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Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:56-63

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:56-63.)
© 1996 American Heart Association, Inc.

Articles

Optimal Antagonism of GPIIb/IIIa Favors Platelet Adhesion by Inhibiting Thrombus Growth

An Ex Vivo Capillary Perfusion Chamber Study in the Guinea Pig

Patrick André; Brigitte Arbeille; Valérie Drouet; Patricia Hainaud; Claire Bal dit Sollier; Jacques P. Caen; Ludovic O. Drouet

From the Institut des Vaisseaux et du Sang (P.A., B.A., V.D., P.H., C.B. dit S., J.P.C., L.O.D.); INSERM U353 (P.A., L.O.D.), Paris; and the Faculté de Médecine (B.A.), Tours, France.

Correspondence to Ludovic O. Drouet, MD, PhD, Institut des Vaisseaux et du Sang, Hôpital Lariboisière, 8 rue Guy Patin, 75010 Paris, France.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
down arrowResults
down arrowDiscussion
down arrowAppendix
down arrowReferences
 
Abstract To evaluate the involvement of the glycoprotein (GP) IIb/IIIa–dependent process in platelet deposition and thrombus growth on capillaries coated with human type III collagen, the effects of incremental doses of Lamifiban, a potent specific synthetic GPIIb/IIIa antagonist, were studied in ex vivo capillary perfusion chambers using guinea pig blood. In this model, nonanticoagulated blood was perfused for 4.5 minutes at three shear rates: 100, 650, and 1600 s-1. Platelet deposition was quantified by computer-assisted morphometry and expressed as platelet adhesion (percentage of capillary surface covered with spread and contact platelets and platelets implicated in thrombus), mean thrombus height, and total thrombus cross-sectional area. In control untreated guinea pigs, platelet adhesion and thrombus height were 63% and 2.5 µm at 100 s-1, 60.5% and 13.8 µm at 650 s-1, and 45% and 28.1 µm at 1600 s-1, respectively. At 100 s-1, Lamifiban had no effect on platelet deposition at any of the three doses administered to the guinea pigs (0.3, 1, and 3 mg/kg). At 0.3 mg/kg and shear rates of 650 and 1600 s-1, Lamifiban had no effect on platelet adhesion or thrombus size, but at 1 and 3 mg/kg and shear rates of 650 and 1600 s-1, it significantly reduced thrombus size. At 1600 s-1, 1 mg/kg Lamifiban significantly increased platelet adhesion from 45% to 62.5%, whereas at 3 mg/kg it induced a significant overall decrease from 45% to 25% and qualitatively increased the ratio of contact to spread platelets. These data suggest that at high shear rates, GPIIb/IIIa participates in platelet spreading and that there is a balance between platelet involvement in adhesion to the thrombogenic surface and the growth of the already formed thrombus. This indicates that important clinical implications of an optimal therapeutic degree of GPIIb/IIIa antagonism could be expected.


Key Words: thrombus • adhesion • guinea pig • GPIIb/IIIa antagonist • capillary


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
down arrowResults
down arrowDiscussion
down arrowAppendix
down arrowReferences
 
One of the main aims in the prevention and treatment of cardiovascular and cerebrovascular diseases is to inhibit the platelet aggregation involved in thrombus growth with minimal inhibition of primary hemostasis. Several groups have studied the relationships between shear rate and platelet deposition and between surface composition and platelet deposition, but very few have studied the possible relationship between platelet adhesion and thrombus formation within platelet deposition.

It is generally recognized that the platelet membrane glycoprotein (GP) IIb/IIIa is involved in platelet-to-platelet interaction (ie, thrombus growth), and this glycoprotein is also thought to be involved in the interaction of platelets with the damaged vessel wall (ie, adhesion).

Several studies have been performed on everted artery subendothelium in an annular perfusion chamber with anticoagulated blood1 2 3 and on purified subendothelial components in various flat chambers4 5 6 with anticoagulated and nonanticoagulated blood. These chambers, which require large amounts of blood, are particularly well adapted to the study of platelet deposition in large animal species and humans. To take into account the circular geometry of vessel flow channels, several laboratories have developed cylindrical perfusion chambers to study thrombogenesis.7 8 9 10 We have developed a new model of the capillary perfusion chamber, in which human type III collagen is exposed to limited amounts of nonanticoagulated blood. Such a model can be applied to small laboratory animals, thus allowing the injection of a limited amount of molecules and avoiding the artifacts of anticoagulation.

The guinea pig was chosen because of its size and homology to human hemostasis. Guinea pig platelet membrane glycoproteins have been found to exhibit a very close homology to human platelet GPIIb/IIIa and GPIb/IX,11 12 and their platelet functions resemble those of human platelets in their responsiveness to ADP and the arachidonic acid cascade.13 In the present study, we evaluated the influence of a growing thrombus on platelet adhesion to the surrounding thrombogenic surfaces. The relationship between thrombus growth and platelet adhesion was studied using Lamifiban, a specific and synthetic GPIIb/IIIa antagonist that is active in the guinea pig,14 15 at three representative shear rates: venous (100 s-1), arterial (650 s-1), and mild stenosed arterial (1600 s-1). The results were analyzed by computer-assisted morphometry.


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
down arrowDiscussion
down arrowAppendix
down arrowReferences
 
Lamifiban (Ro44-9883), kindly provided by Dr S. Roux (F. Hoffmann–La Roche), is a synthetic nonpeptidic GPIIb/IIIa inhibitor with an IC50 of 25 and 450 nmol/L in human and guinea pig platelet-rich plasma,16 respectively. Lamifiban was injected intravenously into guinea pigs as a bolus 10 minutes before blood perfusion.

Animal Selection and Anesthetic Administration
Ninety male Hartley strain guinea pigs (Saint Antoine) weighing 700±100 g were used. Experiments were conducted according to the legislation of the French Ministry of Agriculture, the guidelines of the Institut National de la Santé et de la Recherche Médicale, and the institutional policies of the Institut National de la Recherche Agronomique. Guinea pigs were anesthetized intraperitoneally with 50 mg/kg sodium thiopental (Nesdonal, Specia Rhône Poulenc).

Capillary Perfusion Chamber
The capillary perfusion chambers A, B, and C consisted of glass capillaries that were 64, 100, and 127 mm long with radii of 0.315, 0.4, and 0.78 mm, respectively (Microcaps Drummond, Polylabo). Chamber A capillaries were used to create a mild stenotic arterial shear rate of 1600 s-1, chamber B capillaries to create an arterial shear rate of 650 s-1, and chamber C capillaries to create a venous shear rate of 100 s-1 at blood flow rates of 2.36, 1.96, and 2.06 mL/min for chambers A, B, and C, respectively. The Reynolds numbers were always <22, indicating laminar blood flow conditions in all tubes (Table 1Down, "Appendix").


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  		Table 1. Flow Characteristics of Capillary Perfusion Chambers

Blood entered and left the chambers through Silastic tubes (Sigma Medical) with radii of 0.317, 0.381, and 0.737 mm for the tubes connected to chambers A, B, and C, respectively. The proximal end of the collagen-coated capillary was connected to an identical capillary that was not collagen coated to avoid possible flow turbulence at the entrance to the thrombogenic capillary (Table 1Up, Fig 1Down).



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  		Figure 1. Schematic representation of the ex vivo perfusion apparatus. Two glass capillaries of identical radius were perfused at a fixed flow rate using a roller pump with nonanticoagulated blood drawn directly from the abdominal aorta of a guinea pig. The second capillary (black) was precoated with human type III collagen. Perfusion was performed at a constant temperature (37°C) using a permanent circulating water bath connected to another pump. After 4.5 minutes of perfusion, capillaries were rinsed for 20 seconds in a rinse buffer and then fixed in a fixative solution (see "Methods"). Three different shear rates were created by varying the radius of the capillaries and adapting their respective flow rates.

Coating of the Internal Capillary Surface With Collagen
Human type III collagen (Sigma Chimie) was purified from a lyophilized human placental pepsin extraction by selective salt precipitation.17 Collagen was allowed to polymerize and form fibrils by dialysis for 48 hours against 20 mmol/L Na2HPO4, pH 7.4.18 Internal capillary surfaces were prepared for coating with three washes of 10 minutes each with chromic acid, three rinses with deionized water, and drying at 75°C for 1 hour. Capillaries were filled by capillarity with a 1-mg/mL solution of human type III collagen. To obtain a homogeneous circumferential coating, they were centrifuged for 15 minutes in their longitudinal axis at 2100 rpm and stored at 22°C for 1 hour. They were then connected, undried, to Silastic tubing that was first filled with rinsing buffer ([mmol] 130 NaCl, 2 KCl, 12 NaHCO3, 2.5 CaCl2·2H2O, 0.9 MgCl2·6H2O, and 5 glucose, pH 7.4).

Characterization and Quantitation of Immobilized Collagen
The amount of collagen coating inside the glass capillaries was measured in a spectrophotometer at 230 nm with 125I-labeled collagen. The optical density measured in a fixed volume of buffer that rinsed the filled collagen capillary for 1 minute was compared with the optical density of an equal capillary volume of collagen added directly to the same fixed volume of buffer. Type III 125I-collagen was prepared according to the Bolton and Hunter method.19 The 125I-collagen deposited in the capillaries was pump rinsed with the rinsing buffer for 1 minute at the appropriate shear rate. The amount of collagen deposited in the capillaries was counted in a Beckman model 5500 gamma counter. The degree of removal of coated collagen was assessed by increasing the rinsing period. There were no significant differences between the amounts of collagen deposited in the capillaries after rinsing periods of 1 minute and 5.5 minutes as measured by spectrophotometry with 125I-labeled collagen. The distribution of collagen along the capillary was assessed by dividing the capillaries into 1-cm segments that were individually counted (n=3 for each perfusion period, Table 2Down). The amount of collagen in each proximal capillary section was twice that in the next five sections and varied from 2.52 to 2.79 µg/cm2 (Table 2Down). The thickness of the layer of collagen deposited was revealed by transmission electron microscopy (Philips CM 10), which revealed the deposition of a homogeneous layer along the capillary wall, with a mean thickness of 0.45±0.12 µm at the proximal end of the capillaries (mean±SEM, n=9, P.A., unpublished data, 1995). Some of the glass capillaries were examined with scanning electron microscopy (JEOL 35 C SEM). For this purpose, the internal surface of the capillaries was fixed in 1% glutaraldehyde and 4% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4), post-fixed in 0.1% osmium tetroxide, dehydrated in graded ethanols, critical point–dried with carbon dioxide, and coated by gold sputtering.20


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  		Table 2. Collagen Deposition and Distribution Along Capillary Segments

Perfusion System
Capillary chambers were perfused with nonanticoagulated blood drawn directly from the abdominal aorta punctured with a 19-gauge butterfly infusion set (Venisystems, Abbott Laboratories; Fig 1Up). The infusion set was then connected to the capillary chamber inlet through a T-piece shunt with Silastic tubing. The outlet of the capillary chamber was connected to a roller pump (2120 VarioperpexII Pump, LKB Bromma) to obtain selected blood flow rates. Just before the beginning of blood perfusion, the collagen-coated inner surface of the capillaries was rinsed with 2 mL of buffer at the respective shear rates of chambers A, B, and C. No bubbles were allowed to form before the blood reached the collagen-coated surface. Perfusion apparatus, tubing, and chambers were thermostated by immersion in a permanent circulating water bath at 37°C. The chambers were then perfused with blood for 4.5 minutes. At the end of that time, they were rinsed through the T piece with the rinsing buffer for 20 seconds and then fixed in 2.5% glutaraldehyde cacodylate (0.1 mol/L, pH 7.4) buffer at 4°C for 50 seconds at the appropriate shear rate. The reactive collagen-coated capillary was then separated from the Silastic tubing. Fixation was prolonged by immersing the capillary in freshly prepared fixative for 2 hours at 4°C and storing it in 7% sucrose and 0.1 mol/L cacodylate buffer (pH 7.4) at 4°C until further postfixation (see below). Thus, one capillary chamber was perfused for each guinea pig.

Postfixation, Dehydration, Epon Embedding, Sectioning, and Staining
Capillaries were rinsed with 0.1 mol/L cacodylate, pH 7.4, and then post-fixed with 0.05% KMnO4 for 1 hour. They were dehydrated, embedded in Epon 812, and allowed to polymerize at 50°C for 1 hour and then at 63°C for 16 hours.21 Glass capillaries were broken in water at 4°C under magnifying binoculars. The Epon rods that contained the platelet deposits at their periphery were reembedded in new Epon 812. Cross sections (1 µm) of the embedded preparation were cut perpendicular to the direction of the blood flow at 5 mm (proximal), 30 mm (middle), and 60 mm (distal) from the proximal end. Sections were stained and mounted on microscope slides according to the method of Sakariassen et al.21

Computer-Assisted Morphometric Quantitation of Semithin Sections
The microscope view of the sections was displayed on a color video monitor (Microvitec, HL series) at a final magnification of x1400 by a video camera (Sony, 3CCD) fitted on the photographic pathway of the microscope (Zeiss, Axioplan). Total platelet deposition, percent capillary surface coverage with platelets, percent surface coverage with thrombus >5 µm, mean thrombus height, and total thrombus surface area on the surface coated with collagen were automatically recorded and contrasted by a color-effect generator (NS15000). Data were managed with the Lucie program (Microvision) and a PC 486DX33 computer (Elonex). In accordance with the convention usually adopted by other users of in vitro or ex vivo perfusion chambers,21 thrombi were defined as platelet aggregates >5 µm. Total sectional thrombus surface area was calculated on the basis of the type B capillary cross-section area.

Ex Vivo Platelet Aggregation
Blood samples were collected in 0.129 mol/L trisodium citrate (9:1 vol/vol). Platelet-rich plasma was obtained by centrifugation at 200g for 10 minutes and platelet-poor plasma by centrifugation at 1000g for 20 minutes. Platelet aggregation was monitored by the turbidimetric method on a Chrono-log aggregometer (Coulter). Aggregation was induced by 1.5 µmol/L ADP (Diagnostica Stago), 10 µg/mL human type III collagen (Sigma Chimie), and 1.5 mg/mL ristocetin (Diagnostica Stago). Platelet aggregation was reported as the variation in light transmittance.

Statistical Analysis
Student's t test was used to test the effect of collagen deposition on platelet coverage and thrombus formation. Two-way ANOVA was used to test the effects of Lamifiban on platelet adhesion (percent capillary surface coverage with platelets), thrombus formation (mean thrombus height), and fibrin deposition (percent fibrin). Multiple comparisons were performed with the Tukey test. Values of P<.05 were considered significant.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
*Results
down arrowDiscussion
down arrowAppendix
down arrowReferences
 
Platelet and Fibrin Deposition With Blood From Untreated Animals
Quantitative Analysis
Platelet adhesion (capillary surface coverage with platelets). Maximal adhesion was observed at the proximal part of the type III collagen–coated capillaries. Platelet adhesion at the proximal end of the capillary decreased with increasing shear rates from 63% at 100 s-1 to 60.5% at 650 s-1 and 45% at 1600 s-1. Platelet adhesion decreased with length along the internal capillary surface (Table 3Down).


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  		Table 3. Effects of Shear Rate and Axial Position on Thrombogenesis

Thrombus size. At the proximal part of the capillaries, the percentage of thrombus was shear rate–dependent (0.75% at 100 s-1, 31% at 650 s-1, and 39.8% at 1600 s-1) and so was mean thrombus height (2.5, 13.8, and 28.1 µm at 100, 650, and 1600 s-1, respectively). Both the percentage of thrombus and mean thrombus height decreased along the capillaries from the proximal to distal parts (Table 3Up).

Fibrin deposition. Fibrin deposition was minimal at the proximal part of the three capillaries (value <5%). A significant axial increase in fibrin deposition (P<.001) was observed from the proximal to distal parts of the capillaries for the three shear rates as follows: from 5.1% (proximal) to 80% (distal) at 100 s-1, 3.5% (proximal) to 13% (distal) at 650 s-1, and 2.7% (proximal) to 11.5% (distal) at 1600 s-1.

Qualitative Analysis: Relation Among Platelet Adhesion and Thrombus and Fibrin Formations
Increasing the shear rate increased mean thrombus height at the proximal part of the capillary and reduced platelet adhesion (Table 3Up). The decrease in adhesion was related to a reduction of the number of thrombi, whose size increased (Fig 2ADown through 2D). At the venous shear rate, platelet adhesion dropped along the capillary from 63% (proximal) to 4.5% (distal), which is related to an increase in fibrin formation from 5.1% (proximal) to 80% (distal).



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  		Figure 2. Scanning electron photomicrographs of capillary-coated internal surface after 4.5 minutes of perfusion with nonanticoagulated blood at wall shear rates of 100 (A), 650 (B), and 1600 s-1 (C). At 100 s-1, fibrin strands were parallel to the blood flow. Platelets and red blood cells were captured in the fibrin meshwork. At 650 s-1, fibrin strands were smaller and thrombus size increased. At 1600 s-1, there were no longer any fibrin strands, thrombus size increased, and the number of small thrombi decreased. Thrombi were mainly composed of platelets that had initially adhered inside the collagen network (D). Original magnification: A, x1100; B, x220; C, x220; and D, x22 000.

Effects of Increasing In Vivo Administration of the GPIIb/IIIa Antagonist (Lamifiban) on Ex Vivo Platelet Deposition at the Proximal Part of the Collagen-Coated Capillary
Quantitative Analysis
Platelet adhesion. At shear rates of 100 s-1 and 650 s-1, none of the three doses of Lamifiban (0.3, 1, and 3 mg/kg) significantly affected platelet adhesion. At 1600 s-1, the lowest and highest doses significantly reduced adhesion (30.5% and 25%, respectively; P<.05), but the intermediate dose increased platelet adhesion (62.5%, P<.05) (Fig 3Down).



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  		Figure 3. Graphs show effects of Lamifiban at shear rates of 100, 650, and 1600 s-1 on the percentage of surface coverage with platelets (A), mean thrombus height (B), and total thrombus surface area (C) after 4.5 minutes of perfusion with nonanticoagulated blood. Lamifiban (0.3, 1, and 3 mg/kg) was administered 10 minutes before the perfusion (n=6 for each shear rate and dose). *P<.05, **P<.01, and ***P<.001.

Thrombus size. At the shear rate of 100 s-1, none of the three Lamifiban doses significantly affected thrombus formation. At shear rates of 650 s-1 and 1600 s-1, Lamifiban induced a dose-dependent decrease in thrombus formation (Figs 3BUp and 3CUp and 4A, 4B, and 4C).

At 1600 s-1 and 1 mg/kg, mean thrombus height decreased from 28.1 (control) to 6.5 µm, and this decrease was correlated with a significant increase in platelet adhesion.

Fibrin deposition. Lamifiban did not significantly affect axial fibrin formation along the capillary at any shear rate.

Qualitative Analysis
At 100 s-1, none of the doses of Lamifiban significantly affected the relative distribution of the reactive surfaces covered by contact or spread platelets, or of those involved in thrombus (Fig 5ADown). At 650 s-1, Lamifiban increased the relative percentage of contact platelets. The percentage of spread platelets remained stable, but the thrombus percentage decreased (Fig 5BDown). The greatest effect of Lamifiban was observed at 1600 s-1. At this shear rate, it induced a dose-dependent decrease in the percentage of thrombus, which correlated with a linear increase in the relative surface covered by contact platelets (Fig 5CDown).



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  		Figure 5. Graphs show distribution of platelets with increasing doses of Lamifiban at 100 (A), 650 (B), and 1600 s-1 (C). Platelets were clustered in a thrombus (Thr), spread on the thrombogenic surface (S), or in contact with the thrombogenic surface (C).

Effects of Lamifiban on Ex Vivo Platelet Aggregation
Increasing doses of Lamifiban had no effect on ristocetin-induced ex vivo platelet agglutination (Table 4Down). The highest dose of Lamifiban (3 mg/kg) totally inhibited both ADP- and collagen-induced aggregation. The intermediate dose (1 mg/kg) inhibited ADP-induced aggregation by 84% and collagen-induced aggregation by 78%. The lowest dose (0.3 mg/kg) inhibited ADP-induced aggregation by 55% and collagen-induced aggregation by 49%.


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  		Table 4. Effects of Lamifiban on Platelet Aggregation


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
up arrowResults
*Discussion
down arrowAppendix
down arrowReferences
 
Thrombus composition varies according to shear stress. At high shear stress it is mainly composed of platelets,1 22 23 thus explaining their predominant role in cardiovascular and cerebrovascular diseases. The molecular interactions involved in platelet adhesion and aggregation have been extensively studied from a biochemical point of view, but fewer experiments have been designed to study the hemorheological circulation conditions that might preferentially induce platelet aggregation or adhesion and their interrelationship. Platelet adhesion to subendothelial components24 is mediated by at least one plasma component, von Willebrand factor (vWF),4 and by at least three distinct platelet receptors, ie, GPIb/IX, GPIIb/IIIa,25 26 27 28 and GPIa/IIa.29 30 31 32 33 34 35 36 At high shear rates, GPIb/IX is the specific platelet receptor for subendothelial-bound vWF in the initial contact phase.37 38 39 This phase induces platelet activation and spreading and then thrombus formation, which involves the binding of adhesive molecules including fibrinogen and vWF to activated GPIIb/IIIa.40 41 The predominant involvement of GPIb/IX-vWF in platelet adhesion and GPIIb/IIIa-fibrinogen in thrombus formation has been established in anticoagulated blood,27 42 43 but the in vivo mechanisms governing these processes remain partially unknown. To find out how circulation conditions could favor either thrombus growth or platelet adhesion, the present experiments were performed in an original perfusion chamber with pharmacological modulation of GPIIb/IIIa reactivity by a molecular antagonist, Lamifiban.

For this study, we developed a new type of capillary perfusion chamber that allowed us to study ex vivo thrombogenesis in small animal species. Capillaries of various diameters were chosen to mimic the range of physiological blood shear rates present in the vasculature, and these capillaries were coated with human type III collagen. The guinea pig was chosen because of its small size and the homology exhibited by its platelet glycoprotein receptors to the human receptors involved in thrombogenic mechanisms.11 12 Lamifiban was chosen because it has been shown to be active in the guinea pig by abolishing cyclic flow variations in an in vivo model of arterial thrombosis.14

In the present system, human type III collagen induced ex vivo platelet adhesion and thrombus formation in flowing, native, nonanticoagulated guinea pig blood. Platelet deposition was dependent on reactive surface length. Nevertheless, the axial dependence of platelet-collagen interaction previously reported by Sakariassen and Baumgartner44 was present at all the shear rates tested. This dependence was apparently enormously increased at the venous blood shear rate in our setting. At this shear rate, the drop in platelet adhesion from the proximal to distal end of the capillary did not depend on the upstream consumption of platelets because there were no formed thrombi in the capillary; it was due to the marked increase in fibrin formation. Fibrin reactivity is perhaps species dependent, since it was much higher than what we had observed in humans (P.A., unpublished data, 1995).

At the proximal part of the capillary, raising the shear rate induced a significant increase in thrombus growth, which correlated with a moderate decrease in platelet adhesion. One possible explanation for this observation is that the local microturbulences created by the growing thrombus prevented the platelets from coming into contact with the surrounding collagen. An alternative possibility is that the thrombus surface is more thrombogenic than the collagen surface, which is in agreement with the findings of Badimon et al.45 Our data differ from those of Weiss et al37 and Sakariassen et al,6 who, using anticoagulated and nonanticoagulated blood, found shear rate–dependent increases in both platelet adhesion and thrombus formation. This might be because in our experiment (1) the capillaries were coated with 10 times less collagen than in previous rectangular perfusion chamber studies,6 (2) the collagen never dried, and (3) the species reactivity of platelets may affect the quality of platelet deposition. The shear rate–dependent increase in platelet adhesion at the middle part of the capillary might be related to the decrease in mean thrombus height observed at this level, in accordance with our hypothesis that a growing thrombus slightly reduces platelet adhesion in the guinea pig.

Using incremental doses of Lamifiban, we studied the effects of inhibiting GPIIb/IIIa on the platelet adhesion and thrombus formation induced in type III collagen–coated capillaries at three shear rates in nonanticoagulated blood.

At the venous blood shear rate, Lamifiban had no effect on platelet adhesion in the proximal part of the capillary, where fibrin deposition did not influence platelet deposition. This seems to indicate that at the venous blood shear rate, the involvement of GPIIb/IIIa in platelet contact and spread is minimal. At the higher shear rates, raising the Lamifiban dose significantly reduced thrombus height, but platelet adhesion did not correspond to the degree of inhibition of GPIIb/IIIa. The low dose had a minimal effect on both platelet adhesion and thrombus growth, whereas it induced a 50% inhibition of ADP and collagen-induced ex vivo platelet aggregation. One explanation is that in vivo, numerous mechanisms (such as the vWF-GPIb/IX axis) are implicated in thrombus formation in the guinea pig,46 but such mechanisms did not occur in the aggregometer model. The medium dose significantly inhibited thrombus growth and, due to the balance between adhesion and thrombus formation, apparently increased platelet adhesion; the high dose abolished thrombus formation and impaired adhesion. Our observation of an apparent balance between platelet adhesion and thrombus formation is in agreement with other in vitro and ex vivo observations.47 48 The involvement of the GPIIb/IIIa axis in platelet spreading deduced from our results with Lamifiban is in accordance with the results of experiments with monoclonal antibodies and thrombasthenic platelets.26 49 50

If experimental thrombus growth is indeed related to clinical thrombogenesis and experimental platelet adhesion and spreading are related to clinical hemostasis, the latter observations are of great clinical interest. The GPIIb/IIIa antagonists currently tested in numerous clinical studies have had promising effects on high-risk coronary angioplasty, but these effects were offset by a severe risk of bleeding.51 In our experiments, 1 mg/kg Lamifiban induced a 1.5-fold increase in bleeding time versus the 12-fold increase induced by 3 mg/kg (unpublished data). From the present data, we may justifiably postulate that a specific intermediate dose of GPIIb/IIIa antagonist could partially block the GPIIb/IIIa receptor and induce antithrombotic but not antiadhesive effects, with minor effects on the hemorrhagic risk.



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  		Figure 4. Representative light photomicrographs show the effects of Lamifiban at a wall shear rate of 1600 s-1 after NaCl infusion (A) and boluses of 1 (B) and 3 mg/kg Lamifiban (C). Note that Lamifiban reduced thrombus size and increased platelet adhesion (B) or decreased it (C). Sections were cut perpendicular to the direction of the blood flow 5 mm downstream of the proximal end (P) of the capillary. Original magnification x400.


*    Acknowledgments
 
This study was supported by a scholarship from the Sanofi Foundation, Paris, France (P. André). We thank Dr Françoise F. Lafève for 125I-labeling and Khalid Azzam for expert assistance.


*    Appendix
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
up arrowResults
up arrowDiscussion
*Appendix
down arrowReferences
 
The capillary chamber was used because of its well-known fluid dynamic and mass transport properties and its similarity to blood vessel geometry. With this chamber, fluid movement is well defined and is dependent on the volumetric flow rate (Q), pressure drop (P), tube length (L), radius (R), and fluid viscosity (µ), according to the well-known Hagen-Poiseuille equation52 : Q={Delta}P{pi}R4/8µL. The velocity gradient or shear rate ({gamma}) reflects the local movement of fluid near the wall. At the wall, the shear rate value ({gamma}w) is given by the following equation53 : {gamma}w=4Q/{pi}R3.

Nevertheless, blood is not a homogeneous newtonian fluid. It is a concentrated suspension of cells and proteins and may be treated as homogeneous, with viscosity dependent primarily on the red blood cell concentration. Plasma viscosity and red blood cell deformability and aggregation are relatively minor parameters compared with the red blood cell concentration. For most of the physiological range of shear rates, blood viscosity is relatively constant (in the range of 0.03 to 0.04 mPa/s at 37°C) and does not rise above 100 s-1.53 Guinea pig blood viscosity at 37°C was reported to be 0.035 mPa/s at 100 s-1 and 0.032 mPa/s at >200 s-1.54 Reynolds number (Re) reflects the ratio of inertial to viscous terms and gives further information about flow stability: Re=2Q 114 /{pi}µR. As blood enters a tube from a large reservoir, there is an entrance length (Le) of parabolic profile establishment, in which the shear stress, which is directly proportional to the shear rate ({tau}={eta}{gamma}), increases: Le=0.07R·Ret. In this study, shear rates were calculated according to these rheological parameters.

Received June 19, 1995; accepted October 11, 1995.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
up arrowResults
up arrowDiscussion
up arrowAppendix
*References
 

  1. Baumgartner HR. The role of blood flow in platelet adhesion, fibrin deposition and formation of mural thrombi. Microvasc Res. 1973;5:167-179. [Medline] [Order article via Infotrieve]
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  3. Baumgartner HR. Effect of anticoagulation on the interaction of human platelets with subendothelium in flowing blood. Schweiz Med Wochenschr. 1976;106:1367-1368. [Medline] [Order article via Infotrieve]
  4. Muggli R, Baumgartner HR, Tschopp TB, Keller H. Automated microdensitometry and protein assays as a measure for platelet adhesion and aggregation on collagen-coated slides under controlled flow conditions. J Lab Clin Med. 1980;95:195-207. [Medline] [Order article via Infotrieve]
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