Enhanced Inhibition of in-Vitro Platelet Aggregation by a Targeted Antibody Conjugate Containing an Anti-Rabbit Platelet Glycoprotein Iib/Iiia Antibody

SummaryThe present study was designed to evaluate the effectiveness of the ergoline 5HT2 receptor antagonist, LY53857 in a rabbit model of vascular arterial occlusion. LY53857 (1 and 10 εM) inhibited serotonin amplified platelet aggregation responses to threshold concentrations of ADP in rabbit platelets in vitro. LY53857 (1 εM) not only inhibited the serotonin component of rabbit platelet aggregation, but also inhibited in vitro aggregation induced by ADP (48.7 ± 16.7% inhibition), collagen (76.1 ± 15.9% inhibition) and U46619 (65.2 ± 12.3% inhibition). The effectiveness of this ergoline 5HT2 receptor antagonist in blocking aggregation to ADP, collagen and U46619 may be related to its ability to inhibit a serotonin component of platelet aggregation since rabbit platelets possess high concentrations of serotonin that may be released during aggregation produced by other agents. Based on the effectiveness of LY53857 to inhibit rabbit platelet aggregation, we explored the ability of LY53857 to extend the time to carotid artery occlusion in rabbits following electrical stimulation of the artery. Reproducible carotid artery occlusion was induced in rabbits by moderate stenosis coupled to arterial cross clamping, followed by electrical stimulation. With this procedure, occlusion occurred at 47.0 ± 7 min (n = 30) after initiation of the electrical stimulation. Animals pretreated with LY53857 (50 to 500 εg/kg i.v.) showed a delay in the time to carotid artery occlusion (at 100 εg/kg i.v. occlusion time extended to 164 ± 16 min). Furthermore, ex vivo platelet aggregation from animals treated with LY53857 (300 εg/kg i.v.) resulted in 40.5% inhibition of platelet aggregation in response to the combination of ADP (1 εM) and serotonin (1 εM). These studies document the ability to obtain reproducible arterial occlusion in the rabbit and showed that intravenously administered LY53857 prolonged the time to carotid artery occlusion. Prolongation of carotid artery occlusion time was accompanied by inhibition of serotonin-amplified ADP-induced aggregation in rabbit platelets, an effect observed both in vitro and ex vivo. Thus, the rabbit is a useful model for studying the effectiveness of 5HT2 receptor antagonists in prolonging vascular occlusion induced by insult of the carotid artery.

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Effects of Bupranolol, a New β-Blocker, on Platelet Functions of Rabbit and Human in Vitro

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648052 ◽

1976 ◽

Vol 36 (02) ◽

pp. 376-387 ◽

Cited By ~ 3

Author(s):

Teruhiko Umetsu ◽

Kazuko Sanai ◽

Tadakatsu Kato

Keyword(s):

Platelet Aggregation ◽

Platelet Adhesion ◽

Human Platelets ◽

Rabbit Platelet ◽

Rabbit Platelets ◽

Β Blocker ◽

Platelet Aggregates ◽

Induced Aggregation ◽

Platelet Functions

SummaryThe effects of bupranolol, a new β-blocker, on platelet functions were investigated in vitro in rabbits and humans as compared with propranolol, a well-known β-blocker. At first, the effect of adrenaline on ADP-induced rabbit platelet aggregation was studied because adrenaline alone induces little or no aggregation of rabbit platelets. Enhancement of ADP-induced rabbit platelet aggregation by adrenaline was confirmed, as previously reported by Sinakos and Caen (1967). In addition the degree of the enhancement was proved to be markedly affected by the concentration of ADP and to increase with decreasing concentration of ADP, although the maximum aggregation (percent) was decreased.Bupranolol and propranolol inhibited the (adrenaline-ADP-)induced aggregation of rabbit platelets, bupranolol being approximately 2.4–3.2 times as effective as propranolol. Bupranolol stimulated the disaggregation of platelet aggregates induced by a combination of adrenaline and ADP, but propranolol did not. Platelet adhesion in rabbit was also inhibited by the β-blockers and bupranolol was more active than propranolol. With human platelets, aggregation induced by adrenaline was inhibited by bupranolol about 2.8–3.3 times as effectively as propranolol.From these findings. We would suggest that bupranolol might be useful for prevention or treatment of thrombosis.

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Effects of 1-S replaced and/or decarboxylated latamoxef on rabbit platelet aggregation in vitro.

The Japanese Journal of Pharmacology ◽

10.1254/jjp.46.71 ◽

1988 ◽

Vol 46 (1) ◽

pp. 71-77 ◽

Cited By ~ 2

Author(s):

Kiyohisa UCHIDA ◽

Hisato KAKUSHI ◽

Tsutomu SHIKE

Keyword(s):

Platelet Aggregation ◽

Rabbit Platelet

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Effects of Ethanol on Pathways of Platelet Aggregation In Vitro

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647500 ◽

1988 ◽

Vol 59 (03) ◽

pp. 383-387 ◽

Cited By ~ 42

Author(s):

Margaret L Rand ◽

Marian A Packham ◽

Raelene L Kinlough-Rathbone ◽

J Fraser Mustard

Keyword(s):

Arachidonic Acid ◽

Platelet Aggregation ◽

Platelet Rich Plasma ◽

Secondary Phase ◽

Primary Phase ◽

Rabbit Platelet ◽

Membrane Phospholipids ◽

Rabbit Platelets ◽

Induced Aggregation

SummaryEthanol, at physiologically tolerable concentrations, did not affect the primary phase of ADP-induced aggregation of human or rabbit platelets, which is not associated with the secretion of granule contents. Potentiation by epinephrine of the primary phase of ADP-induced aggregation of rabbit platelets was also not inhibited by ethanol. However, ethanol did inhibit the secondary phase of ADP-induced aggregation which occurs with human platelets in citrated platelet-rich plasma and is dependent on the formation of thromboxane A2. Inhibition by ethanol of thromboxane production by stimulated platelets is likely due to inhibition of the mobilization of arachidonic acid from membrane phospholipids, as ethanol had little or no effect on aggregation and secretion induced by arachidonic acid or the thromboxane mimetic U46619. Rabbit platelet aggregation and secretion in response to low concentrations of collagen, thrombin, or PAF were inhibited by ethanol. Inhibition of the effects of thrombin and PAF was also observed with aspirin-treated platelets. Thus, in addition to inhibiting the mobilization of arachidonate for thromboxane formation that occurs with most agonists, ethanol can also inhibit aggregation and secretion through other effects on platelet responses.

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Studies investigating platelet aggregation and release initiated by sera from patients with thrombotic thrombocytopenic purpura

Blood ◽

10.1182/blood.v69.3.924.bloodjournal693924 ◽

1987 ◽

Vol 69 (3) ◽

pp. 924-928 ◽

Cited By ~ 1

Author(s):

JG Kelton ◽

JC Moore ◽

WG Murphy

Keyword(s):

Platelet Aggregation ◽

Thrombotic Thrombocytopenic Purpura ◽

Disease Process ◽

The Other ◽

Platelet Glycoprotein ◽

Glycoprotein Ib ◽

Thrombocytopenic Purpura ◽

Induced Aggregation

Many patients with thrombotic thrombocytopenic purpura (TTP) have a platelet aggregating factor in their serum that may be pathologically linked with the disease process. To help characterize the type of platelet aggregation and platelet release induced by the sera from seven TTP patients, we measured the ability of a variety of inhibitors of platelet function as well as the ability of monoclonal antibodies (MoAbs) against platelet glycoproteins to inhibit TTP sera-induced platelet aggregation and release. These results were compared with the ability of the same inhibitors to block platelet aggregation induced by ristocetin, collagen, ADP, thrombin, and IgG-immune complexes. Monoclonal antibody directed against platelet glycoprotein Ib totally inhibited ristocetin-induced aggregation and release but had no effect on aggregation and release induced by the TTP sera or by any of the other platelet agonists. However, the MoAb against glycoproteins IIb/IIIa inhibited aggregation and release caused by TTP sera as well as by collagen, thrombin, and ADP but had no effect on aggregation and release induced by ristocetin. The aggregating activity could be abolished by heparin but not by the serine protease inhibitor PMSF (1 mmol/L). And although monomeric human IgG and purified Fc fragments of IgG inhibited IgG-immune complex-induced aggregation and release, they had no effect on TTP sera-induced aggregation and release nor on aggregation and release induced by any of the other agonists. Consistent with these in vitro studies showing no effect of IgG were the in vivo observations that intravenous (IV) IgG was without effect when administered to three patients with TTP. This study indicates that although a von Willebrand factor (vWF)-rich preparation of cryoprecipitate enhances the in vitro platelet aggregation and release caused by sera from the seven TTP patients we studied, the pathway of aggregation and release is not via platelet glycoprotein Ib. Also the aggregating factor of TTP sera is not neutralized in vitro or in vivo by IgG.

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Studies investigating platelet aggregation and release initiated by sera from patients with thrombotic thrombocytopenic purpura

Blood ◽

10.1182/blood.v69.3.924.924 ◽

1987 ◽

Vol 69 (3) ◽

pp. 924-928 ◽

Cited By ~ 35

Author(s):

JG Kelton ◽

JC Moore ◽

WG Murphy

Keyword(s):

Platelet Aggregation ◽

Thrombotic Thrombocytopenic Purpura ◽

Disease Process ◽

The Other ◽

Platelet Glycoprotein ◽

Glycoprotein Ib ◽

Thrombocytopenic Purpura ◽

Induced Aggregation

Abstract Many patients with thrombotic thrombocytopenic purpura (TTP) have a platelet aggregating factor in their serum that may be pathologically linked with the disease process. To help characterize the type of platelet aggregation and platelet release induced by the sera from seven TTP patients, we measured the ability of a variety of inhibitors of platelet function as well as the ability of monoclonal antibodies (MoAbs) against platelet glycoproteins to inhibit TTP sera-induced platelet aggregation and release. These results were compared with the ability of the same inhibitors to block platelet aggregation induced by ristocetin, collagen, ADP, thrombin, and IgG-immune complexes. Monoclonal antibody directed against platelet glycoprotein Ib totally inhibited ristocetin-induced aggregation and release but had no effect on aggregation and release induced by the TTP sera or by any of the other platelet agonists. However, the MoAb against glycoproteins IIb/IIIa inhibited aggregation and release caused by TTP sera as well as by collagen, thrombin, and ADP but had no effect on aggregation and release induced by ristocetin. The aggregating activity could be abolished by heparin but not by the serine protease inhibitor PMSF (1 mmol/L). And although monomeric human IgG and purified Fc fragments of IgG inhibited IgG-immune complex-induced aggregation and release, they had no effect on TTP sera-induced aggregation and release nor on aggregation and release induced by any of the other agonists. Consistent with these in vitro studies showing no effect of IgG were the in vivo observations that intravenous (IV) IgG was without effect when administered to three patients with TTP. This study indicates that although a von Willebrand factor (vWF)-rich preparation of cryoprecipitate enhances the in vitro platelet aggregation and release caused by sera from the seven TTP patients we studied, the pathway of aggregation and release is not via platelet glycoprotein Ib. Also the aggregating factor of TTP sera is not neutralized in vitro or in vivo by IgG.

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Inhibition of Endotoxic Lipopolysaccharide-mediated Platelet Aggregation by Cobra Venom Anticomplementary Factor

Blood ◽

10.1182/blood.v44.3.399.399 ◽

1974 ◽

Vol 44 (3) ◽

pp. 399-409 ◽

Cited By ~ 6

Author(s):

Jack S. C. Fong ◽

James G. White ◽

Robert A. Good

Keyword(s):

Platelet Aggregation ◽

Critical Time ◽

Protein S ◽

Cobra Venom ◽

In Vivo Studies ◽

Rabbit Platelet ◽

Response Curves ◽

Complement Components

Abstract Aggregometry studies on endotoxic lipopolysaccharide (LPS)-mediated rabbit platelet aggregation were performed. Different preparations of LPS showed characteristic aggregometry profiles, and LPS with potent anticomplementary activities generally had a more vigorous platelet aggregation function than did LPS preparations with lesser anticomplementary functions. Cobra venom anticomplementary factor (CVF) inhibited LPS-platelet interaction, and the inhibition was both time and dose dependent. Dose-response curves of CVF inhibition on LPS or zymosan-mediated platelet aggregation were essentially identical. In vitro and in vivo studies showed that CVF inhibition persisted even when hemolytic complement activities reached more than 70% of those originally present. At the critical time of days 5 or 6 following CVF administration, the lack of platelet responses towards LPS could be restored by addition of fresh plasma from normal or C6-deficient rabbits, but not with plasma that had been treated with antigen— antibody complexes, zymosan, or heating at 56° for 30 min. The experimental data indicate that serum protein(s) other than the terminal complement components are involved in LPS-platelet interaction. It seems most likely that the factor(s) perturbed reside in the mechanisms involved in activation of the alternate pathway. Furthermore, it appears quite possible that LPS-platelet interactions can be inhibited by manipulating the humoral factor(s) involved rather than by altering the platelets themselves.

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The Effects of Glycoprotein IIb/IIIa Antagonist RGDS on Platelet Aggregation and Release Reaction In Vitro.

Blood ◽

10.1182/blood.v108.11.3908.3908 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3908-3908

Author(s):

Shuangfeng Xie ◽

Songmei Yin ◽

Danian Nie ◽

Yiqing Li ◽

Xiuju Wang ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Adhesion ◽

Adenosine Diphosphate ◽

Platelet Glycoprotein ◽

High Concentration ◽

Release Reaction ◽

Dense Granules ◽

Negative Effect ◽

Platelet Release

Abstract Platelet activation, including platelet adhesion, platelet aggregation and platelet release reaction, played an important role in thrombogenesis. We all knew that Platelet glycoprotein IIb/IIIa antagonist was the most effective drug for anti-aggregation, while we don’t know clearly its effect on platelet release reaction and the relations between its effects on platelet aggregation and release reaction. Platelet release reactions included α-granules and dense granules releasing. When α-granules were released, its membrane glycoprotein CD62p was expressed in the platelet membrane. We used the CD62p expression as the index of platelet release reaction. In the current study, the 4-peptides RGDS (Arg-Gly-Asp-Ser) was used as glycoprotein IIb/IIIa antagonist. We detected the effects of RGDS on platelet aggregation and CD62p expression induced by adenosine diphosphate (ADP) (finial concentration, 5μmol/L) in vitro. 50, 100, 200, 400 and 800μmol/L RGDS were used separately in the test. RGDS of each concentration could significantly inhibited maximal platelet aggregation (PAG(M)) induced by ADP, the 50% inhibiting concentration was approximately 200μmol/L. 800μmol/L RGDS could inhibited PAG(M) by 80.48±8.18%. Only ≥200μmol/L RGDS could significantly inhibited platelet CD62p expression. 800μmol/L RGDS could inhibit platelet CD62p expression by 27.31±9.74%. The inhibiting effect of RGDS on PAG(M) and platelet CD62p expression had significantly correlation (r =0.976, P<0.05). These results indicated that RGDS in low concentration (<200μmol) had little negative effect on platelet release reaction induced by ATP, while in relatively high concentration (≥200μmol) RGDS could inhibit platelet release reaction. When RGDS concentrations were same its effect on platelet release reaction was much less than that on platelet aggregation, which indicated that platelet glycoprotein IIb/IIIa compound could only partly participated in the platelet release reaction but fully participated in platelet aggregation induced by ADP.

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Vigorous exercise acutely changes platelet and B-lymphocyte CD39 expression

Journal of Applied Physiology ◽

10.1152/japplphysiol.00315.2004 ◽

2005 ◽

Vol 98 (4) ◽

pp. 1414-1419 ◽

Cited By ~ 15

Author(s):

Antonino Coppola ◽

Ludovico Coppola ◽

Liliana dalla Mora ◽

Francesco M. Limongelli ◽

Antonio Grassia ◽

...

Keyword(s):

Platelet Aggregation ◽

B Lymphocytes ◽

Platelet Activation ◽

Critical Role ◽

Strenuous Exercise ◽

Platelet Glycoprotein ◽

Vigorous Exercise ◽

Platelet Aggregates

CD39/ATP diphosphohydrolase is expressed on B lymphocytes, cytotoxic T lymphocytes, monocytes, platelets, and endothelial cells, and it has a critical role in the inhibition of platelet responsiveness. To determine whether strenuous exercise could acutely change expression of CD39 in platelets and lymphocytes, eight healthy sedentary men, 34 yr old (SD 7), and eight physically active men, 34 yr old (SD 6), performed graded upright cycle ergometry to volitional exhaustion. Blood samples collected both at baseline and after exercise test were employed to measure CD39 expression in platelets and lymphocytes. The percentage of circulating platelet-platelet aggregates, the “in vitro” ADP and collagen-induced platelet aggregation, and the expression of both platelet glycoprotein IIb-IIIa (PAC-1) and P-selectin (CD62) were also considered markers of platelet activation. After strenuous exercise, all subjects demonstrated significant platelet activation as judged by the increased percentage of platelet-platelet aggregates. The in vitro ADP-induced platelet aggregation and the expression of CD62P on ADP-stimulated platelets significantly increased in sedentary but not in active subjects. After exercise, all of the subjects showed a significant reduction of CD39 expression in platelet [sedentary: from 2.2 (SD 0.8) to 1.1% (SD 0.8), P = 0.008; active: from 0.6 (SD 0.2) to 0.35% (SD 0.1), P = 0.009] and an increase of CD39 expression in B lymphocytes [sedentary: from 47 (SD 13) to 60% (SD 11), P = 0.0039; active: from 46 (SD 11) to 59% (SD 11), P = 0.0038]. Taken together, these findings confirm the critical role of this ADPase in inhibition of platelet responsiveness, also suggesting a possible role of B lymphocytes in thromboregulation mechanism.

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Novel Antibodies Against Glycoprotein Ibα Inhibit Pulmonary Metastasis By Affecting Vwf-Gpibα Interaction

Blood ◽

10.1182/blood-2018-99-117613 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1133-1133

Author(s):

Qi Yingxue ◽

Wenchun Chen ◽

Ke Xu ◽

Fengying Wu ◽

Xuemei Fan ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Binding Sites ◽

Tumor Metastasis ◽

Cancer Metastasis ◽

Platelet Glycoprotein ◽

Induce Platelet Aggregation ◽

Metastasis Model

Abstract Background: Platelet glycoprotein Ibα (GPIbα) extracellular domain, which is part of the receptor complex GPIb-IX-V, plays an important role in tumor metastasis. However, the mechanism through which GPIbα participates in the metastatic process remains unclear. In addition, potential bleeding complication remains an obstacle for the clinical use of anti-platelet agents in cancer therapy. Methods and Results: To generate antibodies that bind to mouse platelet GPIbα, washed mouse platelet lysate was used as the antigen for rat immunization. Obtained hybridoma clones were screened in ELISA for binding affinity to the GPIb-IX complex. Positive clones were further screened for their abilities to inhibit platelet-cancer cell adhesion. Finally, at static condition, two antibodies, 2B4 and 1D12, had virtually no effect on the activation of integrin αIIbβ3, which is used to indicate platelet activation. Then, we characterized the binding sites of 2B4 and 1D12 by 20 purified recombinant GPIbα fragments binding. Results showed that 2B4 and 1D12 shared the same binding sites with vWF. To determine whether 2B4 and 1D12 affect vWF binding, we tested the binding by flow cytometry using recombined mouse vWF, and then, we investigated platelet aggregation induced by several agonists, including vWF binding agonist ristocetin. Our data demonstrated clearly that 2B4 and 1D12 could inhibit vWF binding. To investigate whether the inhibition of vWF-GPIbα interaction was associated with tumor metastasis, we examined the effect of 2B4 and 1D12 in each of the following interactions in vitro: between activated platelets and tumor cells, platelets and endothelial cells. Meanwhile, We further investigated the inhibitory effect of these antibodies in vivo using the experimental metastasis model and the spontaneous metastasis model. Results showed that 2B4 and 1D12 could potently inhibit the adhesion of cancer cells in vitro, and metastasisin vivo. We next investigated whether 2B4 and 1D12 could affect platelet activation and/or induce thrombocytopenia in vivo. Results showed that the addition of 2B4 or 1D12 to PRP did not induce platelet aggregation and injection of 2B4 or 1D12 Fab at appropriate dose did not affect tail-bleeding time and platelet count. Based on the above findings, we obtained anti-human platelet GPIbα monoclonal antibody YQ3 using the same approach to explore the role of human GPIbα in cancer metastasis. As expected, YQ3 inhibited lung cancer adhesion and demonstrated similar value in metastasis. More importantly, for all three mAbs in our study, none of their Fabs induced thrombocytopenia. Conclusion: Our results therefore supported the hypothesis that GPIbα contributes to tumor metastasis, and suggested potential value of using anti-GPIbα mAb to suppress cancer metastasis. Disclosures Li: Neoletix: Consultancy, Equity Ownership.

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