalent to fluid phase fibrinogen were inactive. Binding of fragment D1 but not fragment E to the GPIIb-GPIIIa complex was also demonstrated by rocket immunoelectrophoresis of the membrane glycoprotein mixture through a gel containing the individual fragments and subsequent autoradiography of the complex following exposure to 125I-anti-fibrinogen. These observations with isolated platelet membrane glycoproteins provide strong evidence that each of the D domains of the fibrinogen molecule interacts directly with the GPIIb-GPIIIa complex on the activated platelet surface, thus allowing formation of a tertiary molecular "bridge" across the surface of two adjacent activated platelets.
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PMID:Complex formation of platelet membrane glycoproteins IIb and IIIa with the fibrinogen D domain. 623 15

Pretreatment of hemoglobin with 50-5000 nmol hydrogen peroxide (H2O2) increased its susceptibility to proteolysis by a number of purified enzymes, including trypsin, chymotrypsin, elastase, and plasmin, and by the neutral protease of rat peritoneal leukocytes. Pretreatment of the protein substrate with catalase-inactivated H2O2 had no effect. Separation of the proteolytic fragments by G-75 Sephadex gel filtration indicated no apparent differences in the size distribution of the fragments produced by treatment with the H2O2/proteolytic enzyme combination as compared with enzyme treatment alone. A partially purified preparation of rat glomerular basement membrane was also treated with proteolytic enzyme alone or in combination with H2O2. As with the hemoglobin, pretreatment of the glomerular basement membrane with H2O2 increased its susceptibility to subsequent proteolytic attack. In addition, treatment of a basement membrane glycoprotein, fibronectin, with H2O2 also increased its sensitivity to subsequent proteolysis. These results suggest that in addition to their other proinflammatory activities, oxygen-derived metabolites may contribute to tissue destruction by altering the susceptibility of proteins to hydrolytic enzymes.
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PMID:Protein degradation following treatment with hydrogen peroxide. 637 92

The deficiency of platelet function is the main defect of the hemostatic mechanism during cardiopulmonary bypass, which greatly exacerbates the postoperative bleeding complications. In this study, we assessed, from basic and clinical perspectives, the mechanism of relieving platelet damage by means of aprotinin. In vitro research confirmed that the addition of urokinase (40 U/ml) to platelet-rich plasma and the addition of plasmin (0.3 U/ml) to washed platelets made ristocetin-induced agglutination decline to 31.6% and 38.5% of control values, respectively. The extent of decline was positively correlated with the concentration of urokinase and plasmin. In addition, the platelet membrane glycoprotein Ib decreased to 76.4% of control value. With the addition of urokinase or plasmin to aprotinin-pretreated platelet-rich plasma or washed platelets, the changes in agglutination are not statistically significant and the decrement in glycoprotein Ib is much less marked. Further in vivo research revealed that cardiopulmonary bypass caused a decrease in plasma alpha 2-antiplasmin, indicating the fibrinolytic system activation. Meanwhile, ristocetin-induced agglutination decreased to 39.6% and platelet glycoprotein Ib decreased to 50% of preoperative values. However, with the administration of aprotinin, plasma alpha 2-antiplasmin during cardiopulmonary bypass did not change; platelet agglutination was improved, platelet glycoprotein Ib was preserved, and this consequently resulted in 46% lower blood loss after the operation. The results showed that fibrinolysis impaired platelet function, and this effect may be associated with the hydrolysis of glycoprotein Ib. Fibrinolytic activation occurred during cardiopulmonary bypass and contributed to postoperative platelet dysfunction to a great extent. Aprotinin may inhibit fibrinolysis during cardiopulmonary bypass and thus relieve the platelet damage and improve the postoperative hemostatic mechanism.
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PMID:Mechanism of the preserving effect of aprotinin on platelet function and its use in cardiac surgery. 768 94

The binding of fibrinogen to membrane glycoprotein GPIIb-IIIa on activated platelets leads to platelet aggregation. This interaction results in conformational changes in fibrinogen as evidenced by the expression of receptor-induced binding sites, RIBS, epitopes which are expressed by the bound but not the free ligand. In the present study, two RIBS epitopes have been localized. One sequence resides at gamma 112-119 and is recognized by mAb 9F9; the second is the RGDF sequence at A alpha 95-98 and is recognized by mAb 155B16. These epitopes are also exposed by adsorption of fibrinogen onto a plastic surface and digestion of the molecule by plasmin. Proteolytic exposure of the epitopes coincides with cleavage of the carboxyl-terminal aspects of the A alpha-chains to form fragment X2. The inaccessibility of the RGDF sequence at A alpha 95-98 in fibrinogen suggests that this sequence does not participate in the initial binding of the molecule to GPIIb-IIIa. The location of these RIBS epitopes suggests a model in which binding of fibrinogen to its receptor alters the conformation of the carboxyl-terminal aspects of the A alpha-chains, exposing the sequences which reside in the coiled-coil connector segments between the D and E domains of the molecule. These sequences may then serve as epitopes and may mediate unique functions of the receptor-bound molecule.
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PMID:Conformational changes in fibrinogen elicited by its interaction with platelet membrane glycoprotein GPIIb-IIIa. 769 5


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