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)

Urokinase-type plasminogen activator (uPA) is a serine protease involved in pericellular proteolysis and tumor cell metastasis via plasmin-mediated degradation of extracellular matrix proteins. Plasma uPA is inhibited by the serine protease inhibitor protein C inhibitor (PCI) by the insertion of PCI's reactive site loop into the active site of the protease. To better understand the structural aspects of this inhibition, 15 reactive-site mutants of recombinant PCI (rPCI) were assayed for differences in uPA inhibition. These assays revealed that substitutions at the P1 Arg354 and P3 Thr352 sites of rPCI were detrimental to inhibitory activity, while P3' Arg357 mutations had little effect upon the inhibition rate. However, replacement of the P2 Phe353 with small residues like Ala and Gly increased the effectiveness of rPCI three- to four fold. To explain these altered rates of inhibition, a computer-derived molecular model of uPA was generated and docked to a model of PCI to simulate complex formation. The changes made by mutagenesis were then recreated in the model of uPA-PCI. In accordance with the kinetic data, the poor performance of P3 variants is primarily attributable to charge repulsion, while alleviation of steric hindrance at P2 produces the observed increase in uPA inhibition. In the model, residues at P3' interact with PCI rather than uPA, consistent with P3' variants demonstrating that little variation from wild-type activity. Ultimately, this combination of mutagenesis and molecular modeling will further refine our understanding of the interaction between PCI and uPA.
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PMID:Assessment of the interaction between urokinase and reactive site mutants of protein C inhibitor. 936 30

Tissue factor pathway inhibitor-2 (TFPI-2), also known as placental protein 5, is a serine protease inhibitor consisting of three tandemly-arranged Kunitz-type protease inhibitor domains. While TFPI-2 is a potent inhibitor of trypsin, plasmin, kallikrein, and factor XIa in the test tube, the function of this inhibitor in vivo remains unclear. In the present study, we investigated the synthesis and secretion of TFPI-2 by cultured endothelial cells derived from human umbilical vein, aorta, saphenous vein, and dermal microvessels to gain insight into its biological function. While all endothelial cells examined synthesized and secreted TFPI-2, dermal microvascular endothelial cells synthesized threefold to sevenfold higher levels of TFPI-2. Approximately 60% to 90% of the TFPI-2 secreted by endothelial cells was directed to the subendothelial extracellular matrix (ECM). When cultured human umbilical vein endothelial cells were stimulated with inflammatory mediators such as phorbol 12-myristate,13-acetate; endotoxin; and tumor necrosis factor-alpha, TFPI-2 synthesis by these cells increased twofold to 14-fold. Recombinant TFPI-2 bound to dermal microvascular endothelial cell monolayers and its ECM in a specific, dose-dependent, and saturable manner with Kd values of 21 and 24 nmol/L, respectively. TFPI-2 interacted with 4.5 X 10(10) sites/cm2 (3 X 10[5] sites/cell) and 2.3 X 10(11) sites/cm2 on endothelial cells and ECM, respectively. In the presence of rabbit anti-TFPI-2 IgG, but not preimmune IgG, endothelial cells dissociated from the culture flask in a time- and IgG concentration-dependent manner. Our findings provide evidence that endothelial cell-derived TFPI-2 is primarily secreted into the abluminal space and presumably plays an important role in maintaining the integrity of the ECM essential for cell attachment.
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PMID:Quantification and characterization of human endothelial cell-derived tissue factor pathway inhibitor-2. 944 54

The urokinase-urokinase receptor system plays a dominant role in the degradation and invasion of extracellular matrix (ECM) by tumor cells. In this system, urokinase bound to its cell receptor converts plasminogen to plasmin, a broad-spectrum serine protease that participates in the degradation and invasion of connective tissues by tumor cells. In this study, we evaluated whether these activities of plasmin are inhibited by a newly characterized human 32 kDa recombinant serine protease inhibitor called trypsin/tissue factor pathway inhibitor-2 (rTFPI-2). We found that rTFPI-2 dose-dependently inhibited fluid-phase plasmin as well as plasmin generated on the ECM and/or the cell surface of HT-1080 fibrosarcoma cells. The degradation of radiolabeled matrix as well as Matrigel invasion by these tumor cells is also inhibited by rTFPI-2 in a dose-dependent fashion. We have reported that rTFPI-2 is identical to 33 kDa extracellular matrix-associated serine protease inhibitor (33 kDa MSPI), whereas the 31 and 27 kDa MSPIs are under-glycosylated forms of the 33 kDa MSPI. We therefore evaluated the ability of MSPIs from the ECM of dermal fibroblasts to inhibit plasmin and found that the plasmin activity was effectively blocked by the MSPIs. We have also evaluated whether the HT-1080 cells synthesize and secrete the MSPIs and found that the synthesis and secretion of the MSPIs was undetectable in these cells. Collectively, our results suggest that rTFPI-2/33 kDa MSPI inhibits plasmin on the tumor cell and ECM surfaces as well as the degradation and invasion of matrix by HT-1080 fibrosarcoma cells.
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PMID:HT-1080 fibrosarcoma cell matrix degradation and invasion are inhibited by the matrix-associated serine protease inhibitor TFPI-2/33 kDa MSPI. 961 Jul 35

Pig conceptuses undergo morphological development from spherical to filamentous forms during days 10 to 12 of pregnancy, coincident with a high content of mRNAs encoding insulin-like growth factor (IGF)-I in the uterine endometrium and secretion of IGF-I into the uterine lumen. The potential regulation by developing conceptuses of the bioavailability of IGF-binding proteins (IGFBPs) within the uterine microenvironment was investigated. Uterine luminal flushings (ULFs) were obtained between days 10 and 18 of pregnancy and the presence of specific IGFBPs was detected by ligand blot analysis. ULFs collected at days 10 and 11 of pregnancy contained 46 and 43 kDa IGFBP-3, several IGFBPs of about 30 kDa including IGFBP-2, and an unidentified 26 kDa IGFBP; IGFBP-3 was the most abundant. By day 12, however, IGFBPs were substantially diminished or undetectable. Examination of the morphology of flushed conceptuses revealed that the loss of IGFBPs in ULF was associated with the transition from spherical to filamentous morphology. The abundance of IGFBP-3 mRNA in uterine endometrium, as monitored by blot-hybridization, was not altered in a similar way, suggesting that lack of IGFBP-3 in 'filamentous' ULF resulted from proteolysis rather than from decreased expression of the IGFBP-3 gene. Consistent with this, incubation of 'spherical' ULF with or without added 'filamentous' ULF at 37 degrees C resulted in the disappearance of endogenous IGFBP-3 only in 'spherical + filamentous' ULF. The protease activity in 'filamentous' ULF was inhibited by EDTA, but unlike matrix metalloproteinases, was not zinc ion-dependent or inhibited by 1,10-phenanthroline. Moreover, this activity was partially inhibited by the serine protease inhibitor aprotinin, but not by 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), a known inhibitor of plasmin. The IGFBP protease activity of ULF may therefore comprise a group of enzymes including an unidentified serine protease. The results suggest that elongating pig conceptuses induce IGFBP protease activity which may increase the intrauterine bioavailability of IGF.
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PMID:Proteolysis of insulin-like growth factor-binding proteins (IGFBPs) within the pig uterine lumen associated with peri-implantation conceptus development. 964 Feb 76

The binding of plasmin to Streptococcus uberis strain 0140 J was optimal in the pH range 5.0-5.5. Plasmin binding decreased exponentially with increasing NaCl concentration (0-0.8 mol l-1), reaching a minimum at NaCl concentrations exceeding 0.55 mol l-1. Neither K+, Mg2+ nor the metal chelator EDTA had any effect on the interaction. Plasmin binding was prevented, in a concentration-dependent manner, by the amino acids lysine, arginine and epsilon-aminocaproic acid. Bound plasmin was also eluted from the bacterial cell using the same amino acids. Bound plasmin was lost from the bacterium in a time- and temperature-dependent fashion, the rate of plasmin loss increased with increasing temperature over the range 4-55 degrees C, and the elution of plasmin from live and heat-killed bacteria was similar. Cell-bound plasmin was only partially inhibited by the physiological inhibitor alpha 2-antiplasmin whereas the serine protease inhibitor aprotinin, and the active site titrant p-nitrophenyl-p-guanidiniobenzoate, inhibited the activity of the cell-bound plasmin by more than 95%.
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PMID:Characterization of the interaction of bovine plasmin with Streptococcus uberis. 971 96

Plasminogen, the pro-enzyme of plasmin, aids various processes essential for normal, acute wound healing, such as fibrinolysis and cell migration. We have investigated if plasminogen is available to perform these functions in chronic wounds such as venous leg ulcers. We report that plasminogen is degraded by fluid from venous leg ulcers to a number of fragments, including kringle domains 1-3, an angiostatin-related protein. The enzyme responsible was inhibited by the serine protease inhibitor phenyl-methylsulfonyl fluoride, but was not inhibited by alpha1-anti-trypsin, an inhibitor of neutrophil elastase, by alpha2-anti-plasmin, an inhibitor of plasmin, or by the matrix metalloprotease inhibitor 1,10 phenanthroline. Plasminogen degraded by wound fluid was a weaker substrate than intact plasminogen for plasmin generation by the keratinocyte cell line HaCaT. These results suggest that serine protease activity in leg ulcer fluid degrades plasminogen and support the hypothesis that keratinocyte migration may be impaired in leg ulcers because of a reduced availability of intact plasminogen for plasmin generation.
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PMID:Wound fluid from venous leg ulcers degrades plasminogen and reduces plasmin generation by keratinocytes. 985 30

P-Selectin, an adhesion molecule expressed on the surfaces of activated platelets and the vascular endothelium, mediates platelet binding to monocytes and neutrophils. Monocytes and neutrophils produce superoxide anion by activated platelets through p-selectin. Aprotinin, a serine protease inhibitor, inhibits plasmin to activate platelets during cardiopulmonary bypass (CPB). A total of 25 patients were studied to clarify the effects of aprotinin on p-selectin expression during CPB. Nine patients were not given aprotinin (control group), and 16 were given aprotinin of 2 million U in the priming solution (aprotinin group). The platelet count and soluble p-selectin in the plasma, p-selectin on the surface membranes of platelets, and leukocyte-platelet conjugate levels were measured during and after CPB. The platelet count was maintained well in the aprotinin group. The increases of soluble p-selectin in the plasma, platelet surface p-selectin, and leukocyte-platelet conjugates were less in the aprotinin group than in the control group (p < 0.05). In conclusion, aprotinin in patients undergoing CPB may reduce the early inflammatory reactions induced by p-selectin.
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PMID:Aprotinin reduces the expression of p-selectin on the surface of platelet and leukocyte-platelet conjugates. 987 93

Tissue factor pathway inhibitor-2 (TFPI-2)/matrix-associated serine protease inhibitor (MSPI), a 32- to 33-kDa Kunitz-type serine protease inhibitor, inhibits plasmin and trypsin. Because plasmin and trypsin are involved in the activation of promatrix metalloproteases proMMP-1 and proMMP-3, we investigated the role of TFPI-2/MSPI in the activation of these proenzymes. Both plasmin and trypsin activated proMMP-1 by converting the 53-kDa proenzyme to the partially active 43-kDa polypeptide; this activity was inhibited by TFPI-2/MSPI. Similarly, TFPI-2/MSPI inhibited the conversion of 66-kDa proMMP-3 to the activated 45- and 30-kDa polypeptides by plasmin and trypsin. Because plasmin is involved in the physiological activation of proMMP-3, we tested whether TFPI-2/MSPI inhibits the activation of proMMP-3 by HT-1080 fibrosarcoma cells and urokinase-charged HeLa cells. We found that the inhibitor inhibited proMMP-3 activation by HT-1080 cells and urokinase-charged HeLa cells. Collectively, our results suggest that TFPI-2/MSPI indirectly regulates MMP-1- and MMP-3-catalyzed matrix proteolysis by regulating the activation of proMMP-1 and proMMP-3.
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PMID:Regulation of ProMMP-1 and ProMMP-3 activation by tissue factor pathway inhibitor-2/matrix-associated serine protease inhibitor. 1008 61

We have recently reported that insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) can significantly increase ceramide-induced apoptosis in an Hs578T breast carcinoma cell line in an IGF-independent manner. It was observed in that study that IGFBP-3 added to the cultures was proteolytically modified, generating a specific pattern of fragmentation. We have also previously reported that almost all of the IGFBP-3 outside the circulation in extravascular fluids is in a fragmented form, apparently due to the activity of a cation-dependent serine protease. The aim of this study was to investigate the role of proteolysis in the IGFBP-3 enhancement of C2-induced apoptosis. In this study we confirmed that preincubation of Hs578T cells with IGFBP-3 enhances the apoptotic effect of the ceramide analog C2. The presence of IGF-I completely inhibited the enhancement effect, apparently by inhibiting cell surface association and proteolytic modification. The presence of a serine protease inhibitor [4-(2-aminoethyl)benesulfonyl fluoride] completely inhibited the enhancement effect of IGFBP-3, and Western immunoblotting of conditioned medium and cell surface-associated IGFBP-3 revealed that proteolytic fragmentation of the IGFBP-3 was reduced. In addition, fragments from the incubation of IGFBP-3 with plasmin were able to enhance the susceptibility of Hs578T cells to C2. The effect of these fragments could, however, also be reduced by 4-(2-aminoethyl)benesulfonyl fluoride despite the fact that IGFBP-3 was already fragmented. This suggests additional roles for serine proteases in the IGFBP-3 effect on C2-induced apoptosis in addition to the cleavage of the binding protein.
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PMID:The role of cell surface attachment and proteolysis in the insulin-like growth factor (IGF)-independent effects of IGF-binding protein-3 on apoptosis in breast epithelial cells. 1046 74

Accelerated thrombin generation is central to the development of hemostatic abnormalities during cardiopulmonary bypass (CPB) that are associated with both thromboembolic complications and serious, abnormal bleeding. Thrombin not only converts fibrinogen to fibrin, but also activates platelets and coagulation factors V, VIII, and XI and causes release of von Willebrand factor from vascular endothelium. Thrombin can also downregulate the hemostatic system by inducing formation of platelet inhibitory agents, such as nitric oxide and prostacyclin, and release of tissue plasminogen activator, facilitating activation of protein C, and releasing tissue factor pathway inhibitor. Excessive thrombin activity may also result in substantial consumption of platelets, fibrinogen, and labile coagulation factors and abnormal bleeding. Elevated tissue plasminogen activator levels secondary to activation of the contact system and surgery catalyze the formation of plasmin, which also consumes or internalizes platelet glycoprotein receptors and coagulation factors V, VIII, and fibrinogen. Heparin can reduce the generation of and mediate neutralization of excessive and CPB-associated thrombin activity. Heparin anticoagulation is commonly monitored with the activated clotting time (ACT). However, the ACT may be prolonged by factors other than heparin during CPB, such as hemodilution and hypothermia, and therefore may not accurately reflect the extent of anticoagulation by heparin. Aprotinin, a nonspecific serine protease inhibitor used with CPB, can also prolong celite-based ACT values, rendering it less reliable for monitoring heparin anticoagulation. Therefore, several alternative anticoagulation strategies have been recommended when aprotinin is used, such as a higher celite ACT trigger (>750 seconds), monitoring of whole blood heparin concentrations (eg, >2.7 U/mL), or administration of heparin based on a CPB duration-dependent, fixed-dose regimen. Administration of heparin doses higher than those generally recommended, as guided by predetermined, patient-specific whole blood heparin concentration measurements during bypass, can reduce excessive thrombin-mediated consumption of platelets and coagulation factors as well as post-CPB blood loss and blood component transfusions. New modalities of improving suppression of excess thrombin generation during CPB include use of heparin-bonded CPB circuits, heparin cofactor II or related analogs, supplemental antithrombin III, direct thrombin inhibitors (eg, hirudin, argatroban), and inhibitors of the contact and tissue factor pathways. The safety and efficacy of these approaches remains to be established by additional, appropriately powered, prospective studies.
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PMID:Anticoagulation and anticoagulation reversal with cardiac surgery involving cardiopulmonary bypass: an update. 1046 45


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