Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.5 (thrombin)
 33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human aortic endothelial cells and smooth cells (SMC) from human aorta and coronary arteries were grown in culture. Subcultured vascular SMC retained several important features of human vascular SMC in situ, for example, vimentin-type intermediate filaments, smooth muscle myosin, a well-developed microfilament system, and expression of caldesmon protein involved in the regulation of contraction in smooth muscle. Aortic endothelial cells were shown to possess functional receptors to histamine, thrombin, serotonin, acetylcholine, bradykinin, platelet activating factor (PAF), angiotensin II, vasopressin, prostaglandin E2 (PGE2), and U46619, a stable analog of thromboxane A2. All these substances stimulated polyphosphoinositide (PPI) breakdown in endothelium. Thrombin, histamine, and PAF were the most potent activators. The response of aortic SMC to the same panel of agonists were different. Serotonin, histamine, and angiotensin II produced higher levels of inositol phosphates (IP, IP2, IP3) in SMC than in endothelium. Responses to acetylcholine, bradykinin, and PGE2 were weak and inferior to those of endothelial cells. Other agents evoked approximately equivalent responses in both cell types. Coronary artery SMC resembled aortic SMC in the high extent of PPI hydrolysis after stimulation with serotonin and histamine. The complete inability of angiotensin II and vasopressin to cause accumulation of inositol phosphates in coronary SMC contrasted with the presence of functional receptors to these hormones on aortic SMC. We conclude that the effect of vasoactive agents on human vascular cells may be realized via activation of PPI hydrolysis. Agonists with reported strong vasoconstrictor action seem to stimulate preferential PPI hydrolysis in SMC, whereas endothelium-dependent relaxers cause more pronounced PPI breakdown in endothelial cells. Peculiarities of angiotensin II and vasopressin receptor expression and/or coupling in human aorta and coronary artery SMC may be relevant for understanding the selective action of agonists on human vessels.
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PMID:Agonist-induced polyphosphoinositide breakdown in cultured human endothelial and vascular smooth muscle cells. 285 52

Platelet proteins which contribute to transglutaminase-catalyzed polymer formation in activated platelets were identified by an immunochemical approach using the Western blot technique. The cross-linked polymer was purified from thrombin- or Ca2+-ionophore A23187-activated platelets by a sucrose density gradient procedure in reducing conditions and in the presence of a non-ionic detergent. Following transblotting of non-crosslinked platelet proteins from a Laemmli-type gel onto nitrocellulose, the platelet protein "lanes" were incubated with unabsorbed antibodies, or antibodies absorbed with purified polymer. Antibodies to native whole platelets as well as specific antibodies were used. The results show that myosin, actin, glycoproteins IIb, IIIa, and tubulin are present in the polymer.
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PMID:Immunochemical characterization of the transglutaminase-catalyzed polymer of activated platelets. 288 77

Platelets are discoidal cytoplasmic particles that respond to a variety of stimuli by developing filopodia and rounding up (shape change), developing the ability to bind fibrinogen from the medium, and, with strong stimuli such as thrombin and PAF-acether, secreting the contents of several types of granules. Arachidonic acid is cleaved from phospholipids by phospholipase A2 and converted by the platelets to endoperoxides, and then to thromboxane A2. The bound dimeric fibrinogen molecules probably cause aggregation by forming bridges between platelets. Aggregation is reinforced by secreted fibrinogen and thrombospondin, which binds the platelets, and by thromboxane A2 and endoperoxides, as well as secreted ADP, which cause additional receptor-mediated activation. The responses to these stimuli are initiated when the agonists bind to specific receptors on the plasma membrane. Subsequent steps resemble those in other types of responsive cells: breakdown of phosphatidylinositol bisphosphate into diacylglycerol, a stimulator of protein kinase C, and inositol-1,4,5-trisphosphate, recently shown to be a potent calcium ionophore. The response of shape change results from increased cytoplasmic Ca2+ which permits phosphorylation of one of the light chains of myosin by a calcium-calmodulin-dependent kinase, with resulting enhanced actin-myosin interaction. Secretion is associated with phosphorylation of a 40,000 to 47,000 dalton protein by the diacylglycerol-activated protein kinase C. These recent findings have increased our understanding of the mechanisms of platelet activation, but much remains to be learned. How do agonist-receptor complexes influence PIP2 breakdown? Is this indeed the first step in activation? What mediates adhesion of platelets to the injured blood vessel wall? Does transduction of this stimulus occur by the same mechanism as transduction of commonly used soluble stimuli? What is the role of the phosphorylated 40-47 K protein in secretion? What change in GP IIb-IIIa promotes their ability to bind fibrinogen? What is the role of calcium-activated protease? Of the phosphorylation of actin-binding protein? Progress is being made rapidly, and these questions may be answered within a few years.
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PMID:Platelet activation. 298 27

Activation of human platelets by different activators resulted in a different extent of degradation of the cytoskeletal proteins actin-binding protein and myosin, as well as of the non-cytoskeletal protein P235. The highest extent of proteolysis was observed with Ca-ionophore A23187 and decreased on going from A23187 greater than collagen plus thrombin greater than collagen greater than thrombin = ADP. The same order of potency has been found previously ((1983) Biochim. Biophys. Acta 736, 57-66) for the ability of platelet activators to induce exposure of aminophospholipids in the outer leaflet of the platelet plasma membrane, and to stimulate platelets to become procoagulant. Degradation of cytoskeletal proteins as a result of platelet stimulation by collagen plus thrombin was prevented in the presence of dibutyryl cAMP or EDTA but not in the presence of aspirin. This also runs in parallel with platelet procoagulant activity. Moreover, platelets from a patient with a partial deficiency in platelet procoagulant activity revealed a diminished extent of degradation of cytoskeletal proteins upon platelet stimulation with collagen plus thrombin. It is concluded that alterations in cytoskeletal organization upon platelet stimulation may lead to alterations in the orientation of (amino)phospholipids in the plasma membrane, and may therefore play a regulatory role in the expression of platelet procoagulant activity.
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PMID:The involvement of cytoskeleton in the regulation of transbilayer movement of phospholipids in human blood platelets. 298 15

The relationship between tension and myosin 20,000-Da light chain phosphorylation in intact nonmuscle cells was investigated using a preparation of thrombin-activated, irreversibly aggregated platelets known as the platelet strip. Steady-state levels of tension generated by the platelet strip were found to be linearly related to the level of myosin phosphorylation. This relationship was observed during dose-dependent relaxation induced by the adenylate cyclase activators prostaglandin (PG) E1 and PGI2, and during contraction induced by ADP, epinephrine, and the prostaglandin endoperoxide analogue U-46619, which did not appreciably alter the basal level of adenosine 3',5'-cyclic monophosphate in the preparation. The fully relaxed platelet strip, in the absence of external Ca2+, was associated with a level of 12% light chain phosphorylation, which increased to 72% on maximal contraction. During both relaxation and contraction, changes in myosin phosphorylation were also found to precede or coincide with tension changes. Furthermore, steady-state contraction induced by ADP was associated with a maintained elevation in the level of myosin phosphorylation. These results support the concept that myosin phosphorylation is an important regulatory mechanism for contractility in platelets.
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PMID:Role of myosin phosphorylation in contractility of a platelet aggregate. 299 87

In primary coculture of endothelial and smooth muscle cells, the muscle cells preserve their specialized phenotype (high myosin content) contrary to a pure smooth muscle cell culture. The addition of thrombin to the coculture-system results in loss of specialized phenotype of smooth muscle cell i.e. the relative myosin content decreases.
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PMID:[Interaction of endothelial and smooth muscle cells of the pig aorta in primary culture]. 309 Aug 38

Glutaraldehyde (GA) and N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ), a hydrophobic, carboxyl group directed, zero-length protein cross-linker, were employed for the chemical cross-linking of the rigor complex between F-actin and the skeletal myosin S-1. The enzymatic properties and structure of the new covalent complexes obtained with both reagents were determined and compared to those known for the EDC-acto-S-1 complex. The GA- or EEDQ-catalyzed covalent attachment of F-actin to the S-1 heavy chain induced an elevated Mg2+-ATPase activity. The turnover rates of the isolated cross-linked complexes were similar to those for EDC-acto-S-1 (30 s-1). The solution stability of the new complexes is also comparable to that exhibited by EDC-acto-S-1. The proteolytic digestion of the isolated AEDANS-labeled covalent complexes and direct cross-linking experiments between actin and various preformed proteolytic S-1 derivatives indicated that, as observed with EDC, the COOH-terminal 20K and the central 50K heavy chain fragments are involved in the cross-linking reactions of GA and EEDQ. KI-depolymerized acto-S-1 complexes cross-linked by EDC, GA, or EEDQ were digested by thrombin which cuts only actin, releasing S-1 heavy chain-actin peptide cross-linked complexes migrating on acrylamide gels with Mr 100K (EDC), 110K and 105K (GA), and 102K (EEDQ); these were fluorescent only when fluorescent S-1 was used. They were identified by immunostaining with specific antibodies directed against selected parts of he NH2-terminal actin segment of residues 1-113.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cross-linking of the skeletal myosin subfragment 1 heavy chain to the N-terminal actin segment of residues 40-113. 314 Aug 94

Actin filament formation during early stages of platelet activation by stimulants was analyzed by determining the distributions of cytoskeletal proteins to a low speed centrifuge pellet (TP), ultracentrifuge pellet (UP), and ultracentrifuge supernatant (US) after lysing platelets with 1% Triton X-100. TP contained actin binding protein, myosin, alpha-actinin and actin, and UP contained these cytoskeletal proteins and other proteins. During thrombin activation, total protein and actin in TP increased with time, while they decreased in UP. Aggregating agents (ADP, PMA, thrombin) and, to a small extent, colchicine and nocodazole, microtubule inhibitors, increased cytoskeletal proteins and total protein in TP, while cytochalsin B, an inhibitor of actin polymerization, had the opposite effect. When the amounts of the respective proteins in TP and UP were summed, these values were not affected by agonists and inhibitors, except in the case of thrombin stimulation. These data suggest that the actin in UP is a form intermediate between the actin filaments in TP and actin monomer in the soluble fraction, and there may be a dynamic conversion between these forms upon platelet activation. UP was also characterized by immunostaining with antibodies against fibrinogen, tubulin, and glycoprotein Ib after SDS-PAGE/electrophoretic transfer to nitrocellulose sheets.
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PMID:Platelet cytoskeletal protein distributions in two triton-insoluble fractions and how they are affected by stimulants and reagents that modify cytoskeletal protein interactions. 341 29

Incubation of human platelets with unilamellar vesicles composed of dilauroylphosphatidylcholine (DLPC) induces shedding of small vesicular structures from the platelet plasma membrane. No significant cell lysis is observed during the process of shedding. Isolated spicules contain the major membrane glycoproteins, Ib, IIb, and IIIa, which are used to define the sidedness of the spicule membrane. These glycoproteins are completely susceptible to chymotrypsin treatment, whereas cytoskeletal proteins are inaccessible towards this enzyme. This demonstrates that the spicule membranes have a right-side-out orientation in as far as membrane proteins are concerned. Isolated spicules were 30-fold more active than platelets in stimulating prothrombin conversion to thrombin by the prothrombinase complex (factors Xa, Va and Ca2+). The increased prothrombinase activity reflects an increased amount of phosphatidylserine in the outer leaflet of the spicule membrane. Protein analysis of platelet spicules and native platelets reveals a number of differences, the most conspicuous of which is the virtual absence of myosin in the spicule preparations. It is proposed that a lack of myosin produces a different cytoskeletal organization in the spicules. This enables phosphatidylserine to become exposed at the outer surface of the spicule membrane.
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PMID:Loss of phospholipid asymmetry in dilauroylphosphatidylcholine induced plasma membrane vesicles from human platelets. 365 53

We studied the cytoskeletal reorganization of saponized human platelets after stimulation by using the quick-freeze deep-etch technique, and examined the localization of myosin in thrombin-treated platelets by immunocytochemistry at the electron microscopic level. In unstimulated saponized platelets we observed cross-bridges between: adjoining microtubules, adjoining actin filaments, microtubules and actin filaments, and actin filaments and plasma membranes. After activation with 1 U/ml thrombin for 3 min, massive arrays of actin filaments with mixed polarity were found in the cytoplasm. Two types of cross-bridges between actin filaments were observed: short cross-bridges (11 +/- 2 nm), just like those observed in the resting platelets, and longer ones (22 +/- 3 nm). Actin filaments were linked with the plasma membrane via fine short filaments and sometimes ended on the membrane. Actin filaments and microtubules frequently ran close to the membrane organelles. We also found that actin filaments were associated by end-on attachments with some organelles. Decoration with subfragment 1 of myosin revealed that all the actin filaments associated end-on with the membrane pointed away in their polarity. Immunocytochemical study revealed that myosin was present in the saponin-extracted cytoskeleton after activation and that myosin was localized on the filamentous network. The results suggest that myosin forms a gel with actin filaments in activated platelets. Close associations between actin filaments and organelles in activated platelets suggests that contraction of this actomyosin gel could bring about the observed centralization of organelles.
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PMID:Cytoskeletal reorganization of human platelets after stimulation revealed by the quick-freeze deep-etch technique. 366 97


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