This Article Abstract Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pedreno, J. Articles by de Castellarnau, C. Search for Related Content PubMed PubMed Citation Articles by Pedreno, J. Articles by de Castellarnau, C.

(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:156-163.)
© 1997 American Heart Association, Inc.

Articles

Platelet Integrin _IIbß3 (GPIIb-IIIa) Is Not Implicated in the Binding of LDL to Intact Resting Platelets

Javier Pedreno; Rosa Fernandez; Cristina Cullare; Antonia Barcelo; Miguel Angel Elorza; Conxita de Castellarnau

the Department of Biochemistry (J.P., R.F., A.B., M.A.E.), Hospital Universitario Son Dureta, Palma de Mallorca, Spain, and the Department of Atherothrombosis and Vascular Biology (C.C., C. de C.), Institut de Recerca del Hospital Santa Creu i Sant Pau, Barcelona, Spain.

Correspondence to Javier Pedreno MD, PhD, Fundacio d'Investigacio Cardiovascular (Pabellon Cardiologia) Hospital de la Santa Creu i Sant Pau, Avenida San Antonio Maria Claret 167, 08025 Barcelona, Spain.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
It has been suggested that the fibrinogen receptor (glycoprotein [GP] IIb-IIIa or platelet integrin _IIbß₃) could be the binding site for low-density lipoprotein (LDL); however, recent data do not support this. Furthermore, GPIIb and not the GPIIb-IIIa complex is the main binding protein for lipoprotein(a) [Lp(a)]. In the present study, we have investigated the interaction between Lp(a) particles and platelet LDL binding sites and whether platelet integrin _IIbß₃ is implicated. Displacement experiments showed that ¹²⁵I-LDL binding to intact resting platelets was inhibited with the same apparent affinity by both unlabeled LDL and apolipoprotein(a)-free lipoprotein particles [Lp(a)^-, an LDL-like particle prepared from Lp(a)]. Hill coefficients for displacement curves suggested that a single set of binding sites was involved. In contrast, both native and oxidized Lp(a) particles were unable to inhibit platelet LDL binding. Furthermore, platelets bound ¹²⁵I-Lp(a)^- particles to a class of saturable binding sites numbering approximately 1958±235 binding sites per platelet with a dissociation constant (K_d) of 48.3±12x10^-9 mol/L. These values were similar to those obtained for LDL. In contrast to Lp(a), evidence indicates that platelet integrin _IIbß₃ was not involved in the interaction of LDL and intact resting platelets. First, specific ligands for platelet integrin _IIbß₃, such as fibrinogen, vitronectin, and fibronectin, were unable to inhibit the binding of LDL to intact resting platelets. Second, similar LDL binding characteristics (K_d and B_max values) were found in platelets from control subjects and patients with type I and type II Glanzmann's thrombasthenia, characterized by total and partial lack of GPIIb-IIIa and fibrinogen, respectively. Third, polyclonal antibodies against the GPIIb-IIIa complex (edu-3 and 5B12), human antiserums against platelet alloantigens (anti-Bak^a/B and anti-PL^A1/2), anti-integrin subunits (anti-_v and anti-ß₃₎, and a wide panel of monoclonal antibodies (mAbs) against well-known epitopes of GPIIb (M3, M4, M5, M6, and M95-2b) and GPIIIa (P23-7, P33, P37, P40, and P97) did not affect platelet LDL binding. Finally, in contrast to the proaggregatory effect of native and oxidized LDL, both native and oxidized Lp(a) particles caused a significant dose-dependent decrease of collagen-induced platelet aggregation. In conclusion, we demonstrate that neither the GPIIb-IIIa complex nor GPIIb and GPIIIa individually are membrane binding proteins for LDL on intact resting platelets. Lp(a) particles do not interact with platelet LDL binding sites, and their biological response is clearly different from that of LDL.

Key Words: binding sites • lipoprotein(a) • platelet integrin _IIbß₃ • oxidized lipoproteins

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Serum cholesterol has been considered to be a systemic thrombogenic risk factor implicated in thrombus formation on ruptured atherosclerotic plaques.¹ Furthermore, the interaction between LDLs and platelets may explain the increased thrombogenicity described in hypercholesterolemia.^{2 3} Moreover, the binding of LDL to specific platelet binding sites seems to be the initial step in the modulation of this process.⁴ Since the first descriptions of specific binding sites for LDL in platelets were given, evidence has indicated that these could be different from the "classical" LDL receptor.^{5 6 7 8 9} In fact, it has been reported that three platelet membrane proteins with an apparent molecular mass of 156, 130, and 115 kD bind LDL specifically.¹⁰ Further studies using ligand and Western blotting techniques identified platelet membrane GPs, such as GPIIb (136 kD) or _IIb subunit and GPIIIa (92 kD) or ß₃ subunit, and also the intact GPIIb-IIIa complex as the main GPs implicated in the binding of LDL.¹¹ Furthermore, we clearly demonstrated that a mAb against the LDL receptor (immunoglobulin G-C7) failed to block LDL binding to intact platelets¹² and that the platelet LDL binding site, in contrast to the classical LDL receptor of human fibroblasts, does not mediate an endocytotic response and recognizes both native and oxidized LDL particles with the same apparent affinity.¹³ All these findings strengthen the idea that the platelet LDL binding site differs from the classical LDL receptor and support the hypothesis that these binding sites might be implicated in the thrombogenic response of native and oxidized LDL.^{14 15 16 17 18} Recently,¹⁹ it has been described that Lp(a), which may also play an important role during thrombogenesis, binds to intact platelets in a saturable and specific manner and inhibits both platelet plasminogen binding and enhancement of platelet surface-mediated plasminogen activation induced by the tissue-type plasminogen activator. Further ligand blotting and binding studies showed that platelet Lp(a) binding was fully blocked by mAbs against GPIIb, GPIIb-IIIa, unlabeled Lp(a), and fibrinogen but not by mAbs against GPIIIa.²⁰ The authors showed that GPIIb and not the GPIIb-IIIa complex is the main platelet binding protein for Lp(a). Overall, these results indicate that fibrinogen, LDL, and Lp(a) binding sites could be related via platelet integrin _IIb ß₃. However, several studies suggest that GPIIb-IIIa apparently may not act as a platelet LDL binding site. In fact, platelet LDL binding characteristics clearly differ from those of fibrinogen.^{4 5 6 7 8 9 10 11 12 13 14 15 16 17 18} Furthermore, instead of inducing inhibition, LDL particles increased the binding of fibrinogen to both ADP and proteolytic enzyme–activated platelets.^{21 22 23} Finally, in contrast to the well-known LDL proaggregatory effect,^{24 25 26} high plasma Lp(a) levels do not alter collagen-induced platelet aggregation,²⁷ indicating that a different biological response is involved in the aggregatory effect of both particles. Therefore, these data have led to conflicting results, and further studies are needed to explore the implication of platelet integrin _IIb ß₃ on LDL binding. The aim of the present study was to investigate the implication of GPIIb-IIIa in the platelet LDL binding and to determine whether Lp(a) particles are involved in this interaction.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Materials
BSA was purchased from Fluka Chemie/AG; fibrinogen, from Kabi; sucrose and EDTA, from E. Merck; ¹²⁵I-Na, from New England Nuclear; Iodo-Gen and amino caproic acid, from Sigma-Aldrich Quimica; Sephadex PD-10 and heparin-Sepharose (HiTrap) columns and lysine- and gelatin-Sepharose 4B, from Pharmacia LKB; and highly purified (>95% by SDS-PAGE) vitronectin and fibronectin, from Calbiochem Corp and Chemicon Intl. Polyclonal antibodies against the GPIIb-IIIa complex (edu-3 and 5B12 [the latter from Dako]) and human antiserums against platelet alloantigens (anti-Bak^a/B [=Lek^a/b, HPA-3, or anti-GPIIb]) and anti-PL^A1/2 [ZW^a/b, HPA-1, or anti-GPIIIa]) were a gift from Dr E. Muniz (Hospital Santa Creu i Sant Pau, Barcelona, Spain). A panel of pure murine mAbs against GPIIb (M3, M4, M5, M6 and M95-2b) and against GPIIIa (P23-7, P33, P37, P40, and P97), which recognize different epitopes of GPIIb and GPIIIa, respectively,^{28 29 30} were a gift from Dr J. Gonzalez-Rodriguez (Instituto de Quimica Fisica "Rocasolano" CSIC, Madrid, Spain); polyclonal antibodies against _v (-vitronectin receptor) and ß₃ (anti-IIIa) integrin subunits and their corresponding nonimmune rabbit sera were generously donated by Dr. E. Dejana (Vascular Biology Laboratory, Mario Negri Institut, Milan, Italy). A mouse immunoglobulin G used as a negative control in binding studies and functional tests was from Menarini Diagnostics.

Lipoproteins
Human LDL (density range, 1.019 to 1.063 g/mL) was obtained from pooled serum of normocholesterolemic [with Lp(a) levels <3 mg/dL] volunteers and isolated by sequential ultracentrifugation³¹ in a T 2060 Centrikon ultracentrifuge (Kontron Instruments). Lp(a) levels, as apo(a) protein, were determined in all LDL preparations, and levels <0.2 µg/mL were considered to be appropriate for functional studies. LDL was dialyzed against buffer A (150 mmol/L NaCl, 20 mmol/L Tris, and 0.3 mmol/L EDTA, pH 7.4) and filtered through a Millipore filter (0.22 µm) before use. Lp(a) from one normocholesterolemic volunteer with a high Lp(a) plasma level (126 mg/dL) was isolated as LDL but at a density range of 1.060 to 1.150 g/mL, followed by affinity chromatography on lysine-Sepharose using amino caproic acid.³² Isolation of apo(a)-free lipoprotein particle [Lp(a)^-, an LDL-like particle prepared from Lp(a)] was achieved by affinity chromatography on heparin-Sepharose by following the procedure previously reported.³³ The absence of apo(a) in Lp(a)^- particles was established by a one-step sandwich-ELISA procedure using a polyclonal antibody against whole Lp(a) particles (Immuno AG). The purity of lipoprotein preparations was assessed by gel filtration in a fast protein liquid chromatographic device from Pharmacia LKB, as previously described,^{12 13} and SDS-PAGE, followed by immunoblot analysis using polyclonal anti-Lp(a) and anti-LDL antibodies.³⁴ All lipoprotein concentrations are expressed in grams of protein per liter measured by the procedure of Bradford,³⁵ with BSA used as the standard. Human fibrinogen was further purified by chromatography on gelatin-Sepharose 4B and lysine-Sepharose columns to remove contaminating fibronectin and plasminogen, respectively. Fibrinogen was routinely subjected to SDS-PAGE under reducing conditions and thrombin coagulation (clottability, >94%), and its capacity to support ADP-induced aggregation of washed platelets was determined to assess the integrity of its three constituent chains. Fibrinogen preparations were used immediately or stored in aliquots at -40°C.

Modification of Lipoproteins
Dialyzed LDL and Lp(a) containing 0.3 mmol/L EDTA were gel-filtered on a Sephadex PD-10 column to remove EDTA and were then immediately oxidized by incubation of 1 mg of protein with 25 µmol/L CuSO₄ in phosphate-buffered saline for 24 hours at 37°C, according to the procedure previously reported.¹³ The degree of oxidation of both particles was assessed by the thiobarbituric acid–reactive substance content and agarose–acrylamide gel electrophoresis mobility methods as previously described.¹³

Iodination of Lipoproteins
Both freshly isolated LDL and Lp(a)^-, obtained as mentioned above, were labeled with ¹²⁵I-Na by the Iodo-Gen method.³⁶ Unbound iodine was removed by chromatography with a Sephadex PD-10 column previously equilibrated with buffer A. Protein fractions were pooled and dialyzed against the same buffer for 24 hours and used immediately. Both final specific radioactivities were 2.1±0.3 Bq/ng protein of LDL (mean±SEM, n=6) and 2.5±0.2 Bq/ng protein of Lp(a)^- (mean±SEM, n=6). Purity of lipoprotein particles was assessed by gel filtration in a fast protein liquid chromatography device (Pharmacia LKB) as previously described.¹²

Platelets
Human platelets were isolated from both freshly drawn citrated (3.8% sodium citrate) blood of healthy volunteers who had not taken any medication for at least 10 days before sampling. Blood from well-characterized patients³⁷ with type I (I AIo, patient 23b) and type II (A Dom, patient 40) GT with less than 5% and 12% of the total platelet content of GPIIb-IIIa, respectively, was kindly provided by Dr M. Pico of the Hospital Vall de Hebron, Barcelona, Spain. Samples were centrifuged at 300g for 10 minutes at room temperature, and the resultant platelet-rich plasma was then sedimented by centrifugation as previously described in detail.¹² Briefly, for binding experiments, the platelet pellet was washed twice and finally resuspended with incubation buffer (20 mmol/L Tris-HCl, pH 7.45, containing 0.15 mol/L NaCl, 1 mmol/L CaCl₂, and 5 g/L BSA) to give a platelet count of 10¹² platelets per liter. For aggregation assays, the first platelet-rich plasma pellets were gently washed in 20 mmol/L Tris-HCl buffer (containing 0.15 mol/L NaCl, 5 mmol/L glucose, and 1 mmol/L EDTA, pH 7.4), centrifuged at 900g for 6 minutes, and finally resuspended at counts in the range of 3 to 5x10¹¹ platelets per liter in Tyrode's buffer (0.14 mol/L NaCl, 2.7 mmol/L KCl, 1 mmol/L CaCl₂, 12 mmol/L NaHCO₃, 0.4 mmol/L NaH₂PO₄, 5.5 mmol/L glucose, 3.5 mg/mL BSA, and 10 mmol/L HEPES, pH 7.4). Platelets washed and resuspended in this manner showed "swirling" as a gross indication of maintenance of discoid shape and were fully responsive to weak agonists in the presence of fibrinogen for at least 2 hours.

LDL Binding Assays and Competition Experiments
Washed platelets (10⁸) from healthy volunteers were incubated at room temperature for 25 minutes with both ¹²⁵I-LDL (up to 1000 µg/mL) and ¹²⁵I-Lp(a)^- (up to 500 µg/mL) particles, in a total volume of 0.25 mL of incubation buffer. ¹²⁵I-LDL and ¹²⁵I-Lp(a)^- binding to platelets was determined as previously described,¹² and nonspecific binding was defined as binding that was not displaced by a 2000-fold molar excess of unlabeled lipoproteins. Displacement of ¹²⁵I-LDL binding (0.01 g/L) in the presence of varying protein concentrations of unlabeled lipoprotein particles (native and oxidized LDL and Lp(a), and Lp(a)^- particles) or the main GPIIb-IIIa ligands (fibrinogen, fibronectin, and vitronectin) was measured as described above. Dissociation constants (K_d) for the competing ligands (nmol/L) were determined according to the method of Cheng and Prussof.³⁸ Displacement curves were also analyzed by Hill plots according to the method reported by Bennett and Yamamura.³⁹ Calculation of bound LDL was based on specific activity of labeled LDL, and the results were expressed as nanograms of protein bound per 10⁸ platelets. Specific binding was evaluated mathematically by Scatchard analysis⁴⁰ to determine the number of binding sites and the dissociation constant by using the KINETIC/EBDA/LIGAND program.⁴¹

To investigate whether platelet GPIIb and GPIIIa are implicated in the binding of LDL to intact resting platelets, the following approaches were used: (1) specificity of platelet LDL binding sites for platelet integrin _IIbß₃ was evaluated by displacement experiments of ¹²⁵I-LDL binding by several purified GPIIb-IIIa ligands, such as fibrinogen, fibronectin, and vitronectin, as mentioned above; (2) with a human model of inherited lack of GPIIb-IIIa, a relationship between LDL binding sites and platelet integrin _IIbß₃ was investigated by binding studies on intact platelets from GT patients (type I and type II); and (3) immunological ligand binding assays were performed after preincubation of washed human platelets with the indicated concentrations of an ample panel of polyclonal antibodies and mAbs or their respective negative antisera for 10 minutes at room temperature before the addition of ¹²⁵I-LDL (500 µg/mL). The polyclonal antibodies tested were the following: anti–GPIIb-IIIa complex (edu-3 and 5B12), anti-integrin subunits (anti-_v or anti–-vitronectin receptor, and anti-ß₃), and human antiserums (anti-Bak^a/B and anti-PL^A1/2). Also, pure murine mAbs that recognize different epitopes of GPIIb (M3, M4, M5, M6, and M95-2b) and GPIIIa (P23-7, P33, P37, P40, and P97) and strongly inhibit both platelet aggregation and fibrinogen binding were tested; some of them (P40, P37, P23-7, and M5) cross-react with ß- and -subunits of the vitronectin receptor in endothelial cells (References 28-30, 42, and, 43; C. de C. et al, unpublished data, 1997).

Aggregation Studies
Platelet aggregation was performed and recorded on a four-channel light aggregometer (Aggrecorder II) as previously described.⁴⁴ Briefly, washed platelets (0.4 mL) with added fibrinogen (600 nmol/L) were stimulated in the aggregometer cuvette at 37°C with the selected low (0.3 to 0.5 µg/mL) and high (0.6 to 1 µg/mL) concentrations of collagen that produced a 30% to 60% or a 40% to 80% change in light transmittance, respectively. Native and oxidized LDL and Lp(a) lipoproteins at 100 and 10 µg protein per milliliter, respectively, were preincubated alone or combined with platelet suspensions before the addition of collagen to trigger aggregation. Blank samples included Tyrode's buffer, fibrinogen, and lipoprotein particles or saline.

Statistical Analysis
Results are expressed as mean±SEM. Differences between means were compared by Student's t test, with P<.05 considered significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Binding of ¹²⁵I-Lp(a)^- and ¹²⁵I-LDL to Washed Human Platelets
Binding of ¹²⁵I-Lp(a)^- (isolated and purified as is indicated in Fig 1) to intact resting platelets in the presence and absence of a 2000-fold molar excess of unlabeled Lp(a)^- is shown in Figure 2A. Binding derived from four independent experiments was saturable and specific at 22°C and similar to that obtained at 37°C (data not shown). A Scatchard plot of the binding data gave a linear correlation coefficient of r=-.92 (Fig 2A, inset). With an assumed molecular weight for apoB of 550 kD, the number of binding sites per platelet was 1958±235, with a K_d of 48.3±12 nmol/L (mean±SEM, n=4). In agreement with our previous publications,^{12 13 22} control experiments performed in parallel using ¹²⁵I-LDL also showed (Fig 2B) that Lp(a)^- particles bind to an extent similar to that of native LDL (B_max, 1367±209 binding sites per platelet with a K_d of 51±8 nmol/L; mean±SEM, n=4).

View larger version (59K): [in this window] [in a new window]   Figure 1. Purification of both Lp(a) and Lp(a)- particles. Elution profiles of Lp(a) isolated by sequential ultracentrifugation (density range, 1.060 to 1.150 g/mL) from both lysine- and heparin-Sepharose fast-flow columns after fast protein liquid chromatography are shown in panels A and B, respectively. A, Peak 1 was eluted at 0.15 mol/L NaCl; peaks 2 and 4 were eluted at 50 mmol/L amino caproic acid. B, Peak 3 represents a pool of peaks 2 and 4 incubated with reductive agents to cleavage intact Lp(a) and reinjected onto the heparin-Sepharose column. Lp(a)- particles were then eluted using a 0 to 1 mol/L NaCl gradient, and the absence of apo(a) was assessed by an ELISA procedure. C, left: Separation of apo(a) isoforms was performed by SDS-PAGE (3.5% slab gels). Lanes 1 through 4 represent peaks eluted from fast-flow columns. C, right: Proteins were electroblotted to nitrocellulose membrane, the strips were then incubated with rabbit polyclonal anti-Lp(a) immunoglobulin G, and the bound antibody was developed by the silver enhancement procedure. Lanes 1 and 3 show the total lack of Lp(a) particles bound in peaks 1 and 3. An immunoblot procedure using anti-LDL polyclonal antibodies shows that both peaks contain only LDL particles; data not shown. Lanes 2 and 4 represent highly purified Lp(a) particles obtained by lysine-Sepharose chromatography and amino caproic acid elution.

View larger version (17K): [in this window] [in a new window]   Figure 2. 125I-LDL (A) and 125I-Lp(a)- (B) binding to intact resting platelets. Washed platelets (108) were incubated with 125I-LDL and with 125I-Lp(a)- at room temperature for 25 minutes, as indicated in "Material and Methods." indicates total binding; , specific binding; and , nonspecific binding (determined in the presence of a 2000-fold molar excess of unlabeled Lp(a)- or LDL). Scatchard analysis is shown in the insets. Results represent mean±SEM of four independent experiments per duplicate. B/F indicates bound-to-free ratio.

To determine whether platelet GPIIb-IIIa or fibrinogen receptors are implicated in the binding of LDL to platelets, we carried out binding studies of ¹²⁵I-LDL to platelets from type I (I AIo) and type II (A Dom) thrombasthenic patients with abnormal levels of GPIIb-IIIa and fibrinogen.³⁷ We found that both type I and type II GT platelets bound ¹²⁵I-LDL and presented similar values (1167 binding sites per platelet with a K_d of 53 nmol/L and 1043 binding sites per platelet with a K_d of 48 nmol/L, respectively) compared with those obtained in control platelets (1348±231 binding sites with a K_d of 51±8 nmol/L).

Competition Experiments
To determine whether increasing concentrations of native lipoproteins [LDL, Lp(a)^-, and Lp(a)], oxidized lipoproteins [LDL and Lp(a)], and specific GPIIB-IIIA ligands (fibrinogen, vitronectin, and fibronectin) interfere with ¹²⁵I-LDL binding, we carried out competition experiments of LDL binding to intact resting platelets at 22°C for 25 minutes. Fig 3 shows that binding of ¹²⁵I-LDL (100 µg of protein per liter) was fully inhibited by unlabeled native and oxidized LDL and also by Lp(a)^- particles. Hill coefficients from displacement curves were -1.07 and -1.10 for native and oxidized LDL, respectively, and -0.89 for Lp(a)^-, suggesting that a single set of binding sites is involved. In contrast, native and oxidized Lp(a) (each at a protein concentration of 500 µg/mL) and purified ligands of GPIIb-IIIa, such as fibrinogen (up to 10 g of protein per milliliter), fibronectin (up to 10 µmol/L), and vitronectin (up to 10 µmol/L), were not effective competitors of ¹²⁵I-LDL binding, and only a slight unspecific diminution of the total binding was found.

View larger version (17K): [in this window] [in a new window]   Figure 3. Inhibition of 125I-LDL binding to intact resting platelets by different types of lipoproteins and other specific ligands of GPIIb-IIIa. Platelets were incubated for 25 minutes at room temperature with 125I-LDL (0.01 g of protein per liter) both in the presence and absence of unlabeled LDL (, up to 500 µg/L), Lp(a)- (, up to 500 µg/L), oxidized LDL (, up to 500 µg/L), Lp(a) (, up to 500 µg/L), oxidized Lp(a) (+, up to 500 µg/L), fibrinogen (, up to 10 000 µg/L), fibronectin (, up to 10 mmol/L), and vitronectin (, up to 10 mmol/L). Specific binding was determined as indicated in "Material and Methods." Results are expressed as means of four independent experiments per duplicate. SEM never exceeds more than 10% of the mean.

Platelet Integrin _IIbß₃ and ¹²⁵I-LDL Binding to Platelets
To further investigate whether the intact GPIIb-IIIa complex is involved, individually (GPIIb and GPIIIa) or as a Ca²⁺-dependent heterodimer (GPIIb-IIIa), in the binding of ¹²⁵I-LDL to intact resting platelets, we carried out ligand binding studies in the absence and presence of a large panel of antibodies against platelet integrin _IIbß₃ and known platelet alloantigens. Fig 4 represents typical line plots showing no inhibition of ¹²⁵I-LDL binding to intact resting platelets by mAbs against GPIIIa (panel A), against GPIIb, and against the GPIIb-IIIa complex (panel B) and human antiserums and polyclonal antibodies (panel C). All experiments (n=7) showed a lack of specific inhibitory effect at any tested protein concentration. However, with some mAbs against GPIIIa, a maximum inhibition (about 40%) was found with low concentrations of P37, and about 25% inhibition was found with P40 and P97. In similar conditions, all mAbs against GPIIb inhibit ¹²⁵I-LDL binding to intact platelets by about 20%, except M95-2b, which inhibits binding by about 30% at all tested concentrations. A similar unspecific effect was found with antiserums against platelet alloantigens and polyclonal antibodies against anti-_v (-vitronectin receptor) and anti-ß₃ (GPIIIa) integrin subunits (panel C). All these experiments clearly indicate that neither the GPIIb-IIIa complex nor GPIIb and GPIIIa individually are the GPs implicated in the binding of LDL to intact resting platelets.

View larger version (20K): [in this window] [in a new window]   Figure 4. Plots showing the effect of several antibodies against the GPIIb-IIIa complex or, individually, GPIIb and GPIIIa on LDL binding to intact resting platelets. Percentages of specifically bound 125I-LDL are plotted against purified mAbs against GPIIIa (A: , P23-7; , P33; +, P37; , P40; and , P97) and against GPIIb (B: , M3 and M4; , M5; +, M6; and , M95-2b) and polyclonal antibodies against the GPIIb-IIIa complex (B: , edu-3. Panel C shows the effect of human antiserums against platelet alloantigens (, anti Baka/B [=Leka/b, HPA-3, or anti-GPIIb]; , anti-PLA1/2 [ZWa/b, HPA-1, or anti-GPIIIa]) and polyclonal antibodies against integrin subunits (, anti-ß3; , anti-v) or against the GPIIb-IIIa complex; +, 5B12 for comparison. Unlabeled LDL () was used as a positive inhibitor in all experiments. Results are mean of seven independent experiments (SEM never exceeds more than 10% of the mean).

Effect of Lipoproteins on Collagen-Induced Aggregation
We found that LDL and Lp(a) deferentially alter the susceptibility of platelets to aggregate with collagen (Figs 5 and 6). Similar results were found on ADP-stimulated platelets (data not shown). Fig 5 shows representative aggregation tracings of 0.3 µg/mL (right) and 0.7 µg/mL (left) collagen alone or collagen combined with 100 µg/mL of native and oxidized LDL and 10 µg/mL of native and oxidized Lp(a). The effect of the combination of LDL and Lp(a) at 100 µg/mL and 10 µg/mL, respectively, is also shown (Fig 5, left). Fig 6 shows dose-response curves of both Lp(a) (panel A) and LDL particles (panel B) on collagen-induced platelet aggregation. When Lp(a) at a protein concentration of 2 to 20 µg/mL was added just before the addition of 0.7 µg/mL collagen, a dose-dependent decrease of collagen-induced platelet aggregation was observed. From seven independent experiments, Lp(a) at a protein concentration of 10 µg/mL decreased collagen-induced platelet aggregation to 37±8% of control (mean±SEM, P<.001). Furthermore, a similar protein concentration of oxidized Lp(a) also caused a significant decrease of similar collagen-induced platelet aggregation (26±5% of control, mean±SEM, n=3, P<.001). However, both native and oxidized LDL particles produced a significant increase in collagen-induced platelet aggregation (62±9% and 55±10% of control, respectively; mean±SEM, n=5, P<.001). Nevertheless, when platelets were preincubated in the presence of a combination of LDL (100 µg/mL) and Lp(a) (10 µg/mL) particles, the result was a strongly antiaggregatory effect, indicating that Lp(a) might inhibit the proaggregatory effect of LDL.

View larger version (19K): [in this window] [in a new window]   Figure 5. Representative aggregation tracings of the effect of native and oxidized (Ox) LDL and Lp(a) on collagen-induced platelet aggregation. The effects of a final concentration of 100 µg/mL of native and Ox LDL and 10 µg/mL of native and Ox Lp(a) or the combination of LDL and Lp(a) added to 0.4 mL of washed platelets and preincubated for 5 minutes at room temperature before the addition of collagen at 0.7 µg/mL (left panel) and 0.3 µg/mL (right panel) are shown. Each aggregation tracing represents one typical experiment out of seven. Bar at lower left indicates 1 minute. C indicates collagen alone (control).

View larger version (10K): [in this window] [in a new window]   Figure 6. Dose-response curve of the effect of Lp(a) (A) and LDL (B) on collagen-induced platelet aggregation. Washed platelets were preincubated with increasing lipoprotein concentrations, and platelet aggregation was determined as indicated in "Material and Methods." Results are mean±SEM of seven independent experiments.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Malle et al²⁰ have recently characterized the binding of ¹²⁵I-Lp(a) to human intact platelets, which was clearly different from that of ¹²⁵I-LDL binding. In fact, LDL particles were much weaker competitors than Lp(a) particles in displacing ¹²⁵I-Lp(a) bound to intact platelets. Since Lp(a) binds to intact platelets only by GPIIb and not by the GPIIb-IIIa complex,²⁰ it can be assumed that platelet LDL binding sites are not related to platelet integrin _IIbß₃. Furthermore, in contrast to the well-known LDL proaggregatory effect,^{4 8 17} the same group has reported²⁷ that collagen-induced aggregation in isolated platelets from subjects with high plasma Lp(a) levels is similar to that from normolipidemic control subjects, indicating that a different biological response mediated by different binding sites could be implicated in the aggregatory effect of both particles. In contrast, Koller et al,¹¹ using ligand blotting methods, have described that LDL binds to platelet integrin _IIbß₃. However, it has recently been demonstrated that instead of inducing inhibition, LDL increases the exposure of fibrinogen receptors in intact activated platelets.^{21 22 23} In order to explore these controversial results, in the present study we investigated the interaction of platelet LDL binding sites and Lp(a) particles, the implication of platelet GPIIb-IIIa or integrin _IIbß₃ in the binding of LDL to intact platelets, and the effect of Lp(a) on agonist-induced platelet aggregation.

Displacement experiments showed that ¹²⁵I-LDL binding to intact resting platelets was inhibited with the same affinity by unlabeled native and oxidized LDL, and Hill coefficients determined from displacement curves showed that a single set of binding sites could be involved. In contrast, Lp(a) particles were unable to inhibit the binding of ¹²⁵I-LDL to intact platelets, even at a 2000-fold molar excess. Furthermore, ligand binding assays showed that human platelets bound Lp(a)^- particles [an LDL-like particle prepared from Lp(a) and deprived of apo(a) by reductive cleavage] to an extent similar to that of LDL particles. Finally, these Lp(a)^- particles were able to inhibit the binding of ¹²⁵I-LDL binding to intact resting platelets with the same affinity. Together, these data provide strong evidence that Lp(a) does not interact with platelet LDL binding sites, and we agree with Malle et al²⁰ that a different binding mechanism in both LDL and Lp(a) binding could be involved in intact resting platelets.

Platelet integrin _IIbß₃ (the GPIIb-IIIa complex) is the most prominent member of the integrin family of platelet adhesive receptors that upon activation mediates platelet aggregation by binding of fibrinogen and recognizes with high affinity several adhesive proteins with the Arg-Gly-Asp (RGD) sequence.⁴⁵ Conversion of platelet membrane GPIIb and GPIIIa from the latent state to the GPIIb-IIIa complex is a calcium-dependent mechanism. The binding of fibrinogen therefore requires this divalent ion, and chelating agents such as EDTA cause the dissociation of the complex at 37°C, fully inhibiting both fibrinogen binding and platelet aggregation.⁴⁵

It is well known that the RGD sequence is not present in apoB-100.⁴⁶ Moreover, we previously demonstrated that platelet LDL binding does not require calcium ions and that EDTA at 37°C does not inhibit the binding of LDL to intact platelets.¹² In the present study, the possible implication of platelet integrin _IIbß₃ on the binding of LDL was investigated using platelets from type I and type II GT and by ligand and immunological binding studies. Our results provide ample evidence that platelet integrin _IIbß₃ is not related to the binding of LDL to intact resting platelets. First, platelets from type I and type II GT patients presented similar LDL binding characteristics (K_d and B_max) compared with control intact resting platelets. Second, assuming that the GPIIb-IIIa complex could bind LDL, its main specific ligands (fibrinogen, vitronectin, and fibronectin) should inhibit the binding of LDL to platelets. However, our results demonstrate a complete lack of inhibitory effect with all of them. These results are in agreement with previous data^{21 22 23} showing that LDL at a physiological protein concentration enhanced fibrinogen binding in activated platelets. Third, in contrast to Koller et al,¹¹ an ample panel of polyclonal, human platelet antiserum, and pure murine mAbs at concentrations that strongly inhibit both platelet aggregation and platelet fibrinogen binding (References 42 and 43; C. de C. et al, unpublished data, 1997) did not block the binding of ¹²⁵I-LDL to intact resting platelets.

Finally, it is well known that atherogenic lipoproteins, such as VLDL and LDL (native and oxidized particles), increase agonist-induced platelet aggregation, whereas nonatherogenic lipoproteins, such as chylomicrons and HDL, cause a significant decrease in platelet aggregation.^{4 8 17} However, little is known about the effect of both native and oxidized Lp(a) particles on platelet aggregation.²⁷ Moreover, "in vitro" effects of these lipoproteins on human platelets are not available. Results from the present study provide evidence for the first time that when both native and oxidized Lp(a) particles were incubated with platelets, a significant dose-dependent antiaggregatory effect was found. Furthermore, an LDL proaggregatory effect was fully inhibited when Lp(a) was present. The mechanism by which Lp(a) particles decrease platelet aggregation remains to be elucidated. However, assuming that individual GPIIb is the main Lp(a) binding protein of intact platelets²⁰ and since platelet aggregation is mediated by the binding of fibrinogen to the GPIIb-IIIa complex,⁴⁵ we speculate that the antiaggregatory effect of Lp(a) found in the present study could be due either to its inhibitory effect on platelet fibrinogen binding or to promoting platelet disaggregation because of its high homology to plasminogen.⁴⁷ In conclusion, we present ample evidence indicating that neither the GPIIb-IIIa complex nor GPIIb and GPIIIa individually are the ligand binding proteins for LDL on intact resting platelets. Furthermore, Lp(a) does not interact with platelet LDL binding sites, and its biological effect is clearly different to those of native and oxidized LDL particles. Further studies are required to identify platelet LDL binding sites.

	Selected Abbreviations and Acronyms

 

BSA = bovine serum albumin ELISA = enzyme-linked immunosorbent assay GP = glycoprotein GT = Glanzmann's thrombasthenia mAb = monoclonal antibody SDS-PAGE = sodium dodecyl sulfate–polyacrylamide gel electrophoresis

	Acknowledgments

 
This study was supported in part by grants from the Comision Interministerial de Ciencia y Tecnologia (CICYT SAF94-0191 to Dr Pedreno), the Spanish Arteriosclerosis Foundation (92/01 to Dr Pedreno), and the Direccion General de Investigacion Cientifica y Tecnica (PM91/0029 to Dr Cullare). We wish to thank Dr Marta Pico for kindly providing access to the GT patients. We also wish to express our gratitude to Magdalena Vila, Rosa Llopart, Rosa Ortin, Agustina Castellvi, and Montserrat Carmona for their excellent technical assistance.

	Footnotes

 
Presented in part at the 64th European Atherosclerosis Society Congress, Utrecht, the Netherlands, June 10-13, 1995, and published in abstract form (Atherosclerosis. 1995;115[suppl]:S88).

Received August 10, 1995; revision received May 20, 1996;

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Fuster V, Badimon L, Badimon JJ, Chesebro JH. The pathogenesis of coronary artery disease and the acute coronary syndromes. N Engl J Med. 1992;326:242-250.[Medline] [Order article via Infotrieve]

2. Badimon JJ, Badimon L, Turitto VT, Fuster V. Platelet deposition at high shear rates is enhanced by high plasma cholesterol levels: in vivo study in the rabbit model. Arterioscler Thromb. 1991;11:395-402.[Abstract/Free Full Text]

3. Cadroy Y, Lemozy S, Diquelou A, Ferrieres J, Douste-Blazy P, Boneu B, Sakariaseen KS. Human type II hyperlipoproteinemia enhances platelet-collagen adhesion in flowing nonanticoagulated blood. Arterioscler Thromb. 1993;13:1650-1653.[Abstract/Free Full Text]

4. Andrews HE, Aitken JW, Hassall DG, Skinner VO, Bruckdofer KR. Intracellular mechanisms in the activation of human platelets by low-density lipoproteins. Biochem J. 1987;242:550-564.

5. Koller E, Koller F, Doleschel W. Specific binding sites on human blood platelets for plasma lipoproteins. Hoppe-Seylers Z Physiol Chem. 1982;363:395-405.[Medline] [Order article via Infotrieve]

6. Aviram M, Brook JG. Platelet interaction with high and low density lipoproteins. Atherosclerosis. 1983;46:259-268.[Medline] [Order article via Infotrieve]

7. Mazurov AV, Preobrazhensky SN, Leytin VL, Repin VS, Smirnov VN. Study of low density lipoprotein interaction with platelets by flow cytofluorometry. FEBS Lett. 1982;137:319-322.[Medline] [Order article via Infotrieve]

8. Aviram M, Brook JG. Platelet activation by plasma lipoproteins. Prog Cardiovasc Dis. 1987;30:61-72.[Medline] [Order article via Infotrieve]

9. Curtiss LK, Plow EF. Interaction of plasma lipoproteins with human platelets. Blood. 1984;64:365-374.[Abstract/Free Full Text]

10. Koller E. Lipoprotein-binding proteins in the human platelet plasma membrane. FEBS Lett. 1986;200:97-102.[Medline] [Order article via Infotrieve]

11. Koller E, Koller F, Binder B. Purification and identification of the lipoprotein-binding proteins from human blood platelet membrane. J Biol Chem. 1989;264:12412-12418.[Abstract/Free Full Text]

12. Pedreno J, de Castellarnau C, Cullare C, Sanchez J, Gomez-Gerique J, Ordonez-Llanos J, Gonzalez-Sastre F. LDL binding sites on platelets differ from the `classical' receptor of nucleated cells. Arterioscler Thromb. 1992;12:1353-1362.[Abstract/Free Full Text]

13. Pedreno J, de Castellarnau C, Cullare C, Ortin R, Sanchez JL, Llopart R, Gonzalez-Sastre F. Platelet LDL receptor recognizes with the same apparent affinity both oxidized and native LDL: evidence that the receptor-ligand complexes are not internalized. Arterioscler Thromb. 1994;14:401-408.[Abstract/Free Full Text]

14. Koller E, Koller F. Binding characteristics of homologous plasma lipoproteins to human platelets. Methods Enzymol. 1992;215:383-398.[Medline] [Order article via Infotrieve]

15. Bochkov VN, Matchin YG, Fuki IV, Lyakishev AA, Tkachuk VA. Platelets in patients with homozygous familial hypercholesterolemia are sensitive to Ca²⁺-mobilizing activity of low density lipoproteins. Atherosclerosis. 1992;96:119-124.[Medline] [Order article via Infotrieve]

16. Virgolini I, Li S, Qiong Y, Koller E, Banyai M, Angelberger P, Sinzinger H. Binding of ¹¹¹In-labeled LDL to platelets of normolipemic volunteers and patients with heterozygous familial hypercholesterolemia. Arterioscler Thromb. 1993;13:536-547.[Abstract/Free Full Text]

17. Malle E, Sattler W. Platelets and the lipoproteins: native, modified and platelet modified lipoproteins. Platelets. 1994;5:70-83.

18. Zhao B, Dierichs R, Liu B, Berkes P. Gold-labelled low density lipoproteins bind to washed human platelets. Platelets. 1994;5:113-120.

19. Ezratty A, Simon DI, Loscalzo J. Lipoprotein(a) binds to human platelets and attenuates plasminogen binding and activation. Biochemistry. 1993;32:4628-4633.[Medline] [Order article via Infotrieve]

20. Malle E, Ibovnik A, Steinmetz A, Kostner GM, Sattler W. Identification of glycoprotein IIb as the lipoprotein(a)-binding protein on platelets: lipoprotein(a) binding is independent of an arginyl-glycyl-aspartate tripeptide located in apolipoprotein(a). Arterioscler Thromb. 1994;14:345-352.[Abstract/Free Full Text]

21. van Willigen G, Gorter G, Akkerman J-WN. LDLs increase the exposure of fibrinogen binding sites on platelets and secretion of dense granules. Arterioscler Thromb. 1994;14:41-46.[Abstract/Free Full Text]

22. Pedreno J, Fernandez R. Proteolytic susceptibility of platelet LDL receptor. Lipids. 1995;30:927-933.[Medline] [Order article via Infotrieve]

23. Koller E, Volf I, Koller F. Binding of low density lipoprotein to human platelets: effects of chymotrypsin. Thromb Haemorrh Disorders. 1992;6:57-65.

24. Hassall DG, Owen JS, Bruckdorfer KR. The aggregation of isolated platelets in the presence of lipoproteins and prostacyclin. Biochem J. 1983;216:43-49.[Medline] [Order article via Infotrieve]

25. Hassall DG, Forrest LA, Bruckdorfer KB. Influence of plasma lipoproteins on platelet aggregation in a normal male population. Arteriosclerosis. 1983;3:332-338.[Abstract/Free Full Text]

26. Aviram M, Brook JG. Characterization of the effect of plasma lipoprotein on platelet function in vitro. Haemostasis. 1983;13:344-350.[Medline] [Order article via Infotrieve]

27. Malle E, Satller W, Prenner E, Leis HJ, Hermetter A, Dries A, Kostner GM. Effects of dietary fish oil supplementation on platelet aggregability and platelet membrane fluidity in normolipemic subjects with and without high plasma Lp(a) concentrations. Atherosclerosis. 1991;88:193-201.[Medline] [Order article via Infotrieve]

28. Calvete JJ, Rivas G, Maruri M, Alvarez M, McGregor JL, Hew CL, Gonzalez-Rodriguez J. Tryptic fragment of human GP IIIa: isolation and biochemical characterization of the 23 kDa N-terminal glycopeptide carrying the antigenic determinant for a monoclonal antibody (P37) which inhibits platelet aggregation. Biochem J. 1988;260:697-704.

29. Calvete JJ, Arias J, Alvarez MV, Lopez MM, Henschen A, Gonzalez-Rodriguez J. Further studies on the topography of human platelet glycoprotein IIb: localization of monoclonal antibody epitopes and the putative glycoprotein IIIa and fibrinogen-binding regions. Biochem J. 1991;273:767-775.

30. Calvete JJ, Arias J, Alvarez MV, Lopez MM, Henschen A, Gonzalez-Rodriguez J. Further studies on the topography of human platelet glycoprotein IIIa: localization of monoclonal antibody epitopes and the putative fibrinogen-binding sites. Biochem J. 1991;274:457-463.

31. Havel RJ, Eder HA, Bragdon JH. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest. 1955;34:1345-1353.

32. Utermann G. The mysteries of lipoprotein(a). Science. 1989;246:904-910.[Abstract/Free Full Text]

33. Armstrong VW, Walli AK, Seidel D. Isolation, characterization, and uptake in human fibroblasts of an apo(a)-free lipoprotein obtained on reduction of lipoprotein(a). J Lipid Res. 1985;26:1314-1323.[Abstract]

34. Utermann G, Menzel HJ, Kraft HG, Duba HC, Kemmler HG, Seitz C. Lp(a) glycoprotein phenotypes: inheritance and relation to Lp(a)-lipoprotein concentrations in plasma. J Clin Invest. 1987;80:458-465.

35. Bradford MA. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye-binding. Anal Biochem. 1976;72:248-254.[Medline] [Order article via Infotrieve]

36. Salacinski PR, McLean C, Sykes JE, Clement-Jones VV, Lowry PJ. Iodination of proteins, glycoproteins, and peptides using a solid-phase oxidizing agent, 1,3,4,6-tetrachloro-3,6-diphenylglycoluril (iodogen). Anal Biochem. 1981;117:136-146.[Medline] [Order article via Infotrieve]

37. James NG, Caen JP, Nurden AT. Glanzmann's thrombasthenia: the spectrum of clinical disease. Blood. 1990;75:1383-1395.[Free Full Text]

38. Cheng YC, Prusoff WH. Relationship between the inhibition constant (K_i) and the concentration of inhibitor which causes 50 percent inhibition (I₅₀) of an enzymatic reaction. Biochem Pharmacol. 1973;22:3099-3108.[Medline] [Order article via Infotrieve]

39. Bennett JP, Yamamura HI. Neurotransmitter, hormone, or drug receptor binding methods. In: Yamamura HI, Enna SJ, Kuhar MJ, eds. Neurotransmitter Receptor Binding. 2nd ed. New York, NY: Raven Press Publishers; 1985:57-90.

40. Scatchard G. The attractions of proteins for small molecules and ions. Ann N Y Acad Sci. 1949;51:660-672.

41. McPherson GA. Analysis of radioligand binding experiments: a collection of computer programs for the IBM PC. J Pharmacol Methods. 1985;14:213-228.[Medline] [Order article via Infotrieve]

42. Cullare C, Muniz-Diaz E, de Castellarnau C, Madoz P, Lluc A, Gonzalez-Rodriguez J. Characterization of monoclonal antibodies against platelet glycoprotein IIb and IIIa involved in platelet aggregation. Thromb Haemost. 1991;65:1148. Abstract.

43. Muniz-Diaz E, de Castellarnau C, Ribera A, Madoz P. Immunologic cross-reactivity between platelet GPIIb and the vitronectin receptor alpha chain using monoclonal anti-IIb antibodies. Blood. 1990;75:318-319.[Free Full Text]

44. Vila L, Cullare C, Sola J, Puig L, de Castellarnau C, de Moragas JM. Cyclooxygenase activity is increased in platelets from psoriatic patients. J Invest Dermatol. 1991;97:1749-1755.

45. Shattil SJ. Function and regulation of the ß₃ integrins in hemostasis and vascular biology. Thromb Haemost. 1995;74:149-155.[Medline] [Order article via Infotrieve]

46. Scanu A, Fless GM. Lipoprotein(a): heterogeneity and biological relevance. J Clin Invest. 1990;85:1709-1715.

47. Loscalzo J, Vaughan DE. Tissue plasminogen activator promotes platelet disaggregation in plasma. J Clin Invest. 1987;79:1749-1755.

This article has been cited by other articles:

				S. Tetik, F. Uras, E. Eksioglu-Demiralp, and K. Turay Yardimci Low-Density Lipoprotein Specifically Binds Glycoprotein IIb/IIIa: A Flow Cytometric Method for Ligand-Receptor Interaction Clinical and Applied Thrombosis/Hemostasis, April 1, 2008; 14(2): 210 - 219. [Abstract] [PDF]

				S. J. A. Korporaal, I. A. M. Relou, M. van Eck, V. Strasser, M. Bezemer, G. Gorter, T. J. C. van Berkel, J. Nimpf, J.-W. N. Akkerman, and P. J. Lenting Binding of Low Density Lipoprotein to Platelet Apolipoprotein E Receptor 2' Results in Phosphorylation of p38MAPK J. Biol. Chem., December 10, 2004; 279(50): 52526 - 52534. [Abstract] [Full Text] [PDF]

				S. Nomura, A. Shouzu, S. Omoto, M. Nishikawa, T. Iwasaka, and S. Fukuhara Activated Platelet and Oxidized LDL Induce Endothelial Membrane Vesiculation: Clinical Significance of Endothelial Cell-Derived Microparticles in Patients With Type 2 Diabetes Clinical and Applied Thrombosis/Hemostasis, July 1, 2004; 10(3): 205 - 215. [Abstract] [PDF]

				L. D Tsironis, J. V Mitsios, H. J Milionis, M. Elisaf, and A. D Tselepis Effect of lipoprotein (a) on platelet activation induced by platelet-activating factor: role of apolipoprotein (a) and endogenous PAF-acetylhydrolase Cardiovasc Res, July 1, 2004; 63(1): 130 - 138. [Abstract] [Full Text] [PDF]

				C. M. Hackeng, M. Huigsloot, M. W. Pladet, H. K. Nieuwenhuis, H. J. M. van Rijn, and J.-W. N. Akkerman Low-Density Lipoprotein Enhances Platelet Secretion Via Integrin-{alpha}IIbß3–Mediated Signaling Arterioscler. Thromb. Vasc. Biol., February 1, 1999; 19(2): 239 - 247. [Abstract] [Full Text] [PDF]

				M. L. Rand, W. Sangrar, M. A. Hancock, D. M. Taylor, S. M. Marcovina, M. A. Packham, and M. L. Koschinsky Apolipoprotein(a) Enhances Platelet Responses to the Thrombin Receptor–Activating Peptide SFLLRN Arterioscler. Thromb. Vasc. Biol., September 1, 1998; 18(9): 1393 - 1399. [Abstract] [Full Text] [PDF]

				P. Maschberger, M. Bauer, J. Baumann-Siemons, K. J. Zangl, E. V. Negrescu, A. J. Reininger, and W. Siess Mildly Oxidized Low Density Lipoprotein Rapidly Stimulates via Activation of the Lysophosphatidic Acid Receptor Src Family and Syk Tyrosine Kinases and Ca2+ Influx in Human Platelets J. Biol. Chem., June 16, 2000; 275(25): 19159 - 19166. [Abstract] [Full Text] [PDF]

This Article Abstract Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pedreno, J. Articles by de Castellarnau, C. Search for Related Content PubMed PubMed Citation Articles by Pedreno, J. Articles by de Castellarnau, C.