Search PubMed⌕ Search

Biomedical subjects

M A Packham

Publications and source records attributed to M A Packham.

At least 91 records · Page 5Linked to original sources
PubMed1986

Isolation and kinetic studies of nucleoside diphosphokinase from human platelets and effects of cAMP phosphodiesterase inhibitors.

Nucleoside diphosphokinase (NDK) of human platelets has been purified by chromatography on Blue Sepharose CL-6B gel (purification factor of 950) and shown to be free of adenylate kinase, ATPase and adenylate cyclase. The molecular weight was 70,000 with subunits of 17,000. The pH optimum was 8.0 Km values for ATP and dTDP were determined in two ways using the pyruvate kinase-lactate dehydrogenase coupled enzyme assay. Values of 0.38 and 0.20 mM were obtained for ATP and 0.29 and 0.21 mM for dTDP. Km values for ADP (0.024 mM) and GTP (0.12 mM) were determined with the hexokinase-glucose-6-phosphate dehydrogenase coupled enzyme assay. These values are in agreement with those reported for NDK from other sources. Theophylline, which inhibits the NDK activity of intact platelets and platelet membrane preparations and inhibits the ADP-induced shape change of platelets, was shown to be a competitive inhibitor of both the free and phosphorylated forms of NDK with competitive inhibition constants (Kic) of 9.3 and 9.6 mM respectively. Papaverine, another cAMP phosphodiesterase inhibitor, which also inhibits the ADP-induced shape change of platelets, had no inhibitory effect on platelet NDK. It was concluded that the inhibitory effect of theophylline on the activity of the purified enzyme was due to the structural similarity between the methylxanthine and the adenine moiety of ADP.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1986

The role of platelets in the development and complications of atherosclerosis.

Although lipids have received most attention in relation to atherosclerosis, vessel injury also has a role in the development of atherosclerotic lesions. Thrombi that form at sites of injury can be incorporated into the wall, causing thickening, and platelets that adhere to damaged vessel walls release a growth factor (PDGF) that stimulates smooth muscle cell proliferation. The early lesions of atherosclerosis are focal and develop around vessel orifices and branches in relation to the patterns of blood flow and areas of increased permeability and endothelial cell damage. Platelets also contribute to the complications of advanced atherosclerosis caused by occlusive thrombi, thromboembolism, and spasm. The causes of vessel wall injury are not established, although there is evidence pointing to disturbed blood flow, hypertension, antigen--antibody complexes, complement, materials originating from platelets and white blood cells, bacteria, endotoxin, viruses, smoking, dietary lipids, homocystinemia, diabetes, other metabolic disorders, and stress. Platelets do not adhere to intact endothelium, but they adhere to the constituents of the subendothelium, release the contents of their granules (including PDGF), and form thromboxanes. If blood flow is disturbed, platelet--fibrin thrombi can form at sites of injury. Platelet adherence to a damaged wall does not require von Willebrand factor except under conditions of high wall shear. Repeated injury of a vessel wall leads to the development of lipid-rich atherosclerotic lesions, even in normocholesterolemic animals, but these lesions do not form if the experimental animals are made thrombocytopenic before injury is induced. Measurable changes in platelets that are associated with the clinical complications of atherosclerosis include shortened survival, release of granule contents (platelet factor 4, beta-thromboglobulin, thrombospondin), formation of thromboxanes, and decreased buoyant density. "Antiplatelet drugs" such as aspirin are proving to be beneficial in selected groups of patients, such as those with unstable angina. Thromboxane synthetase inhibitors and agents that block the thromboxane receptor on platelets are under investigation. Long term administration of "antiplatelet drugs" to affect the rate of development of atherosclerosis seems neither feasible nor desirable. Modification of dietary and smoking habits and control of hypertension are more likely to be beneficial for most individuals.

Animals↗
PubMed1985

Comparison of the interactions of fibrinogen and soluble fibrin with washed rabbit platelets stimulated with ADP.

Because fibrin, formed at a site of vessel wall injury, is involved in the formation and stabilization of a platelet aggregate or thrombus, we have studied reactions of fibrin with rabbit platelets. Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization, was used to prepare soluble fibrin. Fibrin alone did not cause aggregation of washed platelets, but addition of ADP caused aggregation and deaggregation identical to those observed in the presence of fibrinogen. Specific binding of 125I-fibrin to ADP-stimulated platelets was similar to that of 125I-fibrinogen, but 125I-fibrin did not dissociate, even in the presence of high concentrations of apyrase. High non-specific binding of 125I-fibrin was observed that was not associated with aggregation. EDTA, prostaglandin E1 (PGE1) and creatine phosphate/creatine phosphokinase prevented ADP-induced aggregation in the presence of fibrin and caused rapid deaggregation when added after ADP. They also inhibited 125I-fibrin binding when added before ADP, and EDTA or PGE1 caused partial dissociation of bound 125I-fibrin. In vivo, fibrin may bind to stimulated platelets, polymerize, form a gel, and interact with components of the plasma, the platelet aggregate, and the exposed subendothelium.

Adenosine Diphosphate↗
PubMed1985

Deaggregation of human platelets aggregated by thrombin.

Human platelets that have undergone the release reaction do not deaggregate readily. We examined conditions under which washed human platelets can be deaggregated after they have undergone an extensive release reaction induced by thrombin (1 or 5 U/ml). To make fibrinogen receptors unavailable, either CP/CPK (or apyrase) was used to remove released ADP, or PGE1 was used to increase cAMP. Chymotrypsin was used to digest proteins that might link platelets, and heparin to interact with released proteins and interfere with their binding to platelets and to each other. Individually, none of these caused deaggregation; heparin did not inhibit the effect of thrombin because no antithrombin III was present. Platelets exposed to thrombin (1 U/ml) which was neutralized at 90 sec by hirudin, could be deaggregated by combinations of CP/CPK (or apyrase) and chymotrypsin, or PGE1 and chymotrypsin. When a higher concentration of thrombin was used (5 U/ml) these combinations caused platelets to deaggregate only when heparin was added before thrombin induced the release reaction. Thus, when extensive release occurs three mechanisms may come into play to link human platelets: one that requires the fibrinogen receptor; a heparin-sensitive reaction that may involve the binding of released proteins; and a linkage that can be disrupted only by proteolysis, providing the other two mechanisms are also inhibited.

Blood Platelets↗
PubMed1985

Effects of plasmin on rabbit platelets.

The effects of plasmin have been examined because platelets may be exposed to plasmin in vivo and treatment of platelets with plasmin shortens platelet survival. Rabbit plasmin was prepared by urokinase activation of plasminogen immobilized on lysine-Sepharose. Plasmin caused rabbit platelets to aggregate and release the contents of their amine storage granules, but aggregation was slower than in response to ADP or thrombin. EDTA, prostaglandin E1, or creatine phosphate/creatine phosphokinase were inhibitory, but indomethacin was not. Deaggregation did not occur when platelets had been aggregated by a concentration of plasmin that caused extensive release of granule contents. EDTA or prostaglandin E1 caused deaggregation. Low concentrations of ADP and plasmin acted synergistically in causing platelet aggregation. Plasmin decreased the amounts of platelet membrane glycoproteins that stained with periodic acid-Schiff reagent; glycoprotein I was more susceptible than glycoprotein II and III. Concentrations of plasmin that induced the release of amine storage granule contents also released PAS-staining granule glycoproteins. Platelets incubated with plasmin, washed and resuspended, were not aggregated by ADP, but were aggregated strongly by the combination of fibrinogen and ADP, and bound 125I-fibrinogen to a greater extent than untreated platelets. Platelets preincubated with a high concentration of plasmin were unresponsive to thrombin, but were sometimes aggregated by fibrinogen. Plasmin decreased the buoyant density and increased the median size of platelets. Thus plasmin, as well as ADP and thrombin, may contribute to the density shift observed in platelets from rabbits in which thrombosis and continuous vessel injury have been induced.

Adenosine Diphosphate↗
PubMed1985

Measurement of fibrinogen concentrations in suspensions of washed rabbit and human platelets by radioimmunoassays.

Although fibrinogen is a cofactor in platelet aggregation, washed rabbit platelets aggregate when stimulated with ADP even when no fibrinogen is added to the platelet suspension. Washed human platelets usually do not aggregate to a significant extent when stimulated with ADP unless fibrinogen is added. To study this phenomenon, radioimmunoassays for rabbit and human fibrinogens have been developed and used to measure fibrinogen concentrations in suspensions of washed platelets. The fibrinogen concentration in the suspending medium of rabbit platelets was 2.5 +/- 0.9 micrograms/10(9) platelets, and upon stimulation with 9 microM ADP it increased to 10.7 +/- 2.9 micrograms/10(9) platelets. The loss of fibrinogen from the platelets was significantly greater than the loss of 14C-serotonin (11% vs 2%). The presence of prostaglandin E1 reduced the fibrinogen concentration to approximately 1 micrograms/10(9) platelets and prevented aggregation and loss of fibrinogen when the platelets were stimulated with ADP. With human platelets, the extracellular concentrations of fibrinogen and beta-thromboglobulin, expressed as percentages of the amount in the platelets, were similar, and the increase in fibrinogen concentration upon ADP stimulation (approximately 2%) was much lower than with rabbit platelets. We conclude that rabbit platelets may release fibrinogen from their alpha-granules when stimulated with ADP, and that a portion of the released fibrinogen becomes available to support aggregation. Smaller amounts of fibrinogen would become available in the case of human platelets.

Adenosine Diphosphate↗
PubMed1985

Effect of ticlopidine on platelet aggregation, adherence to damaged vessels, thrombus formation and platelet survival.

Ticlopidine (100 mg/kg/day or 400 mg/kg/day) was administered to rats and rabbits for 48 hr before and during the experiments. Aggregation studies of twice-washed platelets resuspended in Tyrode solution containing apyrase and 0.35% albumin showed that inhibition by ticlopidine of aggregation induced by ADP, collagen, sodium arachidonate or thrombin persisted after resuspension, as did inhibition of the release of 14C-serotonin from prelabeled platelets. Thus the inhibitory effect of ticlopidine or its metabolite is not readily reversed. In both species, ticlopidine prolonged platelet survival when it had been shortened by the insertion of an indwelling aortic catheter, although only the higher dose was effective in rabbits. In this species, this dose also prolonged platelet survival in sham-operated animals. Ticlopidine did not have a significant effect on the clearance of rabbit platelets when their survival had been shortened by pretreatment with neuraminidase. Ticlopidine did not affect the number of 51Cr-labeled platelets that accumulated on the injured vessel wall in rats with indwelling aortic catheters or the amount of thrombus that formed around the catheters in the aortas of the rabbits. It also did not affect the accumulation of platelets in vivo on rabbit aortas de-endothelialized with a balloon catheter. Thus, although ticlopidine inhibited platelet aggregation and release and prolonged shortened platelet survival, it did not inhibit platelet adherence to the damaged wall or thrombosis caused by chronic arterial injury. It is evident that effects on platelet survival and thrombosis do not correlate. The reason for the prolongation of platelet survival is unknown.

Animals↗
PubMed1985

Effects on the buoyant density of rabbit platelets of ADP and agents that increase the concentration of cyclic AMP.

Rabbit platelets were aggregated by adenosine diphosphate (ADP), allowed to deaggregate and then separated into density subpopulations by centrifugation through discontinuous Stractan density gradients. Although ADP causes little or no release of the contents of the amine storage granules of rabbit platelets, ADP caused a decrease in platelet density as compared with control platelets subjected to the same procedures except for exposure to ADP. The density change persisted for at least four hours. The apparent size of platelets stimulated with ADP increased initially, but returned to control values during a one-hour period. A similar decrease in platelet density was observed with an albumin density gradient. Under conditions in which aggregation did not occur in response to ADP with ethylenediaminetetraacetic acid (EDTA) in the medium, little or no decrease in platelet density was observed. Agglutination with polylysine did not change platelet density. Thus, not only agents such as thrombin and plasmin that cause the release of the contents of the platelet granules decrease platelet density, but ADP also has this effect. Platelets would be exposed to all of these stimuli during thromboembolic processes, and their effect on platelets may account for the decrease in platelet density observed previously in experiments with rabbits with indwelling aortic catheters. Agents that increase the concentration of cyclic AMP (cAMP) in platelets (PGE1, adenosine, dibutyryl cAMP, forskolin, and papaverine) also decreased platelet density. This effect persisted when the platelets were washed and resuspended in fresh medium and was also demonstrable in plasma. Platelet size was gradually increased by prostaglandin E1 (PGE1) which maintains platelets in a disc shape and does not cause the release of granule contents, indicating that the decrease in platelet density caused by PGE1 may be attributable to platelet swelling.

Adenosine Diphosphate↗
PubMed1985

Platelets, endothelium, and vessel injury.

Injury to the endothelial lining of arteries is an important mechanism in both the early and late stages of the development of atherosclerosis. Platelets can contribute to the early lesions by releasing factors that cause smooth muscle cell migration and proliferation. In the later stages, the formation of large platelet-fibrin thrombi that become organized into the vessel wall contributes to the development of focal atherosclerotic narrowing of arteries. Injury to the vessel wall can also be a factor in causing spasm of coronary arteries, particularly at sites of stenosis. The spasm may cause ischemia, anginal pain, and, in some individuals, ventricular fibrillation and death. In other individuals, the spasm may not cause death but may persist long enough for an occlusive thrombus to form and cause myocardial infarction. The events leading to thrombosis involve not only the release of arachidonic acid and the formation of TXA2, but other pathways that are independent of the arachidonate pathway. In some circumstances thrombin (which causes platelet aggregation and release that are largely independent of the arachidonate pathway and TXA2 formation) is the primary stimulus causing the initiation and growth of the thrombus. The role of products of the arachidonate pathway in causing spasm is not understood. PGI2 produced by the vessel wall could be important in preventing or minimizing coronary artery spasm. The best way to prevent the development of atherosclerosis and its clinical complications is to prevent or minimize injury of the endothelium.

Animals↗
PubMed1985

Duration of the effect of aspirin on the synthesis of thromboxane by density subpopulations of rabbit platelets stimulated with thrombin.

The controversy concerning the relationship between platelet buoyant density and platelet age is unresolved. Our earlier results with rabbit platelets indicate that the most-dense subpopulations are enriched in young platelets and that some platelets become less dense as they age. Other investigators have concluded that platelets either do not change in density upon aging or become more dense. In the present experiments, rabbit platelets were separated on discontinuous gradients of Stractan. Most-dense platelets synthesized significantly more thromboxane B2 (TXB2) (1.27 ng per 10(6) platelets) in response to thrombin (0.75 U/mL) than did least-dense platelets (0.70 ng per 10(6) platelets), indicating that the arachidonate pathway in most-dense platelets is more active than in least-dense platelets. After aspirin administration to rabbits, most-dense platelets recovered their ability to synthesize thromboxane B2 significantly more quickly than did least-dense platelets. Because the platelet cyclooxygenase that is responsible for TXB2 formation is permanently inhibited by aspirin, it is only the new platelets entering the circulation that will be able to form TXB2. These results indicate that, at least in rabbits, the most-dense platelets are enriched in young platelets, and that platelets decrease in density as they age in the circulation.

Animals↗
PubMed1984

Effect of amino sugars on platelet aggregation and on fibrinogen binding.

The amino sugars glucosamine, galactosamine and mannosamine (30 mM) inhibited aggregation of human or rabbit platelets induced by ADP, collagen, thrombin, PAF or high concentrations of sodium arachidonate. 125I-fibrinogen binding during ADP-induced aggregation, and release of amine storage granule contents were also inhibited. Increasing the calcium concentration of the suspending medium to 5 mM did not overcome the inhibitory effect on the release reaction. The amino sugars deaggregated rabbit platelets that had been aggregated by ADP, collagen or thrombin, but deaggregated human platelets readily only when ADP was used as the aggregating agent. Fibrinogen-induced aggregation of chymotrypsin-treated platelets was blocked by the amino sugars. They did not inhibit platelet adherence to a collagen-coated glass surface, nor affect release of granule contents from the adherent platelets. Aggregation and release induced by low concentrations of sodium arachidonate or the divalent cation ionophore A23187 were potentiated, indicating that the effects of the amino sugars on platelets are more complex than simple inhibition of the lectin-like activity that becomes available on the surface of platelets that have undergone the release reaction. One of the effects of the amino sugars, however, is interference with the binding of fibrinogen to platelets. The effects of the amino sugars are shared by other primary amines.

Adenosine Diphosphate↗
PubMed1984

Effects of tris on responses of human and rabbit platelets to aggregating agents.

Despite reports that Tris [tris (hydroxymethyl)aminomethane] affects platelets, it is often used to buffer suspending media. Human or rabbit platelets were washed and resuspended in Tyrode solution containing apyrase and 0.35% albumin. Addition of 15 mM Tris partially inhibited primary aggregation induced by 10 microM ADP and inhibited aggregation and release of 14C-serotonin from prelabelled platelets stimulated with low concentrations of thrombin (0.05-0.2 U/mL), or collagen. Platelets resuspended in 15 mM Tris, 0.15 M NaCl, 0.35% albumin, pH 7.5, did not aggregate in response to 10 microM ADP whereas platelets in Tyrode-albumin aggregated extensively. Ca2+ (5 mM) did not overcome the inhibition of thrombin-induced aggregation. Tris (15 or 1.5 mM) potentiated aggregation and release induced by sodium arachidonate (20-50 microM) or the ionophore A23187 (0.6-1 microM). Pretreatment of platelets with aspirin did not prevent potentiation by A23187, indicating that it is not mediated through activation of the arachidonate pathway. The inhibitory and potentiating effects of Tris are similar to those of amino sugars, lysine, arginine and primary amines such as methylamine and cadaverine, and may represent general effects of amines on platelets. Potentiation of the effects of some aggregating agents and inhibition of others re-emphasizes the concept that there are several different mechanisms through which aggregation can occur. Tris-based buffers are unsuitable for platelet suspending media and their use as solvents for aggregating agents or inhibitors should be limited.

Adenosine Diphosphate↗
PubMed1984

Mechanisms in thrombosis.

A key factor in atherosclerosis, spasm and thrombosis is damage to the endothelium. Prevention of injury to the endothelium will reduce the extent of atherosclerosis, reduce the risk of spasm and prevent thrombosis. Future work in this field will obviously be centered primarily on the endothelium, mechanisms of injury and strategies for prevention of endothelial cell injury. At present, it is not known how useful drugs will be for the long term management of atherosclerosis and its complications. It may be that the more important approach in the future will be to modify risk factors such as smoking and diet since these might injure the endothelium.

Animals↗
PubMed1984

Platelet adhesion.

Platelets do not adhere to surfaces to which flowing blood is normally exposed in vivo. When the lining of a blood vessel is altered or damaged, however, platelets do adhere to the injured site. Platelet adhesion is one of the first events in the formation of hemostatic plugs and thrombi, and plays a part in the development of atherosclerotic lesions. Other surfaces to which platelets adhere include particulate matter in the blood stream, bacteria and other microorganisms, the artificial surfaces of prosthetic devices, and some altered cells in the blood, particularly macrophages. The majority of investigators have studied the interaction of platelets with the subendothelium of normal vessels of young animals, or with isolated vessel wall constituents such as collagen. There are very few studies of platelet adhesion to repeatedly damaged or diseased blood vessels, although it is generally assumed that platelets interact with the connective tissue, fibrin, and cholesterol crystals in atherosclerotic lesions. Underlying the endothelium of blood vessel is the basement membrane, which has been shown to contain type IV collagen, elastin with its associated microfibrils, von Willebrand Factor, fibronectin, thrombospondin, laminin, and heparan sulfate. If only the endothelium is removed, the main structure exposed is the basement membrane with its associated proteins, but deeper injuries expose fibrillar type III collagen and microfibrils. In most studies in which large arteries have been injured by passage of a balloon catheter, basement membrane, type III collagen and the microfibrils around elastin have been exposed. Platelets do not react strongly with basement membrane and the type IV collagen in it is relatively inert. In contrast, platelets adhere firmly to type III (and type I) collagen and spread on it. Although in vitro studies have shown that platelets can interact with collagen in artificial media without plasma proteins, investigations of platelet adhesion at high shear rates indicate that von Willebrand Factor is necessary for firm platelet adhesion under these conditions. Fibronectin and thrombospondin may also have a role in platelet adhesion. However, platelets do not bind von Willebrand Factor or fibronectin until the platelets have been stimulated to release their granule contents, so these binding sites probably do not become available until the platelets have interacted with collagen or another release-inducing agent such as thrombin.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

Effects of variant gamma chains and sialic acid content of fibrinogen upon its interactions with ADP-stimulated human and rabbit platelets.

When platelets are stimulated with adenosine diphosphate (ADP), fibrinogen binds to receptors on the platelet membrane, and the platelets aggregate. The primary platelet recognition sites of human fibrinogen are reported to be at the COOH-terminal ends of the gamma chains, with secondary sites in the A alpha chains. Normal human fibrinogen, which consists of three pairs of disulfide-bonded peptide chains, (A alpha, B beta, gamma)2, is heterogeneous with respect to sialic acid content and also contains a small proportion of molecules with a variant gamma chain (designated gamma'), elongated by a peptide extension at the COOH-terminus of the normal gamma chain. We separated fibrinogen into three fractions by chromatography on DEAE cellulose and tested the interactions of these fractions with ADP-stimulated human and rabbit platelets. Two fractions had the normal chain composition, (A alpha B beta, gamma)2, but different sialic acid contents (6.6 and 7.2 mol/mol), and the third fraction had the chain composition (A alpha, B beta)2 gamma gamma' and a sialic acid content of 7.2 mol/mol, which is similar to that of one of the normal fractions. In binding and aggregation experiments, we detected no significant differences between the reactions of the first two fractions, but ADP-stimulated platelets bound only 50% as much of 125I-fibrinogen from the fraction with the gamma' chains and also aggregated less extensively in the presence of this fraction. We conclude that the sialic acid content of fibrinogen does not significantly affect its interactions with platelets, but the elongated gamma' chains bind less effectively to ADP-stimulated platelets, and thus reduce the ability of fibrinogen to support aggregation. This may result from a conformational change caused by the gamma' extension or from the deletion of a portion of the normal gamma chain recognition site.

Adenosine Diphosphate↗
PubMed1983

The effect of thrombin on platelet accumulation on the vessel wall - influence of heparin and aspirin.

Rabbit aortae were removed from exsanguinated rabbits, washed, everted on probes, treated with thrombin, washed to remove unbound thrombin and used to measure the accumulation of 51Cr-labeled platelets in vitro. Thrombin pretreatment of normal rabbit aortae did not cause platelet accumulation on the endothelium; platelets appeared to accumulate only at sites where the subendothelium had been exposed. The quantitative data obtained with 51Cr-labelled platelets was reinforced by observations by scanning electron microscopy. 125I-labelled thrombin became associated with the endothelium and also with de-endothelialized vessels, and some of it could be displaced by high concentrations of heparin. Exposure of vessels to heparin after thrombin treatment eliminated the enhanced platelet accumulation caused by the thrombin treatment, probably because heparin displaced thrombin from the aortae, as demonstrated in experiments with 125I-thrombin. Inhibition of PGI2 production by aspirin treatment of the vessels did not enhance platelet accumulation on normal or thrombin-treated aortae. Thus, although thrombin treatment of the endothelium does not cause platelets to adhere to it, thrombin does cause increased platelet accumulation on the areas where the subendothelium is exposed or where endothelial cells are damaged.

Animals↗