Natural polyamines are aliphatic bases present in most mammalian tissues in concentrations regulated by hormones, drugs, regenerating activity of tissues, and growth factors. Increasing evidence indicates that these molecules play an important role in many cellular functions, including cell proliferation and signal transduction.1-4 A variety of amines inhibit both the hemagglutination activity of endogenous lectin and the aggregation of platelets caused by various agonists.5-10 However, no definitive conclusions on their mechanism of action have been drawn so far.5-10
Agam et al11 suggested that the polyamine-mediated inhibition of platelet aggregation occurs at the level of platelet-platelet attachment and is independent of the particular agonist tested. In addition, polyamines are more potent inhibitors than monoamines.11 Platelet-platelet attachment results from fibrinogen binding to specific receptors on the platelet surface. These receptors are the dimeric membrane glycoprotein IIb/IIIa (GP IIb/IIIa).12 It has been suggested that amines interfere with the agonist-induced fibrinogen binding to the platelet surface.13,14 Moreover, polyamine binding to activated platelets decreases in the presence of the GP IIb/IIIa antibody LJCP8 or in the presence of decorsin, a peptide known to bind GP IIb/IIIa.15 These results suggest that the polyamine-mediated inhibition of fibrinogen binding to activated platelets is caused by interference with the fibrinogen receptor, ie, glycoprotein IIb/IIIa.
Current evidence supports the concept that platelet activation induces a calcium-dependent conformational change on GP IIb/IIIa that enables it to bind to the fibrinogen molecule.12,16 This work was carried out to investigate the effect of spermine on GPIIb/IIIa activation. The results presented here indicate that the mechanism of action of polyamines involves the suppression of the agonist-induced activation of GP IIb/IIIa, which is a prerequisite for platelet aggregation.
All studies were performed on platelets from healthy individuals who had not taken any medications for 7 days before specimen collection. Blood was obtained via venipuncture from an antecubital vein into trisodium citrate (final concentration 0.4%). Alternatively, blood was collected into test tubes containing low heparin concentrations. To work with a low heparin concentration, the pellet in each commercial vacutainer tube was suspended in 1 mL of saline solution. Then, 0.8 mL was discarded, retaining one fifth of the original heparin concentration.
Preparation of Platelet-Rich Plasma (PRP) and Platelet Count Adjustment
PRP was prepared at room temperature. Blood was centrifuged 10 minutes at 250 × g. The PRP was carefully removed, and platelet-poor plasma (PPP) was obtained by centrifuging the resulting pellet 10 minutes at 1000 × g. Platelet counts were determined using a Coulter T-890 (Coulter Diagnostics, Miami, FL) and adjusted to 225,000 ± 100,000 platelets per microliter using autologous PPP.
Platelet aggregation measurements were performed by the turbidimetric method using an 810-CA Chrono-log aggregometer with an agro-link computer interface (Chrono-log Corporation, Havertown, PA). PRP was incubated with various concentrations of putrescine, spermidine, or spermine for 3 minutes at 37°C. After incubations were completed, baseline aggregations were monitored for 1 minute to exclude the presence of autoaggregation. Platelet aggregation was induced by adding either ADP 10 μM, collagen 2 μg/mL, epinephrine 50 μM, ristocetin 1 mg/mL, arachidonic acid 0.5 mM, or thrombin 1 U/mL. Aggregations were monitored for 4 minutes after agonist addition. In some experiments the agonist was added before the polyamine, as indicated. Control aggregation of platelets was obtained by the addition of identical saline solution volumes instead of polyamine. For each antagonist, the inhibition percentage of aggregation was calculated as follows:
where Tm is the maximal transmittance signal. The concentration of each polyamine that leads to 50% inhibition (IC50) was obtained by nonlinear regression analysis from the respective inhibition curves.
Surface Glycoprotein Expression
Platelet-rich plasma was incubated with saline solution, ADP (100 μM), or thrombin (1 U/mL) without stirring. Then 10 μL of CD42 (GP IX) or CD41 (GP IIb) FITC-coupled specific antibodies (Becton Dickinson, San Jose, CA) was added, and the cell suspension was incubated in the dark for 15 minutes at room temperature. Control suspensions were incubated with an isotype mouse IgM labeled with FITC (Becton Dickinson, San Jose, CA). The platelet suspension was then diluted 30 times with commercial FACS Flow Sheath fluid (Becton Dickinson, San Jose, CA). The labeled preparations were analyzed by standard 1-color flow cytometry in a FACScan flow cytometer (Becton Dickinson, San Jose, CA).
Calibration of fluorescence and light scatter was performed using the manufacturer's standard beds (Calibrite, Becton Dickinson).
Platelets were identified by characteristic light scatter and completely resolved from irrelevant events such as electronic noise and cell debris by the specific FITC-labeled anti-CD42 monoclonal antibody reaction. The platelet population was gated, and the percentage of platelets that displayed a fluorescence higher than 101 was calculated.
Glycoprotein IIb/IIIa Activation
The activation of glycoprotein IIb/IIIa was determined by flow cytometry with the PAC-1 antibody (PharMigen, San Diego, CA). PAC-1 antibody (10 μL) was added to 25 μL of PRP pretreated with either saline, thrombin (1 U), or ADP (100 μM). To prevent platelet aggregation, which would interfere with the cytometric determinations, these assays were performed at room temperature (25°C), and the PAC-1 antibody was added immediately after the agonist. After 15 minutes in the dark, the solution was diluted with 1000 μL FACS Flow Sheath. The samples were analyzed on a Becton Dickinson FACScan flow cytometer. Activation was expressed as positive cell percentage.
Data are expressed as mean ± SD. Differences between measures were assessed by paired t test. A P value of < 0.05 was considered significant.
Putrescine, spermidine, and spermine caused a dose-dependent inhibition of platelet aggregation (Fig. 1). These experiments were conducted in platelets obtained in blood collected in the presence of either citrate (Fig. 1A) or heparin (Fig. 1B). However, as shown in Figure 1, the maximal inhibition of aggregation attained by the most potent polyamine, spermine, could not be achieved by the other 2 polyamines, which may indicate the existence of different binding sites for each of these molecules. In the presence of ADP, the polyamine concentration exhibiting half-maximum inhibition (IC50) is higher for putrescine (7 mM) than for spermidine (5.2 mM) or spermine (3 mM). In the presence of collagen, higher concentrations of each polyamine were needed to inhibit platelet aggregation, such that for putrescine IC50 = 35 mM, for spermidine IC50 = 25 mM, and for spermine IC50 = 11 mM.
In the presence of citrate, the concentration of Ca2+ decreases, probably potentiating the polyamine effect.17 Thus, it was decided to analyze the polyamine effect on platelets obtained from 3 mL of blood collected into 9 U heparin instead of citrate (Fig. 1B). When platelets were from heparin-treated blood, only spermine was capable of promoting the inhibition of aggregation to an extent greater than 60%. Furthermore, at 36 mM it was the only polyamine that exhibited its maximal effect. Spermidine, at a concentration of 45 mM, promoted 50% inhibition, and inhibiton by putrescine was below 30% at any of the concentrations tested (up to 100 mM) (Fig. 1B).
Platelet aggregation is dependent on agonist concentration. Aggregation dependence on ADP, for which we obtained a K0.5 of 4 μM, is extraordinarily increased in the presence of 16 mM spermine (Fig. 2). Maximal aggregation in polyamine-treated platelets never reached that obtained with nontreated platelets irrespective of the ADP concentration; ie, the agonist does not reverse inhibition in these conditions. This further supports the idea that spermine inhibits platelet aggregation by interfering with platelet-platelet attachment, irrespective of the agonist used.
As shown elsewhere,9,10 putrescine, spermidine, and spermine inhibit platelet aggregation in the presence of ADP, collagen, thrombin, epinephrine, arachidonic acid, and ristocetin (Figs. 3 and 4). In these experiments, 16 mM spermine inhibited platelet aggregation almost completely, regardless of the agonist tested. Via et al14 found that spermine caused inhibition of the thrombin-induced platelet aggregation and a concomitant apparent increase in the shape change that normally precedes aggregation. We also observed this phenomenon for thrombin or arachidonic acid-induced aggregation.
To test whether spermine could stop aggregation once it had started, in some experiments the polyamine was added 2 minutes after the corresponding agonist (Figs. 3 and 4B,D,F). Among all agonists tested, spermine arrested aggregation to the greatest extent on its addition. In some individual experiments, disaggregation was also seen, and similar results were obtained with the other polyamines (data not shown). Spermine inhibited platelet aggregation regardless of whether the polyamine was added before or after the agonist. The above results also indicate that inhibition is likely to be exerted on platelet-platelet interaction rather than on specific agonist-receptor binding.
Results in Table 1 indicate that spermine does not reduce the binding of specific antibodies to GP IX (a component of the Ib-IX-V complex, involved in normal platelet adhesion and aggregation), or the IIb component of the fibrinogen receptor. However, when the ADP-induced activation of GP IIb/IIIa was tested with the PAC-1 antibody, it was clearly shown that spermine caused a diminution of active GP IIb/IIIa concentration (Table 1).
Figure 5 shows that spermine causes inhibition of GP IIb/IIIa activation as evaluated with the PAC-1 antibody. Interestingly, when spermine was added 2 minutes after the platelet agonist, the polyamine was also capable of reducing the activation of GP IIb/IIIa. These results indicate that the polyamine caused some “disactivation” of the fibrinogen receptor, which would explain the arrest or reverse of aggregation (Figs. 3 and 4).
Polyamines are potent platelet inhibitors.5-10 However, their mode of action has not been fully elucidated. Early studies concerning polyamine effects on platelet aggregation suggested that the mechanism involved the inhibition of phospholipase C and protein kinase, enzymes involved in platelet aggregation and secretion.18-20 Polyamines are known to inhibit these enzymes in cell-free systems21,22; however, it is now clear that the inhibition of these enzymes is not the mechanism of platelet polyamine inhibition. The responses caused by these enzymes (IP3 production, ATP secretion, cytoplasmic Ca2+ rise) are modified to varying extents in the presence of thrombin but not of the other platelet agonists. This is likely to be caused by some specific spermine-induced inhibition of thrombin binding,14 and the most important reason is the fact that spermine does not enter the platelet membrane.14 This may prevent the polyamine from interacting with cytoplasmic enzymes such as phospholipase C and Ca2+/calmodulin-dependent protein kinases. In this regard, Israelis et al23 reported that spermine did not inhibit the phosphorylation of proteins, inositol phosphates, or phosphatidic acid in platelets occurring in response to arachidonic acid or lysophosphatidic acid.
Polyamines caused a dose-dependent inhibition of platelet aggregation with an order of potency spermine > spermidine > putrescine. We also noted that for aggregation, the sigmoidal dependence on ADP concentration is abolished in the presence of spermine. Thus, it is suggested that the platelet-platelet binding process, being a cooperative phenomenon, loses this characteristic under the influence of spermine. Further analysis of the data suggests that only when Ca2+ concentration was diminished by citrate were the effects of putrescine and spermidine fully prevented (Fig. 1A). However, in the case of spermine, the citrate-mediated decrease in Ca2+ resulted in milder effects, as the IC50 for spermine was 3 mM. Furthermore, in the presence of heparin, where no modifications in Ca2+ concentrations are observed, spermine still exhibited higher inhibitory effects on platelet aggregation than the other polyamines, which were observed at lower concentrations (Fig. 1B).
Spermine inhibits platelet aggregation induced by a variety of agents, ie, ADP, collagen, thrombin, ristocetin, arachidonic acid or epinephrine. However, it is not likely that this polyamine interferes with all agonist's receptors at the same time, instead, a common step in platelet aggregation for most agonist may be arrested by spermine.
Most agonists activate platelets by induction of shape changes, release reactions, and subsequent induction of conformational changes on fibrinogen receptors within the membrane surface. After these activation processes occur, fibrinogen binds to glycoprotein IIb/IIIa, which appears to be a prerequisite for platelet aggregation24 and the common step in the aggregation induced by different agonists.
Spermine is capable of arresting platelet aggregation or even causing disaggregation indicating that spermine inhibition involves platelet-platelet attachment, probably through direct inhibition of GP IIb/IIIa as suggested by Via et al.14
The specific interaction of spermine with GP IIb/IIIa was studied here with the use of specific fluorescent monoclonal antibodies. Binding of anti-CD41 (GP IIb) or anti-CD42 (GP IX) antibodies to platelets was not reduced by spermine.
The CD41 antibody used in this work reacts with GP IIb in the intact complex IIb/IIIa, but not with GP IIb or GP IIIa separately.25,26 Because spermine does not inhibit the binding of this antibody in either resting or activated platelets, it is likely that the polyamines do not bind to the IIb component of the fibrinogen receptor. Spermine, on the other hand, was shown to disaggregate platelets in the spectrophotometric studies; however, because spermine seems not to affect the integrity of the IIb/IIIa complex or inhibit CD41 antibody binding, it is likely that this polyamine does not interfere with the association of the IIb subunit with the IIIa subunit of the fibrinogen receptor.
Current evidence supports the concept that platelet activation induces a calcium-dependent conformational change in GP IIb/IIIa that enables it to bind to fibrinogen.12 With the use of a monoclonal antibody specific for the activated form of GP IIb/IIIa (PAC-1), we demonstrated here that spermine inhibits the activation of GP IIb/IIIa, which is a prerequisite for platelet aggregation.
PAC-1 is am IgM antibody that binds only to activated platelets and appears to be specific for the fibrinogen recognition site within GP IIb/IIIa.27 The specificity of PAC-1 may result from the fact that 1 of its hypervariable regions (CDR3 of heavy chain) contains an arginine-tyrosine-aspartic acid sequence. This hypervariable region may structurally mimic RGD-containing regions in fibrinogen.
As shown here, spermine is not likely to bind to the GP IIb of the IIb/IIIa complex; however, the polyamine clearly inhibits the activation of the receptor. The polyamine also caused an apparent “disactivation” of the IIb/IIIa complex, which may not be caused by disassociation of the IIb and IIIa subunits as proposed before.
The concentration of polyamines needed to cause appreciable effects is far higher than those normally found in plasma. Pakala et al17 demonstrated that millimolar concentrations of polyamines are effective in preventing thrombotic events. The later studies confer relevance to the results presented here, even when these authors do not find different potency between the polyamines (spermine > spermidine > putrescine). Thus, these differences deserve further analysis.
Polyamines are polycationic molecules known to compete with Ca2+ for anionic sites within cells; therefore, because both activation and stabilization of aggregation are Ca2+-dependent phenomena, spermine may interact with Ca2+ binding sites on the platelet surface, including the Ca2+-specific sites on IIb/IIIa involved in its activation. As suggested in the first studies in the field,5 it is possible that polyamine inhibition may involve the abolition of Ca2+ interactions. This is now being studied in our laboratory.
Our results demonstrate that the polyamines putrescine, spermidine, and spermine cause a dose-dependent inhibition of platelet aggregation that is associated with the size and charge of the molecule. The inhibition is not reversed by the agonist used to induce aggregation. The polyamine spermine can reverse platelet aggregation in the presence of thrombin, ADP, collagen, ristocetin, arachidonic acid, or ephineprine as platelet agonists. The mechanisms of this inhibition involves the final step in aggregation, common to all agonists, ie, fibrinogen binding to GPIIb/IIIa through polyamine-inhibition of agonist-induced activation of GP IIb/IIIa.
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Keywords:© 2005 Lippincott Williams & Wilkins, Inc.
platelet aggregation; glycoprotein IIb/IIIa; polyamine; putrescine; spermidine; spermine