Abolition of in vivo platelet thrombus formation in primates with monoclonal antibodies to the platelet GPIIb/IIIa receptor. Correlation with bleeding time, platelet aggregation, and blockade of GPIIb/IIIa receptors.

We previously reported that 0.8 mg/kgof the F(ab’)2 fragment of antibody 7E3, directed at theplatelet GPIIb/IIIa receptor, can abolish periodic platelet thrombus formation on partially stenosed carotid arteries in monkeys (Mnks). The present study was designed to: 1) test another antibody to GPIIb/IIIa (10E5), 2)find the minimum effective dose, and 3) correlate this effect with changesin the template bleeding time (BT) and platelet aggregation (PA). Periodicplatelet thrombi were established in the carotid arteries of 7 anesthetized Mnks after mechanical stenosis (∽70%) and intimal damage. 4 Mnks were treated with 7E3. Mnks 1 and 2 were given 0.2 mg/kg, and this dose: abolished thrombus formation and prevented its return in response to epinephrine infusion and increased intimal damage; abolished PA in response to ADP (10 μM); and increased the BT from 8.5 to 16 min and from 5 to 11 min. Mnk 3 was given 0.1 mg/kg, and this dose abolished the thrombi, inhibited PA by ∽41% and increased the BT only to 10 minfrom 8 min. Mnk 4 was givenincremental doses of 7E3. After 0.1 mg/kg, thrombi were reduced but not abolished, PA was minimally inhibited and the BTwas unchanged (7.5 vs 8 min pre). Afteranother 0.1 mg/kg, thrombi were abolished but could be partially restoredwith extreme provocation, PA was abolished and BT remained 7.5min. Afteranother 0.2 mg/kg, thrombicould not berestored, PA was abolished and the BTincreased to 21 min. After a final0.2mg/kg, the BT increased to 33 min. 3 Mnks were treated with 10E5. Mnk1received 0.4 mg/kg: thrombi and PA werebothabolished, and the BT increased from 5.5 to 14.5 min.Mnk 2 received 0.2mg/kg: thrombi and PA were again abolished while the BT increased to8.5from4.5 min. Mnk 3 received 0.1 mg/kg, and this abolished thrombus formation,butinhibitedPA by only ∽50%and increasedthe BT minimally (7.5 to 8.5 min). Increasedoozing from the neckwounds wasonly observed in animals with significant BT prolongations.We conclude that ∽0.1-0.2 mg/kg of eitherantibody can abolish in vivo thrombusformation, and that it is not necessary to abolish PA or cause marked prolongation ofthe BT in order toabolishthrombus formation in this model.

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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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Antithrombotic Effect of Crotalin, a Platelet Membrane Glycoprotein Ib Antagonist From Venom of Crotalus atrox

Blood ◽

10.1182/blood.v91.5.1582.1582_1582_1589 ◽

1998 ◽

Vol 91 (5) ◽

pp. 1582-1589

Author(s):

Mei-Chi Chang ◽

Hui-Kuan Lin ◽

Hui-Chin Peng ◽

Tur-Fu Huang

Keyword(s):

Platelet Aggregation ◽

Latent Period ◽

Bleeding Time ◽

Thrombus Formation ◽

Platelet Membrane ◽

Dependent Manner ◽

Glycoprotein Ib ◽

Platelet Surface ◽

Dose Dependent

A potent platelet glycoprotein Ib (GPIb) antagonist, crotalin, with a molecular weight of 30 kD was purified from the snake venom ofCrotalus atrox. Crotalin specifically and dose dependently inhibited aggregation of human washed platelets induced by ristocetin with IC50 of 2.4 μg/mL (83 nmol/L). It was also active in inhibiting ristocetin-induced platelet aggregation of platelet-rich plasma (IC50, 6.3 μg/mL). 125I-crotalin bound to human platelets in a saturable and dose-dependent manner with a kd value of 3.2 ± 0.1 × 10−7 mol/L, and its binding site was estimated to be 58,632 ± 3,152 per platelet. Its binding was specifically inhibited by a monoclonal antibody, AP1 raised against platelet GPIb. Crotalin significantly prolonged the latent period in triggering platelet aggregation caused by low concentration of thrombin (0.03 U/mL), and inhibited thromboxane B2formation of platelets stimulated either by ristocetin plus von Willebrand factor (vWF), or by thrombin (0.03 U/mL). When crotalin was intravenously (IV) administered to mice at 100 to 300 μg/kg, a dose-dependent prolongation on tail bleeding time was observed. The duration of crotalin in prolonging tail bleeding time lasted for 4 hours as crotalin was given at 300 μg/kg. In addition, its in vivo antithrombotic activity was evidenced by prolonging the latent period in inducing platelet-rich thrombus formation by irradiating the mesenteric venules of the fluorescein sodium-treated mice. When administered IV at 100 to 300 μg/kg, crotalin dose dependently prolonged the time lapse in inducing platelet-rich thrombus formation. In conclusion, crotalin specifically inhibited vWF-induced platelet agglutination in the presence of ristocetin because crotalin selectively bound to platelet surface receptor-glycoprotein Ib, resulting in the blockade of the interaction of vWF with platelet membrane GPIb. In addition, crotalin is a potent antithrombotic agent because it pronouncedly blocked platelet plug formation in vivo.

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Antithrombotic Effect of Crotalin, a Platelet Membrane Glycoprotein Ib Antagonist From Venom of Crotalus atrox

Blood ◽

10.1182/blood.v91.5.1582 ◽

1998 ◽

Vol 91 (5) ◽

pp. 1582-1589 ◽

Cited By ~ 49

Author(s):

Mei-Chi Chang ◽

Hui-Kuan Lin ◽

Hui-Chin Peng ◽

Tur-Fu Huang

Keyword(s):

Platelet Aggregation ◽

Latent Period ◽

Bleeding Time ◽

Thrombus Formation ◽

Platelet Membrane ◽

Dependent Manner ◽

Glycoprotein Ib ◽

Platelet Surface ◽

Dose Dependent

AbstractA potent platelet glycoprotein Ib (GPIb) antagonist, crotalin, with a molecular weight of 30 kD was purified from the snake venom ofCrotalus atrox. Crotalin specifically and dose dependently inhibited aggregation of human washed platelets induced by ristocetin with IC50 of 2.4 μg/mL (83 nmol/L). It was also active in inhibiting ristocetin-induced platelet aggregation of platelet-rich plasma (IC50, 6.3 μg/mL). 125I-crotalin bound to human platelets in a saturable and dose-dependent manner with a kd value of 3.2 ± 0.1 × 10−7 mol/L, and its binding site was estimated to be 58,632 ± 3,152 per platelet. Its binding was specifically inhibited by a monoclonal antibody, AP1 raised against platelet GPIb. Crotalin significantly prolonged the latent period in triggering platelet aggregation caused by low concentration of thrombin (0.03 U/mL), and inhibited thromboxane B2formation of platelets stimulated either by ristocetin plus von Willebrand factor (vWF), or by thrombin (0.03 U/mL). When crotalin was intravenously (IV) administered to mice at 100 to 300 μg/kg, a dose-dependent prolongation on tail bleeding time was observed. The duration of crotalin in prolonging tail bleeding time lasted for 4 hours as crotalin was given at 300 μg/kg. In addition, its in vivo antithrombotic activity was evidenced by prolonging the latent period in inducing platelet-rich thrombus formation by irradiating the mesenteric venules of the fluorescein sodium-treated mice. When administered IV at 100 to 300 μg/kg, crotalin dose dependently prolonged the time lapse in inducing platelet-rich thrombus formation. In conclusion, crotalin specifically inhibited vWF-induced platelet agglutination in the presence of ristocetin because crotalin selectively bound to platelet surface receptor-glycoprotein Ib, resulting in the blockade of the interaction of vWF with platelet membrane GPIb. In addition, crotalin is a potent antithrombotic agent because it pronouncedly blocked platelet plug formation in vivo.

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Endotoxin enhances microvascular thrombosis in mouse cremaster venules via a TLR4-dependent, neutrophil-independent mechanism

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00305.2005 ◽

2006 ◽

Vol 290 (4) ◽

pp. H1671-H1679 ◽

Cited By ~ 48

Author(s):

Rolando E. Rumbaut ◽

Ricardo V. Bellera ◽

Jaspreet K. Randhawa ◽

Corie N. Shrimpton ◽

Swapan K. Dasgupta ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Adhesion ◽

Ex Vivo ◽

Thrombus Formation ◽

Mouse Strains ◽

Microvascular Endothelium ◽

Microvascular Thrombosis ◽

Platelet Thrombus ◽

Adhesive Interactions

Endotoxemia promotes adhesive interactions between platelets and microvascular endothelium in vivo. We sought to determine whether endotoxin (lipopolysaccharide, LPS) modified platelet thrombus formation in mouse cremaster venules and whether Toll-like receptor 4 (TLR4) and neutrophils were involved in the response. Intravital videomicroscopy was performed in the cremaster microcirculation of pentobarbital-anesthetized mice; venular platelet thrombi were induced with a light/dye endothelial injury model. C57BL/6 mice treated with Escherichia coli endotoxin had enhanced rates of venular platelet thrombus formation: the time to microvessel occlusion was reduced by ∼50% ( P < 0.005) compared with saline-treated animals. Enhanced microvascular thrombosis was evident as early as 2 h after LPS administration. LPS had no effect on thrombosis in either of two mouse strains with altered TLR4 signaling (C57BL/10ScNJ or C3H/HeJ), whereas it enhanced thrombosis in the control strains (C57BL/10J and C3H/HeN). LPS also enhanced platelet adhesion to endothelium in the absence of light/dye injury. Platelet adhesion, but not enhanced thrombosis, was inhibited by depletion of circulating neutrophils. LPS failed to enhance platelet aggregation ex vivo and did not influence platelet P-selectin expression, a marker of platelet activation. These findings support the notion that endotoxemia promotes platelet thrombus formation independent of neutrophils and without enhancement of platelet aggregation, via a TLR4-dependent mechanism.

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Antithrombotic Effect of a Recombinant von Willebrand Factor, VCL, on Nitrogen Laser-Induced Thrombus Formation in Guinea Pig Mesenteric Arteries

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653771 ◽

1995 ◽

Vol 73 (02) ◽

pp. 318-323 ◽

Cited By ~ 20

Author(s):

K Azzam ◽

L I Garfinkel ◽

C Bal dit Sollier ◽

M Cisse Thiam ◽

L Drouet

Keyword(s):

Platelet Aggregation ◽

Bleeding Time ◽

Ex Vivo ◽

Thrombus Formation ◽

Mesenteric Arteries ◽

Antithrombotic Effects ◽

Von Willebrand ◽

Willebrand Factor

SummaryTo assess the antithrombotic effectiveness of blocking the platelet glycoprotein (GP) Ib/IX receptor for von Willebrand factor (vWF), the antiaggregating and antithrombotic effects were studied in guinea pigs using a recombinant fragment of vWF, Leu 504-Lys 728 with a single intrachain disulfide bond linking residues Cys 509-Cys 695. The inhibitory effect of this peptide, named VCL, was tested in vitro on ristocetin- and botrocetin-induced platelet aggregation and compared to the ADP-induced platelet aggregation. In vivo, the antithrombotic effect of VCL was tested in a model of laser-injured mesentery small arteries and correlated to the ex vivo ristocetin-induced platelet aggregation. In this model of laser-induced thrombus formation, five mesenteric arteries were studied in each animal, and the number of recurrent thrombi during 15 min, the time to visualization and time to formation of first thrombus were recorded.In vitro, VCL totally abolished ristocetin- and botrocetin-induced platelet aggregation, but had no effect on ADP-induced platelet aggregation. Ex vivo, VCL (0.5 to 2 mg/kg) administered as a bolus i. v. injection inhibits ristocetin-induced platelet aggregation with a duration of action exceeding 1 h. The maximum inhibition was observed 5 min after injection of VCL and was dose related. The same doses of VCL had no significant effect on platelet count and bleeding time. In vivo, VCL (0.5 to 2 mg/kg) had no effect on the appearance of the thrombi formed but produced dose-dependent inhibition of the mean number of recurrent thrombi (the maximal effect was obtained at 5 min following i. v. injection of the highest dose: 0.8 ± 0.2 thrombi versus 4 ± 0.4 thrombi in controls). The three doses of VCL increased the time in which the first thrombus in a concentration-dependent manner was formed. However, the time to visualize the first thrombus was only prolonged in the higher dose-treated group.These in-vivo studies confirm that VCL induces immediate, potent, and transient antithrombotic effects. Most importantly, this inhibition was achieved without inducing thrombocytopenia nor prolongation of the bleeding time.

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Inhbition of Platelet Thrombus Formation by Chlorpromazine Acting to Diminish Haemodynamically Induced Haemolysis

10.1055/s-0039-1684445 ◽

1979 ◽

Cited By ~ 1

Author(s):

G.V.R. Born ◽

A. Uehmeier

Keyword(s):

Platelet Aggregation ◽

Human Blood ◽

Bleeding Time ◽

Thrombus Formation ◽

Coronary Thrombosis ◽

Internal Diameter ◽

Novel Approach ◽

Platelet Thrombus ◽

Free Haemoglobin ◽

Polyethylene Tubing

The idea that drugs capable of counter-acting hypotonic haemolysis (Seeman, 1972, Pharmacol Rev., 24, 583) might diminish the activating effect of erythrocytes on platelets (Gaarder et al., 1961, Nature, Lond., 192, 531) was suggested by one of us (GVRB) as a novel approach towards inhibiting their intravascular aggregation as thrombi. Indeed, chlorpromazine added to human blood in concentrations which diminish haemolysis but have no direct effect on platelet aggregation (Mills and Roberts, 1967, Nature, Lond., 213, 35) prolong the “bleeding time” from small holes in artificial vessels where extravasation is terminated, as in living arterioles, by aggregated platelets (Born, Bergquist and Arfors, 1976, Nature, Lond., 259, 235). Apyrase had a similar effect, suggesting that it was due to decreased plasma ADP. We now provide evidence that this ADP is released by haemolysis which is diminished by chlorpromazine. Human venous citrated blood at 37° was pumped continuously through polyethylene tubing 280 μm internal diameter. Chlorpromazine caused concentration-related increases in “bleeding times” from a standard cut and decreases in free haemoglobin. The observed haemolysis would provide enough ADP to initiate platelet aggregation. The results support the suggestion (Born et al., 1976) that drugs with this effect of chlorpromazine may prevent haemorrhage-induced, eg. coronary thrombosis.

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Prevention of Canine Coronary Artery Thrombosis with Echistatin, a Potent Inhibitor of Platelet Aggregation from the Venom of the Viper, Echis carmatus

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647361 ◽

1990 ◽

Vol 64 (04) ◽

pp. 576-581 ◽

Cited By ~ 17

Author(s):

Ronald J Shebuski ◽

Denise R Ramjit ◽

Gary R Sitko ◽

Patricia K Lumma ◽

Victor M Garsky

Keyword(s):

Coronary Artery ◽

Platelet Aggregation ◽

Bleeding Time ◽

Ex Vivo ◽

Platelet Rich Plasma ◽

Thrombus Formation ◽

Artery Thrombosis ◽

Coronary Artery Thrombosis ◽

Canine Coronary Artery

SummaryA model of acute, platelet-dependent canine coronary artery thrombosis was utilized to assess the antithrombotic effect of a synthetic, RGD-containing 49-residue protein termed echistatin. This protein is derived from the venom of the viper, Echis carinatus. In vitro, echistatin inhibited ADP (10 ΜM)-induced platelet aggregation with IC50 values in human and canine platelet-rich plasma of 101 ± 4 and 127 ± 32 nM, respectively. In vivo, in the dog, infusion of echistatin for 30 min at 20 pg kg−1 min−1 or 2.6 nM kg−1 min−1 resulted in total abolition of acute platelet-dependent coronary thrombus formation in all dogs tested (n = 5). Infusion of a lower dose (10 pg kg−1 min−1) was not effective in prevention of thrombus formation. Blood samples were taken before and after infusion of echistatin in order to determine ex vivo platelet aggregatory responses. Echistatin (20 pg kg−1 min−1, i.v.) attenuated ex vivo platelet aggregation elicited by ADP, U-46619 and collagen and increased bleeding time by 2.9 ± 0.5-fold over control. Thus, in the dog, echistatin is an effective antithrombotic agent inhibiting both platelet aggregation in vivo in the coronary artery as well as ex vivo with a concomitant increase in bleeding time. Furthermore, the effects of echistatin on platelet aggregation and bleeding time are reversible with restoration to control levels occurring 30-60 min after termination of the infusion.

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The Effect of Prostacyclin (PGI2) on Platelet Behaviour. Thrombus Formation in Vivo and Bleeding Time

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646791 ◽

1979 ◽

Vol 41 (02) ◽

pp. 425-435 ◽

Cited By ~ 69

Author(s):

Fernando B Ubatuba ◽

Salvador Moncada ◽

John R Vane

Keyword(s):

Platelet Aggregation ◽

Prostaglandin E1 ◽

Bleeding Time ◽

Potent Inhibitor ◽

Ex Vivo ◽

Thrombus Formation ◽

Phosphodiesterase Inhibitor ◽

Concomitant Administration ◽

Prostaglandin D2

SummaryProstacyclin (PGI2) infused intravenously into anaesthetized rabbits inhibited electrically-induced thrombus formation in the carotid artery, increased bleeding time and inhibited ex vivo platelet aggregation induced by ADP or arachidonic acid. The increase in bleeding time and the inhibition of ex vivo platelet aggregation lasted for as long as the infusion of PGI2 was maintained but rapidly disappeared after infusion was stopped.Prostacyclin is a more potent inhibitor of platelet function, in vivo than prostaglandin E1 (PGE1) or prostaglandin D2 (PGD2).The effects of prostacyclin on all parameters studied except blood pressure were potentiated by the concomitant administration of theophylline, a phosphodiesterase inhibitor.

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Hemostatic Effects of Fibrinogen-γ Chain Dodecapeptide-Conjugated Polymerized Albumin Particles In Vitro and in Vivo.

Blood ◽

10.1182/blood.v104.11.3883.3883 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3883-3883

Author(s):

Yosuke Okamura ◽

Naohide Watanabe ◽

Shinji Takeoka ◽

Hidenori Suzuki ◽

Mitsuru Murata ◽

...

Keyword(s):

Bleeding Time ◽

Thrombus Formation ◽

Blood Platelet ◽

Becton Dickinson ◽

Platelet Surface ◽

Normal Platelet ◽

Platelet Thrombus ◽

Carboxy Terminal

Abstract We have studied a prototype of platelet substitutes focusing on a dodecapeptide, HHLGGAKQAGDV (H12)1), which is specific for fibrinogen γ-chain carboxy-terminal sequence (γ 400–411). In this study, we conjugated H12 to the surface of polymerized albumin particles (polyAlb) as biocompatible and biodegradable carriers to produce particles having hemostatic ability, and evaluated their in vitro and in vivo effects. H12 (H12; 9.6 x 103 /particles) containing cysteine to the N-terminal was conjugated to polyAlb (260 ± 60 nm), and the effect of H12-polyAlb on platelet thrombus formation was evaluated in vitro with thrombocytopenic whole blood ([platelet] = 2.0 x 104 /μL, anticoagulated with PPACK) under flow conditions (shear rate; 150 s−1). Thrombocytopenic rats were made by busulphan injection at a dose of 20 mg/kg, and a 2.5 mm length x 1.0 mm depth template-guided incision (QuikheelTM, Becton-Dickinson, San Jose, CA) was made 1 cm from the tip of tail. The tail was immersed in a 50 mL cylinder of saline and the time taken to stop bleeding was measured. When thrombocytopenic blood in the presence of H12-polyAlb ([rHSA]=0.14 mg/mL) was flowed on collagen-plate, the surface coverage of DiOC6-labeled platelets evaluated by fluorescence microscopy was increased to 3.9 ± 1.1 % (n=3) from 2.1 ± 0.4 % (n=3) obtained in the absence of H12-polyAlb. In the same experiments, rhodamine-labeled H12-polyAlb was found to be involved in platelet-platelet interactions by binding to activated platelet surface, thus enhancing platelet thrombus formation. Next, in vivo hemostatic effect was tested by measuring tail bleeding time of thrombocytopenic rats 5 minutes after the intravenous administration of H12-polyAlb. The bleeding times of normal ([platelet] = 8.1 ±0.9 x 105 /μL) and thrombocytopenic rats ([platelet] = 2.0 ±0.3 x 105 /μL) were 187 ± 51 s and 609 ± 153 s (n=6), respectively. H12-polyAlb administration at a dose of 4 mg/kg significantly shortened the bleeding time to 342 ± 73 s (n=10), whereas polyAlb was without effects (553 ± 104 s, n=6). These results indicate that H12-polyAlb can be a suitable candidate for an alternative to human platelet concentrates infused into thrombocytopenic patients.

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