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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The coagulation changes observed in acute experimental pancreatitis were compared with those after the intravenous infusion of pancreatic juice and ascitic fluid exudate obtained from bile-induced pancreatitis in dogs. The coagulation changes after acute pancreatitis was induced by the intraductal injection of autologous bile, trypsin or elastase showed decreased platelet counts, decreased plasma fibrinogen levels, prolonged partial thromboplastin and prothrombin times, shortened euglobulin clot lysis time and increased fibrin degradation products. Multiple microemboli were observed in the lung and, occasionally, in the kidney, an indication of consumption coagulopathy. The effects upon blood coagulation after the intravenous injection of pancreatic juice included decreased platelet counts, decreased plasma fibrinogen levels and prolonged partial thromboplastin and prothrombin times. The intravenous injection of pancreatic exudate produced greater changes than did those of an equal amount of pancreatic juice. There was a shortening of euglobulin clot lysis time and a marked increase in fibrin degradation products. Pancreatic exudate which accumulates during acute pancreatitis may contain a toxic substance or substances which contribute to the consumption of coagulation factors during acute pancreatitis.
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PMID:The effects upon blood coagulation in dogs of experimentally induced pancreatitis and the infusion of pancreatic juice. 726 8

beta/gamma-thrombin containing 25% of beta-form and 75% of gamma-form was obtained from alpha-thrombin by limited trypsin proteolysis. As compared to the parent alpha-thrombin, these preparations preserved less than 0.02% of fibrinogen-clotting activity, the entire esterase activity and 27% of the amidolytic activity toward N-benzoyl-Phe-Val-Arg-p-nitroanilide. Like alpha-thrombin, beta/gamma-thrombin splits prothrombin to prethrombin I (Mr 58000) and fragment I of prothrombin (Mr 25000) possessing antithrombin activity. Kinetics of prothrombin hydrolysis was studied by different concentrations of enzymes. It was shown that alpha-form is more specific than beta/gamma-form that preserved approximately 5% of prothrombinolytic activity of alpha-thrombin. It is emphasized that beta/gamma-form devoid of clotting activity retains regulatory functions.
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PMID:[Proteolysis of prothrombin by beta/gamma thrombin]. 738 59

The tick Boophilus microplus contains a protein that inhibits a range of proteolytic enzymes. Variations in the concentration of this protein throughtout the life cycle were followed by measuring simultaneously the inhibition of trypsin and chymotrypsin and reaction with an antiserum to the purified inhibitor. The protein is present in large amounts in eggs and in unfed larvae, but its concentration falls very rapidly after the start of the parasitic stage of the life cycle. This, together with previous evidence, suggests that the inhibitor is important both in eggs and in the initial establishment of the parasite on its host. The activity of the protein towards several enzymes has been measured as an indication of its possible function. Bovine trypsin, chymotrypsin and plasmin and pig pancreatic kallikrein are all inhibited. The protein also affects the blood-coagulation system at several points, since it prolongs both activated-partial-thromboplastin time and prothrombin time. It inhibits the complement-dependent lysis of erythrocytes, but is without significant effect on mitogen-induced lymphocyte stimulation. Thus the inhibitor could have several effects on the host that would be beneficial to the parasite.
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PMID:On the biological role of a proteolytic-enzyme inhibitor from the ectoparasitic tick Boophilus microplus. 745 13

Two forms of Factor C cDNAs: CrFC21 (3448 bp) and CrFC26 (4182 bp) have been cloned into lambda gt22. CrFC26 includes 568 nucleotides of 5' untranslated region (5' UTR) containing seven ATGs before the real initiation site, an open reading frame (ORF) of 3249 nucleotides, a stop codon, and 365 nucleotides of 3' untranslated sequence. There are four polyadenylation signals and six potential glycosylation sites. The ORF codes for a signal peptide of 24 amino acids and a Factor C zymogen of 1059 residues. The CrFC21 lacks most of the 5' UTR, and has some base changes in its ORF. The predicted secondary mRNA structures of the 5' end of CrFC26 showed numerous stem-and-loop structures, thus obscuring its real start codon. In contrast, CrFC21 has a well-exposed AUG start site, and expresses Factor C in transcription-translation reactions in vitro. There is a typical serine protease catalytic triad of Asp-His-Ser, which is structurally like prothrombin, but catalytically more similar to trypsin. Although an overall homology of 97.7% was observed in comparison with the Tachypleus tridentatus Factor C (TtFC) cDNA, there were notable differences in the restriction sites and subtle base substitutions in the CrFC cDNA. The high degree of homology between Factor C from T. tridentatus and C. rotundicauda substantiates, at the molecular level, the proximity of these two species in the course of evolution. This finding contravenes the apparent disparities with respect to their morphology, ecological habitat, and taxonomical classification.
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PMID:Molecular cloning and sequence analysis of factor C cDNA from the Singapore horseshoe crab, Carcinoscorpius rotundicauda. 753 1

Hereditary deficiency of factor IXa (fIXa), a key enzyme in blood coagulation, causes hemophilia B, a severe X chromosome-linked bleeding disorder afflicting 1 in 30,000 males; clinical studies have identified nearly 500 deleterious variants. The x-ray structure of porcine fIXa described here shows the atomic origins of the disease, while the spatial distribution of mutation sites suggests a structural model for factor X activation by phospholipid-bound fIXa and cofactor VIIIa. The 3.0-A-resolution diffraction data clearly show the structures of the serine proteinase module and the two preceding epidermal growth factor (EGF)-like modules; the N-terminal Gla module is partially disordered. The catalytic module, with covalent inhibitor D-Phe-1I-Pro-2I-Arg-3I chloromethyl ketone, most closely resembles fXa but differs significantly at several positions. Particularly noteworthy is the strained conformation of Glu-388, a residue strictly conserved in known fIXa sequences but conserved as Gly among other trypsin-like serine proteinases. Flexibility apparent in electron density together with modeling studies suggests that this may cause incomplete active site formation, even after zymogen, and hence the low catalytic activity of fIXa. The principal axes of the oblong EGF-like domains define an angle of 110 degrees, stabilized by a strictly conserved and fIX-specific interdomain salt bridge. The disorder of the Gla module, whose hydrophobic helix is apparent in electron density, can be attributed to the absence of calcium in the crystals; we have modeled the Gla module in its calcium form by using prothrombin fragment 1. The arched module arrangement agrees with fluorescence energy transfer experiments. Most hemophilic mutation sites of surface fIX residues occur on the concave surface of the bent molecule and suggest a plausible model for the membrane-bound ternary fIXa-FVIIIa-fX complex structure: fIXa and an equivalently arranged fX arch across an underlying fVIIIa subdomain from opposite sides; the stabilizing fVIIIa interactions force the catalytic modules together, completing fIXa active site formation and catalytic enhancement.
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PMID:X-ray structure of clotting factor IXa: active site and module structure related to Xase activity and hemophilia B. 756 20

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

Cerastocytin, a thrombin-like enzyme from the venom of the desert viper, Cerastes cerastes, has been purified to homogeneity by fast performance liquid chromatography (FPLC) on Mono-Q and Mono-S columns. It is a basic protein (isoelectric point higher than 9) made of a single polypeptide chain of 38 kDa. Its N-terminal polypeptide sequence shows strong similarities with other thrombin-like enzymes from snake venoms. Nanomolar concentrations of cerastocytin induce aggregation of blood platelets. This activity is inhibited by chlorpromazine, theophylline and mepacrine, as in the case of platelet aggregation stimulated by low doses of thrombin. Cerastocytin also possesses an amidolytic activity measured with the thrombin chromogenic substrate S-2238. The platelet aggregating activity and the amidolytic activity of cerastocytin were inhibited by PMSF, TPCK, TLCK and soybean trypsin inhibitors, suggesting that cerastocytin is a serine proteinase. On the other hand, both amidolytic activity and platelet aggregating activity of cerastocytin were unaffected by hirudin or by antithrombin III in the presence of heparin. High concentrations of cerastocytin (1-10 microM) also cleaved prothrombin and Factor X.
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PMID:Cerastocytin, a new thrombin-like platelet activator from the venom of the Tunisian viper Cerastes cerastes. 776 51

Prothrombin is a vitamin K-dependent plasma protein composed of several functional domains, which is proteolytically activated into thrombin by factor Xa in the presence of factor Va, Ca2+, and phospholipids. During the activation, prothrombin is cleaved into three fragments: fragment 1, containing a domain rich in gamma-carboxyglutamic acid residues and kringle 1 domain; fragment 2, containing the kringle 2 domain; and a protease catalytic domain, thrombin. Here we studied the interaction site for factor Xa in human prothrombin during the activation. The isolated fragment 2 inhibited the activation of prothrombin by either prothrombinase complex or factor Xa alone in a dose-dependent manner, whereas fragment 1 and diisopropylphosphate (DIP)-thrombin did not. Factor Xa directly bound to fragment 2 immobilized to microwell plates with a Kd of 9.0 x 10(-8) M, but not to fragment 1 or DIP-thrombin. Factor Xa also bound to immobilized prothrombin and prethrombin 1 with Kds of 2.0 x 10(-7) and 1.5 x 10(-7) M, respectively, suggesting that factor Xa interacts with the kringle 2 domain in these molecules. The binding of factor Xa to immobilized fragment 2 was Ca(2+)-dependent with an optimal concentration at 6 mM. In the presence of Ca2+, the interaction was enhanced by phospholipids in a concentration-dependent manner. To localize the factor Xa-binding site in the kringle 2 domain, fragment 2 was digested with lysyl endopeptidase and then trypsin after reduction and S-carboxymethylation. The resulting peptides were immobilized to microwell plates and assayed for factor Xa binding ability. The amino acid sequence of the peptide positive in the assay was determined to be residues His205 to Arg220. Factor Xa bound to a synthetic peptide corresponding to the residues His205 to Arg220 immobilized to microwell plates. The peptide inhibited factor Xa-catalyzed activation of prothrombin, but a peptide with the reversed sequence of His205 to Arg220 did not. These findings indicate that factor Xa interacts with at least a linear sequence, His205 to Arg220, in the kringle 2 domain of prothrombin during its activation into thrombin.
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PMID:Blood coagulation factor Xa interacts with a linear sequence of the kringle 2 domain of prothrombin. 785 76

To study the role of a putative high affinity Ca2+ binding site in the protease domain of factor X, we prepared a deletion mutant (E2FX) lacking the Gla and first epidermal growth factor-like domains. E2FX possesses a single high affinity Ca2+ binding site (Kd = 154 microM). Asp-70 or Glu-80 (chymotrypsin numbering system) was then converted to Lys. These mutants, E2FXD70K and E2FXE80K, allow examination of the Ca(2+)-dependent assembly of activation complexes involving substrate and enzyme forms of factor X that do not bind Ca2+. E2FXD70K and E2FXE80K share similar properties. They retain functional activity, but the D70K mutant no longer binds Ca2+, and neither mutant exhibits a Ca(2+)-dependent increase in peptide chromogenic substrate activity. Activation of E2FXD70K by the soluble tissue factor-factor VIIa complex was still Ca(2+)-dependent (Kd(app) = 133 +/- 38 microM), but the Kd(app) for Ca2+ was decreased relative to E2FX (Kd(app) = 205 +/- 53 microM). Prothrombin or prethrombin 1 activation by the E2FXaD70K in the presence of factor Va was also Ca(2+)-dependent (Kd(app) = 124 +/- 47 microM and 102 +/- 38 microM, respectively), and the Kd(app) was again lower than that of E2FXa (272 +/- 79 microM and 179 +/- 54 microM, respectively). In the absence of factor Va, activation of prethrombin 1 by E2FXaD70K was not influenced by Ca2+, but activation by E2FXa was enhanced (Kd(app) = 161 +/- 32 microM). The results suggest the presence of functionally important Ca2+ binding sites in components of the factor X and prothrombin activation complexes that do not involve factor X. Furthermore, the Ca2+ binding sites in factor X and trypsin appear to be structurally similar. The Asp or Glu-->Lys mutations within this site may create an internal salt bridge that conserves factor X (Xa) function even in the absence of Ca2+.
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PMID:Asp-70-->Lys mutant of factor X lacks high affinity Ca2+ binding site yet retains function. 806 84

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


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