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

---

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

The event that initiates the extrinsic pathway of blood coagulation is the association of coagulation factor VIIa (VIIa) with its cell-bound receptor, tissue factor (TF), exposed to blood circulation following tissue injury and/or vascular damage. The natural inhibitor of the TF.VIIa complex is the first Kunitz domain of tissue factor pathway inhibitor (TFPI-K1). The structure of TF. VIIa reversibly inhibited with a potent (Ki=0.4 nM) bovine pancreatic trypsin inhibitor (BPTI) mutant (5L15), a homolog of TFPI-K1, has been determined at 2.1 A resolution. When bound to TF, the four domain VIIa molecule assumes an extended conformation with its light chain wrapping around the framework of the two domain TF cofactor. The 5L15 inhibitor associates with the active site of VIIa similar to trypsin-bound BPTI, but makes several unique interactions near the perimeter of the site that are not observed in the latter. Most of the interactions are polar and involve mutated positions of 5L15. Of the eight rationally engineered mutations distinguishing 5L15 from BPTI, seven are involved in productive interactions stabilizing the enzyme-inhibitor association with four contributing contacts unique to the VIIa.5L15 complex. Two additional unique interactions are due to distinguishing residues in the VIIa sequence: a salt bridge between Arg20 of 5L15 and Asp60 of an insertion loop of VIIa, and a hydrogen bond between Tyr34O of the inhibitor and Lys192NZ of the enzyme. These interactions were used further to model binding of TFPI-K1 to VIIa and TFPI-K2 to factor Xa, the principal activation product of TF.VIIa. The structure of the ternary protein complex identifies the determinants important for binding within and near the active site of VIIa, and provides cogent information for addressing the manner in which substrates of VIIa are bound and hydrolyzed in blood coagulation. It should also provide guidance in structure-aided drug design for the discovery of potent and selective small molecule VIIa inhibitors.
...
PMID:Structure of extracellular tissue factor complexed with factor VIIa inhibited with a BPTI mutant. 992 87

Thromboembolic events may complicate clinical course of chronic pancreatitis. It is accepted that trypsin plays a role in the pathogenesis of these abnormalities. However there is a lack of information about local activation of blood coagulation in pancreatic tissue in chronic inflammation and contribution of tissue factor (TF) to this process. Immunohistochemistry was applied to AMeX-fixed sections of tissues of ten cases of chronic pancreatitis to explore the presence and distribution of components of the coagulation system in situ. TF antigen was present in cells of ductules. Fibrinogen and fibrin were detected in the inflammatory infiltrates of the pancreatic tissue. The data suggest that there is local activation of blood coagulation in pancreatic tissue in chronic inflammation that depends on tissue factor.
...
PMID:[Local activation of blood coagulation in pancreatic tissue in chronic pancreatitis]. 1010 56

A second protease-activated receptor (PAR-2) that could be activated by trypsin or more physiologically by mast cell tryptase has been recently cloned. Both the structure and activation mechanism of PAR-2 was similar to the functional thrombin receptor (PAR-1). Although many effects of the coagulation protease thrombin on the vascular endothelium could be attributed to PAR-1 activation, very little is known about the physiological and pathophysiological role of PAR-2. We investigated whether stimulation of PAR-2 on endothelial cells induced two cellular responses that play a central role in primary and secondary haemostasis: the release of high molecular weight von Willebrand factor (hmw-VWF) from Weibel-Palade bodies and the de novo synthesis of tissue factor (TF) mRNA and protein. Human umbilical vein endothelial cells (HUVEC) were incubated with agonists for PAR-2 at 37 degrees C. Both trypsin and SLIGKV increased TF mRNA and activity and induced the release of hmw-VWF due to elevated levels of cytosolic Ca2+. Trypsin (10 nm) induced a 6-fold increase of TF mRNA and reduced time until fibrin clot formation to 37%, indicating trebling of the cell surface located TF activity. Stimulation of HUVEC with the PAR-2 agonist peptide SLIGKV induced a dose-dependent increase of TF mRNA up to 6 times and TF activity up to 3 times. Release of hmw-VWF was achieved both after incubation of HUVEC with trypsin and SLIGKV and was directly depending on intracellular Ca2+ mobilization. To make results comparable to the functional thrombin receptor, homologous experiments were carried out using the PAR-1 agonists thrombin and SFLLRN.
...
PMID:Endothelial protease-activated receptor-2 induces tissue factor expression and von Willebrand factor release. 1023 35

Human type-2 tissue factor pathway inhibitor (TFPI-2), also known as placental protein 5, is a 32-kDa serine proteinase inhibitor consisting of three tandemly arranged Kunitz-type domains homologous to tissue factor pathway inhibitor. TFPI-2 inhibits a variety of serine proteinases involved in coagulation and fibrinolysis through an arginine residue (R24) in its first Kunitz-type domain, which constitutes a putative P1 residue for the substrate recognition sites of these proteinases. As recent studies have shown that this P1 residue to be a glutamine in murine TFPI-2, we constructed, expressed, and purified a human TFPI-2 mutant with glutamine substituted for arginine at position 24 (R24Q TFPI-2). R24Q TFPI-2 lost approximately 90% of its inhibitory activity towards bovine trypsin and virtually all inhibitory activity towards human plasmin and the factor VIIa-tissue factor complex, emphasizing the importance of the P1 Arg24 residue in the inhibition of these serine proteinases. However, whereas wild-type TFPI-2 is a relatively weak inhibitor of human factor Xa amidolytic activity (IC50 approximately 1 microM), R24Q TFPI-2 exhibited enhanced inhibitory activity towards the amidolytic and coagulant activities of this proteinase with a Ki of 18 nM. While the molecular basis for the enhanced inhibition of human factor Xa by R24Q TFPI-2 is unknown, these data provide suggestive evidence that murine TFPI-2 may function as a serine proteinase inhibitor in spite of the absence of a P1 Arg or Lys residue.
...
PMID:Inhibitory properties of human recombinant Arg24-->Gln type-2 tissue factor pathway inhibitor (R24Q TFPI-2). 1032 61

Proteolytically activated receptors define a new subclass among the G-protein coupled receptors. Proteinase activated receptor-2 (PAR-2), the second member to be identified of this growing receptor subclass, can be activated by trypsin and trypsin-like serine proteases such as mast cell tryptase. PAR-2 is expressed in endothelial cells. Here we have studied if activation of PAR-2 changes the coagulation properties of cultured human umbilical vein endothelial cells. We show that activation of PAR-2 induces rapid and transient formation of tissue factor mRNA with a maximum level 1 hour after receptor stimulation. The increased mRNA level was accompanied by an increased tissue factor activity at the endothelial cell surface, shortening coagulation time in a standard clotting assay. The level of tissue factor activity after PAR-2 activation was comparable with the effects of thrombin receptor (PAR-1) activation although neither of the two protease receptors were as strong inducers of tissue factor as tumor necrosis factor-alpha.
...
PMID:Stimulation of proteinase activated receptor-2 causes endothelial cells to promote blood coagulation in vitro. 1040 79

The upstream coagulation enzymes are homologous trypsin-like serine proteases that typically function in enzyme-cofactor complexes, exemplified by coagulation factor VIIa (VIIa), which is allosterically activated upon binding to its cell surface receptor tissue factor (TF). TF cooperates with VIIa to create a bimolecular recognition surface that serves as an exosite for factor X binding. This study analyzes to what extent scissile bond docking to the catalytic cleft contributes to macromolecular substrate affinity. Mutation of the P1 Arg residue in factor X to Gln prevented activation by the TF.VIIa complex but did not reduce macromolecular substrate affinity for TF.VIIa. Similarly, mutations of the S and S' subsites in the catalytic cleft of the enzyme VIIa failed to reduce affinity for factor X, although the affinity for small chromogenic substrates and the efficiency of factor X scissile bond cleavage were reduced. Thus, docking of the activation peptide bond to the catalytic cleft of this enzyme-cofactor complex does not significantly contribute to affinity for macromolecular substrate. Rather, it appears that the creation of an extended macromolecular substrate recognition surface involving enzyme and cofactor is utilized to generate substrate specificity between the highly homologous, regulatory proteases of the coagulation cascade.
...
PMID:Macromolecular substrate affinity for the tissue factor-factor VIIa complex is independent of scissile bond docking. 1044 91

Human tissue factor pathway inhibitor-2 (TFPI-2)/matrix-associated serine protease inhibitor (MSPI), a Kunitz-type serine protease inhibitor, inhibits plasmin, trypsin, chymotrypsin, plasma kallikrein, cathepsin G, and factor VIIa-tissue factor complex. The mature protein has a molecular mass of 32-33 kDa, but exists in vivo as two smaller, underglycosylated species of 31 and 27 kDa. TFPI-2/MSPI triplet is synthesized and secreted by a variety of cell types that include epithelial, endothelial, and mesenchymal cells. Because the majority (75-90%) of TFPI-2/MSPI is associated with the extracellular matrix (ECM), we examined which components of the ECM bind TFPI-2/MSPI. We found that TFPI-2/MSPI bound specifically to heparin and dermatan sulfate. Interaction of these two glycosaminoglycans (GAGs) with TFPI-2/MSPI involved one or more common protein domains, as evidenced by cross-competition experiments. However, binding affinity for TFPI-2/MSPI with heparin was 250-300 times greater than that for TFPI-2/MSPI with dermatan sulfate. Binding of TFPI-2/MSPI to GAGs was inhibited by NaCl or arginine but not by glucose, mannose, galactose, 6-aminohexanoic acid, or urea, suggesting that arginine-mediated ionic interactions participate in the GAG binding of TFPI-2/MSPI. This supposition was supported by the observation that only NaCl or arginine could elute the TFPI-2/MSPI protein triplet from an ECM derived from human dermal fibroblasts. Reduced TFPI-2/MSPI did not bind to heparin, suggesting that proper disulfide pairings and conformation are essential for matrix binding. To determine whether heparin modulates the activity of TFPI-2/MSPI, we determined the rate of inhibition of plasmin by the inhibitor with and without heparin and found that TFPI-2/MSPI is more active in the presence of heparin. Collectively, our results demonstrate that conformation-dependent arginine-mediated ionic interactions are responsible for the TFPI-2/MSPI triplet binding to fibroblast ECM, heparin, and dermatan sulfate and that heparin augmented the rate of inhibition of plasmin by TFPI-2/MSPI.
...
PMID:Matrix localization of tissue factor pathway inhibitor-2/matrix-associated serine protease inhibitor (TFPI-2/MSPI) involves arginine-mediated ionic interactions with heparin and dermatan sulfate: heparin accelerates the activity of TFPI-2/MSPI toward plasmin. 1049 84

The protease activity is mandatory for intracellular activities induced by coagulation factor VIIa (FVIIa), and in this way it resembles signal transduction induced by thrombin and trypsin caused by specific, proteolytic cleavage of protease activated receptors (PARs). The mechanism for FVIIa-induced signal transduction is, however, not known although a mechanism involving PAR cleavage has been deduced from studies of cytosolic Ca2+ release and p44/p42 mitogen activated protein kinase (MAPK) activation. In the present work we have examined the possibilities that i) FVIIa-induced signal transduction involves the activation of one of the four known PARs, or ii) exposure of cells to FVIIa releases a soluble ligand that is responsible for MAPK activation. For this purpose, we evaluated the effects of FVIIa, thrombin, FXa, trypsin and PAR agonist peptides on the Ca2+ release and MAPK activation in tissue factor-(TF) transfected baby hamster kidney (BHK[+TF]) cells and Madin-Darby canine kidney (MDCK) cells. FVIIa induced a significant MAPK signal in BHK(+TF) cells and in MDCK-I and -II cells whereas no MAPK activation was observed with thrombin, FXa or PAR agonist peptides. Thrombin, trypsin, PAR-1 and PAR-2 agonist peptides induced a prominent Ca2+ response in both cell types. In contrast the cells did not respond with a detectable Ca2+ signal when treated with FVIIa. These results suggest that the intracellular activity induced by FVIIa is distinctly different from that induced by trypsin, thrombin and FXa not involving any of the known PARs. Conditioned medium from BHK(+TF) cells treated with FVIIa failed to induce a MAPK response in untreated BHK(+TF) cells when FVIIa was removed by immunoadsorption from the medium prior to its transfer to the untreated BHK(+TF) cells. Although it is not possible entirely to exclude a transient response close to the cell surface, the data suggest that the intracellular response was not induced by an autocrine release of a soluble mediator to the medium.
...
PMID:Exclusion of known protease-activated receptors in factor VIIa-induced signal transduction. 1078 Mar 19

Activated factor VII (FVIIa) is a trypsin-like serine protease that plays a key role in the initiation of the blood coagulation cascade. FVIIa, which comprises a light chain (152 residues) and a heavy chain (254 residues) linked by a disulphide bond, is generated by the cleavage of the Arg152-Ile153 peptide bond in factor (F)VII. While a corresponding internal peptide bond cleavage unleashes the activity of other trypsin-like enzymes, FVIIa is unusual in that it remains in a zymogen-like state after cleavage and only becomes an efficient catalyst when associated with tissue factor, its protein cofactor and allosteric regulator. We have determined the structure of free FVIIa lacking the gamma-carboxyglutamic acid (Gla) domain as a means to elucidate the molecular reasons for its poor activity when not bound to tissue factor and the conformational changes induced by its association with tissue factor.
...
PMID:Structure of human coagulation activated factor VII. 1085 May 58

The protease domain of coagulation factor VIIa (FVIIa) is homologous to trypsin with a similar active site architecture. The catalytic function of FVIIa is regulated by allosteric modulations induced by binding of divalent metal ions and the cofactor tissue factor (TF). To further elucidate the mechanisms behind these transformations, the effects of Zn2+ binding to FVIIa in the free form and in complex with TF were investigated. Equilibrium dialysis suggested that two Zn2+ bind with high affinity to FVIIa outside the N-terminal gamma-carboxyglutamic acid (Gla) domain. Binding of Zn2+ to FVIIa, which was influenced by the presence of Ca2+, resulted in decreased amidolytic activity and slightly reduced affinity for TF. After binding to TF, FVIIa was less susceptible to zinc inhibition. Alanine substitutions for either of two histidine residues unique for FVIIa, His216, and His257, produced FVIIa variants with decreased sensitivity to Zn2+ inhibition. A search for putative Zn2+ binding sites in the crystal structure of the FVIIa protease domain was performed by Grid calculations. We identified a pair of Zn2+ binding sites in the Glu210-Glu220 Ca2+ binding loop adjacent to the so-called activation domain canonical to serine proteases. Based on our results, we propose a model that describes the conformational changes underlying the Zn2+-mediated allosteric down-regulation of FVIIa's activity.
...
PMID:Binding of Zn2+ to a Ca2+ loop allosterically attenuates the activity of factor VIIa and reduces its affinity for tissue factor. 1085 Jul 95


<< Previous 1 2 3 4 5 6 Next >>

---