scholarly journals Platelet Antiheparin Activity: Storage Site and Release Mechanism

Blood ◽  
1974 ◽  
Vol 44 (2) ◽  
pp. 157-168 ◽  
Author(s):  
Peter N. Walsh ◽  
Giovanna Gagnatelli
Keyword(s):  
Lysosomal Enzymes ◽  
Time Course ◽  
Adenosine Diphosphate ◽  
Normal Subjects ◽  
Human Platelets ◽  
Release Mechanism ◽  
Storage Site ◽  
Dense Granules ◽  
Storage Pool ◽  
Platelet Storage

Abstract The platelet storage and release mechanisms for the heparin-neutralizing activity (HNA), adenosine diphosphate (ADP), serotonin, and lysosomal enzymes were investigated in normal human platelets and in platelets with defective storage or release of ADP and serotonin. The time course of release of HNA from normal washed platelets by thrombin and collagen was slower than that of serotonin. Lysosomal enzymes were not released by collagen from normal washed platelets, whereas under the same conditions HNA was released. In four of six patients with storage pool deficiency, the platelets contained normal amounts of HNA but definitely decreased amounts of ADP and serotonin, whereas in the remaining two patients the total contents of ADP, serotonin, and HNA were all definitely lower than normal. In four of six patients with storage pool deficiency, the amounts and per cents of total HNA released by collagen were normal, whereas the amounts and per cents of total ADP released were diminished compared with normal. Platelets from patients with the aspirinlike platelet release defect and from aspirin-treated normal subjects contained normal quantities of ADP, serotonin, and HNA, but HNA and ADP were not released in response to collagen. It is concluded that either HNA is stored in and released from dense granules by mechanisms different from those for ADP and serotonin, or that HNA is stored in and released from granules other than the dense granules, which contain ADP and serotonin and the α-granules, which contain lysosomal enzymes.

scholarly journals The response of platelets to epinephrine in storage pool deficiency-- evidence pertaining to the role of adenosine diphosphate in mediating primary and secondary aggregation

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1717-1725 ◽  
Author(s):  
HJ Weiss ◽  
B Lages
Keyword(s):  
Platelet Aggregation ◽  
Receptor Agonist ◽  
Adenosine Diphosphate ◽  
Normal Subjects ◽  
Dense Granule ◽  
Binding Studies ◽  
Dense Granules ◽  
Storage Pool ◽  
Receptor Sites

Abstract Aggregation responses and thromboxane (Tx) formation in ten patients with storage pool deficiency (SPD) specific to the dense granules (delta-SPD) were studied to assess further the role of dense granule adenosine diphosphate (ADP) in mediating platelet aggregation by epinephrine. The ability of epinephrine to elicit secondary aggregation (SA) responses was highly variable in delta-SPD when tested at 5 mumol/L epinephrine, but was consistently abnormal when tested over a range of concentrations. The occurrence of SA in both delta-SPD patients and normal subjects was correlated with the magnitude of the rate of primary aggregation (PA). This PA rate was normal, on average, for the entire patient group but was greater in patients with more consistent SA responses. The PA findings were related to the Kd value obtained in binding studies with 3H-yohimbine, but not with the number of alpha 2-receptor sites. Studies on Tx production (assessed by radioimmunoassay of TxB2) showed that the ability to synthesize Tx from arachidonate was not impaired in delta-SPD, and that there was an absolute positive correlation between epinephrine-induced SA and Tx production. Aggregation in delta-SPD platelets in response to the Tx receptor agonist U44069 was consistently decreased, but could be corrected by addition of ADP. The results of the study suggest that dense granule-derived ADP is not required for PA by epinephrine, but mediates SA as a synergistic agonist with TxA2. This role of ADP in SA may be elucidated more precisely by further studies on platelet activation processes in delta-SPD.

scholarly journals The response of platelets to epinephrine in storage pool deficiency-- evidence pertaining to the role of adenosine diphosphate in mediating primary and secondary aggregation

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1717-1725 ◽  
Author(s):  
HJ Weiss ◽  
B Lages
Keyword(s):  
Platelet Aggregation ◽  
Receptor Agonist ◽  
Adenosine Diphosphate ◽  
Normal Subjects ◽  
Dense Granule ◽  
Binding Studies ◽  
Dense Granules ◽  
Storage Pool ◽  
Receptor Sites

Aggregation responses and thromboxane (Tx) formation in ten patients with storage pool deficiency (SPD) specific to the dense granules (delta-SPD) were studied to assess further the role of dense granule adenosine diphosphate (ADP) in mediating platelet aggregation by epinephrine. The ability of epinephrine to elicit secondary aggregation (SA) responses was highly variable in delta-SPD when tested at 5 mumol/L epinephrine, but was consistently abnormal when tested over a range of concentrations. The occurrence of SA in both delta-SPD patients and normal subjects was correlated with the magnitude of the rate of primary aggregation (PA). This PA rate was normal, on average, for the entire patient group but was greater in patients with more consistent SA responses. The PA findings were related to the Kd value obtained in binding studies with 3H-yohimbine, but not with the number of alpha 2-receptor sites. Studies on Tx production (assessed by radioimmunoassay of TxB2) showed that the ability to synthesize Tx from arachidonate was not impaired in delta-SPD, and that there was an absolute positive correlation between epinephrine-induced SA and Tx production. Aggregation in delta-SPD platelets in response to the Tx receptor agonist U44069 was consistently decreased, but could be corrected by addition of ADP. The results of the study suggest that dense granule-derived ADP is not required for PA by epinephrine, but mediates SA as a synergistic agonist with TxA2. This role of ADP in SA may be elucidated more precisely by further studies on platelet activation processes in delta-SPD.

Secreted Dense Granule Adenine Nucleotides Promote Calcium Influx and the Maintenance of Elevated Cytosolic Calcium Levels in Stimulated Human Platelets

1999 ◽  
Vol 81 (02) ◽  
pp. 286-292 ◽  
Author(s):  
Harvey Weiss ◽  
Bruce Lages
Keyword(s):  
Calcium Influx ◽  
Adenine Nucleotides ◽  
Normal Subjects ◽  
Cytosolic Calcium ◽  
Human Platelets ◽  
Dense Granule ◽  
Specific Component ◽  
Fura 2 ◽  
Dense Granules ◽  
Platelet Storage

SummaryEvidence that secreted dense granule adenine nucleotides mediate part of the agonist-induced cytosolic calcium ([Ca2+]i) responses in human platelets was obtained from comparisons of fura-2-loaded platelets from normal subjects and from patients with a form of platelet storage pool deficiency (SPD) in which the secretory dense granules and their contents are virtually absent. SPD platelets had normal initial [Ca2+]i in creases induced by thrombin and the endoperoxide analog U46619, but a significantly enhanced decay of elevated [Ca2+]i levels following the initial increases. With thrombin, this enhanced [Ca2+]i decay was associated with decreased Ca2+ influx, as measured by Mn2+ quench of fura-2 fluorescence. Addition of micromolar concentrations of ADP, alone or together with ATP, after stimulation reversed the enhanced [Ca2+]I decay and increased Mn2+ quench in SPD platelets, but had no effect on these responses in normal platelets, while addition of 100-fold higher concentrations of ATP or apyrase before stimulation increased [Ca2+]I decay and decreased Mn2+ quench in normal platelets, but had little effect in SPD platelets. ATP and α,β-methylene ATP, a specific agonist for P2X1 receptors, at micromolar concentrations also increased Mn2+ quench, but to lesser extents than did ADP, in SPD platelets isolated and loaded with fura-2 in the presence of apyrase. Similar effects of ADP and excess ATP were seen in U46619-stimulated platelets, but decreased Ca2+ influx could not be measured directly in SPD platelets, presumably due to the very transient influx response seen with U46619. These results suggest that secreted dense granule ADP and ATP contribute to the maintenance of elevated [Ca2+]i levels, but not to the initial [Ca2+]i increases, in stimulated human platelets, most likely via a nucleotide-specific component of Ca2+ influx which may be mediated by interactions with both P2X1 and P2Y1 purinoceptors.

scholarly journals Platelet storage pool deficiency in mouse pigment mutations associated with seven distinct genetic loci

Blood ◽  
1984 ◽  
Vol 63 (3) ◽  
pp. 536-544 ◽  
Author(s):  
EK Novak ◽  
SW Hui ◽  
RT Swank
Keyword(s):  
Lysosomal Enzyme ◽  
Human Platelet ◽  
Adenosine Diphosphate ◽  
Dense Granule ◽  
Enzyme Secretion ◽  
Dense Granules ◽  
Storage Pool ◽  
Platelet Storage ◽  
Storage Pool Disease ◽  
Experimental Injury

Abstract Seven mouse pigment mutants, which have alterations at distinct genes, are known to have a defect in kidney lysosomal enzyme secretion. Two of these, beige and pale ear, have a bleeding abnormality associated with a deficiency in the number of platelet dense granules. In the present study, five other mutants with defective lysosomal enzyme secretion-- pearl, pallid, light ear, maroon, and ruby-eye--were likewise found to have abnormally prolonged bleeding times after experimental injury. Platelet counts were similar to those of normal mice, but the platelet dense granule components serotonin, adenosine triphosphate (ATP), and adenosine diphosphate (ADP) and morphologically identifiable dense granules were markedly reduced in these mutants. The capacity to accumulate exogenous 3H-serotonin in platelets was reduced 2–3-fold. Thrombin-stimulated secretion of 3H-serotonin was slightly decreased in all mutants. However, the seven mutants could be subdivided into three groups based on the degree of secretion of lysosomal enzymes after thrombin stimulation. Thus, all seven mouse pigment mutants have symptoms consistent with platelet storage pool deficiency and may serve as useful animal models for specific types of human platelet storage pool disease. Also, the results emphasize the genetic, morphological, and functional interrelatedness of three organelles: melanosomes, lysosomes, and platelet dense granules.

scholarly journals Platelet storage pool deficiency in mouse pigment mutations associated with seven distinct genetic loci

Blood ◽  
1984 ◽  
Vol 63 (3) ◽  
pp. 536-544 ◽  
Author(s):  
EK Novak ◽  
SW Hui ◽  
RT Swank
Keyword(s):  
Lysosomal Enzyme ◽  
Human Platelet ◽  
Adenosine Diphosphate ◽  
Dense Granule ◽  
Enzyme Secretion ◽  
Dense Granules ◽  
Storage Pool ◽  
Platelet Storage ◽  
Storage Pool Disease ◽  
Experimental Injury

Seven mouse pigment mutants, which have alterations at distinct genes, are known to have a defect in kidney lysosomal enzyme secretion. Two of these, beige and pale ear, have a bleeding abnormality associated with a deficiency in the number of platelet dense granules. In the present study, five other mutants with defective lysosomal enzyme secretion-- pearl, pallid, light ear, maroon, and ruby-eye--were likewise found to have abnormally prolonged bleeding times after experimental injury. Platelet counts were similar to those of normal mice, but the platelet dense granule components serotonin, adenosine triphosphate (ATP), and adenosine diphosphate (ADP) and morphologically identifiable dense granules were markedly reduced in these mutants. The capacity to accumulate exogenous 3H-serotonin in platelets was reduced 2–3-fold. Thrombin-stimulated secretion of 3H-serotonin was slightly decreased in all mutants. However, the seven mutants could be subdivided into three groups based on the degree of secretion of lysosomal enzymes after thrombin stimulation. Thus, all seven mouse pigment mutants have symptoms consistent with platelet storage pool deficiency and may serve as useful animal models for specific types of human platelet storage pool disease. Also, the results emphasize the genetic, morphological, and functional interrelatedness of three organelles: melanosomes, lysosomes, and platelet dense granules.

Collagen-Induced Platelet Aggregation: -Evidence Against the Essential Role of Platelet Adenosine Diphosphate

1979 ◽  
Vol 42 (04) ◽  
pp. 1193-1206 ◽  
Author(s):  
Barbara Nunn
Keyword(s):  
Platelet Aggregation ◽  
Time Course ◽  
Essential Role ◽  
Atp Release ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
High Doses ◽  
Induced Aggregation

SummaryThe hypothesis that platelet ADP is responsible for collagen-induced aggregation has been re-examined. It was found that the concentration of ADP obtaining in human PRP at the onset of aggregation was not sufficient to account for that aggregation. Furthermore, the time-course of collagen-induced release in human PRP was the same as that in sheep PRP where ADP does not cause release. These findings are not consistent with claims that ADP alone perpetuates a collagen-initiated release-aggregation-release sequence. The effects of high doses of collagen, which released 4-5 μM ADP, were not inhibited by 500 pM adenosine, a concentration that greatly reduced the effect of 300 μM ADP. Collagen caused aggregation in ADP-refractory PRP and in platelet suspensions unresponsive to 1 mM ADP. Thus human platelets can aggregate in response to collagen under circumstances in which they cannot respond to ADP. Apyrase inhibited aggregation and ATP release in platelet suspensions but not in human PRP. Evidence is presented that the means currently used to examine the role of ADP in aggregation require investigation.

Correlation Between Noradrenaline Fluxes, Metabolism and Compartmentalisation in Human Platelets

1982 ◽  
Vol 48 (01) ◽  
pp. 062-066 ◽  
Author(s):  
Chantal Legrand ◽  
Véronique Dubernard ◽  
Philippe Meyer
Keyword(s):  
Noradrenaline Release ◽  
Human Platelets ◽  
Release Of Noradrenaline ◽  
Storage Pool ◽  
Platelet Storage ◽  
Efflux Of Noradrenaline ◽  
Preferential Localization

Summary(3H) noradrenaline was taken up by human platelets and partially converted into sulfoconjugated noradrenaline. This uptake was inhibited by drugs which have been previously shown to impair the uptake of 5-HT (ouabain, chlorimipramine) or the storage of 5-HT (tyramine, reserpine) by platelets. In addition, tyramine and reserpine stimulated the formation of sulfoconjugated noradrenaline. The efflux of noradrenaline from platelets was measured in parallel and was found to be directly related to the proportion of non metabolized to metabolized noradrenaline in the cells. Unlike tyramine, which induced a similar release of noradrenaline and 5-HT, reserpine was less effective at inducing noradrenaline release than 5-HT release. This study indicates a preferential localization of noradrenaline in the granular pool of human platelets with the existence of an extragranular sulfoconjugated pool which is increased when the granular storage of noradrenaline is impaired. Studies of noradrenaline fluxes and metabolism may be useful in the understanding of both acquired and inherited platelet storage pool defects.

scholarly journals Immature dense granules in platelets from mice with platelet storage pool disease

Blood ◽  
1987 ◽  
Vol 69 (5) ◽  
pp. 1300-1306 ◽  
Author(s):  
M Reddington ◽  
EK Novak ◽  
E Hurley ◽  
C Medda ◽  
MP McGarry ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Dense Granule ◽  
Mutant Mice ◽  
Normal Numbers ◽  
Group Of Seven ◽  
Dense Granules ◽  
Storage Pool ◽  
Platelet Storage ◽  
Transmission Electron ◽  
Storage Pool Disease

Mepacrine uptake into platelets and bone marrow megakaryocytes was analyzed to further characterize the dense granule defects in a group of seven mouse pigment mutants that have characteristics of platelet storage pool disease (SPD). In contrast to our previous studies using electron microscopy, this method revealed that all mutants had normal numbers of dense granules. However, total mepacrine uptake in all mutant platelets was significantly diminished to less than 50% of normal uptake. Also, the flashing phenomenon observed when normal dense granules are irradiated with ultraviolet light was either greatly diminished or absent when platelets of individual mutants were similarly irradiated. Therefore the principal defect in the mutant platelets is an inability to accumulate dense granule contents rather than an absence of the granules. Mepacrine uptake into megakaryocytes was indistinguishable in normal and mutant mice. This indicates the mutant dense granule defects appear either very late in megakaryocyte development or early in platelet formation in correlation with development of the mature dense granule. By standard transmission electron microscopy we have not been able to detect gross structural or subcellular abnormalities in either platelets or megakaryocytes of mutant mice. It appears all seven mutants produce immature or functionally abnormal dense granules.

A Functional Basis For The Platelet Storage Pool Deficiency In Chediak-Higashi Cattle

1981 ◽  
Author(s):  
K M Meyers ◽  
M Fukami ◽  
H Holmsen
Keyword(s):  
Freeze Fracture ◽  
Subcellular Fractionation ◽  
Dense Granule ◽  
Bivalent Cation ◽  
Fibrous Matrix ◽  
Dense Granules ◽  
Storage Pool ◽  
Common Basis ◽  
Platelet Storage ◽  
Amine Storage

Platelets from cattle with the morphologic homolog of the Chediak-Higashi (CH) syndrome are essentially devoid of secretable nucleotides and serotonin. There are reduced but still substantial amounts of secretable calcium and magnesium. The storage pool deficiency may be, in part, due to a functional granule defect. Platelets from CH cattle take up serotonin and protect it from degradation for several hours. If these platelets are treated with thrombin, serotonin and bivalent cations are released by mechanisms similar to that of secretion, suggesting a granule location for the released serotonin and cations. This suggestion is verified by subcellular fractionation studies where platelets are first incubated with 14C-serotonin then lysed using a French press. Organelles were then separated on a sucrose gradient by centrifugation. Serotonin in normal bovine platelets is associated with the dense granule or pellet while in CH platelets serotonin is primarily found in a region of the sucrose density zone that in normal platelets contain alpha granules. These findings suggested that some granules in CH platelets are able to acquire the bivalent cation and amine but not the nucleotide component of the bivalent cation-nucleotide-amine storage complex that is thought to occur in normal dense granules.Ultrastructural identification of the serotonin-containing CH granule is not known. There are 2 identifiable granule populations in CH platelets, alpha granules and fibrous matrix granules. Based on serial sectioning freeze fracture and morphometric studies, there are less than 4 of these granules/platelet. Mepacrine studies also demonstrate 2 granule populations. One population has an incidence of 2 per granule and characteristics of normal dense granules. Since the number of fibrous matrix granules and mepacrine granules is similar, a common basis for these granules which has at least some dense granule characteristics, i.e., mepacrine storage, is suggested.

scholarly journals Content and thrombin-induced release of acid hydrolases in gel-filtered platelets from patients with storage pool disease

Blood ◽  
1975 ◽  
Vol 46 (1) ◽  
pp. 131-142 ◽  
Author(s):  
H Holmsen ◽  
CA Setkowsky ◽  
B Lages ◽  
HJ Day ◽  
HJ Weiss ◽  
...  
Keyword(s):  
Acid Phosphatase ◽  
Normal Subjects ◽  
Acid Hydrolases ◽  
Release Reaction ◽  
Dense Granules ◽  
Storage Pool ◽  
Low Concentrations ◽  
Storage Pool Disease ◽  
Beta Galactosidase

Abstract The levels of four acid hydrolases, beta-N-acetyl glucosaminidase, beta- glucuronidase, beta-galactosidase, and acid phosphatase, and the extent of their release (release II) by thrombin was determined in platelets from nine normal subjects, nine patients with storage pool disease, and in normal platelets which had been exposed to aspirin. The levels of all four hydrolases were normal in patients with SPD. However, release of three of these hydrolases (acid phosphatase was an exception) by low concentrations of thrombin (0.015 and 0.04 U/ml) was decreased in the patients as a group, although considerable variation in the extent of release of each enzyme was noted. In contrast, aspirin failed to inhibit release II in normal platelets (except for a slight impairment in the release of beta-N-acetyl glucosaminidase), although release I (serotonin, ATP and ADP) was inhibited. All release defects could be overcome by using higher concentrations of thrombin (0.2 U/ml). The normal levels of acid hydrolases in the platelets of patients with SPD (who are deficient in the platelet dense granules) suggest that these enzymes are not normally stored in the dense granules, but rather in alpha-granules. The findings also support the conclusions of previous studies that the release reaction is impaired in SPD. This release defect appears to be different from that seen in normal platelets after exposure to aspirin.

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