The calcium inhibitor SR33805 reduces intimal formation following injury of the porcine carotid artery.

We studied the effect of SR33805, a calcium channel blocker, in vitro on the proliferation of vascular smooth muscle cells (SMC) stimulated by foetal calf serum, basic fibroblast growth factor and platelet derived growth factor, and in vivo with regard to SMC migration and proliferation which occurred following injury of the porcine carotid artery. The intimal lesion was induced by a silasten collar surgically positioned around the carotid artery and by a stenosis reducing blood flow by 50% for 30 days. Animals received SR33805 (5 mg/kg/day) 8 days before the induction of the lesion and up to 30 days after. In vitro, SR33805 inhibited in a dose-dependent manner growth factor-induced proliferation of SMC (0.20

[The role of thrombotic and hemostatic mechanisms in the initial phases of atherosclerosis]. / Participation des mécanismes de la thrombose et de l'hémostase aux étapes initiales de l'athérosclérose.

The factors of thrombosis (endothelium, haemostasis, coagulation, fibrinolysis) are implicated from the initiating phase of atherosclerotic lesions. Their participation is more established (and studied) in the later phases of intraluminal evolution of atherosclerosis, of thromboembolic complications of the lesions and interventional procedures. The traditional theory of response to physical lesions of the endothelium as an initiating factor of atherosclerotic lesions, which gave platelets an essential role, has been replaced by that linking an early functional lesion of the endothelium and a cellular response by monocytes infiltrating the vessel wall, becoming macrophages. The macrophages participate in changes of the LDL in the wall, ingest the lipids at the same time as the smooth muscle cells which have migrated and proliferated from the media to the intima. The lipid overload, especially with oxidised LDL, is intracellular at first in these foam cells, then extracellular as the cells die. During the early stages, all the tissue factors of activation and development of coagulation are present in the vessel wall and then within the lesion. This intra-cellular coagulation results in the production of thrombi in the tissues and the transformation of fibrinogen to fibrin. These stages precede and participate in cellular proliferation and extracellular lipid deposits. Factors of tissular thrombolysis (the uPA pathway) play a part in cellular immigration and proliferation. It is only at a later stage that the lesion activates intravascular coagulation and fibrinolysis which, in conditions of variable equilibrium, will result in the clinical complications of the atherosclerotic process. All these factors therefore participate firstly in the tissues and then within the lumen, in the progression and complications of atherosclerosis which for these reasons is often called atherothrombotic disease. The comprehension of these mechanisms is essential for the development and interpretation of tests and treatment applied to different stages of the disease, which is all the more complex given that in a given patient at a given time, lesions at different stages are present in the arterial network.

Role of plasma and platelet von Willebrand factor in arterial thrombogenesis and hemostasis in the pig.

To evaluate the relative role of plasma and platelet von Willebrand factor (vWF) pools in hemostasis and arterial thrombogenesis, pigs with vW disease (vWD) were injected with vWF concentrate and/or grafted with bone marrow from a normal pig. Hemostasis was assessed by measurement of ear immersion bleeding time, factor VIII (FVIII) activity, and plasma and platelet vWF antigen levels. The thrombotic process was explored at 650 s(-1) and 1600 s(-1) in an ex vivo cylindrical perfusion chamber. Pigs with vWD exhibited a prolonged bleeding time (>30 minutes) compared with normal pigs (<5 minutes); in addition, they showed normal platelet adhesion and thrombus formation at 650 s(-1) but profoundly reduced platelet adhesion and thrombus formation at 1600 s(-1). Each experiment was performed before and 3 and 24 hours after injection of vWF concentrate. In our bleeding time study, only plasma vWF restoration induced a partial but delayed correction (24 hours postinjection), which was correlated with the highest measured level of FVIII activity. In the perfusion chamber model, restoration of plasma or platelet vWF pools resulted in similar partial correction of platelet adhesion and average thrombus size. In the perfused pigs, the maximum correction occurred 3 hours postinjection. Platelet deposition reached normal values after vWF concentrate was injected into a grafted pig. The present results suggest that when both plasma and platelet vWF levels are restored in vWD pigs, bleeding time and the thrombotic process are normalized according to different kinetics and with differing degrees of effectiveness.

Relative involvement of GPIb/IX-vWF axis and GPIIb/IIIa in thrombus growth at high shear rates in the guinea pig.

The relative involvement of the glycoprotein (GP) Ib/IX-von Willebrand factor (vWF) axis and GPIIb/IIIa in thrombus growth at high shear rates was assessed and compared by testing the pharmacological effects of VCL, a recombinant GPIb-binding fragment of vWF (residues 504-728), aurintricarboxylic acid (ATA), which binds to the 509-695 disulfide loop of vWF, and lamifiban, a specific synthetic GPIIb/IIIa antagonist. In vivo, their effects were evaluated in guinea pig mesenteric arteries, in a model of a laser-induced cyclic thrombotic process, and ex vivo, at a shear rate of 1800 s(-1), in a capillary perfusion chamber model, in which collagen-adherent platelets are exposed to nonanticoagulated guinea pig blood. In vivo, VCL, ATA, and lamifiban administered 2 minutes after intimal injuries stopped thrombus growth, prevented the cyclic thrombotic process, and induced gradual thrombus dissolution. Ex vivo, at 1800 s(-1), collagen exposure to untreated blood for 2 minutes, 4 minutes, or two consecutive periods of 2 minutes each resulted in similar platelet adhesion, 56%, 59%, and 61%, respectively, with an average thrombus volume of 6, 19, and 17.5 microm3/microm2, respectively, without any fibrin formation. This indicated that the two consecutive perfusions did not affect the dynamic process of thrombus growth. When collagen-adherent platelets deposited after the first 2-minute perfusion were perfused for 2 minutes with VCL-, ATA-, or lamifiban-treated blood, thrombus growth was prevented and platelet adhesion remained unchanged, but fibrin formation increased on and around the predeposited platelets. These results suggest that both the GPIb/IX-vWF axis and GPIIb/IIIa are involved in in vivo platelet-to-platelet interactions at high shear rates in the guinea pig.

[What are the criteria for choosing a model for evaluating arterial antithrombotic therapy?]. / Sur quels critères choisir un modèle pour évaluer une thérapeutique antithrombotique artérielle?

The use of models of experimental arterial thrombosis both in vivo and ex vivo in animals and ex vivo in humans is an obligatory step to the understanding of mechanisms involved in thrombogenesis as well as in the evaluation of anti-thrombotic therapeutics. Arterial thrombogenesis is a complex phenomenon which involves multiple systems, mechanisms and parameters. Therefore studies of thrombogenesis from a pathological as well as a therapeutic point are necessary for understanding this problem in its entirety. For these reasons, it is necessary to use models as representative as possible of the human pathological condition. Besides these theoretical requirements, practical needs have also to be fulfilled (accessibility of the models, adaptation to the type of the technique to different animal model and/or of the size of the animal to the amount of molecule available, cost ...) which necessary lead to some promises. In this review we have tried to underline the criteria for the choice, characteristics, advantages and disadvantages of the major models commonly accepted and used, in such a form that the reader who may not be an expert in the field would be led either to choose a particular model for a specific purpose or to appreciate a paper or a report based on an experimental model of arterial thrombosis. In vitro models of arterial thrombosis are so far removed from reality and due to their nature can generate so much artifacts thus we have omitted their discussion from this paper.

Which experimental model to choose to study arterial thrombosis and evaluate potentially useful therapeutics?

The use of experimental models of arterial thrombosis both in vivo and ex vivo in animals and ex vivo in humans is an obligatory step for the understanding of mechanisms involved in thrombogenesis as well as in the evaluation of anti-thrombotic therapeutics. Arterial thrombogenesis is a complex phenomenon which involves multiple systems, mechanisms and parameters. Therefore studies of thrombogenesis from a pathological as well as a therapeutic point are necessary for understanding this problem in its entirety. For these reasons, it is necessary to use models as representative as possible of the human pathological condition. Besides these theoretical requirements, practical needs have also to be fulfilled (accessibility of the models, adaptation to the type of the technique to different animal model and/or of the size of the animal to the amount of molecule available, cost...) which necessarily lead to some compromises. In this review we have tried to underline the criteria for the choice, characteristics, advantages and disadvantages of the major models commonly accepted and used, in such a form that the reader who may not be an expert in the field would be led either to a choice of a particular model for a specific purpose or to appreciate a paper or a report based on an experimental model of arterial thrombosis. In vitro models of arterial thrombosis are so far removed from reality and due to their nature can generate so much artifacts that we have omitted their discussion from this paper.

Optimal antagonism of GPIIb/IIIa favors platelet adhesion by inhibiting thrombus growth. An ex vivo capillary perfusion chamber study in the guinea pig.

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 microns at 100 s-1, 60.5% and 13.8 microns at 650 s-1, and 45% and 28.1 microns 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.