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.

The Appearance Of PF1 And PF3 In Activated Human Platelets

1981 ◽  
Author(s):  
H Sandberg ◽  
A P Bodet ◽  
F A Dombrosei ◽  
L O Andersson ◽  
B R Lentz
Keyword(s):  
Human Platelets ◽  
Dense Granule ◽  
Void Volume ◽  
Factor V ◽  
Dense Granules ◽  
Protein Particles ◽  
Hydrolysis Of ◽  
Platelet Pellet ◽  
Stimulation Of

Collagen and thrombin induced platelet activation were examined, in vitro, with regard to the appearance of surface-associated Factor V-like activity (PF1) and catalytic phospholipid-like surface activity (PF3). Two test systems were used: a clotting assay (a modified KAPTT) and a chromogenic substrate assay (maximum hydrolysis of S-2238). Following stimulation of normal platelets, both PF1 and PF3 appeared simultaneously in the supernatant and platelet pellet. When normal platelets were collected and carefully washed in a buffer containing adenosine, PGE1, and theophylline, the appearance of both PF1 and PF3 was blocked, as was the release of ATP from dense granules, the release of β-TG and PF4 from α-granules, and the occurrence of aggregation. When platelets were collected in this same inhibitor-containing buffer, and then gel filtered/centrifuge-washed in an inhibitor-free buffer, the appearance of PF1 and PF3 was still blocked. This occurred even though release of ATP, β-TG and PF4 as well as aggregation followed a pattern equivalent to platelets never exposed to these inhibitors. When the release supernatant from normal platelets isolated in the absence of inhibitors was gel filtered on Sepharose CL-4B in the presence of EDTA, the carbohydrate-free, lipid- protein particles (70-170nm diam.) that provide PF3 appeared in the void volume. When the release supernatant from normal platelets was gel filtered in the presence of Ca2+, both, PF1 and PF3 eluted in the void volume. With platelets isolated from severe F.V-deficient donors, only PF3 was found in the void volume, in the presence or absence of Ca2+. It seems that the appearance of PF1 and PF3 as coagulant activities is completely separate from both the release of dense granule and α-granule contents as well as platelet aggregation and that the appearance of PF1 requires the presence of Ca2+.

POSSIBLE ROLE FOR THE CA2+-DEPENDENT PROTEASE (CALPAIN I) AS AN IRREVERSIBLE ACTIVATOR OF CA2+/CALMODULIN-MEDIATED REACTIONS IN THE HUMAN PLATELET

1987 ◽  
Author(s):  
Robert W Wallace ◽  
E Ann Tallant ◽  
Lynn M Brumley
Keyword(s):  
Myosin Light Chain ◽  
Binding Proteins ◽  
Human Platelet ◽  
Limited Proteolysis ◽  
Physiological Role ◽  
Human Platelets ◽  
Dependent Manner ◽  
Protease Activation ◽  
Stimulation Of

Calmodulin (CaM)-binding proteins have been identified in human platelets using Western blotting techniques and 125I-CaM. Ten proteins of 245, 225. 175, 150, 90. 82(2), 60 and 41(2) kilodaltons (kDa) bind 125I-CaM in a Ca2+-dependent manner; the binding is blocked by both trifluoperazine and nonradiolabeled CaM. The 225 and 90 kDa proteins are labeled by antisera against myosin light chain kinase (MLCK); the 60 kDa and one of the 82 kDa proteins have been identified as the CaM-dependent phosphatase (calcineurin) and caldesmon. The other proteins are presumed to be other Ca2+/CaM regulated enzymes and proteins which may be important in platelet function. Most of the CaM-binding proteins are degraded upon addition of Ca2+ to a platelet homogenate; the degradation may be blocked by either EGTA, leupeptin or N-ethylmaleimide which suggests that the degradation is due to a Ca2+-dependent protease. Activation of intact platelets under conditions which promote platelet aggregation (i.e. stirring with extracellular Ca2+) also results in limited proteolysis of CaM-binding proteins including those labeled with anti sera against MLCK and the phosphatase. In vitro studies utilizing purified phosphatase and calpain I indicate that the phosphatase is irreversibly activated upon Ca2+-dependent proteolysis. The proteolytically-activated enzyme is insensitive to either Ca2+ or Ca2+/CaM; in addition, its activity in the absence of Ca2+ is even greater than the activity of the unproteolyzed enzyme in the presence of Ca2+ and CaM. Proteolytic stimulation of the phosphatase is accompanied by degradation of the 60 kDa subunit of the enzyme (subunit A) to 56, 52 and 45 kDa fragments, sequentially; proteolysis results in the loss of CaM binding to the enzyme. These results suggest that the Ca2+-dependent protease may have a physiological role in platelet activation as an irreversible activator of Ca2+/ CaM-dependent reactions. Supported by NIH grant HL29766.

Endotoxin-Induced Platelet Activation in Human Whole Blood In Vitro

1988 ◽  
Vol 59 (03) ◽  
pp. 378-382 ◽  
Author(s):  
Gyorgy Csako ◽  
Eva A Suba ◽  
Ronald J Elin
Keyword(s):  
Platelet Aggregation ◽  
Whole Blood ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Atp Release ◽  
Human Platelets ◽  
Lag Phase ◽  
Human Whole Blood ◽  
Dose Dependent

SummaryThe effect of purified bacterial endotoxin was studied on human platelets in vitro. In adding up to 1 μg/mL of a highly purified endotoxin, we found neither aggregation nor ATP release in heparinized or citrated human platelet-rich plasma. On the other hand, endotoxin at concentrations as low as a few ng/mL (as may be found in septic patients) caused platelet aggregation in both heparinized and citrated human whole blood, as monitored by change in impedance, free platelet count, and size. Unlike collagen, the platelet aggregation with endotoxin occurred after a long lag phase, developed slowly, and was rarely coupled with measurable release of ATP. The platelet aggregating effect of endotoxin was dose-dependent and modified by exposure of the endotoxin to ionizing radiation. Thus, the activation of human platelets by “solubilized” endotoxin in plasma requires the presence of other blood cells. We propose that the platelet effect is mediated by monocytes and/or neutrophils stimulated by endotoxin.

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 Release of human platelet factor V activity is induced by both collagen and ADP and is inhibited by aspirin

Blood ◽  
1980 ◽  
Vol 56 (3) ◽  
pp. 448-455
Author(s):  
WJ Vicic ◽  
B Lages ◽  
HJ Weiss
Keyword(s):  
Human Platelet ◽  
Initial Response ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Dense Granule ◽  
Antimycin A ◽  
Factor V ◽  
Platelet Factor ◽  
Gradient Separation ◽  
Density Gradient Separation

Factor V activity in suspensions of human platelets washed by albumin density gradient separation increased in response to stimulation by both collagen and adenosine diphosphate (ADP). The appearance of factor V activity extracellularly had the characteristics of platelet secretion and was partially inhibited by aspirin and by the antimetabolites 2-deoxyglucose and antimycin A. Some increase in factor V activity was also observed in platelet suspensions during the initial response to ADP; this activity was not detected extracellularly, but remained associated with the platelets. Patients with storage pool deficiency (SPD) whose platelets are deficient only in dense granule substances released normal amounts of factor V activity, whereas decreased amounts were released in a patient whose platelets have both dense and alpha granule deficiencies. These findings suggest that a portion of platelet factor V is associated with, and released from, alpha granules.

scholarly journals Assessment of the hemostatic effectiveness of human platelets treated with aminomethyltrimethyl psoralen and UV A light using a rabbit ear bleeding time technique

Blood ◽  
1993 ◽  
Vol 82 (11) ◽  
pp. 3489-3492
Author(s):  
SJ Wagner ◽  
L Bardossy ◽  
G Moroff ◽  
RY Dodd ◽  
MA Blajchman
Keyword(s):  
Bleeding Time ◽  
Human Platelet ◽  
Human Platelets ◽  
Ethyl Palmitate ◽  
Rabbit Ear ◽  
Immunodeficiency Virus ◽  
New Zealand White Rabbits ◽  
The Mean ◽  
Human Immunodeficiency Virus 1

The photochemical aminomethyltrimethyl psoralen (AMT), in conjunction with UV A light (UVA), has been shown to inactivate human immunodeficiency virus-1 and model viruses in platelet suspensions under conditions that have only a minimal effect on in vitro platelet properties. A rabbit ear bleeding time technique was used to assess the hemostatic effectiveness of human platelet suspensions treated with AMT/UVA. New Zealand White rabbits were made thrombocytopenic by a combination of irradiation and heterologous antirabbit platelet antiserum. Reticuloendothelial function in these rabbits was suppressed by the intravenous administration of ethyl palmitate. The hemostatic function of 1- and 5-day-old human platelet suspensions (14.5% plasma) that had been treated on day 1 with 40 micrograms/mL AMT and 24 kJ/m2 UVA (1 x UVA) was evaluated by measuring microvascular bleeding times after a standard incision. Comparable bleeding times were observed after infusion with both control and AMT/UVA-treated platelets stored for either 1 or 5 days. With the transfusion of AMT/1 x UVA-treated platelets stored for 5 days, the mean (+/- SD) bleeding time was 156.3 +/- 39.2 seconds (n = 10). With untreated platelets (no AMT/no UVA), stored for 5 days, the mean bleeding time was 189.2 +/- 36.4 seconds (n = 10). Neither AMT nor 1 x UVA treatment alone influenced the observed bleeding times. In contrast, the hemostatic effectiveness of human platelet suspensions was diminished if they were exposed to three times the standard UVA dose (72 kJ/m2) on day 1 and stored for 4 more days, regardless of whether AMT was present, with the mean bleeding time increasing to 442.2 +/- 122.6 seconds (n = 15, AMT present) or 396.0 +/- 45.9 seconds (n = 10, AMT absent). These results are consistent with data obtained from in vitro studies and indicate that virucidal AMT/1 x UVA treatment does not influence platelet hemostatic function. However, the final conditions to achieve these results must be carefully controlled.

Activation of Human Platelets by Endotoxin

1975 ◽  
Author(s):  
John Das ◽  
Dianna Ausprunk ◽  
Judah Folkman
Keyword(s):  
Platelet Count ◽  
Human Platelet ◽  
Human Platelets ◽  
Gram Negative ◽  
Platelet Counts ◽  
Membrane Changes ◽  
The One ◽  
Limulus Test

Endotoxin-like activity (ELA) can be detected in platelets in human Gram-negative sepsis1. With our photometric version of the Limulus test, we quantified the ELA of platelets in 12 children with gastroenteritis of Gram-negative origin. 5 severe cases had thrombocytopenia (41 × 103/μl), and an ELA-equivalent of 10.4 ± 6.9 ng in platelets from 1 ml PRP; 4 died of DIC. 7 patients wore moderately ill, with platelets 138 × 103/μl, and an ELA of 5.3 ± 3.9 ng. In fi controls with a background platelet-ELA of 1.9 ng, incubation with endotoxin (50 ng/ml PRP) raised the ELA by 3 ng. In the one infant surviving severe enteritis, a similar incubation raised the platelet-ELA by 16 ng, the platelet count being 26 × 103/μl. In DIC, we routinely find platelet-ELA at 20 ng and platelet counts of 25 χ 103/μl. In in-vitro EM studies, endotoxin complexed with Cu2+ adhered to human platelet surfaces; pseudopods and degranulation were also seen along with fragmentation of endotoxin particles. The membrane changes suggested peroxidation.We postulate that endotoxin adheres to an activates human platelets to release materials with procoagulant properties in the Limulus test, and capable of precipitating DIC. As sensitized platelets are phagocytized by the RES, platelets may prove to be a system for clearing circulating endotoxin. 1 Das, Schwartz and Folkman: Surgery 74, 235, 1973.

scholarly journals P38 mitogen activated protein kinase inhibitor improves platelet in vitro parameters and in vivo survival in a SCID mouse model of transfusion for platelets stored at cold or temperature cycled conditions for 14 days

PLoS ONE ◽  
2021 ◽  
Vol 16 (5) ◽  
pp. e0250120
Author(s):  
Andrey Skripchenko ◽  
Monique P. Gelderman ◽  
Jaroslav G. Vostal
Keyword(s):  
Mouse Model ◽  
Cold Storage ◽  
Scid Mouse ◽  
Room Temperature ◽  
Human Platelet ◽  
Human Platelets ◽  
Bacterial Proliferation ◽  
Scid Mouse Model

Platelets for transfusion are stored at room temperature (20–24°C) up to 7 days but decline in biochemical and morphological parameters during storage and can support bacterial proliferation. This decline is reduced with p38MAPK inhibitor, VX-702. Storage of platelets in the cold (4–6°C) can reduce bacterial proliferation but platelets get activated and have reduced circulation when transfused. Thermocycling (cold storage with brief periodic warm ups) reduces some of the effects of cold storage. We evaluated in vitro properties and in vivo circulation in SCID mouse model of human platelet transfusion of platelets stored in cold or thermocycled for 14 days with and without VX-702. Apheresis platelet units (N = 15) were each aliquoted into five storage bags and stored under different conditions: room temperature; cold temperature; thermocycled temperature; cold temperature with VX-702; thermocycled temperature with VX-702. Platelet in vitro parameters were evaluated at 1, 7 and 14 days. On day 14, platelets were infused into SCID mice to assess their retention in circulation by flow cytometry. VX-702 reduced negative platelet parameters associated with cold and thermocycled storage such as an increase in expression of activation markers CD62, CD63 and of phosphatidylserine (marker of apoptosis measured by Annexin binding) and lowered the rise in lactate (marker of increase in anaerobic metabolism). However, VX-702 did not inhibit agonist-induced platelet aggregation indicating that it does not interfere with platelet hemostatic function. In vivo, VX-702 improved initial recovery and area under the curve in circulation of human platelets infused into a mouse model that has been previously validated against a human platelet infusion clinical trial. In conclusion, inhibition of p38MAPK during 14-days platelet storage in cold or thermocycling conditions improved in vitro platelet parameters and platelet circulation in the mouse model indicating that VX-702 may improve cell physiology and clinical performance of human platelets stored in cold conditions.

Impairment of Human Platelet Aggregation and Serotonin Release Caused in Vitro by Echis Colorata Venom

1973 ◽  
Vol 30 (01) ◽  
pp. 191-198 ◽  
Author(s):  
Haim Biran ◽  
Alexander Dvilansky ◽  
Ilana Nathan ◽  
Avinoam Livne
Keyword(s):  
Platelet Aggregation ◽  
Functional Impairment ◽  
Human Platelet ◽  
Human Platelets ◽  
Endothelial Damage ◽  
Serotonin Release ◽  
Bleeding Tendency ◽  
Human Platelet Aggregation

SummaryAggregation of washed human platelets, induced by either ADP, thrombin or collagen, was decreased by Echis colorata venom (EVC). With ADP as an inducer, the inhibition of aggregation was proportional to the venom concentration, starting from 0.27 μg/ml and attaining full inhibition with venom concentration of 9 μg/ml. Higher concentrations were required for comparable venom effects when collagen or thrombin were used as inducers. Based on serotonin release measurements and platelet counting, it is concluded that the ECV-diminished aggregation is not due to platelet lysis. Thrombin-dependent serotonin release was inhibited by the venom to an extent proportional to the log ECV concentration at a range of 0.27 to 90 μg/ml. ECV effeces on serotonin release are apparently independent on its effects on aggregation, since similar results were obtained either with or without EDTA.Endothelial damage and defibrination are already known to be associated with the bleeding tendency caused by ECV. The present data disclose a functional impairment of platelets as an additional antihemostatic effect of this venom.

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.

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