Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.7 (plasmin)
 9,023 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An in vitro model is described which utilizes human umbilical vein endothelial cells cultured on plastic microcarrier spheres and perfused with serum-free medium. This model was used to study the acute release of von Willebrand factor following stimulation of the cells with putative agonists. Thrombin, plasmin and interleukin-1 were found to release von Willebrand factor. Adrenaline and bradykinin also stimulated release but only at high dosage. 1-desamino-8-D-arginine vasopressin (DDAVP) was inactive.
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PMID:An in vitro model for the study of acute release of von Willebrand factor from human endothelial cells. 349 73

The effects of Staphylococcus aureus V8 protease (V8) on the multimeric structure of human von Willebrand factor (vWF) were studied to test and expand our model for the substructure of vWF. Electron microscopy of V8 digests of vWF revealed that the multimers were cleaved where the flexible rod (R) domains join the large elongated globular (G) domains. The resulting two major fragments, which were purified by affinity and hydrophobic interaction chromatography and by glycerol-gradient ultracentrifugation, are disulfide-linked homodimers of these domains (i.e. RR and GG) and are morphologically identical to the alternating RR and GG domains of intact vWF. The glycoprotein fragment GG (6.5 X 35 nm) has mass 343 kDa by sedimentation equilibrium and the amino-terminal sequence of intact plasma vWF. It contains the binding site for heparin within 300 residues of its amino terminus and a separate site for the platelet GPIb receptor responsible for platelet agglutination in the presence of ristocetin. With approximately 18% alpha-helix and approximately 15% beta-pleated sheet, fragment GG accounts for most of the ordered secondary structure present in whole vWF. The two thin flexible rod domains (1.8-2.0 X 30-34 nm) of fragment RR are joined at a small central nodule (approximately 5 nm diameter) and also have a small nodule at each free end. Fragment RR contains an extraordinarily high cystine content, lower than average amounts of other hydrophobic residues, and essentially no alpha-helix, as judged by circular dichroism. The amino-terminal sequence and amino acid composition of fragment RR corresponded to that of the COOH-terminal 685 residues of the intact vWF subunit (Titani, K., Kumar, S., Takio, K., Ericsson, L. H., Wade, R. D., Ashida, K., Walsh, K. A., Chopek, M. W., Sadler, J. E., and Fujikawa, K. (1986) Biochemistry 25, 3171-3184). This sequence analysis gives a mass of 180 kDa for glycosylated fragment RR, somewhat higher than the 130 kDa we obtained by sedimentation equilibrium. Our sequence analysis of a 110-kDa plasmic vWF peptide also permitted identification of a major plasmin cleavage site 705 residues from the COOH terminus and a half-cystine residue (1360) involved in maintaining the multimeric structure of plasmin-degraded vWF.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Substructure of human von Willebrand factor. Proteolysis by V8 and characterization of two functional domains. 353 10

Factor VIII (FVIII) procoagulant activity is the function of a plasma glycoprotein that is missing or inactive in patients with classic hemophilia. Numerous studies have shown that trace thrombin causes rapid enhancement followed by gradual inactivation of FVIII procoagulant activity. Recent evidence suggests that thrombin activation of the FVIII/von Willebrand factor (vWF) protein is required for inactivation to occur. All of these studies have used the one-stage partial thromboplastin time to assay FVIII activity. Other investigators have used the two-stage assay of FVIII activity and have been unable to demonstrate thrombin-induced enhancement of FVIII activity, although inactivation has consistently occurred. We performed experiments designed to help resolve this disagreement, using the two-stage assay specifically modified to detect thrombin potentiation of FVIII activity. The length of the first-stage incubation time was found to be critical in demonstrating the initial effect of thrombin on FVIII activity. Taking advantage of this finding we were able to show a 4.1 +/- 0.5-fold enhancement of FVIII activity upon incubating purified FVIII/vWF with 0.04 NIH unit thrombin per ml. The apparent enhancement of FVIII activity declined with increasing thrombin concentration. Incubation with 0.08, 0.16, and 0.32 NIH unit thrombin per ml resulted in only 3.2 +/- 0.5, 2.6 +/- 0.5 and 1.5 +/- 0.3-fold enhancement, respectively, of FVIII activity. As with results from the one-stage assay, activation was followed by slow inactivation of FVIII/vWF. Using the two-stage assay we also showed 100% inactivation and 100% inhibition of FVIII activity by plasmin and human anti-FVIII IgG, respectively. Plasmin inactivation of FVIII activity showed a dose-response effect. Thrombin was unable to activate plasmin-degraded FVIII/vWF. Our results show that thrombin potentiation of FVIII activity is easily demonstrable in the two-stage assay. These findings support the contention that activation of FVIII activity by thrombin is prerequisite for inactivation and underscore the importance of thrombin activation of FVIII/vWF in the intrinsic clotting system.
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PMID:Thrombin potentiation of factor VIII procoagulant activity: assessment by the two-stage assay. 621 66

To better define the role of carbohydrate in the structure and ristocetin cofactor activity of von Willebrand factor, we have removed up to 83% of total hexose by sequential treatment of the molecule with endo-beta-N-acetyl-glucosaminidase F (endo F), neuraminidase, and beta-galactosidase. Endo F alone removed 69% of total hexose and D-galactose, and 71% of sialic acid. However, there was no discernible loss of large multimers and the ristocetin cofactor activity was decreased by only 11%. The reduced von Willebrand factor subunit migrated more rapidly in polyacrylamide gels containing SDS, consistent with a 10% decrease of molecular mass. All multimers of unreduced carbohydrate-modified von Willebrand factor migrated more rapidly in SDS-agarose, but the triplet pattern of individual multimers was unchanged. This alteration in multimer migration rate did not resemble alterations found so far in von Willebrand disease variants. Further treatment of von Willebrand factor with neuraminidase and beta-galactosidase reduced the D-galactose to 15% and ristocetin cofactor activity to 57%. A similar decrease in ristocetin cofactor activity was seen if von Willebrand factor was treated only with neuraminidase and beta-galactosidase. In contrast, treating von Willebrand factor with neuraminidase and beta-galactosidase in the presence of protease inhibitors (20 mM benzamidine, 20 U/ml aprotonin, 15 micrograms/ml leupeptin) resulted in a comparable removal of carbohydrate with no change in ristocetin cofactor activity. Moreover, the multimeric structure remained intact in spite of 80% removal of D-galactose. This suggested that carbohydrate was protecting von Willebrand factor against traces of one or more protease contaminants. Evidence in support of this hypothesis was obtained by exposing von Willebrand factor to plasmin after pretreatment with neuraminidase alone or with neuraminidase and beta-galactosidase. A loss of large multimers was observed from von Willebrand factor that had been pretreated with neuraminidase, but this was even greater if pretreatment was also with beta-galactosidase. In contrast, the multimeric structure of von Willebrand factor with intact carbohydrate was not affected by plasmin under similar conditions. These studies suggest that carbohydrate protects von Willebrand factor from disaggregation occurring secondarily to proteolytic attack but does not play a direct role in maintaining its multimeric structure or ristocetin cofactor activity.
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PMID:Carbohydrate moiety of von Willebrand factor is not necessary for maintaining multimeric structure and ristocetin cofactor activity but protects from proteolytic degradation. 623 76

Understanding the effect of fibrinolysis on platelet function is of clinical importance. Plasmin is recognized to affect platelet adhesive function by reducing the interaction of platelet glycoprotein (GP) Ib with von Willebrand factor (vWF) bound to the subendothelium. This platelet function is commonly explored in vitro by the ristocetin-induced agglutination test. Our previous study demonstrated a plasmin-induced redistribution of GP Ib molecules from the platelet surface to the linings of the surface-connected canalicular system (SCCS), a critical mechanism for understanding plasmin-induced GP Ib dysfunction. Here, we demonstrate that neutralization of plasmin by its inhibitors, aprotinin or tripeptide Val-Phe-Lys-CH2Cl, permits a time dependent recovery (within 30 min) of ristocetin-induced agglutination in the platelets which were stimulated by plasmin at < 1 CU ml-1. This functional recovery was accompanied with a restoration of a normal amount of GP Ib on the platelet surface, as measured by the binding of both monoclonal anti-GP Ib antibody SZ 2 and 125I-labelled vWF to the platelets. Cytochalasin D did not inhibit this recovery, suggesting that this process may be due to passive actin depolymerization. These findings were further confirmed by immunoelectron microscopic study. Utilizing the platelets pre-labelled with anti-GP Ib antibody prior to plasmin stimulation, it was demonstrated that the observed recovery is due to a reverse translocation from the SCCS to the plasma membrane of the same GP Ib molecules which were present initially at the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Reversible translocation of glycoprotein Ib in plasmin-treated platelets: consequences for platelet function. 751 34

Activated protein C (APC)-protein C inhibitor (PCI) complex level was examined in 35 patients with acute pulmonary embolism (PE) and in 20 healthy volunteers. Thrombin-antithrombin III complex, plasmin alpha 2 plasmin inhibitor complex, and fibrin-D-dimer levels were significantly increased in the patients with PE compared to levels in healthy volunteers. Levels of plasminogen activator inhibitor-I, tissue type plasminogen activator, and von Willebrand factor antigens were also significantly increased in patients with PE. Plasma level of APC-PCI complex was increased in most patients with PE and APC-alpha 1 antitrypsin complex level was increased in 13 patients. These complexes were not detected in healthy volunteers. These findings suggested that plasma protein C was activated in patients with PE, and that PCI was the major inhibitor of APC generated in this condition. Thus, regulation of the protein C pathway might play an important role in the pathogenesis of PE.
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PMID:Increased activated protein C-protein C inhibitor complex levels in patients with pulmonary embolism. 751 16

In 1,800 patients undergoing cardiac surgery over a 2-year period, 11 incidents of abnormal inlet pressure elevations occurred before the membrane oxygenators. In 3 patients, the oxygenators had to be changed during cardiopulmonary bypass. This complication was found to be caused by fibrin formation possibly secondary to precipitation of fibrinogen with other coagulation factors in the heat exchangers of the oxygenators during the cooling phase. Large amounts of fibrin were demonstrated in the heat exchanger of the oxygenators. After careful washing of the apparatus, plasmin was added and fibrin was detected by measuring D-dimer levels. In heat exchangers from uneventful operations, only trace amounts of fibrin were found. Because there were no cold agglutinins demonstrated in the patients before surgery, cryoprecipitation studies were performed soon after surgery. When the patients' plasma samples were studied at different temperatures, from 37 degrees C down to 3 degrees C, cryoprecipitates or a gel (in 1 patient only) were formed. This indicated that there might be something abnormal with regard to fibrinogen-fibrin formation. The study patients were therefore investigated after the acute phase of the operation had ended for various coagulation factors, as well as for fibrin gel network characteristics. The results were compared with those of a control group (n = 10) with uneventful operations. There were no differences between the groups with regard to levels of coagulation factors VII and VIII and von Willebrand factor, although they were increased in both groups. The mean levels of coagulation inhibitors, antithrombin and Protein S, were slightly lower in the study patients. All of these patients had a highly pathologic, ie, tight fibrin gel network, except for the patient in whose sample a gel formed, despite being treated with aspirin or oral anticoagulants. The network was also tighter in some of the controls (v middle-aged reference individuals), although it was significantly tighter in the patients. It is concluded that some individuals who have an increased tendency to form tighter fibrin gel networks might be at increased risk for a severe complication during cardiac surgery performed under hypothermia.
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PMID:Pathologic fibrin formation and cold-induced clotting of membrane oxygenators during cardiopulmonary bypass. 771 53

The aim of this study was to compare the secretory response of the vascular wall in vivo to DDAVP (i.v. 0.3 microgram/kg, 30 min) and to venous occlusion (VO, 20 min) in control healthy subjects, patients with von Willebrand's disease type I (vWd I) and patients with von Willebrand's disease type III (vWd III). In controls (n = 10) and vWd I (n = 12), DDAVP induced a 2 to 3-fold rise in plasma von Willebrand factor antigen (vWf: Ag), factor VIII coagulant activity (VIII: C) and tissue--type plasminogen activator antigen (t-PA:Ag). VO was less effective in increasing vWf: Ag and VIII:C but produced a greater rise in t-PA:Ag. Large increments (over 10-fold) were observed in plasmin-alpha 2-antiplasmin complexes following both stimuli. In vWd III (n = 10), DDAVP and VO failed to increase vWf:Ag, VIII:C and t-PA:Ag. No significant changes in plasmin-alpha 2-antiplasmin complexes were observed in this group. Moreover, the baseline t-PA:Ag values were significantly lower in vWd III (2.17 +/- 1.13 ng/ml) than in controls (4.84 +/- 1.97 ng/ml, p < 0.001). A significant increase in urokinase--type plasminogen activator antigen (u-PA:Ag) was found only in controls after VO. Neither controls nor patients with vWd showed any changes in plasma fibronectin levels following DDAVP. The low t-PA:Ag results and the abnormal fibrinolytic response to DDAVP and VO in patients with severe (type III) vWd indicate that their endothelial cell abnormality is more extensive than the defect in the synthesis or release of vWf.
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PMID:Secretory response of the vessel wall to DDAVP and venous occlusion in von Willebrand's disease. 799 99

We studied the effect of antiplatelet therapy not only on the secondary prevention of stroke but also on the suppression of vascular damages in patients with cerebral thrombosis at the chronic phase. We measured von Willebrand factor (vWF) as a marker for the endothelial system, and coagulation and fibrinolytic parameters in addition to platelet functions. The platelet aggregation and markers for platelet activation were monitored for the adequate inhibition of platelets. Twenty-one patients were treated with 200 mg ticlopidine. 9 patients with 100 mg ticlopidine and 60-150 mg acetylsalicylic acid, and 18 patients with 200 mg cilostazol daily. The mean duration of follow up was 8.4 +/- 3.0 months. A patient was attacked by a recurrent stroke, but no fatal vascular events occurred during the period. A significant decrease was observed in the collagen- and ADP-induced platelet aggregation and markers for platelet activation such as platelet factor 4 (PF4) and beta-thromboglobulin (beta TG) by the antiplatelet therapy. In addition, the activities of coagulation factor VIII (FVIII) and vWF, markers for vascular damages, showed a significant decrease. The results suggest that the antiplatelet therapy could ameliorate the vascular damage through the inhibition of platelet function. Moreover, thrombin-antithrombin III complex (TAT) and alpha 2-plasmin inhibitor-plasmin complex (PIC), markers for the activation of coagulation and fibrinolytic systems, decreased significantly, suggesting that the treatment inhibits the activation of coagulation and fibrinolytic systems induced by the platelet activation. The activities of FVIII and vWF decreased significantly when the level of beta TG or that of PF4 lowered sufficiently by the treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[Antiplatelet therapy in patients with cerebral thrombosis at the chronic phase--assessment of its effect on coagulation and fibrinolytic parameters]. 799 82

High-molecular-weight von Willebrand factor (vWf) multimers were separated from their smaller multimers by molecular-sieve chromatography and were measured by several sandwich enzyme-linked immunosorbent assays (ELISA) with monoclonal antibodies (moABs) against human vWf. The epitopes of these moABs were mapped partially using fragments generated by V-8 protease digests of native vWf. Large multimers were more immunoreactive with the sandwich ELISA using immobilized VW28-1 and enzyme-labeled VW92-3 than with any other ELISA. The epitopes recognized by these two moABs were sensitive to trypsin and plasmin digestion, and the other moABs appeared to be reactive with the extensively digested antigen. Relative reactivities of this ELISA to plasma vWf multimers in diabetes mellitus were significantly reduced compared to those in healthy subjects. These data demonstrate that molecular abnormalities of vWf circulating in diabetic patients may be caused by some plasma proteases that increase in the microcirculation of patients with diabetic vascular diseases.
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PMID:Immunoenzymometric analysis for plasma von Willebrand factor degradation in diabetes mellitus using monoclonal antibodies recognizing protease-sensitive sites. 807 70


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