The death of human platelets during incubation in citrated plasma involves shedding of CD42b and aggregation of dead platelets

2006 ◽  
Vol 95 (01) ◽  
pp. 100-106 ◽  
Author(s):  
John Savill ◽  
Simon Brown ◽  
Paul Hartley
Keyword(s):  
Platelet Rich Plasma ◽  
Vital Staining ◽  
Human Platelets ◽  
Staining Procedure ◽  
Aggregate Formation ◽  
Factors Affecting ◽  
Transmission Electron ◽  
Reliable Assessment ◽  
Vital Stains ◽  
Calcein Am

SummaryThe ability to readily identify dead platelets is invaluable to studies examining the means of their death, factors affecting their lifespan and their means of clearance by phagocytes. The aim of the present work was to develop a vital staining procedure for the rapid and objective discrimination of live from dead platelets that accrued in citrated platelet rich plasma (cPRP) incubated at 37°C for several days. By transmission electron microscopy it was noted that platelet death was morphologically similar to necrosis and associated with aggregate formation. The vital dyes calcein-AM and FM 4–64 were found to robustly report the death of platelets and indicated that the aggregates which formed during incubation were populated exclusively by dead platelets. Additionally, platelet death was associated with the shedding of CD42b. Microscopic and cytometric analyses of incubated cPRP indicated that shedding of CD42b and aggregate formation by dead platelets were completely inhibited by the metalloproteinase inhibitor GM6001. Automated counting of platelets incubated in the presence of GM6001 revealed that death did not lead to a loss in cellularity. It is proposed that calcein-AM and FM4–64 are effective as vital stains for the reliable assessment of platelet viability and that platelet aggregation can occur by a novel mechanism dependent upon platelet death and metalloproteinase activity.

scholarly journals Factors Affecting the Factor X Activator Activity of Human Platelets

1977 ◽  
Author(s):  
J. Vermylen ◽  
N. Semeraro
Keyword(s):  
Factor Viii ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Activate Factor ◽  
Factor Viii Inhibitor ◽  
Factor X ◽  
Factors Affecting ◽  
Coagulant Activity ◽  
Viii Inhibitor

Several recent studies have indicated that patients with a thrombotic tendency have enhanced platelet coagulant activity. It therefore is of importance to attempt to identify factors which modify platelet coagulant activities. Recent work in our laboratory has provided evidence that human platelets possess the capacity to directly activate factor X.Adenosine-5'-diphosphate, collagen, acetylsalicylic acid and prostaglandin E1 do not modify this activity. All endotoxins studied however clearly enhanced this activity.Furthermore, infusion of ‘activated’ prothrombin concentrates in haemophiliacs with or without factor VIII inhibitor enhanced the activity of this platelet activator of factor X. The hypothesis has been put forward that this may be the mechanism through which ‘activated’ prothrombin concentrates ‘bypass’ factor VIII of IX inhibitors. Platelet isolated from platelet-rich plasma to which the ‘activated’ prothrombin concentrate had been added at a concentration approaching the maximal concentration achieved following in vivo infusion, also showed an increase in platelet coagulant activity. Work is in progress to identify the component in ‘activated’ prothrombin concentrates which enhances platelet coagulant activity.

A MORPHOLOGICAL APPROACH TO THE MECHANISMS OF RISTOCETIN INDUCED PLATELET AGGLUTINATION(RIPA)

1987 ◽  
Author(s):  
G Escolar ◽  
J Monteagudo ◽  
N Villamor ◽  
M Garrido ◽  
R Castillo
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibody ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Complex Interaction ◽  
Electron Dense Deposit ◽  
Transmission Electron ◽  
Dense Deposit ◽  
Morphological Approach ◽  
Adhesive Proteins

Changes in the morphology of human platelets induced by Ristocetin (RIPA) have been analyzed at ultraestructural level by means of a tannine acid procedure. Studies were completed with aggregation and binding experiments. Modificationsof these tests induced by apyrase,a monoclonal antibody (Mab) to GPIIb/IIIa and EDTA were also investigated.Transmission electron microscopy reveals that ristocetin precipitates adhesive proteins on plateletmembrane. An electron-dense deposit was noticeable within 20 secondsafter ristocetin was added. When experiments were carried out in theaggregometer cuvette under stirring, groups of platelets become activated, change shape, and finally aggregate releasing part of their content. The morphology of aggregates did not differ from those formed in the presence of ADP.Aggregation studies demonstrated that a Mab to GPIIb/IIIa modifies the extent and the rate of the aggregation curve when RIPA was performed in citrated platelet rich plasma (c-PRP). Apyrase modified the extent but not the slope of the curve. Neither the antibody nor apyrase modified RIPA when it was performed in PRP obtained in presence of EDTA. Binding experiments confirmed that I-vWF bound to platelets in presence of ristocetin was not modified by apyrase or anti-GPIIb/IIIa Mab. All these facts together suggest that RIPA,when performed in c-PRP besides reflecting the interaction of GPI with vWF, is also testing other mechanisms of the platelet function including exposure of GPIIb/IIIa complex, interaction of fibrinogen with this glycoprotein, and the contribution of the release reaction.

RELATIONSHIP BETWEEN ELEVATION OF CYCLIC-3∲,5∲-GMP (cGMP) AND AGGREGATE FORMATION IN HUMAN PLATELETS

1987 ◽  
Author(s):  
M Edgecombe ◽  
M C Scrutton ◽  
R Kerry
Keyword(s):  
Dose Response ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Light Transmittance ◽  
Aggregate Formation ◽  
Response Curves ◽  
Specific Radioimmunoassay ◽  
A Chain ◽  
The Right ◽  
Stimulation Of

Maximal 3- to 5-fold increases in platelet cGMP levels as measured by specific radioimmunoassay are observed on stimulation of aspirin-treated platelet-rich plasma with saturating doses of ADP, adrenaline, 5HT, PAF, thrombin, 9,11-epoxymethano-PGH2 (U44069), collagen, ristocetin and the Ca2+-ionophore ionomycin. The dose/response curves for the elevation in cGMP induced by these agents are either superimposable on, or lie to the right of, those describing the rate or extent of the aggregatory response as measured by an increase in light transmittance. The increase in cGMP induced by ADP is totally inhibited by addition of PGI2 or forskolin with dose/response relationships superimposable on those observed for inhibition of the aggregatory response. No increase in cGMP is observed if platelets are stimulated by PAF or ionomycin in an unstirred system or when aggregation induced by ADP is prevented by addition of a monoclonal antibody which recognises the glycoprotein IIb/IIIa complex.Addition of the fibrinogen γ-chain C-terminal decapeptide (γ402-411) or α-Chain tetrapeptide ARG-GLY-ASP-SER prevents aggregation and the increase in cGMP induced by PAF. The γ-chain decapeptide also completely prevents the increase in cGMP induced by ristocetin, but the a-chain tetrapeptide is ineffective in this respect. Both peptides inhibit to some extent aggregate formation induced by ristocetin.The data demonstrates a strong correlation between aggregate formaticfn and the increase in the platelet cGMP levels and support the previous postulate that platelet-platelet contact causes, activation of guanylate cyclase. No relationship is apparent between the effects of the various agents tested on cGMP levels and their known ability to increase cytosolic Ca2+ concentration. (Supported by SERC and Ciba-Geigy.)

Functional Characterization of PM6/13, a β3-specific (GPIIIa/CD61) Monoclonal Antibody that Shows Preferential Inhibition of Fibrinogen Binding over Fibronectin Binding to Activated Human Platelets

1998 ◽  
Vol 79 (01) ◽  
pp. 177-185 ◽  
Author(s):  
Ashia Siddiqua ◽  
Michael Wilkinson ◽  
Vijay Kakkar ◽  
Yatin Patel ◽  
Salman Rahman ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Functional Characterization ◽  
Human Platelets ◽  
Western Blots ◽  
Activated Platelets ◽  
Reducing Conditions ◽  
Fibronectin Binding

SummaryWe report the characterization of a monoclonal antibody (MAb) PM6/13 which recognises glycoprotein IIIa (GPIIIa) on platelet membranes and in functional studies inhibits platelet aggregation induced by all agonists examined. In platelet-rich plasma, inhibition of aggregation induced by ADP or low concentrations of collagen was accompanied by inhibition of 5-hydroxytryptamine secretion. EC50 values were 10 and 9 [H9262]g/ml antibody against ADP and collagen induced responses respectively. In washed platelets treated with the cyclooxygenase inhibitor, indomethacin, PM6/13 inhibited platelet aggregation induced by thrombin (0.2 U/ml), collagen (10 [H9262]g/ml) and U46619 (3 [H9262]M) with EC50 = 4, 8 and 4 [H9262]g/ml respectively, without affecting [14C]5-hydroxytryptamine secretion or [3H]arachidonate release in appropriately labelled cells. Studies in Fura 2-labelled platelets revealed that elevation of intracellular calcium by ADP, thrombin or U46619 was unaffected by PM6/13 suggesting that the epitope recognised by the antibody did not influence Ca2+ regulation. In agreement with the results from the platelet aggregation studies, PM6/13 was found to potently inhibit binding of 125I-fibrinogen to ADP activated platelets. Binding of this ligand was also inhibited by two other MAbs tested, namely SZ-21 (also to GPIIIa) and PM6/248 (to the GPIIb-IIIa complex). However when tested against binding of 125I-fibronectin to thrombin stimulated platelets, PM6/13 was ineffective in contrast with SZ-21 and PM6/248, that were both potent inhibitors. This suggested that the epitopes recognised by PM6/13 and SZ-21 on GPIIIa were distinct. Studies employing proteolytic dissection of 125I-labelled GPIIIa by trypsin followed by immunoprecipitation with PM6/13 and analysis by SDS-PAGE, revealed the presence of four fragments at 70, 55, 30 and 28 kDa. PM6/13 did not recognize any protein bands on Western blots performed under reducing conditions. However Western blotting analysis with PM6/13 under non-reducing conditions revealed strong detection of the parent GP IIIa molecule, of trypsin treated samples revealed recognition of an 80 kDa fragment at 1 min, faint recognition of a 60 kDa fragment at 60 min and no recognition of any product at 18 h treatment. Under similar conditions, SZ-21 recognized fragments at 80, 75 and 55 kDa with the 55kDa species persisting even after 18 h trypsin treatment. These studies confirm the epitopes recognised by PM6/13 and SZ-21 to be distinct and that PM6/13 represents a useful tool to differentiate the characteristics of fibrinogen and fibronectin binding to the GPIIb-IIIa complex on activated platelets.

Conditions Affecting the Responses of Human Platelets to Epinephrine

1988 ◽  
Vol 60 (02) ◽  
pp. 209-216 ◽  
Author(s):  
Chantal Lalau Keraly ◽  
Raelene L Kinlough-Rathbone ◽  
Marian A Packham ◽  
Hidenori Suzuki ◽  
J Fraser Mustard
Keyword(s):  
Platelet Rich Plasma ◽  
Shape Change ◽  
Human Platelets ◽  
Dense Granule ◽  
Primary Phase ◽  
Lag Phase ◽  
Acid Citrate Dextrose ◽  
Two Phases ◽  
Citrate Dextrose ◽  
Thromboxane Formation

SummaryConditions affecting the responses of human platelets to epinephrine were examined. In platelet-rich plasma prepared from blood anticoagulated with hirudin or PPACK (D-pheny- lalanyl-L-prolyl-L-arginine chloromethyl ketone), epinephrine did not cause shape change or aggregation. In a Tyrode-albumin- apyrase solution containing a concentration of Ca2+ in the physiological range, and fibrinogen, epinephrine in concentrations as high as 40 μM did not induce platelet shape change, caused either no primary aggregation or very slight primary aggregation, and did not induce thromboxane formation, release of dense granule contents, or secondary aggregation. In contrast, in citrated platelet-rich plasma, epinephrine induced two phases of aggregation. This is not attributable to the generation of traces of thrombin since the same effects were evident when blood was taken into a combined citrate-hirudin anticoagulant or a combined citrate-PPACK anticoagulant. In a modified Tyrode-albu- min-apyrase solution containing approximately 20 μM Ca2+, 1 mM Mg2+, and fibrinogen, epinephrine induced extensive aggregation after a lag phase, but no primary phase was evident; thromboxane formation and release of dense granule contents accompanied the aggregation response. These responses were also observed when PPACK was included with the acid-citrate- dextrose anticoagulant, and in the washing and resuspending fluids. In the presence of aspirin or the thromboxane receptor blocker BM 13.177 a few small aggregates were detected by particle counting and by scanning electron microscopy; with the latter inhibitor, the platelets in the aggregates retained their disc shape; secondary aggregation and the responses associated with it did not occur. Thus thromboxane A2 formation is not necessary for the formation of these small aggregates, but is required for extensive aggregation and release. As with other weak agonists, the close platelet-to-platelet contact in the low Ca2+ medium appears to be necessary for full secondary aggregation. Omission of fibrinogen from the low Ca2+ medium prevented both primary and secondary aggregation in response to epinephrine. An antibody (10E5) to the glycoprotein Ilb/IIIa complex was completely inhibitory in the presence of fibrinogen. Thus the response of human platelets to epinephrine is influenced by the concentration of Ca2+ and the presence of fibrinogen in the medium in which they are suspended.

Extraction of Protein-Bound ATP and ADP from Human Platelets in Plasma

1990 ◽  
Vol 63 (02) ◽  
pp. 286-290 ◽  
Author(s):  
Christina Beurling-Harbury ◽  
Pehr B Harbury
Keyword(s):  
Specific Activity ◽  
Platelet Rich Plasma ◽  
Binding Protein ◽  
Human Platelets ◽  
Luciferase Assay ◽  
First Time ◽  
Plasma Extraction

SummaryActin is the major ATP and ADP binding protein in platelets, 0.9–1.3 nmol/108 cells, 50–70% in the unpolymerized state. The goal of these experiments was to develop a method for extracting all protein-bound ATP and ADP from undisturbed platelets in plasma. Extraction of actin-bound ADP is routine while extraction of actin-bound ATP from platelets in buffer has been unsuccessful. Prior to extraction the platelets were exposed to 14-C adenine, to label the metabolic and actin pools of ATP and ADP. The specific activity was determined from the actin-bound ADP in the 43% ethanol precipitate. Sequential ethanol and perchlorate extractions of platelet rich plasma, and the derived supernatants and precipitates were performed. ATP concentrations were determined with the luciferase assay, and radioactive nucleotides separated by TLC. A total of 1.18 nmol/108 cells of protein-bound ATP and ADP was recovered, 52% ATP (0.61 nmol). The recovery of protein-bound ADP was increased from 0.3 to 0.57 nmol/108 cells. This approach for the first time successfully recovered protein bound ATP and ADP from platelets in a concentration expected for actin.

The Effect of Red Blood Cells on the ADP-induced Aggregation of Human Platelets in Flow Through Tubes

1990 ◽  
Vol 63 (01) ◽  
pp. 112-121 ◽  
Author(s):  
David N Bell ◽  
Samira Spain ◽  
Harry L Goldsmith
Keyword(s):  
Platelet Aggregation ◽  
Shear Rate ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Platelet Rich Plasma ◽  
Mean Transit Time ◽  
White Blood Cells ◽  
Aggregate Size ◽  
Human Platelets ◽  
Induced Aggregation

SummaryThe effect of red blood cells, rbc, and shear rate on the ADPinduced aggregation of platelets in whole blood, WB, flowing through polyethylene tubing was studied using a previously described technique (1). Effluent WB was collected into 0.5% glutaraldehyde and the red blood cells removed by centrifugation through Percoll. At 23°C the rate of single platelet aggregtion was upt to 9× greater in WB than previously found in platelet-rich plasma (2) at mean tube shear rates Ḡ = 41.9,335, and 1,920 s−1, and at both 0.2 and 1.0 µM ADP. At 0.2 pM ADP, the rate of aggregation was greatest at Ḡ = 41.9 s−1 over the first 1.7 s mean transit time through the flow tube, t, but decreased steadily with time. At Ḡ ≥335 s−1 the rate of aggregation increased between t = 1.7 and 8.6 s; however, aggregate size decreased with increasing shear rate. At 1.0 µM ADP, the initial rate of single platelet aggregation was still highest at Ḡ = 41.9 s1 where large aggregates up to several millimeters in diameter containing rbc formed by t = 43 s. At this ADP concentration, aggregate size was still limited at Ḡ ≥335 s−1 but the rate of single platelet aggregation was markedly greater than at 0.2 pM ADP. By t = 43 s, no single platelets remained and rbc were not incorporated into aggregates. Although aggregate size increased slowly, large aggregates eventually formed. White blood cells were not significantly incorporated into aggregates at any shear rate or ADP concentration. Since the present technique did not induce platelet thromboxane A2 formation or cause cell lysis, these experiments provide evidence for a purely mechanical effect of rbc in augmenting platelet aggregation in WB.

Reaction of Acetaldehyde with Human Platelets

1992 ◽  
Vol 67 (01) ◽  
pp. 126-130 ◽  
Author(s):  
Olivier Spertini ◽  
Jacques Hauert ◽  
Fedor Bachmann
Keyword(s):  
Platelet Aggregation ◽  
Inhibitory Action ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Reaction Medium ◽  
Human Platelets ◽  
Membrane Glycoproteins ◽  
Neutral Ph

SummaryPlatelet function defects observed in chronic alcoholics are not wholly explained by the inhibitory action of ethanol on platelet aggregation; they are not completely reproduced either in vivo by short-term ethanol perfusion into volunteers or in vitro by the addition of ethanol to platelet-rich plasma. As acetaldehyde (AcH) binds to many proteins and impairs cellular activities, we investigated the effect of this early degradation product of ethanol on platelets. AcH formed adducts with human platelets at neutral pH at 37° C which were stable to extensive washing, trichloracetic acid hydrolysis and heating at 100° C, and were not reduced by sodium borohydride. The amount of platelet adducts formed was a function of the incubation time and of the concentration of AcH in the reaction medium. At low AcH concentrations (<0.2 mM), platelet bound AcH was directly proportional to the concentration of AcH in the reaction medium. At higher concentrations (≥0.2 mM), AcH uptake by platelets tended to reach a plateau. The amount of adducts was also proportional to the number of exposures of platelets to pulses of 20 pM AcH.AcH adducts formation severely impaired platelet aggregation and shape change induced by ADP, collagen and thrombin. A positive correlation was established between platelet-bound AcH and inhibition of aggregation.SDS-PAGE analysis of AcH adducts at neutral pH demonstrated the binding of [14C]acetaldehyde to many platelet proteins. AcH adduct formation with membrane glycoproteins, cytoskeleton and enzymes might interfere with several steps of platelet activation and impair platelet aggregation.This in vitro study shows that AcH has a major inhibitory action on platelet aggregation and may account for the prolonged ex vivo inhibition of aggregation observed in chronic alcoholics even in the absence of alcoholemia.

Effects of Halothane on Platelet Function

1980 ◽  
Vol 44 (03) ◽  
pp. 143-145 ◽  
Author(s):  
J Dalsgaard-Nielsen ◽  
J Gormsen
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Halothane Anaesthesia ◽  
Inhibition Of Platelet Aggregation ◽  
Transient Impairment ◽  
High Concentrations ◽  
Uptake And Release ◽  
Platelet Functions

SummaryHuman platelets in platelet rich plasma (PRP) incubated at 37° C with 0.3–2% halothane for 5–10 min lost the ability to aggregate with ADP, epinephrine and collagen.At the same time uptake and release of 14C-serotonin was inhibited. When halothane supply was removed, platelet functions rapidly returned to normal. However, after high concentrations of halothane, the inhibition of platelet aggregation was irreversible or only partially reversible.The results suggest that halothane anaesthesia produces a transient impairment of platelet function.

Interactions of Staphylokinase with Human Platelets

1995 ◽  
Vol 73 (03) ◽  
pp. 472-477 ◽  
Author(s):  
H R Lijnen ◽  
B Van Hoef ◽  
D Collen
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Specific Binding ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Normal Plasma ◽  
Atp Secretion ◽  
Platelet Disaggregation ◽  
Activation Rate

SummaryThe interactions of recombinant staphylokinase (SakSTAR) with human platelets were investigated in a buffer milieu, in a human plasma milieu in vitro, and in plasma from patients with acute myocardial infarction (AMI) treated with SakSTAR.In a buffer milieu, the activation rate of plasminogen by SakSTAR or streptokinase (SK) was not significantly altered by addition of platelets. Specific binding of SakSTAR or SK to either resting or thrombin- activated platelets was very low. ADP-induced or collagen-induced platelet aggregation in platelet-rich plasma (PRP) was 94 ± 2.7% or 101 ± 1.7% of control in the presence of 0.1 to 20 μM SakSTAR, with corresponding values of 95 ± 2.8% or 90 ± 4.6% of control in the presence of 0.1 to 4 μM SK. No effects were observed on platelet disaggregation. ATP secretion following collagen-induced platelet aggregation was 4.3 ± 0.26 μM for SakSTAR (at concentrations of 0.1 to 20 μM) and 4.4 ± 0.35 μM for SK (at concentrations of 0.1 to 4 μM), as compared to 3.4 ± 0.70 μM in the absence of plasminogen activator.Fifty % lysis in 2 h (C50) of 60 μl 125I-fibrin labeled platelet-poor plasma (PPP) clots prepared from normal plasma or from plasma of patients with Glanzmann thrombasthenia and immersed in 0.5 ml normal plasma, was obtained with 12 or 16 nM SakSTAR and with 49 or 40 nM SK, respectively. C50 values for lysis of 60 μl PRP clots prepared from normal or patient plasma were also comparable for SakSTAR (19 or 21 nM), whereas SK was 2-fold more potent toward PRP clots prepared from Glanzmann plasma as compared to normal plasma (C50 of 130 versus 270 nM).No significant effect of SakSTAR on platelet function was observed in plasma from patients with AMI treated with SakSTAR, as revealed by unaltered platelet count, platelet aggregation and ATP secretion.Thus, no effects of high SakSTAR concentrations were observed on human platelets in vitro, nor of therapeutic SakSTAR concentrations on platelet function in plasma.

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