J. F. Mustard

Preparation of Suspensions of Washed Platelets from Humans

Summary: Methods have been developed for the preparation of suspensions of washed platelets from humans. Heparin is used in the washing fluids to prevent: thrombin generation and apyrase is used to prevent adenine nucleotide accumulation. Platelets suspended in Eagles tissue culture medium containing albumin were more responsive to ADP than platelets in Tyrodes‐albumin solution. Addition of fibrinogen is required for maximum sensitivity to ADP‐induced aggregation. These platelets can be stored for 4 hr or more at 37°C in the presence of apyrase and maintain their ability to aggregate upon the addition of low concentrations of ADP.

Thromboembolism A Manifestation of the Response of Blood to Injury

Thromboembolism may be considered as one of the manifestations of the response of blood to injury. Other manifestations of this include hemostasis, increased vessel permeability, and vasculitis; disturbances of the microcirculation may lead to tissue injury and organ dysfunction. The factors that can initiate these changes by stimulation of platelets are exposure of subendothelial tissue (collagen, basement membrane) and intravascular stimuli such as antigen-antibody complexes, viruses, bacteria, and endotoxin. These stimuli have a number of effects on blood; the interaction of the platelets with the above stimuli leads to the release of platelet constituents including ADP; the ADP causes the platelets to adhere to each other; the aggregated platelets cause acceleration of clotting; this and changes in blood flow promote the formation of fibrin around the platelet aggregates. Some of the stimuli such as collagen and antigen-antibody complexes also activate blood coagulation through factor XII; some of the materials released from these platelets affect the vessel wall. The initial platelet mass is transformed to a fibrin mass. There are compounds that inhibit the platelet release reaction, platelet aggregation, and blood coagulation and activate the fibrinolytic mechanism. It appears that by the selective use of these compounds we can improve our management of all aspects of thromboembolic disease related to vascular and intravascular stimuli.

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.

Platelet Economy (Platelet Survival and Turnover)

Investigators studying blood platelet survival and turnover have used the term ‘thrombo‐kinetics’ to describe the subject, by analogy with the usages of thermodynamics. Inasmuch as these phenomena reflect the interactions and hazards which the platelets encounter while discharging their function in the circulation, we prefer the term ‘platelet economy’ and we consider that the function and fate of the platelet cannot usefully be separated as objects of study.

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.

Changes in 32P-content of phosphatidic acid and the phosphoinositides of rabbit platelets during aggregation induced by collagen or thrombin.

Summary. During the ADP‐induced shape change and aggregation of washed platelets that have been labelled with 32PO4 there is increased incorporation of 32P into phosphatidic acid (PA) and the phosphoinositides of the platelets. When added to suspensions of washed platelets, collagen and thrombin cause not only a change in shape and aggregation, but also the release reaction. The present experiments were carried out to study the effect of collagen and thrombin on the 32P‐content of PA and the phosphoinositides of washed platelets from rabbits. Both collagen and thrombin caused an increase in the labelling of PA that was apparent when the platelets had changed shape and before aggregation of platelets was detected. Unlike the change in labelling of PA that occurs during ADP‐induced aggregation, there was a marked increase in labelling of PA after the commencement of platelet aggregation. Comparison with the maximum response caused by ADP showed that both collagen and thrombin caused a greater increase in the 32P‐content of PA than can be obtained with ADP. Like ADP, collagen and thrombin also caused an increase in labelling of mono‐phosphatidylinositol (MPI). Collagen caused an increase in labelling of diphosphatidylinositol (DPI) but not triphosphatidylinositol (TPI). As with ADP in previously reported experiments, the increase in labelling of DPI occurred after the increase in labelling of PA but preceded the increase in labelling of MPI. An increase in the 32P‐content of DPI has not been demonstrated previously in similar in vitro experiments with other cells using other stimuli. The changes in extent of incorporation of 32P into PA and phosphoinositides may reflect changes in these phospholipids which are important in the mechanism of the change in platelet shape and the release reaction.

Effect of ADP-induced Shape Change on Incorporation of 32P into Platelet Phosphatidic Acid and Mono-, Di- and Triphosphatidyl Inositol

Summary. Experiments were carried out to determine whether the ADP‐induced increase in 32P‐content of phosphatidic acid (PA), monophosphatidylinositol (MPI), diphosphatidylinositol (DPI) or triphosphatidylinositol (TPI) of washed 32PO4 labelled platelets is related to platelet aggregation or to the change in shape induced by ADP. Washed platelets were labelled with 32PO4, washed, and resuspended in medium containing unlabelled PO4. With rabbit platelets, either the omission of Ca++ from the suspending medium or the addition of ethyleneglycoltetraacetic acid (EGTA) in amounts sufficient to chelate Ca ++ in the suspending medium, prevented ADP‐induced platelet aggregation. Lack of free Ca++ in the suspending medium did not prevent the ADP‐induced shape change or the increased labelling of PA and DPI. In suspensions containing EGTA, the increase in 32P‐content of PA induced by varying amounts of ADP was compared to the extent of the shape change. ADP in a final concentration of 10−6m or greater, produced both the maximum increase in 32P‐content of PA and the maximum degree of shape change. Washed platelets from pigs or humans without fibrinogen in the suspending medium showed a shape change following the addition of ADP but no aggregation, whereas in suspensions containing fibrinogen, the platelets showed both a shape change and aggregation. In suspension with and without fibrinogen, the ADP induced increase in 32P‐content of PA in human platelets and of PA+ MPI and DPI in pig platelets was the same. Prostaglandin E1 (PGE1), 10−4m final concentration, added to suspensions of rabbit platelets prevented both the ADP‐induced shape change and the increase in 32P incorporation into PA and DPI. PGE1, 10−4m, also caused a decrease in labelling of TPI. PGE1, 10−7m final concentration, prevented platelet aggregation but did not prevent the ADP‐induced shape change or the increase in 32P‐content of PA. Dibutyrylcyclic AMP (DBcAMP) inhibited both the ADP‐induced shape change and the increase in 32P‐content of PA. With rabbit platelets, the time course of the ADP‐induced shape change was compared to the time course of the increase in 32P‐content of PA, MPI and DPI. The increase in labelling of PA was detected 2 s after the addition of ADP and was near maximum at 8 s, the time of maximum shape change. The increase in labelling of DPI was not significant until 30 s. The regaining of the disc shape was associated with an increase in labelling of MPI and a decrease in labelling of PA and DPI. The results show that the changes in platelet PA are related to the ADP‐induced shape change rather than to 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.

Changes in 32P-Labelling of Platelet Phospholipids in Response to ADP

Summary. Suspensions of washed platelets from rabbits were labelled in vitro with 32PO4, washed, and then stimulated with ADP. Thirty seconds after the addition of ADP or of a control solution, samples of the suspension were subjected to lipid extraction. The phospholipid classes were isolated by thin layer chromatography. The results of phosphate estimation on the isolated phospholipids were in agreement with the previously described amounts of diphosphatidylinositol (DPI) and triphosphatidylinositol (TPI) in human platelets. In both ADP‐treated and control plateletes the label in phospholipid was mainly in phosphatidic acid (PA), phosphatidylinositol (MPI), DPI and TPI. Thirty seconds after the addition of ADP there was an increase in labelling of PA, DPI and TPI but the amounts of PA, DPI and TPI in platelets did not change. The results indicate that ADP probably induces an increase in turnover of the phosphate moieties of these phospholipids. The changes observed represent changes in the metabolism of the phospholipids of the platelet membrane which may be important in the mechanism of platelet aggregation.

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.