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 generated a series of libraries having variants of the first Kunitz domain of human lipoprotein-associated coagulation inhibitor (LACI-D1, also known as tissue-factor pathway inhibitor-I) displayed on bacteriophage M13 as pIII-fusions. We varied LACI-DI iteratively in two regions: the P1 region (positions 10-21) and the "second loop", (positions 31-39), which together form one end of the domain. Display-phage library Lib#1 allows 31 200 amino-acid sequences in P1 region (residues 13, 16-19). Preliminary, we screened Lib#1 against human plasmin (PLA, EC 3.4.21.7) immobolized on agarose to enrich for phage displaying variants with PLA affinity. We introduced a 1600-fold increase in second-loop diversity (residues 31, 32, 34, 39) into the population of selectants from Lib#1, yielding Lib#2. Lib#2 (allowing approximately 50 million amino-acid sequences) was screened against PLA-agarose to isolate highest affinity binders. Protein EPI-P211, derived from the best isolate of Lib#2, inhibits PLA with Ki = 2 nM (at least 500-fold better than LACI-D1) and with high specificity. We used amino-acid sequences of PLA-binding selectants to design a PLA-biased library (Lib#3) which we screened against PLA. The protein EPI-P302 (derived from the best binder obtained from Lib#3) has Ki for PLA inhibition of 87 pM, which is 25-fold better than the first-round best binder and > or = 12 500-fold better than LACI-D1. EPI-P302 also shows very high specificity for PLA vs other human proteases and is resistant to inactivation by oxidants and extremes of temperature or pH. Thus, one can use selectants from one library to design target-tailored combinatorial libraries and obtain quite stable, highly specific, very high-affinity binding molecules while maintaining an essentially human framework.
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PMID:Iterative optimization of high-affinity proteases inhibitors using phage display. 1. Plasmin. 867 9

As discussed in the accompanying paper [Markland, W., Ley, A. C., & Ladner, R. C. (1996) Biochemistry 35, 8045-8057], we generated libraries from the first Kunitz domain of human lipoprotein-associated coagulation inhibitor (LACI-D1) using multivalent M13 III display and derived potent inhibitors of human plasmin (PLA) by iterative variegation and selection. Here, we show that high-affinity, high-specificity binders to human plasma kallikrein (pKAL) and human thrombin (THBN) can be obtained starting from the identical library and employing the same iterative variegation procedures used to obtain PLA inhibitors. Lib#1 (allowing 31 200 variants involving five positions near the P1 residue of LACI-D1) and its pKAL-biased derivative, Lib#4 (allowing an additional 1600 variants at residues 31, 32, 34, and 39), were screened against pKAL, yielding potent inhibitors. One of these, EPI-K401, has Ki = 284 pM, very high specificity, and excellent stability. We used information from Lib#4 selectants to design Lib#5 (allowing 1.5 x 10(6) amino-acid sequences involving nine varied positions) from which we obtained an inhibitor (EPI-K503) having high affinity for pKAL (Ki = 40 pM) and retaining the high specificity of EPI-K401. When we screened Lib#1 and its THBN-tailored derivative, Lib#6, against THBN, we obtained a different and very homogeneous population of selected molecules. The purified proteins derived from Lib#6 selectants bound to THBN-agarose beads but did not inhibit proteolytic activity of THBN, suggesting that these selectants bind to a site on THBN other than the catalytic site. Thus, a single large combinatorial library can serve as a source to obtain highly specific, high-affinity binding molecules for each of several targets. Furthermore, the results with THBN show that the binding of Kunitz domains to other proteins is not limited to the catalytic sites of trypsin-homologous proteases.
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PMID:Iterative optimization of high-affinity protease inhibitors using phage display. 2. Plasma kallikrein and thrombin. 867 10

Human lipoprotein-associated coagulation inhibitor (LACI) is a serum protein containing three Kunitz domains. We displayed the first domain (LACI-D1) on the III protein of phage M13 and made libraries of this domain. We iteratively varied 13 residues in the region corresponding to the BPTI-trypsin interface and selected for binding to human plasmin (PLA) and human plasma kallikrein (pKAL). For PLA, our first-round best binder, EPI-P211, had KD = 2 nM. Using information from the first selection, we made a PLA-biased library containing approximately 500,000 proteins and selected from these a protein, EPI-P302, having a KD for PLA of 87 pM. EPI-P302 inhibits pKAL with KD approximately 250 nM (approximately 2800-fold higher than for PLA) and KD values for other proteases are higher yet. From the same initial LACI-D1 library, we selected an inhibitor of pKAL, EPI-K401, with a KD for pKAL of 287 pM. We used information from this selection to construct a pKAL-biased library from which we selected EPI-K502, which has a KD for pKAL of 40 pM. EPI-K502 inhibits PLA with KD approximately 20 nM (500-fold higher than for pKAL); KD values for other proteases are much higher. For both targets and for both selections, there are families of proteins having a few differences and a range of affinities for their targets. These proteins are candidate drugs and imaging agents for indications involving excess PLA or pKAL. Structure-activity relationships of PLA and pKAL binders will allow design of small molecules that are specific for these targets.
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PMID:Obtaining a family of high-affinity, high-specificity protein inhibitors of plasmin and plasma kallikrein. 923 42

We examined hemostatic abnormalities in 23 patients with acute myocardial infarction (AMI), 10 with pulmonary embolism (PE), and 10 with deep vein thrombosis (DVT). At the onset of AMI, plasma levels of tissue-type plasminogen activator (t-PA), PA inhibitor-I (PAI-I), fibrin-D-dimer, thrombin-antithrombin complex (TAT), and plasmin-plasmin inhibitor complex (PPIC) were significantly increased. Both the plasma total TFPI and free-TFPI levels in the AMI patients were significantly higher than those in the healthy volunteers, PE patients, and DVT patients. There was no significant difference in total TFPI or free-TFPI among patients with PE, those with DVT, and healthy volunteers. One hour after percutaneous transluminal coronary angioplasty (PTCA) in the AMI group, the total TFPI level was further increased, and it was significantly reduced 24 hr after PTCA, to a level similar to that in healthy volunteers. Free-TFPI showed a pattern similar to that of total TFPI. The ratio of free-TFPI/total TFPI was highest 1 hr after PTCA. Increased TFPI in AMI patients might be released from ischemic tissues.
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PMID:Increased tissue factor pathway inhibitor in patients with acute myocardial infarction. 925 77

Thromboregulatory physiology is essentially a function of the blood vessel wall. Constitutive endothelial cell activities maintain blood fluidity by down regulating the initiation as well as, the propagation of blood coagulation. The major systems involved include: the Protein C, TFPI, plasmin generating and antithrombin pathways, all of which are focused on the cell membrane. Altered regulation of these endothelial functions forms the basis of the pathophysiologic events associated with the inherited primary hypercoagulable states. Secondary hypercoagulable syndromes occurring in various clinical states with conversion to a vascular thrombogenic phenotype reflect non constitutive activated endothelial cell functions with concomitant down regulation of the constitutive anticoagulant surface activity. So called idiopathic clinical thrombosis in most circumstances represents multi hit events in which specific genetic abnormalities or polymorphisms together with specific acquired alterations in geographically distinct endothelial cell beds culminate in a recognizable coagulation phenotype.
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PMID:The Jeremiah Metzger Lecture. Hypercoagulable states: challenges and opportunities. 1141 75

Plasmin is an important protease that mediates clot fibrinolysis and vessel wall extracellular matrix proteolysis. Recently, in vitro studies have suggested that plasmin can cleave and inactivate recombinant TFPI, a major inhibitor of TF-mediated coagulation. We hypothesized that such an interaction may occur in vascular cells expressing TFPI, or in the vessel wall, with implications for thrombolysis. In a series of experiments, we examined the effects of plasmin on cell surface and extracellular matrix (ECM) associated TFPI in endothelial cells (EC) in culture and on EC and smooth muscle cells (SMC) in the vessel wall. Plasmin (0.2 microM) decreased cell surface and matrix associated TFPI activity in cultured endothelial cells by 77 +/- 5% and 69 +/- 6% respectively (p < 0.01). Plasminogen, the proenzyme form of plasmin had no such effect on cell surface TFPI or matrix TFPI. Cell surface TFPI antigen measured by fluorescence activated cell sorter (FACS) was also significantly reduced by plasmin. Proteolysis of conditioned medium TFPI was suggested by loss of a approximately 45kD TFPI on Western Blot analysis following plasmin treatment. Plasmin also proteolysed a approximately 45kD TFPI protein in the intact ECM of EC, an effect which was inhibited by preincubation with aprotinin, a plasmin inhibitor. Incubation of similar concentrations of plasmin, with homogenates of normal vessel decreased a approximately 45kD TFPI immunoreactive band on Western blot analysis. Plasmin also decreased surface TFPI activity on frozen sections of normal vessel as measured by an amidolytic assay. Finally, plasmin treatment of atherosclerotic plaque sections caused complete removal of TFPI immunoreactivity associated with luminal EC and intimal SMC, when compared to control treated plaque (n = 3). Together these data suggest that plasmin proteolyses the majority of EC-associated (surface and matrix) TFPI and may remove TFPI from the luminal surface and intima of the vessel wall. TFPI proteolysis in cultured EC was associated with significant reduction in TFPI anticoagulant activity. These data provide evidence that plasmin degradation of TFPI occurs in vascular cells and in the vessel wall and may have implications for re-thrombosis following thrombolysis in vivo.
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PMID:Plasmin proteolysis of endothelial cell and vessel wall associated tissue factor pathway inhibitor. 1158 28

Activation of the extrinsic pathway of blood coagulation plays the key role in the process of blood coagulation. The hemostasis is influenced by the activity of procoagulant factors and its inhibitors. In this work we summarize existing data on tissue factor pathway inhibitors: TFPI and TFPI-2. Despite structural similarity between them, they exhibit many differences in synthesis, distribution and mechanism of action. TFPI inhibits the activity of factor Xa and the complex of tissue factor and factor VIIa (TF/VIIa). Conversely, TFPI-2 does not inhibits factor Xa, but it is a strong inhibitor of factor XIa, plasma kallikrein, plasmin and trypsin.
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PMID:[Tissue factor pathway inhibitors]. 1236 12

Vasculogenic mimicry (VM), the formation of matrix-rich vascular-like networks in three-dimensional culture corresponding with the expression of vascular cell-associated genes, and the lining of matrix-rich networks in situ, has been observed in highly aggressive and malignant melanoma. However, little is known about the molecular underpinnings of this phenomenon. On the basis of gene profiling, protein detection, and immunohistochemistry, aggressive relative to poorly aggressive melanoma showed up-regulation of tissue factor (TF), TF pathway inhibitor 1 (TFPI-1) and 2 (TFPI-2), critical genes that initiate and regulate the coagulation pathways. The procoagulant function of TF on highly aggressive melanoma is shown to be regulated by TFPI-1 but not by TFPI-2. Thus, aggressive melanoma exhibits endothelial cell-like anticoagulant mechanisms that may contribute to the fluid-conducting potential of melanoma cell-lined networks, as studied by correlative in vivo Doppler flow measurements. Antibody inhibition experiments reveal that TFPI-2 is required for VM in vitro, but plasmin is an unlikely target protease of TFPI-2. Blockade of TFPI-2 suppressed matrix metalloproteinase-2 activation, and, therefore, TFPI-2 appears to regulate an essential pathway of VM. TFPI-2 is synthesized by endothelial and tumor cells, which deposit TFPI-2 into extracellular matrices. Culturing poorly aggressive melanoma cells on three-dimensional matrix containing recombinant TFPI-2 produces some of the phenotypic changes associated with aggressive, vasculogenic melanoma cells. Thus, TFPI-2 contributes to VM plasticity, whereas TFPI-1 has anticoagulant functions of relevance for perfusion of VM channels formed by TF-expressing melanoma cells.
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PMID:Differential role of tissue factor pathway inhibitors 1 and 2 in melanoma vasculogenic mimicry. 1450 Mar 72

In normal pregnancy, there is a marked increase in the procoagulant activity in maternal blood characterized by elevation of factors VII, X, VIII, fibrinogen and von Willebrand factor, which is maximal around term. This is associated with an increase in prothrombin fragments (PF1+2) and thrombin-antithrombin complexes. There is a decrease in physiological anticoagulants manifested by a significant reduction in protein S activity and by acquired activated protein C (APC) resistance. The overall fibrinolytic activity is impaired during pregnancy, but returns rapidly to normal following delivery. This is largely due to placental derived plasminogen activator inhibitor type 2 (PAI-2), which is present in substantial quantities during pregnancy. D-dimer, a specific marker of fibrinolysis resulting from breakdown of cross-linked fibrin polymer by plasmin, increases as pregnancy progresses. Overall, there is a 4- to 10-fold increased thrombotic risk throughout gestation and the postpartum period. Local haemostasis at the placental throphoblast level is characterized by increased tissue factor (TF) expression and low expression of the inhibitor TFPI. Microparticles derived from maternal endothelial cells and platelets, and from placental throphoblasts may contribute to the procoagulant effect. Local anticoagulant mechanisms on placental throphoblasts are important for counterbalance of the procoagulant milieu. Disruption of anticoagulant mechanisms, for example, autoantibodies, to annexin V may increase pregnancy complications in patients with antiphospholipid antibodies (APLA).
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PMID:Haemostatic changes in pregnancy. 1550 71

Disturbances in hemostasis and abnormal angiogenesis are components in the plaque growth and destabilization. The role of the vascular endothelial growth factor (VEGF) in the perturbation of hemostasis in chronic kidney disease (CKD) is still unknown. In this preliminary study, we investigate the relation between VEGF and the parameters of coagulation: tissue factor (TF), its inhibitor (TFPI), and fibrinolytic system: urokinase-type plasminogen activator (uPA), its soluble receptor (suPAR), tissue-type plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), plasmin/antiplasmin complexes (PAP) in the patients with mild-to-moderate, and severe CKD and healthy controls. All indices (except TFPI) were raised in CKD patients, particularly in those with severe CKD, compared with controls. The strong positive correlations were between VEGF and some parameters, both coagulation (TF, TFPI, TF/TFPI ratio) and fibrinolytic system (uPA, suPAR, PAP). The relationships were also between the individual hemostatic parameters. In multiple regression analysis, VEGF and kidney dysfunction markers (urea and creatinine levels) were independently associated with uPA, and VEGF was independently associated with suPAR levels. Moreover, PAP was independently associated with age and suPAR. This study represents the first to investigate the relation between VEGF and the activation both coagulation and fibrinolysis in CKD patients. VEGF and the parameters of hemostatic system activation were higher in the CKD group than in the controls with a significant correlation between them. VEGF was independently associated with uPA/suPAR system, whereas suPAR was independently related to PAP levels, suggesting a new link between abnormal angiogenesis and hyperfibrinolysis in this population.
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PMID:Vascular endothelial growth factor and uPA/suPAR system in early and advanced chronic kidney disease patients: a new link between angiogenesis and hyperfibrinolysis? 2268 25


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