scholarly journals Platelet Aggregation and Secretion: Effects of Anticoagulants and Divalent Cation Chelators

1977 ◽  
Author(s):  
B.A. Lages ◽  
H.J. Weiss
Keyword(s):  
Platelet Aggregation ◽  
Divalent Cation ◽  
Chelating Agents ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Concentration Addition ◽  
Single Wave ◽  
Cation Concentration ◽  
Aggregation Response ◽  
Low Levels

The aggregation and secretion responses of human platelets to ADP and epinephrine have been studied in citrated platelet-rich plasma (cPRP) and in heparinized platelet-rich plasma (hPRP) containing varying concentrations of divalent cation chelating agents. ADP induced a single wave of aggregation in hPRP while E induced biphasic aggregation in both hPRP and cPRP. Secretion of 14C-serotonin (5HT*) occurred in both hPRP and cPRP in response to ADP and E, but generally to a lesser extent in hPRP. The extent of 5HT* secretion in hPRP, relative to that in cPRP, decreased with increasing ADP concentration but remained essentially constant oyer the same range of E concentration. Addition of citrate or EDTA to hPRP produced a clear maximum in ADP-induced 5HT* secretion at levels of 5-20mM citrate or 2mM EDTA and, in some cases, was associated with the appearance of a biphasic aggregation response. With citrate, but not with EDTA, the chelator concentration corresponding to maximum secretion varied directly with the concentration of ADP. These results indicate that ADP-induced secretion is a function of divalent cation concentration but does not occur only at the low levels of divalent cation present in cPRP and that divalent cation may be more directly associated with ADP-platelet interactions than in interactions of platelets and E.

Dependence of Human Platelet Functional Responses on Divalent Cations: Aggregation and Secretion in Heparin- and Hirudin-Anticoagulated Platelet-Rich Plasma and the Effects of Chelating Agents

1981 ◽  
Vol 45 (02) ◽  
pp. 173-179 ◽  
Author(s):  
Bruce Lages ◽  
Harvey J Weiss
Keyword(s):  
Platelet Aggregation ◽  
Chelating Agents ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Divalent Cations ◽  
Normal Subjects ◽  
Concentration Addition ◽  
Functional Responses ◽  
Induced Aggregation

SummaryThe dependence of ADP- and epinephrine-induced platelet aggregation and secretion on extracellular divalent cations was examined by quantitating these responses in citrate-, heparin-, and hirudin-anticoagulated platelet-rich plasma. ADP-induced 14C-5HT secretion in heparin-PRP and hirudin-PRP was generally decreased, relative to that in citrate-PRP, without corresponding reductions in aggregation, whereas in response to epinephrine, both aggregation and secretion were decreased in heparin-PRP, and abolished in hirudin-PRP. In heparin-PRP, but not in hirudin-PRP, the degree to which these responses were altered was highly variable among normal subjects, and was dependent on the anticoagulant concentration. Addition of citrate restored the extent of ADP-induced secretion and of epinephrine-induced aggregation and secretion in heparin-PRP to that observed in citrate-PRP, and increased the extent of ADP-induced secretion in hirudin-PRP. Addition of EDTA or EGTA, however, had no effect on ADP-induced secretion in heparin-PRP. These results suggest that ADP-induced aggregation and secretion, as well as responses to ADP vs. epinephrine, have different dependencies on extracellular or surface-bound divalent cations. The variable responses observed in heparin-PRP may reflect direct interactions of heparin with platelets, and this variability may account for the conflicting results of previous studies.

COllagen-induced Platelet Behaviour

1979 ◽  
Author(s):  
Patricia M. Taylor ◽  
S. Heptinstall
Keyword(s):  
Platelet Aggregation ◽  
Divalent Cation ◽  
Platelet Rich Plasma ◽  
Divalent Cations ◽  
Human Platelets ◽  
Chelating Resin ◽  
Release Reaction ◽  
Chelex 100 ◽  
Light Absorbance

A comparison has been made of the extent to which collagen induces human platelets to aggregate (change in light absorbance) and to undergo the release reaction (release of 3H-serotonin) in heparinised platelet rich plasma which had been depleted of divalent cations by treating with Chelex 100 chelating resin (Ch.PRHP) and in Ch.PRHP to which different concentrations of CaCl2 or MgCl2 had been added.Platelet aggregation required extracellular Ca++; Mg++ could only support aggregation when Ca++ was also present. The release reaction occurred via Ca++-dependent and Ca++-independent pathways. The divalent cation requirement for Ca++-dependent release was the same as for aggregation.The extent to which platelets aggregated and were induced to undergo release varied with the concentration of extracellular Ca++ or Mg++. A plot of extent of aggregation or of release against concentration of Ca++ or Mg++ has an inverted ‘U’ shape. In any one experiment there was good correlation between extent of aggregation and of release.It is suggested that collagen induces a Ca++-independent release reaction that leads to aggregation and that Ca++-dependent release is a result of the platelets aggregating.

STUDIES ON THE INTERACTIONS BETWEEN THE HEPARIN-LIKE SUBSTANCES AND HUMAN PLATELETS USING THE MONOCLONAL ANTIBODIES TO PLATELET GLYCOPROTEINS

1987 ◽  
Author(s):  
C Ruan ◽  
H Wan ◽  
W Zhang
Keyword(s):  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Binding Sites ◽  
Platelet Rich Plasma ◽  
Biological Effects ◽  
Factor Xa ◽  
Human Platelets ◽  
Final Concentration ◽  
Single Wave ◽  
Second Wave

We reported the new sulphated polysaccharides, SJAMP and HLAMP that have been isolated from the sea cucumbers and compared their biological effects with those of standard heparin. (Med. J. Australia, 1986; 144: HS 17-21). SJAMP and HLAMP have an action that is independent of the antithrombin III and do not inhibit factor Xa, although they have potent antithrombin effect. SJAMP has been used as an antithrombin agent. As does heparin, SJAMP and HLAMP cause platelet aggregation of platelet-rich plasma. At the final concentration of SJAMP or HLAMP of 25μg/ml, a single wave of aggregation was observed.A second wave of aggregation can be induced at a concentration of SJAMP or HLAMP of 50μg/ml. In washed platelets, the platelet aggregation that was induced by SJAMP or HLAMP was fibrinogen-dependent. Furthermore ,plate let aggregation and the secretion of serotonin that is induced by ADP or arachidonic acid were enhanced by the prior addition of SJAMP or HLAMP.This effect may explain the cases of thrombocytopenia that may be observed in patients who are undergoing treatment with SJAMP. Using the monoclonal antibodies ( MoAb ) to human platelet glycoproteins (GP), we studied the SJAMP-p late let and heparin-platelet interactions. SZ-21 is a MoAb directed against GPIIIa and the epitope for SZ-21 is essential for the binding sites of fibrinogen on GPIIb/lIIa complex.SZ-2 is a MoAb to GPIb and recognize an epitope located on the peptide tail (Mr=35K) of the α-chain of GPIb. The platelet aggregation and the secretion of serotonin induced by SJAMP were inhibited by SZ-21, but not by SZ-2. However the preincubation of platelets with SJAMP at the final concentration of 25μg/ml or with heparin at 5μ/ml prevented the binding of radioiodinated-SZ-2 to human platelets. But SJAMP or heparin did not compete for the binding of radioiodinated-SZ-21 to human platelets. These results suggest that the platelet aggregation induced by SJAMP is dependent of the binding sites of fibrinogen on GPIIb/IIIa complex, and that the binding sites of SJAMP and heparin on human platelets are related with GPIb, but not with GPIIb/IIIa.

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.

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.

In Vitro Incorporation and Metabolism of Icosapentaenoic and Docosahexaenoic Acids in Human Platelets - Effect on Aggregation

1986 ◽  
Vol 56 (01) ◽  
pp. 057-062 ◽  
Author(s):  
Martine Croset ◽  
M Lagarde
Keyword(s):  
Arachidonic Acid ◽  
Fatty Acid ◽  
Platelet Aggregation ◽  
Fatty Acid Distribution ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Aggregation Response ◽  
Docosahexaenoic Acids ◽  
Membrane Level

SummaryWashed human platelets were pre-loaded with icosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or EPA + DHA and tested for their aggregation response in comparison with control platelets. In fatty acid-rich platelets, an inhibition of the aggregation could be observed when induced by thrombin, collagen or U-46619. The strongest inhibition was observed with DHA-rich platelets and it was reduced when DHA was incorporated in the presence of EPA.Study of fatty acid distribution in cell lipids after loading showed that around 90% of EPA or DHA taken up was acylated into phospholipids and a very small amount (less than 2%) remained in their free and hydroxylated forms. DHA was more efficiently acylated into phosphatidylethanolamine (PE) than into phosphatidylinositol (PI) in contrast to what observed with EPA, and both acids were preferentially incorporated into phosphatidylcholine (PC). EPA inhibited total incorporation of DHA and increased its relative acylation into PE at the expense of PC. In contrast, DHA did not affect the acylation of EPA. Upon stimulation with, thrombin, EPA was liberated from phospholipids and oxygenated (as judged by the formation of its monohydroxy derivative) whereas DHA was much less metabolized, although consistently transferred into PE.It is concluded that EPA and DHA might affect platelet aggregation via different mechanisms when pre-loaded in phospholipids. Whereas EPA is known to alter thromboxane A2 metabolism from endogenous arachidonic acid, by competing with it, DHA might act directly at the membrane level for inhibiting aggregation.

scholarly journals Activation of human platelets by immune complexes prepared with cationized human IgG

Blood ◽  
1993 ◽  
Vol 82 (10) ◽  
pp. 3045-3051
Author(s):  
M Schattner ◽  
M Lazzari ◽  
AS Trevani ◽  
E Malchiodi ◽  
AC Kempfer ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Immune Complexes ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Human Igg ◽  
Experimental Conditions ◽  
Induce Platelet Aggregation ◽  
Soluble Immune Complexes ◽  
High Concentrations

The present study shows that the ability of soluble immune complexes (IC), prepared with human IgG and rabbit IgG antibodies against human IgG, to trigger platelet activation was markedly higher for IC prepared with cationized human IgG (catIC) compared with those prepared with untreated human IgG (cIC). CatIC induced platelet aggregation and adenosine triphosphate release in washed platelets (WP), gel-filtered platelets (GFP), or platelet-rich plasma (PRP) at physiologic concentrations of platelets (3 x 10(8)/mL) and at low concentrations of catIC (1 to 30 micrograms/mL). On the contrary, under similar experimental conditions, cIC did not induce aggregation in PRP, WP, or GFP. Low aggregation responses were only observed using high concentrations of both WP (9 x 10(8)/mL) and cIC (500 micrograms/mL). Interestingly, catIC were also able to induce platelet activation under nonaggregating conditions, as evidenced by P-selectin expression. Cationized human IgG alone did not induce platelet aggregation in PRP but triggered either WP or GFP aggregation. However, the concentration needed to induce these responses, was about eightfold higher than those required for catIC. The responses induced either by catIC or cationized human IgG were completely inhibited by treatment with heparin, dextran sulphate, EDTA, prostaglandin E1, or IV3, a monoclonal antibody against the receptor II for the Fc portion of IgG (Fc gamma RII). The data presented in this study suggest that IgG charge constitutes a critical property that conditions the ability of IC to trigger platelet activation.

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