The relative importance of antithrombin and anti-factor Xa activities of heparin fractions required to achieve optimal antithrombotic effects is unknown. To study this, we measured the effects of standard heparin, an octasaccharide heparin fraction (anti-factor Xa activity only), and dermatan sulfate (antithrombin activity only) on the prevention of thrombosis and related this to their anticoagulant effects in vivo in rabbits. Thrombosis was measured as the incorporation of 125I- fibrinogen into tissue thromboplastin-induced thrombi using a Wessler- type model. Ex vivo changes in thrombin clotting time (TCT) were used as an index of antithrombin activity, and a chromogenic anti-factor Xa assay was used to measure anti-factor Xa activity. In addition, the ability of the three sulfated polysaccharides to simultaneously inhibit the generation of thrombin activity and to enhance the inactivation of the factor Xa added to initiate thrombin generation in plasma was determined. Standard heparin, in a dose of 10 anti-factor Xa U/kg, inhibited thrombus formation by 90%, prolonged the TCT by two seconds, and resulted in an anti-factor Xa level of 0.32 U/mL. The octasaccharide heparin fraction, in a dose of 10 anti-factor Xa U/kg, inhibited thrombus formation by 41%, had no effect on the TCT, and resulted in an anti-factor Xa level of 0.28 U/mL. Higher doses of the octasaccharide resulted in a further increase in the anti-factor Xa levels but had no further effect on thrombus formation. Dermatan sulfate, in a dose of 500 micrograms/kg, inhibited thrombus formation by 95%, but had no affect on the TCT. These results indicate that the antithrombotic effect achieved by inhibiting factor Xa is limited and that better antithrombotic effects are achieved by heparin or heparin- like substances capable of influencing the inactivation and/or the generation of thrombin.

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It is possible to prepare suspensions of washed platelets from rabbit blood which respond normally to stimuli that cause platelet aggregation and the release reaction (1). These platelets are disc-shaped and appear to retain their electron density and organeiles (1). The purpose of the study reported in this paper was to determine whether these platelets survived normally in vivo by comparing their survival with that of platelets labeled in vivo.Materials and Methods. Twice-washed rabbit platelets were prepared by the method of Ardlie et al. (1). Blood from 3 rabbits anesthetized with sodium pentobarbital was collected by carotid artery cannulation. The platelets were labeled in the first washing solution by incubation for 1 min with 5 pCi tritiated diisopropylfluorophosphate ( 3H-DFP; specific activity 3.3 Ci/mmole; Amersham/Searle Corp., Des Plaines, Ill.) in 0.1 ml propylene glycol. The platelet count in the final suspension averaged 4.6 X 106/mm3. The specific activities of the platelet preparations were between 3,000 and 4,000 cpm/mg washed and dried platelets. Approximately 1O1O washed platelets in 2 .O-2.5 ml of platelet suspending medium, corresponding to about 1576 of the total number of platelets in the recipient rabbit were injected into a marginal ear vein of each of 4 animals. In each experiment a control group of 4 rabbits received 30 pCi 3H-DFP in 0.03 ml propylene glycol/kg iv. The 3H-DFP was diluted 1:20 in 0.85% saline just before administration.A total of 16 male New Zealand rabbits weighing 3,160 t 640 g (2 +-SD) were used. 1This study was supported by the Medical Research Council of Canada, Grant MT 1309. 2 Research Fellow of the Canadian Heart Foundation.There was no statistical difference in the body weight between the experimental groups. The rabbits received a standard rabbit chow and water ad libitum. In two experiments 4 rabbits were injected with platelets labeled with 3H-DFP in vitro and 4 other rabbits were injected intravenously at the same time with 3H-DFP. The first blood sample was taken 8 hr after the injection of either in vitro labeled platelets or 3H-DFP. Blood samples were drawn daily thereafter for four days.For each determination of platelet radioactivity 6 ml of blood was drawn from an ear vein into a plastic syringe containing 1 ml acid-citrate-dextrose.Platelets were counted by the method of Brecher and Cronkite (2). Platelets were isolated for radioactivity determination by the method described previously (3, 4). Ten microliters of water was added to the dried and weighed (Cahn electrobalance, Cahn Motor Co., Paramount, Calif.) platelets in an aluminum boat followed ten minutes later by 0.2 ml of NCS solubilizer ( Amersham/Searle) . After 30 min incubation at room temperature 10 ml of toluene-fluor solution was added and the radioactivity determined in a Philips liquid scintillation analyzer (efficiency 2 5 % ) . Each sample was counted for 10,000 counts or 100 min. The specific activities of the platelets were expressed as cpm/mg dry weight.Results were evaluated by t...

Blood/vessel wall cell interactions depend, in part, on the expression of adhesion receptors on cell surfaces, such as expression of the vitronectin receptor (VnR) on the apical surface of endothelial cells (ECs) for platelet/EC adhesion. However, it is unclear how receptor expression is regulated from within cells. In previous studies, we found that ECs metabolize linoleic acid into the lipoxygenase monohydroxide, 13-hydroxyoctadecadienoic acid (13-HODE), and that the intracellular level of 13-HODE correlates inversely with VnR expression and platelet adhesion to the EC apical surface. In this study, we determined the physical associations of 13-HODE and VnR in unstimulated and stimulated ECs, ie, at times when ECs were and were not adhesive for specific ligands and platelets, using double antibody immunofluorescent staining techniques and binding assays. 13-HODE and the VnR were colocalized within unstimulated ECs. When ECs were stimulated, 13-HODE was no longer detectable, either in or outside the ECs, and the VnR was detected on the apical surface of the ECs. These changes were paralleled by increased vitronectin binding and increased platelet adhesion to the ECs. We suggest that colocalization of 13-HODE with VnR reflects a 13-HODE/VnR interaction, confining the VnR in a nonadhesive form inside unstimulated ECs, and, as a result, the ECs are nonadhesive. When the ECs are stimulated, 13-HODE and VnR dissociate, allowing the VnR to relocate on the EC surface, where the VnR undergoes a conformational change resulting in increased EC adhesivity.

Platelet turnover is increased when platelets interact with prosthetic surfaces and damaged vessel wall. To determine whether the resulting increase in young platelets is associated with an increased tendency to thrombosis, we induced a state of increased platelet turnover in rabbits by inserting a sterile cannula into the abdominal aorta and tested for platelet thrombogenecity by measuring the deposition of circulating platelets onto a second injury site in the carotid arteries. Platelet half-life was decreased and platelet turnover was increased after the aortic cannulation, although the circulating platelet count remained unchanged. Platelet thrombogenecity determined 20 hr after cannulation was significantly decreased when compared to sham-operated animals. Ear bleeding studies demonstrated that the platelets circulating in cannulated animals were hemostatically less effective than those in sham-operated animals. This effect was intrinsic to the platelet and was associated with a platelet function defect. These data suggest that platelets exposed to a damaged or foreign surface interact with the surface and then circulate in a less reactive state.