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Department of Biology, National Cheng Kung University, Tainan, Taiwan, 701 and * Department of Neurosurgery, Chang Gung University and Memorial Hospital, Taipei, Taiwan, 105
2To whom correspondence should be addressed. E-mail: sjchang{at}mail.ncku.edu.tw.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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80% of the GPII/IIIa receptor. Platelet aggregation was inhibited by B-6 vitamers via the occupancy of GPIIb/IIIa with the potency of PLP > PN > PM > PL.
KEY WORDS: • vitamin B-6 • GPIIb/IIIa • platelet aggregation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Fibrinogen binding to the activated platelet receptor GPIIb/IIIa plays a fundamental role in the formation of thrombus. Blockage of fibrinogen binding was addressed recently as a new therapeutic approach (2
–4
). Thus, there is great interest in the clinical development of agents that can bind to platelet GPIIb/IIIa, block fibrinogen binding and be used in the prevention and management of thrombotic disease states.
Vitamin B-6 in its coenzyme form of pyridoxal-phosphate (PLP), has been shown to inhibit platelet aggregation in studies done in our laboratory (5
) and those of others (6
,7
). Although we found that vitamin B-6 down-regulated the gene expression of the GPIIb promoter (8
), the mechanism by which it mediated platelet aggregation is still uncertain. Recently, we found four major PLP binding proteins on the membrane of both inactivated and activated platelets. The PLP binding proteins, with molecular weights of 123 and 105 kDa, were suggested to be GPIIb and GPIIIa, respectively. A competitive ELISA using an anti-GPIIb/IIIa antibody indicated that PLP inhibited the binding of fibrinogen to GPIIb/IIIa (9
). However, the effect of the occupancy of GPIIb/IIIa by PLP on platelet aggregation is unknown. Therefore, the aim of this study was to quantify GPIIb/IIIa binding in platelets treated with B-6 vitamers [PLP; pyridoxal (PL); pyridoxine (PN); pyridoxamine (PM)] and to determine whether the final common pathway of platelet aggregation could be prevented by the binding of vitamin B-6 to GPIIb/IIIa. A quantitative flow cytometry assay was used to evaluate the kinetics of the platelet blockade after vitamin B-6 treatment compared with aggregometry assays.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The monoclonal fluorescein isothiocyanate-conjugated antibodies CD41a to GPIIb (Ancell, Bayport, MN), CD61 to GPIIIa (Biodesign, Saco, ME), CD41 to GPIIb/IIIa complex (Biodesign, Saco, ME), isotype mouse immunoglobulin G1 (Ancell, Bayport, MN) as well as platelet GPIIb/IIIa occupancy kit (Biocytex, Marseille, France) were used to quantify the occupancy of GPIIb/IIIa. B-6 vitamers (PLP, PL, PM and PN) and ADP were purchased from Sigma Chemical (St. Louis, MO).
Blood collection.
Blood was collected from healthy volunteers who had not taken aspirin or any other antiplatelet agent in the previous 7 d, by vein puncture using a 22-gauge needle and minimal stasis. The blood was anticoagulated with 38 g/L sodium citrate (9:1 v/v), centrifuged at 250 x g for 15 min and the supernatant was aspirated as platelet-rich plasma (PRP). After PRP removal, the remaining plasma was centrifuged at 2400 x g for 10 min and the supernatant was aspirated to procure platelet-poor plasma (PPP). The PRP was diluted to 2 x 1010 platelets/L with PPP for flow cytometry assays and diluted to 2–3 x 1011 platelets/L with PPP for the platelet aggregation assay. Flow cytometry and aggregation assays were performed within 1 h of blood collection. Platelet counts were performed using a Coulter Counter (Coulter Counter ZM, Coulter, Luton, UK). The study protocol was approved by the National Science Council, Executive Yuan, Taiwan, and was explained to the donors before the study.
Ligand binding assays.
Aliquots of 50 µL PRP were treated with 600 µL of different concentrations (0–60 mmol/L) of PLP, PL, PM and PN for 45 min, then with 100 µL ADP (200 µmol/L) for 30 min. PRP treated with PBS were used as both positive and isoptype controls. Aliquots of treated PRP were incubated with CD41a (anti-GPIIb), CD61 (anti-GPIIIa) and CD41 (anti-GPIIb/IIIa) at room temperature for 45 min. The samples were fixed with 100 µL of 1% formaldehyde after a 30-min incubation and then analyzed by flow cytometry (Coulter Epics XL-MCLTM; Miami, FL) at 488 nm excitation. Platelet populations were gated according to their forward and side scatter. Histograms were generated using 10,000 counts, and mean fluorescence was calculated using EXPO 2 analysis software of the Coulter Epics system (Coulter). The binding of an isotypic control antibody was considered nonspecific binding and was subtracted from the observed mean fluorescence.
GPIIb/IIIa receptor number and occupancy.
GPIIb/IIIa receptor number and occupancy were quantified using the GPIIb/IIIa receptor occupancy kit (Biocytex), which contains the anti-GPIIIa monoclonal antibodies mAb1 (clone LYP18), mAb2 (clone 4F8), isotypic control antibody and calibration beads. Calibration beads, consisting of a mixture of four different populations of 2-µm diameter latex beads, each with a different defined amount of murine antibody per bead were used to estimate the number of antibody molecules bound per platelet as described by Quinn et al. (10
).
Platelet aggregation.
Platelet aggregation was studied at 37°C by light transmission using a platelet aggregation chromogenic kinetic system (Helona Laboratories, PACK-4, Beaumont, TX). Response of the platelets to various doses of ADP was measured to determine the threshold aggregating concentration (defined as the minimum concentration of the agent that results in maximal aggregation). PLP, PL, PN and PM from 0 to 60 mmol/L in a volume of 50 µL were added to 400 µL of PRP in the aggregometer cuvette and incubated for 10 min before adding the threshold concentration of ADP. Platelet aggregation was quantified as the percentage of light transmission after the addition of 50 µL of ADP.
Statistical analysis.
Data are means ± SEM and were analyzed by SigmaStat (11
) within the B-6 vitamers. Data from the different concentrations were compared by an initial one-way ANOVA and then with a subsequent Duncan’s test when ANOVA indicated significant differences among means at P < 0.05. The percentage of baseline B-6 vitamer binding and the percentage of baseline aggregation were evaluated by linear regression, and Pearson’s correlation coefficients are reported.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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PLP bound to <30% of GPIIb, GPIIIa and GPIIb/IIIa complexes at concentrations 
0.6 mmol/L. The binding of PLP to GPIIb, GPIIIa and GPIIb/IIIa complexes was >80% at 3 mmol/L. At concentrations of 0.6–3 mmol/L, PLP binding to GPIIb, GPIIIa and GPIIb/IIIa was dose dependent (Fig. 1
).
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18 mmol/L. At concentrations of 3–30 mmol/L, PL binding to GPIIb and GPIIb/IIIa was dose dependent (Fig. 1)
.
PN bound to <30% of GPIIb, GPIIIa and GPIIb/IIIa complexes at concentrations between 1.8 and 3 mmol/L. The binding of PN to GPIIb, GPIIIa and GPIIb/IIIa was close to 80% at concentrations 6 mmol/L (Fig. 1)
.
PM bound to <30% of GPIIb, GPIIIa and GPIIb/IIIa complexes at concentrations of 3 mmol/L. The binding of PM to GPIIb, GPIIIa and GPIIb/IIIa complexes was >80% at concentrations between 12 and 18 mmol/L and saturable at concentrations 18 mmol/L. At concentrations of 3–18 mmol/L, PM binding to GPIIb and GPIIb/IIIa was dose dependent (Fig. 1)
.
Relationship between occupancy of GPIIb/IIIa and aggregation by B-6 vitamers.
Platelet aggregation was significantly inhibited by PLP, PL, PN and PM at 1.8, 6, 3 and 6 mmol/L, respectively (Table 1
). The GPIIb/IIIa receptor numbers were also significantly decreased after platelets were treated with 1.8, 6, 3 and 6 mmol/L of PLP, PL, PN and PM, respectively (Fig. 2
). These results indicated that the occupancies of GPIIb/IIIa complex were significantly increased by PLP, PL, PN and PM additions at these concentrations. Occupancies of the GPIIb/IIIa receptor by PLP, PL, PN and PM were negatively correlated with platelet aggregation (r = -0.93, -0.92, -0.90 and -0.94, respectively, P < 0.0001).
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The ability of B-6 vitamers to bind to GPIIb/IIIa increased with increasing concentrations and appeared to be the highest for PLP, followed by PN, then PM and PL (Fig. 3
). Binding affinities to GPIIb/IIIa were PLP > PN > PM > PL with Kd of 1.83 ± 1.15, 3.63 ± 1.67, 10.89 ± 2.93 and 19.4 ± 7.86, respectively.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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0.6 mmol/L, PL 3 mmol/L, PN 1.8 mmol/L and PM 3 mmol/L (Fig. 1)
, their bindings to the GPIIb, GPIIa and GPIIb/IIIa complexes were all <30% and platelet aggregation was not affected (Table 1)
. These findings confirmed another study in which platelet aggregation was unaffected at receptor occupancies of <30% to <50% (12
).
The >80% GPIIb/IIIa receptor occupancy achieved at 3, 18, 6 and 12 mmol/L of PLP, PL, PN and PM, respectively (Fig. 1)
, was sufficient to eliminate platelet aggregation (Table 1)
. This result confirmed the suggestion of Coller et al. (13
) that as receptor occupancy exceeds 80%, aggregation might be completely inhibited despite the presence of unoccupied receptors.
PLP was reported to inhibit ADP-induced platelet aggregation (5
–7
). PN, PL, and PM have also been shown to inhibit platelet aggregation. Platelet aggregation was reported to be inhibited by B-6 vitamers at the following concentrations: deoxypyridoxine, 10 mmol/L; PLP, 3 mmol/L; PM, 7 mmol/L; pyridoxamine phosphate, 6 mmol/L; and PN, 9 mmol/L (14
). In the present study, platelet aggregation was significantly inhibited by PLP, PL, PN and PM at concentrations of 1.8, 6, 3 and 6 mmol/L, respectively (Table 1)
. The occupancies of GPIIb/IIIa complex were also significantly increased by PLP, PL, PN and PM at these same concentrations (Fig. 2)
. Concentrations of B-6 vitamers required to inhibit platelet aggregation (Table 1)
were consistent with the levels needed to occupy the GPIIb/IIIa receptor (Fig 2)
. This was shown by the strong negative correlations between occupancy of the GPIIb/IIIa receptor and platelet aggregation after the treatment of platelets with the four B-6 vitamers in this study (r = -0.90 to -0.94, P < 0.001).
Attempts to explain the mechanism of inhibition by PLP include Schiff base formation with platelet membrane proteins (15
), inhibition of agonist-receptor binding (15
,16
) or interaction with the platelet surface of GPIIb/IIIa (9
,17
). In addition, down-regulation of GPIIb promoter activity to 54, 35 and 63% in the presence of PN, PL and PLP, respectively, may also contribute to the mechanism of inhibition by B-6 vitamers (8
). Recently, we found four major PLP binding proteins on the membrane of activated and inactivated platelets; two of them were GPIIb and GPIIIa (9
). In the present study, we further demonstrated that the occupancy of the GPIIb/IIIa receptor plays a major role in antiplatelet aggregation.
The other major finding of this study was that either a subunit or a complex of a membrane receptor responsible for platelet aggregation could be occupied by all B-6 vitamers tested (Fig. 1)
. The results of the binding affinity (Kd) to the GPIIb/IIIa complex (PLP > PN > PM > PL) paralleled the results of the percentage of binding for the four B-6 vitamers to the GPIIb/IIIa complex (PLP > PN > PM > PL) (Fig. 3)
. The variation in binding affinity among the four B-6 vitamers may be attributed to the structural differences that affect the interactions between B-6 vitamers and platelet receptor proteins. PLP was found to inhibit many enzymes through Schiff base formation with 
-amino groups of lysyl residues in enzyme proteins (18
). The phosphate group on PLP, which can donate and accept a proton during the formation of a Schiff base, is close to the aldehyde group of the PLP, and can provide and accept protons simultaneously during proton transfer, resulting in the rapid formation of a Schiff base between the -amino and aldehyde groups (19
). PL itself can form a hemiacetal form that is not accessible for Schiff base formation (20
). Additionally, PL formed a Schiff base with cytosolic protein, whereas PLP could do so only with platelet membrane protein (14
). These facts explain why the binding affinity of PLP to the platelet membrane protein was higher than that of PL. PN and PM have been found to penetrate the platelet membrane (14
), which may decrease their availibility for GPIIb/IIIa occupancy. In addition, PN, with its OH group, may interact with the COOH group of amino acids in GPIIb/IIIa to form a covalent bound. The amine (NH2) group on PM may form an amide with the COOH group of amino acids in GPIIb/IIIa via hydrogen-bound formation. These different abilities of the B-6 vitamers to bind may explain their different binding affinities to GPIIb/IIIa.
The interactions of individual B-6 vitamers with platelet membrane proteins explain their different potencies for inhibitory action. However, ligand binding to GPIIb/IIIa requires an intact heterodimer to exhibit the cross-link of platelet aggregation. Therefore, we suggest that platelet aggregation was inhibited by B-6 vitamers via the occupancy of GPIIb/IIIa receptor with the potency of PLP > PN > PM > PL.
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FOOTNOTES |
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3 Abbreviations used: GP, glycoprotein; PL, pyridoxal; PLP, pyridoxal phosphate; PM, pyridoxamine; PN, pyridoxine; PPP, platelet-poor plasma; PRP, platelet-rich plasma.
Manuscript received 17 July 2002. Initial review completed 6 August 2002. Revision accepted 12 September 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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