J. Fraser Mustard

Differences in inhibition of PGI2 production by aspirin in rabbit artery and vein segments.

Abstract We have studied the prostaglandin I 2 production by endothelial and subendothelial cells of rabbit aortae and vena cavae. Rabbits were injected intravenously with 0, 10 or 100 mg/kg aspirin. One, 3, 6 or 30 hours later, segments of thoracic aorta or inferior vena cava were removed and their capacity to produce prostaglandin I 2 was measured, using a bioassay system. Both endothelial and subendothelial cells of the arteries and veins produced prostaglandin I 2 which was inhibited by aspirin in doses of 10 and 100 mg/kg. The inhibitory effect of aspirin on venous prostaglandin I 2 production was relatively short-lived and returned to baseline levels within 3 to 6 hours. In contrast, the inhibitory effect of aspirin on prostaglandin I 2 production by arteries lasted for at least 6 hours and by 20 hours had only returned to 50–70% of baseline. When experiments were performed in vitro, a similar difference in the duration of aspirin effect on venous and arterial prostaglandin I 2 production was also observed. These data are consistent with the hypothesis that the turnover of cyclo-oxygenase is more rapid in venous than in arterial cells.

Adhesion of Fibroblasts to Polymerizing Fibrin and Retraction of Fibrin Induced by Fibroblasts

Summary Fibroblasts readily adhere to polymerizing fibrin, but not to fibrinogen or fully polymerized fibrin. Conditions for optimal interaction are similar to those known for the platelet-fibrin interaction. Fibroblasts retract fibrin formed by thrombin but not by reptilase. Fibrin retraction induced by fibroblasts is inhibited by agents inhibiting clot retraction induced by platelets. We thank Dr. L. Prevec for generous supplies of fibroblasts and Messrs. A. F. Senyi, K. L. Wong and T. Bistricki for skilled technical assistance.

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.

Serotonin transport and storage in rabbit blood platelets—The effects of reserpine and imipramine☆

Abstract Serotonin transport and storage in suspensions of washed rabbit platelets were investigated by following the exchange of platelet-bound [ 3 H]serotonin and [ 14 C]serotonin added to the suspending medium. Assuming a three-compartment system (suspending medium, platelet cytoplasm and platelet storage organelles), the transfer rates between the different compartments were calculated from the exchange data by statistical analysis. Reserpine reduced the storage organelle serotonin content by inhibiting the transfer of serotonin from the cytoplasm into the amine storage organelles. It also reduced the fraction of serotonin in the cytoplasm transferred per unit of time into the suspending medium. Imipramine (20 μM) inhibited the uptake of serotonin across the platelet plasma membrane into the cytoplasm and reduced the fraction of cytoplasmic serotonin transferred per unit of time into the suspending medium. At this concentration it had no effect on serotonin transport across the amine storage organelle membrane in either direction. The method used allows the serotonin transfer rates across the platelet plasma membrane to be distinguished from those across the amine storage organelle membrane in intact cells, and permits these transfer rates to be estimated simultaneously. The method may be used for determining the effects of drugs that interfere with transport and storage of biogenic amines and in defining the defects in diseases with abnormal transport or storage of biogenic amines.

Survival of washed rabbit platelets in vivo.

Summary Survival of rabbit platelets washed twice and labeled with 3H-DFP in vitro was compared to the survival of rabbit platelets labeled in vivo with 3H-DFP. There was no significant difference in the platelet survival between these two groups.

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.

The relation among vessel injury, thrombus formation, and platelet survival in rabbits

Continuous or repeated injury of rabbit aortae by indwelling vascular catheters caused the deposition of platelets on the injured vessels and the formation of thrombi rich in platelets and fibrin at sites where flow was most disturbed and injury was most extensive. Incorporation of 51Cr platelets into the thrombi reached a maximum between 3 and 24 hr. The platelet-fibrin-rich thrombi remained reactive to circulating platelets for at least 14 days. Continuing reactivity of thrombi and the turnover of platelets in the thrombi were accompanied by an increase in the proportion of platelets that separated in the least dense fraction on Stractan density gradients. Platelet survival was also shortened (43.5 +/- 5.9 hr in animals with catheters, compared with 62.6 +/- 4.5 hr in animals with a sham operation), indicating that some platelets that had taken part in thrombus formation or had interacted with the injured vessel wall were rapidly cleared from the circulation. Platelets from rabbits that had had indwelling aortic catheters in place for 3 or 6 days survived significantly longer than those from animals with a sham operation upon injection of the platelets into normal animals; thus, continuous turnover of platelets on injured vessels and thrombi, and the clearance of altered platelets, leads to a population of younger platelets that survive longer. The continuing reactivity of thrombi may in part account for repeated occlusive episodes in vascular disease. The contribution of thrombin generation and fibrin formation to the platelet-rich thrombi is substantial and warrants the ongoing evaluation of treatment with a combination of anticoagulant and antiplatelet agents in arterial thrombosis and in thrombus formation on vascular catheters.

Effects of reserpine on rabbit platelet aggregation and adherence to collagen or injured rabbit aorta

Abstract Effects of reserpine in vivo and in vitro on rabbit platelets in citrated platelet-rich plasma and in suspensions of washed platelets have been studied. Administration of reserpine ( 5 mg kg ) intraperitoneally 18 hr before platelets were isolated caused inhibition of collagen-induced aggregation but not of aggregation induced by ADP or thrombin. Thrombin-induced aggregation was slightly enhanced. Platelets from reserpine-treated rabbits were less adherent than control platelets to collagen-coated glass surfaces or to the subendothelium of the rabbit thoracic aorta. Similar effects on aggregation were obtained when reserpine (0.2 to 10 μM) was added to suspensions of washed rabbit platelets as little as 2 sec before the addition of collagen. Collagen-induced release of nucleotides and [ 14 C]serotonin from prelabeled washed rabbit platelets was not affected by the presence of reserpine, whereas thrombin-induced release was slightly enhanced. Inhibition by reserpine (2–10 μM) of platelet adherence to a collagen-coated surface or to the subendothelium was also observed within a time interval too short for the reserpine to have caused depletion of platelet granule contents. Thus, reserpine has an immediate effect on the plasma membrane of the platelets which is responsible for inhibition of platelet adherence to collagen and hence of collagen-induced aggregation. This inhibitory effect differs from a much slower effect of reserpine at the granule membrane which results in the depletion of the granule contents of serotonin and adenine nucleotides. The effect of reserpine is not abolished by washing and resuspending platelets that have been exposed to reserpine in vivo . By inhibiting the interaction of platelets with collagen, reserpine may interfere with one of the components of hemostatic plug and thrombus formation.

In vitro Methods and the Study of Platelet Mechanisms

To be hemostatically effective, platelets should be able to adhere to the subendothelium, release their granule contents (include ADP, serotonin, platelet factor 4, P-thromboglobulin and many other materials), form the prostaglandin endoperoxides PGG, and PGH2 and the related substance thromboxane A2 (TXA2), aggregate, and accelerate the generation of thrombin around the mass of aggregated platelets. Thrombin causes further platelet aggregation, release of granule contents, formation of PGG2, PGH2 and TXA2, and also converts fibrinogen to fibrin to stabilize the platelet mass. If the platelets are unable to function effectively in these reactions, hemostasis is likely to be impaired [I, 21.

PATHOPHYSIOLOGY, BIOCHEMISTRY, AND PHARMACOLOGY OF PLATELETS

This subject will be presented in relation to the study of platelets and vascular disease. Platelets are involved not only in thrombosis but also in atherosclerosis, which is a key factor in the development of arterial thrombosis. The study of arterial disease and thrombosis in living subjects has been difficult.