Functional Characterization of PM6/13, a β3-specific (GPIIIa/CD61) Monoclonal Antibody that Shows Preferential Inhibition of Fibrinogen Binding over Fibronectin Binding to Activated Human Platelets

SummaryWe report the characterization of a monoclonal antibody (MAb) PM6/13 which recognises glycoprotein IIIa (GPIIIa) on platelet membranes and in functional studies inhibits platelet aggregation induced by all agonists examined. In platelet-rich plasma, inhibition of aggregation induced by ADP or low concentrations of collagen was accompanied by inhibition of 5-hydroxytryptamine secretion. EC50 values were 10 and 9 [H9262]g/ml antibody against ADP and collagen induced responses respectively. In washed platelets treated with the cyclooxygenase inhibitor, indomethacin, PM6/13 inhibited platelet aggregation induced by thrombin (0.2 U/ml), collagen (10 [H9262]g/ml) and U46619 (3 [H9262]M) with EC50 = 4, 8 and 4 [H9262]g/ml respectively, without affecting [14C]5-hydroxytryptamine secretion or [3H]arachidonate release in appropriately labelled cells. Studies in Fura 2-labelled platelets revealed that elevation of intracellular calcium by ADP, thrombin or U46619 was unaffected by PM6/13 suggesting that the epitope recognised by the antibody did not influence Ca2+ regulation. In agreement with the results from the platelet aggregation studies, PM6/13 was found to potently inhibit binding of 125I-fibrinogen to ADP activated platelets. Binding of this ligand was also inhibited by two other MAbs tested, namely SZ-21 (also to GPIIIa) and PM6/248 (to the GPIIb-IIIa complex). However when tested against binding of 125I-fibronectin to thrombin stimulated platelets, PM6/13 was ineffective in contrast with SZ-21 and PM6/248, that were both potent inhibitors. This suggested that the epitopes recognised by PM6/13 and SZ-21 on GPIIIa were distinct. Studies employing proteolytic dissection of 125I-labelled GPIIIa by trypsin followed by immunoprecipitation with PM6/13 and analysis by SDS-PAGE, revealed the presence of four fragments at 70, 55, 30 and 28 kDa. PM6/13 did not recognize any protein bands on Western blots performed under reducing conditions. However Western blotting analysis with PM6/13 under non-reducing conditions revealed strong detection of the parent GP IIIa molecule, of trypsin treated samples revealed recognition of an 80 kDa fragment at 1 min, faint recognition of a 60 kDa fragment at 60 min and no recognition of any product at 18 h treatment. Under similar conditions, SZ-21 recognized fragments at 80, 75 and 55 kDa with the 55kDa species persisting even after 18 h trypsin treatment. These studies confirm the epitopes recognised by PM6/13 and SZ-21 to be distinct and that PM6/13 represents a useful tool to differentiate the characteristics of fibrinogen and fibronectin binding to the GPIIb-IIIa complex on activated platelets.

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A New Platelet-Aggregation-Inhibiting Factor Isolated fromBothrops moojeniSnake Venom

BioMed Research International ◽

10.1155/2017/4315832 ◽

2017 ◽

Vol 2017 ◽

pp. 1-9 ◽

Cited By ~ 2

Author(s):

Bruna Barbosa de Sousa ◽

Carla Cristine Neves Mamede ◽

Mariana Santos Matias ◽

Déborah Fernanda da Cunha Pereira ◽

Mayara Ribeiro de Queiroz ◽

...

Keyword(s):

Platelet Aggregation ◽

Snake Venom ◽

Platelet Rich Plasma ◽

Functional Characterization ◽

Adenosine Diphosphate ◽

Inhibitory Effect ◽

Single Chain ◽

Inhibiting Factor ◽

Reducing Conditions ◽

Bothrops Moojeni

This work reports the purification and functional characterization of BmooPAi, a platelet-aggregation-inhibiting factor fromBothrops moojenisnake venom. The toxin was purified by a combination of three chromatographic steps (ion-exchange on DEAE-Sephacel, molecular exclusion on Sephadex G-75, and affinity chromatography on HiTrap™ Heparin HP). BmooPAi was found to be a single-chain protein with an apparent molecular mass of 32 kDa on 14% SDS-PAGE, under reducing conditions. Sequencing of BmooPAi by Edman degradation revealed the amino acid sequence LGPDIVPPNELLEVM. The toxin was devoid of proteolytic, haemorrhagic, defibrinating, or coagulant activities and induced no significant oedema or hyperalgesia. BmooPAi showed a rather specific inhibitory effect on ristocetin-induced platelet aggregation in human platelet-rich plasma, whereas it had little or no effect on platelet aggregation induced by collagen and adenosine diphosphate. The results presented in this work suggest that BmooPAi is a toxin comprised of disintegrin-like and cysteine-rich domains, originating from autolysis/proteolysis of PIII SVMPs fromB. moojenisnake venom. This toxin may be of medical interest because it is a platelet aggregation inhibitor, which could potentially be developed as a novel therapeutic agent to prevent and/or treat patients with thrombotic disorders.

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

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615656 ◽

2001 ◽

Vol 85 (04) ◽

pp. 702-709 ◽

Cited By ~ 3

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.

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The Effect of Red Blood Cells on the ADP-induced Aggregation of Human Platelets in Flow Through Tubes

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645696 ◽

1990 ◽

Vol 63 (01) ◽

pp. 112-121 ◽

Cited By ~ 18

Author(s):

David N Bell ◽

Samira Spain ◽

Harry L Goldsmith

Keyword(s):

Platelet Aggregation ◽

Shear Rate ◽

Red Blood Cells ◽

Blood Cells ◽

Platelet Rich Plasma ◽

Mean Transit Time ◽

White Blood Cells ◽

Aggregate Size ◽

Human Platelets ◽

Induced Aggregation

SummaryThe effect of red blood cells, rbc, and shear rate on the ADPinduced aggregation of platelets in whole blood, WB, flowing through polyethylene tubing was studied using a previously described technique (1). Effluent WB was collected into 0.5% glutaraldehyde and the red blood cells removed by centrifugation through Percoll. At 23°C the rate of single platelet aggregtion was upt to 9× greater in WB than previously found in platelet-rich plasma (2) at mean tube shear rates Ḡ = 41.9,335, and 1,920 s−1, and at both 0.2 and 1.0 µM ADP. At 0.2 pM ADP, the rate of aggregation was greatest at Ḡ = 41.9 s−1 over the first 1.7 s mean transit time through the flow tube, t, but decreased steadily with time. At Ḡ ≥335 s−1 the rate of aggregation increased between t = 1.7 and 8.6 s; however, aggregate size decreased with increasing shear rate. At 1.0 µM ADP, the initial rate of single platelet aggregation was still highest at Ḡ = 41.9 s1 where large aggregates up to several millimeters in diameter containing rbc formed by t = 43 s. At this ADP concentration, aggregate size was still limited at Ḡ ≥335 s−1 but the rate of single platelet aggregation was markedly greater than at 0.2 pM ADP. By t = 43 s, no single platelets remained and rbc were not incorporated into aggregates. Although aggregate size increased slowly, large aggregates eventually formed. White blood cells were not significantly incorporated into aggregates at any shear rate or ADP concentration. Since the present technique did not induce platelet thromboxane A2 formation or cause cell lysis, these experiments provide evidence for a purely mechanical effect of rbc in augmenting platelet aggregation in WB.

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

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648166 ◽

1991 ◽

Vol 65 (04) ◽

pp. 432-437 ◽

Cited By ~ 9

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

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Specific Cross-reaction of IgG Anti-phospholipid Antibody with Platelet Glycoprotein IIIa

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650238 ◽

1996 ◽

Vol 75 (01) ◽

pp. 168-174 ◽

Cited By ~ 13

Author(s):

Shigeru Tokita ◽

Morio Arai ◽

Naomasa Yamamoto ◽

Yasuhiro Katagiri ◽

Kenjiro Tanoue ◽

...

Keyword(s):

Platelet Aggregation ◽

Heparan Sulfate ◽

Disulfide Bonds ◽

Dextran Sulfate ◽

Human Platelets ◽

Platelet Glycoprotein ◽

Activated Platelets ◽

Fibrinogen Binding ◽

Dose Dependent Fashion ◽

Glycoprotein Iiia

SummaryTo study the pathological functions of anti-phospholipid (anti-PL) antibodies, we have analyzed their effect on platelet function. We identified an IgG anti-PL mAb, designated PSG3, which cross-reacted specifically with glycoprotein (GP) IIIa in human platelets and inhibited platelet aggregation. PSG3 bound also to certain polyanionic substances, such as double-stranded DNA, heparan sulfate, dextran sulfate and acetylated-LDL, but not to other polyanionic substances. The binding of PSG3 to GPIIIa was completely inhibited by heparan sulfate and dextran sulfate, indicating that PSG3 recognizes a particular array of negative charges expressed on both GPIIIa and the specified polyanionic substances. Since neither neuraminidase- nor endoglycopeptidase F-treatment of GPIIIa had any significant effect on the binding of PSG3, this array must be located within the amino acid sequence of GPIIIa but not in the carbohydrate moiety. Reduction of the disulfide bonds in GPIIIa greatly reduced its reactivity, suggesting that the negative charges in the epitope are arranged in a particular conformation. PSG3 inhibited platelet aggregation induced by either ADP or collagen, it also inhibited fibrinogen binding to activated platelets in a dose-dependent fashion. PSG3, however, did not inhibit the binding of GRGDSP peptide to activated platelets. These results suggest that the PSG3 epitope on GPIIIa contains a particular array of negative charges, and possibly affects the fibrinogen binding to GPIIb/IIIa complex necessary for platelet aggregation.

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Reaction of Acetaldehyde with Human Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648393 ◽

1992 ◽

Vol 67 (01) ◽

pp. 126-130 ◽

Cited By ~ 8

Author(s):

Olivier Spertini ◽

Jacques Hauert ◽

Fedor Bachmann

Keyword(s):

Platelet Aggregation ◽

Inhibitory Action ◽

Ex Vivo ◽

Platelet Rich Plasma ◽

Shape Change ◽

Reaction Medium ◽

Human Platelets ◽

Membrane Glycoproteins ◽

Neutral Ph

SummaryPlatelet function defects observed in chronic alcoholics are not wholly explained by the inhibitory action of ethanol on platelet aggregation; they are not completely reproduced either in vivo by short-term ethanol perfusion into volunteers or in vitro by the addition of ethanol to platelet-rich plasma. As acetaldehyde (AcH) binds to many proteins and impairs cellular activities, we investigated the effect of this early degradation product of ethanol on platelets. AcH formed adducts with human platelets at neutral pH at 37° C which were stable to extensive washing, trichloracetic acid hydrolysis and heating at 100° C, and were not reduced by sodium borohydride. The amount of platelet adducts formed was a function of the incubation time and of the concentration of AcH in the reaction medium. At low AcH concentrations (<0.2 mM), platelet bound AcH was directly proportional to the concentration of AcH in the reaction medium. At higher concentrations (≥0.2 mM), AcH uptake by platelets tended to reach a plateau. The amount of adducts was also proportional to the number of exposures of platelets to pulses of 20 pM AcH.AcH adducts formation severely impaired platelet aggregation and shape change induced by ADP, collagen and thrombin. A positive correlation was established between platelet-bound AcH and inhibition of aggregation.SDS-PAGE analysis of AcH adducts at neutral pH demonstrated the binding of [14C]acetaldehyde to many platelet proteins. AcH adduct formation with membrane glycoproteins, cytoskeleton and enzymes might interfere with several steps of platelet activation and impair platelet aggregation.This in vitro study shows that AcH has a major inhibitory action on platelet aggregation and may account for the prolonged ex vivo inhibition of aggregation observed in chronic alcoholics even in the absence of alcoholemia.

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Effects of Halothane on Platelet Function

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650105 ◽

1980 ◽

Vol 44 (03) ◽

pp. 143-145 ◽

Cited By ~ 19

Author(s):

J Dalsgaard-Nielsen ◽

J Gormsen

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Platelet Rich Plasma ◽

Human Platelets ◽

Halothane Anaesthesia ◽

Inhibition Of Platelet Aggregation ◽

Transient Impairment ◽

High Concentrations ◽

Uptake And Release ◽

Platelet Functions

SummaryHuman platelets in platelet rich plasma (PRP) incubated at 37° C with 0.3–2% halothane for 5–10 min lost the ability to aggregate with ADP, epinephrine and collagen.At the same time uptake and release of 14C-serotonin was inhibited. When halothane supply was removed, platelet functions rapidly returned to normal. However, after high concentrations of halothane, the inhibition of platelet aggregation was irreversible or only partially reversible.The results suggest that halothane anaesthesia produces a transient impairment of platelet function.

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Interactions of Staphylokinase with Human Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653799 ◽

1995 ◽

Vol 73 (03) ◽

pp. 472-477 ◽

Cited By ~ 5

Author(s):

H R Lijnen ◽

B Van Hoef ◽

D Collen

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Specific Binding ◽

Platelet Rich Plasma ◽

Human Platelets ◽

Normal Plasma ◽

Atp Secretion ◽

Platelet Disaggregation ◽

Activation Rate

SummaryThe interactions of recombinant staphylokinase (SakSTAR) with human platelets were investigated in a buffer milieu, in a human plasma milieu in vitro, and in plasma from patients with acute myocardial infarction (AMI) treated with SakSTAR.In a buffer milieu, the activation rate of plasminogen by SakSTAR or streptokinase (SK) was not significantly altered by addition of platelets. Specific binding of SakSTAR or SK to either resting or thrombin- activated platelets was very low. ADP-induced or collagen-induced platelet aggregation in platelet-rich plasma (PRP) was 94 ± 2.7% or 101 ± 1.7% of control in the presence of 0.1 to 20 μM SakSTAR, with corresponding values of 95 ± 2.8% or 90 ± 4.6% of control in the presence of 0.1 to 4 μM SK. No effects were observed on platelet disaggregation. ATP secretion following collagen-induced platelet aggregation was 4.3 ± 0.26 μM for SakSTAR (at concentrations of 0.1 to 20 μM) and 4.4 ± 0.35 μM for SK (at concentrations of 0.1 to 4 μM), as compared to 3.4 ± 0.70 μM in the absence of plasminogen activator.Fifty % lysis in 2 h (C50) of 60 μl 125I-fibrin labeled platelet-poor plasma (PPP) clots prepared from normal plasma or from plasma of patients with Glanzmann thrombasthenia and immersed in 0.5 ml normal plasma, was obtained with 12 or 16 nM SakSTAR and with 49 or 40 nM SK, respectively. C50 values for lysis of 60 μl PRP clots prepared from normal or patient plasma were also comparable for SakSTAR (19 or 21 nM), whereas SK was 2-fold more potent toward PRP clots prepared from Glanzmann plasma as compared to normal plasma (C50 of 130 versus 270 nM).No significant effect of SakSTAR on platelet function was observed in plasma from patients with AMI treated with SakSTAR, as revealed by unaltered platelet count, platelet aggregation and ATP secretion.Thus, no effects of high SakSTAR concentrations were observed on human platelets in vitro, nor of therapeutic SakSTAR concentrations on platelet function in plasma.

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The Effect of Collagen Mediated Platelet Release on Plasma Prekallikrein Activation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661014 ◽

1984 ◽

Vol 51 (01) ◽

pp. 037-041 ◽

Cited By ~ 2

Author(s):

K M Weerasinghe ◽

M F Scully ◽

V V Kakkar

Keyword(s):

Platelet Aggregation ◽

Healthy Volunteers ◽

Platelet Rich Plasma ◽

Age Groups ◽

Inhibitory Effect ◽

Age Group ◽

Platelet Factor ◽

Activated Platelets ◽

Platelet Release ◽

Collagen Treatment

SummaryCollagen mediated platelet aggregation caused -5.6 ± 6.7% inhibition and +39.1 ± 15.2% potentiation of prekallikrein activation in plasma from normal healthy volunteers between 20–40 and 50–65 years of age, respectively (n = 15, p <0.01). The amouns of platelet factor-four (PF4) released in the two groups were not significantly different. Collagen treatment in the presence of indomethacin caused +11.5 ± 3.6% and +59.6 ± 19.5% potentiation in the 20–40 and 50–65 age groups respectively (p <0.02). Adrenaline mediated platelet aggregation caused -55.2 ± 7.1% and -35.2 ± 8.3% inhibition in the 20–40 and 50–65 age groups, respectively. Collagen treatment of platelet-deficient-plasma and platelet-rich-plasma in EDTA also caused potentiation of prekallikrein activation.The results indicate that the observed degree of prekallikrein activation after platelet aggregation is a net result of the inhibitory effect of PF4 and the potentiatory effect of activated platelets. The potentiatory effect was greater after collagen treatment as compared to adrenaline treatment, and in the 50–65 age group as compared to the 20–40 age group.

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Activation of human platelets by immune complexes prepared with cationized human IgG

Blood ◽

10.1182/blood.v82.10.3045.bloodjournal82103045 ◽

1993 ◽

Vol 82 (10) ◽

pp. 3045-3051

Author(s):

M Schattner ◽

M Lazzari ◽

AS Trevani ◽

E Malchiodi ◽

AC Kempfer ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Immune Complexes ◽

Platelet Rich Plasma ◽

Human Platelets ◽

Human Igg ◽

Experimental Conditions ◽

Induce Platelet Aggregation ◽

Soluble Immune Complexes ◽

High Concentrations

The present study shows that the ability of soluble immune complexes (IC), prepared with human IgG and rabbit IgG antibodies against human IgG, to trigger platelet activation was markedly higher for IC prepared with cationized human IgG (catIC) compared with those prepared with untreated human IgG (cIC). CatIC induced platelet aggregation and adenosine triphosphate release in washed platelets (WP), gel-filtered platelets (GFP), or platelet-rich plasma (PRP) at physiologic concentrations of platelets (3 x 10(8)/mL) and at low concentrations of catIC (1 to 30 micrograms/mL). On the contrary, under similar experimental conditions, cIC did not induce aggregation in PRP, WP, or GFP. Low aggregation responses were only observed using high concentrations of both WP (9 x 10(8)/mL) and cIC (500 micrograms/mL). Interestingly, catIC were also able to induce platelet activation under nonaggregating conditions, as evidenced by P-selectin expression. Cationized human IgG alone did not induce platelet aggregation in PRP but triggered either WP or GFP aggregation. However, the concentration needed to induce these responses, was about eightfold higher than those required for catIC. The responses induced either by catIC or cationized human IgG were completely inhibited by treatment with heparin, dextran sulphate, EDTA, prostaglandin E1, or IV3, a monoclonal antibody against the receptor II for the Fc portion of IgG (Fc gamma RII). The data presented in this study suggest that IgG charge constitutes a critical property that conditions the ability of IC to trigger platelet activation.

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