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)

Platelets are heterogeneous in the content of membrane glycoprotein (GP)IIb/IIIa complex. To determine whether this heterogeneity is related to changes associated with platelet aging in the circulation, newly released platelets, obtained during recovery from nonimmune-mediated acute experimental thrombocytopenia in baboons, were studied. Monoclonal antibody (MoAb) binding to epitopes expressed on GPIIb/IIIa complex (LJ-CP8), GMP-140 (S12), and GPIa/IIa (12F1) was measured on control platelets (comprising platelets with a normal age distribution; mean age 60 to 72 hours) and newly formed platelets (mean age 12 hours), both in the resting state and after thrombin stimulation. Whereas LJ-CP8 binding to resting control platelets increased by 34% upon stimulation by gamma-thrombin from 30,885 +/- 1,171 to 41,458 +/- 1,311 molecules/platelet at saturating concentrations of antibody, LJ-CP8 binding to resting young platelets did not increase significantly upon thrombin stimulation (31,878 +/- 3,330 and 33,791 +/- 3,486 molecules/platelet, respectively). Similarly, binding of antibody S12 in response to maximal thrombin stimulation was reduced by 42% from 10,246 +/- 834 molecules/platelet at saturating concentrations of S12 for control platelets to 5,971 +/- 665 molecules/platelet for young platelets (P = .001). S12 binding to unstimulated platelets was less than 10% of the binding observed after thrombin stimulation at all concentrations of S12 for both control and young platelets. However, maximal binding of antibody 12F1 to resting control platelets did not differ significantly from that observed with resting young platelets (2,926 +/- 167 and 2,857 +/- 208 molecules/platelet, respectively), and 12F1 binding was unchanged after thrombin stimulation for both control and young platelets. We conclude that the thrombin-induced increase in the expression of epitopes on platelet membrane GPIIb/IIIa complex and GMP-140 is a function of platelet age.
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PMID:Thrombin-induced increase in surface expression of epitopes on platelet membrane glycoprotein IIb/IIIa complex and GMP-140 is a function of platelet age. 247 8

Polyclonal antiserum prepared to purified type I collagen receptor, a 65,000 molecular weight protein, isolated from human platelets reacted with two other proteins with molecular weights of 90,000 and 58,000 in immunoblots of solubilized platelet membranes. The immunoreactive proteins were purified to homogeneity with molecular sieve chromatography and preparative gel electrophoreses. Periodic acid Schiff stain showed that both the 90,000 and 65,000 proteins were glycoproteins. These purified proteins reacted with immunoglobulin G (IgG) fractions isolated from antiserum raised against the 65,000 protein and poly- and monoclonal antibody specific for the glycoprotein IIb-IIIa suggesting that these three proteins are immunocross reactive with GPIIb-IIIa. To further examine the immunocross-reactivity of these proteins, a monoclonal antibody was raised against the 90,000 glycoprotein. This monoclonal antibody also reacted with all three proteins in enzyme-linked immunosorbent assays and transblot experiments suggesting that these three possess a common antigenic determinant. The monoclonal antibody prepared to the 90,000 glycoprotein also inhibited platelet aggregation induced by the addition of collagen and ADP but not alpha-thrombin and epinephrine-induced aggregation. This suggests that the monoclonal antibody binds a protein on the platelet surface which plays a role in platelet aggregation induced by the addition of these agonists.
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PMID:Collagen-platelet interaction: inhibition by a monoclonal antibody which binds a 90,000 dalton platelet glycoprotein. 249 84

Three murine monoclonal antibodies (anti-CD9: ALB6, anti-CD41: VI-PL3 and PL2-49/GPIIb - final concentration: 7.5 micrograms/mL) are shown to elicit after a lag time aggregation of washed platelets and a calcium signal (as detected by light emitted by loaded aequorin), which is only partially inhibited by aspirin. By comparison the rise induced by thrombin is greater and almost instantaneous. In the presence of EGTA a calcium mobilization from internal stores can be detected with thrombin and with ALB6, but neither with PL2-49 nor with VI-PL3, whereas platelets still change their shape and release ATP. It is tempting to speculate that although all the antibodies induce a calcium change, they activate platelets by different pathways: calcium may be not primarily involved in the activation induced by the anti-CD41 antibodies.
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PMID:Calcium rise in human platelets elicited by anti-CD9 and -CD41 murine monoclonal antibodies. 251 Mar 59

The proteolytic digestion of GPIIIa on intact platelets by chymotrypsin, thrombin, plasmin, trypsin, and staphylococcal V8 protease was monitored in immunoblot studies employing three different antibodies to GPIIIa, one of which was made against a 13-residue synthetic peptide containing the amino terminus of GPIIIa. Chymotrypsin, plasmin, and trypsin gave similar patterns, from which it could be inferred that the major products after extensive digestion were two-chain molecules composed of amino-terminal fragments of Mr approximately 17,000-18,000 disulfide bonded to carboxyl-terminal remnants of Mr approximately 58,000-70,000. These patterns suggest that GPIIIa contains a large disulfide-bonded loop of at least 325 amino acids that is susceptible to proteolytic cleavage, and that the 4 cysteine residues between residues 177 and 273 bond with each other. Such a structure can also account for the presence of the PIA1 epitope, which has recently been localized to a polymorphism at position 33 on these late digestion products. Thrombin did not proteolyze GPIIIa even at 2.5 units/ml. Still to be resolved is whether the minor immunoreactive GPIIIa bands found on normal platelets are related to in vivo or in vitro proteolysis and whether GPIIIa proteolysis plays a role in chymotrypsin-induced exposure of the GPIIb/IIIa receptor.
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PMID:Evidence that platelet glycoprotein IIIa has a large disulfide-bonded loop that is susceptible to proteolytic cleavage. 252 61

Platelets contain a pool of endogenous platelet-von Willebrand factor (vWF) that becomes expressed on the platelet surface when platelets are stimulated by a variety of agonists. Maximal platelet-vWF expression occurs in concert with platelet alpha-granule secretion. Aspirin (ASA) is known to impair platelet activation and alpha-granule secretion by irreversible inhibition of platelet cyclo-oxygenase. We studied native and ASA-treated platelets for their ability to mobilize and to express platelet-vWF in response to adenosine diphosphate (ADP) or thrombin. We found that each agonist was effective in promoting increased platelet-vWF surface expression on native and ASA-treated platelets. ASA-treated platelets responded identically to native platelets to low (0.01 U/mL) and high (1.0 U/mL) concentrations of thrombin, while the ADP-induced increase in ASA-treated platelets was only 50% to 60% of that for control platelets. Measurement of secreted platelet-vWF and beta-thromboglobulin indicated that the increase seen with ADP was largely independent of alpha-granule secretion. Using monoclonal antibodies (MoAbs) against the platelet glycoproteins (GP) IIb/IIIa and Ib (MoAbs 10E5 and 6D1, respectively), we demonstrated that the ADP-induced increase in platelet-vWF expression on control platelets primarily involved the binding of secreted platelet-vWF to the platelet GPIIb/IIIa. In contrast, the increase in platelet-vWF that occurred following ADP stimulation of ASA-treated platelets was largely insensitive to GPIIb/IIIa blockade. No effect of GPIb blockade in platelet-vWf expression was noted for either control or ASA-treated platelets. When platelet shape change was prevented by the addition of cytochalasin D, ADP-induced platelet-vWf surface expression on ASA-treated platelets was reduced by more than 80%. Our data indicate that platelets in which the cyclooxygenase pathway is blocked by the action of aspirin can increase surface expression of platelet-vWf as a consequence of platelet shape change. We speculate that this process exposes platelet-vWf bound to GPIIb/IIIa, or possibly GPIb, within the surface connected canalicular system.
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PMID:Effect of aspirin on platelet-von Willebrand factor surface expression on thrombin and ADP-stimulated platelets. 252 23

Affinity purified anti-fibrinogen (anti-Fg) Fab fragments were used to study the mechanism of expression of alpha-granule fibrinogen on activated platelets. Low amounts of the radiolabeled anti-Fg Fab bound to unstimulated or adenosine diphosphate (ADP)-stimulated cells. They readily bound to platelets stimulated with collagen, alpha-thrombin or gamma-thrombin in the presence of divalent cations. At 1 n mol/L alpha-thrombin or 25 nmol/L gamma-thrombin, platelet fibrinogen was expressed on the surface of the cells notwithstanding the presence of AP-2, a monoclonal antibody to the glycoprotein (GP) IIb-IIIa complex, or the synthetic peptides Arg-Gly-Asp-Ser and gamma 400-411, all substances that prevented the binding of plasma fibrinogen to platelets. These results suggest that platelet fibrinogen may interact with its receptors during its translocation from the alpha-granules to the plasma membrane and, thus, not occupy the same sites as those available for plasma fibrinogen on the surface of the cell. Furthermore, we found that platelet fibrinogen was expressed on the thrombin-stimulated platelets of a Glanzmann's thrombasthenia variant that failed to bind plasma fibrinogen. Normal platelets stimulated with 5 nmol/L alpha-thrombin bound increased amounts of the anti-fg Fab, the additional expression being inhibited by the anti-GP IIb-IIIa monoclonal antibody or by Gly-Pro-Arg-Pro, an inhibitor of fibrin polymer formation. This suggests that rebinding to externally located GP IIb-IIIa complexes becomes important once fibrin is formed.
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PMID:Studies on the mechanism of expression of secreted fibrinogen on the surface of activated human platelets. 253 13

Synthetic peptides containing the adhesion site recognition sequences present on the A alpha and gamma chains of fibrinogen were studied for their effect on the binding of endogenous platelet-von Willebrand factor (vWF) and exogenous plasma-vWf to thrombin-stimulated platelets. In agreement with previously reported data, the tetrapeptide consisting of the RGDS sequence was a more potent inhibitor of plasma-vWf binding to platelets than was the pentadecapeptide of the carboxy terminus of the fibrinogen gamma-chain (IC50 10.6 mumol/L for the RGDS tetrapeptide v 44.9 mumol/L for the gamma-chain pentadecapeptide). No apparent synergy in the inhibition of plasma-vWf binding was noted when the RGDS and gamma-chain peptides were used together (IC50 15.2 mumol/L). In contrast, the gamma-chain peptide was significantly more inhibitory than was the RGDS tetrapeptide on the binding of platelet-vWf to platelets (IC50 1.4 mumol/L for the gamma-chain pentadecapeptide v 4.5 mumol/L for the RGDS tetrapeptide, P less than .05), and there was significant synergy in the inhibition of platelet-vWf binding noted when the gamma-chain and RGDS peptides were used together (IC50 0.04 mumol/L). These results indicate that the binding of platelet-vWf to its receptor on the platelet glycoprotein IIb/IIIa complex involves both the RGDS and gamma-chain recognition sites. In contrast to the results with plasma-vWf binding, the gamma-chain recognition site appears to be more important than the RGDS recognition site in platelet-vWf binding to platelets.
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PMID:Inhibition of platelet-von Willebrand factor binding to platelets by adhesion site peptides. 254 39

We used flow cytometry to investigate the change of platelet membrane glycoproteins (GPIb and GP IIb/IIIa) and the distributions of fibrinogen (Fbg), thrombospondin (TSP) and fibronectin (Fn) on the surface of thrombin-stimulated platelets. The binding of a monoclonal antibody directed at the von Willebrand factor binding site on GPIb decreased in thrombin-stimulated platelets. This antibody caused a reactive delay in thrombin-induced aggregation, but had little influence on aggregability. Slight thrombin-induced aggregation was observed even after blocking the binding of Fbg to GP II b/IIIa. The new expression of GP II b/IIIa was detected on the surface of thrombin-stimulated platelets, whereas there was little increase of Fbg dependent on this GP II b/IIIa. An increase of TSP after thrombin stimulation was observed on the surface of platelets of healthy controls and patients with Glanzmann's thrombasthenia (Type I). The level of on platelet surface was slightly increased by thrombin stimulation. The mechanism involved in thrombin-induced aggregation appears to differ from that in ADP-induced aggregation.
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PMID:[Analysis of platelet surface conformation in thrombin-induced aggregation]. 258 48

A patient is described with a disturbance of platelet function comparable to that in Glanzmann's thrombasthenia. Platelet aggregation and binding of fibrinogen to the patient's platelets were defective and thrombin-induced clot retraction was absent. The platelet fibrinogen content was only moderately reduced. As measured by monoclonal antibody binding in the presence of divalent cations, the platelets contained about 15% of the normal amount of GPIIb and GPIIIa and only 6% of the normal amount of intact GPIIb/IIIa complex. The residual GPIIb/IIIa complex exhibited a decreased stability as shown by the lack of binding of a complex-dependent anti-GPIIb/IIIa antibody to platelets incubated with ethylene diamine tetraacetic acid (EDTA) at 22 degrees C. Crossed immunoelectrophoresis (CIE) in the presence of divalent cations showed partial dissociation of GPIIb/IIIa as well as the presence of two forms of the residual intact GPIIb/IIIa complex. In addition, both CIE in the presence of the EDTA and two-dimensional sodium dodecyl sulphate (SDS) gel electrophoresis showed the presence of two forms of GPIIb. This form of thrombasthenia is characterized by a defective platelet function, a marked reduction of GPIIb and GPIIIa, decreased stability of the residual GPIIb/IIIa complex and structural heterogeneity of GPIIb.
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PMID:Decreased stability and structural heterogeneity of the residual platelet glycoprotein IIb/IIIa complex in a variant of Glanzmann's thrombasthenia. 261 Nov 37

Four types of anti-human platelet glycoprotein IIb/IIIa (GP IIb/IIIa)monoclonal antibodies (McAb), termed HIP2, HIP4, HIP7, HIP8, were obtained in our laboratory. All of them recognized GP IIb/IIIa complex except that HIP2 recognized GP IIb. As to the effect of the four McAbs on platelet aggregation, the results showed: 1) HIP4 and HIP8 can completely inhibit platelet aggregation induced by ADP or collagen, but not that induced by thrombin or ristocetin. 2) HIP2 was able to activate platelet aggregation directly. But it is Ca++ and complement dependent; 3) HIP7 did not show any effect on platelet aggregation induced by collagen, ADP, thrombin or ristocetin. When clinically studied, some cases of platelet-associated diseases such as thrombocytasthenia and thrombocytopenia had abnormal responses to the above McAbs. In addition, we also found that these McAbs can stimulate hematopoiesis in CFU-E assay.
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PMID:[Production and application of anti-human platelet GP IIb/IIIa monoclonal antibodies]. 262 Feb 70


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