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 characterized a murine IgG monoclonal antibody, OP-G2, specific for platelet glycoprotein (GP) IIb-IIIa (alpha IIb beta 3). OP-G2 Fab fragments inhibit fibrinogen-mediated platelet aggregation and competitively inhibit adenosine diphosphate-induced binding of 125I-fibrinogen to washed platelets. OP-G2 binding to GPIIb-IIIa is specifically inhibited by RGD-containing peptides but not the fibrinogen gamma-chain carboxy-terminal peptide, and OP-G2 Fab fragments, like RGD-containing peptides, alter the conformation of GPIIb-IIIa resulting in the expression of a ligand-induced binding site (LIBS) recognized by PMI-1. OP-G2 fails to bind to the recombinant Cam variant of GPIIb-IIIa (alpha III beta 3Cam) wherein an Asp119 to Tyr119 substitution in GPIIIa abrogates the ability to recognize RGD. These data indicate that OP-G2 recognizes an epitope at or in very close proximity to the RGD recognition site of GPIIb-IIIa and that, in every aspect tested, OP-G2 behaves like a macromolecular RGD ligand. Interestingly, two-color flow cytometry shows that OP-G2 IgG can bind to nonactivated platelets. Quantitative binding assays indicate that nonactivated platelets bind approximately 50,000 125I-OP-G2 molecules/platelet. Furthermore, the affinity of OP-G2 for platelets activated with thrombin is roughly fivefold higher (nonactivated, kd = 24.8 nmol/L; activated, kd = 4.9 nmol/L). These results suggest that the RGD recognition site of GPIIb-IIIa is available to macromolecules that contain RGD even on nonactivated platelets, provided that the affinity of the ligand is adequate.
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PMID:The Arg-Gly-Asp (RGD) recognition site of platelet glycoprotein IIb-IIIa on nonactivated platelets is accessible to high-affinity macromolecules. 137 72

A unique murine monoclonal antibody (LK-4) is described which differentiates PLA1/PLA1 platelet extracts from PLA2/PLA2 and PLA1/PLA2 platelet extracts on solid phase ELISA and immunoblot at the 100kD GPIIIa location, but not on intact platelets. LK-4 reacts equally with intact PLA1/PLA2 and PLA2/PLA2 platelets. Adsorbtion of LK-4 with PLA1/PLA1 platelets results in loss of reactivity for intact platelets as well as platelet extracts on ELISA or immunoblot. LK-4 inhibits platelet aggregation induced by ADP, epinephrine, collagen and thrombin, suggesting reactivity at or near the fibrinogen binding site on GPIIIa. It is suggested the LK-4 reacts with a conformation-induced common epitope for PLA1 and PLA2 on GPIIIa, with loss of this conformation for PLA2 GPIIIa following solubilization with Triton X-100.
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PMID:A monoclonal antibody (LK-4) which differentiates PLA1 from PLA2 platelet extracts but not intact platelets. 138 61

Glycoprotein (GP) IIb and IIIa are major constituents of the platelet membrane which are involved in forming the fibrinogen receptor on activated platelets. We used flow cytometry to study the effects of ethylene-diamine tetraacetic acid (EDTA) on the membrane GPIIb/IIIa complexes of platelets and microparticles, and to study the effects of cations on dissociated GP complexes. Microparticles were detected by both the volume signal and by fluorescence using an FITC-conjugated anti-GPIb antibody (NNKY5-5). When platelets were stimulated with ADP, calcium ionophore A23187, or thrombin, fibrinogen binding to the platelet surface increased markedly. However, fibrinogen binding to microparticles showed little increase in response to such agonists. Microparticle GPIIb/IIIa complexes were dissociated by incubation with EDTA at 37 degrees C but did not reassociate after treatment with divalent cations (Ca2+, Mg2+, and Mn2+) in contrast to platelet GPIIb/IIIa complexes. These results suggest that some interaction of GPIIb/IIIa and linked structures like the platelet cytoskeleton may be involved in the reassociation of dissociated GPIIb and GPIIIa, perhaps explaining the failure of reassociation of microparticle GPIIb/IIIa (i.e., the fibrinogen binding to microparticles).
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PMID:Differences between platelet and microparticle glycoprotein IIb/IIIa. 145 94

Seven different tumor cell lines (human melanoma SK MEL 28; hamster melanoma HM29; murine melanomas B16F10 and amelanotic melanoma B16a; human colon carcinoma HCT8; murine colon carcinoma CT26; and murine Lewis lung carcinoma) were treated with thrombin at 0.5-1 unit/ml and examined for their ability to bind to adherent platelets; HM29 was studied for its ability to bind to fibronectin and von Willebrand factor; CT26, B16F1, B16F10, and B16a were studied for their ability to form pulmonary metastasis after i.v. injection of thrombin-treated tumor cells; CT26 was studied for its ability to grow s.c. Five of 7 thrombin-treated tumor cell lines increased their adhesion to adherent platelets 2-to 3-fold. HM29 increased its adherence to fibronectin and von Willebrand factor 2- to 3-fold. CT26, B16F1, B16F10, and B16a increased experimental pulmonary metastasis 10- to 156-fold. Thrombin-treated CT26 cells demonstrated 2-fold greater growth in vivo after s.c. injection. The mechanism of enhanced adhesion of thrombin-treated tumor cells to platelets required the platelet integrin GPIIb-GPIIIa since it could be inhibited by agents known to block adhesion of ligands to GPIIb-GPIIIa (monoclonal antibody 10E5, tetrapeptide RGDS, disintegrin Albolabrin); as well as a "GPIIb-GPIIIa-like" structure on tumor cells since it could be inhibited by treatment of thrombin-treated tumor cells with 10E5 and RGDS. The thrombin effect on tumor cells was optimum at 1 h of incubation with thrombin, did not require active thrombin on the tumor cell surface, and did not require protein synthesis (not inhibited by cycloheximide). Thus, thrombin-treated tumor cells markedly enhance pulmonary metastasis. It is suggested that this may be secondary to thrombin-induced enhanced adhesion as well as growth of tumor cells.
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PMID:Effect of thrombin treatment of tumor cells on adhesion of tumor cells to platelets in vitro and tumor metastasis in vivo. 159 84

We describe a new variant of Glanzmann's thrombasthenia (variant Strasbourg I). The patient (M.S.) showed an absence of platelet aggregation to ADP, thrombin, and collagen, and a decreased clot retraction. Platelet fibrinogen was approximately 20% of normal levels. ADP-stimulated platelets bound markedly reduced amounts of soluble fibrinogen and platelet adhesion to surface-bound fibrinogen was defective. Normal to subnormal amounts of glycoprotein (GP) IIb-IIIa (alpha IIb beta 3) complexes, the platelet fibrinogen receptor, were revealed by SDS-PAGE, crossed immunoelectrophoresis, and antibody binding. However, the complexes were unusually sensitive to dissociation with EDTA at room temperature. Furthermore, flow cytometry showed that the platelets failed to bind the activation-dependent monoclonal antibody, PAC-1, after stimulation. In contrast, an RGDS-containing peptide induced significant binding of the anti-ligand-induced binding site antibody, D3GP3, suggesting the presence of a functional RGD binding domain on the patient's GPIIb-IIIa complex. Sequence analysis was performed after polymerase chain reaction amplification of selected patient's GPIIIa exons, and of the patient's platelet GPIIb and GPIIIa mRNAs. A point mutation (C to T) was localized in exon D (iv) of GPIIIa that resulted in an 214Arg to 214Trp amino acid substitution. The defect has been inherited from the parents who are heterozygous for the same mutation. This substitution points to an essential amino acid in a region of GPIIIa involved in the binding of fibrinogen and influencing the Ca(2+)-dependent stability of the GPIIb-IIIa complex.
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PMID:A new variant of Glanzmann's thrombasthenia (Strasbourg I). Platelets with functionally defective glycoprotein IIb-IIIa complexes and a glycoprotein IIIa 214Arg----214Trp mutation. 160 6

Cerebral deposition of the amyloid beta-protein (A beta P), approximately 40 residue fragment of the integral membrane protein, beta-amyloid precursor protein (beta APP), has been implicated as the probable cause of some cases of familial Alzheimer's disease (AD). The parallels between A beta P deposition in AD and the deposition of certain plasma proteins in systemic amyloid diseases has heightened interest in the analysis of beta APP in circulating cells and plasma. Here, we describe distinct isoform patterns of beta APP in peripheral platelets and lymphocytes. PCR-mediated amplification of mRNA from purified platelets demonstrated the expression of all three major beta APP transcripts (beta APP770,751,695). The full-length, approximately 140 kDa form of beta APP751,770 was detected in membranes of resting and activated platelets but very little immature, approximately 122 kDa beta APP751,770 was found, suggesting a different processing of beta APP in platelets than that described in a variety of cultured cells and tissues. Platelets stimulated with thrombin, calcium ionophore, or collagen released the soluble, carboxyl-truncated form of beta APP (protease nexin-II), but no evidence for the shedding of full-length beta APP associated with platelet microparticles was found, in contrast to previous reports. As a positive control marker for microparticles, the fibrinogen receptor subunit, GPIIIa, was readily detected in platelet releasates. Resting and activated platelets contained similar amounts of the approximately 10 kDa carboxyl terminal beta APP fragment that is retained in platelet membranes following the constitutive cleavage of protease nexin-II. Nonstimulated peripheral B and T lymphocytes contained small amounts of membrane-associated mature and immature beta APP751,770. The potentially amyloidogenic full-length beta APP molecules present in circulating platelets and lymphocytes but not in microparticles could serve as a source of the microvascular A beta P deposited during aging and particularly in AD.
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PMID:Detection of distinct isoform patterns of the beta-amyloid precursor protein in human platelets and lymphocytes. 162 72

Glycoprotein IIb-IIIa (GPIIb-IIIa) is the fibrinogen receptor on activated platelets. GPIIIa is phosphorylated in resting platelets and the incorporation of 32Pi increases with platelet activation. To address the functional significance of this modification, the stoichiometry of GPIIIa phosphorylation was determined in resting and activated platelets by estimating the specific activity of metabolic [gamma-32P]ATP from the specific activity of phosphatidic acid. Approximately 0.01 mol of P/mol of GPIIIa was phosphorylated in resting platelets and 0.03 mol of P/mol of GPIIIa was phosphorylated in thrombin-, phorbol ester-, or U46619-treated platelets. Myosin light chain (MLC) phosphorylation served as a positive control for this method (1.2 mol of P/mol of MLC). Phosphorylation of purified GPIIb-IIIa by human platelet protein kinase C (PKC) resulted in levels of GPIIIa phosphorylation similar to that in platelets (0.05 mol of P/mol of GPIIIa). However, while GPIIIa in platelets was phosphorylated primarily on threonine, purified GPIIIa treated with PKC was phosphorylated primarily on serine. These results suggest that PKC may not directly phosphorylate GPIIIa in intact platelets. Ca2+/calmodulin-dependent kinase II phosphorylated purified GPIIIa to higher levels (0.5 mol of P/mol of GPIIIa) with phosphorylation on both threonine and serine. The limited phosphorylation of GPIIIa in intact platelets suggests that this event is unlikely to affect functions involving large populations of GPIIb-IIIa, such as its conversion to a fibrinogen receptor. However, these results may suggest the existence of a more readily phosphorylated subpopulation of GPIIb-IIIa with potentially distinct structural or functional properties.
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PMID:Phosphorylation of human platelet glycoprotein IIIa (GPIIIa). Dissociation from fibrinogen receptor activation and phosphorylation of GPIIIa in vitro. 165 Mar 65

We confirm the recent report (J Clin Invest 83:1778, 1989) of a polymorphism at amino acid 33 of platelet GPIIIa associated with the PLA1/PLA2 phenotype by using the polymerase chain reaction on cDNA derived from platelet RNA, using the base-pair primers 105-129 and 452-428. Platelet cDNA from three PLA2-homozygous individuals, when digested with Nci I, gave two bands of 256 bp and 91 bp, whereas eight PLA1 cDNAs gave a single band of 347 bp. Two 13-mer amino acid peptides straddling the amino acid polymorphism: SDEALP (L/P) GSPRCD were synthesized for epitope studies. Two mouse polyclonal antibodies were raised: one against the PLA1-associated peptide, the other against the PLA2 peptide. Both antibodies react with either peptide, as well as with both PLA1 and PLA2 platelets. The PLA1 peptide did not block the binding of two different human anti-PLA1 antibodies to the 100-Kd GPIIIa band on immunoblot of platelet extracts; neither did it block the binding of the same antibodies to PLA1-platelet extracts in an enzyme-linked immunosorbent assay. Further studies were performed on the PLA1 epitope following subtilisin digestion of purified GPIIIa. A 55-Kd fragment was obtained that retained the PLA1 epitope as well as the first 13 N-terminal amino acids of GPIIIa. Reduction of the 55-Kd fragment resulted in loss of the PLA1 epitope with production of a 67-Kd, 21-Kd, and 10-Kd band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 55-Kd band does not react with LK-2, a monoclonal antibody versus GPIIIa that inhibits adenosine diphosphate, collagen, epinephrine, and thrombin-induced aggregation. Thus, the PLA1 epitope is conformation-induced, resides on an N-terminal 55-Kd fragment composed of two or more peptides held together by -SH bonds, and is not required for platelet aggregation.
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PMID:A 13-mer peptide straddling the leucine33/proline33 polymorphism in glycoprotein IIIa does not define the PLA1 epitope. 170 95

Recent studies have revealed a role for platelets and the platelet-adhesive proteins, fibronectin and von Willebrand factor (vWF) in platelet-tumor cell interaction in vitro and metastasis in vivo. The present report documents the effect of thrombin treatment of platelets on this interaction in vitro and in vivo. In vitro, thrombin at 100-1,000 mU/ml maximally stimulated the adhesion of six different tumor cell lines from three different species two- to fivefold. As little as 1-10 mU/ml was effective. The effect of thrombin was specific (inhibitable by hirudin, dansyl-arginine N-(3-ethyl-1,5 pentanediyl) amide and unreactive with the inactive thrombin analogue N-P-tosyl-L-phenylchloromethylketone-thrombin and D-phenylalanyl-L-propyl-L-arginine chloromethylketone-thrombin (PPACK-thrombin), and required high-affinity thrombin receptors (competition with PPACK-thrombin but not with N-P-tosyl-L-lysine-chloromethyl-ketone-thrombin). Functionally active thrombin was required on the platelet surface. Binding of tumor cells to thrombin-activated platelets was inhibitable by agents known to interfere with the platelet GPIIb-GPIIIa integrin: monoclonal antibody 10E5, tetrapeptide RGDS and gamma chain fibrinogen decapeptide LGGAKQAGDV, as well as polyclonal antibodies against the platelet adhesive ligands, fibronectin and vWF. In vivo, thrombin at 250-500 mU per animal increased murine pulmonary metastases fourfold with CT26 colon carcinoma cells and 68-413-fold with B16 amelanotic melanoma cells. Thus, thrombin amplifies tumor-platelet adhesion in vitro two- to fivefold via occupancy of high-affinity platelet thrombin receptors, and modulation of GPIIb-GPIIIa adhesion via an RGD-dependent mechanism. In vivo, thrombin enhances tumor metastases 4-413-fold with two different tumor cell lines.
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PMID:Thrombin stimulates tumor-platelet adhesion in vitro and metastasis in vivo. 184 69

Using an immunogold staining technique and electron microscopy, we investigated the localization of the alpha-granule pool of glycoprotein (GP) IIb-IIIa in normal platelets and maturing megakaryocytes (MK), in pathologic platelets from a patient with type I Glanzmann's thrombasthenia (GT), and from three patients with the gray platelet syndrome (GPS). In normal resting platelets, GPIIb-IIIa was observed on the plasmatic side of the plasma membrane, the open canicular system (OCS) membranes, and along the internal face of the alpha-granule membrane. This location was found with three monospecific polyclonal antibodies: one anti-GPIIb-IIIa antibody, the second specific for GPIIb, and the third specific for GPIIIa. After thrombin stimulation, the alpha-granule labeling disappeared whereas membrane labeling increased. Platelets from GT did not display labeling on plasma membranes, OCS membranes, or alpha-granule membranes. Platelets from the three patients with GPS displayed intense labeling of the plasma membrane and the OCS membrane, as well as the abnormal small alpha-granules and along the inside of large vacuoles (which contain the granule membrane protein [GMP]-140). In cultured immature MK from normal progenitors, both peptide components of GPIIb-IIIa appeared in the Golgi saccules and vesicles, and in the small precursors of alpha-granules, labeling both their membranes and their matrix. It was then observed only on the membrane of the mature MK alpha-granules, although labeling was less consistent than on the platelet granules. The MK plasma membrane and demarcation membrane system also displayed GPIIb-IIIa labeling. In conclusion, this study demonstrates that GPIIb-IIIa is present on the internal face of the alpha-granule membranes of platelets (where it appears early during MK maturation) as well as in the abnormal alpha-granules of gray platelets; it is absent from GT type I platelets.
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PMID:Alpha-granule pool of glycoprotein IIb-IIIa in normal and pathologic platelets and megakaryocytes. 231 Aug 22


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