Antiplatelet Effect of FK633, a Platelet Glycoprotein Ilb/IIIa Antagonist, on Thrombus Formation and Vascular Patency after Thrombolysis in the Injured Hamster Carotid Artery

SummaryThe antithrombotic and restenosis-preventing effects of FK633, an inhibitor of platelet aggregation via binding to the glycoprotein (GP) Ilb/IIIa receptor, were studied. IC50 value of FK633 against platelet aggregation ex vivo induced by 2.5 |iM adenosine diphosphate (ADP) was 5.4 X 10"7 M as determined using hamster platelet rich plasma. The inhibitory effect was also investigated in vivo on thrombus formation at the carotid arterial wall injured by a modified catheter. As a control, the left carotid artery was injured and the time required to develop a thrombotic occlusion (3.9 ±1.1 min, mean ± S.E.M., n = 18) was determined. Then, the right carotid artery of the same animal was injured while a continuous intravenous (i.v.) infusion of FK633 was administered at doses of 0 (saline), 0.1,0.3 or 1.0 mg/kg/h. The time to occlusion was dose-dependently prolonged. In a separate experiment, 10% of the total tPA dose (0.52 mg/kg) was injected into the injured artery as a bolus and the remaining was infused i.v. at a constant rate for 30 min. When FK633 (0.3 or 1.0 mg/kg/h) was infused together with tPA, late patency of the reperfused artery was much improved as compared with that of treatment with tPA alone. Bleeding time, measured at the end of the tPA infusion, was markedly prolonged when the higher dose of FK633 (1.0 mg/kg/h) was coadministered, however coadministration of the lower dose of FK633 (0.3 mg/kg/h) was almost without prolongation on the bleeding time, despite a significant effect on the vascular patency after thrombolysis. Next, neointima formation was evaluated 2 weeks after the vascular injury. When FK633 (0.3 mg/kg/h) was continuously infused i. v. by an implanted osmotic pump for 3,7 or 14 days after the vascular injury, the neointimal area formation was significantly suppressed in the treatment groups for 7 or 14 days. These findings suggest that FK633 inhibits platelet activation in the injured artery and improves vascular patency after thrombolysis with tPA with a concomitant suppression of neointima formation.

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Combined effects of a factor Xa inhibitor YM466 and a GPIIb/IIIa antagonist YM128 on thrombosis and neointima formation in mice

Thrombosis and Haemostasis ◽

10.1160/th04-04-0206 ◽

2004 ◽

Vol 92 (12) ◽

pp. 1221-1228 ◽

Cited By ~ 8

Author(s):

Yoshiyuki Iwatsuki ◽

Kazumi Hayashi ◽

Yumiko Moritani ◽

Tomoko Nii ◽

Keiji Miyata ◽

...

Keyword(s):

Platelet Aggregation ◽

Vascular Injury ◽

Coronary Angioplasty ◽

Bleeding Time ◽

Factor Xa ◽

Neointima Formation ◽

Combined Effects ◽

Test Drug ◽

Thrombotic Occlusion ◽

Acute Thrombosis

SummaryThrombosis and neointima formation limit the efficacy of coronary angioplasty. Factor Xa inhibitors and GPIIb/IIIa antagonists have shown to be effective on acute thrombosis and late neointima formation, however, their combined effects remain to be elucidated. Vascular injury was induced by FeCl3 in the carotid artery in mice. For thrombosis studies, the test drug was orally administered 1 hour before vascular injury. For neointima studies, the test drug was orally administered 1 hour before and twice daily for 1 week after vascular injury, and then histological analysis was performed 3 weeks after vascular injury. YM466 inhibited thrombotic occlusion at 30 mg/kg with prolongation of prothrombin time (PT), and tail transection bleeding time (BT) was affected at 100 mg/kg. YM466 also inhibited neointima formation at 10 mg/kg. YM128 inhibited thrombotic occlusion and neointima formation at 10 and 30 mg/kg, respectively, with inhibition of platelet aggregation and prolongation of BT. In contrast, the combination of 10 mg/kg YM466 and 3 mg/kg YM128 inhibited thrombotic occlusion and neointima formation without affecting PT, platelet aggregation and BT. Concomitant inhibition of factor Xa and GPIIb/IIIa may provide a safer and more effective therapeutic regimen for treatment of coronary angioplasty.

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In Vivo Relevance for Platelet Glycoprotein Ibα Residue Tyr276 in Thrombus Formation.

Blood ◽

10.1182/blood.v108.11.216.216 ◽

2006 ◽

Vol 108 (11) ◽

pp. 216-216

Author(s):

Jose A. Guerrero ◽

Taisuke Kanaji ◽

Junling Liu ◽

Susan Russell ◽

T.K. Gartner ◽

...

Keyword(s):

Blood Flow ◽

Arachidonic Acid ◽

Platelet Aggregation ◽

Carotid Artery ◽

Bleeding Time ◽

Thrombus Formation ◽

Type I ◽

Complete Occlusion ◽

Platelet Counts ◽

Platelet Receptor

Abstract Background: GPIbα is a critically important platelet receptor best recognized for its ability to bind vWF and mediate platelet adhesion. In addition, a GP Ibα binding site for thrombin has been described and characterized for several decades. However, the physiologic relevance of GPIbα/thrombin binding has remained elusive. Site-directed mutagenesis has shown that a sulfated GP Ibα residue, Tyr276, is essential for thrombin binding to GP Ibα but not critical for vWF binding. The importance of Tyr276 was further substantiated in the crystal structure of a GP Ibα/thrombin complex confirming the sulfated Tyr276 GP Ibα residue directly participates with other neighboring negatively charged residues in establishing a first contact with the anion-binding exosite II of an α-thrombin molecule. To determine the physiologic relevance, if any, of the GP Ibα Tyr276 residue we generated mice with platelets containing a mutant human GP Ibα subunit containing a Tyr276 to Phe276 substitution (Y276F) and did comparative studies with mice expressing a normal human (WT) GP Ibα subunit. Methods: Mouse colonies expressing the normal or variant GP Ibα subunits were established by transgenic technology and bred into a mouse colony devoid of murine GP Ibα. Surface expression of WT or Y276F GP Ibα subunits was measured by flow cytometry. Relevant assays included platelet counts, FeCl3-induced thrombus formation, tail bleeding times, hemoglobin content following tail resection (measured as 575 nm absorbance of lysed erythrocytes), and washed platelet aggregation induced by different agonists (fibrillar type I collagen, thrombin, ristocetin, arachidonic acid, and PAR activating peptides). Results: Founder mice were selected with nearly identical levels of surface-expressed WT and Y276F GPIbα subunits. Circulating platelet counts were similar between the 2 colonies. Stirred platelet aggregation assays induced by 0.05, 0.1, 0.5 units/ml thrombin, 10 and 20 mg/ml fibrillar collagen, 1.25 mg/ml ristocetin, 0.25 mM arachidonic acid, 100 mM PAR3 activating peptide and 100 mM PAR4 activating peptide revealed no significant differences between WT and Y276F samples. No statistically significant values were obtained in tail bleeding time assays or hemoglobin loss comparing WT and Y276F animals. However, the time required for a reduction in carotid artery blood flow following FeCl3 injury was significantly prolonged in Y276F animals (10.6±1.6 min vs. 5.8±1.4 min). All WT animals displayed complete occlusion following the initial reduction in blood flow. In contrast, 3 out of 6 Y276F animals did not show complete occlusion and had evidence of fluctuating blood flow suggestive of embolization. Discussion: FeCl3 injury of the mouse carotid artery revealed an essential role for GP Ibα residue Tyr276. In contrast, no differences were seen in tail bleeding time assays or in any platelet aggregation assay. These results are suggestive that thrombin binding to human GPIbα may be relevant for the pathological development of a thrombus. This conclusion is based on both the failure of some carotid arteries from Y276F animals to fully occlude and our observation that all Y276F animals showed an increased length of time for the reduction of blood flow. The results suggest thrombin binding to GPIbα could be significant in terms of enhancing fibrinogen cleavage and therefore stabilizing the development of a platelet-rich thrombus.

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Comparison of Antithrombotic Effects of GPIIb-IIIa Receptor Antagonist and TXA2 Receptor Antagonist in the Guinea-pig Thrombosis Model: Possible Role of TXA2 in Reocclusion after Thrombolysis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653841 ◽

1995 ◽

Vol 73 (04) ◽

pp. 683-688 ◽

Cited By ~ 11

Author(s):

Yoshiharu Takiguchi ◽

Fumitoshi Asai ◽

Kouichirou Wada ◽

Mitsuyoshi Nakashima

Keyword(s):

Platelet Aggregation ◽

Guinea Pig ◽

Receptor Antagonist ◽

Thrombus Formation ◽

Tissue Type ◽

Platelet Glycoprotein ◽

Dependent Manner ◽

Thrombosis Model ◽

Vascular Patency ◽

Higher Doses

SummaryThe effects of Ro 44-9883, a new specific antagonist of platelet glycoprotein IIb-IIIa receptor, on thrombus formation and reocclusion after thrombolysis induced by tissue-type plasminogen activator (t-PA) were compared with those of vapiprost, a thromboxane (TX) A2 receptor antagonist, using a photochemically-induced thrombosis model in the guinea-pig femoral artery. Pretreatment with Ro 44-9883 (5,10 and 20 μg/kg/min, i. v.) prolonged the time required to occlude the artery in a dose-dependent manner. Ro 44-9883 at 10 and 20 μg/kg/min significantly inhibited ex vivo platelet aggregation in whole blood induced by collagen, ADP or U46619. Vapiprost 0.3 mg/kg inhibited thrombus formation and platelet aggregation induced by collagen or U46619, to the same extent as Ro 44-9883 at the higher doses. In the thrombolysis study, Ro 44-9883 at the higher doses given as comedication with t-PA reduced the time to achieve reperfusion and increased the vascular patency after successful reperfusion. Vapiprost also significantly reduced the time to reperfusion and prevented reocclusion. However, the vascular patency after thrombolysis by t-PA with vapiprost was significantly increased compared with Ro 44-9883. Ro 44-9883 inhibited platelet aggregation, but did not prevent TXA2 formation in platelets. Thus, vascular contraction mediated by platelet-derived TXA2 may be responsible for lower efficacy of Ro 44-9883 against reocclusion compared with vapiprost. These results indicate that not only platelet aggregation but also vasoconstriction may contribute to reocclusion after t-PA-induced thrombolysis in the guinea-pig.

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Plasmin Generation Plays different Roles in the Formation and Removal of Arterial and Venous Thrombus in Mice

Thrombosis and Haemostasis ◽

10.1055/s-0037-1612950 ◽

2002 ◽

Vol 87 (01) ◽

pp. 98-104 ◽

Cited By ~ 16

Author(s):

Osamu Kozawa ◽

Kiyotaka Okada ◽

Shigeru Ueshima ◽

Osamu Matsuo ◽

Toshihiko Uematsu ◽

...

Keyword(s):

Carotid Artery ◽

Bleeding Time ◽

Jugular Vein ◽

Thrombus Formation ◽

Endothelial Injury ◽

Wild Type ◽

Venous Thrombus ◽

Vascular Patency ◽

The Times

SummaryThe role of plasminogen (Plg) and α2-antiplasmin (α2-AP) in vascular thrombolysis in vivo was investigated in mice deficient in plasminogen (Plg−/−) or α2-AP (α2-AP−/−) or their wild type (PAI-1+/+, α2AP+/+). A thrombus was induced in the murine carotid artery or the internal jugular vein by endothelial injury. Blood flow was continuously monitored for 90 min and for 6 h 30 min after the initiation of endothelial injury. The times to occlusion by the developing thrombus in the carotid artery and the jugular vein of wild type mice were 12 ± 1.8 and 7.2 ± 1.9 min, respectively. The arterial thrombus formation in α2AP−/− mice was indistinguishable from the one in wild type mice, whereas the time to occlusion in Plg−/− was significantly shortened to 5.9 ± 1.7 min. Vascular patency after spontaneous reperfusion was markedly improved in α2-AP−/− mice. On the contrary, arterial patency in Plg−/− mice was aggravated. In venous thrombus formation, the time to occlusion in α2-AP−/− mice was significantly prolonged (27.1 ± 5.2 min), whereas in Plg−/− it was slightly shortened to 6.5 ± 2.5 min. Vascular patency after spontaneous reperfusion was also improved in α2-AP−/− mice, but not in Plg−/− mice. Histological observations using SEM indicated that fibrin nets were firmly fixed on the injured area in Plg−/− mice, but not in α2-AP−/− mice. The tail bleeding time was not different in any type of mice. However, re-bleeding time using a template bleeding device was significantly prolonged in α2-AP−/− as compared with that of wild type mice. In conclusion, lack of plasminogen markedly reduces the antithrombotic activities in vivo, whereas α2-AP plays a more important role in the formation and removal of venous thrombus in mice. Consequently, the inhibition of α2-AP could be a useful tool for the therapy of venous thrombosis and the prevention of re-thrombus formation.

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ADP Plays a Key Role in Thrombogenesis in Rats

Thrombosis and Haemostasis ◽

10.1055/s-0038-1642759 ◽

1988 ◽

Vol 59 (02) ◽

pp. 225-230 ◽

Cited By ~ 63

Author(s):

J P Maffrand ◽

A Bernat ◽

D Delebassée ◽

G Defreyn ◽

J P Cazenave ◽

...

Keyword(s):

Platelet Aggregation ◽

Bleeding Time ◽

Thrombus Formation ◽

Vascular Wall ◽

Cyclooxygenase Inhibitors ◽

High Dose ◽

Silk Thread ◽

Dense Granules ◽

Prolonged Bleeding Time ◽

Venous Shunt

SummaryThe relative importance of ADP, arachidonic acid metabolites and serotonin as thrombogenic factors was evaluated in rats by comparing, after oral administration, the effects of two inhibitors of ADP-induced platelet aggregation (ticlopidine and PCR 4099), three cyclo-oxygenase inhibitors (aspirin, triflusal and indobufen) and a selective serotonin 5HT2 receptor antagonist (ketanserin) on platelet aggregation, in four platelet-dependent thrombosis models and on bleeding time. Platelet aggregation induced by ADP and collagen was completely inhibited by ticlopidine and PCR 4099 whereas only the collagen aggregation was reduced by the cyclo-oxygenase inhibitors. Ketanserin or a depletion of platelet serotonin by reserpine did not affect platelet aggregation. Ticlopidine and PCR 4099 greatly prolonged rat tail transection bleeding time. This is probably related to their known ability to inhibit ADP-mediated platelet aggregation. In contrast, the cyclooxygenase inhibitors did not affect bleeding time at all. Reserpine and ketanserin prolonged bleeding time by interfering with the action of serotonin on the vascular wall. Ticlopidine and PCR4099 were very potent antithrombotics in all the models. Aspirin, only at a high dose, inhibited poorly thrombus formation on a silk thread in an arterio-venous shunt, suggesting that the inhibition of cyclo-oxygenase was not responsible. Triflusal was inactive in all models while indobufen slightly reduced thrombus formation in the silk thread and metallic coil models. Ketanserin and reserpine reduced thrombus only in the metallic coil model. Thrombus formation was greatly reduced in fawn-hooded rats, which lack ADP in their platelet dense granules because of a genetic storage pool deficiency. Taken together, the results obtained with the drugs and with the fawn-hooded rats support the concept that ADP plays a key role in thrombogenesis in rats.

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Antithrombotic Effects and Bleeding Time Prolongation with Synthetic Platelet GPIIb/llla Inhibitors in Animal Models of Platelet-Mediated Thrombosis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1642390 ◽

1994 ◽

Vol 71 (01) ◽

pp. 095-102 ◽

Cited By ~ 43

Author(s):

Désiré Collen ◽

Hua Rong Lu ◽

Jean-Marie Stassen ◽

Ingrid Vreys ◽

Tsunehiro Yasuda ◽

...

Keyword(s):

Platelet Aggregation ◽

Bleeding Time ◽

Bolus Injection ◽

Cell Injury ◽

Ex Vivo ◽

Thrombus Formation ◽

Femoral Vein ◽

Thrombotic Occlusion ◽

Antithrombotic Effects

SummaryCyclic Arg-Gly-Asp (RGD) containing synthetic peptides such as L-cysteine, N-(mercaptoacetyl)-D-tyrosyl-L-arginylglycyl-L-a-aspartyl-cyclic (1→5)-sulfide, 5-oxide (G4120) and acetyl-L-cysteinyl-L-asparaginyl-L-prolyl-L-arginyl-glycyl-L-α-aspartyl-[0-methyltyrosyl]-L-arginyl-L-cysteinamide, cyclic 1→9-sulfide (TP9201) bind with high affinity to the platelet GPIIb/IIIa receptor.The relationship between antithrombotic effect, ex vivo platelet aggregation and bleeding time prolongation with both agents was studied in hamsters with a standardized femoral vein endothelial cell injury predisposing to platelet-rich mural thrombosis, and in dogs with a carotid arterial eversion graft inserted in the femoral artery. Intravenous administration of G4120 in hamsters inhibited in vivo thrombus formation with a 50% inhibitory bolus dose (ID50) of approximately 20 μg/kg, ex vivo ADP-induccd platelet aggregation with ID50 of 10 μg/kg, and bolus injection of 1 mg/kg prolonged the bleeding time from 38 ± 9 to 1,100 ± 330 s. Administration of TP9201 in hamsters inhibited in vivo thrombus formation with ID50 of 30 μg/kg, ex vivo platelet aggregation with an ID50 of 50 μg/kg and bolus injection of 1 mg/kg did not prolong the template bleeding time. In the dog eversion graft model, infusion of 100 μg/kg of G4120 over 60 min did not fully inhibit platelet-mediated thrombotic occlusion but was associated with inhibition of ADP-induccd ex vivo platelet aggregation and with prolongation of the template bleeding time from 1.3 ± 0.4 to 12 ± 2 min. Infusion of 300 μg/kg of TP9201 over 60 min completely prevented thrombotic occlusion, inhibited ex vivo platelet aggregation, but was not associated with prolongation of the template bleeding time.TP9201, unlike G4120, inhibits in vivo platelet-mediated thrombus formation without associated prolongation of the template bleeding time.

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Pharmacological properties of YM-254890, a specific Gαq/11 inhibitor, on thrombosis and neointima formation in mice

Thrombosis and Haemostasis ◽

10.1160/th04-09-0635 ◽

2005 ◽

Vol 94 (07) ◽

pp. 184-192 ◽

Cited By ~ 28

Author(s):

Masatoshi Taniguchi ◽

Yumiko Moritani ◽

Toshio Uemura ◽

Takeshi Shigenaga ◽

Hajime Takamatsu ◽

...

Keyword(s):

Oral Administration ◽

Vascular Injury ◽

Bolus Injection ◽

Thrombus Formation ◽

Therapeutic Window ◽

Pharmacological Properties ◽

Neointima Formation ◽

Intravenous Bolus ◽

Test Drug ◽

Intravenous Bolus Injection

SummaryThe pharmacological properties of YM-254890,a specific Gαq/11 inhibitor, on acute thrombosis and chronic neointima formation after vascular injury have been investigated. FeCl3 was used to induce vascular injury in the carotid artery of mice. For the thrombosis studies, the test drug was either intravenously or orally administered before vascular injury. For the neointima studies, the test drug was orally administered 1 h before and twice daily for 1 week after vascular injury. Histological analysis was then performed 3 weeks later. YM-254890 significantly inhibited ex vivo platelet aggregation 5 min after intravenous bolus injection at 0.03 mg/kg or more, and 1 h after oral administration at 1 mg/kg. YM-254890 significantly inhibited thrombus formation after intravenous bolus injection at 0.03 mg/kg as well as after oral administration at 1 mg/kg, but tail transection bleeding time was significantly prolonged at 0.1 mg/kg for intravenous injection and 3 mg/kg for oral administration. Furthermore, oral administration of YM-254890 dose-dependently inhibited neointima formation 3 weeks after vascular injury with significant effects at 1 mg/kg twice daily for 1 week. Clopidogrel also significantly inhibited neointima formation at its antithrombotic dose, but its inhibitory potency was less than that of YM-254890. However, YM-254890 significantly reduced systemic blood pressure at doses 3 times higher than those that produced significant inhibitory effects on thrombosis and neointima formation. Though the systemic use of YM-254890 may be limited, owing to its narrow therapeutic window, this unique compound is a useful research tool for investigating the physiological roles of Gαq/11.

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The Antiaggregating and Antithrombotic Activity of Clopidogrel Is Potentiated by Aspirin in Several Experimental Models in the Rabbit

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615238 ◽

1998 ◽

Vol 80 (09) ◽

pp. 512-518 ◽

Cited By ~ 75

Author(s):

Frédérique Dol ◽

André Bernat ◽

Robert Falotico ◽

Alain Lalé ◽

Pierre Savi ◽

...

Keyword(s):

Platelet Aggregation ◽

Carotid Artery ◽

Ex Vivo ◽

Thrombus Formation ◽

Experimental Models ◽

Arteriovenous Shunt ◽

Thrombotic Occlusion ◽

Antithrombotic Activity ◽

Rabbit Carotid Artery ◽

Antithrombotic Efficacy

SummaryIt is unknown whether the addition of aspirin might increase both the efficacy and the potency of clopidogrel, a potent and selective ADP blocker. For that purpose, the efficacy of clopidogrel (1–20 mg/kg, p.o.) administered orally to rabbits alone or in combination with aspirin (0.1–10 mg/kg, p.o.) was determined in several experimental models. A potent synergistic effect of the clopidogrel/aspirin association was demonstrated with regard to collagen-induced platelet aggregation measured ex vivo. Similarly, aspirin potentiated the antithrombotic activity of clopidogrel measured with regard to experimental thrombosis induced by a silk thread or on stents placed in an arteriovenous shunt, thrombus formation following electrical stimulation of the rabbit carotid artery and with regard to 111In-labeled platelet deposition on a stent implanted in an arteriovenous shunt or on the subendothelium following air drying injury of the rabbit carotid artery. A similar potentiating effect of aspirin was obtained with regard to myointimal proliferation (restenosis) in the femoral arteries of atherosclerotic rabbits which occurred as a consequence of stent placement. The clopidogrel/aspirin combination showed only additive-type effects on bleeding time prolongation induced by ear transection in the rabbit, therefore showing that combined inhibition of cyclooxygenase and ADP‘s effects provide a marked enhanced antithrombotic efficacy. Such a combination may provide substantial protection against platelet aggregation leading to thrombotic occlusion at sites of endothelial injuries and coronary artery stenosis in humans.

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Local delivery of soluble platelet collagen receptor glycoprotein VI inhibits thrombus formation in vivo

Thrombosis and Haemostasis ◽

10.1160/th05-11-0731 ◽

2006 ◽

Vol 95 (05) ◽

pp. 763-766 ◽

Cited By ~ 31

Author(s):

Andreas Bültmann ◽

Christian Herdeg ◽

Zhongmin Li ◽

Götz Münch ◽

Christine Baumgartner ◽

...

Keyword(s):

Carotid Artery ◽

Vascular Injury ◽

Carotid Arteries ◽

Thrombus Formation ◽

Critical Role ◽

Local Delivery ◽

Computer Assisted ◽

Glycoprotein Vi ◽

Collagen Receptor

SummaryPlatelet-mediated thrombus formation at the site of vascular injury isa major trigger for thrombo-ischemic complications after coronary interventions. The platelet collagen receptor glycoprotein VI (GPVI) plays a critical role in the initiation of arterial thrombus formation. Endothelial denudation of the right carotid artery in rabbits was induced through balloon injury. Subsequently, local delivery of soluble, dimeric fusion protein of GPVI (GPVI-Fc) (n=7) or control Fc (n=7) at the site of vascular injury was performed with a modified double-balloon drugdelivery catheter.Thrombus area within the injured carotid artery was quantified using a computer-assisted image analysis and was used as index of thrombus formation.The extent of thrombus formation was significantly reduced in GPVI-Fc- compared with control Fc-treated carotid arteries (relative thrombus area, GPVI-Fc vs. Fc: 9.3 ± 4.2 vs. 2.3 ± 1.7, p<0.001). Local delivery of soluble GPVI resulted in reduced thrombus formation after catheter-induced vascular injury.These data suggest a selective pharmacological modulation of GPVI-collagen interactions to be important for controlling onset and progression of pathological arterial thrombosis, predominantly or even exclusively at sites of injured carotid arteries in the absence of systemic platelet therapy.

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