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D W Perry

Publications and source records attributed to D W Perry.

At least 37 records · Page 2Linked to original sources
PubMed1993

Degranulation of human platelets by the thrombin receptor peptide SFLLRN: comparison with degranulation by thrombin.

A new, simplified method of degranulating human platelets using the thrombin receptor peptide SFLLRN (20 microM) is described; released fibrinogen cannot be converted to fibrin, and the platelets are not exposed to a proteolytic enzyme, as they are when thrombin is used for degranulation. The peptide-degranulated platelets regain their disc shape and are recovered as single platelets which have released approximately 90% of the contents of their dense granules. Their procoagulant activity is greater than that of control platelets, but somewhat less than that of thrombin-degranulated platelets. Without added fibrinogen, the peptide-degranulated platelets aggregate slightly in response to 50 microM SFLLRN, and to collagen, arachidonic acid, the thromboxane A2 mimetic U46619, platelet activating factor, ADP, and the divalent cation ionophore A23187; added fibrinogen enhances aggregation caused by these agonists. Extensive aggregation of peptide-degranulated platelets is caused by thrombin in the absence of added fibrinogen; it may be that the alternative thrombin receptor that is not activated by SFLLRN is responsible for the strong response to thrombin. Aggregation responses to most of the agonists are greater than those observed previously with thrombin-degranulated platelets. By this method, platelets are obtained that have been degranulated in a way that is similar to in vivo degranulation. They are useful for studies of platelet responses without the complicating effects of released granule contents, and for investigation of the characteristics and functions of platelets that have come in contact with release-inducing agents in vivo.

Amino Acid Sequence↗
PubMed1993

A significant portion of the aequorin luminescent signal from stimulated human and rabbit platelets is due to exposure of the aequorin to calcium in the suspending medium.

We have examined in unstimulated and thrombin-stimulated human and rabbit platelets the localization and behavior of aequorin loaded by a variety of published methods. When platelets were suspended at 37 degrees C in Tyrode-albumin medium containing 2 mM Ca2+ and apyrase, we found with all preparations that total aequorin revealed by addition of Triton X-100 decreased by more than 50% over one hour. Incubation in the presence of 5 mM EGTA followed by addition of Ca2+ to restore the concentration to 2 mM showed that some aequorin had entered the medium; subsequent addition of Triton X-100 showed that the increase in aequorin in the medium matched the decrease in aequorin in the platelets, such that total aequorin remained unchanged. However, comparison of aequorin in platelets incubated in media with and without Ca2+ showed a larger decrease in platelets incubated in the presence of Ca2+; this finding may indicate the presence of an intracellular pool of Ca2+ which is more dependent on external Ca2+. Stimulation of platelets with thrombin in the presence of EGTA resulted in a smaller luminescent signal than in the presence of Ca2+. Subsequent addition of Ca2+ to 2 mM in the platelet suspension that originally contained EGTA or to its supernate (after centrifugation of the platelet suspension), resulted in a larger luminescent signal compared with controls, indicating that stimulation of the platelets had increased loss of the aequorin into the medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Aequorin↗
PubMed1993

Rabbit lung macrophages stimulate platelets in vitro as observed by density-gradient centrifugation and transmission electron microscopy.

Both platelets and macrophages play a role in the pathogenesis of atherosclerosis. To examine whether they may interact and, if they do, to elucidate the mechanisms of such an interaction, suspensions of the two cell types from rabbits were mixed together, then subjected to Stractan density-gradient centrifugation and transmission electron microscopy. Suspensions of only one cell type served as controls. When otherwise unstimulated platelets and macrophages came into contact with each other, the platelets became less dense. Ultrastructurally, the platelets underwent shape changes without losing their granules, and were often arranged around the macrophages like a rosette. The processes of the macrophages became elongated. ADP caused a similar shift in platelet density and, when the cell types were together, increased this shift. With ADP the rosetting was abolished, but platelet aggregates were found to be in superficial contact with the macrophages. With thrombin the contact between the platelet aggregates and macrophages was close. Addition of platelet antagonists showed that the shift in platelet density and the rosetting upon contact with macrophages are dependent on divalent cations. Neither ADP, nor thrombin, nor PAF seem to be involved in the reactions.

Adenosine Diphosphate↗
PubMed1992

Decreased platelet membrane fluidity due to glycation or acetylation of membrane proteins.

Platelets from diabetic subjects and animals are hypersensitive to agonists in vitro. Membrane fluidity modulates cell function and previously we observed reduced membrane fluidity in platelets from diabetic patients associated with hypersensitivity to thrombin. We previously reported that decreased fluidity of isolated platelet membranes from diabetic patients is associated with increased glycation of platelet membrane proteins, but not with any change in the cholesterol to phospholipid molar ratio. We have now examined in vitro whether incubation of platelet membranes in a high glucose medium causes sufficient glycation to reduce membrane fluidity. Incubation of platelet membranes from control subjects in a high glucose (16.1 mM) medium for 10 days at 37 degrees C led to an increase in the extent of glycation of membrane proteins and a decrease in membrane fluidity (indicated by an increase in steady state fluorescence polarization); most of the changes occurred within the first 3 days of incubation. Incubation of platelet membranes with 5.4 mM glucose had less effect. In contrast, incubation of platelet membranes with the same concentrations of 1-0-methylglucose did not cause a change in either the extent of glycation of proteins or membrane fluidity. We also determined if acetylation by aspirin or acetyl chloride of the sites available for glycation on platelet membrane proteins leads to a similar reduction in membrane fluidity. Pretreatment of platelet membranes with aspirin or acetyl chloride diminished the extent of glycation that occurred when platelet membranes were subsequently incubated with glucose, but membrane fluidity was reduced even in the absence of glucose; subsequent incubation with glucose caused no further reduction in membrane fluidity.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylation↗
PubMed1992

Lack of stability of aggregates after thrombin-induced reaggregation of thrombin-degranulated platelets.

The stability of platelet aggregates is influenced by the extent of the release of granule contents; if release is extensive and aggregation is prolonged, deaggregation is difficult to achieve. The relative importance of the contributions of released substances to aggregate stability are not known, although stable thrombin-induced aggregates form in platelet-rich plasma from patients with barely detectable plasma or platelet fibrinogen, and ADP stabilizes thrombin-induced aggregates of platelets from patients with delta storage pool deficiency which otherwise deaggregate more readily than normal platelets. We degranulated platelets with thrombin (0.9 U/ml caused greater than 90% loss of delta and alpha granule contents) and recovered them as individual platelets in fresh medium. The degranulated platelets were reaggregated by thrombin (2 U/ml). To prevent continuing effects of thrombin, FPRCH2Cl was added when thrombin-induced aggregation of thrombin-degranulated platelets reached its maximum. EDTA (5 mM) or EGTA (5 mM) added at maximum aggregation did not deaggregate these platelets, indicating that the stability of these aggregates does not depend on Ca2+ in the medium. Whereas with control platelets a combination of PGE1 (10 microM) and chymotrypsin (10 U/ml) was required for deaggregation, with thrombin-degranulated platelets either PGE1 or chymotrypsin alone caused extensive deaggregation. The rate and extent of deaggregation of thrombin-degranulated platelets by a combination of PGE1 and chymotrypsin was greater than with control platelets.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗
PubMed1992

Hypersensitivity to ADP of platelets from diabetic rats associated with enhanced fibrinogen binding.

Platelets from diabetic humans and animals are hypersensitive to ADP. The hypersensitivity to ADP of platelets from diabetic rats occurs independently of activation of the arachidonate pathway or release of dense granule contents. During platelet aggregation by ADP, fibrinogen binds to its receptor on platelets. We examined if the hypersensitivity to ADP of platelets from diabetic rats is associated with enhanced early binding of fibrinogen to its receptor on these platelets. Fibrinogen association with platelets from rats with spontaneous or streptozotocin-induced diabetes was significantly greater 10 s or 1 min after addition of ADP (10 microM) than with platelets from their corresponding control rats. Since enhanced fibrinogen association occurred with platelets from insulin-treated rats with spontaneous diabetes, and from rats with streptozotocin-induced diabetes that did not receive insulin, the enhanced fibrinogen binding is likely due to the diabetic state rather than to the administration of insulin or the mechanism responsible for the diabetes. Therefore, enhanced early fibrinogen association with platelets from diabetic rats is associated with their hypersensitivity to ADP.

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

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↗
PubMed1983

Factors influencing the deaggregation of human and rabbit platelets.

The mechanisms involved in platelet deaggregation are unclear. Washed platelets from rabbits or humans aggregated by ADP can be deaggregated by EDTA or PGI2 if the release reaction has not occurred; during deaggregation 125I-fibrinogen dissociates from the platelets. Human platelets suspended in a medium without calcium undergo the release reaction during ADP-induced aggregation; EDTA, PGE1 or PGI2 do not deaggregate these platelets although EDTA displaces much of the 125I-fibrinogen that associates with them during aggregation. Rabbit platelets aggregated by low concentrations of release-inducing stimuli (sodium arachidonate, collagen or thrombin) can be deaggregated by EDTA, PGI2 or PGE1 and 125I-fibrinogen dissociates from them; with high concentrations of collagen or thrombin, deaggregation and dissociation of 125I-fibrinogen is slower. Human platelets that have undergone the release reaction in response to thrombin, collagen or a combination of sodium arachidonate and ADP are not readily deaggregated by EDTA or PGE1. Since aggregation and fibrinogen binding involving the glycoprotein IIb/IIIa complex are readily reversed by EDTA, and since Ca2+ is required for thrombospondin binding to activated platelets, there may be a third type of platelet-platelet adherence that is not disrupted by EDTA; this type of binding plays a greater role with human than with rabbit platelets.

Adenosine Diphosphate↗
PubMed1983

Changes in the properties of platelets from rats with experimentally induced shortened platelet survival.

Platelet survival is shortened in experimental animals in which indwelling aortic catheters have been placed. We have examined the properties of platelets harvested from rats on day 1 or day 6 after insertion of indwelling aortic catheters. Platelets taken from these animals at 6 days (but not at 1 day) survived in normal rats or in rats with indwelling catheters for a significantly longer time than platelets from sham-operated rats. The catheters caused a persistent fall in the platelet count, an increase in the proportion of platelets in the most dense fraction after separation on discontinuous Stractan density gradients, and a decrease in mean sialic acid and protein per 10(9) platelets, but not change in modal size as determined with a Coulter Counter Channelyzer. No significant differences in the sensitivity to aggregating and release-inducing agents (ADP, thrombin, or collagen) were observed. It seems likely that chronic damage of the vessel wall shortened platelet survival and increased platelet turnover, although the increase in platelet production was not sufficient to maintain the platelet counts at the same values as in the sham-operated animals. The prolonged survival times in recipient rats of platelets from rats with indwelling aortic catheters indicate that a population of predominantly young platelets exists in these animals. Their increased density supports this conclusion.

Animals↗
PubMed1982

The inhibitory effects of exogenous arachidonic acid on rabbit platelet aggregation and the release reaction.

Although arachidonic acid causes rabbit platelet aggregation and the release of granule contents in suspensions of washed platelets when used in concentrations of approximately 50-300 microM, higher concentrations (500 microM) cause neither aggregation nor release. Suspensions of platelets from rabbits wee exposed to arachidonic acid (250 microM) for 15 min, allowed to recover in the presence of PGE1 for 30 min, washed, and resuspended; in some experiments, the platelets were treated with aspirin before being exposed to arachidonic acid. Aggregation of platelets pretreated with arachidonic acid was inhibited in response to ADP; this effect was greater with the non-aspirin-treated platelets and persisted for at least 4 hr after resuspension. The association of 125I-fibrinogen with the platelets as a result of ADP stimulation was also inhibited. Aggregation and release of granule contents in response to collagen and low concentrations of thrombin was inhibited, but the inhibition could be overcome by higher concentrations. Thrombin induced further release of granule contents from platelets exposed to arachidonic acid without pretreatment with aspirin. Platelets that had been exposed to arachidonic acid, either with or without pretreatment with aspirin, did not aggregate or undergo further release upon stimulation with arachidonic acid after they were washed and resuspended. Inhibition of the lipoxygenase pathway with eicosatetraynoic acid (ETYA) or nordihydroguaiaretic acid (NDGA) did not affect the inhibition caused by arachidonic acid, so it is unlikely that a product of this pathway is responsible for the inhibition. Mixing experiments indicated that the pretreated platelets did not form a thromboxane-A2-like activity, and that they were unresponsive to aggregation and release induced by products formed from arachidonic acid. Experiments with 3H-arachidonic acid showed that after 45 min of incubation with platelets, only 1.1% of the 3H-arachidonic acid remained as free arachidonic acid in the platelets. Although cyclic-AMP was slightly increased 1 min after the addition of arachidonic acid, the cyclic-AMP concentration was the same as that of control platelets after the platelets were washed and resuspended, indicating that increased cyclic-AMP is not likely to be responsible for the persistent inhibitory effect. Thus, the inhibitory effect of pretreatment with arachidonic acid is a general effect on responses to a variety of aggregating agents that act through different mechanisms, and the inhibition is not related to thromboxane-A2 formation. The possibility of membrane perturbation resulting in the unavailability of receptors may explain the persistent inhibitory effect, but the responsible reactions have not been identified.

Adenosine Diphosphate↗
PubMed1979

Comparison of fibrinogen association with normal and thrombasthenic platelets on exposure to ADP or chymotrypsin.

Although 125I-fibrinogen becomes associated with washed platelets from normal human subjects during ADP-induced shape change and aggregation, 125I-fibrinogen did not become associated with washed plateletes from a thrombasthenic subject during ADP-induced shape change and the platelets did not aggregate. Platelets from control and thrombasthenic subjects were treated with chymotrypsin, which is known to degrade platelet membrane glycoproteins. More 125I-fibrinogen became associated with chymotrypsin-pretreated platelets from normal subejcts than with untreated platelets, and fibrinogen caused the enzyme-treated platelets to aggregate. 125I-fibrinogen did not become associated with chymotrypsin-pretreated thromobasthenic platelets, and fibrinogen did not aggregate them. Thus, there appears to be a defect in thrombasthenic platelets that prevents the association of fibrinogen with them.

Adenosine Diphosphate↗
PubMed1977

Properties of washed human platelets.

We have shown previously that washed human platelets resuspended in Tyrode solution containing albumin and apyrase maintain their disc shape and their ability to aggregate upon the addition of low concentration of ADP, providing fibrinogen is added to the suspending medium. We have now examined their responses to other aggregating and release-inducing agents. Collagen, arachidonate, thrombin, immune serum globulin, the ionophore A23, 187 and phytohaemagglutinin from Phaseolus vulgaris caused aggregation and release of granule contents. The response to adrenaline was variable. Serotonin caused the platelets to change shape but no aggregation or release occurred. Addition of a small amount of plasma was necessary for ristocetin-induced aggregation. Polylysine caused immediate platelet-to-platelet adherence with little or no release of granule contents. Responses to collagen or thrombin were greater in a modified medium containing magnesium but no calcium; in this medium, aggregation caused by ADP or polylysine was followed by the release of granule contents whereas these agents caused aggregation without release in a medium with both calcium and magnesium. When protein was omitted from the suspending medium, platelet aggregation in response to ADP was variable. In this medium, collagen and thrombin caused more extensive release than in the albumin-containing medium. Aggregation by polylysine was accompanied by release and extensive lysis in the protein-free medium. Thus, the composition of the final resuspending medium has a major effect on the responses of washed human platelets to aggregating agents.

Arachidonic Acids↗
PubMed1976

Reactions of polylysine with human platelets in plasma and in suspensions of washed platelets.

The effects of polylysine on human platelets have been examined in citrated platelet-rich plasma (PRP) and in suspensions of washed platelets in various media. In PRP, polylysine caused aggregation after a lag phase. Heparin inhibited this completely. At certain concentrations of polylysine, two phases of aggregation occurred, the second being associated with release of 14C-serotonin from prelabelled platelets; this phase was inhibitable with prostaglandin E1, acetylsalicylic acid, sulphinpyrazone, adenosine, apyrase, or creatine phosphate/creatine phosphokinase. Polylysine-induced release also occurred in PRP with EDTA or hirudin as anticoagulant. In suspensions of washed platelets in Tyrode solution containing 0.35% or 4% albumin, or 1% gelatin, polylysine caused immediate platelet-to-platelet adherence and very little release of 14C-serotonin or platelet lysis. Heparin inhibited aggregation, but acetylsalicylic acid, prostaglandin E1, adenosine, apyrase, creatine phosphate/creatine phosphokinase or EDTA did not. In a modified Tyrode-albumin medium containing 1 mM magnesium but no calcium, polylysine-induced aggregation was associated with the release of 14C-serotonin which could be inhibited by acetylsalicylic acid or indomethacin; this is similar to the effect of ADP in this medium. In Tyrode solution without albumin or gelatin, polylysine-induced platelet aggregation was associated with release of a large percentage of 14C-serotonin, together with as much as 18% lysis; indomethacin inhibited this release reaction.

Albumins↗