scholarly journals Exposure of platelet fibrinogen-binding sites by collagen, arachidonic acid, and ADP: inhibition by a monoclonal antibody to the glycoprotein IIb-IIIa complex

Blood ◽  
1983 ◽  
Vol 61 (1) ◽  
pp. 140-148
Author(s):  
G Di Minno ◽  
P Thiagarajan ◽  
B Perussia ◽  
J Martinez ◽  
S Shapiro ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Arachidonic Acid ◽  
Binding Sites ◽  
Human Platelet ◽  
Adenosine Diphosphate ◽  
Scatchard Analysis ◽  
Single Class ◽  
Fibrinogen Binding ◽  
Binding Data ◽  
High Affinity Site

Following stimulation with adenosine diphosphate (ADP), collagen, or arachidonic acid, unstirred human platelet suspensions bind 125I- fibrinogen in a reaction that reaches completion within 30 min. Scatchard analysis of these binding data reveals two sets of binding sites with all 3 agents: a high affinity site (Kd 0.029–0.045 microM) binding 1000–1600 fibrinogen molecules per platelet, and a lower affinity site (Kd 1.2–2.0 microM) binding 46,000–76,000 fibrinogen molecules per platelet. At a concentration of apyrase that inhibited ADP-induced fibrinogen binding by greater than 85%, fibrinogen binding induced by collagen and arachidonic acid was only partially affected. This suggests that fibrinogen binding induced by collagen or arachidonic acid does not require released ADP. We isolated a monoclonal antibody, B59.2, which precipitated the glycoprotein IIb- IIIa complex from solubilized platelet membranes. Binding of labeled antibody to platelets before or after exposure to ADP, collagen, or arachidonic acid showed a single class of approximately 22,000 binding sites with Kd 0.019 microM. Binding of B59.2 was complete within 1 min and was not inhibited by EDTA. Preincubation of platelet suspensions with a 2.1 microM concentration of B59.2 caused inhibition of secretion and aggregation, but not of thromboxane-B2 synthesis, in response to 1 microgram/ml collagen, 40 microM arachidonic acid, or 4 microM ADP, concentrations of aggregating agents that produced complete aggregation and secretion in the absence of B59.2. At this concentration of B59.2, fibrinogen binding to stimulated platelets was inhibited by approximately 45%-55%. These data demonstrate that collagen and arachidonic acid can expose fibrinogen binding sites independently of released ADP; and that the glycoprotein IIb-IIIa complex is involved in secretion, aggregation, and fibrinogen binding, but not in thromboxane synthesis occurring in response to collagen, arachidonic acid, or ADP.

scholarly journals Exposure of platelet fibrinogen-binding sites by collagen, arachidonic acid, and ADP: inhibition by a monoclonal antibody to the glycoprotein IIb-IIIa complex

Blood ◽  
1983 ◽  
Vol 61 (1) ◽  
pp. 140-148 ◽  
Author(s):  
G Di Minno ◽  
P Thiagarajan ◽  
B Perussia ◽  
J Martinez ◽  
S Shapiro ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Arachidonic Acid ◽  
Binding Sites ◽  
Human Platelet ◽  
Adenosine Diphosphate ◽  
Scatchard Analysis ◽  
Single Class ◽  
Fibrinogen Binding ◽  
Binding Data ◽  
High Affinity Site

Abstract Following stimulation with adenosine diphosphate (ADP), collagen, or arachidonic acid, unstirred human platelet suspensions bind 125I- fibrinogen in a reaction that reaches completion within 30 min. Scatchard analysis of these binding data reveals two sets of binding sites with all 3 agents: a high affinity site (Kd 0.029–0.045 microM) binding 1000–1600 fibrinogen molecules per platelet, and a lower affinity site (Kd 1.2–2.0 microM) binding 46,000–76,000 fibrinogen molecules per platelet. At a concentration of apyrase that inhibited ADP-induced fibrinogen binding by greater than 85%, fibrinogen binding induced by collagen and arachidonic acid was only partially affected. This suggests that fibrinogen binding induced by collagen or arachidonic acid does not require released ADP. We isolated a monoclonal antibody, B59.2, which precipitated the glycoprotein IIb- IIIa complex from solubilized platelet membranes. Binding of labeled antibody to platelets before or after exposure to ADP, collagen, or arachidonic acid showed a single class of approximately 22,000 binding sites with Kd 0.019 microM. Binding of B59.2 was complete within 1 min and was not inhibited by EDTA. Preincubation of platelet suspensions with a 2.1 microM concentration of B59.2 caused inhibition of secretion and aggregation, but not of thromboxane-B2 synthesis, in response to 1 microgram/ml collagen, 40 microM arachidonic acid, or 4 microM ADP, concentrations of aggregating agents that produced complete aggregation and secretion in the absence of B59.2. At this concentration of B59.2, fibrinogen binding to stimulated platelets was inhibited by approximately 45%-55%. These data demonstrate that collagen and arachidonic acid can expose fibrinogen binding sites independently of released ADP; and that the glycoprotein IIb-IIIa complex is involved in secretion, aggregation, and fibrinogen binding, but not in thromboxane synthesis occurring in response to collagen, arachidonic acid, or ADP.

scholarly journals Further studies on the topography of the N-terminal region of human platelet glycoprotein IIIa. Localization of monoclonal antibody epitopes and the putative fibrinogen-binding sites

1991 ◽  
Vol 274 (2) ◽  
pp. 457-463 ◽  
Author(s):  
J J Calvete ◽  
J Arias ◽  
M V Alvarez ◽  
M M Lopez ◽  
A Henschen ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Binding Sites ◽  
Human Platelet ◽  
Binding Domain ◽  
Terminal Region ◽  
Platelet Glycoprotein ◽  
Gamma Chain ◽  
Fibrinogen Binding ◽  
Glycoprotein Iiia ◽  
Membrane Bound

The precise localization of the epitopes for six monoclonal antibodies specific for the N-terminal region of human platelet glycoprotein IIIa (GPIIIa) was determined. The epitope for P37, a monoclonal antibody that inhibits platelet aggregation, was found at GPIIIa 101-109, flanked by the epitopes for P23-3 (GPIIIa 16-28), P23-4 (GPIIIa 83-91), P23-5 (GPIIIa 67-73), P23-7 (GPIIIa 114-122) and P40 (GPIIIa 262-302), and very close to the early chymotryptic cleavage site of GPIIIa in whole platelets (Phe-100). When the amino acid sequence of GPIIIa was searched for peptide sequences hydropathically complementary to the fibrinogen gamma-chain C-terminal (gamma 400-411) and A alpha-chain RGD-containing peptides, none was found for the gamma 400-411, two (GPIIIa 128-132 and 380-384) were found complementary to fibrinogen A alpha 571-575 and two (GPIIIa 109-113 and 129-133) were found for A alpha 94-99. Two of these putative fibrinogen-binding sites overlap with each other, and a third one overlaps with the epitope for P37. These findings reinforce the earlier suggestion that the N-terminal region of GPIIIa is involved in fibrinogen binding, and suggest the existence in GPIIIa of either multiple or alternative RGD-binding sites or one RGD-binding domain with several moieties. Finally, early chymotryptic cleavage of GPIIIa in whole platelets liberates to the soluble fraction the peptide stretch Ser-101-Tyr-348, which carries the epitope for P37 and the putative binding sites for fibrinogen. The rest of the molecule, together with the GPIIb-resistant moiety, remains membrane-bound. This leads us to propose that the fibrinogen-binding domain of GPIIIa is not involved in the binding to GPIIb to form the Ca2(+)-dependent GPIIb-GPIIIa complex.

scholarly journals Platelet membrane alterations induced by the local anesthetic dibucaine

Blood ◽  
1986 ◽  
Vol 68 (2) ◽  
pp. 463-471 ◽  
Author(s):  
EI Peerschke
Keyword(s):  
Monoclonal Antibody ◽  
Binding Sites ◽  
Periodic Acid ◽  
Platelet Membrane ◽  
Actin Binding ◽  
Scatchard Analysis ◽  
Affinity Binding ◽  
High Affinity ◽  
Fibrinogen Binding ◽  
Induced Aggregation

Abstract Tertiary amine local anesthetics modify a variety of platelet membrane- related functions. The present study explored dibucaine (DB)-induced inhibition of platelet cohesion by examining structural and functional alterations of the human platelet membrane glycoprotein IIb-IIIa complex (GPIIb-IIIa) and platelet Ca2+ homeostasis. Complete inhibition of ADP-induced aggregation was achieved five minutes after platelet exposure to 0.10 to 0.25 mmol/L of DB when fibrinogen binding was reduced by 50%. At higher concentrations of DB (approximately 1 mmol/L), ADP-induced fibrinogen binding was completely blocked. Scatchard analysis revealed loss of high-affinity binding sites in addition to reduction in Bmax. In contrast, chymotrypsin-treated platelets sustained 50% inhibition of fibrinogen binding when incubated with 0.4 to 0.5 mmol/L DB, and kinetic analysis showed that the high- affinity platelet-fibrinogen interactions were reduced but not absent. Fibrinogen binding to chymotrypsin-treated platelets could not be completely inhibited even at high DB concentrations (1 mmol/L). The inhibition of fibrinogen binding to chymotrypsin-treated platelets correlated with changes in binding of a monoclonal antibody (10E5) specific for an epitope on the GPIIb-IIIa complex. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and radioelectroimmunoassay of DB-treated platelets, however, showed no evidence of a reduction or degradation of GP IIb or IIIa. Platelet incubation with DB (five minutes, 0.1 to 1.0 mmol/L) was also accompanied by: increased platelet membrane-associated Ca2+ involving low-affinity binding sites [Kd = 5 X 10(-5) mol/L-]; increased 45Ca2+ uptake which correlated with degradation of actin-binding protein (ABP) and digestion of GPIb as visualized on periodic-acid Schiff (PAS)- stained SDS gels and as inferred from decreased binding of a monoclonal antibody (6D1) directed against this glycoprotein; and enhanced Ca2+ exchange. Thus, exposure of platelets to DB results in membrane-related alterations that may contribute to inhibition of platelet cohesion: Decreased fibrinogen receptor exposure by traditional agonists and diminished accessibility of the GPIIb-IIIa complex to extracellular ligands correlate with DB-induced inhibition of platelet aggregation; and increased calcium uptake and exchange across the platelet membrane likely leads to activation of the calcium-dependent protease(s) which was previously shown to correlate with DB-induced inhibition of ristocetin-induced platelet agglutination.

scholarly journals A murine antiglycoprotein Ib complex monoclonal antibody, SZ 2, inhibits platelet aggregation induced by both ristocetin and collagen

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 570-577 ◽  
Author(s):  
CG Ruan ◽  
XP Du ◽  
XD Xi ◽  
PA Castaldi ◽  
MC Berndt
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Binding Sites ◽  
Human Platelet ◽  
Platelet Glycoprotein ◽  
Scatchard Analysis ◽  
Type I ◽  
Glycoprotein Ib ◽  
Induced Aggregation ◽  
Bernard Soulier Syndrome

Abstract A new monoclonal antibody (MoAb), SZ 2, reactive with the human platelet glycoprotein Ib complex has been produced by the hybridoma technique. SZ 2 immunoprecipitated the components of the glycoprotein Ib complex, glycoprotein Ib and glycoprotein IX, from Triton-X-100- solubilized, periodate-labeled platelets. Western blot analysis indicated that the epitope for SZ 2 was on the alpha-subunit of glycoprotein Ib. Scatchard analysis of SZ 2 binding to formaldehyde- fixed, washed platelets revealed a single class of binding sites with Kd = 6.6 +/- 3.3 X 10(-10) mol/L and 15,200 +/- 4,100 binding sites per platelet (mean +/- SD, n = 10). Intact antibody and its purified (Fab')2 fragments not only inhibited the ristocetin-dependent binding of von Willebrand factor to platelets and ristocetin-induced platelet agglutination but also inhibited platelet aggregation induced by Type I collagen and platelet-activating factor (PAF). SZ 2 inhibited platelet serotonin and beta-thromboglobulin release in response to these stimuli and also platelet thromboxane A2 formation in response to ristocetin and collagen. SZ 2 was without effect on platelet aggregation or release in response to other platelet stimuli such as ADP, thrombin, or arachidonic acid. The inhibition by SZ 2 of collagen- and PAF-induced platelet aggregation is surprising in that Bernard-Soulier syndrome platelets, which lack the glycoprotein Ib complex, respond normally to both these stimuli. SZ 2 was unreactive toward Bernard-Soulier syndrome platelets, as evaluated by fluorescence-associated cell sorting, and had no effect on the collagen- and PAF-induced aggregation of Bernard- Soulier syndrome platelets. The combined results suggest that the inhibition by SZ 2 of collagen- and PAF-induced aggregation of normal platelets is steric and are consistent with the glycoprotein Ib complex and the platelet collagen and PAF receptor(s) being adjacent in the human platelet plasma membrane.

scholarly journals Platelet membrane alterations induced by the local anesthetic dibucaine

Blood ◽  
1986 ◽  
Vol 68 (2) ◽  
pp. 463-471
Author(s):  
EI Peerschke
Keyword(s):  
Monoclonal Antibody ◽  
Binding Sites ◽  
Periodic Acid ◽  
Platelet Membrane ◽  
Actin Binding ◽  
Scatchard Analysis ◽  
Affinity Binding ◽  
High Affinity ◽  
Fibrinogen Binding ◽  
Induced Aggregation

Tertiary amine local anesthetics modify a variety of platelet membrane- related functions. The present study explored dibucaine (DB)-induced inhibition of platelet cohesion by examining structural and functional alterations of the human platelet membrane glycoprotein IIb-IIIa complex (GPIIb-IIIa) and platelet Ca2+ homeostasis. Complete inhibition of ADP-induced aggregation was achieved five minutes after platelet exposure to 0.10 to 0.25 mmol/L of DB when fibrinogen binding was reduced by 50%. At higher concentrations of DB (approximately 1 mmol/L), ADP-induced fibrinogen binding was completely blocked. Scatchard analysis revealed loss of high-affinity binding sites in addition to reduction in Bmax. In contrast, chymotrypsin-treated platelets sustained 50% inhibition of fibrinogen binding when incubated with 0.4 to 0.5 mmol/L DB, and kinetic analysis showed that the high- affinity platelet-fibrinogen interactions were reduced but not absent. Fibrinogen binding to chymotrypsin-treated platelets could not be completely inhibited even at high DB concentrations (1 mmol/L). The inhibition of fibrinogen binding to chymotrypsin-treated platelets correlated with changes in binding of a monoclonal antibody (10E5) specific for an epitope on the GPIIb-IIIa complex. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and radioelectroimmunoassay of DB-treated platelets, however, showed no evidence of a reduction or degradation of GP IIb or IIIa. Platelet incubation with DB (five minutes, 0.1 to 1.0 mmol/L) was also accompanied by: increased platelet membrane-associated Ca2+ involving low-affinity binding sites [Kd = 5 X 10(-5) mol/L-]; increased 45Ca2+ uptake which correlated with degradation of actin-binding protein (ABP) and digestion of GPIb as visualized on periodic-acid Schiff (PAS)- stained SDS gels and as inferred from decreased binding of a monoclonal antibody (6D1) directed against this glycoprotein; and enhanced Ca2+ exchange. Thus, exposure of platelets to DB results in membrane-related alterations that may contribute to inhibition of platelet cohesion: Decreased fibrinogen receptor exposure by traditional agonists and diminished accessibility of the GPIIb-IIIa complex to extracellular ligands correlate with DB-induced inhibition of platelet aggregation; and increased calcium uptake and exchange across the platelet membrane likely leads to activation of the calcium-dependent protease(s) which was previously shown to correlate with DB-induced inhibition of ristocetin-induced platelet agglutination.

scholarly journals The role of ADP secretion and thromboxane synthesis in factor VIII binding to platelets

Blood ◽  
1983 ◽  
Vol 62 (1) ◽  
pp. 186-190
Author(s):  
G Di Minno ◽  
SS Shapiro ◽  
PM Catalano ◽  
L De Marco ◽  
S Murphy
Keyword(s):  
Arachidonic Acid ◽  
Factor Viii ◽  
Binding Sites ◽  
Human Platelet ◽  
Thromboxane A2 ◽  
Adenosine Diphosphate ◽  
Prostaglandin Endoperoxide ◽  
Platelet Thromboxane

Following stimulation with arachidonic acid, collagen, U-46619 (a stable analogue of prostaglandin endoperoxide/thromboxane-A2), thrombin, or adenosine diphosphate (ADP), unstirred human platelet suspensions bound labeled factor VIII in a reaction that reached equilibrium within 10 min. Apyrase inhibited binding induced by arachidonic acid, collagen, U-46619, and thrombin by less than 40%, but inhibited ADP-induced binding by 95%. Binding to aspirin-treated platelets was normal in response to U-46619, reduced by 60%-70% in response to ADP, collagen, and thrombin, and absent in response to arachidonic acid. Binding in response to U-46619 was not altered by the combination of apyrase and aspirin. Binding of factor VIII was decreased by 90% when 10 mM EDTA was added before each agonist, but it was inhibited less than 30% when EDTA was added following platelet stimulation. We conclude that arachidonic acid, collagen, and thrombin can expose binding sites for factor VIII independently of released ADP; that Ca++ is required for activation but probably not for binding of factor VIII to platelets; and that platelet thromboxane synthesis plays a major role in the binding of factor VIII to platelets induced by thrombin, ADP, or collagen.

Ontogeny of ACTH(1–24) receptors in rat adrenal glands during the perinatal period

1989 ◽  
Vol 123 (3) ◽  
pp. 421-428 ◽  
Author(s):  
A. Chatelain ◽  
P. Durand ◽  
E. Naaman ◽  
J. P. Dupouy
Keyword(s):  
Binding Sites ◽  
Adrenal Glands ◽  
Perinatal Period ◽  
Scatchard Analysis ◽  
Newborn Rats ◽  
Excellent Correlation ◽  
Temperature Dependent ◽  
Single Class ◽  
Binding Data ◽  
Cytoplasmic Structures

ABSTRACT Binding of ACTH to receptors was studied on crude adrenal membranes from fetal and newborn rats. 125I-Labelled ACTH(1–24) was used as the radioligand, the steroidogenic potency of which was 100-fold lower than that of unlabelled ACTH(1–24). Binding was specific, rapidly equilibrated and temperature dependent. Scatchard analysis of the binding data revealed a single class of binding sites with a dissociation constant of about 100 nmol/l at all stages of development studied. The concentration of ACTH receptors expressed per mg membrane proteins decreased in fetuses between days 17 and 21 of gestation and remained stable in newborn rats from weeks 1 to 4. The number of ACTH receptors expressed per adrenal increased regularly in fetal and newborn rats. The perinatal evolution of these concentrations of ACTH receptors is related to the increase in the size of the adrenals and the changes in cytoplasmic structures of the adrenocortical cells. When the number of ACTH-binding sites was expressed per μg DNA, maximum values occurred in fetuses on day 19 of gestation, and minimum values in newborn rats, 1 week after birth. There was an excellent correlation between the plasma levels of immunoreactive ACTH and corticosterone and the number of ACTH receptors per μg DNA during the perinatal period. Other results suggest that ACTH is able to up-regulate the number of its own receptors. Journal of Endocrinology (1989) 123, 421–428

scholarly journals A murine antiglycoprotein Ib complex monoclonal antibody, SZ 2, inhibits platelet aggregation induced by both ristocetin and collagen

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 570-577 ◽  
Author(s):  
CG Ruan ◽  
XP Du ◽  
XD Xi ◽  
PA Castaldi ◽  
MC Berndt
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Binding Sites ◽  
Human Platelet ◽  
Platelet Glycoprotein ◽  
Scatchard Analysis ◽  
Type I ◽  
Glycoprotein Ib ◽  
Induced Aggregation ◽  
Bernard Soulier Syndrome

A new monoclonal antibody (MoAb), SZ 2, reactive with the human platelet glycoprotein Ib complex has been produced by the hybridoma technique. SZ 2 immunoprecipitated the components of the glycoprotein Ib complex, glycoprotein Ib and glycoprotein IX, from Triton-X-100- solubilized, periodate-labeled platelets. Western blot analysis indicated that the epitope for SZ 2 was on the alpha-subunit of glycoprotein Ib. Scatchard analysis of SZ 2 binding to formaldehyde- fixed, washed platelets revealed a single class of binding sites with Kd = 6.6 +/- 3.3 X 10(-10) mol/L and 15,200 +/- 4,100 binding sites per platelet (mean +/- SD, n = 10). Intact antibody and its purified (Fab')2 fragments not only inhibited the ristocetin-dependent binding of von Willebrand factor to platelets and ristocetin-induced platelet agglutination but also inhibited platelet aggregation induced by Type I collagen and platelet-activating factor (PAF). SZ 2 inhibited platelet serotonin and beta-thromboglobulin release in response to these stimuli and also platelet thromboxane A2 formation in response to ristocetin and collagen. SZ 2 was without effect on platelet aggregation or release in response to other platelet stimuli such as ADP, thrombin, or arachidonic acid. The inhibition by SZ 2 of collagen- and PAF-induced platelet aggregation is surprising in that Bernard-Soulier syndrome platelets, which lack the glycoprotein Ib complex, respond normally to both these stimuli. SZ 2 was unreactive toward Bernard-Soulier syndrome platelets, as evaluated by fluorescence-associated cell sorting, and had no effect on the collagen- and PAF-induced aggregation of Bernard- Soulier syndrome platelets. The combined results suggest that the inhibition by SZ 2 of collagen- and PAF-induced aggregation of normal platelets is steric and are consistent with the glycoprotein Ib complex and the platelet collagen and PAF receptor(s) being adjacent in the human platelet plasma membrane.

scholarly journals The role of ADP secretion and thromboxane synthesis in factor VIII binding to platelets

Blood ◽  
1983 ◽  
Vol 62 (1) ◽  
pp. 186-190 ◽  
Author(s):  
G Di Minno ◽  
SS Shapiro ◽  
PM Catalano ◽  
L De Marco ◽  
S Murphy
Keyword(s):  
Arachidonic Acid ◽  
Factor Viii ◽  
Binding Sites ◽  
Human Platelet ◽  
Thromboxane A2 ◽  
Adenosine Diphosphate ◽  
Prostaglandin Endoperoxide ◽  
Platelet Thromboxane

Abstract Following stimulation with arachidonic acid, collagen, U-46619 (a stable analogue of prostaglandin endoperoxide/thromboxane-A2), thrombin, or adenosine diphosphate (ADP), unstirred human platelet suspensions bound labeled factor VIII in a reaction that reached equilibrium within 10 min. Apyrase inhibited binding induced by arachidonic acid, collagen, U-46619, and thrombin by less than 40%, but inhibited ADP-induced binding by 95%. Binding to aspirin-treated platelets was normal in response to U-46619, reduced by 60%-70% in response to ADP, collagen, and thrombin, and absent in response to arachidonic acid. Binding in response to U-46619 was not altered by the combination of apyrase and aspirin. Binding of factor VIII was decreased by 90% when 10 mM EDTA was added before each agonist, but it was inhibited less than 30% when EDTA was added following platelet stimulation. We conclude that arachidonic acid, collagen, and thrombin can expose binding sites for factor VIII independently of released ADP; that Ca++ is required for activation but probably not for binding of factor VIII to platelets; and that platelet thromboxane synthesis plays a major role in the binding of factor VIII to platelets induced by thrombin, ADP, or collagen.

Binding of Recombinant Apolipoprotein(a) to Human Platelets and Effect on Platelet Aggregation

2001 ◽  
Vol 85 (04) ◽  
pp. 686-693 ◽  
Author(s):  
C. Martínez ◽  
J. Rivera ◽  
S. Loyau ◽  
J. Corral ◽  
R. González-Conejero ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Binding Sites ◽  
Human Platelets ◽  
Scatchard Analysis ◽  
High Concentration ◽  
Single Class ◽  
Apo B ◽  
Platelet Surface ◽  
The Individual

SummaryThe interaction of lipoprotein(a) [Lp(a)] with platelets is not well defined, particularly with regards to the individual contribution of the protein components of Lp(a), the apo B-100 and the apolipoprotein apo(a). This study investigated the binding of different recombinant apo(a) [r-apo(a)] isoforms, to human platelets and its effect on platelet aggregation. Scatchard analysis of saturation binding experiments demonstrated that human platelets display a single class of high affinity r-apo(a) binding sites (71 ± 46 molec./platelet, Kd = 5.6 ± 2.0 nmol/L). Platelet activation with strong agonists (thrombin, arachidonic acid) increased 2- to 10-fold the r-apo(a) binding, without affecting the affinity. Competition assays showed that the binding sites are highly specific for r-apo(a) and Lp(a). At high concentration t-PA could also bind to the r-apo(a) binding sites. By contrast, neither fibrinogen nor plasminogen inhibited to the r-apo(a) binding. The lysine analogue EACA inhibits the binding of r-apo(a) to platelets, thus suggesting the involvement of lysine residues in that interaction. Moreover, the r-apo(a) binding to platelets is unlikely mediated by GPIIb/IIIa-attached fibrin since it is not affected by platelet treatment with either LJ-CP8, a monoclonal antibody that specifically blocks fibrinogen binding to GPIIb/IIIa, nor GPRP, an inhibitor of fibrin polymerisation. Finally, we show that the distinct recombinant apo(a) proteins, as well as native Lp(a), promote an aggregation response of platelets to otherwise subaggregant doses of arachidonic acid. This proaggregant effect of r-apo(a) is dependent on its binding to platelets since it requires a minimum incubation time, and it is prevented by EACA at concentration inhibiting the r-apo(a)-platelet interaction.These results suggest that the prothrombotic action of Lp(a) may be in part mediated by modulating the platelet function through the interaction of its apo(a) subunit with a specific receptor at the platelet surface.

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