Effects of a Monoclonal Antibody to Glycoprotein IIb/IIIa (P256) and of Enzymically Derived Fragments of P256 on Human Platelets

1991 ◽  
Vol 65 (04) ◽  
pp. 432-437 ◽  
Author(s):  
A W J Stuttle ◽  
M J Powling ◽  
J M Ritter ◽  
R M Hardisty
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Platelet Aggregation ◽  
Calcium Ions ◽  
Human Platelet ◽  
Human Platelets ◽  
In Vivo Detection ◽  
Fibrinogen Binding ◽  
Specific Antagonist

SummaryThe anti-platelet monoclonal antibody P256 is currently undergoing development for in vivo detection of thrombus. We have examined the actions of P256 and two fragments on human platelet function. P256, and its divalent fragment, caused aggregation at concentrations of 10−9−3 × 10−8 M. A monovalent fragment of P256 did not cause aggregation at concentrations up to 10−7 M. P256–induced platelet aggregation was dependent upon extracellular calcium ions as assessed by quin2 fluorescence. Indomethacin partially inhibited platelet aggregation and completely inhibited intracellular calcium mobilisation. Apyrase caused partial inhibition of aggregation. Aggregation induced by the divalent fragment was dependent upon fibrinogen and was inhibited by prostacyclin. Aggregation induced by the whole antibody was only partially dependent upon fibrinogen, but was also inhibited by prostacyclin. P256 whole antibody was shown, by flow cytometry, to induce fibrinogen binding to indomethacin treated platelets. Monovalent P256 was shown to be a specific antagonist for aggregation induced by the divalent forms. In–111–labelled monovalent fragment bound to gel-filtered platelets in a saturable and displaceable manner. Monovalent P256 represents a safer form for in vivo applications

Studies on the Binding of 3H-SR121566, an Inhibitor of Gp IIb-IIIa Activation

2001 ◽  
Vol 85 (04) ◽  
pp. 702-709 ◽  
Author(s):  
P. Savi ◽  
G. Zamboni ◽  
O. Rescanières ◽  
J. M. Herbert
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Platelet Aggregation ◽  
Binding Sites ◽  
Human Platelets ◽  
Gamma Chain ◽  
Activated Platelets ◽  
Fibrinogen Binding ◽  
Direct Competition ◽  
Stimulation Of

SummarySR121566 is a new synthetic agent which inhibits the binding of fibrinogen to activated platelets, and platelet aggregation. 3H-SR121566 bound with nanomolar affinity (KD ranging from 45 to 72 nM) to Gp IIb-IIIa expressing cells only. On activated human platelets, this ligand allowed the detection of a maximal number of 100-140,000 binding sites. The binding of SR121566 to platelets, was displaced by several agents including RGD-containing peptides and synthetic RGD mimetics, but not by ReoPro®, a humanised monoclonal antibody which inhibits the binding of fibrinogen to the Gp IIb-IIIa complex. Neither the fibrinogen dodecapeptide nor fibrinogen itself were able to compete with SR121566 whether platelets were activated or not.Flow cytometry studies indicated that SR121566 which did not activate Gp IIb-IIIa by itself, dose-dependently prevented the detection of activation-induced binding sites on TRAP-stimulated platelets in the presence or absence of exogenous fibrinogen, indicating a direct effect on the activation state of the Gp IIb-IIIa complex. Moreover, SR121566 was able to reverse the activation of Gp IIb-IIIa and to displace the binding of fibrinogen when added up to 5 min after TRAP stimulation of platelets. When added at later times (15 to 30 min), SR121566 failed to displace fibrinogen binding, even if SR121566 binding sites were still accessible and the Gp IIb-IIIa complex not activated.In conclusion, our study is in accordance with the finding that fibrinogen is recognised by the activated Gp IIb-IIIa complex through the dodecapeptide sequence present on its gamma chain, and that this interaction is inhibited by SR121566 by preventing and reversing the activated conformation of Gp IIb-IIIa and not by direct competition with fibrinogen.

scholarly journals Inhaled Nitric Oxide Inhibits Human Platelet Aggregation, P-Selectin Expression, and Fibrinogen Binding In Vitro and In Vivo

Circulation ◽  
1998 ◽  
Vol 97 (15) ◽  
pp. 1481-1487 ◽  
Author(s):  
André Gries ◽  
Christoph Bode ◽  
Karlheinz Peter ◽  
Axel Herr ◽  
Hubert Böhrer ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Platelet Aggregation ◽  
Human Platelet ◽  
Inhaled Nitric Oxide ◽  
Human Platelet Aggregation ◽  
Fibrinogen Binding

A SCID Mouse Model for Monitoring in Vivo Viability of Human Platelet Concentrates Using Flow Cytometry

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4549-4549
Author(s):  
Jia-hua Ding ◽  
Cheng-yin Huang ◽  
Shiyun Xu
Keyword(s):  
Flow Cytometry ◽  
Whole Blood ◽  
Human Platelet ◽  
Human Platelets ◽  
Scid Mice ◽  
Test Method ◽  
Platelet Concentrates ◽  
Tail Vein

Abstract Objective To develop an animal test method for evaluating the in vivo quality of human platelet concentrates. Methods Human platelets were transfused to mice by tail vein with a 1mL insulin syring fitted with a 29-gauge ultra-fine needle. Blood samples were taken at 30 minutes,2,4,6,8,12, and 24hours after infusion with a tail vein nick technique, whole blood was collected into heparinized capillary tubes. Human platelets in mouse whole blood were detected by flow cytometry with monoclonal anti-human CD61-PE–conjugated antibodies. All subsequent recoveries were calculated as a percentage of the initial collection. Results The survival time of human platelets were significantly prolonged in SCID than in BALB/c,FVB mice. Recoveries at 4 hours after transfusion in SCID, BALB/c,FVB mice were 68.6%±8.1%(n =10),29.9%±6.5%(n =8),28.1%±5.5%(n =8), respectively, and with a T½ estimate of 8 hours for SCID, 2.5 hours for BALB/c and 2 hours for FVB mice. platelet storage lesions either by chemical treatment or by suboptimal conditions storage exhibited decreased recoveries in SCID mice. Conclusion The quality of platelet Products can be evaluated by assessing the survival of human platelets in SCID mice using flow cytometry.

Thromboxane A2-Stimulated Human Platelet Aggregation Is Potentiated By Epinephrine Acting VIA Alpha Adrenergic Receptors

1981 ◽  
Author(s):  
G J Johnson ◽  
G H R Rao ◽  
J G White
Keyword(s):  
Platelet Aggregation ◽  
Adrenergic Receptors ◽  
Human Platelet ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Adrenergic Agents ◽  
Alpha Adrenergic Receptors ◽  
Human Platelet Aggregation ◽  
Induced Aggregation

Epinephrine (E) potentiates arachidonate (A)-induced aggregation of human platelets. A-insensitive dog platelets (AIP), that form thromboxane A2 (T) but do not aggregate when stirred with A alone, aggregate when exposed to E + A. Therefore, we studied the effect of E on T-stimu- lated human platelet aggregation. AIP stirred with A formed T which was confirmed by TLC. 1/100 to 1/200 volume of AIP was removed 30 sec. after A, and transferred to gel- filtered, aspirin-incubated human platelets. Recipient platelet aggregation was proportional to the volume of AIP transferred. The addition of the thromboxane synthetase inhibitor, Azo Analog I, abolished the aggregating activity of AIP. Transfer of an aliquot of AIP that was inadequate to aggregate human gel-filtered, aspirin-incubated platelets resulted in irreversible aggregation in the presence of ≥0.5nM E. E potentiated aggregation when added 3 min. before but not 3 min. after aliquot transfer. T-stimulated aggregation was abolished by the T-antagonist, 13 azapro- stenoic acid (APA), but E added after APA and before T restored aggregation. E potentiation of T-stimulated aggregation was abolished by prior exposure to equimolar yohimbine, dihydroergocryptine and phentolamine, agents that bind to alpha2 adrenergic receptors, but not by prazosin an alpha1 antagonist. Higher concentrations of E reversed the inhibitory effects of the alpha2 adrenergic agents. All of these agents in higher concentrations (1-100μM) also blocked aggregation induced by T alone. Therefore T-induced platelet aggregation is potentiated by E, in concentrations attained in vivo, by a mechanism linked to platelet alpha adrenergic receptors. Platelet alpha2 receptors have a close functional relationship to the postulated T receptor. E may initiate platelet aggregation in vivo when T is formed in quantities inadequate to alone induce aggregation.

A Novel Regulatory Epitope Defined by a Murine Monoclonal Antibody to the Platelet GPIIb-IIIa Complex (αIIbβ3 Integrin)

1996 ◽  
Vol 76 (06) ◽  
pp. 1038-1046 ◽  
Author(s):  
Michihide Tokuhira ◽  
Makoto Handa ◽  
Tetsuji Kamata ◽  
Atsushi Oda ◽  
Masahiko Katayama ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Human Platelets ◽  
Antibody Binding ◽  
Murine Monoclonal Antibody ◽  
Conformational Structure ◽  
Dependent Manner ◽  
Binding Parameters ◽  
Fibrinogen Binding ◽  
Αiibβ3 Integrin

SummaryWe characterized a murine monoclonal antibody, PT25-2 (IgG1), raised against washed human platelets. The antibody and its Fab fragments were both capable of inducing platelet aggregation in a fibrinogen-dependent manner and induced 125I-fibrinogen binding to unstimulated platelets (120,000 molecules/platelet at a 100 nM IgG concentration). The antibody immunoprecipitated the αIIbβ3 complex from lysates of iodinated platelets but did not react with the respective subunits when complex formation was disrupted by treatment with 5 mM EDTA at 37°C for 30 min. However, simply removing the extracellular divalent cation with EDTA had no effect on antibody binding indicating that the antibody’s epitope depends upon a conformational structure maintained by αβ subunit association. Antibody binding to unstimulated, washed platelets yielded binding parameters (Kd = 40 nM, Bmax = 100,000 molecules/platelet), which were found to be virtually unchanged when binding was performed using thrombin or RGDS-peptide-stimulated platelets. Thus, the PT25-2 antibody defines a novel regulatory epitope expressed by the αIIbβ3 integrin on unstimulated, quiescent platelets.

TRYPTOPHAN PYROLYSIS PRODUCTS FOUND IN COOKED FOODS INHIBIT HUMAN PLATELET AGGREGATION BY INHIBITING CYCLOOXYGENASE

1987 ◽  
Author(s):  
S Manabe ◽  
H Yanagisawa ◽  
S Ishikawa ◽  
Y Kitagawa ◽  
K Tohyama ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Cell Function ◽  
Human Platelet ◽  
Human Platelets ◽  
Heterocyclic Amines ◽  
Inhibitory Effects ◽  
Pyrolysis Products ◽  
Human Platelet Aggregation ◽  
Leukocyte Aggregation

Humans are exposed to numerous toxic compounds in foods. During the past decade, several carcinogenic heterocyclic amines have been reported to be present in the cooked foods. Recently, we reported that some of the carcinogenic heterocyclic amines isolated from foods were present in human plasma. In order to know the effects of the carcinogens isolated from foods on the cell function, we investigated the effects of the carcinogenic heterocyclic amines including Trp-P-1(3-amino-l,4-dimethyl-5H-pyrido❘4,3-b❘indole) and Trp-P-2(3-amino-1-methyl-5H-pyrido❘4,3-b❘indole) on human platelet aggregation and polymorphonuclear leukocyte aggregation. Only tryptophan pyrolysis products, Trp-P-1 and Trp-P-2, had potent inhibitory effects on human platelet aggregation when platelets were preincubated with the carcinogens for 15 min. Other carcinogenic heterocyclic amines such as glutamic acid pyrolysates (Glu-P-1 and Glu-P-2) and 3H-imidazo ❘4,5-f❘quinoline-2-amines(IQ and MelQ) did show no effect on platelet aggregation even at 100 μM.The autoradiogram demonstrated that Tryptophan pyrolysis products, Trp-P-1 and Trp-P-2, dose-dependently inhibited the formation of HHT,PGD2,PGE2 and TXB2 induced by sodium arachidonate in human platelets labeled with ❘ 14c❘ arachidonic acid. Moreover, Trp-P-1 and Trp-P-2 did not show significant effects on leukocyte aggregation induced by sodium arachidonate (0.75mM) even at lOOnM. It is concluded that Trp-P-1 and Trp-P-2 isolated from cooked foodstuffs have potent inhibitory effects on the cyclo-oxygenase pathway of the platelet. Therefore, human platelet function might be affected with daily foods containing tryptophan pyrolysis products in vivo.

scholarly journals A murine monoclonal antibody that blocks fibrinogen binding to normal platelets also inhibits fibrinogen interactions with chymotrypsin- treated platelets

Blood ◽  
1984 ◽  
Vol 64 (1) ◽  
pp. 59-63 ◽  
Author(s):  
EI Peerschke ◽  
BS Coller
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Membrane Glycoproteins ◽  
Molecular Weights ◽  
Fibrinogen Receptor ◽  
Fibrinogen Binding ◽  
Unstimulated Control ◽  
Lines Of Evidence

We recently described a monoclonal antibody, 10E5 , that completely blocks adenosine diphosphate (ADP) induced fibrinogen binding to platelets and aggregation induced by ADP, epinephrine, and thrombin. Multiple lines of evidence indicate that 10E5 binds to platelet membrane glycoproteins IIb and/or IIIa. Because it has been reported that platelets treated with chymotrypsin aggregate when fibrinogen is added, we tested the effect of 10E5 antibody on chymotrypsin-induced fibrinogen binding and platelet aggregation. Aspirin-treated human platelets were washed in modified Tyrode's buffer (pH 7.5), incubated for 5 minutes at 22 degrees C with 300 micrograms/mL chymotrypsin, and washed again. The amount of 10E5 antibody bound to these platelets (37,232 +/- 2,928 molecules/platelet; mean +/- SEM, N=9) was similar to that bound to unstimulated control platelets (36,910 +/- 2,669) and did not differ significantly from the amount of antibody bound to ADP- treated platelets (P less than .01, N = 5). The amount of 10E5 bound to chymotrypsin-treated platelets correlated directly with the amount of fibrinogen bound to separate aliquots of the same platelet samples (r = .876, P less than .001). The 10E5 antibody caused virtually complete inhibition of both the binding of fibrinogen to chymotrypsin-treated platelets and the aggregation induced by exogenous fibrinogen. Immunoprecipitation studies of 125I-labeled chymotrypsin-treated platelets revealed that the 10E5 antibody bound proteins with molecular weights characteristic of glycoproteins IIb and IIIa. These data suggest that the fibrinogen receptor on chymotrypsin-treated platelets is identical to that on ADP-treated platelets and that this receptor is either near to, or on, the glycoprotein IIb/IIIa complex.

scholarly journals A murine monoclonal antibody that blocks fibrinogen binding to normal platelets also inhibits fibrinogen interactions with chymotrypsin- treated platelets

Blood ◽  
1984 ◽  
Vol 64 (1) ◽  
pp. 59-63 ◽  
Author(s):  
EI Peerschke ◽  
BS Coller
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Membrane Glycoproteins ◽  
Molecular Weights ◽  
Fibrinogen Receptor ◽  
Fibrinogen Binding ◽  
Unstimulated Control ◽  
Lines Of Evidence

Abstract We recently described a monoclonal antibody, 10E5 , that completely blocks adenosine diphosphate (ADP) induced fibrinogen binding to platelets and aggregation induced by ADP, epinephrine, and thrombin. Multiple lines of evidence indicate that 10E5 binds to platelet membrane glycoproteins IIb and/or IIIa. Because it has been reported that platelets treated with chymotrypsin aggregate when fibrinogen is added, we tested the effect of 10E5 antibody on chymotrypsin-induced fibrinogen binding and platelet aggregation. Aspirin-treated human platelets were washed in modified Tyrode's buffer (pH 7.5), incubated for 5 minutes at 22 degrees C with 300 micrograms/mL chymotrypsin, and washed again. The amount of 10E5 antibody bound to these platelets (37,232 +/- 2,928 molecules/platelet; mean +/- SEM, N=9) was similar to that bound to unstimulated control platelets (36,910 +/- 2,669) and did not differ significantly from the amount of antibody bound to ADP- treated platelets (P less than .01, N = 5). The amount of 10E5 bound to chymotrypsin-treated platelets correlated directly with the amount of fibrinogen bound to separate aliquots of the same platelet samples (r = .876, P less than .001). The 10E5 antibody caused virtually complete inhibition of both the binding of fibrinogen to chymotrypsin-treated platelets and the aggregation induced by exogenous fibrinogen. Immunoprecipitation studies of 125I-labeled chymotrypsin-treated platelets revealed that the 10E5 antibody bound proteins with molecular weights characteristic of glycoproteins IIb and IIIa. These data suggest that the fibrinogen receptor on chymotrypsin-treated platelets is identical to that on ADP-treated platelets and that this receptor is either near to, or on, the glycoprotein IIb/IIIa complex.

scholarly journals Antiplatelet effect of cudraxanthone B is related to inhibition of calcium mobilization, αIIbβ3 activation, and clot retraction

2021 ◽  
Vol 64 (1) ◽  
Author(s):  
Jung-Hae Shin ◽  
Muhammad Irfan ◽  
Man Hee Rhee ◽  
Hyuk-Woo Kwon
Keyword(s):  
Platelet Aggregation ◽  
Human Platelet ◽  
Human Platelets ◽  
Adhesion Assay ◽  
Inhibitory Effects ◽  
Clot Retraction ◽  
Cudrania Tricuspidata ◽  
Antiplatelet Effect ◽  
Human Platelet Aggregation ◽  
Fibrinogen Binding

AbstractCudrania tricuspidata (C. tricuspidata) is widespread throughout Asia and has known to have various physiological activities such as, inflammation, diabetes, obesity and tumor. Cudrania tricuspidata, a rich source of xanthones and flavonoids, have been investigated phytochemically and biologically. However, research of these compounds on platelets is limited. Therefore, we searched for a new substance from various xanthones and flavonoids in C. tricuspidata. We confirmed the results that steppogenin and isoderrone suppress human platelets among the various components isolated from C. tricuspidata, and as a result of analyzing the antiplatelet effect using additional new samples, we found that cudraxanthone B (CXB) has the effect of suppressing human platelets. Therefore, we studied the potential efficacies of CXB on human platelet aggregation and its inhibitory mechanism. Inhibitory effects of CXB on platelet aggregation were assessed using washed platelets, followed by measurement of [Ca2+]i mobilization and dense granule release, fibrinogen binding, fibronectin adhesion assay, and clot retraction. Our data showed that CXB suppressed collagen-induced human platelet aggregation, [Ca2+]i mobilization, fibrinogen binding, fibronectin adhesion and clot retraction without cytotoxicity. Thus, our results show that inhibitory effects of CXB on human platelet activation and thrombus formation, suggesting its potential use as a natural substance for preventing platelet-induced thrombosis.

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