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)

A new cell line (LC-1/sq) of human lung squamous-cell carcinoma was established from a surgically resected specimen of primary lung cancer. Upon continuous propagation in serum-free culture medium, it secreted trypsin inhibitors into the conditioned medium. The major fraction of the trypsin inhibitor (T1-1) was purified to apparent homogeneity by anion-exchange and gel-filtration high-performance liquid chromatography (HPLC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by transblotting to Immobilon. T1-1 effectively inhibited trypsin. Chymotrypsin, plasmin and kallikrein were inhibited to a lesser extent, but urokinase-type plasminogen activator, elastase, thrombin and papain were not inhibited. The activity of T1-1 was acid-stable and heat-resistant, and its molecular weight was 115 kDa by SDS-PAGE. It exhibited single NH2-terminal sequence, and its first 20 NH2-terminal amino-acid residues were identical with those of protease nexin-II (PN-II)/amyloid beta-protein precursor (APP). These characteristics of T1-1 suggest that the major trypsin inhibitor secreted by LC-1/sq is indistinguishable from PN-II/APP. LC-1/sq is the first lung squamous carcinoma cell line that secretes functionally active trypsin inhibitor, PN-II/APP, in vitro and is useful for studying its biological significance in malignant tumor.
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PMID:Establishment of a new human cancer cell line secreting protease nexin-II/amyloid beta protein precursor derived from squamous-cell carcinoma of lung. 191 42

Protease nexin-2 (PN-2) is a protease inhibitor that is synthesized and secreted by a variety of extravascular cells including human fibroblasts. It forms sodium dodecyl sulfate-stable complexes with trypsin, the epidermal growth factor binding protein and the gamma-subunit of nerve growth factor. Recently we reported that PN-2 is the secreted form of the amyloid beta-protein precursor (APP) and is a potent inhibitor of chymotrypsin. Here we describe a two-step procedure to purify PN-2/APP using a monoclonal antibody immunoaffinity column. We also quantitated the protease inhibitory properties of purified PN-2/APP on a number of serine proteases. PN-2/APP was a potent inhibitor of coagulation factor XIa with a Ki = 2.9 x 10(-10). The inhibition of factor XIa by PN-2/APP was augmented by heparin and resulted in a Ki = 5.5 x 10(-11) M. Trypsin and chymotrypsin were also effectively inhibited with a Ki = 4.2 x 10(-10) and 1.6 x 10(-9), respectively. PN-2/APP also inhibited the epidermal growth factor binding protein, the gamma-subunit of nerve growth factor, and chymase and plasmin to a lesser extent. In view of recent findings that PN-2/APP is contained in alpha-granules of platelets and is secreted upon platelet activation, the potent inhibition of factor XIa suggests that PN-2/APP may play a regulatory role in the coagulation pathway at vascular wound sites. In addition, these studies define biochemical activities of PN-2/APP which may be involved in regulating proteases that lead to the generation and deposition of the beta-protein in neurodegenerative lesions associated with Alzheimer's disease and Down's syndrome.
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PMID:Immunopurification and protease inhibitory properties of protease nexin-2/amyloid beta-protein precursor. 211 43

Senile plaques, often surrounded by abnormally grown neurites, are characteristic of Alzheimer's diseased brain. The core of the plaque is mainly composed of amyloid beta protein (beta-AP), two of whose three precursors (APP) have serine proteinase inhibitor regions (APPI). APPI derivatives containing 60, 72 or 88 amino-acid fragments (APPI-60, APPI-72 and APPI-88, respectively) of the longest APP were produced in COS-1 cell culture medium, with the APPI cDNA ligated to the signal sequence of tissue plasminogen activator. The secreted APPIs were purified by sequential acetone precipitation followed by affinity chromatography using immobilized trypsin. These three APPIs and O-glycosylation-site-mutated APPI showed similar inhibitory activity against trypsin, chymotrypsin and plasmin. The purified APPI-72 was found to inhibit trypsin (Ki = 1.1 x 10(-10) M) and chymotrypsin (Ki = 5.8 x 10(-9) M) most strongly, and to inhibit leukocyte elastase (Ki = 7.9 x 10(-7) M) and several blood coagulation proteinases (Ki = 0.46-12 x 10(-7) M), but not urokinase or thrombin. The observed inhibition pattern was quite different from that of protease nexin I, one of serine proteinase inhibitors possessing neurite outgrowth activity. This suggests that the physiological roles of APPI are different from those of protease nexin I, and that APPI could not cause aberrant growth of neurite into the plaque. The presence of APPI having strong inhibitory activity in the brain might lead to the formation of amyloid deposits by preventing complete degradation of APPs.
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PMID:Enzyme specificity of proteinase inhibitor region in amyloid precursor protein of Alzheimer's disease: different properties compared with protease nexin I. 218 Apr 85

The Alzheimer's disease related protein, amyloid beta-protein precursor (A beta PP), contains a domain homologous to Kunitz-type serine protease inhibitors (KPI). The recombinant KPI domain of A beta PP is a potent inhibitor of coagulation factors XIa and IXa and functions as an anticoagulant in vitro. Here we report the expression, purification, and characterization of a reactive center lysine mutant of the KPI domain of A beta PP (KPI-Lys17). An expression plasmid for the KPI-Lys17 domain of A beta PP encoded amino acids 285-345 of the A beta PP cDNA containing a lysine substitution at arginine 17 in the KPI domain. The secreted 61-amino acid product was purified to homogeneity and functionally characterized. The protease inhibitory properties of the KPI-Lys17 domain were compared to those of the native KPI domain of A beta PP. Both KPI domains equally inhibited trypsin, chymotrypsin, and coagulation factors IXa and Xa. However, the KPI-Lys17 domain was an approximately 25-fold less effective inhibitor of coagulation factor XIa resulting in markedly less prolongation of the activated partial thromboplastin time compared to the native KPI domain of A beta PP. On the other hand, the KPI-Lys17 domain was an approximately 10- and 5-fold better inhibitor of plasmin in a chromogenic substrate assay and in a fibrinolytic assay, respectively, than the native KPI domain of A beta PP. Together, these studies suggest that the KPI-Lys17 domain has enhanced anti-fibrinolytic and diminished factor XIa inhibitory properties compared to the native KPI domain of A beta PP.
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PMID:Enhanced plasmin inhibition by a reactive center lysine mutant of the Kunitz-type protease inhibitor domain of the amyloid beta-protein precursor. 755 14

Phage displaying APPI Kunitz domain libraries have been used to design potent and selective active site inhibitors of human plasma kallikrein, a serine protease that plays an important role in both inflammation and coagulation. Selected clones from two Kunitz domain libraries randomized at or near the binding loop (positions 11-13, 15-19, and 34) were sequenced following five rounds of selection on immobilized plasma kallikrein. Invariant preferences for Arg at position 15 and His at position 18 were found, whereas His, Ala, Ala, and Pro were highly preferred residues at positions 13, 16, 17, and 19, respectively. At position 11 Pro, Asp, and Glu were favored, while hydrophobic residues were preferred at position 34. Selected variants, purified by trypsin affinity chromatography and reverse phase high performance liquid chromatography, potently inhibited plasma kallikrein, with apparent equilibrium dissociation constants (Ki*) ranging from approximately 75 to 300 pM. From sequence and activity data, consensus mutants were constructed by site directed mutagenesis. One such mutant, KALI-DY, which differed from APPI at 6 key residues (T11D, P13H, M17A, I18H, S19P, and F34Y), inhibited plasma kallikrein with a Ki* = 15 +/- 14 pM, representing an increase in binding affinity of more than 10,000-fold compared to APPI. Similar to APPI, the variants also inhibited Factor XIa with high affinity, with Ki* values ranging from approximately 0.3 to 15 nM; KALI-DY inhibited Factor XIa with a Ki* = 8.2 +/- 3.5 nM. KALI-DY did not inhibit plasmin, thrombin, Factor Xa, Factor XIIa, activated protein C, or tissue factor. Factor VIIa. Consistent with the protease specificity profile, KALI-DY did not prolong the clotting time in a prothrombin time assay, but did prolong the clotting time in an activated partial thromboplastin time assay > 3.5-fold at 1 microM.
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PMID:Potent and selective Kunitz domain inhibitors of plasma kallikrein designed by phage display. 759 8

In this report we describe the use of the methylotrophic industrial yeast Pichia pastoris as a host system for the large scale production of the Kunitz-type proteinase inhibitor (KPI) domain of the amyloid beta-protein precursor-like protein-2 (APLP-2). The expression plasmid for the KPI domain of APLP-2 encoded amino acids 305-364 of the APLP-2 cDNA (Slunt et al. (1994) J. Biol. Chem. 269, 2637-2644). The secreted 60 amino-acid product was purified to homogeneity and biochemically characterized. Amino-acid sequencing of the expressed KPI domain of APLP-2 verified its integrity. The proteinase inhibitory properties of the KPI domain of APLP-2 were compared to those of the KPI domain of proteinase nexin-2/amyloid beta-protein precursor (PN-2/A beta PP). Both KPI domains potently inhibited trypsin and, to a lesser extent, chymotrypsin, plasmin, and coagulation factors XIa and IXa. However, the KPI domain of APLP-2 was a approximately 20-fold less effective inhibitor of coagulation factor XIa compared to the KPI domain of PN-2/A beta PP. Similarly, the KPI domain of APLP-2 was a less effective anticoagulant in coagulation based assays than the KPI domain of PN-2/A beta PP. These studies indicate that the KPI domains of PN-2/A beta PP and APLP-2 form a family of proteinase inhibitors although the former is a better inhibitor of factor XIa and a more potent anticoagulant than the latter.
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PMID:Expression, purification, and characterization of the Kunitz-type proteinase inhibitor domain of the amyloid beta-protein precursor-like protein-2. 781 86

Amyloid precursor protein forms that contain Kunitz protease inhibitor domains are released from activated platelets, T-lymphocytes, and leukocytes and inhibit trypsin, plasmin, and activated factor XI. We investigated the effects of amyloid precursor protein isoforms on activated Hageman factor (factor XII), activated factor X (Stuart factor), and thrombin. Recombinant amyloid precursor proteins with or without the Kunitz domain, 770 and 695 amino acids, respectively, were produced in insect cells by Baculovirus expression (BAC770 and BAC695). Neither BAC695 nor BAC770 inhibited human alpha-thrombin or activated factor X. The partial thromboplastin time was prolonged by both amyloid precursor proteins, only one of which, BAC770, contains the Kunitz protease inhibitor domain. Both forms of amyloid precursor proteins inhibited ellagic acid-induced activation of Hageman factor but did not inhibit activated Hageman factor. Bismuth subgallate, which is an insoluble analog of ellagic acid, lost its ability to activate Hageman factor on being exposed to BAC770. Inhibition of ellagic acid-induced activation of Hageman factor by both forms of amyloid precursor protein was enhanced by heparin. These findings suggested that the heparin-binding domain of amyloid precursor proteins is not in the Kunitz domain. This heparin-binding domain may block the activation of Hageman factor by negatively charged agents. Thus, amyloid precursor proteins may be involved in the control of hemostasis, properties not all dependent on the Kunitz domain.
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PMID:Inhibitory action of amyloid precursor protein against human Hageman factor (factor XII). 784 73

Amyloid beta protein (beta/A4) is deposited in senile plaques of patients with Alzheimer's disease. This protein is derived from a larger membrane-associated protein, termed amyloid precursor protein (APP). The constitutive processing of APP occurs at the central portion of beta/A4, resulting in the release of large N-terminal peptides. We have purified these peptides from the culture medium of cDNA-transfected COS-1 cells. Some of the isoforms contain the Kunitz-type protease inhibitor (KPI) domain and strongly inhibit trypsin, chymotrypsin and plasmin, but do not inhibit kallikrein, prolyl endopeptidase or granzyme A. The peptides also do not inhibit cysteine proteases such as cathepsin B or calpain. Soluble APPs lacking the KPI domain fail to inhibit any of these proteases. The results indicate that the KPI domain in soluble APPs has protease inhibitory activity against certain serine proteases.
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PMID:Inhibitory spectra of purified protease nexin-II and related proteins towards cellular proteinases. 790 50

Potent active-site inhibitors of human tissue factor-Factor VIIa (TF.FVIIa) have been selected from Alzheimer's amyloid beta-protein precursor inhibitor (APPI) Kunitz domain libraries displayed on phage. Eight randomized positions on the extended primary binding loop (P5 through P4') and positions 34 and 39 were examined in three separate libraries. Libraries contained from 3.2 x 10(5) to 3.2 x 10(6) potential variants resulting from replacing up to 5 positions with all 20 amino acids. Following 4 rounds of selection against FVIIa associated with immobilized tissue factor (TF), 12 clones from each library were sequenced. Variants were purified by trypsin affinity chromatography and reverse-phase high performance liquid chromatography, and characterized for their ability to inhibit TF.FVIIa chromogenic activity. Measured apparent equilibrium dissociation constants (Ki*) ranged from about 10 to 500 nM. From sequence and activity data, an overall consensus sequence, TF7I-C, was constructed by site-directed mutagenesis. TF7I-C differed from APPI at 4 key residues, T11P, M17L, S19L, and G39Y, and inhibited TF.FVIIa with a Ki* = 1.9 +/- 0.4 nM, which represented an increase in binding affinity of more than 150-fold compared to APPI. At 40 microM, TF7I-C prolonged the clotting times 3.5-fold in a prothrombin time assay and > 10-fold at 7 microM in an activated partial thromboplastin time assay. Prolongation of the activated partial thromboplastin time correlates with potent inhibition of FXIa (Ki* = 0.8 nM) and plasma kallikrein (Ki* = 1.2 nM). TF7I-C also inhibited plasmin (Ki* = 40 nM) and FXa (Ki* = 55 nM), but not activated protein C, thrombin, or FXIIa (Ki* > 10 microM each).
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PMID:Kunitz domain inhibitors of tissue factor-factor VIIa. I. Potent inhibitors selected from libraries by phage display. 807 37

Active-site inhibitors of the human tissue factor-Factor VIIa complex (TF.FVIIa) that are both potent and specific have been selected from Alzheimer's amyloid beta-protein precursor inhibitor (APPI) Kunitz domain libraries using a strategy termed competitive phage selection. Phage display Libraries I-III, previously sorted by direct selection, were sorted by competitive selection against immobilized TF.FVIIa in which increasing amounts of Factor XIa (FXIa) were added to the selection buffer to remove phage with high affinity for FXIa. The most striking difference in the selected Kunitz domain sequences was at the P4' position where Lys was highly preferred instead of Leu which was found here by direct selection (Dennis, M. S., and Lazarus, R. A. (1994) J. Biol. Chem. 269, 22129-22136). In addition, both Lys and Arg were selected in the P1 position as opposed to Arg alone. A new library (Library IV) was constructed which contained all previously observed amino acids at 9 positions in the primary and secondary binding loops of APPI. Comparable results were obtained by sorting Library IV against immobilized TF.FVIIa in the presence of either FXIa alone or FXIa, plasma kallikrein, and plasmin. The Kunitz domains obtained by either competitive selection strategy potently inhibited TF.FVIIa, with Ki* values ranging from about 2 to 20 nM; the Ki* values were generally > 1 microM for FXIa and plasma kallikrein and ranged from 4 to 200 nM for plasmin. Variants selective for TF.FVIIa were assayed for free FVIIa and FXa inhibitory activity and characterized in coagulation assays. A rationale for the selection of Lys at both the P4' and P1 positions based upon comparison of sequences and structures of relevant serine proteases and inhibitors is presented.
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PMID:Kunitz domain inhibitors of tissue factor-factor VIIa. II. Potent and specific inhibitors by competitive phage selection. 807 38


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