P.D. Winocour

Reduced Membrane Fluidity in Platelets From Diabetic Patients

Platelets from diabetic patients are hypersensitive to agonists in vitro. Membrane fluidity modulates cell function, and reduced membrane fluidity in cholesterol-enriched platelets is associated with platelet hypersensitivity to agonists, including thrombin. Decreased membrane fluidity of these platelets is attributed to an increased cholesterolphospholipid molar ratio in platelet membranes. We examined the response of platelets from diabetic subjects to thrombin, platelet membrane fluidity, and platelet cholesterol-phospholipid molar ratio. Twelve poorly controlled diabetic subjects were compared with 12 age- and sex-matched control subjects. In response to a low concentration of thrombin, mean values for release of [14C]serotonin from washed prelabeled platelets were not significantly different between diabetic and control subjects, but in 8 of 12 diabetic subjects, the release response was greater than in their paired control subjects. Mean steady-state fluorescence polarization values in 1,6-diphenyl-1,3,5- hexatriene-labeled platelets prepared from diabetic subjects were significantly greater than in control subjects; this indicates a decreased membrane fluidity in platelets from diabetic subjects. Total or very-low-density (VLDL), low-density (LDL), or highdensity (HDL2, HDL3) lipoprotein cholesterol concentrations in plasma were not significantly different between groups; however, the ratio of VLDL + LDL to HDL2 + HDL3 was significantly greater in diabetic than in control subjects. There was no difference in the total platelet cholesterol-phospholipid molar ratio between groups. Thus, reduced membrane fluidity of platelets from diabetic patients could account for their increased sensitivity to agonists; reduced membrane fluidity does not appear to result from a change in the plasma or platelet cholesterol content but is associated with an increase in the ratio of plasma VLDL + LDL to HDL2 + HDL3.

Intimal alterations in rabbit aortas during the first 6 months of alloxan-induced diabetes.

Diabetes mellitus is a major risk factor for atherosclerosis. Since endothelial alteration is probably associated with the development of atherosclerosis, we questioned whether morphological evidence of endothelial injury could be observed during the first 6 months of diabetes induced by a single intravenous injection of alloxan in normally fed rabbits compared with age-matched controls. Diabetes (plasma glucose greater than 16 mM) was established by 5 days after alloxan injection. Endothelial alterations consistent with injury, including adhesion of white blood cells, platelets, and fibrin-like material to the endothelial surface, were seen in diabetic rabbit aortas by 2 weeks. These alterations became more severe during the next 6 months. Increased endothelial replication in diabetic vessels was shown by the uptake of tritium-labeled thymidine at 2 weeks and at 3 and 6 months. Hyperplasia of intimal smooth muscle cells progressed during 3 months after treatment. About one third of the diabetic rabbits also showed an elevated plasma cholesterol level, which correlated with increased intimal proliferation but not with endothelial injury or replication. The onset of alloxan-induced diabetes in rabbits is associated with nondenuding endothelial injury and subsequent intimal hypertrophy, changes that are consistent with atherogenesis.

Platelet turnover in advanced diabetes

Abstract. There is considerable information to indicate that when there is vessel injury, platelets can play a major role in the development of atherosclerosis and its thromboembolic complications in the non‐diabetic population. Diabetes is a disease associated with an increased risk of vascular complications, but the nature of this association is unclear. Platelets from diabetic humans and animals are hypersensitive to agonists in vitro, but it is unclear if this hypersensitivity persists in vivo. Platelet survival and turnover in the circulation have been used as indicators of platelet behaviour in vivo, but platelet survival measurements do not necessarily correlate with platelet sensitivity in vitro and these measurements may be too insensitive to reflect platelet turnover on injured vessels or in thrombi, unless there is extensive vascular disease. Studies in rats and rabbits indicate that diabetes causes increased platelet interaction with experimentally‐injured vessels and accumulation in thrombi. It is unclear, however, whether this altered behaviour of platelets at the injured vessel surface reflects changes in the platelets or in the vessel wall and whether it contributes to the increased risk for vascular complications in diabetic patients.

The effect of dietary saturated fat and cholesterol on platelet function, platelet survival and response to continuous aortic injury in rats

Induction of hypercholesterolemia in rats by diets containing milk fat, cholesterol and taurocholate caused increased sensitivity of platelets to thrombin-induced aggregation and release, but not to ADP- or collagen-induced aggregation or release. This hypersensitivity to thrombin persisted in the presence of CP/CPK to convert released ADP to ATP, and aspirin to block formation of thromboxane A2. The increased sensitivity of platelets to thrombin in hypercholesterolemic animals was associated with an increase in 18:1 omega 9, 18:2 omega 6 and 20:3 omega 6 and a decrease in 20:4 omega 6 and 22:4 omega 6 in their phospholipids. Hypercholesterolemic animals also had a shortened platelet survival that did not appear to be due to an alteration in the lipid composition of the platelets. The diet-induced changes in platelet function were not associated with enhanced thrombosis in animals with indwelling aortic catheters, but were associated with increased platelet accumulation on the exposed subendothelium.

Platelet survival and thrombosis.

This study examined the relation among platelet survival, thrombosis, and repeated vessel injury. With the use of 51 Cr-labeled platelets, indwelling aortic catheters were shown to reduce platelet survival in rabbits and rats. In rabbits, thrombi were observed mainly at the aortic bifurcation and at the tip of the catheter. The amount of thrombus that formed in rabbits with short and long catheters was similar, but platelet survival was shortened only in rabbits with long indwelling aortic catheters. In rats, the aortic catheters did not cause thrombosis, and platelet survival was shortened significantly in rats with both short and long catheters, but was more pronounced in animals with longer catheters. In both rabbits and rats, long aortic catheters caused more extensive vessel injury than the short catheters and this was associated with greater platelet interaction with the vessel wall. Platelet survival cannot be used as an estimate of thrombus formation, but may reflect the extent and frequency of vessel wall injury. Thus, shortened platelet survival may represent increased platelet interaction with the damaged arterial wall and increased platelet consumption.

Hypersensitivity to ADP of platelets from diabetic rats associated with enhanced fibrinogen binding

Abstract. Platelets from diabetic humans and animals are hypersensitive to ADP. The hypersensitivity to ADP of platelets from diabetic rats occurs independently of activation of the arachidonate pathway or release of dense granule contents. During platelet aggregation by ADP, fibrinogen binds to its receptor on platelets. We examined if the hypersensitivity to ADP of platelets from diabetic rats is associated with enhanced early binding of fibrinogen to its receptor on these platelets. Fibrinogen association with platelets from rats with spontaneous or streptozotocin‐induced diabetes was significantly greater 10 s or 1 min after addition of ADP (10μm) than with platelets from their corresponding control rats. Since enhanced fibrinogen association occurred with platelets from insulin‐treated rats with spontaneous diabetes, and from rats with streptozotocin‐induced diabetes that did not receive insulin, the enhanced fibrinogen binding is likely due to the diabetic state rather than to the administration of insulin or the mechanism responsible for the diabetes. Therefore, enhanced early fibrinogen association with platelets from diabetic rats is associated with their hypersensitivity to ADP.

Platelet function and survival in rats with genetically determined hypercholesterolaemia.

Platelets from rats with genetically determined hypercholesterolaemia are hypersensitive to aggregation induced by thrombin compared with platelets from their genetic controls without hypercholesterolaemia. Aggregation or release induced by thrombin of platelets from hypercholesterolaemic and control rats correlated significantly with plasma cholesterol concentrations. Platelet responses to ADP or collagen were not different between the groups. The hypersensitivity to thrombin-induced aggregation was independent of released ADP or products of arachidonic acid metabolism. The changes in platelet sensitivity occurred with only moderate increases in plasma cholesterol concentration and with no detectable changes in total platelet cholesterol. The hypersensitivity of platelets from hypercholesterolaemic rats was not associated with a reduction in platelet survival or any significant injury to the aortic endothelium in these animals. Platelets from hypercholesterolaemic rats were smaller than platelets from controls. Thus, platelets from rats with genetically determined hypercholesterolaemia have alterations in function similar to those found with platelets from rats with diet-induced hypercholesterolaemia indicating that this strain can be used to study the mechanisms by which cholesterol can change platelet function without the possible complicating effects of dietary factors. Since platelet hypersensitivity occurred in rats with genetically determined hypercholesterolaemia without a reduction in platelet survival, these studies are also consistent with the reduced platelet survival found in animals with diet-induced hypercholesterolaemia being independent of platelet changes.

Thrombin binding to platelets from hypercholesterolaemic rats

Platelets from rats made hypercholesterolaemic with a diet enriched with milk fat and cholesterol and containing taurocholate to promote hypercholesterolaemia aggregated more extensively to a low concentration of thrombin than platelets from rats given a milk fat-enriched diet containing sitosterol. Total and specific binding of thrombin to platelets from hypercholesterolaemic rats was significantly greater than in controls when expressed per mg platelet protein, per mumol platelet cholesterol, or per unit relative surface area. Total and specific binding of thrombin per platelet were not different between the groups. However, platelets from hypercholesterolaemic rats had less protein and cholesterol, were smaller and had less surface area than control platelets; platelet cholesterol content expressed per mg platelet protein was not different. Thus, the increase in thrombin-binding to the smaller platelets from hypercholesterolaemic rats during the first 10 s after its addition may be responsible, at least in part, for the hypersensitivity of these platelets to thrombin.

Effect of the amount and type of dietary fat on platelet function, platelet survival and response to continuous aortic injury in rats

The effect of giving diets containing 1.5 or 16% safflower or corn oil or 16% milk fat for 15 weeks on changes in the fatty acid composition of platelet phospholipids, in vitro platelet function, platelet survival and thrombosis was examined in rats. The mean plasma cholesterol concentration was not different among the groups. Diets containing 1.5% safflower or corn oil or 16% milk fat were associated with a decrease in 18:2n - 6 and an increase in 18:1n - 9 and the 20:4n - 6/18:2n - 6 ratio in the platelet phospholipids compared with the 16% safflower or corn oil diets. The 16% milk fat diet was associated with an increase in 14:0, 20:3n - 9, 22:3n - 9 and a decrease in 22:4n - 6 in platelet phospholipids compared with the other groups. There were no differences among the groups in the sensitivity of washed platelets to ADP-, thrombin- or collagen-induced aggregation, or thrombin- or collagen-induced release of granule contents or loss of arachidonate from platelet phospholipids. Platelet survival and turnover in rats given the diets were not different among the groups. In response to indwelling aortic catheters neither the percentage reduction in platelet survival nor the platelet accumulation on injured aortae and catheters were different among the groups. No macroscopic thrombi were seen in rats given any of the diets. The results of these studies provide no evidence that diet-induced alterations in fatty acid content (increases in 18:1n - 9, 20:3n - 9, 22:3n - 9, 20:3n - 6, and 20:4n - 6/18:2n - 6 ratio and a decrease in 22:4n - 6) of platelet phospholipids modify in vitro platelet function, platelet survival or turnover or influence thrombosis in rats.

Increased platelet, but unaltered fibrinogen, accumulation in experimental thrombi in alloxan-induced diabetic rabbits

Platelets from diabetic humans and animals have been found previously to be hypersensitive to agonists, including thrombin, in vitro but it is unclear if this hypersensitivity also occurs in vivo and leads to a greater thrombotic tendency. In the present study, the effect of diabetes was examined on thrombus formation and vessel wall responses which result from continuous intimal injury induced by indwelling aortic catheters in rabbits. Platelet and fibrin(ogen) associated with the thrombus and damaged aortae were examined. Control or alloxan-induced diabetic rabbits (9-12 months after initial treatment) were injected with 51Cr-labeled autologous platelets and 125I-labeled fibrinogen (prepared from control rabbits) before insertion of indwelling aortic catheters. The anesthetized rabbits were perfused-fixed after 20 hr or 4 days. The dry weight of thrombus that formed was determined and platelet and fibrin(ogen) accumulation in thrombi and on injured aortae were calculated from the associated 51Cr and 125I, respectively. In diabetic rabbits, more platelets accumulated in the thrombi which formed after either 20 hr or 4 days, although the weight of thrombus and net fibrin(ogen) incorporation into the thrombus were not different from corresponding control rabbits. Net platelet and fibrin(ogen) association with the injured aortae were not different between control and diabetic rabbits. It is likely that the increased platelet accumulation in arterial thrombi in diabetic rabbits which results from continuous injury to aortae is a consequence of hypersensitivity of these platelets to thrombin generated in the thrombus and at the sites of vessel injury.