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)

Triflavin, an antiplatelet peptide from Trimeresurus flavoviridis snake venom, inhibits aggregation of human platelets stimulated by a variety of agonists. However, triflavin does not affect the shape change and release reaction of platelets stimulated by thrombin and collagen. In this paper, we further investigate its effect on the intracellular events occurring after the activation of platelets. Triflavin does not inhibit the intracellular free calcium rise of Quin 2-AM loaded platelets stimulated by thrombin and it also has no significant effect on thromboxane B2 formation of platelets stimulated by thrombin. Triflavin does not affect the 3(H)-inositol monophosphate formation of the 3(H)-myoinositol loaded platelets. However, triflavin dose-dependently inhibits fibrinogen-induced aggregation and 125I-fibrinogen binding of ADP-stimulated platelets. In addition, triflavin dose-dependently blocks fibrinogen-induced aggregation of elastase-treated platelets. It is concluded that triflavin specifically inhibits fibrinogen binding to fibrinogen receptors associated with glycoprotein IIb/IIIa complex on platelet membrane surface without any inhibitory effect on the platelet-activation process.
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PMID:Mechanism of action of a potent antiplatelet peptide, triflavin from Trimeresurus flavoviridis snake venom. 166 95

Vitronectin is one of the glycoproteins that mediate cell adhesion and spreading of a variety of cells through the RGD(S) sequence. Vitronectin is demonstrated to bind to glycoprotein IIb-IIIa and play a role in platelet aggregation. Synthetic peptides containing the RGD(S) sequence can inhibit vitronectin binding to platelets, but the affinity of these peptides is less than 1/100th that of native vitronectin. The present study thus examined the ability of modified RGD(S)-containing peptide to inhibit vitronectin binding to thrombin-stimulated platelets. The cyclicization of GRGDSPA peptide was done by the linkage of NH2-terminal glycin and the COOH-terminal alanin. The circular dichroism spectrum of cyclic GRGDSPA peptide only showed negative minimum at approximately 220 nm, but those of other linear peptides such as GRGDSPA and GRGESPA had no effect. This result indicated that only the cyclic GRGDSPA peptide retained some conformational structure to restrict its flexibility. Inhibition experiments revealed that the affinities of the ligands for the receptor decreased in the order of vitronectin = fibronectin = fibrinogen = von Willebrand factor (vWF) greater than cyclic GRGDSPA peptide greater than GRGDSPA peptide. GRGESPA peptide had no effect. These results demonstrate that the conformational structure of the RGD(S) sequence plays the important role for the affinity of vitronectin binding to activated platelets and the increased affinity of the modified peptide is a prerequisite for the potential antithrombotic use.
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PMID:Novel effect of cyclicization of the Arg-Gly-Asp-containing peptide on vitronectin binding to platelets. 170 43

Platelets contain a pool of endogenous adhesive proteins that can be released and may bind to surface membrane receptors under appropriate conditions. Because the binding of exogenous fibrinogen to platelets was shown previously to be accompanied by a time-dependent decrease in fibrinogen accessibility to antibody and enzymes, studies were performed to evaluate changes in the expression of endogenous fibrinogen released from thrombin-stimulated platelets using monospecific polyclonal and monoclonal antibody F(ab')2 fragments. Parallel studies were performed to compare the expression of released fibronectin and von Willebrand factor (vWF). Binding of polyclonal antibody F(ab')2 fragments directed against individual adhesive proteins was inhibited by EDTA or the 10E5 monoclonal antibody, suggesting that fibrinogen, fibronectin, and vWF expression was mediated, in large part, by divalent cation-dependent interactions with the glycoprotein IIb-IIIa complex. Interestingly, when polyclonal antibody F(ab')2 fragments were added to platelet suspensions at discrete times after thrombin stimulation, antifibrinogen F(ab')2 binding decreased by 72% +/- 15% (mean +/- SD, n = 22) over a 60-minute time course, whereas antifibronectin and anti-vWF antibody F(ab')2 fragment binding changed minimally (6% +/- 23%, n = 22 and 3% +/- 26%, n = 14, respectively). Similar observations were made with monoclonal antibodies. Parallel experiments using 125I-labeled fibrinogen as a marker indicated that the observed decrease in antifibrinogen F(ab')2 binding was not accompanied by fibrinogen dissociation. Moreover, antibody accessibility to platelet-bound fibrinogen could be restored after Triton X-100 platelet lysis. The data suggest that fibrinogen, fibronectin, and vWF are not coordinately expressed on thrombin-stimulated platelets. Rather, fibrinogen expression appears transient compared with the expression of fibronectin and vWF. The ability of platelets to secrete and organize adhesive proteins on their surface is likely to have important implications for hemostasis and thrombosis.
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PMID:Adhesive protein expression on thrombin-stimulated platelets: time-dependent modulation of anti-fibrinogen, -fibronectin, and -von Willebrand factor antibody binding. 173 4

Intracellular platelet fibrinogen surface expression was studied in arabinogalactan-purified, resting, and thrombin-stimulated platelets. Platelet fibrinogen is derived from endocytosis of plasma fibrinogen by megakaryocytes. Like a variety of other adhesive proteins, it is stored in the platelet alpha-granule. Platelet fibrinogen surface expression was studied by using the antigen-binding fragments of a murine monoclonal antibody to platelet fibrinogen, F26, and an immunopurified polyclonal antifibrinogen antibody. Studies correlating platelet fibrinogen surface expression with the presence of the glycoprotein IIb-IIIa (GPIIb-IIIa) complex showed that in the presence of ethylene glycol tetraacetic acid (EGTA) at 37 degrees C, neither the GPIIb-IIIa complex nor platelet fibrinogen was expressed on the surface of thrombin-activated platelets. Similar experiments performed in the presence of EGTA and calcium showed proportional expression of the GPIIb-IIIa complex and platelet fibrinogen. The addition of Arg-Gly-Asp-Ser-containing peptides, the pentadecapeptide of the fibrinogen gamma-chain carboxy terminus, or the monoclonal antibody 10E5, when directed against the GPIIb-IIIa complex before thrombin activation, inhibited 65% to 94% of the platelet fibrinogen expression, as determined with the polyclonal and monoclonal antigen-binding fragments. When these same inhibitory agents were added immediately after or 5 minutes after thrombin, the amount of inhibition decreased significantly. Similar studies with a washed platelet system revealed that when the inhibitors of platelet fibrinogen expression were added before thrombin stimulation, the degree of inhibition observed was only 24% to 38%. This suggests that the major portion of platelet fibrinogen expression involves the release of platelet fibrinogen and its subsequent binding to GPIIb-IIIa. This binding may occur within the open canalicular system or on the platelet surface; in either case, wherever the site of released platelet fibrinogen binding occurs, it can be markedly inhibited by the RGD-containing peptides and the gamma-chain fibrinogen peptides. Approximately 10% to 30% of platelet fibrinogen may be expressed prebound to a platelet receptor, or else it is released and binds to a platelet receptor other than the GPIIb-IIIa complex.
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PMID:Endogenous platelet fibrinogen surface expression on activated platelets. 174 9

Triflavin, an antiplatelet peptide containing Arg-Gly-Asp, purified from Trimeresurus flavoviridis venom, inhibits aggregation of human platelets stimulated by a variety of agonists. It blocks aggregation through interference with fibrinogen binding to its specific receptor on the platelet surface membrane in a competitive manner, but it has no apparent effect on intracellular events, such as thromboxane B2 formation, phosphoinositides breakdown and intracellular Ca2+ mobilization of thrombin-activated platelets. In this study, we determined the complete sequence of triflavin, which is composed of a single polypeptide chain of 70 amino acids. Its sequence is rich in cysteine and contains Arg-Gly-Asp at residues 49-51 in the carboxy-terminal domain. Triflavin shows about 68% identity of amino acid sequence with trigramin, which is a specific antagonist of the fibrinogen receptor associated with glycoprotein IIb/IIIa complex. [125I]Triflavin binds to unstimulated and ADP-stimulated platelets in a saturable manner and its Kd values are estimated to be 76 and 74 nM, respectively; the corresponding numbers of binding sites are 31,029 and 34,863 per platelet, respectively. [125I]Triflavin binding is blocked by Gly-Arg-Gly-Asp-Ser in a competitive manner. EDTA, the Arg-Gly-Asp-containing peptides (including naturally occurring polypeptides, trigramin and rhodostomin), and monoclonal antibody, 7E3, raised against GP IIb/IIIa complex, inhibit [125I]triflavin binding to unstimulated and ADP-stimulated human platelets. In conclusion, triflavin specifically binds to fibrinogen receptor associated with GP IIb/IIIa complex and its binding site is located at or near GP IIb/IIIa complex, overlapping with those of 7E3 and another Arg-Gly-Asp-containing polypeptide, rhodostomin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Triflavin, an antiplatelet Arg-Gly-Asp-containing peptide, is a specific antagonist of platelet membrane glycoprotein IIb-IIIa complex. 186 44

By means of Sephadex G-75 and CM-Sephadex C-50 column chromatography and reverse-phase HPLC, a low molecular weight (Mr = 7500), cysteine-rich peptide, halysin, was purified from Agkistrodon halys (mamushi) snake venom. Halysin is a potent platelet aggregation inhibitor that concentration-dependently inhibited human platelet aggregation stimulated by ADP, thrombin and collagen (IC50 = 0.16 to 0.36 microM) without affecting platelet secretion. It was active in inhibiting platelet aggregation of platelet-rich plasma and whole blood. Halysin had no effect on thromboxane B2 formation of platelets or intracellular Ca2+ mobilization of Quin 2-AM loaded platelets stimulated by thrombin. It inhibited the fibrinogen-induced aggregation of elastase-treated platelets. Halysin concentration-dependently inhibited the 125I-fibrinogen binding to ADP-stimulated platelets in a competitive manner (IC50 = 0.16 microM). 125I-Halysin bound to resting platelets (Kd = 1.6 x 10(-7) M) and to ADP-stimulated platelets (Kd = 3.4 x 10(-8) M) in a saturable manner. EDTA, the Arg-Gly-Asp (RGD)-containing snake venom peptides trigamin and rhodostomin, Arg-Gly-Asp-Ser (RGDS), and Gly-Gln-Gln-His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val blocked both 125I-fibrinogen binding and 125I-halysin binding to ADP-stimulated platelets. The monoclonal antibody, 7E3, raised against glycoprotein IIb-IIIa complex blocked both 125I-fibrinogen and 125I-halysin binding, whereas 10E5 had no significant effect on halysin binding to ADP-stimulated platelets, indicating that 7E3 and halysin bind to an epitope which is different from that of 10E5. RGDS concentration-dependently inhibited 125I-halysin binding in a competitive manner. We determined the primary structure of halysin which is a single peptide chain of 71 amino acid residues. An RGD sequence appeared in the carboxy-terminal domain of halysin. Halysin showed about an 85% identical sequence with trigamin which is a specific antagonist of fibrinogen receptor associated with glycoprotein IIb-IIIa complex. In conclusion, halysin inhibited platelet aggregation by interfering with fibrinogen binding to the fibrinogen receptor of the activated platelets. The RGD sequence of halysin plays an important role in the expression of its biological activity.
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PMID:Halysin, an antiplatelet Arg-Gly-Asp-containing snake venom peptide, as fibrinogen receptor antagonist. 188 30

To characterize the interaction between thrombospondin and human platelets, thrombospondin was purified from the supernatant of thrombin-activated human platelets, labeled with iodine 125, and allowed to interact with the washed platelets. With concentrations of 10 to 50 micrograms/ml, only minute amounts of 125I-labeled thrombospondin bound to resting platelets or to platelets activated by adenosine diphosphate. In contrast, when platelets were stimulated with thrombin, binding increased fivefold to sixfold in a time-dependent and 125I-labeled thrombospondin concentration-dependent manner. Binding of 125I-labeled thrombospondin to thrombin-activated platelets required the presence of divalent cations, proceeded concomitantly with platelet release, and at a concentration of 1 nmol/L thrombin, reached a maximum of 2200 +/- 260 molecules of 125I-labeled thrombospondin bound per platelet. After its binding to platelets, 125I-labeled thrombospondin was not internalized, because up to 85% of the 125I-labeled thrombospondin was dissociated from the cell surface by adding ethylenediaminetetraacetic acid. Using various experimental approaches, including studies with severe type I thrombasthenic platelets, we further demonstrated that the interaction of 125I-labeled thrombospondin with thrombin-stimulated platelets occurred as a fibrinogen- and fibrin-independent process, and that the glycoprotein IIb-IIIa complex did not function as a physiologic plasma membrane receptor for 125I-labeled thrombospondin. Last, about 60% of the 125I-labeled thrombospondin molecules bound to the platelet surface were found to be associated with the platelet cytoskeleton recovered from platelets solubilized with Triton X-100. On Western blot analysis, this cytoskeletal fraction lacked detectable glycoprotein IV, the putative platelet receptor for thrombospondin. These results suggest that on the surface of thrombin-activated platelets, a fraction of 125I-labeled thrombospondin does not associate with glycoprotein IV but instead with other plasma membrane components that have yet to be identified.
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PMID:Characterization of the binding of thrombospondin to human platelets and its association with the platelet cytoskeleton. 194 May 85

Two platelet mechanisms contribute to haemostasis and thrombosis. (1) Compounds such as thrombin activate glycoprotein IIb/IIIa; fibrinogen is the ligand. The cyclooxygenase pathway is involved and so this process is aspirin sensitive. (2) Shearing forces alone activate a different domain on glycoprotein IIb/IIIa; von Willebrand's factor is the ligand. This process is probably non-enzymatic and is aspirin insensitive. The prevention of shear-induced platelet activation may prove to be more rewarding therapeutically than inhibition of aspirin sensitive pathways.
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PMID:Shear-induced platelet aggregation. 196 70

Arginine-glycine-aspartic acid (RGD) is the minimal sequence in fibrinogen that leads to recognition and binding to the glycoprotein IIb/IIIa platelet receptor during aggregation. Analogs of tetrapeptides containing the RGD sequence have been previously shown to block fibrinogen binding to activated platelets in vitro. SC-46749 is an analog of arginine-glycine-aspartic acid-phenylalanine in which the phenylalanine is replaced by O-methyltyrosine. In this study the biological activities of SC-46749 were examined and its actions compared with the tetrapeptide arginine-glycine-aspartic acid-serine (RGDS), one of the natural sequences on the fibrinogen alpha chain that binds to platelets. In vitro, SC-46749 was more potent than RGDS in inhibiting fibrinogen binding (IC50: SC-46749, 27 microM; RGDS, 47 microM), in preventing ADP-induced aggregation in human platelet-rich plasma (IC50: SC-46749, 32 microM; RGDS, 95 microM) and in inhibiting thrombin-induced aggregation in washed human platelets (IC50: SC-46749, 23 microM; RGDS, 64 microM). In rats, SC-46749 prevented collagen-induced thrombocytopenia with an ED50 of 0.87 mg/kg whereas RGDS did not inhibit the response by 50% at doses up to 10 mg/kg. SC-46749 inhibited thrombus formation in an electrically damaged rat carotid artery in a dose-dependent fashion whereas the effects of RGDS were biphasic. RGDS appeared to delay thrombus formation at lower doses but had no effect at higher doses. When infused in dogs for 15 min, SC-46749 prevented ex vivo collagen-induced aggregation at 4 mg/kg/min. These data demonstrate that SC-46749 is a potent inhibitor of platelet aggregation and platelet-dependent thrombus formation.
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PMID:Antiplatelet and antithrombotic effects of platelet glycoprotein IIb/IIIa (GPIIb/IIIa) inhibition by arginine-glycine-aspartic acid-serine (RGDS) and arginine-glycine-aspartic acid (RGD) (O-me)Y (SC-46749). 200 85

Arietin, an Arg-Gly-Asp containing peptide from venom of Bitis arietans, inhibited aggregation of platelets stimulated by a variety of agonists with a similar IC50, 1.3-2.7.10(-7) M. It blocked aggregation through the interference of fibrinogen binding to fibrinogen receptors on platelet surface. In this paper, we further demonstrated that arietin had no significant effect on the intracellular mobilization of Ca2+ in Quin2-AM-loaded platelets stimulated by thrombin. It inhibited 125I-fibrinogen binding to ADP-stimulated platelets in a competitive manner (IC50, 1.1.10(-7) M). 125I-arietin bound to unstimulated, ADP-stimulated and elastase-treated platelets in a saturable manner and its Kd values were estimated to be 3.4.10(-7), 3.4.10(-8) and 6.5.10(-8) M, respectively, while the corresponding binding sites were 46,904, 48,958 and 34,817 per platelet, respectively. Arg-Gly-Asp-Ser (RGDS) inhibited 125I-arietin binding to ADP-stimulated platelets in a competitive manner. RGD-containing peptides, including trigramin and rhodostomin, EDTA and monoclonal antibody, 7E3, raised against glycoprotein IIb-IIIa complex, inhibited 125I-arietin binding to ADP-stimulated platelets, indicating that the binding sites of arietin appear to be located at or near glycoprotein IIb-IIIa complex. In conclusion, arietin and other RGD-containing trigramin-like peptides preferentially bind to the fibrinogen receptors associated with glycoprotein IIb-IIIa complex of the activated platelets, thus leading to the blockade of fibrinogen binding to its receptors and subsequent aggregation. The presence of RGD of arietin is essential for the expression of its biological activity. Its binding sites are overlapped with those of trigramin, rhodostomin and the monoclonal antibody, 7E3.
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PMID:Mechanism of action of the antiplatelet peptide, arietin, from Bitis arietans venom. 204 64


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