R. Andries

The effect of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (paf-acether) on the arterial wall.

The effect of a topical paf-acether superfusion over an injured arterial segment was assessed in the guinea-pig, using an opto-electronic in vivo thrombosis model allowing on-line quantification of small platelet thrombus dynamics. As compared to control, ADP-induced, thromboformation and behaviour, exogenous paf-acether causes a large, dense platelet thrombus, invaded and surrounded by numerous leukocytes, spreading widely over the adjoining, vacuolized, endothelium. Its embolization has to be forced with prostanoids, mepacrine, EDTA, or with a specific paf-acether antagonist (BN 52021). A few minutes after such forced embolization, a new thrombus starts growing at the same site, without renewal of the paf-acether superfusion. This phenomenon of spontaneous reappearance after forced embolization can be followed during several hours. Experiments with labelled paf-acether and the paf-acether antagonist indicate a possible endogenous paf-acether (or paf-acether-like) production triggered by superfusion with exogenous paf-acether.

Modulation of prostacycline synthetase by cicletanine and drugs which affect ion transport

Cicletanine, a drug which affects membrane ion transport, induces a marked increase of the liberation of PGI2 as demonstrated by the increase of the stable metabolites in the plasma following intravenous administration of arachidonic acid. Furthermore, the inhibiting effect of tranylcypromine on prostacyclin synthetase is completely removed by this pharmacon. These observations are suggestive that this drug presents a scope for treatment of thrombotic disorders as well as hypertension.

Thrombus induction by endogenic PAF-acether and its inhibition by Ginkgo Biloba extracts in the guinea pig

The anti-thrombotic effects of specific paf-acether antagonist BN 52021 were compared to the effects of Ginkgo Biloba extracts A, B, (A + B), and C. Local superfusion of BN 52021 over an experimentally injured arterial segment embolizes an existent paf-acether induced platelet thrombus. When applied before paf-acether, BN 52021 prevents local thromboformation in this model. Applied intravenously, BN 52021 reduces local thromboformation in a significant way. As compared to this BN 52021 standard, only Ginkgo Biloba B and the (A + B)-mixture present major thromboreductive activity.

Endogenic PAF-acether production by Guinea pig endothelial cells in experimental arterial thrombosis

Superfusion of PAF-acether over a branch of the mesenteric artery in the guinea pig invariably results in local endothelial injury and thrombus formation within 3-10 minutes. The thrombotic phenomena do not disappear when PAF-acether superfusion is discontinued, and even when forced embolization is induced. Within a very short interval renewal of thrombosis occurs at the same site. Several data point to a mechanism involving generation and release of endogenic PAF-acether. Recents findings on PAF-acether release by cultured endothelial cells indicate that in the in vivo situation this phenomenon could well be responsible for maintaining the thrombotic status as demonstrated by ultrastructural analysis. In a later stadium polymorphonuclear leukocytes are also involved in total thromboformation.

Endothelial Injury and Platelet Thrombosis in Mesenteric Arteries of Rats: A Scanning Electron Microscopy Study

For several years, an in vivo model for the induction and on-line quantification of arterial platelet thrombosis in mesenteric arteries of a small laboratory animal species has been developed in our laboratory. In the present paper, we further document the intimal lesions and the ADP superfusion-induced local platelet thrombus as seen in the scanning electron microscope. The surface morphology of the intimal lesion, induced by electric current, shows a circular or slightly oval denuded area, affecting about 15-20 endothelial cells. The edge of this lesion is often occupied by partially disrupted and detached endothelial cells. The successive embolizations of several ADP thrombi clean this edge and augment the denuded area. The final lesion never exceeds the area of 30-40 endothelial cells. ADP-induced platelet thrombi in invariably appear as loose, sponge-like platelet aggregates, very bloodstream-lined, anchored on the denuded subendothelium. There is an excellent correlation between the in vivo light microscopic observations and the actual ultrastructure of this platelet mass.

PAF-Acether Induced Arterial Thrombosis and the Effect of Specific Antagonists

Platelet-vessel wall interactions and local thrombosis are investigated in vivo in a branch of the mesenteric artery of the guinea pig, using optoelectronic registration and ultrastructural control. Following an electrical challenge resulting in changes of cell membrane polarization, subsequent superfusion by PAF-acether or a stable analogue, (1-O-alkyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine, 10(-8) M focal concentration (f.c.)) for a restricted period results in endothelial cell retraction and bleb formation followed by platelet adhesion and the development of a thrombus which over time becomes invaded by leukocytes and eventually occludes the vascular lumen. It was demonstrated in a previous investigation that these phenomena are triggered by the generation of endogenous PAF-acether by the endothelial cells. Specific PAF-acether-antagonists, such as BN 52021 a ginkgolide, but also synthetic molecules, derivatives of the triazolo-pyridino-diazepine group (BN 50727, BN 50755 and BN 50789), significantly inhibit platelet-vessel wall interactions and thrombosis, but not the formation of blebs in the endothelial cells. Hydrogen peroxide (10(-5)M f.c.) not only primes the effect of PAF-acether, but is by itself capable of inducing thrombosis through a PAF-acether-mediated mechanism. Inhibition of acetyl hydrolase by PMSF (phenyl-methyl-sulfonyl-fluoride, 10(-5)M f.c.) invariably results in a significant enhancement of thrombosis, while conversely, inhibition of acetyl transferase by 27584 RP (4-(naphtylvinyl)pyridine hydrochloride, 10(-6)M f.c.) inhibits thromboformation indicating that the remodeling pathway is involved.

The effect of inhibition of endothelial cell cyclooxygenase on arterial thrombosis.

Arterial platelet thrombosis was induced in vivo in a branch of the mesenteric artery of the white Wistar rat by topical superfusion by adenosine diphosphate following local de-endothelialization of the dissected segment by means of a small electrical current. Detection of the thrombotic phenomena was performed by projection of the arterial segment onto a set of light sensitive elements, which allows the registration of several discriminating parameters. The addition of arachidonic acid to the superfusing mixture results in an increase in thromboformation; this increasing effect can be blocked completely by prior inhibition of the vessel wall cyclooxygenase activity by inhibitors such as flurbiprofen or indomethacin. We therefore concluded that the arachidonate cascade, triggered within the endothelial cells, is involved in platelet-vessel wall interaction leading ultimately to local thrombosis.

In vivo arterial platelet-vessel wall interaction and thrombosis: induction, on-line registration and ultrastructural control.

A technique for induction and on-line quantification of local platelet thrombi in mesenteric arteries of small laboratory animals was developed and standardized in our laboratory. In the past, this model was used to study the nature of platelet-vessel wall interaction in the living animal. The ultrastructure of the experimental intimal lesion and the vessel wall regeneration were assessed by transmission electron microscopy (TEM), both in normal and pathologic conditions. Scanning electron microscopy (SEM) now shows the ultramorphology of platelet thrombi on the experimentally injured arterial segment following topical superfusion with ADP, mepacrine or platelet-activating factor (PAF). The application of these substances, each with proper bio-activity, leads to distinct types of platelet thrombi. Mepacrine or PAF superfusion causes large thrombotic masses, as compared to control, ADP induced thrombi, and seems toxic for the endothelial cells. Mepacrine thrombi differ significantly from PAF thrombi in their platelet density, degree of platelet activation and in their relation to the endothelium that surrounds the experimental lesion. Furthermore, PAF superfusion induces a phenomenon of spontaneous regeneration of the thrombus after its forced embolization. This is probably due to some unknown bio-action of PAF in the vessel wall.

Early morphologic changes in coronary arteries of experimental diabetic rats

SummaryDiabetes was induced in 24 adult male Wistar rats by a single intravenous injection of streptozotocin. The ultramorphology of the coronary arterial wall was studied by scanning and transmission electron microscopy up to 10 weeks following this diabetes induction. At that moment, several ultrastructural characteristics of primary atherogenesis could be observed, i.e. important myointimal thickening with extensive medial smooth muscle cell migration towards and into the intima, and synthesis of an abundant mass of intercellular ground substance in the media and in subendothelial areas. Lipid deposition or foam cell formation was not observed. These results confirm that experimental diabetes is a risk factor for the coronary arterial wall as it induces typical atherogenic phenomena.

Arterial Obstruction Induced by PAF-Acether (1-0-Alkyl-SN-Glycero-3-Phosphocholine)

Platelet activating factor (PAF-acether) is a very potent mediator which is involved in many inflammatory and allergic reactions (Demopoulos, Pinckard and Hanahan, 1979; Vargaftig et al., 1981). Since its discovery in 1972 by Benveniste, Henson and Cochrane it has been demonstrated that this agent can induce leukopenia (O’Flaherty et al., 1981) and plasma leakage at the venular site of the vascular compartment (Bjork et al., 1983) followed eventually by arterial hypotension and death. In a previous paper (Bourgain et al., 1985), we demonstrated the effect of PAF-acether on the arterial wall in the guinea-pig following topical superfusion of the mediator in well standardized conditions. It became evident from these investigations that within two to three minutes following a three minute application of the mediator (10−7 M), a platelet thrombus developed at the site of superfusion. Over time, this platelet thrombus recruited leukocytes; these invaded the thrombotic mass and simultaneously underwent diapedesis. It happened that fragments of the thrombotic mass detached from the main structure and embolized; nevertheless the thrombus increased in size and occlusion of the vascular lumen occasionally occurred, leading to peripheral ischaemia and tissue hypoxia.