Dissociation of antithrombotic effect and bleeding time prolongation in rabbits by inhibiting tissue factor function.

Inhibition of the tissue factor/factor VIIa (TF/F.VIIa) complex attenuates thrombosis in different animal models of arterial thrombosis. However, it remains unclear to what extent the antithrombotic effects are associated with changes in hemostatic functions and how this compares with inhibition of thrombin, an enzyme acting at a later stage in the coagulation cascade. The antithrombotic and the antihemostatic effects of a monoclonal anti-TF antibody (AP-1) were compared in a model of arterial thrombosis to those of a direct thrombin inhibitor (napsagatran) and heparin. In anesthetized rabbits transient arterial thrombi were induced by mechanical damage to the subendothelium of a moderately stenosed carotid artery. Recurrent formation and dislodgement of thrombi resulted in cyclic flow variations (CFVs) which were monitored over 2 hours. Rabbits received intravenously either a placebo (control), a monoclonal anti-rabbit TF antibody (AP-1, 0.05 mg/kg as an i.v. bolus repeated every 15 min, a specific low molecular weight thrombin inhibitor (napsagatran, 3 microg/kg/min) or heparin (3 and 13 microg/kg/min). The effect of the inhibitors on the hemostatic system was studied in a separate set of rabbits by measuring template bleeding times (BT) in the ear arterioles, marginal ear vein and the nail cuticle of the foreleg. AP-1 and napsagatran showed a similar antithrombotic activity (78% and 80% abolition of the CFVs, respectively), whereas either low or high dose heparin was poorly effective (43% and 40% inhibition of CFVs, respectively). At these antithrombotic doses and even at 4-fold higher dosage, AP-1 did not significantly alter the BT, whereas napsagatran and heparin prolonged the ear vessels and cuticle BT in a dose-dependent manner. These results suggest that in contrast to direct thrombin inhibition, the blockade of the TF/F. VIIa function did not result in a concomitant prolongation of the bleeding time. Thus, dissociation of antithrombotic and antihemostatic effects indicates that inhibition of the coagulation system at its initial stage represents a promising approach for the development of new anticoagulants.

Unilateral axillary adenopathy secondary to a silicone wrist implant: report of a case detected at screening mammography.

At routine screening mammography, asymmetrically enlarged left axillary lymph nodes were seen in a healthy 70-year-old woman. Ultrasound (US)-guided fine-needle aspiration biopsy of the largest node revealed a foreign body reaction. The patient recalled a previous silicone implantation in her wrist. On the basis of cytologic study, normal findings of physical examination, and clinical history, a benign foreign body reaction to silicone implant material was diagnosed. Consideration of regional silicone lymphadenopathy is recommended in the differential diagnosis of unilateral enlarged lymph nodes noted on mammograms. US-guided fine-needle aspiration biopsy is a simple, rapid, inexpensive, and relatively nontraumatic means of ruling out malignancy.

Expression of human P450c17 as an export protein in Saccharomyces cerevisiae.

Cytochrome P450c17 (P450c17), together with cytochrome P450c21 (P450c21), plays an important role in progesterone metabolism in the mammalian adrenal cortex. Low levels of expression and the presence of other steroidogenic enzymes in adrenal cortex endoplasmic reticulum (ER) impedes purification and characterisation of wild type as well as mutant forms of the hemoprotein. Heterologous gene expression systems have previously been used successfully to express active P450c17. Heterologous expression can also be used for the preparation of anti-P450c17-IgG. For antibody production larger amounts of pure P450c17 peptide, rather than the active protein, is, however, desirable. If the expressed protein can be affinity tagged and secreted into the medium, isolation and purification will be facilitated. Saccharomyces cerevisiae, YPH259, was transformed with a modified YCplac111 yeast expression-secretion vector (pPRL2). The gene coding for a truncated human P450c17 (signal anchor sequence 1-18 was removed) was inserted, in reading frame, downstream from the leader sequence MF alpha. A histidine tag was incorporated at the C-terminus. The modified yeast expression vector was expressed in yeast, the secreted P450c17-peptide purified by affinity chromatography and identified by immunoblot analysis.

Effects of heparin, aspirin and a synthetic platelet glycoprotein IIb-IIIa receptor antagonist (Ro 43-5054) on coronary artery reperfusion and reocclusion after thrombolysis with tissue-type plasminogen activator in the dog.

Adjuncts to thrombolytic agents have improved coronary patency and prevented early reocclusion after thrombolysis in acute myocardial infarction. The aim of this study was to compare in a canine thrombolysis model the effects of Ro 43-5054 = N-(N-[N-(p-amidinobenzoyl)-beta-alanyl]-L-alpha-aspartyl)-3- phenyl-L-alanine-trifluor-acetate, a new glycoprotein IIb-IIIa receptor antagonist with aspirin or heparin. Six groups of 10 dogs each were studied. A platelet-rich coronary thrombus was induced in open-chest dogs by electrical stimulation. In addition to recombinant tissue-type plasminogen activator (30 micrograms/kg.min during 60 min), the dogs received 1) saline, 2) heparin 200 U/kg + 50 U/kg.hr i.v., 3) aspirin 10 mg/kg i.v., 4) heparin+aspirin, 5) Ro 43-5054 (3 micrograms/kg.min) and 6) heparin+Ro 43-5054. The overall reperfusion rate was 70% (range, 60-90%) and comparable in all the six groups. During the 120-min observation period, episodes of reocclusion were observed in the absence of antiplatelet therapy (group 1 and 2) irrespective of heparin treatment. Aspirin prevented coronary reocclusion in half of the reperfused dogs (group 3 and 4). However, after reinforcement of the thrombogenic stimulus, 80% of the reperfused dogs treated with aspirin showed reocclusion, whereas none of them reoccluded when treated with Ro 43-5054. Thus, inhibition of platelet activation by the selective, nonpeptidic glycoprotein IIb-IIIa receptor antagonist Ro 43-5054, although without effect on the time to reperfusion, better protected than aspirin against early reocclusion after thrombolytic therapy.

Effects of chronic aortic coarctation on atherosclerosis and arterial lipid accumulation in the Watanabe hereditary hyperlipidemic (WHHL) rabbit.

The effects of high blood pressure on atherosclerosis were examined in the Watanabe heritable hyperlipidemic (WHHL) rabbit. For this purpose, the subdiaphragmatic aorta of rabbits was partially ligated (coarctation) to increase blood pressure. Atherosclerosis was assessed 4 months later by morphometric analyses and quantitation of arterial lipids. Results were compared to control WHHL rabbits with matched plasma triglycerides and cholesterol levels. A marked increase in atherosclerotic lesions was observed in the thoracic aorta of the hypertensive rabbits without qualitative changes in its morphometric features. The cross sectional area of the atherosclerotic plaques of the ascending and descending aorta in the hypertensive rabbits was two- and six-times larger than in normotensive rabbits, respectively. Lesions represented 12.0% +/- 3.5% of the total medial cross sectional area of the descending aorta of normotensive rabbits, versus 45.0% +/- 5.7% in hypertensive rabbits. No lesions were observed downstream of the coarctation in hypertensive rabbits, nor in the normotensive rabbits. Accumulation of cholesterol and choline-containing phospholipids in the descending aorta of hypertensive rabbits was increased 3.2- and 1.5-fold, respectively, when compared to normotensive rabbits. Hypertension did not change the unesterified cholesterol/total cholesterol and sphingomyelin/lecithin + lysolecithin molar ratios. In conclusion, chronic coarctation enhances the atherosclerotic response in WHHL rabbits in the high blood pressure compartment, and reduces the variability of this response.

Cilazapril inhibits wall thickening of vein bypass graft in the rat.

Venous grafts implanted in the systemic circulation progressively undergo wall thickening, a phenomenon thought to be responsible for late saphenous graft disease after bypass surgery. Angiotensin converting enzyme (ACE) inhibitors recently have been shown to prevent myointimal thickening after arterial injury. Thus, the goal of the present study was to test the effects of ACE inhibition in a rat model of venous graft. For this purpose, a segment of jugular vein was interposed in the right carotid artery of normotensive rats that received either placebo or 10 mg/kg/day cilazapril, a long-acting ACE inhibitor. Three weeks later, the venous grafts and the implanted carotid arteries were fixed under perfusion and embedded for morphometric analysis. In the control group, the wall of the venous graft thickened dramatically compared with the nonimplanted contralateral jugular vein (up to 20 times increase in total cross-sectional area). On the arterial side, thickening of the wall and a neointima also were observed, most likely because of the surgical arterial injury. Cilazapril decreased by 33% (p less than 0.05) and 26% (p less than 0.01) the total cross-sectional area of the wall of the venous grafts and of the carotid arteries, respectively. Thus, these results suggest a role of the renin-angiotensin system in the early process of venous graft thickening. This study also suggests that ACE inhibitors could be a new therapeutic means to prevent late saphenous graft disease.

Effect of aspirin and epinephrine on experimentally induced thrombogenesis in dogs. A parallelism between in vivo and ex vivo thrombosis models.

Thrombosis on the damaged or ruptured vascular wall in a stenotic coronary artery is believed to be the precipitating factor leading to unstable angina. Little is known about the nature of the interactions among platelets, fluid dynamic factors, and vessel wall properties under such conditions. In the present investigation we have compared two experimental models of thrombosis simultaneously in anesthetized dogs. The first was an in vivo model of unstable angina, in which a fixed circumflex coronary artery stenosis was produced and the resultant cyclic blood flow reductions (CFRs) through the vessel were investigated after infusion of aspirin and a combination of aspirin and epinephrine. As previously reported, aspirin inhibited the CFRs, but the continuous infusion of epinephrine reestablished the appearance of CFRs. The second was an ex vivo model, in which thrombus formation on a type III collagen surface was investigated in a parallel-plate perfusion system under controlled conditions of exposure time and flow; morphological evaluation of thrombus volume, platelet adhesion, and fibrin deposition was performed. The chamber was positioned in an extracorporeal shunt between the carotid artery and the jugular vein of anesthetized dogs and exposed to nonanticoagulated blood at a shear rate of 1,600 sec-1. Thirty minutes after establishment of the CFRs, a blood sample for platelet aggregation was collected and a bleeding time and a first ex vivo perfusion were performed. At the end of this perfusion, animals were subjected either to no treatment (n = 10) or to an intravenous bolus of 10 mg/kg aspirin (n = 7), and a second perfusion was conducted 30 minutes later. Additional untreated animals (n = 6) were given aspirin followed by a continuous intravenous infusion of 10 micrograms/ml epinephrine, and a third perfusion was conducted. Results with respect to platelet adhesion, thrombus volume, and fibrin deposition were similar in the two perfusions in untreated animals. Treatment with aspirin abolished the CFRs in all dogs and concomitantly reduced the ex vivo thrombus volume by 84% (p less than 0.01) without affecting platelet adhesion and fibrin deposition. Bleeding time increased by 40% (p less than 0.05), and collagen-induced platelet aggregation was virtually abolished (p less than 0.01). However, infusion of epinephrine in dogs after aspirin treatment restored the CFRs, and the ex vivo thrombus volumes were not statistically different from predrug values. Thus, the ex vivo model satisfactorily reflects the more complicated in vivo model events with respect to intracoronary thrombosis and substantiates the view that aspirin interrupts coronary thrombogenesis in the dog by interfering with platelet cohesion.