Washed human platelets bound radioiodinated low density lipoprotein ( 125 I-LDL) to a class of saturable binding sites; they numbered 1,348±126 per platelet, and the dissociation constant (K D ) was 50.7±9 nM. I25I-LDL binding to platelets was reversible, and apparent equilibrium was attained within 25 minutes at 22°C and was characterized by forward and reverse rate constants of 1.47xlO4 Xsec"'xM" 1 and 8xlO~4xsec~' xM" 1 , respectively. Such binding was largely unaltered by temperature, divalent ions, and chelating agents. In addition, neither did receptor regulation (up or down) occur when platelets were loaded with cholesterol, nor did prostaglandin E, (PGE,) increase the binding of 12S I-LDL to platelets. On the other hand, the specificity of LDL binding was not typical of the LDL receptor of nucleated cells. Lipoproteins competed for the occupancy of LDL binding sites in platelets with the following order of potency: very low density >> intermediate density > high density subfraction 2. High density lipoprotein subfraction 3, heparin, and PGE, had no effect on this binding.

We have recently demonstrated that the platelet low-density lipoprotein (LDL) receptor is immunologically different from the "classic" receptor of nucleated cells. We undertook the current studies to investigate the interaction of this receptor with oxidized LDL and to determine whether an endocytosis-mediated response is involved in the binding of LDL to platelets. The platelet LDL receptor recognized with the same affinity both native and oxidized LDL particles (IQo, 0.045 and 0.054 g/L; K a , 45.8 and 65.9 nmol/L, respectively). The Hill coefficients of the displacement of t is widely accepted that platelets are implicated in the initiation of atherosclerotic lesions and that they play a major role in the later thrombotic complications that are expressed as clinical evidence of the disease process.12 Moreover, the evidence, both clinical and experimental, leaves no doubt that lipoproteins are important in the development of atherosclerotic lesions. oxidized LDL at a protein concentration of 0.5 g/L enhanced ADP-and collagen-induced platelet aggregation in a manner similar to native LDL. We present ample evidence to show that LDL binding to platelets is not mediated by an endocytotic process. First, dissociation studies showed that at 4°C, 22°C, and 37°C approximately 93% of the surface-bound LDL was dissociated and that in the absence and presence of an ATPase inhibitor such as sodium azide, similar values for the reverse rate constant (K_ u 6±2 and 8.9±3xlO~4-s~' • mol/L" 1 ) and half-time (25±5 and 30±4 minutes) were found. Second, the lack of a blocking effect on LDL binding to platelets of microtubule inhibitors (colchicine), acidotropic agents (ammonium chloride and chloroquine), and Golgi apparatus disrupters (monensin) was found. Third, the inability of platelets to degrade IM I-LDL was shown; finally, imipramine and dopamine, which fully prevented platelet [

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It has been suggested that the fibrinogen receptor (glycoprotein [GP] IIb-IIIa or platelet integrin alpha IIb beta 3) 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 alpha IIb beta 3 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 (Kd) of 48.3 +/- 12 x 10(-9) mol/L. These values were similar to those obtained for LDL. In contrast to Lp(a), evidence indicates that platelet integrin alpha IIb beta 3 was not involved in the interaction of LDL and intact resting platelets. First, specific ligands for platelet integrin alpha IIb beta 3, such as fibrinogen, vitronectin, and fibronectin, were unable to inhibit the binding of LDL to intact resting platelets. Second, similar LDL binding characteristics (Kd and Bmax 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-Baka/B and anti-PLA1/2), anti-integrin subunits (anti-alpha v and anti-beta 3), 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.

In this work human platelet aggregation induced in vitro by ADP, collagen, arachidonic acid and U-46619 (a thromboxane A(2) analogue) was used as a functional test to characterize 19 anti-GPIIb (M series) and anti2 GPIIIa (P series) monoclonal antibodies whose epitope location is known for most of them. Additionally, flow cytofluorimetry was applied to study the epitope expression of these antibodies in resting, EDTA-treated and SFLLRN peptide (thrombin receptor agonist)-activated platelets. Antibodies M6 (epitope located at GPIIbH 657-665), P23-7 (GPIIIa 114-122) and P40 (GPIIIa 262-303) bind weakly to only 43%, 70% and 66%, respectively, of the resting platelet population. This binding was enhanced in EDTA-treated and in activated platelets. Platelet activation enhances the apparent binding of most of the other antibodies. Further evidence on the existence of agonist-specific activated states of GPIIb/IIIa was provided by the agonist-dependent immunochemical inhibition in vitro of platelet aggregation by some of the anti-subunit antibodies studied here. The most notable cases are those of P40 and M6, which at 140 nM inhibit most, the platelet aggregation induced by arachidonic acid and U-46619. On the other hand, three of the most strong and agonist-independent inhibitors, P37 (GPIIIa 101-109), P97 and P95-2 (GPIIIa N-terminal half) bind to resting platelets with high affinity (5-8 nM), compete with each other for binding to GPIIb-IIIa and their epitopes are located at the N-terminal domain of GPIIIa, where the receptor ligand binding site(s) have been found. Given that the formation of activated GPIIb-IIIa (GPIIb-IIIa*) is the first step at which the anti-subunit antibodies can intervene as inhibitors and that agonist-specific inhibitors should block only agonist-specific steps, while nonspecific inhibitors should block steps common to all the agonists, then our present work support the hypothesis that there are different agonist-specific GPIIb-IIIa*s or, alternatively, different receptor environments, that can be specifically blocked by some of the antibodies. These results add to earlier evidence on agonist-dependent ligand specificity and activated states found for this and other integrins. Finally, the correlation between the in vitro inhibition of platelet aggregation and the antithrombotic activity in vivo is discussed for these antibodies.

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SummaryVascular prostacyclin (PGI2) production is different in the arteries and veins of the dog. Experiments were performed to determine whether chronic grafting of the femoral vein into the arterial circulation would alter the normal PGI2 and thromboxane (TxA2) synthesis of the “arterialized” veins. Spontaneous and arachidonic acid (AA) stimulated PGI2 and TxA2 production (measured by radioimmunoassay of 6-keto PGF1α and TxB2 respectively) were analysed in full thickness punch biopsies of the middle part of the grafts after 3 and 16 months and compared with unoperated veins and arteries. PGI2 production was significantly higher in arteries than in veins but no significant difference in TxB2 production was found. Middle “arterialized” venous graft produced significantly lower amounts of PGI2 and higher amounts of TxB2 than unoperated vessels. PGI2 production was more reduced in the distal than in the middle or the proximal parts of the venous grafts especially when stimulated with AA. These findings do not support the concept that the venous graft was biochemically adapted or “arterialized” in terms of PGI2 production when implanted for 3 months or longer. Rather the markedly decreased PGI2/TxB2 ratio in the middle of the graft may be a contributory cause of thrombogenicity and may be implicated in the pathogenesis of neointimal hyperplasia.