Marian A. Packham

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.

[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.

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.

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.

Effects of Penicillin G on Platelet Aggregation, Release, and Adherence to Collagen

Summary Penicillin G (1000–8000 units/ml) inhibits ADP, collagen or thrombin-induced platelet aggregation and the release reaction in human PRP and in suspensions of washed platelets from rabbits or pigs. Adherence of washed platelets from all three species to a collagen-coated surface is also inhibited by penicillin G in this concentration range. Penicillin G may coat platelets, as it does red cells, block the sites where the aggregating agents interact with the platelets, and thus inhibit the response of platelets to them. Since these concentrations are achieved in vivo when massive penicillin therapy is used or penicillin is applied locally, these observations may be relevant to alterations of platelet function during such treatment.

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.

Platelet Function and Myocardial Infarction

Platelet thromboemboli may be important not only in the development of some forms of atherosclerosis and occlusive thrombi, but also in initiating disturbances of the microcirculation. The latter mechanism may be significant in those cardiac deaths that are not directly attributable to advanced atherosclerotic disease or occlusive thrombi in coronary arteries. In addition, it is possible that mural thrombi and atherosclerotic lesions may fragment and shower the microcirculation with emboli. Such a mechanism appears to occur in the cerebrovascular and renal circulations, and it seems unlikely that the myocardial tissue would be spared the effects of such a mechanism. Thus, in examining the relation between platelets, thrombosis, and the complications of vascular disease, it is important to emphasize that consideration must be given to platelet thromboemboli and disturbances of the microcirculation as well as to occlusive coronary artery thrombi.

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 ++ .

Effects of cephalothin and penicillin g on platelet function in vitro

High concentrations of cephalothin or penicillin G inhibit a number of the functions of human or rabbit platelets in citrated platelet‐rich plasma (PRP) and in suspensions of washed platelets. The reactions shown to be inhibited are: ADP‐induced shape change and the primary and secondary phases of aggregation and release induced by ADP or adrenaline in human citrated PRP; release and aggregation of washed human platelets exposed to collagen, thrombin, vasopressin, or the ionophore A23,187; aggregation of washed human platelets exposed to phytohaemagglutinin from Phaseolus vulgaris (PHA) or polylysine; release induced by concanavalin A or PHA in suspensions of washed platelets from rabbits; platelet adherence to a collagen‐coated surface or to the damaged intimal surface of the rabbit aorta; platelet factor 3 availability; lysis of rabbit platelets by an antiserum directed against them; and clot retraction. Neither antibiotic affected serotonin‐induced aggregation; a high concentration of cephalothin slightly inhibited the initial rate of serotonin uptake. Penicilloic acid showed about half the inhibitory effect of penicillin G on ADP‐induced aggregation. In citrated human platelet‐rich plasma, ampicillin and oxacillin inhibited ADP‐induced aggregation to the same extent as similar concentrations of penicillin G; in suspensions of washed platelets, however, ampicillin was less inhibitory than penicillin G or oxacillin. Platelet ultrastructure, assessed by transmission electron microscopy, was not visibly altered. Evidence that the antibiotics become bound to platelets is the finding that platelets incubated with the antibiotics and resuspended in fresh media showed less response to aggregating agents compared with control platelets. Penicillin G and related antibiotics may be inhibitory because they coat the platelet surface. Their effects on platelet functions are probably responsible for excessive bleeding and increased bleeding times observed in patients and volunteers receiving high doses of these antibiotics.