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)

The formation of proplatelet-like processes on megakaryocytes cultured in vitro has been shown to be inhibited by prothrombin, found residually in human serum, which is converted in culture to thrombin. This study reports that another factor found in human serum will counter this inhibition and permit proplatelet-like process formation to occur in vitro even in the presence of inhibitory concentrations of thrombin. The factor was purified from human platelet lysates and identified by amino acid sequence analysis as the proteoglycan serglycin. A similar, if not identical, factor was found at elevated levels in the plasma of thrombocytopenic rabbits. Serglycin probably functions as a proplatelet potentiator by virtue of a tendency to complex with thrombin. Thrombin in complex with serglycin retains its enzymatic properties, but is apparently sterically hindered from interacting with the megakaryocyte cell surface. In preliminary studies, the in vivo administration of serglycin in mice resulted in an increased number of circulating platelets when given in combination with interleukin-6 (IL-6).
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PMID:The effect of the platelet-derived glycosaminoglycan serglycin on in vitro proplatelet-like process formation. 833 Jun 53

Platelet-derived growth factor (PDGF) is a potent mitogen consisting of heterodimers of two distinct but homologous polypeptide chains, denoted A and B. PDGF-like homodimers of the A- and B-chains have been isolated, as well as two distinct receptor types (alpha and beta), which discriminate among the PDGF isoforms. The PDGF A- and B-chains are encoded by distinct genes located on human chromosomes 7 and 22, respectively. Although PDGF has been implicated as an important participant in development, tissue repair, and numerous pathologic states including tumorigenesis, atherosclerosis and inflammation, the mechanisms which determine the rate of its synthesis are only beginning to be understood. Basal expression of the PDGF A- and B-chain genes has been characterized in a number of cell types and is directed in part by elements in the respective proximal promoter-regulatory regions of the two genes. In addition, the first intron of PDGF-B has been shown to contain both positive and negative regulatory elements. Transcription of the PDGF subunit genes is inducible by a wide variety of mitogenic growth factors, cytokines and other agonists. These agents produce a rapid increase in steady-state concentrations of PDGF A- and B-chain mRNAs, peaking within 4-8 h of stimulation. The inductive effects of protein kinase C-activating phorbol 12-myristate 13-acetate (PMA), thrombin and transforming growth factor-beta (TGF-beta) are mediated through increases in the transcription rates of both genes. In addition, cAMP blocks the increases in transcription of the B-chain gene induced by thrombin and TGF-beta. Studies have demonstrated the importance of sequences immediately upstream of the B-chain transcription start site for induction in response to PMA-initiated megakaryocyte differentiation, an effect which is dependent on protein synthesis. However, cis-acting elements which mediate more rapid transcriptional induction seen in endothelial cells and astrocytes have yet to be identified in the proximal 5'-flanking sequences of either the A- or B-chain genes, suggesting that such events may be mediated by elements located outside of this region.
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PMID:Transcriptional control of the platelet-derived growth factor subunit genes. 834 77

Vascular diseases and related complications still represent the main cause of death in diabetic patients. Neuropathy, nephropathy, retinopathy, and disturbed nutritive tissue perfusion may result from reduced capillary microcirculation. These disturbances are diabetes specific. Macroangiopathy does not differ structurally from atherosclerotic lesions of nondiabetic subjects, but leads to accelerated cerebral, coronary, and peripheral artery disease. Occurrence of life-terminating thrombotic events, which are superimposed on those vascular lesions, are increased. Thus, morbidity and mortality of diabetics depend mainly on vascular complications. Normal blood flow is a prerequisite of adequate organ perfusion and results from vasomotion, plasma components, corpuscular blood elements, vascular architecture, and the undisturbed interaction of these components at the endothelial interface. Functional thromboresistance of the endothelial layer is reduced in the diabetic state. Increased intravascular thrombin generation, reduced fibrinolytic potential, and hyperactive platelets lead to a prethrombotic state. This thrombotic diathesis increases the permanent danger of acute flow interruption. Activated platelets operate by three mechanisms: (1) Microembolization of the capillaries; (2) local progression of preexisting vascular lesions by secretion of constrictive, mitogenic, and oxidative substances; (3) trigger of the prognosis-limiting arterial thrombotic event. We were able to show that the increased functional properties of diabetic platelets result from the primary release of larger platelets with enhanced thromboxane formation capacity and increased numbers of functional glycoprotein receptors GPIb and GPIIb/IIIa, which are synthesized in the megakaryocytes. The megakaryocyte-platelet system is turned on in diabetes mellitus. It could be demonstrated with the Duesseldorf III method of flow cytometric activation marker testing (CD62, CD63, thrombospondin) that predominantly large platelets circulate in an activated state in diabetes mellitus.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Platelets in diabetes: the role in the hemostatic regulation in atherosclerosis. 835 57

There are several species of alternatively spliced fibronectin (FN). One of these, FN EIIIB, is primarily present in embryonic and in proliferating and migrating cells and is believed to be important for cell maturation. We have studied the synthesis, localization, and secretion of this FN isoform in isolated guinea pig megakaryocytes, nonmegakaryocytic bone marrow cells, and platelets. There was 7.5 times more general FN in megakaryocytes than in nonmegakaryocytic cells based on the analysis of equivalent amounts of protein. FN EIIIB was detected by Western blotting in megakaryocytes but not in nonmegakaryocytic cells present in bone marrow. Neither megakaryocytes nor platelets secreted FN EIIIB, while megakaryocytes secreted 25.3% +/- 4.6% general FN and platelets secreted about 61% general FN in response to thrombin. Analysis of immunostained cells by confocal microscopy revealed that FN EIIIB had been redistributed to the surface of megakaryocytes in response to thrombin. Synthesis was studied by metabolic labeling, and megakaryocytes were shown to synthesize FN and FN EIIIB. Thus, megakaryocytes and platelets are among a small number of adult cells and tissues that synthesize and contain FN EIIIB. The expression of FN EIIIB on the megakaryocyte surface may influence migration and maturation.
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PMID:The synthesis and localization of alternatively spliced fibronectin EIIIB in resting and thrombin-treated megakaryocytes. 863 28

The migration of integrins to sites of cell-cell and cell-matrix contact is thought to be important for adhesion strengthening. We studied the lateral diffusion of integrin alpha IIb beta 3 (glycoprotein IIb/IIIa) in the plasma membrane of a cultured human megakaryocyte by fluorescence recovery after photobleaching of FITC-labelled monovalent Fab fragments directed against the beta 3 subunit. The diffusion of beta 3 on the unstimulated megakaryocyte showed a lateral diffusion coefficient (D) of 0.37 x 10(-9) cm2/s and a mobile fraction of about 50%. Stimulation with ADP (20 microM) or alpha-thrombin (10 U/ml) at 22 degrees C induced transient decreases in both parameters reducing D to 0.21 x 10(-9) cm2/s and the mobile fraction to about 25%. The fall in D was observed within 1 min after stimulation but the fall in mobile fraction showed a lag phase of 5 min. The lag phase was absent in the presence of Calpain I inhibitor, where-as cytochalasin D completely abolished the decreased in mobile fraction. The data are compatible with the concept that cell activation induces anchorage of 50% of the mobile alpha IIb beta 3 (25% of the whole population of receptor) to the cytoplasmic actin filaments, although, as discussed, other rationals are not ruled out.
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PMID:Lateral mobility of integrin alpha IIb beta 3 (glycoprotein IIb/IIIa) in the plasma membrane of a human megakaryocyte. 903 65

Megakaryocytopoiesis concerns the commitment of hematopoietic stem cells towards the megakaryocyte lineage, the proliferation and maturation of megakaryocyte progenitors, leading to platelet formation. Normal megakaryocytopoiesis requires an equilibrium between positive and negative regulators. Thrombopoietin, recently cloned, is the main growth factor for the megakaryocyte lineage, enhancing all the steps of megakaryocyte development: proliferation, maturation, ploidisation, platelet formation. Several other factors have a direct (interleukin 3, 6, 11, 13, GMCSF, erythropoietin) or indirect (interleukin 1, stem cell factor) positive effect. Factors inhibiting megakaryocytopoiesis are synthetized by the megakaryocytes themselves (platelet factor 4, transforming growth factor beta), or have many other effects (alpha-interferon, thrombin) or correspond to a novel molecule (anagrelide). Heparin and other glycosaminoglycans positively modulate megakaryocytopoiesis by acting synergistically with some growth factors and by neutralizing some inhibitors. These progresses in the knowledge of normal and pathological megakaryocytopoiesis should lead to considerable therapeutic advances.
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PMID:[Positive and negative regulation of megakaryocytopoiesis]. 907 17

At sites of vascular injury thrombin is generated via prothrombinase, a stoichiometric (1:1), Ca2+-dependent, and membrane-bound complex consisting of the nonenzymatic cofactor factor Va and the serine protease factor Xa. While the importance of anionic platelet membrane phospholipids in regulating thrombin generation is well recognized, the identification of regulatory protein receptors has eluded investigators. This study reports the first description of a human platelet membrane protein that regulates prothrombinase complex assembly and function. Direct platelet-protein binding studies indicated that, although required, platelet-bound factor Va alone is insufficient to mediate factor Xa binding, and that factor Va and factor Xa bind to discrete sites on activated platelets for which expression is independently regulated as a function of the agonist concentration. When specific monoclonal antibodies against effector cell protease receptor-1 (EPR-1, a 65-kDa membrane receptor for factor Xa) were used in Western blotting, immunohistochemical staining, and/or flow cytometric analyses, activated platelets and their precursors, megakaryocytes, were shown to express EPR-1. These results were confirmed by reverse transcription-polymerase chain reaction of mRNA extracted from megakaryocyte-like cell lines. Additional flow cytometric studies demonstrated that a platelet-bound factor Va/factor Xa complex precluded binding of the anti-EPR-1 antibody, B6, to activated platelets by approximately 50%. Likewise, the anti-EPR-1 antibody was shown to inhibit prothrombinase-catalyzed thrombin generation on activated platelets in a dose- and platelet donor-dependent manner, indicating that platelet-expressed EPR-1 mediates factor Xa assembly into the prothrombinase complex. These collective data indicate that both EPR-1 and membrane-bound factor Va are required to mediate factor Xa binding to the activated platelet to form a functional prothrombinase complex.
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PMID:Effector cell protease receptor-1, a platelet activation-dependent membrane protein, regulates prothrombinase-catalyzed thrombin generation. 908 58

Platelet glycoprotein (GP) V is a major surface protein cleaved during thrombin-induced platelet activation. GPV associates noncovalently with the GPIb-IX complex to form GPIb-V-IX, a receptor for von Willebrand factor and thrombin. We describe the cloning of the genes coding for rat and mouse GPV and compare them with the human gene. The two rodent genes have a similar structure and resemble the human GPV gene with a coding sequence (approximately 1,700 nucleotides) entirely contained in one exon and a single intron (approximately 900 nucleotides) in the 5' untranslated region. Both genes have megakaryocyte-type promoters with conserved tandem Ets and GATA recognition motifs and lack a TATA box. The mature rat and mouse proteins comprise 551 amino acids, have 70% sequence identity, and contain an additional 8-amino acid intracellular segment as compared with the human protein. As in human GPV, there is an NH2-terminal leucine-rich region of 15 repeats and a thrombin cleavage recognition sequence. Whereas the rat and human thrombin cleavage sites are similar, the mouse cleavage site resembles that of the human thrombin receptor. Functionality of these sites was demonstrated by thrombin cleavage of synthetic peptides and analysis by high-performance liquid chromatography (HPLC) or mass spectrometry. Cleavage of native rat GPV was confirmed by means of a polyclonal antibody directed against the new NH2-terminal peptide exposed after thrombin cleavage. This antibody specifically recognized thrombin-activated rat platelets by fluorescence-activated cell sorting (FACS) analysis. In addition, we raised monoclonal antibodies specific for rat GPV (88 kD), which recognized the NH2-terminal soluble fragment (70 kD) liberated after thrombin cleavage. Knowledge of these rodent GPV genes and availability of species-specific peptides and antibodies will be essential to further studies aiming to define the exact in vivo function of platelet GPV using animal models of thrombosis and gene inactivation experiments.
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PMID:Gene cloning of rat and mouse platelet glycoprotein V: identification of megakaryocyte-specific promoters and demonstration of functional thrombin cleavage. 912 30

1. A combination of single cell fluorescence and patch clamp techniques were used to study the mechanisms underlying thrombin-evoked Ca2+ signals in human erythroleukaemia (HEL) cells, a leukaemic cell line of platelet-megakaryocyte lineage. 2. Thrombin caused a transient increase in intracellular Ca2+ ([Ca2+]i), consisting of both release of Ca2+ from intracellular stores and influx of extracellular Ca2+. Mn2+ quench studies indicated that the thrombin-evoked divalent cation-permeable pathway was activated during, but not prior to, release from internal stores. 3. Thapsigargin (1 microM) irreversibly released internal Ca2+ from the same store as that released by thrombin and continuously activated a Ca(2+)-influx mechanism. The amplitude of the thrombin- and thapsigargin-induced Ca2+ influx displayed a marked single cell heterogeneity which showed no correlation with the size of the store Ca2+ transient. 4. In whole-cell patch clamp recordings, both thrombin and thapsigargin evoked an inwardly rectifying Ca2+ current which developed with little or no increase in current noise, showed no reversal in the voltage range -110 to +60 mV and was blocked by 1 mM Zn2+. The apparent divalent cation permeability sequence of this pathway was Ca2+ > > Ba2+ > Mn2+, Mg2+. The thapsigargin-evoked current density at -100 mV varied between 0.42 and 2.1 pA pF-1 in different cells. Thrombin failed to activate additional Ca2+ current if it was added after the thapsigargin-induced inward current had fully developed. 5. These studies indicate that thrombin activates Ca2+ influx in HEL cells entirely via a Ca(2+)-store-release-activated Ca2+ current (Icrac) rather than via receptor-operated or second messenger-dependent Ca2+ channels. The level of expression of Icrac appears to be a major factor in determining the duration of the thrombin-evoked [Ca2+]i response and therefore represents a means by which cells can exert control over [Ca2+]i-dependent events.
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PMID:Thrombin-dependent calcium signalling in single human erythroleukaemia cells. 921 9

Stimulation of platelets by collagen leads to activation of a tyrosine kinase cascade resulting in secretion and aggregation. We have recently shown that this pathway involves rapid tyrosine phosphorylation of an Fc receptor gamma chain, which contains an immunoreceptor tyrosine-based activation motif (ITAM), enabling interaction with the tandem SH2 domains of the tyrosine kinase Syk. Activation of Syk lies upstream of tyrosine phosphorylation of phospholipase Cgamma2. In the present study we sought to test directly the role of the ITAM/Syk interaction and the role of the Src-related kinases in collagen receptor signaling using mouse megakaryocytes. We demonstrate that the calcium-mobilizing action of a collagen-related peptide (CRP) is kinase-dependent, inhibited by the microinjection of the tandem SH2 domains of Syk and abolished in Syk-deficient mice. Furthermore, the CRP response is abolished by the Src family kinase inhibitor PP1 and inhibited in Fyn-deficient mice. In contrast, the calcium response to the G-protein-linked receptor agonist thrombin is not significantly altered under these conditions. These results provide direct evidence of the functional importance of Fyn and Syk in collagen receptor signaling and support the megakaryocyte as a model for the study of proteins involved in this pathway.
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PMID:Syk and Fyn are required by mouse megakaryocytes for the rise in intracellular calcium induced by a collagen-related peptide. 934 87


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