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)

Thrombin forms sodium dodecyl sulfate stable complexes of 77 and greater than 450 kDa with proteins secreted by activated platelets. The kinetics of formation of these complexes were investigated by addition of 125I-thrombin to the supernatant solution of A23187-activated platelets. Complexes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis either with or without reduction of disulfide bonds. When analyzed on nonreduced gels, the 77-kDa complex reached a maximum at about 3 min and then declined as the greater than 450-kDa complex increased. On reduced gels (on which there was no greater than 450-kDa complex) the 77-kDa complex approached the level of the greater than 450-kDa complex on nonreduced gels. The half-time of formation was less than 1 min for the 77-kDa complex and about 15 min for the greater than 450-kDa complex. These time courses suggested that the 77-kDa complex was incorporated into the greater than 450-kDa complex as an essential precursor. Formation of complexes was inhibited by a competitive inhibitor or a noncompetitive inhibitor of thrombin, and the pH dependence of formation of both complexes was similar to the pH dependence for catalytic activity of thrombin. Ca2+ inhibited formation of the greater than 450-kDa complex but not of the 77-kDa complex. A model is presented in which thrombin and a secreted protein form a 77-kDa complex by a process that involves the active site of thrombin. The 77-kDa complex is then incorporated into a greater than 450-kDa complex by thiol-disulfide exchange with thrombospondin, a process that is inhibited by Ca2+. Thrombin in the greater than 450-kDa complex had no catalytic activity.
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PMID:Kinetics of the formation of thrombin-thrombospondin complexes: involvement of a 77-kDa intermediate. 284 22

Glycoprotein IIIb (also known as glycoprotein IV) is a major glycoprotein present on the surface of human platelets. Recent studies suggest that glycoprotein IIIb may be a receptor site for thrombospondin. Thrombospondin, a multifunctional adhesive glycoprotein released from stimulated platelets, plays an important role in the stabilization of platelet aggregates. In this study, a new method for the purification of glycoprotein IIIb is described. Glycoprotein IIIb was isolated from Triton X-114 platelet membrane extracts, under nondenaturing conditions, by tandem anion-exchange and size exclusion fast protein liquid chromatography. The purified glycoprotein had the same apparent molecular mass (88 kDa) under nonreducing or reducing conditions. The tryptic peptide map of the purified protein was identical to that of bona fide glycoprotein IIIb as isolated from two-dimensional polyacrylamide gels of platelet membrane proteins. In addition, the purified glycoprotein was recognized by an anti-GPIIIb monoclonal antibody (OKM5). The purified glycoprotein specifically bound to thrombospondin in the presence of calcium. Monospecific anti-GPIIIb antibodies interfered with the expression of endogenous thrombospondin on thrombin-activated platelets and partially inhibited collagen- and thrombin-induced platelet aggregation without a significant effect on platelet secretion. Glycoprotein IIIb, by interacting with thrombospondin on the activated platelet surface, may play an important role in the platelet aggregation process.
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PMID:Rapid purification and partial characterization of human platelet glycoprotein IIIb. Interaction with thrombospondin and its role in platelet aggregation. 290 35

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

The distribution of platelet thrombospondin (TSP), fibrinogen, and glycoproteins IIb-IIIa (GPIIb-IIIa) and GPIb were studied in resting and activated human platelets using frozen thin-section immunoelectron microscopy. In resting platelets, TSP and fibrinogen were found within alpha granules and not on the platelet surface. In unstimulated platelets, GPIIb-IIIa and GPIb were distributed diffusely over the platelet membrane as well as within the body of the platelets. Upon thrombin or A23187 stimulation, TSP, fibrinogen, and GPIIb-IIIa colocalized on the platelet membrane and the canalicular system as well as on pseudopodia and between adherent platelets. GPIb distribution was unchanged by platelet activation. The findings support the hypothesis that a macromolecular complex of TSP-fibrinogen and GPIIb-IIIa forms on the activated platelet membrane.
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PMID:Platelet membrane topography: colocalization of thrombospondin and fibrinogen with the glycoprotein IIb-IIIa complex. 299 82

We have developed a monospecific antiserum directed against a major glycoprotein in the elastin-associated microfibrils with an apparent molecular mass of 128 kDa (GP 128). When immunoblotting or enzyme-linked immunosorbent microassay was used, its IgGs recognized thrombospondin in a platelet lysate, but did not react with several basement-membrane-derived macromolecules, nor with plasma fibronectin. Similar patterns of immunofluorescence and immunoperoxidase were found after incubation of endothelial cells with either anti-GP 128 or anti-(platelet thrombospondin) IgGs. Both antibodies inhibited the microfibrils- and thrombin-induced platelet aggregation, and were without effect on the aggregation by other inducers. These results confirm that there is an antigenic homology between GP 128 and thrombospondin.
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PMID:Characterization of an antibody directed against a 128 kDa glycoprotein involved in the thrombogenicity of the elastin-associated microfibrils. 305 19

The interaction of isolated and radioiodinated thrombospondin with washed human platelets has been characterized. The ligand bound to nonstimulated and thrombin-stimulated platelets in a time-dependent manner, and apparent steady state was reached within 25 min. Binding was not due to iodination of the ligand and was inhibited by nonlabeled thrombospondin but not by unrelated proteins, and bound ligand was identical with thrombospondin in terms of subunit structure. Nonlinear curve-fitting analyses of binding to resting platelets suggested the presence of a single class of sites which bound 3,100 +/- 1,000 molecules/platelet with an apparent Kd of 50 +/- 20 nM. This interaction was not attributable to contaminating cells or inadvertant platelet activation. Binding to thrombin-stimulated platelets had a lower apparent affinity (Kd = 250 +/- 100 nM) and higher apparent capacity (35,600 +/- 9,600 molecules/platelet). Thrombin-enhanced binding was dependent upon agonist dose and platelet stimulation. Fibrinogen, a monoclonal antibody to GPIIb-IIIa, temperature, and divalent ions had differential effects upon thrombospondin binding to resting and stimulated platelets, suggesting the presence of two distinct mechanisms of thrombospondin binding to platelets. While thrombospondin binding to thrombin-stimulated platelets occurs with characteristics similar to those observed for fibrinogen, fibronectin, and von Willebrand Factor, its high affinity interaction with resting platelets is unique to this adhesive glycoprotein.
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PMID:Interaction of thrombospondin with resting and stimulated human platelets. 308 90

The radiolabelled monoclonal antibody, 5G11, directed against native thrombospondin, has been used to assess the surface expression of secreted thrombospondin on human blood platelets. Emphasis has been placed on studying the role of fibrinogen in this process. Unstimulated platelets bound low amounts of 5G11 (about 2000 molecules/platelet). Binding increased 2-fold and 5-7-fold after stimulation of platelets with ADP or thrombin (or ionophore A23187) respectively. Unstimulated platelets from patients deficient in alpha-granule proteins (gray platelet syndrome) bound baseline levels of 5G11. However, binding was not increased after activation. Thrombospondin expression on thrombin-stimulated normal platelets was for a large part divalent-cation-dependent and was not affected by AP-2, a monoclonal antibody to GPIIb-IIIa complexes. However, binding of 5G11 was some 50% lower when platelets were stimulated in the presence of Fab fragments of a polyclonal rabbit antibody to fibrinogen. This suggested either a direct binding of thrombospondin to surface-bound fibrinogen or a steric inhibition due to a close proximity of the two proteins. The fact that binding of 5G11 was at the lower limit of the normal range to the stimulated platelets of an afibrinogenaemic patient specifically lacking detectable fibrinogen favoured the latter explanation. Thus, a major fibrinogen-independent pathway for thrombospondin expression must exist.
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PMID:Use of a monoclonal antibody to measure the surface expression of thrombospondin following platelet activation. 312 35

A labeled 77-kDa complex formed when 125I-thrombin was added to platelet suspensions or to the supernatant solution of ionophore-activated platelets. Prostacyclin inhibited complex formation with whole platelets but not with the supernatant solution of ionophore-activated platelets. This is evidence that the complex formed with a factor secreted from activated platelets. Smaller complexes of 70 and 58 kDa formed between labeled thrombin and lysed platelets. The 77-kDa complex was necessary for the formation of a thrombin-thrombospondin complex.
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PMID:Complexes of thrombin with secreted platelet proteins. 312 41

Platelet membrane glycoprotein V (GPV) was hydrolyzed during thrombin-induced platelet aggregation releasing a fragment GPVfl into the supernatant. Hydrolysis of GPV required catalytically active thrombin and was diminished by chemical modification of the fibrinogen binding site of thrombin. Half-maximal liberation of GPVfl occurred at a 10-fold higher concentration of thrombin than was required for half-maximal release. Time course studies at several thrombin concentrations showed disparate release of GPVfl and thrombospondin. These results emphasize the complexity of the initial events in thrombin-induced platelet activation.
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PMID:Glycoprotein V hydrolysis by thrombin. Lack of correlation with secretion. 316 Dec 10

Stimulated human blood platelets release thrombospondin, an alpha-granule glycoprotein of 450 kDa. The aim of this work was to characterize an anti-thrombospondin monoclonal antibody (P8) in order to study the role of thrombospondin in platelet functions. The presence of thrombospondin receptor sites on resting and thrombin-stimulated platelets of three Glanzmann's thrombasthenia patients and normal donors was investigated using the P8 monoclonal antibody. Monoclonal antibody P8 was extensively characterized using ELISA, immunoprecipitation, immunoadsorbent affinity chromatography combined with tryptic peptide map analysis and crossed immunoelectrophoretic techniques. Labelled P8 bound strongly to thrombin-stimulated normal platelets (n = 14917 +/- 420, mean +/- SD) (Kd = 9.2 +/- 3.0 nM) and poorly to resting platelets (n = 2697 +/- 1278) (Kd = 24.8 +/- 18.6 nM). Moreover, the number of binding sites for P8 on thrombin-stimulated platelets from three Glanzmann's thrombasthenia patients, lacking the IIb-IIIa glycoprotein complex, were found similar to normal samples. F(ab')2 fragments of P8 inhibited aggregation of, and reduced secretion from, washed platelets stimulated by low concentrations of thrombin (0.05-0.06 U/ml) and collagen (0.5-0.6 microgram/ml). F(ab')2 fragments of P8 inhibited thrombin-induced platelet aggregation, but did not reduce fibrinogen binding (n) nor affect its dissociation constant (Kd). Inhibition of platelet aggregation by P8 suggests that thrombospondin plays an active role in promoting platelet aggregation, at low concentrations of thrombin and collagen. Normal binding of P8 to thrombin-stimulated Glanzmann thrombasthenic platelets indicates the presence of a thrombospondin receptor on the platelet surface distinct from the GPIIb-IIIa complex.
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PMID:Characterization of an anti-thrombospondin monoclonal antibody (P8) that inhibits human blood platelet functions. Normal binding of P8 to thrombin-activated Glanzmann thrombasthenic platelets. 333 71


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