Failure of Preactivated Human Blood Platelets to Restore Defective Thromboxane Synthesis despite Prolonged Incubation in Plasma

1989 ◽  
Vol 62 (03) ◽  
pp. 1016-1022 ◽  
Author(s):  
Marcus Stockschläder ◽  
Rüdiger E Scharf
Keyword(s):  
Blood Platelets ◽  
Human Platelets ◽  
Autologous Plasma ◽  
Prolonged Incubation ◽  
Platelet Storage ◽  
Thromboxane Synthetase ◽  
Responsible Mechanism ◽  
Short Time ◽  
Storage Pool Disease

SummaryAcquired platelet storage pool disease has been shown to be associated with reduced platelet thromboxane synthesis. However, the mechanisms for this dysfunction are incompletely understood. The present experiments were designed to evaluate some of the possible defects which may account for impaired thromboxane formation in human platelets previously exposed to thrombin in vitro. Washed platelets pretreated with 0.5 U/ml thrombin for 20 sec and subsequently recovered as single degranulated platelets were incapable of forming normal amounts of thromboxane upon a second stimulation with thrombin (as compared to Tyrode-pretreated control platelets). In contrast, thrombin-degranulated platelets released additional amounts of thromboxane in response to arachidonate, or collagen, indicating that short-time exposure to thrombin does not irreversibly inactivate platelet cyclooxygenase or thromboxane synthetase. Upon incubation of the thrombin-pretreated platelets in autologous plasma in the presence of 14C-arachidonate, the label became associated with the platelets to the same extent as with control platelets. However, the platelets did not recover their ability to synthesize normal amounts of thromboxane upon restimulation with thrombin, and only about half of the label was lost from the thrombin-pretreated platelets as compared to control platelets in response to thrombin. The ability of collagen to cause loss of 14C-arachidonate and formation of thromboxane was the same regardless of whether or not the platelets had been pretreated with thrombin. Incubation of platelets in plasma in the presence of added arachidonate for 90 min resulted in complete refractoriness to a second stimulation with thrombin but not with collagen. However, the control platelets also lost most of their ability to synthesize thromboxane when incubated with arachidonate for 90 min and thereafter stimulated with thrombin. Thus, the presence of added arachidonate affects the thrombin-inducible thromboxane synthesis after prolonged incubation of human platelets in plasma. Our observations suggest that depletion of endogenous arachidonate is not a major cause for the defective thrombin-induced thromboxane synthesis in thrombin-pretreated platelets. It is more likely that impaired mobilization of endogenous arachidonic acid explains this dysfunction. Defective mobilization of arachidonate in thrombin-degranulated platelets may be due to agonists-specific receptor desensitization, but the responsible mechanism has not been identified.

scholarly journals Lecithin Analog as a New Platelet Aggregating Agent

1977 ◽  
Author(s):  
J. Hawiqer ◽  
H. W. Hooper
Keyword(s):  
Platelet Aggregation ◽  
Blood Platelets ◽  
Human Platelets ◽  
Phospholipase A ◽  
Synthetic Analog ◽  
Autologous Plasma ◽  
Vitro Effect ◽  
Dose Dependent ◽  
Phospholipase A Inhibitor

Lecithin comprises 32% of the human platelet phospholipids which are the source of arachidonic acid required for biosynthesis of prostaglandin endoperoxides, potent inducers of platelet aggregation. A synthetic analog of lecithin, dimethly-DL-2,3-distearoyloxypropyl-2’-hydroxy-ethylammonium acetate known as a phospholipase A inhibitor, showed a profound in vitro effect upon human platelets. Blood platelets obtained from normal, healthy, fasting volunteers and suspended in autologous plasma aggregated in response to lecithin analog. Platelet aggregation was dose-dependent within the range of 10-5 to 10-4M of lecithin analog and accompanied by release of [3H] serotonin. In contrast, addition of equimolar or higher amounts of lecithin to human platelets in vitro remained without a measurable effect on their function. Sensitivity of human platelets to the lecithin analog used was increased at least 10-fold by separation of platelets from the bulk of plasma proteins by gel filtration on Sepharose 2B. Release of [3H] serotonin from gel-filtered platelets induced by the lecithin analog studied was dose-dependent and partly reversible due to reuptake of serotonin. Thus, the lecithin analog used in our experiments is a new platelet activating agent which is a “false phospholipid” and phospholipase A inhibitor acting through a previously unrecognized mechanism triggering membrane-mediated functions of human platelets.

The Interrelationship between Thromboxane Biosynthesis, Aggregation and 5-Hydroxytryptamine Secretion in Human Platelets in Vitro

1980 ◽  
Vol 43 (01) ◽  
pp. 038-040 ◽  
Author(s):  
L C Best ◽  
T K Holland ◽  
P B B Jones ◽  
R G G Russell
Keyword(s):  
Human Platelet ◽  
Human Platelets ◽  
Dense Granule ◽  
Thromboxane B2 ◽  
Essential Requirement ◽  
Direct Mechanism ◽  
Thromboxane Synthetase ◽  
Thromboxane Production ◽  
Higher Doses

SummaryPlatelet aggregation, secretion of 5-hydroxy tryptamine and production of thromboxane B2 were monitored simultaneously in human platelet suspensions in the absence and presence of cyclooxygenase or thromboxane synthetase inhibitors. Aggregation, secretion and thromboxane B2 formation in response to either sodium arachidonate or epinephrine were blocked by aspirin or by 1-N-butyl imidazole suggesting that thromboxane biosynthesis was an essential requirement for platelet activation by these agents. In contrast, thrombin and collagen could apparently induce aggregation and secretion via two pathways: at low doses involving thromboxane production, but at higher doses by a direct mechanism independent of thromboxane biosynthesis. In the case of ADP, inhibition of thromboxane production blocked secretion but had little effect on aggregation, indicating that secretion was probably dependent on thromboxane biosynthesis which probably occurred as a result of aggregation. Thus it appears that although the processes of thromboxane production, release of dense granule constituents and aggregation may often be intimately linked, each process can occur independently of the other, depending upon the stimulus used.

Preparation of a Viable Population of Indium-111- Labelled Human Blood Platelets

1979 ◽  
Vol 42 (05) ◽  
pp. 1473-1482 ◽  
Author(s):  
A Dup Heyns ◽  
P N Badenhorst ◽  
H Pieters ◽  
M G Lötter ◽  
P C Minnaar ◽  
...  
Keyword(s):  
Platelet Rich Plasma ◽  
Blood Platelets ◽  
Kinetic Studies ◽  
Physiological Saline ◽  
Human Platelets ◽  
Autologous Plasma ◽  
Platelet Suspension ◽  
Viable Population ◽  
Indium 111

SummaryFactors influencing labelling of human platelets with 111Indium-8-hydroxyquinoline ([111In]-oxine) in a physiological saline medium were investigated. The efficiency of labelling is influenced by time of incubation, concentration of oxine, and pH of the incubating medium. It was found that a viable platelet population could be labelled under the following conditions: (1) centrifugation of platelet rich plasma in polystyrene conical tubes at 800 g for 15 min; (2) resuspension of the platelet pellet in saline, pH 5.5; (3) incubating for 30 min at 22°C with [111In]-oxine at a concentration of 6.25 mg oxine/litre platelet suspension; (4) washing once with platelet poor autologous plasma (PPP); and (5) finally resuspending the platelets in PPP. The labelled platelets aggregated normally with collagen and ADP. Electron microscopy, done immediately after labelling, showed internal organelle reorganization characteristic of activated platelets. These ultrastructural features were reversible on incubation in PPP at 37°C for 30 min. The 111In is not released from aggregated platelets and the label does not elute from incubated platelets for at least five hr. We conclude that human platelets thus labelled are suitable for in vivo kinetic studies.

Interactions Between Blood Platelets and Vascular Endothelium in vitro Studies

1979 ◽  
Author(s):  
M.A. Gimbrone ◽  
K.D. Curwen ◽  
R. I. Handin
Keyword(s):  
Platelet Aggregation ◽  
Vascular Endothelium ◽  
Potent Inhibitor ◽  
Platelet Reactivity ◽  
Blood Platelets ◽  
Primary Cultures ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Platelet Adherence

Endothelial cells (EC) can actively influence the hemostatic response at sites of vascular injury through multiple mechanisms. For example, EC can degrade adenosine diphosphate, release plasminogen activator, and synthesize prostacyclin (PGI2), a potent inhibitor of platelet aggregation. We have examined whether PGI2 also might account for the normal lack of platelet adherence to the uninjured EC surface. In a monolayer adherence assay, radiolabeled human platelets in citrated plasma showed minimal interaction with primary cultures of human EC (<1 platelet adhering per cell). Platelets from aspirin-treated and untreated donors behaved similarly. However, aspirin pretreatment of EC consistently resulted in ~2-fold increases in platelet adherence which could be completely abolished by exogenous PGI2 (0.5–1.0 μg/ml). SV40-transformed human EC (SVHEC), which are deficient in PGI2 production compared to primary EC, showed 10-30 times more platelet adherence. Exogenous PGI2 produced a dose - related (.001-1.0 μg/ml) decrease in platelet adherence to SVHEC but did not result in the basal levels observed with normal EC monolayers. These data suggest that : 1) In addition to its effects on platelet aggregation, PGI2 can influence platelet endothelial cell interactions; 2) The increased platelet reactivity of transformed EC is associated with, but not completely attributable, to decreased PGI2 production; and 3) Factors other than PGI2 may play a role in the thromboresistance of normal vascular endothelium.

Effect Of Solvents On Indium-111 Oxine Transport In Human Platelets And On Platelet Function In Vitro

1981 ◽  
Author(s):  
T Tsukada
Keyword(s):  
Platelet Function ◽  
Kinetic Constant ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Polysorbate 80 ◽  
Autologous Plasma ◽  
Indium 111 ◽  
Induced Aggregation ◽  
Water Space

Mechanism of Indium-111 oxine(In) transport in human platelets in buffered saline and the effect of In-labeling on platelet function were studied using In dissolved in 25% of ethanol in saline (In-ES) or 0.01% of polysorbate 80 in HEPES buffer(In-PH). Increase in temperature up to 37° C progressively enhanced the transport of In-ES, while transport of In-PH reached to plateau at 15°C. A states of equilibrium was not reached during 2 hr incubation at 22°C in In-ES. Uptake of In-PH reached to plateau after only 15 min of incubation. Distribution of In taken up by platelets in InES was 57% in cytosol and 27% in stroma, while in In-PH 69% in stroma and 22% in cytosol. 88% of In in cytosol was bound to lipids(46% in cholesterol and 27% in PS+PI). 82% of In in stroma was found in PS+PI fraction.The fact that the ratio of free In between the platelet water space and the outside medium after 30 min of incubation at up to 0.1 uM of In exceeded unity, suggests satura- , ble component of In transport prevails at this concentration in In-ES and In-PH. Kinetic constant could be calculated, Kt= 2nM, Vmax= 2.5 pmol/min/ml in In-ES, and Kt= InM, Vmax=0.7 pmol/min/ml in In-PH.Elution of In from radiolableled platelets in autologous plasma incubated at 37°C for 5 hr was less than 10% in the case of In-ES and 56% in the case of In-PH. Less than 3% of labeled-In was eluated from platelets in collagen-induced aggregation and 4-7% of In was eluated in thrombin-induced aggregation.Although 0.3% of ethanol and/or 6nM of oxine have no inhibitory effect of platelet aggregation, collagen-induced aggregation and release reaction of In-labeled platelet was impaired. 0.003% of polysorbate 80 itself abolished completely the aggregability of platelets by collagen or thrombin.It is concluded In-PH is unsuitable for platelet labeling. In-111 oxine also seems to have problems which Cr-51 has, i.e. inhomogenous distribution of In in a platelet population, elution of In from labeled platelets in circulation.

scholarly journals Survival and recovery of human platelets stored for five days in a non- plasma medium

Blood ◽  
1986 ◽  
Vol 67 (3) ◽  
pp. 672-675 ◽  
Author(s):  
GA Adams ◽  
SD Swenson ◽  
G Rock
Keyword(s):  
Platelet Aggregation ◽  
Human Blood ◽  
Blood Platelets ◽  
Human Platelets ◽  
Plasma Medium ◽  
Release Reaction ◽  
In Vivo Recovery ◽  
Human Blood Platelets

Abstract Human blood platelets were stored for five days as concentrates in 60 mL of: (a) plasma; (b) non-plasma medium with anticoagulant; and (c) non-plasma medium without anticoagulant. All preparations were equally functional when tested for platelet aggregation and release reaction in response to single agonist or synergistic pairs of agonists in vitro. Platelets stored in non-plasma medium with anti-coagulant had lower kallikrein, fibrino(gen)peptide A, lactate, and beta-thromboglobulin than did plasma controls after five days. In vivo recovery and survival of platelets stored in non-plasma medium with anticoagulant were 51.2% +/- 4.3% and 8.7 +/- 0.3 days, respectively, which were not statistically different from plasma controls of 39.2% +/- 4.9% and 7.2 +/- 0.8 days, respectively. It is concluded that platelets can be stored for five days in a non-plasma medium and still have good in vivo recoveries and survivals.

scholarly journals Labeling of the releasable adenine nucleotides of washed human platelets

Blood ◽  
1977 ◽  
Vol 49 (1) ◽  
pp. 89-99 ◽  
Author(s):  
HJ Reimers ◽  
MA Packham ◽  
JF Mustard
Keyword(s):  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Energy Charge ◽  
Human Platelets ◽  
Transport Processes ◽  
Adenylate Energy Charge ◽  
Prolonged Incubation ◽  
Atp Pool ◽  
Adenine Nucleotide Pool

Abstract In rabbit platelets, the metabolically active ATP pool equilibrates with the releasable ATP pool within 1 day. The studies showing this have now been extended to human platelets. Human platelets labeled with 14C-adenosine or 14C-adenine were incubated for up to 10 hr in vitro at 37 degrees C. After 10 hr, about 12% of the total platelet 14C-ATP and 14C-ADP had become releasable with thrombin (4.2 units/ml). Lysis of platelets did not occur, since less than 1% of the platelet-bound 51Cr from platelets labeled with this radioisotope appeared in the ambient fluid upon thrombin treatment. The 14C-ATP/14C-ADP ratio of the released adenine nucleotides (7.6) was similar to the 14C-ATP/14C-ADP ratio of the nonreleasable adenine nucleotides (7.1) 2 hr after the labeling with 14C-adenosine. However, upon prolonged incubation (10 hr) in vitro, the 14C-ATP/14C-ADP ratio of the releasable adenine nucleotides decreased to 2.7. The adenylate energy charge and the 14C- ATP/14C-ADP ratio of the metabolic adenine nucleotide pool did not change significantly during the time of observation. The 14C-ATP content of the platelets decreased by less than 1% hr of incubation at 37 degrees C. These observations are interpreted to mean that the 14C is transferred from the metabolically active, nonreleasable adenine nucleotide pool of human platelets into the releasable adenine nucleotide pool as ATP and is partially hydrolyzed there to yield ADP. The transfer of ATP across the storage organelle membrane of platelets may be similar to transport processes in the chromaffin cells of the adrenal medulla and may represent a general phenomenon in cells that possess storage organelles containing adenine nucleotides.

Human platelet chemotaxis: requirement for plasma factor(s) and the role of collagen

1978 ◽  
Vol 235 (1) ◽  
pp. H23-H28 ◽  
Author(s):  
R. W. Lowenhaupt
Keyword(s):  
Structural Integrity ◽  
Human Platelet ◽  
Human Platelets ◽  
Factor Xi ◽  
Normal Plasma ◽  
Plasma Factor ◽  
Plasma Factors ◽  
Short Time

Platelets are actively mobile in plasma in vitro and, in addition, they migrate specifically and directionally toward added intact collagen (chemotaxis). Native human, bovine, and equine collagen, suspended in plasma, induce a chemotactic response in human platelets. However, heat-denatured and dinitrofluorobenzene-treated collagen fail to attract platelets. Platelets migrate directionally and specifically to intact native collagen incubated in plasma over a large distance (6 mm) in a very short time (total 15 min), as observed in a newly designed micromaze apparatus. Platelets obtained from donors deficient in plasma factors XII, IX, and VIII showed normal migration and chemotaxis in normal plasma and in their respective factor-deficient plasmas. Although nondirectional movement (mobility) was normal, platelets from a donor deficient in factor XI did not exhibit chemotaxis toward collagen in either factor XI-deficient plasma or in normal plasma. The results indicate that 1) collagen is a physiological substrate for the chemotactic phenomenon, 2) intact chemical and/or structural integrity of collagen is required for the induction of platelet chemotaxis, 3) at least one plasma constituent, factor XI, plays an essential role in the chemotactic phenomenon, and 4) contact between collagen and a plasma factor is essential for normal chemotaxis.

scholarly journals Potassium uptake and release by human blood platelets

Blood ◽  
1976 ◽  
Vol 48 (2) ◽  
pp. 185-197
Author(s):  
JS Wiley ◽  
J Kuchibhotla ◽  
CC Shaller ◽  
RW Colman
Keyword(s):  
Time Course ◽  
Specific Activity ◽  
Blood Platelets ◽  
Gel Filtration ◽  
Lactic Dehydrogenase ◽  
Human Platelets ◽  
First Order Kinetics ◽  
A Minor ◽  
Human Blood Platelets

Thrombin is known to reduce the K+ content of human platelets, but the subcellular origin of the lost K+ is not known. The effect of aggregating agents on K+ release was studied in platelets labeled in plasma by preincubation with 42KCI. Platelets were separated from plasma by gel filtration through Sepharose 2B equilibrated with K+ - free Tyrode's buffer. Platelet K+ was 116nEq/10(8) platelets, of which 23% was found to be extracellular immediately after gel filtration. K+ influx was 65 nEq/10(8) platelets/hr at pH 7.5 and was more rapid at pH 7.9. About 70% of cell K+ exchanged with plasma in 4 hr with first- order kinetics, while a minor fraction of about 30% exchanged with a slower time course. This slowly exchanging fraction of platelet K+ was thought to arise from heterogeneity in the platelet population. Epinephrine and ADP aggregated gel-filtered platelets and released serotonin, but with loss of only 5%-10% of cell K+ and no beta- glucuronidase. In contrast, thrombin released up to 30% of platelet K+, whether aggregation occurred or was prevented by not stirring the cells. The specific activity of K+ released by all aggregating agents was identical to the specific activity of total platelet K+. Thrombin (0.01–0.2 NIH U/ml) released serotonin and also beta-glucuronidase (an enzyme of the alpha-granule), and there was a linear relation between release of K+ and this enzyme (r = 0.88). No lysis of platelets occurred, since lactic dehydrogenase was not detected. Pretreatment of platelets with aspirin in vitro inhibited thrombin-induced release of serotonin but had no effect on the loss of K+ or beta-glucuronidase. In contrast, the ingestion of aspirin by mouth inhibited the release of serotonin, beta-glucuronidase, and K+ by thrombin. The data suggested that the K+ loss induced by thrombin was primarily derived from release of alpha-granules and that these organelles contained about 20% of the total platelet K+ in a freely exchangeable and nonsequestered state.

scholarly journals The effect of thrombin on the density distribution of blood platelets: detection of activated platelets in the circulation

Blood ◽  
1983 ◽  
Vol 62 (2) ◽  
pp. 433-438
Author(s):  
B van Oost ◽  
IH van Hien-Hagg ◽  
AP Timmermans ◽  
JJ Sixma
Keyword(s):  
Cardiopulmonary Bypass ◽  
Density Distribution ◽  
Density Gradient Centrifugation ◽  
Blood Platelets ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Human Platelets ◽  
Activated Platelets

The buoyant density of human platelets is decreased after they have been aggregated and induced to secrete their granule content by thrombin. This change in density was detected by discontinuous density gradient centrifugation using arabinogalactan (Stractan) solutions. The density decrease was dependent on the thrombin concentration and paralleled the extent of serotonin and beta-thromboglobulin secretion. The degranulated platelets maintained their integrity, and many of their functional properties. Mixtures of degranulated platelets and normal platelets could be resolved by Stractan gradient centrifugation and the number of degranulated platelets quantitated. Using this method, increased levels of less dense platelets were shown to occur after cardiopulmonary bypass. Assay of changes in platelet density by Stractan gradient centrifugation is a useful method for detection of activated platelets in vitro and in vivo.

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