Selective inhibition of the platelet phosphoinositide 3-kinase p110β as promising new strategy for platelet protection during extracorporeal circulation

2008 ◽  
Vol 99 (03) ◽  
pp. 609-615 ◽  
Author(s):  
Hans Wendel ◽  
Klaus Dietz ◽  
Daniela Schiebold ◽  
Karlheinz Peter ◽  
Simone Schoenwaelder ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Platelet Function ◽  
Extracorporeal Circulation ◽  
Ventricular Assist Devices ◽  
Platelet Glycoprotein ◽  
Promising Alternative ◽  
Phosphoinositide 3 Kinase

SummaryExtracorporeal circulation (ECC) is used in cardiac surgery for cardiopulmonary bypass as well as in ventricular assist devices and for extracorporeal membrane oxygenation. Blood contact with the artificial surface and shear stress of ECC activates platelets and leukocytes resulting in a coagulopathy and proinflammatory events. Blockers of the platelet glycoprotein (GP) IIb/IIIa (CD41/CD61) can protect platelet function during ECC, a phenomenon called “platelet anaesthesia”, but may be involved in post-ECC bleeding. We hypothesized that the new selective phosphoinositide 3-kinase p110β inhibitor TGX-221 that inhibits shear-induced platelet activation without prolonging the bleeding time in vivo may also protect platelet function during ECC. Heparinized blood of healthy volunteers (n=6) was treated in vitro with either the GP IIb/IIIa blocker tirofiban, TGX-221 or as control and circulated in an ECC moand after 30 minutes circulation CD41 expression on the ECCtubing as measure for platelet-ECC binding and generation of the platelet activation marker β-thromboglobulin were determined using ELISA. Platelet aggregation and platelet-granulocyte binding were analysed in flow cytometry. After log-transforming the data statistical evaluation was performed using multifactor ANOVA in combination with Tukey’s HSD test (global alpha = 5%).Tirofiban and TGX-221 inhibited platelet-ECC interaction, platelet aggregation and platelet-granulocyte binding. Tirofiban also inhibited ECC-induced β-thromboglobulin release.The observed inhibition of platelet-ECC interaction and platelet activation by tirofiban contributes to explain the mechanism of“platelet anaesthesia”.TGX-221 represents a promising alternative to GP IIb/IIIa blockade and should be further investigated for use during ECC in vivo.

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

Blood ◽  
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.

Platelet Activation Markers in Patients with Peripheral Arterial Disease

1997 ◽  
Vol 78 (06) ◽  
pp. 1434-1437 ◽  
Author(s):  
Paolo Gresele ◽  
Mariella Catalano ◽  
Carlo Giammarresi ◽  
Raul Volpato ◽  
Rosanna Termini ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Platelet Function ◽  
Arterial Disease ◽  
Platelet Function Test ◽  
Activation Markers ◽  
Significant Difference ◽  
Peripheral Arterial

SummaryPeripheral vascular disease (PVD) is an indicator of diffuse atherosclerosis and is associated with a greatly increased incidence of coronary heart and cerebrovascular disease. Although several studies have assessed whether in vivo platelet activation takes place in patients with PVD, no data are available comparing different platelet function tests in this patient population.We have compared prospectively four tests for the measurement of in vivo platelet activation (plasma βTG, plasma PF4, intraplatelet (βTG and urinary excretion of 11-dehydro-TXB2) and one in vitro platelet function test (ADP-induced platelet aggregation) in 63 well-characterized patients with intermittent claudication and in 18 age- and sex- matched healthy volunteers.No statistically significant difference was found between patients and controls for plasma βTG (20.0 ± 11.8 vs. 18.8 ± 9.0 ng/ml, respectively), plasma PF4 (5.2 ± 2.9 vs. 6.3 ± 3.5 ng/ml), βTG/PF4 ratio (4.0 ± 2.9 vs. 3.6 ± 1.8), intraplatelet pTG (4503 ± 1482 vs. 4059 ± 1065 ng/ml), and threshold aggregatory concentration of ADP (1.7 ± 0.72 vs. 1.45 ± 0.56 μM).Urinary 11-dehydro-TXB2 was instead significantly higher in the PVD group (55.4 ± 27.5 vs. 26.7 ± 7.0 ng/h, p <0.001).Our study shows that urinary 11-dehydro-TXB2 is a more sensitive index of in vivo platelet activation than the measurement of either platelet specific proteins or of in vitro platelet aggregation in patients with PVD.

scholarly journals The role of polyphenolic compounds in the diet as inhibitors of platelet function

2003 ◽  
Vol 62 (2) ◽  
pp. 469-478 ◽  
Author(s):  
Gary P. Hubbard ◽  
Siegfried Wolffram ◽  
Julie A. Lovegrove ◽  
Jonathan M. Gibbins
Keyword(s):  
Cardiovascular Disease ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Platelet Function ◽  
Dietary Intervention ◽  
Ex Vivo ◽  
Polyphenolic Compounds ◽  
Dietary Fats

Platelets play a substantial role in cardiovascular disease, and for many years there has been a search for dietary components that are able to inhibit platelet function and therefore decrease the risk of cardiovascular disease. Platelets can be inhibited by alcohol, dietary fats and some antioxidants, including a group of compounds, the polyphenols, found in fruits and vegetables. A number of these compounds have been shown to inhibit platelet function both in vitro and in vivo. In the present study the effects of the hydroxycinnamates and the flavonoid quercetin on platelet activation and cell signalling in vitro were investigated. The hydroxycinnamates inhibited platelet function, although not at levels that can be achieved in human plasma by dietary intervention. However, quercetin inhibited platelet aggregation at levels lower than those previously reported. Quercetin was also found to inhibit intracellular Ca mobilisation and whole-cell tyrosine protein phosphorylation in platelets, which are both processes essential for platelet activation. The effect of polyphenols on platelet aggregation in vivo was also investigated. Twenty subjects followed a low-polyphenol diet for 3 d before and also during supplementation. All subjects were supplemented with a polyphenol-rich meal every lunchtime for 5 d. Platelet aggregation and plasma flavonols were measured at baseline and after 5 d of dietary supplementation. Total plasma flavonoids increased significantly after the dietary intervention period (P=0·001). However, no significant changes in ex vivo platelet aggregation were observed. Further investigation of the effects of individual polyphenolic compounds on platelet function, both in vitro and in vivo, is required in order to elucidate their role in the relationship between diet and the risk of cardiovascular disease.

A Comparison of the Effect of Decorsin and Two Disintegrins, Albolabrin and Eristostatin, on Platelet Function

1995 ◽  
Vol 74 (05) ◽  
pp. 1316-1322 ◽  
Author(s):  
Mary Ann McLane ◽  
Jagadeesh Gabbeta ◽  
A Koneti Rao ◽  
Lucia Beviglia ◽  
Robert A Lazarus ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Sequence Similarity ◽  
Platelet Aggregation Inhibitors ◽  
Antiplatelet Drug ◽  
Rgd Sequence ◽  
Fibrinogen Receptor ◽  
Activated Platelets

SummaryNaturally-occurring fibrinogen receptor antagonists and platelet aggregation inhibitors that are found in snake venom (disintegrins) and leeches share many common features, including an RGD sequence, high cysteine content, and low molecular weight. There are, however, significant selectivity and potency differences. We compared the effect of three proteins on platelet function: albolabrin, a 7.5 kDa disintegrin, eristostatin, a 5.4 kDa disintegrin in which part of the disintegrin domain is deleted, and decorsin, a 4.5 kDa non-disintegrin derived from the leech Macrobdella decora, which has very little sequence similarity with either disintegrin. Decorsin was about two times less potent than albolabrin and six times less potent than eristostatin in inhibiting ADP- induced human platelet aggregation. It had a different pattern of interaction with glycoprotein IIb/IIIa as compared to the two disintegrins. Decorsin bound with a low affinity to resting platelets (409 nM) and to ADP-activated platelets (270 nM), and with high affinity to thrombin- activated platelets (74 nM). At concentrations up to 685 nM, it did not cause expression of a ligand-induced binding site epitope on the (β3 subunit of the GPIIb/IIIa complex. It did not significantly inhibit isolated GPIIb/IIIa binding to immobilized von Willebrand Factor. At low doses (1.5-3.0 μg/mouse), decorsin protected mice against death from pulmonary thromboembolism, showing an effect similar to eristostatin. This suggested that decorsin is a much more potent inhibitor of platelet aggregation in vivo than in vitro, and it may have potential as an antiplatelet drug.

The Effect of Barbituric Acid Derivatives on Platelet Function in Vitro and in Vivo

1973 ◽  
Vol 30 (02) ◽  
pp. 315-326
Author(s):  
J. Heinz Joist ◽  
Jean-Pierre Cazenave ◽  
J. Fraser Mustard
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Barbituric Acid ◽  
Platelet Rich Plasma ◽  
Inhibitory Effect ◽  
Primary Response ◽  
Sodium Pentobarbital ◽  
Barbituric Acid Derivatives

SummarySodium pentobarbital (SPB) and three other barbituric acid derivatives were found to inhibit platelet function in vitro. SPB had no effect on the primary response to ADP of platelets in platelet-rich plasma (PRP) or washed platelets but inhibited secondary aggregation induced by ADP in human PRP. The drug inhibited both phases of aggregation induced by epinephrine. SPB suppressed aggregation and the release reaction induced by collagen or low concentrations of thrombin, and platelet adherence to collagen-coated glass tubes. The inhibition by SPB of platelet aggregation was readily reversible and isotopically labeled SPB did not become firmly bound to platelets. No inhibitory effect on platelet aggregation induced by ADP, collagen, or thrombin could be detected in PRP obtained from rabbits after induction of SPB-anesthesia.

Comparison of the effect of coagulation and platelet function impairments on various mouse bleeding models

2014 ◽  
Vol 112 (08) ◽  
pp. 412-418 ◽  
Author(s):  
Nima Vaezzadeh ◽  
Ran Ni ◽  
Paul Y. Kim ◽  
Jeffrey I. Weitz ◽  
Peter L. Gross
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Saphenous Vein ◽  
Ex Vivo ◽  
Arterial Injury ◽  
Inhibitory Antibody ◽  
Study Objective ◽  
Tail Tip

SummaryHaemostatic impairments are studied in vivo using one of several murine bleeding models. However it is not known whether these models are equally appropriate for assessing coagulation or platelet function defects. It was our study objective to assess the performance of arterial, venous and combined arterial and venous murine bleeding models towards impaired coagulation or platelet function. Unfractionated heparin (UFH) or αIIbβ3 inhibitory antibody (Leo.H4) were administered to mice, and their effects on bleeding in saphenous vein, artery, and tail tip transection models were quantified and correlated with their effects on plasma clotting and ADP-induced platelet aggregation, respectively. All models exhibited similar sensitivity with UFH (EC50 dose = 0.19, 0.13 and 0.07 U/g, respectively) (95% CI = 0.14 – 0.27, 0.08 – 0.20, and 0.03 – 0.16 U/g, respectively). Maximal inhibition of ex vivo plasma clotting could be achieved with UFH doses as low as 0.03 U/g. In contrast, the saphenous vein bleeding model was less sensitive to αIIbβ3 inhibition (EC50 = 6.9 µg/ml) than tail transection or saphenous artery bleeding models (EC50 = 0.12 and 0.37 µg/ml, respectively) (95% CI = 2.4 – 20, 0.05 – 0.33, and 0.06 – 2.2 µg/ml, respectively). The EC50 of Leo.H4 for ADP-induced platelet aggregation in vitro (8.0 µg/ml) was at least 20-fold higher than that of the tail and arterial, but not the venous bleeding model. In conclusion, venous, arterial and tail bleeding models are similarly affected by impaired coagulation, while platelet function defects have a greater influence in models incorporating arterial injury.

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

Blood ◽  
1987 ◽  
Vol 69 (3) ◽  
pp. 924-928 ◽  
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.

scholarly journals Are Platelet Aggregation Tests the Best Way to Assess New Drugs for Arterial Disease

2018 ◽  
Vol 1 (1) ◽  
pp. 01-03
Author(s):  
Mark I. M. Noble
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Platelet Rich Plasma ◽  
New Drugs ◽  
In Vitro Test ◽  
In Vivo Efficacy ◽  
Experimental Animals ◽  
Stenosed Artery

Over many years, laboratory testing of platelet aggregability have been carried out in attempts to develop drugs that would prevent thrombosis in arteries. The problems encountered included the question of methodology. Blood samples have to be anticoagulated in order to study the platelets. Anti-coagulation with citrate and tests on derived platelet rich plasma did not correlate at all well with thrombus growth in the stenosed coronary arteries of experimental animals and citrate removes the calcium ions which are vital for platelet function. Anticoagulation with heparin also interfered with platelet function, so that now, hirudins are the preferred anticoagulant. However it was observed that if, instead of stimulating platelet aggregation with adrenaline or ADP, serotonin was applied to the preparation, very little aggregation took place in spite of serotonin 5HT2A antagonists being the most potent inhibitors of thrombus growth in experimental animals. Another indicator that primary platelet agggregation is not a predictor of in vivo efficacy was the finding that 5HT2A antagonism inhibited aggregate growth. In a stenosed artery the platelets are activated by increased shear stress and blood turbulence with release of platelet serotonin causing positive feedback activation of more platelets. At present, there does not seem to be a bench in vitro test that accurately predicts in vivo efficacy in stenosed artery occlusive thrombosis.

A Key Role of Adenosine Diphosphate in the Irreversible Platelet Aggregation Induced by the PAR1-Activating Peptide Through the Late Activation of Phosphoinositide 3-Kinase

Blood ◽  
1999 ◽  
Vol 94 (12) ◽  
pp. 4156-4165 ◽  
Author(s):  
Catherine Trumel ◽  
Bernard Payrastre ◽  
Monique Plantavid ◽  
Béatrice Hechler ◽  
Cécile Viala ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Kinase Inhibitors ◽  
Adenosine Diphosphate ◽  
Blood Platelet ◽  
Activation Process ◽  
Irreversible Aggregation ◽  
Stable Association ◽  
Phosphoinositide 3 Kinase

Abstract Although adenosine diphosphate (ADP), per se, is a weak platelet agonist, its role as a crucial cofactor in human blood platelet functions has now been clearly demonstrated in vitro and in vivo. The molecular basis of the ADP-induced platelet activation is starting to be understood since the discovery that 2 separate P2 purinergic receptors may be involved simultaneously in the activation process. However, little is known about how ADP plays its role as a cofactor in platelet activation and which signaling pathway initiated by a specific agonist can be modulated by the released ADP. To investigate these points, we took advantage of a model of platelet activation through the thrombin receptor PAR1 in which both ADP scavengers and phosphoinositide 3-kinase (PI 3-kinase) inhibitors have been shown to transform the classical irreversible aggregation into a reversible one. We have observed that, among the different PI 3-kinase products, the accumulation of phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2] was dramatically and specifically attenuated when ADP was removed by apyrase treatment. A comparison between the effects of PI 3-kinase inhibitors and apyrase strongly suggest that the late, ADP-dependent, PtdIns(3,4)P2accumulation is necessary for PAR1-induced irreversible aggregation. Using selective antagonists, we found that the effect of ADP was due to the ADP receptor coupled to inhibition of adenylyl cyclase. Finally, we found that both ADP and PI 3-kinase play an important role in PAR1-dependent reorganization of the cytoskeleton through a control of myosin heavy chain translocation and the stable association of signaling complexes with the actin cytoskeleton.

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

Blood ◽  
1987 ◽  
Vol 69 (3) ◽  
pp. 924-928 ◽  
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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