Robert G. Schaub

Human protein c induces anticoagulation and increased fibrinolytic activity in the cat

The effect of activated human Protein C (PCa) infusion on the coagulation and fibrinolytic systems of the cat was examined. Cats received bolus i.v. injections of PCa of 3 micrograms/mL (n = 7), 10 micrograms/mL (n = 2) or 16 micrograms/mL (n = 1) of calculated total blood volume. Control cats (n = 7) received either unactivated Protein C (10 micrograms/mL) or the activation vehicle containing heparin and antithrombin in tris-saline buffer. Citrated blood samples were drawn prior to infusion and 5, 10, 20, 40, 60, 120 and 180 minutes post infusion. PCa caused an immediate, dose dependent increase in anticoagulation as measured by APTT which began to normalize within 20 minutes. PCa also increased fibrinolytic activity measured by euglobulin clot lysis time and an 125I whole blood clot lysis assay. However, the profibrinolytic effect of PCa did not parallel the anticoagulant effect. In all treated cats, maximum fibrinolytic activity did not occur until 40-120 minutes after infusion. Control cats demonstrated no significant change in coagulation or fibrinolytic activity. Our results demonstrate that infusion of activated human Protein C can induce anticoagulation and fibrinolytic activity in the cat.

Acetyl glyceryl ether phosphorylcholine (PAF-acether) and leukotriene B4-mediated neutrophil chemotaxis through an intact endothelial cell monolayer

Human endothelial cell monolayers were grown on nucleopore filters, and used to partition the two halves of a modified Boyden chamber. Human neutrophil chemotaxis through the monolayer was studied in response to leukotriene B4 and acetyl glyceryl ether phosphorylcholine (PAF-acether). Both leukotriene B4 and PAF-acether concentration-dependently stimulated neutrophil chemotaxis through intact monolayer. The biologically inactive lyso-PAF, and leukotriene C4 and D4 were inactive as chemotactic agents. Leukotriene A4 was weakly chemotactic. In the absence of chemotaxin, little penetration of the monolayer by neutrophils was observed. Agents that elevate neutrophil cyclic AMP levels inhibit both leukotriene B4 and PAF-acether-stimulated chemotaxis through the endothelial cell monolayer. The specific 5-lipoxygenase inhibitor, 6,8-de-epoxy-6,9-(phenylimino) delta 6,8-prostaglandin I1 (U-60257), inhibits PAF-acether, but not leukotriene B4-mediated chemotaxis. These data suggest that an intact 5-lipoxygenase may be required for normal PAF-acether-mediated chemotaxis, but leukotriene B4-mediated chemotaxis is independent of 5-lipoxygenase activity. This system may prove to be a useful model for the study of neutrophil-endothelial cell interactions.

Feline polymorphonuclear leukocytes respond chemotactically to leukotriene B4 and activated serum but not to F-Met-Leu-Phe.

The chemotactic response of feline polymorphonuclear leukocytes (PMNs) to three types of chemoattractants was studied. Feline PMNs responded to leukotriene B4 as well as to agarose-activated autologous and homologous serum. However, no response was obtained to N-formylmethionylleucylphenylalanine (FMLP), and four similar peptides that activate the FMLP receptor (N-formylnorleucylleucylphenylalanine, N-formylmethionylphenylalanine, methionylleucylphenylalanine, and pepstatin.) Thus, feline PMNs are similar to equine, porcine, bovine and canine PMNs which also do not respond chemotactically to these peptides.

Leukocyte mediated vein injury and thrombosis is reduced by a lipoxygenase inhibitor

We have previously demonstrated an in vivo model of deep vein thrombosis which suggests that the neutrophil promotes vascular injury and thrombosis following blood flow stasis. Since leukotrienes are potent mediators of vascular injury and neutrophil (PMN) chemotaxis, we wished to determine if in vivo inhibition of 5-lipoxygenase would reduce neutrophil mediated events in our model. Lipoxygenase was inhibited in vivo with 2,3-diethyl-4-methoxy,1-naphthalenol acetate (U-66,855). The in vivo activity of U-66,855 was demonstrated in 4 cats. Each animal was treated with 5 mg/kg of U-66,855 intravenously. Blood cell leukotriene B4 (LTB4) and thromboxane A2, via its metabolite thromboxane B2 (TBX2) was assessed before and 30, 60, and 120 min after dosing. Blood cell LTB4 and TBX2 production was stimulated by A23187 (24 microM) and assayed by radioimmunoassay. We exposed and isolated a 3-cm segment of the jugular veins from 10 additional cats 5 of which were treated with U-66,855 (5 mg/kg, iv). In order to assess the effect of stasis, the jugular veins were ligated at the thoracic inlet for 2 hr after which the veins were perfused, fixed in 2.5% glutaraldehyde, and prepared for electron microscopy. U-66,855 reduced LTB4 production significantly (P less than 0.01), but not TBX2. In untreated cats, PMNs adhered to and migrated underneath the venous endothelium. Additionally, platelets, fibrin and formed thrombi were found on the basement membrane exposed by the migrating neutrophils. In contrast, we observed significantly reduced PMN adhesion as well as no fibrin deposition in veins obtained from cats treated with U-66,855. The results suggest that 5-lipoxygenase inhibition can significantly reduce undesirable neutrophil/vessel wall interactions.

Correlation of leukocyte interleukin-1 production with the stimulation of prostaglandin and tissue factor synthesis by human umbilical vein endothelial cells.

Human leukocyte suspensions (neutrophils 80–85%, monocyte 15–20%) were incubated alone or with cultured human umbilical vein endothelial cells. Leukocytes were either directly added to the endothelial cell cultures or separated from them by a 0.4 micron insert filter. Supernatants or cell lysates were obtained at 0.5, 1, 2, and 4 hours of incubation. Supernatants were assayed for the prostacyclin (PGI2) metabolite 6-keto prostaglandin F1α and prostaglandin E2 (PGE2) by radioimmunoassay and for interleukin-1 (IL-1) by the thymocyte co-mitogen assay. Cell lysates were analyzed for cell-associated procoagulant activity (PCA). Co-incubation of endothelial cells with leukocytes stimulated the synthesis of PGI2, PGE2, and PCA. These biochemical changes correlated partially with the release of IL-1 beta. The results suggest that IL-1 released in monocyte/neutrophil co-cultures can produce prothrombotic (increased PCA expression) and inflammatory changes (increased synthesis of vasodilatory and permeability enhancing PGI2 and PGE2) in endothelial cells. Neutrophils may represent a source of the released IL-1 and/or may act to stimulate monocyte release of this cytokine and thus play an important role in vascular pathology by a mechanism unrelated to their more direct cytotoxic activity.

Comparison of lipid accumulation and metabolism in carrageenan-induced granulomas to aorta and blood monocytes of normal and cholesterol-fed rabbits

New Zealand rabbits (six each) were either maintained on a standard chow diet (ND) or the chow diet supplemented with cholesterol/peanut oil (HD) for 2 weeks. After 2 weeks, each animal had 15 ml of a 1% carrageenan gel injected subcutaneously into the midabdominal area. Rabbits were maintained on the diets for an additional 4 weeks. At sacrifice, blood was collected both for serum and for monocyte isolation and granulomas and aorta were excised. Tissues were assayed for lipid composition and lipid metabolism. Electron and light microscopies were performed on granuloma tissue. Granulomas from ND animals did not stain with oil red O. Granulomas from HD animals had homogenous oil red O staining indicating lipid accumulation. Granulomas from both ND and HD animals consisted of macrophages. Macrophages from ND rabbits accumulated follicular carrageenan but not lipid, while HD macrophages had the appearance of foam cells. Granuloma lipid content and metabolism closely paralleled the aorta and blood monocytes. The HD tissue had increased acylCoA:cholesterol acetyltransferase (ACAT) activity and lipid composition changes reflective of the atherosclerotic process. ND granulomas had no elevation of lipid content or ACAT. The carrageenan-induced granulomas provide a useful model for studying the biochemical and morphologic changes characteristic of arteries undergoing atherogenic change.

Heparin inhibits fibrin, but not leukocytes, in a model of deep-vein thrombosis

Previous studies with models of deep-vein thrombosis (DVT) have demonstrated that leukocyte (PMN)-mediated vein injury may be an initiating event in DVT (14, 17). Since heparin (H) can prevent DVT, we studied its effect on vascular injury and thrombosis in our model. Three groups of rabbits were treated with H either sc (73 and 147 U/kg) or iv (662 U/kg). Scanning electron microscopy revealed that the 73 U/kg sc dose was ineffective. All veins had PMN accumulation, fibrin deposition and complex thrombus formation. There was no increase in anti-Xa activity; activated partial thromboplastin times (APTT) and whole blood clotting times were normal. The 147 U/kg sc and the intravenous dose did not inhibit PMN-mediated vein injury. The endothelium was sloughed by migrating PMNs, basement membrane was exposed, and platelets adhered to it. Thrombosis was completely absent in the iv dose group. This correlated with increased anti-Xa activity and prolonged APTT and whole blood clotting times. Our results indicate that heparin does not inhibit the PMN adhesion and migration which produces vascular injury. However, the anticoagulant activity of heparin effectively reduces fibrin deposition and complex thrombus formation.

Ciprostene, a stable prostacyclin analog, produces peripheral vasodilation, platelet inhibition and increased clot dissolution in the cat

The effect of the stable prostacyclin analog ciprostene on hemodynamic parameters, platelet aggregation and clot dissolution was examined in the sodium pentobarbital anesthetized cat. Hemodynamic and platelet aggregation effects were measured in 5 cats following infusion of 5, 10, 20, 40 and 80 micrograms/kg/min of ciprostene. Drug was dissolved in Tyrodes buffer (pH 7.4) and all doses were infused for 20 minute intervals in ascending order. The hemodynamic data were consistent with peripheral vasodilation. The total peripheral resistance and mean aortic pressure decreased with increasing dose. No change in heart rate, cardiac index, or left ventricle dP/dt (contractility) was observed. All doses infused produced inhibition of ADP induced platelet aggregation. In vivo fibrinolytic activity was assessed with an aortic thrombus positioned at the bifurcation of the aorta. Five cats were infused with vehicle and 5 cats each were infused with 8 and 20 micrograms/kg/min ciprostene respectively. All infusions were via a 4F catheter positioned in the aorta proximal to the thrombus. Infusion time was 3 hours. Infusion of 8 micrograms/kg/min did not enhance dissolution of the aortic thrombus. However, the 20 micrograms/kg/min infusion significantly reduced the thrombus weight (mean = 13.2 mg) compared to vehicle (mean = 38.7 mg) (p less than 0.03). The results suggest that ciprostene is a potent vasodilator and platelet inhibitor with clot dissolution properties.

In vivo demonstration of enhanced arterial constrictor response to serotonin following focal endothelial cell loss

Arterial sensitivity to vasospasm was assessed prior to and 2 weeks following a 15-min period of external compression of the superficial femoral artery in dogs. Compression was achieved by placing a plastic cuff around the artery to produce a 40-60% reduction in the artery cross-sectional area. An additional six dogs were used to assess morphologic changes produced in the artery immediately and at 2 weeks after compression. Angiography following intraarterial infusions of serotonin (10 and 30 micrograms/min), norepinephrine (0.1 microgram/min), ergonovine (20 micrograms/min), and the thromboxane mimic U-46,619 (0.1 microgram/min) demonstrated a specific sensitivity to serotonin 2 weeks after the 15-min application of external arterial compression. The serotonin response was antagonized by the specific serotonin (5-HT2) receptor antagonist, ketanserin. Scanning electron microscopy of the acutely injured luminal surface revealed loss of endothelium and deposition of platelets. Patchy areas with intact endothelium and migrating leukocytes were located within the denuded sites. Two weeks after constrictor placement, the compressed area appeared as a raised or semiraised lesion in which the orientation and shape of the luminal cells were distinctly delineated from the adjacent noninjured segments. However, the luminal cells appeared to be endothelium that had regrown over the previously denuded area. The results of this study demonstrate, in an in vivo model, an enhancement in serotonin-mediated vasoconstriction following intimal injury and repair and support the suggestion that endothelial damage or dysfunction may play a role in the pathophysiology of arterial spasm.

Interleukin-1 induced vascular pathology "in vivo": A scanning electron-microscopy study

Ethylene vinyl acetate (EVA) discs containing either 50 U interleukin-1 (IL-1) or 250 mg bovine serum albumin as control were implanted around the exposed jugular veins of rabbits. After 24 hr, the veins were examined by scanning electron-microscopy. Compared with minor changes in control vein lumena, those of EVA-IL-1 treated veins showed extensive endothelial cell denudation and exposure of basement membrane associated with platelet accumulation and adherence/subendothelial migration of leucocytes. these observations suggest that locally-released IL-1 provokes significant vascular pathological changes “in vivo” and emphasise the importance of this cytokine as a mediator of inflammatory and thrombotic/atherosclerotic diseases.