Raelene L. Kinlough-Rathbone

[1] Isolation of human platelets from plasma by centrifugation and washing

Publisher Summary This chapter describes the process of isolating human platelets from plasma by centrifugation and washing. Through the centrifugation and washing methods, large volumes of blood can be handled readily and the platelet count in the final suspension can be adjusted to any desired number. By using these methods, platelets respond to aggregating and release-inducing agents in a manner similar to platelets in native plasma, or plasma anticoagulated with hirudin, and maintain this responsiveness for hours. The constituents of a medium can be varied or additions can be made, as required, for experimental purposes. Thrombin can be used as an agonist without the problem of the formation of large amounts of fibrin that occurs when thrombin is added to platelet-rich plasma. The morphological appearance of platelets by electron microscopy is similar to that of platelets in plasma. Because platelet suspensions are kept at 37°, experiments can readily be done at this temperature so that conditions more closely resemble an in vivo situation.

Effect of Repeated Treatment of Rabbit Platelets with Low Concentrations of Thrombin on their Function, Metabolism and Survival

Summary. Low concentrations of thrombin (< 0.05 u/ml) cause reversible aggregation of washed rabbit platelets in Tyrode‐albumin solution containing apyrase. The same platelets were aggregated several times by addition of the same concentration of thrombin with washing and resuspension after each aggregation step. Deaggregation of rabbit platelets aggregated by thrombin was enhanced by the addition of prostaglandin E1.

The Effect of Prostaglandin E1 on Platelet Function in Vitro and in Vivo

Summary Low concentrations of prostaglandin E1 (PGE1) inhibit ADP‐induced aggregation in pig and rabbit citrated platelet‐rich plasma and in suspensions of washed platelets. Higher concentrations also inhibit the initial change in shape induced by ADP and the release of platelet ATP, ADP and serotonin caused by stimuli such as collagen, thrombin, antigen‐antibody complexes and gamma‐globulin‐coated polystyrene particles. PGE1 is not taken up by platelets and its effects can be removed by resuspending platelets in fresh medium. Immediately following an intra‐arterial injection, ADP‐induced platelet aggregation is suppressed, but after 5 min the response returns to normal. PGE1 inhibits haemostasis in rabbits when given as a continuous infusion. It is concluded that the effect of PGE1 on haemostasis involves inhibition of the release of ADP from platelets exposed to collagen and thrombin, and inhibition of ADP‐induced aggregation.

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.

Aspirin in the treatment of cardiovascular disease: A review

Large-scale clinical trials of the use of aspirin in post-myocardial infarction patients were based on the assumption that inhibition of platelet activity would reduce thromboembolism associated with atherosclerosis, and that thromboembolism is a major cause of the clinical complications of atherosclerosis. However, spasm and occlusive thrombi may also contribute to this picture, and thus thromboembolism is probably only one of the mechanisms that cause the clinical complications. Aspirin inhibits thrombosis only if thromboxane A2 formation by platelets plays a major part in the growth of thrombi; aspirin has little effect on thrombosis when thrombin generation and fibrin formation are dominant factors. Nevertheless, analysis of the combined data from the six clinical trials indicates a highly significant (21 percent) reduction in reinfarction rate and a 16 percent reduction in cardiovascular mortality rate in patients treated with aspirin. Aspirin may be most useful in treating an as-yet-unidentified subgroup of patients.

Effect of ionophore A23, 187 on thrombin-degranulated washed rabbit platelets

Abstract The ionophore A23,187 causes platelets to release their granule contents and aggregate. To determine whether aggregation is a direct effect of A23,187 or is secondary to the release reaction, washed rabbit platelets were treated with thrombin (0.45 U/ml) to cause release of 99% of their granule contents. These degranulated platelets were recovered as disc-shaped platelets which aggregated upon the addition of ADP in the presepce of fibrinogen, were unresponsive to further additions of thrombin and adhered to collagen. A23,187 caused shape change and aggregation of the thrombin-degranulated platelets in the presence of Ca ++ (2 mM) + Mg ++ (1 mM) or Ca ++ alone; only shape change occurred in the presence of Mg ++ alone, EGTA or EDTA. A23,187 caused aggregation in the absence of fibrinogen, but the presence of fibrinogen increased the extent of aggregation. PGE 1 , theophylline or caffeine inhibited aggregation of thrombin-degranulated platelets by A23,187 but had no effect on platelet shape change. AMP, adenosine or apyrase had no effect. Thus, A23,187 causes platelet shape change and aggregation independent of ADP release. Ionophore-induced aggregation depends on external calcium but shape change does not depend on external Ca ++ or Mg ++ . Shape change may be caused by A23,187-induced modulation of the intracellular distribution of Ca ++ or Ca ++ and Mg ++ .

Conditions influencing release of granule contents from human platelets in citrated plasma induced by ADP or the thrombin receptor activating peptide SFLLRN: Direct measurement of percent release of β‐thromboglobulin and assessment by flow cytometry of P‐selectin expression

Contrary to a recent report [Rinder et al.: Blood 82:505, 1993], aspirin does inhibit the release of α‐granule contents as well as inhibiting the release of dense granule contents by human platelets during ADP‐induced aggregation in citrated platelet‐rich plasma (PRP). Measurements were: percent release of 14C‐serotonin from prelabeled platelets, radio‐immunoassay of β‐thromboglobulin (βTG), and expression on the platelet surface of the α‐granule constituent, P‐selectin, by flow cytometry. During the second phase of ADP‐induced aggregation, 69.0 ± 8.3% of βTG and 54.1 ± 4.6% of 14C‐serotonin were released (means ± SEM, n = 13); aspirin treatment reduced these values to 6.0 ± 1.2 and 1.0 ± 0.3%, respectively. In contrast, incubation of platelets with ADP without stirring caused only 6.7 ± 1.7% release of βTG and 2.1 ± 0.4% release of 14C‐serotonin; these low values were not appreciably affected by aspirin. During ADP‐induced primary aggregation in PRP anticoagulated with FPRCH2Cl (PPACK), only 4.7 ± 0.9% release of βTG and no detectable release of 14C‐serotonin occurred; aspirin had no effect. In both stirred and unstirred PRP, the thrombin receptor activating peptide, SFLLRN (50 μM), caused at least 75% release of the contents of both granules, which was partially inhibited by aspirin. Upon incubation of platelets with ADP (2–10 μM), the mean fluorescence intensity due to P‐selectin was <14% of that induced by SFLLRN. In this unstirred system used for flow cytometry, aspirin treatment caused no significant inhibition of P‐selectin expression. Thus, under conditions in which ADP does not cause secondary aggregation (physiological Ca2??? concentration or unstirred citrated PRP) release of the contents of both types of granules is less than 7% and aspirin is not inhibitory; the P‐selectin expression associated with this low percent release is also unaffected by aspirin. However, aspirin does strongly inhibit the extensive release of both α‐granule and dense granule contents during ADP‐induced secondary aggregation in citrated PRP.

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.

The Influence of Red Blood Cells on the Effects of Aspirin or Sulphinpyrazone on Platelet Adherence to Damaged Rabbit Aorta

Summary. The effects of acetylsalicylic acid (ASA; aspirin) or sulphinpyrazone (SP) on the adherence of washed rabbit platelets to the subendothelial surface of an everted aorta mounted on a probe or to the subendothelial surface of a rabbit aorta attached to a perfusion apparatus were examined. ASA had no effect on platelet adherence to a damaged aorta perfused with a suspension of washed platelets in a medium containing 10% red blood cells (RBC); SP was slightly inhibitory at high concentration. When damaged rabbit aortae were everted on a probe and rotated in a suspension of washed platelets to which RBC were added to a packed cell volume of 10%, both ASA and SP inhibited platelet adherence to the damaged vessel wall. When the PCV was 40%, ASA was not inhibitory and SP reduced platelet adherence only at very high concentrations. It is therefore unlikely that, at the concentrations achieved in man, SP exerts an effect on platelet adherence. The different effects of ASA and SP on platelet survival do not appear attributable to their effects on platelet adherence.

Effect of diet-induced hyperlipidemia on in vitro-function of rabbit platelets

Abstract Hyperlipidemia was induced in rabbits by feeding them a diet enriched with egg yolk for a minimum period of sixteen weeks. Platelets isolated from the blood of these rabbits and resuspended in Tyrode-albumin solution showed a significant increase in collagen- or thrombin-induced aggregation and serotonin release but not in aggregation induced by ADP. Collagen-induced platelet factor 3-availability was also greater with platelets from hyperlipidemic rabbits compared with platelets from control rabbits. There were no significant differences between the diet and control groups in platelet aggregation induced by ADP, collagen, or thrombin or serotonin release induced by collagen or thrombin in citrated platelet-rich plasma. With washed platelets there was no correlation between the percentage increase in aggregation induced by collagen or thrombin and the plasma cholesterol or triglyceride levels in hyperlipidemic or control rabbits. When washed normal rabbit platelets were resuspended in citrated plasma from a hyperlipidemic rabbit their response to aggregating or release-inducing stimuli was not significantly different from that observed when platelets from normal rabbits were resuspended in normal plasma. When washed normal platelets were incubated for 30 min in hyperlipidemic plasma at 37°C, isolated from the plasma and resuspended in Tyrode-albumin solution, their response to aggregating or release-inducing stimuli was not significantly different from that observed with normal platelets incubated in normolipidemic plasma. The findings indicate that diet-induced hyperlipidemia in rabbits may induce an intrinsic functional alteration of the platelets which cannot be readily reproduced by short-term in vitro exposure of platelets to hyperlipidemic plasma.