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Biomedical subjects

M J Silver

Publications and source records attributed to M J Silver.

At least 37 records · Page 2Linked to original sources
PubMed1986

Trial of repeated low-dose aspirin in diabetic angiopathy.

We compared the ability of aspirin to suppress platelet aggregation and thromboxane synthesis in ten normal subjects and ten patients with diabetic angiopathy and high rate of entry of new platelets into the circulation. When single doses of 100 to 1,000 mg aspirin were ingested daily for 1 month, there were time gaps between doses in which platelets from diabetics and normals aggregated and formed thromboxane ex vivo in response to the combination of arachidonic acid plus collagen. Similar gaps were also found for diabetics, but not for normals, following four daily doses (every six hours) of 25 or 100 mg. Our data show that dose schedules of aspirin which may suffice in normals are not effective in patients with diabetic angiopathy, presumably because these patients have a high rate of entry of new platelets into the circulation. We suggest that continual suppression of platelet thromboxane synthesis and aggregation by low-dose, "slow-release" preparations of aspirin would be an ideal long-term approach for the prevention of thrombosis in patients with a high rate of entry of new platelets into the circulation.

Adenosine Triphosphate↗
PubMed1985

Phospholipid biosynthesis in human platelets. The acylation of lyso-platelet-activating factor.

Acyl-CoA:1-alkyl-sn-glycero-3-phosphocholine acyltransferase of human platelets is membrane-bound, has a pH optimum of 7.5, is insensitive to 1 mM-Mg2+, is inhibited by 1 mM-Ca2+, and is stimulated slightly by 1 mM-EDTA. Maximal formation of 1-alkyl-2-acyl-sn-glycero-3-phosphocholine is observed at 150 microM-1-alkyl-sn-glycero-3-phosphocholine and 20 microM unsaturated fatty acyl-CoA. The transfer of unsaturated fatty acyl groups to 1-alkyl-sn-glycero-3-phosphocholine is 3-14 times slower than to 1-acyl-sn-glycero-3-phosphocholine. The CoA esters of linoleate and arachidonate, two unsaturated fatty acyl groups commonly found in platelet phospholipids, are the preferred fatty acyl group donors.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMed1985

Increased binding of fibrinogen to platelets in diabetes: the role of prostaglandins and thromboxane.

Previous studies suggested a role for prostaglandins or thromboxane A2, or both in the exposure of fibrinogen receptors on normal platelets in response to several aggregating agents. Platelets from diabetics are known to be more sensitive to aggregating agents and to produce more prostaglandins and thromboxane than platelets from normal subjects. We compared fibrinogen binding to platelets from diabetic subjects with binding to platelets from normal subjects and determined whether aspirin (which inhibits the formation of prostaglandins and thromboxane) would inhibit the binding of fibrinogen to platelets from diabetic subjects and whether this correlated with its effects on platelet aggregation. We found the following: Aspirin suppressed thromboxane formation and rendered the platelets less sensitive to the induction of aggregation by adenosine diphosphate (ADP) or collagen. The amount of U-46619 [( 15s]-hydroxy-11-alpha, 9-alpha [epoxy-methano]-prosta[5Z,13E]-dienoic acid, a stable analog of prostaglandin endoperoxide/thromboxane A2) necessary to induce aggregation, was similar in normal and diabetic subjects and was unchanged after ingestion of aspirin. Binding of 125I-fibrinogen following stimulation of platelets by ADP or collagen was greater in diabetic (because more binding sites were exposed) than in normal subjects. However, following stimulation by U-46619, binding was similar in diabetic and normal subjects. Aspirin caused a reduction in the exposure of binding sites on both platelets from diabetic and normal subjects, so that (in this respect) platelets from diabetic subjects became more like those from normal subjects. Effects of the monoclonal antibody B59.2, which is specific for the platelet glycoprotein IIb-IIIa complex (the presumed receptor for fibrinogen on the platelet surface) were also studied. The amount of this antibody that bound to platelets was the same for normal and diabetic subjects both before and after aspirin and with or without stimulation by ADP or collagen. In addition, B59.2 inhibited aggregation and fibrinogen binding in both platelets from diabetic and normal subjects. The combined data suggest that the glycoprotein IIb-IIIa complex of platelets from diabetic subjects is similar to that of platelets from normal subjects and that the increased fibrinogen binding and aggregation of platelets from diabetic subjects in response to ADP or collagen is mediated by increased formation of prostaglandin endoperoxide or thromboxane A2, or both.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1984

A quick method for screening platelet dysfunctions using the whole blood lumi-aggregometer.

Patients who present with a clinical history suggesting a bleeding disorder are often tested initially for a clotting defect rather than for platelet dysfunction, due to the length of time necessary to complete a platelet function study in platelet-rich plasma. We have developed a sensitive method for measuring platelet aggregation and release of ATP employing the Whole Blood Lumi -Aggregometer. This method makes it possible to quickly detect patients who require further study for possible platelet function disorders such as cyclooxygenase deficiency, storage-pool defect, thrombasthenia, and von Willebrand's disease. The results obtained with this electrical impedance instrument do not differ from those obtained with the conventional optical method. However, it is now possible to recognize a platelet function defect within 30 min of obtaining a 5 ml sample of citrated whole blood. Further, platelets of unusual size or density are not lost to testing through centrifugation.

Adenosine Triphosphate↗
PubMed1984

Model system to study interaction of platelets with damaged arterial wall. I. Inhibition of platelet adhesion to subendothelium by aspirin and dipyridamole.

An experimental model system has been developed to study the interaction of platelets with a damaged vessel wall, in vivo, without deep surgical intervention. Endothelium of the central ear artery of an anesthetized rabbit is damaged by placing artery forceps on the ear directly over the vessel. Forceps are removed 30 min later and blood flow resumes. After 30 min, blood is washed out with Tyrode's solution and the vessel is perfused with 1% glutaraldehyde solution. Tissue containing the vessel is then removed and further prepared for scanning and transmission electron microscopy. Damage to the endothelium observed by scanning electron microscopy included separation of adjacent endothelial cells (EC); partial destruction and lifting up of some EC, exposing subendothelium; and denudation of larger areas of endothelium. Disc-shaped platelets were seen clinging to some damaged endothelial cells. Platelets adhered, formed pseudopods, and spread over the surface of some areas of exposed subendothelium. The extent of platelet adhesion to exposed subendothelium was estimated by eight "blind" evaluators and the average taken. Aspirin (8 mg/kg, ip) significantly inhibited adhesion (P less than 0.05) when compared to controls. No other agent tested gave significant inhibition after a single treatment. Dipyridamole (1.5 mg/kg, ip) given five times on 3 successive days, inhibited adhesion significantly (P less than 0.001). Heparin (800 U/kg, iv) or dipyridamole (0.7 mg/kg, ip) enhanced the inhibitory effect of aspirin (8 mg/kg, ip), with either combined treatment giving P less than 0.001.

Animals↗
PubMed1983

Human and rabbit platelets form platelet-activating factor in response to calcium ionophore.

Homogenous preparations of human and rabbit platelets synthesized platelet-activating factor (PAF) when stimulated by calcium ionophore A23187. PAF was isolated by high performance liquid chromatography (HPLC) on a Lichrosorb Si60 column. PAF was well separated from other phospholipids especially sphingomyelin and lysolecithin. The retention times of PAF derived from human or rabbit platelets were identical to the commercially available semisynthetic PAF and radiolabeled PAF. The amount of PAF was determined by the extent to which it induced the aggregation of washed rabbit platelets.

Animals↗
PubMed1983

Metabolism of platelet-activating factor by blood platelets and plasma.

High performance liquid chromatography in combination with a radioactivity detector was used to study the metabolism of platelet-activating factor (1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by washed platelets, platelet-free plasma and platelet-rich plasma obtained from rabbits and humans. Degradation of platelet-activating factor to its 2-lyso derivative was observed in rabbit and human plasma. This degradation of platelet-activating factor in plasma was completely inhibited by diisopropylfluorophosphate and was partially inhibited by ethylenediamine tetraacetic acid. Washed platelets metabolized platelet-activating factor not only to the 2-lyso compound but also, by reacylation of this lyso intermediate, to an analogue of platelet-activating factor probably containing a long-chain acyl group at the sn-2 position. These transformations occurred, but to a lesser extent, in platelet-rich plasma.

Animals↗
PubMed1983

Early morphological changes in the endothelium of a peripheral artery of rabbits fed an atherogenic diet.

The effect of an atherogenic diet on the endothelium of the central artery of the rabbit ear was studied by scanning and transmission electron microscopy. Examination of the inner surface of the artery after only 5 weeks on the diet revealed morphological changes including irregularly shaped cells, breaks at intercellular junctions, swelling of the membrane over the central part of the cells, and occasional holes in the cells. By 9 weeks more severe damage was seen including lifting off of cells and some holes. In addition, arrays of dark spots were seen by scanning electron microscopy in some cells in arteries of rabbits fed the diet for 5 to 9 weeks. The dark spots may correspond to abnormally large vacuoles seen inside endothelial cells by transmission electron microscopy. The central ear artery and aorta of some rabbits were tested for their ability to convert arachidonic acid into prostacyclin and thromboxane A2 by radioimmunoassay of the stable metabolites 6-keto-prostaglandin F1 alpha and thromboxane B2. The vessels from rabbits fed the atherogenic diet did not differ from vessels of rabbits on the control diet in their production of either metabolite.

6-Ketoprostaglandin F1 alpha↗
PubMed1983

Backscattered electron imaging to visualize arterial endothelial detachment in the scanning electron microscope.

The endothelium of the central ear artery of an anesthetized rabbit was damaged by placing artery forceps on the ear over the vessel for 30 min. After removal of the forceps, blood was allowed to flow through the injured vessel for 30 min. Blood flow was then stopped, blood was washed out of the artery in situ and initial fixation with 1% glutaraldehyde was begun. Specimens of the vessel containing the damaged region were postfixed in 3% cacodylate buffered glutaraldehyde and prepared for SEM examination after methenamine silver staining or following post fixation with osmium. Backscattered electron imaging (BEI) of silver stained specimens with inverted signal polarity showed "black" endothelial nuclei similar to darkly stained nuclei of conventional light microscope slide preparations. Secondary electron imaging (SEI) of corresponding sites stained with silver also showed endothelial nuclei. An interruption of the normal pattern of nuclei indicated a detached area of endothelium where subendothelium was exposed. Platelets were observed adhering to the surface of the exposed subendothelium. Injury to the endothelium was seen more readily in silver stained preparations as voids in the nuclear pattern as compared with conventional SEI preparations, postfixed with osmium, which did not show well defined nuclei.

Animals↗
PubMed1983

Evaluation of electrical aggregometry: comparison with optical aggregometry, secretion of ATP, and accumulation of radiolabeled platelets.

Platelet aggregation has been most commonly studied in vitro by measuring increases in light transmission as platelets aggregate in PRP. Recently, an electrical impedance method for measuring platelet aggregation has been introduced. This method can be used with either PRP or whole blood and measures an increase in impedance across electrodes placed in the blood samples as platelets accumulate on them. We have compared the results obtained by the two methods, using ADP and collagen as aggregating agents, and also have measured the secretion of platelet ATP simultaneously. Although the aggregometry results were similar, recordings obtained by the electrical method did not distinguish two waves of platelet aggregation or correlate with secretion as well as recordings obtained by the optical method. When PGI2 or PGE1 was added to the PRP, both the rate and extent of the increase in light transmittance were inhibited, but the main effect on the increase in impedance was a decrease in its rate and not in its extent. Increases in impedance and secretion of ATP were also measured in whole blood after the platelets had been labeled with a 125I-containing antibody specific for platelet surface glycoproteins. It appeared that the increases in impedance lagged several minutes behind the formation of platelet aggregates and the secretion of platelet ATP. It is suggested that there are advantages and disadvantages to both methods of measurement of platelet aggregation and that the parameters being measured must be clearly understood to properly interpret the results.

Adenosine Triphosphate↗
PubMed1983

Monitoring the entry of new platelets into the circulation after ingestion of aspirin.

There have been reports of a 24-48-hr delay in the recovery of platelet cyclooxygenase activity and platelet function after the ingestion of aspirin. However, these studies employed a single aggregating agent to stimulate enzymatic or functional activity. We investigated the effects of some pairs of aggregating agents on 14 platelet-rich plasmas (PRP) from normal subjects 2 and 4 hr after ingestion of 650 mg aspirin and daily up to 72 hr. We studied platelet aggregation and secretion with a lumiaggregometer and thromboxane-B2 formation by radioimmunoassay. Aggregation and secretion occurred as early as 4 hr after aspirin ingestion in response to combinations of arachidonic acid with epinephrine, collagen, or adenosine diphosphate (ADP). Thromboxane formation was detected as early as 4 hr after ingestion of aspirin in response to 1 mM arachidonic acid in combination with 1 microgram/ml collagen. Up to 72 hr, there was a linear return of thromboxane formation stimulated by this combination, reflecting the entry of new platelets into the circulation. In vitro experiments with mixtures of aspirin-free and aspirin-treated platelets showed that the combination of collagen and arachidonic acid (AA) could produce full aggregation and secretion when only 2.5% of aspirin-free platelets were present. Use of the combination of collagen plus AA demonstrates the early entry into the circulation of platelets originating from megakaryocytes whose cyclooxygenase has not been completely acetylated.

Adenosine Triphosphate↗
PubMed1983

Mouse antithrombotic assay: a simple method for the evaluation of antithrombotic agents in vivo. Potentiation of antithrombotic activity by ethyl alcohol.

We present a simple method for screening antithrombotic agents. When male Swiss-Webster mice (25-34 g; N = 143) were given an i.v. injection of 0.1 ml of a mixture of collagen (dose, 15 micrograms/mouse) and epinephrine (dose, 1.8 micrograms/mouse), 94% died within 5 min or remained paralyzed for more than 15 min. Examples of the use of the system for the study of antithrombotic agents are given. Solutions of agents were administered to the animals i.p. about 1 hr before the thrombotic challenge. Aspirin (20 mg/kg), OKY-1581 (30 mg/kg) and ethanol (2 gm/kg), administered as single agents in aqueous medium, protected (P less than .01, chi 2 test) 40, 50 and 35% of the animals, respectively. Heparin (150 U/kg) was ineffective. Combinations of ethanol with indomethacin or indobufen provided complete protection, whereas ethanol plus aspirin protected 84% of the animals and ethanol plus OKY-1581 protected 70% of the animals. Dipyridamole alone (3 mg/kg), dipyridamole (1.65 mg/kg) plus ethanol or dipyridamole (1.65 mg/kg) plus aspirin were ineffective. The method appears well suited for screening potential antithrombotic agents which act primarily against platelet thromboembolism.

Adenosine Triphosphate↗
PubMed1982

Phospholipid biosynthesis in human platelets. Formation of phosphatidylcholine from 1-acyl lysophosphatidylcholine by acyl-CoA:1-acyl-sn-glycero-3-phosphocholine acyltransferase.

Arachidonic acid and other fatty acids are taken up by human platelets from plasma and incorporated into membrane phospholipids. However, little is known about the mechanism and specificity of the various steps of fatty acid insertion into phospholipid. Previous findings from this laboratory have shown that the incorporation of radioactive C20-unsaturated fatty acids (arachidonic, 8,11,14-eicosatrienoic, and 5,8,11,14,17-eicosapentaenoic) into the phospholipids of "resting"p platelets is more rapid than that of the radioactive C16- and C18-saturated and unsaturated fatty acids. We now provide evidence that human platelet microsomes contain acyl-CoA:1-acyl-sn-glycero-3-phosphocholine acyltransferase. The enzyme preparation has a pH optimum of 7.0. Activity is insensitive to 1 mM EDTA and is inhibited 37% by 1 mM Ca2+ and 20% by 1 mM Mg2+. Maximal activity is observed at 100 microM 1-acyl lysophosphatidylcholine at several concentrations of fatty acyl-CoA esters. Apparent Km values from 1.05 to 5.70 microM were obtained for saturated and unsaturated fatty acyl group donors in the presence of 100 microM 1-acyl lysophosphatidylcholine as fatty acyl group acceptor. Comparison of the apparent Vmax values showed that unsaturated CoA esters were transferred more rapidly to 1-acyl lysophosphatidylcholine than saturated CoA esters. Oleate, linoleate, and arachidonate, the major unsaturated fatty acids in platelet phosphatidylcholine, were transferred at similar rates. 8,11,14-eicosatrienoate was transferred about two times faster than these three fatty acyl groups. The data indicate that the incorporation of unsaturated fatty acids into phosphatidylcholine by human platelets occurs via reacylation of 1-acyl lysophosphatidylcholine.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMed1982

Regulation of arachidonate-induced platelet aggregation by the lipoxygenase product, 12-hydroperoxyeicosatetraenoic acid.

The lipoxygenase product, 12-hydroperoxyeicosatetraenoic acid (12-HPETE) was biosynthesized, purified and incubated with washed human platelet. It inhibited arachidonic acid, azo-prostaglandin H2 or U-46619-induced aggregation and secretion in a concentration-dependent fashion (IC50 = 2-3 microM). Collagen-induced aggregation and secretion were also inhibited (IC50 = 6 microM). 12-HPETE inhibited malondialdehyde and thromboxane B2 formation in platelets stimulated with arachidonic acid or thrombin. While thrombin-induced aggregation was unaffected by 50 microM 12-HPETE, thrombin-induced secretion was inhibited. Inhibiton of secretion by 12-HPETE was observed in platelets from untreated as well as aspirin-treated donors, indicating that 12-HPETE inhibits secretion independently of its ability to inhibit prostaglandin formation. Aggregation of washed human platelets by arachidonic acid yielded a bell-shaped concentration-response curve. Diminished aggregation at higher concentrations was associated with an increase in the ratio of lipoxygenase products to thromboxane B2. The data suggest that 12-HPETE formation may regulate platelet aggregation and secretion and that its primary effect, at low concentrations, is inhibition of endoperoxide-induced responses. At higher concentrations 12-HPETE also inhibits arachidonic acid metabolism. Thus, the combined inhibitory effects on endoperoxide-induced aggregation and thromboxane formation would explain the diminished aggregation observed in response to high concentrations of arachidonic acid.

Adenosine Triphosphate↗
PubMed1982

Arachidonic acid induces human platelet-fibrin retraction: the role of platelet cyclic endoperoxides.

Arachidonic acid (0.2-0.8 mM) retracts clots formed in human citrated platelet-rich plasma by batroxobin. Extracellular calcium ions, but not the secretion of ADP by platelets, are required. AA-induced clot-retraction requires cyclo-oxygenase but not thromboxane synthetase activity since the retraction is inhibited by aspirin but not by selective inhibitors of thromboxane synthesis. The data indicate that endogenous cyclic endoperoxides mediate the retraction. Moreover, intact endoperoxide/thromboxane receptors also seem to be necessary because clot retraction is inhibited by thromboxane receptor antagonists.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗