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:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The half-life of activated protein C (APC) was 31 min in citrated blood and 18 min in whole blood. Immunoblotting analysis of citrated blood identified APC-protein C inhibitor (APC-PCI) and APC-alpha 1-antitrypsin complexes. Whole blood contained two additional APC-inhibitor complexes, one stimulated by Ca2+ and another by Mg2+. The former was identified as APC-alpha 2-macroglobulin (APC-alpha 2M) while the latter was not identified. APC-alpha 2-antiplasmin complexes (APC-alpha 2AP) were identified, comigrating with APC-PCI complexes. Purified alpha 2M and alpha 2AP inhibited APC in the presence of Ca2+ (k2 = 99 and 100 M-1 S-1, respectively. Inhibition of APC and Factor Xa by alpha 2M and inhibition of APC by alpha 2AP was stimulated by Ca2+, Mn2+, and Mg2+. Inhibition of thrombin by alpha 2M and of plasmin by alpha 2AP was not altered by EDTA or Ca2+, suggesting divalent metal ions affect APC and Factor Xa rather than the inhibitors. k2 values for the APC inhibitors and their plasma concentrations suggest that PCI and alpha 1-antitrypsin are the more important APC inhibitors and that alpha 2M and alpha 2AP are metal ion-dependent auxiliary inhibitors. Inhibitors can account for the in vivo half-life of APC.
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PMID:Identification of divalent metal ion-dependent inhibition of activated protein C by alpha 2-macroglobulin and alpha 2-antiplasmin in blood and comparisons to inhibition of factor Xa, thrombin, and plasmin. 171 32

Pediatric patients with acute lymphoblastic leukemia (ALL) are at an increased risk of thromboembolic events. Potential responsible mechanisms include the disease process itself, treatment with chemotherapeutic agents (particularly L-Asparaginase [ASP]), or a combination of the disease and treatment. We studied thrombin regulation in 26 consecutive children with ALL and 14 healthy age-matched controls by: (1) plasma concentrations of prothrombin; (2) plasma inhibition of 125I-alpha-thrombin; and (3) four biochemical markers of in vivo thrombin activation (thrombin complexed to its inhibitor antithrombin III [ATIII; TAT], prothrombin fragment 1.2 (F1.2), activated protein C complexed to the inhibitors alpha 1 antitrypsin [APCAT]), and protein C inhibitor (APC-PCI). Measurements were made at presentation before treatment, after treatment with ASP alone, and during combination chemotherapy with and without ASP. At presentation, the capacity to generate thrombin (reflected by plasma prothrombin concentrations) and the capacity to inhibit thrombin (125I-alpha-thrombin--inhibitor complex formation) were similar in children with ALL compared with that for healthy children. After ASP alone or as part of combination chemotherapy, prothrombin levels were preserved, whereas plasma inhibition of 125I-alpha-thrombin decreased significantly because of a decrease in plasma concentrations of inhibitors, most importantly ATIII. After combination chemotherapy without ASP, plasma concentrations of ATIII and the capacity to inhibit 125I-alpha-thrombin returned to normal values, whereas prothrombin levels increased above control values. Thrombin generation in vivo also differed from healthy controls. At presentation, plasma concentrations of three of four markers of in vivo thrombin activity (TAT, F1.2, APCAT, but not APC-PCI) were increased in children with ALL. Neither ASP alone nor combination chemotherapy with or without ASP significantly altered values of these three markers. In summary, although the in vitro capacity to generate thrombin was preserved, the in vitro capacity to inhibit 125I-alpha-thrombin decreased after ASP therapy. Evidence for increased endogenous thrombin generation was documented in children with ALL at presentation and throughout treatment. We speculate that poor regulation of this thrombin may contribute to thrombotic complications in children with ALL.
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PMID:Increased endogenous thrombin generation in children with acute lymphoblastic leukemia: risk of thrombotic complications in L'Asparaginase-induced antithrombin III deficiency. 828 39

A recent study indicated that Tyr99 (chymotrypsin numbering) of factor Xa and Thr99 of activated protein C are S2 subsite residues that determine the P2 specificity of their substrates and inhibitors. To investigate the contribution of Leu99 to the P2 binding specificity of thrombin, three mutants of thrombin were prepared in which Leu99 was substituted with Tyr (L99Y), Thr (L99T), or Gly (L99G). Kinetic analysis indicated that antithrombin (AT with P2 Gly) inhibited thrombin L99Y, 14.1- and 5.5-fold slower than thrombin in the absence and presence of heparin, respectively. The L99Y mutation increased the stoichiometry of AT inhibition in the presence of heparin from approximately 1.6 to approximately 4.6, indicating that L99Y recognized AT as a substrate. The inhibition rates of L99T and L99G by AT, respectively, were 500.0- and 916.7-fold slower than thrombin in the absence of heparin but only 41.8- and 64.5-fold slower than thrombin in the presence of heparin. Resolution of the two-step reactions of AT with the mutant thrombins revealed that the impaired reactivities occurred in the second reaction step in which a non-covalent AT-thrombin encounter complex is converted to a stable, covalent complex. In reactions with protein C inhibitor (PCI with P2 Phe), L99Y was inhibited 3.5-fold slower than thrombin, L99T was inhibited at a similar or faster rate, and L99G was inhibited 23.9-fold faster than thrombin. The epidermal growth factor-like domains 4-6 of thrombomodulin (TM4-6) accelerated the PCI inhibition of wild-type and L99G thrombins 73.9- and 5.3-fold, respectively. Further studies indicated that the fibrinogen clotting and protein C activation rates by the mutants were impaired, but the cofactor function of TM was not affected as TM4-6 bound to wild-type [Kd(app) = 5.9 nM] and mutant thrombins with similar affinities [Kd(app) = 4.4-6.9 nM] and enhanced protein C activation rates by all mutants effectively. These results indicate that (1) Leu99 of thrombin is critical for determination of the P2 specificity of serpins, AT and PCI, (2) increasing the polarity of the S2 pocket of thrombin by introduction of a hydrophilic residue into this pocket is detrimental for reaction with AT, but it is tolerated in reaction with PCI, so that only the size of the S2 pocket of thrombin determines the P2 specificity of PCI, and (3) the thrombomodulin-induced conformational change that results in acceleration of thrombin inhibition by PCI involves Leu99.
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PMID:Role of Leu99 of thrombin in determining the P2 specificity of serpins. 920 Jun 92

Excessive procoagulant activity in the alveolar space may play a relevant role in the pathogenesis of pulmonary fibrosis. Hypercoagulability results from the disruption of the balance between the procoagulant and anticoagulant factors. The aim of this study was to assess the levels of molecular markers of the anticoagulant protein C (PC) pathway in the bronchoalveolar lavage fluid (BALF) and plasma of 11 patients with idiopathic pulmonary fibrosis (IPF), 14 with sarcoidosis and 16 with collagen vascular disease (CVD)-associated interstitial lung disease (CVD-ILD). Six healthy nonsmoking volunteers served as control subjects. BALF concentrations of the marker of clotting activation, thrombin- antithrombin III complex (TAT), in patients with sarcoidosis and CVD-ILD were significantly greater than those in control subjects. PC levels in BALF were markedly higher in patients with IPF (610 +/- 150 ng/ml), sarcoidosis (680 +/- 170 ng/ml), and CVD-ILD (1,580 +/- 600 ng/ml) than in control subjects (230 +/- 140 ng/ml). BALF concentrations of activated PC-PC inhibitor (APC-PCI) complex were significantly decreased in IPF (0.46 +/- 0.16 ng/ml), sarcoidosis (0. 43 +/- 0.11 ng/ml), and CVD-ILD (0.50 +/- 0.15 ng/ml) patients as compared with control subjects (1.08 +/- 0.23 ng/ml). APC-PCI/PC ratios were significantly lower in patients with IPF (2.70 +/- 1.74 ng/microg), sarcoidosis (1.94 +/- 0.82 ng/microg), and CVD-ILD (1.89 +/- 0.68 ng/microg) than in control subjects (15.91 +/- 8.45 ng/microg). Plasma levels of APC- PCI and the APC-PCI/PC ratio were also significantly decreased in patients with CVD-ILD as compared with control subjects. Overall, these findings suggest that decreased PC activation with increased procoagulant activity occurs in patients with ILD.
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PMID:Protein C anticoagulant system in patients with interstitial lung disease. 962 Sep 17

The N-glycosylation site mutants of human protein C inhibitor (PCI; N230S, N243Q, N319Q, N230S/N243Q, and N230S/N319Q) were prepared by amino acid replacement of the asparagine residue with a serine or glutamine residue using site-directed mutagenesis and expressed in the baculovirus/insect cell expression system. To examine the importance of each Asn-linked glycan in the activity of PCI, we compared wtPCI with the mutants of N-glycosylation site(s) in terms of the procoagulant protease-inhibitory and anticoagulant activities. The inhibitory activities of N230S, N319Q, and N230S/N319Q toward human thrombin and plasma kallikrein were significantly increased compared with wtPCI, but those of N243Q and N230S/N243Q were reduced. The inhibitory activity of N230S toward human plasma coagulation was significantly increased compared with wtPCI, and that of N230S/N319Q was also significantly increased compared with N319Q. Furthermore, the procoagulant protease-inhibitory and anticoagulant activities of N230S/N319Q (glycosylated on Asn243 only) compared favorably with those of N230S, and both of the mutants possessed highest activities in the purified mutants. These results suggest that the Asn243-linked glycan in PCI molecule possesses critical roles for its anticoagulant activity in the circulation, and the Asn230-linked glycan down-regulates the activity of PCI.
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PMID:Role of each Asn-linked glycan in the anticoagulant activity of human protein C inhibitor. 1186 13

Antithrombin (ATIII), heparin cofactor II (HCII) and protein C inhibitor (PCI; also named plasminogen activator inhibitor-3) are serine protease inhibitors (serpins) whose thrombin inhibition activity is accelerated in the presence of glycosaminoglycans. We compared the inhibition properties of PCI and HCII to ATIII using R93A/R97A/R101A thrombin, an anion-binding exosite-2 (exosite-2) mutant that has greatly reduced heparin-binding properties. Heparin-enhanced PCI inhibition of R93A/R97A/R101A thrombin was only approximately 2-fold compared to 40-fold enhancement with wild-type recombinant thrombin. Thrombomodulin (TM) (with or without the chondroitin sulfate moiety) accelerated PCI inhibition of both wild-type and R93A/R97A/R101A thrombins. HCII achieved the same maximum activity in the presence of heparin with both wild-type and R93A/R97A/R101A thrombins; however, the optimum heparin concentration was 20 times greater than the reaction with wild-type thrombin, indicative of a decrease in heparin affinity. Dermatan sulfate (DSO4)-catalyzed HCII thrombin inhibition was unchanged in R93A/R97A/R101A thrombin compared to wild-type recombinant thrombin. These results suggest that PCI is similar to ATIII and depends upon ternary complex formation with heparin and these specific thrombin exosite-2 residues to accelerate thrombin inhibition. In contrast, HCII does not require Arg(93), Arg(97) and Arg(101) of thrombin exosite-2 and further supports the hypothesis that HCII uses an allosteric process following glycosaminoglycan binding to inhibit thrombin.
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PMID:Inhibition of a thrombin anion-binding exosite-2 mutant by the glycosaminoglycan-dependent serpins protein C inhibitor and heparin cofactor II. 1241 92

Activated protein C (APC) generation strongly affects sepsis and thrombosis by inhibition of thrombin generation. However, it is unclear if there are age-related differences in effectiveness of protein C (PC). We studied age effects on plasma APC generation +/- endothelium. Defibrinated (Ancrod) plasma (from adults or newborns (umbilical cord)) was recalcified with buffer containing tissue factor +/- thrombomodulin (TM) on either plastic or endothelium (HUVEC) at 37 degrees C. Timed subsamples of reaction mixture were taken into either heparin-EDTA or FFRCMK-EDTA solutions and analyzed for APC-PC inhibitor (APC-PCI) or APC-alpha1 antitrypsin (APC-alpha1 AT) by ELISAs. Since heparin converts free APC to APC-PCI, the difference in APC-PCI measured in heparin-EDTA and FFRCMK-EDTA samples was equal to free active APC. APC-alpha2 macroglobulin (APC-alpha2M) was measured as remaining chromogenic activity in heparin-EDTA. Free APC, APC-PCI and APC-alpha1 AT were decreased in newborn compared to adult plasma on plastic. However, APC-alpha2M made up a larger fraction of inhibitor complexes in new-born plasma. On endothelium, significantly more APC, APC-PCI and APC-alpha1AT were generated in either plasma compared to that on plastic with excess added TM. APC, APC-PCI and APC-alpha1AT were also reduced and total APC-alpha2M increased in newborn plasma on HUVEC. Addition of PC to newborn plasma gave APC generation similar to adult plasma. Thus, free APC, APC-PCI and APC-alpha1AT generation is reduced in newborn compared to adult plasma with or without endothelium, likely due to reduced plasma PC levels. Endothelium enhances APC generation, regardless of plasma type, possibly because of cell surface factors such as TM, phospholipid and endothelial PC receptor.
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PMID:Activated protein C generation is greatly decreased in plasma from newborns compared to adults in the presence or absence of endothelium. 1496 Nov 49

Primary PCI is an effective reperfusion strategy for acute MI patients, which has evolved significantly in the last decade. While many adjunctive therapies have contributed to its success, substantial obstacles remain before optimal reperfusion can be achieved. Anti-platelet therapy with aspirin, clopidogrel and GP IIb/IIIa inhibitors reduces early ischemic complications, improves microvascular function and, potentially, affects the inflammatory response to ischemic injury. Current anti-thrombin therapy with UFH can be improved with LMWH, and, possibly with direct thrombin inhibitors. A number of important aspects of this strategy, though, need still to be elucidated. We need to optimize microvascular protection before and during PCI in order to capitalize on the myocardial sparing effects of reperfusion therapy. This will be probably achieved with a combination of pharmacological interventions and mechanical emboli protection devices. Improved and more targeted anti-inflammatory therapy should decrease the effects of neutrophil-related reperfusion injury, while a variety of metabolic interventions might preserve myocardial function during ischemia and after reperfusion.
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PMID:Adjunctive therapy for percutaneous revascularization in acute myocardial infarction. 1496 1

Previously, we observed a positive association of prothrombin concentrations with thrombin generation (fragment 1 + 2) and thrombin activity (fibrinopeptide A), but no association with protein C activation peptide levels. We further evaluated a potential beneficial effect of increased prothrombin concentrations on activated protein C generation by assessing the plasma concentration of activated protein C in complex with protein C inhibitor (APC-PCI). Blood samples were used from 195 family members of a large French-Canadian kindred with type I protein C deficiency due to a 3363C insertion in the protein C gene. We utilized a new and highly sensitive assay for measuring the concentration of APC-PCI complex as a measure of the level of activation of protein C. Means of the plasma concentrations of the APC-PCI complex were compared among carriers and non-carriers of the prothrombin G20210A mutation. Protein C activity levels were positively associated with APC-PCI complex plasma concentrations; however, APC-PCI complex levels were not different for carriers of the prothrombin G20210A mutation than for non-carriers. Thus, carriers of the prothrombin G20210A mutation do not have increased protein C activation despite the increased thrombin generation resulting from the higher prothrombin concentrations associated with the G20210A mutation.
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PMID:No effect of the prothrombin G20210A mutation on protein C activation in a large kindred with type I protein C deficiency. 1538 24

The serine protease inhibitor (serpin) protein C inhibitor (PCI; also named plasminogen activator inhibitor-3) regulates serine proteases in hemostasis, fibrinolysis, and reproduction. The biochemical activity of PCI is not fully defined partly due to the lack of a convenient expression system for active rPCI. Using pET-15b plasmid, Ni(2+)-chelate and heparin-Sepharose affinity chromatography steps, we describe here the expression, purification and characterization of wild-type recombinant (wt-rPCI) and two inactive mutants, R354A (P1 residue) and T341R (P14 residue), expressed in Escherichia coli. Wild-type rPCI, but not the two mutants, formed a stable bimolecular complex with thrombin, activated protein C and urokinase. In the absence of heparin, wt-rPCI-thrombin, -activated protein C, and -urokinase inhibition rates were 56.7, 3.4, and 2.3 x 10(4) M(-1) min(-1), respectively, and the inhibition rates were accelerated 25-, 71-, and 265-fold in the presence of 10 mug/mL heparin for each respective inhibition reaction. The stoichiometry of inhibition (SI) for wt-rPCI-thrombin was 2.0, which is comparable to plasma-derived PCI. The present report describes for the first time the expression and characterization of recombinant PCI in a bacterial expression system and demonstrates the feasibility of using this system to obtain adequate amounts of biologically active rPCI for future structure-function studies.
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PMID:Characterization of recombinant human protein C inhibitor expressed in Escherichia coli. 1575 93


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