Antithrombotic Activity of SR 46349, a Novel, Potent and Selective 5-HT2 Receptor Antagonist

1993 ◽  
Vol 69 (03) ◽  
pp. 262-267 ◽  
Author(s):  
J M Herbert ◽  
A Bernat ◽  
G Barthelemy ◽  
F Dol ◽  
M Rinaldi
Keyword(s):  
Oral Administration ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Dependent Manner ◽  
Antithrombotic Agent ◽  
Venous Shunt ◽  
Induced Aggregation ◽  
Dose Dependent

SummarySR 46349 (trans-4-[(3Z)3-(2-dimethylaminoethyl)oxyimino-3(2-fluorophenyl)propen-1-yl] phenol, hemifumarate) is the first member of a newly-developed 5-HT2 antagonist series. SR 46349 potently inhibited serotonin-induced aggregation of rabbit and human platelets (IC50 = 1 and 3.9 nM respectively) but had no effect on the action of other platelet aggregating agents. SR 46349 was 118 and 25 times more potent than ketanserin against 5-HT + epinephrine-induced aggregation of rabbit and human platelets respectively.A single per os administration of SR 46349 (1 mg/kg) resulted in a strong inhibition of 5-HT + epinephrine-induced platelet aggregation in the rabbit as measured ex vivo (67% inhibition, 6 h after the administration). Intravenous or oral administration of SR 46346 inhibited in a dose-dependent manner venous thrombosis induced by ligature of the jugular vein of rabbits whose blood was made hypercoagulable by i.v. administration of tissue thromboplastin. The doses of SR 46349 which inhibited 50% of thrombus formation were 1.5 ± 0.8 mg/kg and 17 ± 0.5 mg/kg after i.v. or oral administration respectively. When given i.v. to rabbits, SR 46349 exhibited a dose-dependent antithrombotic effect in an arterio-venous shunt model. Significant increase of the bleeding time was observed after the i.v. administration of 5 mg/kg of SR 46349 (3-fold increase). In dogs, SR 46349 inhibited cyclic coronary artery blood flow variations, complete abolition of CFVs being achieved after the i.v. administration of 0.5 mg/kg.In conclusion, SR 46349 is a highly potent, selective antagonist of serotonin in vitro and is to be considered as a potent, orally active antithrombotic agent.

Thrombosomes Show Dose-Dependent Increase in Thrombus Formation in a Model of Deep Arterial Injury

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2319-2319
Author(s):  
Nikhil Vilas Joshi ◽  
Jennifer Raftis ◽  
AJ Lucking ◽  
Narendra Tandon ◽  
M Fitzpatrick ◽  
...  
Keyword(s):  
Whole Blood ◽  
Immunohistochemical Staining ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Arterial Injury ◽  
Dependent Manner ◽  
Flow Conditions ◽  
Structural And Functional Properties ◽  
Dose Dependent

Abstract Abstract 2319 Introduction Thrombosomes are novel lyophilized human platelet derivatives that retain a number of key platelet structural and functional properties. Building on preliminary in vitro studies, we here sought to investigate whether Thrombosomes, suspended in platelet free plasma (PFP), would enhance and be incorporated in thrombus generated under flow conditions within a validated and well-characterised model of deep arterial injury. Methods PFP was obtained by centrifuging citrated whole blood from six healthy non-smoking volunteers and filtering with a 0.22μm filter. Thrombosomes were suspended in 60 mL of PFP to generate final concentrations of 20 and 200 × 106Thrombosomes /mL. Immediately prior to use, 1.2 mL of 1M CaCl2 was added to permit fibrin generation. Thrombus formation was assessed using the Badimon Chamber at low (212 s−1) and high (1690 s−1) shear rates with porcine aortic tunica media as the thrombogenic substrate. Total thrombus area and platelet-rich area were measured histomorphometrically using conventional and immunohistochemical staining respectively. Fluorescent labeled Thrombosomes were added to the extracorporeal circuit in the Badimon chamber to study the ex vivo thrombus generation in the whole blood. Electron microscopy of Thrombosomes and platelets was undertaken. Results Thrombosomes contributed towards thrombus formation in whole human blood as evidenced by incorporation of fluorescent-labeled Thrombosomes into the thrombus. Immunohistochemical staining of the glycoprotein IIb/IIIa receptor confirmed incorporation of Thrombosomes into the thrombus. In the high shear chamber, mean thrombus area increased in a dose-dependent manner following the addition of Thrombosomes (704 μm2 [95% CI, 224 – 1184 μm2], 1511 μm2 [95% CI, 687 – 2336 μm2] and 2378 μm2 [95% CI, 1567 – 3189 μm2] for PFP and Thrombosomes at concentrations of 20 and 200 × 106/mL respectively [P= 0.003]). In the low shear chamber, total thrombus area for the PFP was 4962 μm2 (95% CI, 2489 – 7434 μm2). The addition of Thrombosomes at concentrations of 20 and 200 × 106/mL led to a numerical increase in mean thrombus area to 6170 μm2 (95% CI, 3944 – 8397 μm2) and 7504 μm2 (95% CI, 3864 – 11144 μm2) respectively, although this was not statistically significant (P= 0.2969). Conclusions Thrombosomes suspended in PFP and exposed to injured arterial tissue under physiologically relevant flow conditions are incorporated into thrombus and enhance thrombus formation in a dose dependent manner. These findings act as impetus to undertake clinical studies of this rehydrated, lyophilized infusible hemostatic platelet product. Disclosures: Fitzpatrick: Cellphire Inc.: Employment, Equity Ownership. Feuerstein:Cellphire: Consultancy. Newby:Cellphire: Research Funding.

scholarly journals Pharmacologic targeting of Cdc42 GTPase by a small molecule Cdc42 activity-specific inhibitor prevents platelet activation and thrombosis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Xin Duan ◽  
Rehana Perveen ◽  
Akhila Dandamudi ◽  
Reheman Adili ◽  
James Johnson ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Small Molecule ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Intraperitoneal Administration ◽  
Specific Inhibitor ◽  
Human Platelets ◽  
Induced Aggregation ◽  
Inactive Analog

AbstractGene targeting of Cdc42 GTPase has been shown to inhibit platelet activation. In this study, we investigated a hypothesis that inhibition of Cdc42 activity by CASIN, a small molecule Cdc42 Activity-Specific INhibitor, may down regulate platelet activation and thrombus formation. We investigated the effects of CASIN on platelet activation in vitro and thrombosis in vivo. In human platelets, CASIN, but not its inactive analog Pirl7, blocked collagen induced activation of Cdc42 and inhibited phosphorylation of its downstream effector, PAK1/2. Moreover, addition of CASIN to washed human platelets inhibited platelet spreading on immobilized fibrinogen. Treatment of human platelets with CASIN inhibited collagen or thrombin induced: (a) ATP secretion and platelet aggregation; and (b) phosphorylation of Akt, ERK and p38-MAPK. Pre-incubation of platelets with Pirl7, an inactive analog of CASIN, failed to inhibit collagen induced aggregation. Washing of human platelets after incubation with CASIN eliminated its inhibitory effect on collagen induced aggregation. Intraperitoneal administration of CASIN to wild type mice inhibited ex vivo aggregation induced by collagen but did not affect the murine tail bleeding times. CASIN administration, prior to laser-induced injury in murine cremaster muscle arterioles, resulted in formation of smaller and unstable thrombi compared to control mice without CASIN treatment. These data suggest that pharmacologic targeting of Cdc42 by specific and reversible inhibitors may lead to the discovery of novel antithrombotic agents.

scholarly journals Binding of Dipyridamole of Human Platelets and to α-Acid Glycoprotein and its Significance for the Inhibition of Adenosine Uptake

1977 ◽  
Author(s):  
K. Subbarao ◽  
B. Rucinski ◽  
A. Summers ◽  
S. Niewiarowski
Keyword(s):  
Binding Sites ◽  
Ex Vivo ◽  
Human Platelets ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Acid Glycoprotein ◽  
High Affinity ◽  
Adenosine Uptake ◽  
Adenosine Transport

The interactions of dipyridamole with α1-acid glycoprotein of plasma and with human platelets are related to inhibition of adenosine uptake by platelets. One mole of dipyridamole binds to one mole of α1-acid glycoprotein with a dissociation constant (Kd) of 1.3 μM. It was found that platelets contain both high and low affinity binding sites for the drug. The binding of dipyridamole to the high affinity sites follows a Michaelis Menten binding pattern with a Kd of 0.04 μM. Approximately 2x104 dipyridamole molecules are bound at the high affinity sites of each platelet. The lower affinity sites bind the drug with a Kd of 4 μM. In the presence of α1acid glycoprotein the binding of dipyridamole to platelets is inhibited. Correspondingly, the dipyridamole inhibition of adenosine uptake by platelets is reduced 1000-fold by α1acid glycoprotein. Binding of dipyridamole to human platelets is essential for its inhibition of adenosine uptake by platelets. Dipyridamole reduced the [14C]-ATP to [14C]-ADP ratio in the platelets. Purified α1acid glycoprotein reversed these effects of dipyridamole on adenosine metabolism of platelets in a concentration dependent manner. A correlationwas observed between the level of circulating dipyridamole in plasma and the inhibition of [14C]-adenosine uptake by platelets of PRP samples of 12 human volunteers given different amounts of dipyridamole. The in vitro and ex vivo effects of dipyridamole on the [14C]-adenosine uptake by platelets were found to be identical. Our data suggest the presence of dipyridamole binding sites in platelets that regulate adenosine transport across the cell surface.

Anticoagulant and antithrombotic properties of platelet protease nexin-1

Blood ◽  
2010 ◽  
Vol 115 (1) ◽  
pp. 97-106 ◽  
Author(s):  
Yacine Boulaftali ◽  
Frédéric Adam ◽  
Laurence Venisse ◽  
Véronique Ollivier ◽  
Benjamin Richard ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Platelet Surface ◽  
Type Plasminogen Activator ◽  
Healthy Donors ◽  
Protease Nexin ◽  
Deficient Mice

AbstractProtease nexin–1 (PN-1) is a serpin that inhibits plasminogen activators, plasmin, and thrombin. PN-1 is barely detectable in plasma but is expressed by platelets. Here, we studied platelet PN-1 in resting and activated conditions and its function in thrombosis. Studies on human platelets from healthy donors and from patients with a Gray platelet syndrome demonstrate that PN-1 is present both at the platelet surface and in α-granules. The role of PN-1 was investigated in vitro using human platelets incubated with a blocking antibody and using platelets from PN-1–deficient mice. Both approaches indicate that platelet PN-1 is active on thrombin and urokinase-type plasminogen activator. Blockade and deficiency of platelet PN-1 result in accelerated and increased tissue factor-induced thrombin generation as indicated by calibrated automated thrombography. Moreover, platelets from PN-1–deficient mice respond to subthreshold doses of thrombin, as assessed by P-selectin expression and platelet aggregation. Thrombus formation, induced ex vivo by collagen in blood flow conditions and in vivo by FeCl3-induced injury, is significantly increased in PN-1–deficient mice, demonstrating the antithrombotic properties of platelet PN-1. Platelet PN-1 is thus a key player in the thrombotic process, whose negative regulatory role has been, up to now, markedly underestimated.

The human renin infused rat: use as an in vivo model for the biological evaluation of human renin inhibitors

1999 ◽  
Vol 77 (11) ◽  
pp. 886-895 ◽  
Author(s):  
Gordon Bolger ◽  
Jean-Claude Vigeant ◽  
Francine Liard ◽  
Bruno Simoneau ◽  
Diane Thibeault ◽  
...  
Keyword(s):  
Oral Administration ◽  
Pressor Response ◽  
Biological Evaluation ◽  
In Vivo Model ◽  
Dependent Manner ◽  
Renin Inhibitors ◽  
Human Renin ◽  
Dose Dependent

The human renin infused rat model (HRIRM) was used as an in vivo small-animal model for evaluating the efficacy of a collection of inhibitors of human renin. The intravenous infusion of recombinant human renin (2.4 µg·kg-1·min-1) in the ganglion-blocked, nephrectomized rat produced a mean blood pressor response of 47 ± 3 mmHg (1 mmHg = 133.3 Pa), which was reduced by captopril, enalkiren, and losartan in a dose-dependent manner following oral administration, with ED50 values of 0.3 ± 0.1, 2.5 ± 0.9, and 5.2 ± 1.6 mg/kg, respectively. A series of peptidomimetic P2-P3 butanediamide renin inhibitors inhibited purified recombinant human renin in vitro in a concentration-dependent manner, with IC50 values ranging from 0.4 to 20 nM at pH 6.0, with a higher range of IC50 values (0.8-80 nM) observed at pH 7.4. Following i.v. administration of renin inhibitors, the pressor response to infused human renin in the HRIRM was inhibited in a dose-dependent manner, with ED50 values ranging from 4 to 600 µg/kg. The in vivo inhibition of human renin following i.v. administration in the rat correlated significantly better with the in vitro inhibition of human renin at pH 7.4 (r = 0.8) compared with pH 6.0 (r = 0.5). Oral administration of renin inhibitors also resulted in a dose-dependent inhibition of the pressor response to infused human renin, with ED50 values ranging from 0.4 to 6.0 mg/kg and the identification of six renin inhibitors with an oral potency of <1 mg/kg. The ED50 of renin inhibitors for inhibition of angiotensin I formation in vivo was highly correlated (r = 0.9) with the ED50 for inhibition of the pressor response. These results demonstrate the high potency, dose dependence, and availability following oral administration of the butanediamide series of renin inhibitors.Key words: renin-angiotensin system, recombinant human renin, rat, renin inhibitors.

The In Vitro Effect of Ticlopidine on Fibrinogen and Factor VIII Binding to Human Platelets

1981 ◽  
Vol 46 (03) ◽  
pp. 590-592 ◽  
Author(s):  
Helen Lee ◽  
R C Paton ◽  
C Ruan ◽  
J P Caen
Keyword(s):  
Factor Viii ◽  
Antiplatelet Agent ◽  
Human Platelets ◽  
Dependent Manner ◽  
Fibrinogen Binding ◽  
The Mean ◽  
In Vitro Effects ◽  
Vitro Effect ◽  
Induced Aggregation

SummaryThe mode of action of the antiplatelet agent ticlopidine is not yet fully understood. Its multiple effects on platelet function include prolongation of the bleeding time, reduction in primary and secondary Waves of ADP-induced aggregation and inhibition of collagen and thrombin-induced aggregation. We have studied the in vitro effects of ticlopidine on fibrinogen binding induced by ADP and adrenaline as well as factor VIII/vWF binding induced by ristocetin. 125I-fibrinogen binding was measured in suspensions of freshly washed normal platelets stimulated by 10 μM ADP or 10 μM adrenaline. The binding of 125I-factor VIII/vWF in the presence of 1 mg/ml ristocetin was measured in both washed and paraformaldehyde-fixed platelets. Ticlopidine at final concentrations of 200, 100, 50 and 25 μM inhibited both ADP and adrenaline-induced fibrinogen binding in a dose-dependent manner. The mean % inhibition of ADP-induced fibrinogen binding was 82, 73, 42 and 32 respectively. The mean % inhibition of adrenaline induced fibrinogen binding was 86, 82, 60 and 35 respectively. In contrast, the factor VIII/vWF binding was unaffected by ticlopidine at all concentrations except at 200 μM using fresh platelets where a slight inhibition (19%) was observed.These results suggest that ticlopidine either inhibits platelet activation and consequently fibrinogen binding, or inhibits the binding directly, presumably by having an effect on the specific configuration of the platelet membrane required for normal fibrinogen binding.

Inhibitory Effect of Piracetam on Platelet-rich Thrombus Formation in an Animal Model

1998 ◽  
Vol 79 (01) ◽  
pp. 222-227 ◽  
Author(s):  
F. Stockmans ◽  
W. Deberdt ◽  
Å. Nyström ◽  
E. Nyström ◽  
J. M. Stassen ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Ex Vivo ◽  
Peak Plasma ◽  
Thrombus Formation ◽  
Plasma Concentrations ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Cell Deformability

SummaryIntravenous administration of piracetam to hamsters reduced the formation of a platelet-rich venous thrombus induced by a standardised crush injury, in a dose-dependent fashion with an IC50 of 68 ± 8 mg/kg. 200 mg/kg piracetam also significantly reduced in vivo thrombus formation in rats. However, in vitro aggregation of rat platelets was only inhibited with piracetam-concentrations at least 10-fold higher than plasma concentrations (6.2 ± 1.1 mM) obtained in the treated animals. No effects were seen on clotting tests.In vitro human platelet aggregation, induced by a variety of agonists, was inhibited by piracetam, with IC50’s of 25-60 mM. The broad inhibition spectrum could be explained by the capacity of piracetam to prevent fibrinogen binding to activated human platelets. Ex vivo aggregations and bleeding times were only minimally affected after administration of 400 mg/kg piracetam i.v. to healthy male volunteers, resulting in peak plasma levels of 5.8 ± 0.3 mM.A possible antiplatelet effect of piracetam could be due to the documented beneficial effect on red blood cell deformability leading to a putative reduction of ADP release by damaged erythrocytes. However similarly high concentrations were needed to prevent stirring-induced “spontaneous” platelet aggregation in human whole blood.It is concluded that the observed antithrombotic action of piracetam cannot satisfactorily be explained by an isolated direct effect on platelets. An additional influence of piracetam on the rheology of the circulating blood and/or on the vessel wall itself must therefore be taken into consideration.

Abstract 33: Platelet 12-lipoxygenase is a Key Regulator of Platelet Reactivity and Thrombus Formation in vivo

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Reheman Adili ◽  
Katherine Mast ◽  
Theodore R Holman ◽  
Michael Holinstat
Keyword(s):  
Platelet Aggregation ◽  
Ex Vivo ◽  
Platelet Reactivity ◽  
Thrombus Formation ◽  
High Shear ◽  
Vessel Occlusion ◽  
12 Lipoxygenase ◽  
Induced Aggregation

Background: Platelet reactivity is required to maintain hemostasis, however high platelet reactivity leads to thrombus formation, myocardial infarction, and stroke. Platelet 12-lipoxygenase (12-LOX) has been demonstrated by our lab and others to regulate agonist-mediated platelet reactivity suggesting a role for 12-LOX in regulation of in vivo thrombosis. The ability to target 12-LOX in vivo has not been established to date. Therefore, we sought to determine if 12-LOX regulates platelet reactivity and thrombus formation in vivo using the selective 12-LOX inhibitor ML355 to determine whether platelet 12-LOX is an effective target for anti-platelet therapeutics. Methods: ML355 effects on human platelet function was assessed in vitro by platelet aggregometry, ex vivo by perfusion chamber, and in vivo by thrombus formation and vessel occlusion in small and large vessels in 12-LOX -/- , WT mice, and mice treated with ML355 via intravital microscopy using the FeCl 3 and laser injury models. Results: In in vitro platelet aggregation, ML355 dose-dependently inhibited agonist-induced aggregation. In ex vivo flow chamber assays, platelet adhesion and thrombus formation on collagen-coated surfaces at high shear was attenuated in both mouse and human whole blood after incubation with ML355. Further, platelet aggregation and thrombus growth in 12-LOX -/- mice were impaired in both laser and FeCl 3 -induced mesenteric, carotid artery and cremaster arteriole thrombosis models. Thrombi in 12-LOX -/- mice were unstable and frequently formed emboli, which resulted in impaired vessel occlusion or reopening. Additionally, thrombus formation and vessel occlusion was impaired in ML355 treated WT mice. Conclusions: The 12-LOX inhibitor ML355 inhibits platelet aggregation induced by a number of platelet agonists. Ex vivo high shear conditions in both mice and human was attenuated in the presence of ML355. Thrombus formation and vessel occlusion were impaired in mice deficient in 12-LOX. Finally, ML355 attenuates thrombus formation and prevents vessel occlusion in vivo . Our data strongly indicates 12-LOX is an important determinant of platelet reactivity and inhibition of platelet 12-LOX may represent a new target for anti-platelet therapeutics.

STAT3 Regulates Collagen-Induced Platelet Aggregation Independent of Its Transcription Factor Activity

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 157-157
Author(s):  
Zhou Zhou ◽  
Francisca C. Gushiken ◽  
Angela Bergeron ◽  
Vinod K. Vijayan ◽  
Rolando Rumbaut ◽  
...  
Keyword(s):  
Shear Stress ◽  
Transcription Factor ◽  
Platelet Aggregation ◽  
Tyrosine Phosphorylation ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Dependent Manner ◽  
Transcription Factor Activity ◽  
Factor Activity ◽  
Dose Dependent

Abstract Abstract 157 Signal Transducer and Activator of Transcription 3 (STAT3) serves as a transcription factor activated by cytokine-induced intracellular signals, which are critical in megakaryopoiesis. This signaling pathway may also be active in anucleated platelets that are primed by proinflammatory cytokines, suggesting that STAT3 plays a role in platelet hyperactivity associated with inflammation. We have recently found that three different classes of STAT3 inhibitors each selectively inhibited collagen-induced aggregation of human platelets by ∼50%. They also blocked thrombus formation (∼80%) on immobilized collagen under an arterial shear stress of 62.5 dyn/cm2. These STAT3 inhibitors also blocked platelet aggregation induced by collagen-related peptide, suggesting that they acted on GP VI-mediated intracellular signaling in platelets. These in vitro results were further verified in two sets of experiments in mouse models. First, an oligonucleotide G-quartet STAT3 inhibitor (1 mg/ml) or a scrambled control oligonucleotide were infused into C57/BJ6 mice daily for three days. Collagen-induced platelet aggregation was then induced and found to be reduced by up to 60% in mice infused with the STAT3 inhibitor, but not with the control oligonucleotide. Photochemical injury-induced thrombosis in the cremaster arterioles was also significantly delayed in the inhibitor-infused mice as compared to control mice. Second, infusing STAT3 inhibitor could result in platelet inhibitor indirectly by acting endothelial cells. To address this concern, we have generated platelet-specific STAT3 null mice that have developed normally and have normal platelet counts. The collagen-, but not TRAP-induced platelet aggregation in the platelet STAT3 KO mice was reduced as compared to their littermates. Platelets from the platelet-specific STAT3 KO mice were also significantly defective in thrombus formation on immobilized collagen under 62.5 dyn/cm2 of fluid shear stress that was generated in a parallel-plate flow chamber system. Consistent with results from these functional assays, collagen induced rapid (peaked at 5 min after stimulation) and dose-dependent tyrosine phosphorylation of STAT3, but not of STAT1 or STAT5 in washed human platelets. The phosphorylation was blocked dose-dependently by two STAT3 inhibitors. Syk inhibitors also blocked collagen-induced STAT3 phosphorylation in a dose-dependent manner, but STAT3 inhibitors had no effect on Syk phosphorylation, suggesting that Syk acts upstream of STAT3. Furthermore, STAT3 inhibitors also dose-dependently reduced collagen-induced tyrosine phosphorylation of PLCγ2, which is a known substrate of Syk. Consistent with this temporal interaction among STAT3, Syk and PLCγ2, activated STAT3 co-immunoprecipitated phosphorylated Syk and PLCγ2 in collagen-activated human platelets. The tri-molecular complex was also immunoprecipitated by an antibody to PLCγ2. Taken together, these data suggest that STAT3 regulates collagen-induced platelet aggregation, independent of its transcription factor activity. The regulation is potentially achieved by STAT3 serving as a protein scaffold linking the kinase Syk with its substrate PLCγ2 to enhance the signal relay in collagen-activated platelets. This cross-talk between collagen and cytokine signaling pathways provides a mechanism for how proinflammatory mediators could prime platelets for activation by hemostatic ligands. Disclosures: No relevant conflicts of interest to declare.

The Fibrinogen γ Chain Dodecapeptide Inhibits Agonist-induced Aggregation of Rabbit Platelets and Fibrinogen Binding to Rabbit Glycoprotein IIb-IIIa

1999 ◽  
Vol 82 (12) ◽  
pp. 1680-1686 ◽  
Author(s):  
Marian Packham ◽  
Desirée Taylor ◽  
Erik Yeo ◽  
Cynthia Gemmell ◽  
Sonali Patil ◽  
...  
Keyword(s):  
Direct Interaction ◽  
Human Platelets ◽  
Rabbit Platelet ◽  
Dependent Manner ◽  
Affinity Matrix ◽  
Human Fibrinogen ◽  
Fibrinogen Binding ◽  
Rabbit Platelets ◽  
Induced Aggregation ◽  
Dose Dependent

SummaryThe HHLGGAKQAGDV (H12) sequence at the carboxyl termini of the γ chains and the RGD sequences in the Aα chains of human fibrinogen are potential recognition sites for the binding of soluble fibrinogen to glycoprotein IIb-IIIa (GPIIb-IIIa) on activated human platelets. Thus, addition of either H12 or RGD-containing peptides inhibits aggregation of and fibrinogen binding to human platelets. In contrast, we reported previously that RGDS had relatively little inhibitory effect on these functions of rabbit platelets. In the present study, we found that H12 inhibited ADP- and thrombin-induced aggregation of rabbit platelets in a dose-dependent manner. Specificity was demonstrated by the failure of the variant HHLGGAKQAGEV peptide to inhibit ADP-induced aggregation. Furthermore, flow cytometric analyses demonstrated that H12 inhibited the binding of FITC-fibrinogen to ADP-activated rabbit platelets in a dose-dependent manner. To examine the direct interaction of H12 with rabbit GPIIb-IIIa, we performed affinity chromatography by applying an octylglucoside extract of rabbit platelet proteins onto an affinity matrix containing the fibrinogen γ chain sequence. Proteins of ∼135 kDa and ∼95 kDa were specifically eluted by soluble H12, and the 95 kDa protein band was immunoblotted by anti-LIBS1, a monoclonal antibody against human GPIIIa. In control samples, no detectable protein from rabbit platelet lysates was eluted from an RGD affinity matrix by GRGDSP. Collectively, our results demonstrated that H12 inhibits aggregation of and fibrinogen binding to rabbit platelets by directly interacting with rabbit GPIIb-IIIa. These findings suggest that rabbit platelets would serve as a suitable thrombosis model for testing the efficacy of peptide mimetics derived from H12.

Sign in / Sign up

Export Citation Format

Share Document