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)

We have previously demonstrated that platelets in polycythaemia vera (PV) exhibit decreased aggregation after stimulation with platelet activating factor (PAF) and reduced expression of GPIIIa on both resting and stimulated platelets. In the present study, we investigated if these results were related to changes in the mobilization of intracellular calcium, activation of phospholipase D (PLD) or amounts of GPIIIa and the intracellular tyrosine kinases Fak, Syk, Grb2, Shc and rhoA. Intracellular calcium levels were not different in resting platelets from 14 PV patients and 15 healthy controls (median 43 nmol/L, range 10-114, vs. 36 nmol/L, range 10-119). After stimulation with PAF (1 micromol/L) an equal increase was seen (125 nmol/L for PV platelets, range 67-257, vs. 113 nmol/L for controls, range 60-250). Also formation of phosphatidyl ethanol (PEt) was similar after exposure to 0.5 U/ml thrombin (0.28% PEt of total phospholipid, range 0.16-1.10, vs. 0.24 for controls, range 0.11-2.3) and 1 micromol/L PMA (0.25, range 0.16-0.32, vs. 0.14, range 0.09-0.6). In contrast to the reduced amount of GPIIIa on the surface of PV platelets, immunoblotting on whole cell lysates showed no reduction in PV patients compared to controls, indicating the possibility of an impaired incorporation of GPIIIa to the cell membrane. Levels of Fak, Syk, Shc, Grb2 and rhoA appeared equal in patients and controls. Similar intracellular proteins were tyrosine phosphorylated after stimulation with thrombin, PAF and PMA. In summary, defective platelet aggregation after stimulation with PAF is caused by neither defective mobilization of intracellular calcium nor, in contrast to the situation in PV granulocytes, an impaired activation of PLD. Moreover, no apparent differences in the intracellular amounts of Fak, Syk Shc, Grb2 and rhoA could be detected between PV and control platelets.
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PMID:Defective platelet aggregation in polycythaemia vera is not caused by impaired calcium signaling, phospholipase D activation or decreased amounts of focal adhesion proteins. 1109 63

Defects in glycoprotein (GP)IIb-IIIa or in its activation may cause abnormal platelet aggregation and a bleeding diathesis. We report studies in a 67-year-old man with a myeloproliferative disease and markedly abnormal platelet responses. By flow cytometry, platelet binding of two complex-specific anti-GPIIb-IIIa monoclonal antibodies (mAbs), A2A9 and 10E5, was approximately 50% of normal. An enzyme-linked immunosorbent assay (ELISA) using immobilized kistrin showed 18% of normal membrane GPIIb-IIIa complex. By immunoblot analysis, GPIIb and GPIIIa levels in platelet lysates and membranes were near normal. Activation of GPIIb-IIIa, monitored with mAb PAC-1, was markedly decreased (< 20% of normal) in response to ADP, thrombin and platelet-activating factor (PAF); expression of ligand-induced binding sites (LIBS) was < or = 30% of normal. Signal transduction-independent LIBS expression, induced by echistatin, was approximately 60% of normal, suggesting that the integrin present had intact ligand-binding capability. Sequence analysis of GPIIb and GPIIIa cDNA, and platelet mRNA levels for both subunits, were normal. These findings document an acquired combined defect in membrane expression (secondary to a defect in post-translational processing of the complex) and inside-out signalling-dependent activation of the GPIIb-IIIa complex.
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PMID:Combined defect in membrane expression and activation of platelet GPIIb--IIIa complex without primary sequence abnormalities in myeloproliferative disease. 1112 60

Early studies considered that fibrinogen receptor (glycoprotein [GP] IIb-IIIa or platelet integrin alpha(IIb)beta(3)) is the binding site for low-density lipoprotein (LDL) and high-density lipoprotein type 3 (HDL(3)). Recent data, however, do not support the hypothesis that the binding of LDL to human intact resting platelets is related to integrin alpha(IIb)beta(3). In this study we present evidence that platelet integrin alpha(IIb)beta(3) is also not involved in the interaction of HDL(3) and human intact resting platelets. Firstly, specific ligands for platelet integrin alpha(IIb)beta(3), such as fibrinogen, vitronectin, von Willebrand factor and fibronectin, were unable to inhibit the binding of HDL(3) to intact resting platelets. Secondly, the HDL(3) binding characteristics (K(d) and B(max) values), the activation of protein kinase C (PKC) and the inhibition of thrombin-induced inositoltriphosphate (IP(3)) formation and calcium (Ca(2+)) mobilization mediated by HDL(3) particles were similar in platelets from control subjects and patients with type I and type II Glanzmann's thrombasthenia, which are characterized by total and partial lack of GPIIb-IIIa and fibrinogen, respectively. In contrast, nitrosylation of tyrosine residues of HDL(3) by tetranitromethane fully abolished both the ability of particles to interact with its specific binding sites and the functional effects. Thirdly, polyclonal antibodies against the GPIIb-IIIa complex (edu-3 and 5B12), human antiserums against platelet alloantigens (anti-Bak(a/B) and anti-PL(A1/2)), anti-integrin subunits (anti-alpha(V) and anti-beta(3)), and a wide panel of monoclonal antibodies (mAbs) against well-known epitopes of GPIIb (M3, M4, M5, M6, M8 and M95-2b) and GPIIIa (P23-7, P33, P37, P40, and P97) did not affect the binding of HDL(3) particles to human intact resting platelets. Overall results show that neither the GPIIb-IIIa complex nor GPIIb or GPIIIa individually are the membrane binding proteins for HDL(3)on intact resting platelets.
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PMID:Platelet HDL(3) binding sites are not related to integrin alpha(IIb)beta(3) (GPIIb-IIIa). 1113 79

Store-regulated Ca(2+) entry (SOCE) is an important mechanism of elevating cytosolic [Ca(2+)]i in platelets, though the Ca(2+) influx channels involved are still unclear. We screened human platelets and their precursor cells (human stem cells and megakaryocytes) for the presence of candidate influx channels, i.e., isoforms of the Trp family of proteins. Primary stem cells were cultured with thrombopoietin to allow differentiation into megakaryocytes. The undifferentiated stem cells (CD34(+)) showed mRNA expression of only a spliced variant Trp1A. Immature (CD61(+)/CD42b(low)) and mature (CD61(+)/CD42b(high)) megakaryocytes as well as platelets expressed in addition unspliced Trp1 as well as Trp4 (less abundant) and Trp6 isoforms. This unspliced isoform appeared to be specific for cells of the megakaryocyte/platelet lineage, since immature (CD14(+)/CD61(-)/CD42b(-)) and mature monocytes expressed only the Trp1A isoform. This conclusion was confirmed by the presence of Trp1A, 3, 4 and 6 transcripts in the immature megakaryocytic Dami cell line, and of Trp1, 1A, 4 and 6 transcripts in the more mature CHRF-288 cell line. The up-regulation of Trp1, 4 and 6 in the lineage from primary stem cells to mature megakaryocytes and platelets was accompanied by increased influx of extracellular Ca(2+) after pretreatment of the cells with thapsigargin or thrombin. Expression of new Trp isoforms in the differentiated cells is thus accompanied by increased SOCE.
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PMID:Expression of transient receptor potential mRNA isoforms and Ca(2+) influx in differentiating human stem cells and platelets. 1142 Jan 22

Enumerating and phenotyping of platelets, resting and activated, from whole blood is important for both the identification and verification of many disease states. Microvolume laser scanning cytometry (MLSC) has been shown to be a simple method for enumerating and phenotyping peripheral blood cells. Here, the utility of MLSC, in conjunction with an anticoagulant containing platelet activation inhibitors, for simultaneously measuring platelet count, phenotype and responsiveness directly from non-fixed whole blood was examined. CTAD or EDTA anticoagulated blood was collected from five to 20 healthy volunteers, stained with fluorescence-labeled antibodies specific for platelet antigens, and run on an in-house modified MSLC device. MLSC was able to measure antigens CD9, CD29, CD36, CD41, CD42a, CD42b, and CD61 on platelets and determine an average of 2.3 x 10(5) +/- 7 x 10(4) platelets per microliter. Counts correlated well with those obtained from the Cell-Dyn 3500 (r(2)=0.84). Agreeing with previous data, less than 2% of platelets from peripheral blood of normal individuals expressed the activation markers CD62P or CD63. After in vitro thrombin activation, >93% of the platelets expressed activation markers. Data presented here shows the benefits of using MLSC in combination with platelet inhibitors to quantitate and phenotype platelets while maintaining a viable responsive state.
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PMID:Whole blood microvolume laser scanning cytometry for monitoring resting and activated platelets. 1148 84

This article reports a Glanzmann thrombasthenia (GT) patient, N.M., with a point mutation in the third cysteine-rich repeat of beta3-integrin or platelet glycoprotein (GP) IIIa, leading to the expression of a constitutively activated fibrinogen receptor. The diagnosis of GT was based on a severely reduced platelet-aggregation response to a series of agonists and approximately 20% of surface-expressed GPIIb-IIIa. The patient's GPIIb-IIIa constitutively expressed epitopes recognized by antibodies to ligand-induced binding sites (LIBS) and also spontaneously bound the fibrinogen-mimetic antibody, PAC-1. Furthermore, significant amounts of bound fibrinogen were detected on his platelets ex vivo. No signs of platelet activation were observed on sections of unstimulated platelets from N.M. by electron microscopy. Immunogold labeling highlighted the presence of surface-bound fibrinogen but revealed platelet heterogeneity with regard to the surface density. When the patient's platelets were stimulated by thrombin-receptor activating peptide, amounts of surface-expressed GPIIb-IIIa increased and the aggregation response improved, although it failed to normalize. Platelets from N.M. were able to adhere and spread on immobilized fibrinogen. Sequence analysis of genomic DNA from N.M. revealed a homozygous g1776T>C mutation in GPIIIa, leading to a Cys560Arg amino acid substitution. A stable Chinese hamster ovary (CHO) cell line was prepared expressing surface GPIIb-Arg560IIIa. Like platelets from the patient, GPIIb-Arg560IIIa-transfected CHO cells constitutively bound LIBS antibodies and PAC-1. They also showed an enhanced ability to adhere on surface-bound fibrinogen. Overall, these data demonstrate that a gain-of-function mutation can still be associated with a thrombasthenic phenotype even though platelets show spontaneous fibrinogen binding.
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PMID:A point mutation in the cysteine-rich domain of glycoprotein (GP) IIIa results in the expression of a GPIIb-IIIa (alphaIIbbeta3) integrin receptor locked in a high-affinity state and a Glanzmann thrombasthenia-like phenotype. 1158 40

Research on normal human megakaryopoiesis has been limited by technical problems in obtaining megakaryocytic cells in sufficient quantities for experimental purposes. We describe here an ex vivo serum-free liquid culture system to expand normal human megakaryoblasts from purified bone marrow-, cord blood- or peripheral blood-derived CD34(+) cells. The early megakaryocytic cells are expanded in the presence of recombinant thrombopoietin (TpO) and interleukin-3 (IL-3), and if necessary further purified by employing anti-CD61 immunomagnetic beads. Our expansion system generates normal human megakaryoblasts in quantities sufficient to perform various functional studies on these cells as well as to isolate from them proteins and mRNA for molecular analysis. Megakaryocytic cells isolated from these cultures (i) express several markers characteristic of this lineage (CD41, CD61, CD62 P, CXCR-4, PAR-1, etc.), (ii) respond by calcium flux and phosphorylation of various intracellular proteins to stimulation by thrombin and (iii) adhere to fibrinogen and vitronectin. However, human megakaryoblasts derived from the cultures supplemented with TpO + IL-3, in contrast to murine megakaryocytic cells cultured under similar conditions, display poor polyploidization and do not release platelets. Since IL-3 has been reported to inhibit final maturation of megakaryocytic cells, we recently modified our expansion strategy. In this new approach CD34(+) cells are first expanded for 11 days in the presence of TpO + IL-3. Then megakaryoblasts derived are expanded for an additional 7 days supplemented with TpO only. We found that megakaryocytic cells expanded in this 'two step culture' model are more differentiated, are polyploid and release platelets. The model described here provides normal human megakaryoblasts in adequate numbers, to study megakaryopoiesis and megakaryocyte function.
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PMID:In vitro expansion of human megakaryocytes as a tool for studying megakaryocytic development and function. 1167 71

To understand how platelet signal transduction pathways develop during megakaryocytopoiesis, we isolated human stem cells from umbilical cord blood and cultured the cells in the presence of thrombopoietin (TPO). Based on the early expression of CD61 and late expression of CD42b, immature (CD61+/CD42b(low)) and mature (CD61+/ CD42b(high)) megakaryocytes were immunomagnetically purified and, together with stem cells (CD34+), characterized for Galpha-protein expression and agonist-induced [Ca2+]i increases. Megakaryocytopoiesis was accompanied by down-regulation of the 43 kDa and 46 kDa variants of G16alpha, constant expression of Gsalpha, and up-regulation of Gqalpha and Gialpha1/2. The increase in Gqalpha and Gialpha1/2 expression was accompanied by an increase in Ca2+ signaling triggered by thrombin and other agonists known to signal to Ca2+ via these G-proteins in platelets. The prostacyclin analog iloprost and TPO also induced [Ca2+]i increases, and the iloprost-induced Ca2+ response disappeared during maturation. These data reveal sharp changes in Ca2+ regulation during megakaryocytopoiesis.
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PMID:Biogenesis of G-protein mediated calcium signaling in human megakaryocytes. 1168 31

Because the initial decrease in light transmission in platelet aggregometry is attributed to platelet shape change, it is widely held that platelet shape change is a prerequisite for platelet aggregation. We conducted this study to determine the basis of this initial optical effect in aggregometry. Platelets were activated with ADP, thrombin, or the thrombin receptor agonist peptide SFLLRN (TRAP(1-6)). In every case the initial decrease in light transmission occurred with the concomitant formation of microaggregates. This was also seen when preactivated platelets, which cannot undergo further morphological changes, were used, and when platelets were activated in the presence of shape-change inhibitors such as cytochalasin D and vincristine. Microscopy analysis of samples fixed at minimum light transmission in the aggregometer, which is generally assumed to signal shape change, always showed the presence of microaggregates. Microaggregation appeared to be distinct from full aggregation, as it was not inhibited by the addition of CD61, an antibody to the beta(3) integrin. To model these findings, fibrinogen-coated latex spheres, which cannot change shape, were aggregated with thrombin; the initial decrease in light transmission was still seen, and microaggregates formed at this time. These results indicate that platelet shape change is not a prerequisite for aggregation and that the signal widely believed to represent shape change reflects platelet microaggregation instead. We conclude that platelet aggregation occurs independently of shape change and that shape change is not necessarily followed by aggregation. These observations suggest an alternative role for platelet shape change of single platelets.
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PMID:Platelet aggregation is not initiated by platelet shape change. 1170 59

Genetic background has been shown to affect phenotype in transgenic mice with hemostatic defects. We present comparative data on various platelet function tests, as well as on microparticle and thrombin generation capacity in three mouse strains most commonly used in transgenic applications. Normal, inbred 129Sv (n = 24), C57BL/6 (n = 14) and BALB/c (n = 22) mice were used. BALB/c mice showed statistically significantly longer tail-bleeding times (158 +/- 42 s, P<0.02) than 129Sv (113 +/- 37) and C57BL/6 (122 +/- 29) and paradoxically increased velocity of platelet aggregation (P<0.01) with ADP, collagen, ionophore and thrombin. ATP-release did not differ between strains, it was weakest with ADP and strongest with thrombin. 129Sv platelets were less responsive to thrombin. Generation of platelet microparticles did not differ between strains either and could efficiently be inhibited by EDTA but not by aspirin or alprostadil. Two anti-GPIIIa monoclonal antibodies did not inhibit microparticles either, although they could block aggregation. Thrombin generation was equivalent in C57BL/6 and BALB/c, but slower in 129Sv. Interestingly, the reaction in all strains was immediate and different from the human model in that no lag-phase was seen after triggering. In summary these mouse strains show similar patterns of ex vivo platelet aggregation, bleeding times as well as microparticle and thrombin generation. Subtle differences were found, which should be taken into consideration when interpreting data from wild-type or transgenic models.
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PMID:Influence of the genetic background on platelet function, microparticle and thrombin generation in the common laboratory mouse. 1179 99


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