Cyclooxygenase activity is increased in platelets from psoriatic patients.

The aim of the present study was to ascertain the relationship between in vitro hyper-aggregability and alterations in arachidonic acid metabolism in platelets from psoriatic patients. We have studied the response to several concentrations of ADP, collagen, and arachidonic acid of intact platelets from psoriatic patients and normal subjects, with and without irreversible inhibition of platelet cyclooxygenase by aspirin. Apparent kinetic constants (apparent Michaelis constant [Km] and apparent maximum velocity [Vmax]) of cyclooxygenase in platelets from both controls and psoriatic patients were also studied. The maximum percentage and slope of aggregation induced by collagen or sodium arachidonate were significantly greater (p less than 0.05) in platelets from psoriasis patients, whereas lag time was significantly shorter in the psoriasis group in response to arachidonic acid only when compared to controls. Cyclooxygenase pathway blockade inhibited the response to aggregation inducers in the following order: sodium arachidonate greater than collagen greater than ADP. When platelets were pre-treated with aspirin no significant differences were observed between controls and psoriasis patients. We also found a significant increase of the apparent Vmax value (p less than 0.05) for cyclooxygenase activity in platelets from psoriatic patients in comparison with controls. Our results indicate that platelet hyperaggregation in psoriatic patients is related to enhanced cyclooxygenase activity in their platelets.

Prostacyclin and thromboxane production by autogenous femoral veins grafted into the arterial circulation of the dog.

Vascular 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 PGF1 alpha 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.

Platelet integrin alpha IIb beta 3 (GPIIb-IIIa) is not implicated in the binding of LDL to intact resting platelets.

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.

Functional characterization of GPIIb- and GPIIIa-specific monoclonal antibodies. Further evidence for the existence of agonist-specific activated states of the platelet fibrinogen receptor.

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.

LDL binding sites on platelets differ from the "classical" receptor of nucleated cells.

Washed human platelets bound radioiodinated low density lipoprotein (125I-LDL) to a class of saturable binding sites; they numbered 1,348 +/- 126 per platelet, and the dissociation constant (KD) was 50.7 +/- 9 nM. 125I-LDL binding to platelets was reversible, and apparent equilibrium was attained within 25 minutes at 22 degrees C and was characterized by forward and reverse rate constants of 1.47 x 10(4) x sec-1 x M-1 and 8 x 10(-4) x sec-1 x M-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 E1 (PGE1) increase the binding of 125I-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 PGE1 had no effect on this binding. 125I-LDL binding to lymphocytes and fibroblasts and proteolytic degradation of 125I-LDL by lymphocytes was inhibited by the monoclonal antibody IgG-C7 directed against the LDL receptor to 88%, 85%, and 85% (p < 0.001), respectively. However, with this monoclonal antibody, a blocking effect on neither 125I-LDL binding to platelets nor on LDL-enhanced platelet aggregation induced by ADP and collagen was found. Moreover, we confirmed the existence of LDL binding in platelets from patients with familial hypercholesterolemia. Our results indicate that human platelets bind LDL by saturable sites, which clearly differ from the "classical" LDL receptor in their binding properties, absence of receptor regulation, presence in platelets of familial hypercholesterolemia patients, and the lack of a blocking effect of IgG-C7 on LDL binding and LDL biological activity.

Platelet LDL receptor recognizes with the same apparent affinity both oxidized and native LDL. Evidence that the receptor-ligand complexes are not internalized.

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 (IC50, 0.045 and 0.054 g/L; Kd, 45.8 and 65.9 nmol/L, respectively). The Hill coefficients of the displacement of 125I-LDL binding were -1.10 and -1.05 for unlabeled native and oxidized LDL, respectively, thereby suggesting a single set of binding sites. To ascertain whether human platelets bind oxidized LDL, we performed ligand binding assays with 125I-oxidized LDL. Saturation curves of 125I-oxidized LDL binding at 22 degrees C showed that human platelets bound these modified particles to a class of saturable binding sites, numbering approximately 3895 +/- 241 per platelet with a dissociation constant (Kd) of 96.2 +/- 10.3 nmol/L. Displacement experiments showed that 125I-oxidized LDL binding was inhibited with the same affinity by both oxidized and native LDL (IC50, 0.055 and 0.065 g/L; Kd, 88 and 64 nmol/L, respectively). The Hill coefficients of the displacement of the 125I-oxidized LDL binding were -1.02 and -1.07 for unlabeled oxidized and native LDL, respectively, suggesting that a single set of binding sites is implicated. Moreover, 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.(ABSTRACT TRUNCATED AT 250 WORDS)