Presence of Epstein-Barr virus (EBV) antigens detected by sensitive methods has no influence on local immune environment in diffuse large B cell lymphoma.

EBV+ diffuse large B cell lymphoma (DLBCL) not otherwise specified (NOS) is a new entity confirmed by the World Health Organization (WHO) in 2017. In this new entity, the virus may contribute to a tolerogenic microenvironment. Traces of the virus have been described in DLBCL with more sensitive methods, in cases that were originally diagnosed as negative. The aim of this study was to analyze the expression of immune response genes in the tumor microenvironment to disclose the role of the virus and its traces in DLBCL. In 48 DLBCL cases, the expression of immune response genes and the presence of molecules that induce tolerance, such as TIM3, LAG3 and PDL1 by immunohistochemistry (IHC), were studied. To broaden the study of the microenvironment, tumor-associated macrophages (TMAs) were also explored. No significant differences were observed in the expression of immune response genes in the EBV+ DLBCL and those cases that were EBV- DLBCL but that exhibited viral traces, assessed by ViewRNA assay. Only the EBV+ DLBCL cases displayed a significantly higher increase in the expression of CD8 and cytotoxic T cells detected by gene expression analysis, and of PDL1 in tumor cells and in the expression of CD68 in the tumor microenvironment detected by IHC, not observed in those cases with viral traces. The increase in CD8 and cytotoxic T cells, PDL1 and CD68 markers only in EBV+ DLBCL may indicate that traces of viral infection might not have influence in immune response markers.

Epstein-Barr virus recruits PDL1-positive cells at the microenvironment in pediatric Hodgkin lymphoma.

BACKGROUND: Classic Hodgkin lymphoma (cHL) is a lymphoid malignancy in which the microenvironment, where the neoplastic cells are immersed, contributes to the lymphomagenesis process. Epstein-Barr virus (EBV) presence also influences cHL microenvironment composition and contributes to pathogenesis. An increase in PDL1 expression in tumor cells and at the microenvironment was demonstrated in adult cHL. Therefore, our aim was to assess PD1/PDL1 pathway and EBV influence on this pathway in pediatric cHL, given that in Argentina, our group proved a higher incidence of EBV-associated pediatric lymphoma in children. METHODS: For that purpose, EBV presence was assessed by in situ hybridization, whereas PD1 and PDL1 expressions were studied by immunohistochemistry. PDL1 genetic alterations were analyzed by FISH, and survival was evaluated for PD1 and PDL1 expressions. RESULTS: EBV presence demonstrated no influence neither on PD1 expression at the microenvironment nor on PDL1 expression at HRS tumor cells. Unexpectedly, only 38% pediatric cHL displayed PDL1 genetic alterations by FISH, and no difference was observed regarding EBV presence. However, in EBV-related cHL cases, a higher number of PDL1 + cells were detected at the microenvironment. CONCLUSION: Even though a high cytotoxic environment was previously described in EBV-related pediatric cHL, it might be counterbalanced by an immunoregulatory micro-environmental PDL1 + niche. This regulation may render a cytotoxic milieu that unsuccessfully try to eliminate EBV + Hodgkin Reed Sternberg tumor cells in pediatric patients.

Inhibition of transglutaminase 2 reduces efferocytosis in human macrophages: Role of CD14 and SR-AI receptors.

BACKGROUND AND AIMS: Transglutaminase 2 (TGM2), a member of the transglutaminase family of enzymes, is a multifunctional protein involved in numerous events spanning from cell differentiation, to signal transduction, apoptosis, and wound healing. It is expressed in a variety of cells, macrophages included. Macrophage TGM2 promotes the clearance of apoptotic cells (efferocytosis) and emerging evidence suggests that defective efferocytosis contributes to the consequences of inflammation-associated diseases, including atherosclerotic lesion progression and its sequelae. Of interest, active TGM2 identified in human atherosclerotic lesions plays critical roles in plaque stability through effects on matrix cross-linking and TGFß activity. This study explores the mechanisms by which TGM2 controls efferocytosis in human macrophages. METHODS AND RESULTS: Herein we show that TGM2 increases progressively during monocyte differentiation towards macrophages and controls their efferocytic potential as well as morphology and viability. Two experimental approaches that took advantage of the inhibition of TGM2 activity and protein silencing give proof that TGM2 reduction significantly impairs macrophage efferocytosis. Among the mechanisms involved we highlighted a role of the receptors CD14 and SR-AI whose levels were markedly reduced by TGM2 inhibition. Conversely, CD36 receptor and αvß3 integrin levels were not influenced. Of note, lipid accumulation and IL-10 secretion were reduced in macrophages displaying defective efferocytosis. CONCLUSION: Overall, our data define a crucial role of TGM2 activity during macrophage differentiation via mechanisms involving CD14 and SR-AI receptors and show that TGM2 inhibition triggers a pro-inflammatory phenotype.

Data for proteomic analysis of Human monocyte-derived macrophages.

This data article is referred to the research article entitled Human monocyte-derived macrophages are heterogeneous: proteomic profile of different phenotypes by Eligini et al. Eligini S., Brioschi M., Fiorelli S., Tremoli E., Banfi C., Colli S. Human monocyte-derived macrophages are heterogeneous: proteomic profile of different phenotypes. J. Proteomics 124, 2015, 112-123. Macrophages obtained in vitro from blood monocytes are largely used as surrogate model of tissue macrophages that are heterogeneous and not easy to obtain and handle. Under spontaneous differentiation in vitro, monocyte-derived macrophages (MDMs) display two dominant subsets (round and spindle) that show different transcriptional, antigenic, and functional profiles mimicking, at least in part, the heterogeneity of tissue macrophages. This article reports the nano-LC-MS(E) analysis of the proteome of round and spindle MDMs allowing a deeper comprehension of macrophage heterogeneity.

Human monocyte-derived macrophages are heterogenous: Proteomic profile of different phenotypes.

Tissue macrophages play a key role in many aspects of human physiology and pathology. These cells are heterogeneous both in term of morphology and function. As an example, heterogeneity has been reported within the atherosclerotic lesions where distinct populations exert opposite functions driving plaque progression or stability. Tissue macrophages are not easily obtained and differentiated blood-derived monocytes are largely used as surrogate model. We previously reported that human macrophages spontaneously differentiated from adherent monocytes show two dominant subsets, distinct for morphology (spindle and round) and functions. The aim of this study was to evaluate the intracellular proteome of these two macrophage subsets by means of a microproteomic workflow properly set up to simultaneously identify and quantify proteins from a minimal number of morphotypically heterogeneous cells in culture. We report two distinct proteomic profiles that distinguish round from spindle macrophages. In particular, differential abundances were observed for proteins involved in membrane traffic regulation, lipid handling, efferocytosis, and protection against stress conditions. Results reinforce and extend previous data on the functional and antigenic profile of these macrophage phenotypes strengthening the suitability of our model to focus on macrophage heterogeneity. BIOLOGICAL SIGNIFICANCE: Tissue macrophages patrol homeostatic functions, immune surveillance, and resolution of inflammation. The spectrum of macrophage activation states is, therefore, wide and gives ground for the heterogeneity of these cells, documented in health and disease. This study provides knowledge of the distinct proteome that characterises the two dominant morphotypes (round and spindle) of human macrophages that, in our culture condition, are generated by spontaneous differentiation from blood-derived monocytes. Results extend previous data about the different antigenic, transcriptional, and functional profiles of these morphotypes and further strengthen the suitability of this in vitro model to study macrophage heterogeneity and to address the effects of environmental challenges and drugs.

Mitochondrial reactive oxygen species: a common pathway for PAR1- and PAR2-mediated tissue factor induction in human endothelial cells.

BACKGROUND: Protease-activated receptors (PARs) comprise a family of G-protein-coupled receptors with a unique proteolytic activation mechanism. PARs regulate a broad range of cellular functions and are involved in the pathogenesis of inflammatory disorders. Moreover, PAR1 and PAR2 activation in the endothelium shifts it toward a prothrombotic condition. OBJECTIVES: To assess the relevance of intracellular reactive oxygen species (ROS) in the signaling events underlying tissue factor (TF) expression elicited by PAR1 and PAR2 occupancy in endothelial cells, and to investigate their source. METHODS: Human umbilical vein endothelial cells (HUVEC) were exposed to specific PAR1 and PAR2 agonist peptides. TF expression was determined by real-time reverse transcription polymerase chain reaction analysis and measurement of procoagulant activity. ROS generation was determined by a fluorometric assay after cell loading with 2'-7'-dichlorofluorescein diacetate. RESULTS: ROS generated by the mitochondrial chain, mostly from complex III, provide a pathway through which PAR1 and PAR2 occupancy induces TF. Other sources of ROS do not participate in TF induction. Activation of both ERK1/2 and p38 MAPK is critical for mitochondrial ROS generation. In addition to these pathways shared by the two PARs, mechanisms downstream from PAR1 and PAR2 activation, different for the two receptors, also induced TF. A module that sensitively regulates PAR1 signaling and ultimately involves NF-kappaB activation has been identified. CONCLUSIONS: Our data identify ROS originating in mitochondria as key mediators of the signaling pathways triggered by PAR1 and PAR2 engagement in endothelial cells and show that downstream from receptor activation occur cascades that are mechanistically coupled to procoagulant activity.

Fatty acid composition of plasma, blood cells and whole blood: relevance for the assessment of the fatty acid status in humans.

UNLABELLED: The composition and incorporation of fatty acids (FA) in plasma and blood cells is the result of distinct processes: intake, metabolism and peripheral utilization. AIM OF THE STUDY: was to compare the FA profile in plasma, lipoproteins and blood cells with that in whole blood (WB) from healthy volunteers; to assess the quantitative distribution of selected FA in triacylglycerols, cholesteryl esters and phospholipids. Lipid FA profiles are comparable in plasma and lipoproteins but differ from those in blood cells. In WB, the FA profile results from the balanced proportion of FA pools in plasma and cells. The contribution of each lipid class to the total amount of FA differs among blood specimens. Phospholipids of plasma and red blood cell are the major contributors to the FA amount and profile in WB. In conclusion, the FA profile of WB reflects the FA status and WB could be an adequate specimen for the assessment of FA intakes.

15-deoxy-delta12,14-Prostaglandin J2 inhibits tissue factor expression in human macrophages and endothelial cells: evidence for ERK1/2 signaling pathway blockade.

Basic and clinical evidence has provided insight into the molecular events that link inflammation and coagulation. Increased expression of tissue factor (TF) by circulating and vascular cells has been indicated as responsible for the thrombotic complications associated with acute and chronic inflammation. TF is indeed inducible in circulating and vascular cells by cytokines and bacterial lipopolysaccharide (LPS) and its expression triggers the coagulation. The cyclopentenone prostaglandins are naturally occurring prostaglandin D2 (PGD2) derivatives that comprises prostaglandin J2 (PGJ2) and its metabolites delta12-PGJ2 and 15-deoxy- delta12,14-prostaglandin J2 (15d-PGJ2). These compounds, detected in vivo in a later phase of the inflammatory response, are characterized by anti-inflammatory activity and participate to the resolution of inflammation. We have here investigated the effect of 15d-PGJ2 on TF expression in human macrophages and endothelial cells (HUVEC). Our results indicate that 15d-PGJ2 down-regulates LPS- and TNFalpha-induced TF activity, protein and mRNA through inhibition of TF gene transcription. The effect of 15d-PGJ2 is targeted to the NF-kappaB/I-kappaB pathway and to the mitogen activated protein kinase ERK1/2. A role of PPAR-gamma activation in TF inhibition by 15d-PGJ2 was excluded. We conclude that 15d-PGJ2 negatively affects TF expression in macrophages and endothelial cells through a PPARgamma-independent mechanism. This down-regulation may be crucial to limit excessive blood clotting activation in immuno-inflammatory diseases.

Oxidized LDLs influence thrombotic response and cyclooxygenase 2.

Oxidative modification of low-density lipoproteins (LDLs) plays a key role in the development of atherosclerosis and the onset of coronary artery disease. LDL oxidation alters the antithrombotic balance of human endothelial cells inducing surface tissue factor (TF) pathway activity, which results in enhanced fibrin deposition. Fibrinolysis, which is strictly regulated by plasminogen activator inhibitor-1 (PAL-1) and tissue-type plasminogen activator (tPA). Is also dysregulated by LDL oxidation with a net increase in the inhibitory rate. Oxidized LDLs (oxLDLs) also affect many aspects of macrophage function linked to the inflammatory response of these cells, In particular, oxLDLs downregulate inducible cyclooxigenase (Cox-2) in human monocyte-derived macrophages exposed to bacterial lipopolysaccharide. This observation may support the hypothesis that, within atheromata, the transformation macrophages into foam cells results in the attenuation of the inflammatory response, thus contributing to the progression of athrogenesis. Among lipid constituents of oxLDLs, Ox-PAPC, a mixture of oxidized arachidonic acid-containing phospholipids, prevents Cox-2 expression, suggesting that it could be considered responsible for the biological activity of oxLDLs.

In vitro measurement of platelet glycoprotein IIb/IIIa receptor blockade by abciximab: interindividual variation and increased platelet secretion.

BACKGROUND AND OBJECTIVES: Inhibition of soluble fibrinogen binding to activated platelets represents the target of pharmacologic approach with antagonists of the glycoprotein IIb/IIIa (GPIIb/IIIa) complex. In this study we assessed the effects of abciximab, a recombinant chimeric Fab fraction of the antibody against GPIIb/IIIa, on several markers of platelet activation. DESIGN AND METHODS: The platelet surface expression of GPIIb/IIIa was measured by a flow cytometry technique using a two-color assay. GPIIb/IIIa was detected by FITC-conjugated antibodies in whole blood, either unstimulated or exposed to platelet stimuli. The following antibodies were used: CD41, which recognizes the IIb/IIIa complex both in activated and non-activated conformers, and PAC-1, which is directed toward the activated conformer of GPIIb/IIIa. In addition, the same blood sample was incubated with CD62 antibody to measure P-selectin, as a marker of a-granule degranulation. The effect of abciximab was also assessed by experiments carried out on shear stress-induced platelet aggregation, a test that appears to be a predictor of platelet hemostatic function. RESULTS: Abciximab inhibited CD41 binding to glycoprotein IIb (GPIIb) in a concentration-dependent manner and also inhibited the binding of PAC-1 to active GPIIb/IIIa. In contrast, membrane-associated P-selectin was significantly increased by the drug, which suggests that blockade of GPIIb/IIIa receptors results in an increased platelet degranulation in response to agonists. Shear stress-induced platelet aggregation was inhibited by abciximab, with a more pronounced effect on blood filtration, which represents an index of platelet aggregate formation. INTERPRETATION AND CONCLUSIONS: Our results indicate that GPIIb/IIIa blockade by abciximab is accompanied by an increase of a-granule secretion, suggesting that different mechanisms regulate these aspects of platelet activation. The described flow cytometry technique, that allows the simultaneous in vitro detection of several platelet markers, is a suitable method for assessing the effects of agents which interfere with platelet function.

Magnesium inhibits arterial thrombi after vascular injury in rat: in vivo impairment of coagulation.

Magnesium deficiency is associated with a high frequency of cardiac arrhythmia, hypertension and sudden ischemic death. We investigated the if vivo effects of intravenous magnesium administration in a rat model of chemically induced (FeCl3) carotid thrombosis. The infusion of magnesium sulfate (MgSO4) before the topical application of FeCl5 prevented thrombus formation at concentrations of 0.3 M and 0.6 M, and delayed it even at 0.15 M. Similar results were obtained with MgCl2. The infusion of MgSO4 0.6 M seven minutes after FeCl3 application delayed but did not prevent thrombus formation. MgSO4 slightly reduced platelet aggregation ex vivo without affecting plasma clotting tests, but in vivo blood clotting time was markedly prolonged (tail transection method), thus indicating profoundly impaired coagulation. These data provide a rationale for the use of magnesium as an antithrombotic agent. but its pharmacological effect critically depends on the timing of administration.

Synthesis and anti-aggregatory activity of linear retro-inverso RGD peptides.

Six retro-inverso tri- and tetrapeptide analogues of RGD were prepared and their anti-aggregatory activity was determined by platelet aggregation tests in comparison with the corresponding parent peptides. An efficient method for the introduction of a malonyl-aspartic residue into a peptide chain is described for the first time. A 2-3-fold decrease in potency or total loss of bioactivity was observed with the new peptides; structure-activity relationships are discussed.

Oxidized low density lipoprotein suppresses expression of inducible cyclooxygenase in human macrophages.

Atherogenesis involves several aspects of chronic inflammation and wound healing. Indeed, the atheroma is considered a special case of tissue response to injury. Injurious stimuli may include lipoproteins trapped within lesions where protein and lipid moieties have undergone chemical modifications. We have studied the effect of oxidized low density lipoproteins (ox-LDL) on inducible cyclooxygenase (Cox-2) in human monocyte-derived macrophages exposed to bacterial lipopolysaccharide (LPS). Levels of both Cox-2 and constitutive cyclooxygenase (Cox-1) were assessed using Western blot analysis. Prior incubation of macrophages with ox-LDL resulted in a strong inhibition of Cox-2 induced by LPS, without effect on Cox-1. The inhibitory effect was dependent on ox-LDL concentration and its onset was early in time (already detectable 1 hour after macrophage exposure to ox-LDL). Native LDL, and other forms of modified LDL, were without effect. The inhibition was dependent on endocytosis of ox-LDL and could be reproduced using the lipid extract from ox-LDL. Lysophosphatidylcholine, 7beta-hydroxycholesterol, and 7-oxocholesterol failed to mimic the inhibition, but oxidized arachidonic acid-containing phospholipids, produced by autoxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, markedly inhibited Cox-2. The observation that ox-LDL downregulates Cox-2 in human macrophages may explain the fact that, within atheromata, the transformation of macrophages into foam cells results in attenuation of the inflammatory response, thus contributing to progression of atherogenesis.

Platelet function and anesthetics in cardiac surgery: an in vitro and ex vivo study.

UNLABELLED: We studied the effects of the anesthetics commonly used in cardiac surgery on platelet function. Fentanyl, droperidol, succinylcholine, pancuronium, thiopental, and diazepam at therapeutic concentrations were tested for their in vitro effects on the expression of platelet membrane glycoproteins Ib and IIbIIIa (GpIb, GpIIb-IIIa) and of P-selectin in anticoagulated whole blood by flow cytometry. The expression of P-selectin was determined under basal conditions, after the incubation of blood with adenosine diphosphate (ADP) 10 micromol/L, and the stable prostaglandin endoperoxide analog U46619 1 micromol/L. No drug affected the expression of P-selectin in unstimulated and ADP- or U46619-stimulated platelets, with the exception of thiopental, which markedly decreased the U46619-induced expression of P-selectin. Thiopental concentration-dependently inhibited U46619-induced and ADP-induced platelet aggregation, with effects on U46619-induced aggregation at therapeutic concentrations. To assess ex vivo effects, the same platelet markers were also assessed in blood obtained from 10 patients undergoing elective coronary surgery. Compared with basal values, platelet response to U46619 was significantly reduced just after the administration of anesthetic drugs, and the effect persisted for 48 h after surgery. Our study suggests that, at therapeutic concentrations, thiopental inhibits U46619-induced platelet activation both in vitro and ex vivo. The mechanisms responsible of this effect, together with its clinical significance, require further investigation. IMPLICATIONS: Thiopental inhibited prostaglandin-induced platelet activation at therapeutic concentrations both in vitro and ex vivo in cardiac surgical patients whereas adenosine diphosphate-induced activation was affected only at supratherapeutic drug concentrations. Thus, administration of sodium thiopental may contribute to the in vivo impairment of platelet function in patients undergoing elective cardiac surgery.

Tissue factor in atherosclerosis.

Thrombosis is a key feature of the initiation and progression of atherosclerosis and its clinical sequelae. Acute thrombosis can lead to arterial occlusion and consequently provoke myocardial infarction, unstable angina, stroke and sudden death. Acute thrombosis can also be a complication of arterial bypass surgery, balloon angioplasty, atherectomy, or coronary artery stenting. The thrombotic response is influenced by several factors, among them the thrombogenicity of the vessel wall and of certain blood components as well as their interaction with the lipid pool. Tissue factor (TF) is considered to be the primary cofactor of cellular origin that is involved in activation of the coagulation pathway. The active form of TF has been shown to be present in specimens of human coronary artery in association both with acellular lipid areas and with macrophages and smooth muscle cells, which suggests that TF plays a major role in determining plaque thrombogenicity. We discuss here what is currently known about the role of tissue factor in atherogenesis, and focus attention on pharmacological approaches in this area.

Increased thrombogenic potential of human monocyte-derived macrophages spontaneously transformed into foam cells.

This study investigated whether spontaneous lipid enrichment of human macrophages affects their thrombogenic potential as measured by increased production of tissue factor (TF) and plasminogen activation inhibitor types 1 and 2 (PAI-1 and PAI-2). Macrophages were obtained following a 7-day culture period of monocytes, isolated from the same donor, in autologous serum (HS) or in fetal bovine serum (FBS). Those cultured in HS underwent marked lipid accumulation relative to those cultured in FBS that was accompanied by increased production of TF and PAI-1, but not of PAI-2, and decreased production of interleukin-1beta. They also contained more arachidonic and linoleic acid and lower amounts of n-3 polyunsaturated fatty acids, particularly docosahexaenoic acid (22: 6). These data indicate that the transformation of macrophages into foam cells results in an increase in their thrombogenic and antifibrinolytic potential and provide a possible explanation of the thrombotic sequelae frequently consequent on plaque fissuring and disruption.

Endothelial tissue factor induction by T lymphocytes in systemic sclerosis.

We evaluated the ability of circulating T lymphocytes obtained from patients with systemic sclerosis (SSc) to induce the expression of tissue factor (TF) by human umbilical vein endothelial cells (HUVECs), and compared the results with those obtained from healthy controls. Nine patients with SSc and 10 sex- and age-matched healthy subjects were studied. Peripheral T lymphocytes obtained from SSc patients induced TF activity from HUVECs in a dose-dependent manner. A significant induction of endothelial TF was observed when 2 x 10(6) lymphocytes per well (TF values, mean+/-SD: 0.34+0.21U/microg of cell protein vs 0.04+/-0.03; n = 9, p = 0.001) or 1 x 10(6) lymphocytes per well (0.13+/-0.06 vs 0.04+/-0.04; n = 8, p<0.001) were added to HUVEC cultures. Lower concentrations of T lymphocytes were ineffective. Similar results were obtained with control lymphocytes. There were no differences in endothelial TF induction between patients and controls at any lymphocyte concentration tested. Within the SSc group, there were no correlations between TF activity and clinical features or disease duration.

Heterogeneous platelet-activating factor (PAF) receptors and calcium increase in platelets and macrophages.

We used the increase in cytosolic Ca2+ levels, [Ca2+]i, as a way to characterize PAF (platelet-activating factor, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) receptors in human platelets and rat and human macrophages. [Ca2+] was measured by means of the fluorescent probe fura-2/acetoxymethylester. PAF recognized heterogeneous receptors in human macrophages only (curve slope <1). The PAF antagonist SCH 37370 (1-acetyl-4(8-chloro-5,6-dihydro-11H-benzo[5.6]cyclohepta[1,2-b]pyridine -11-ylidine)piperidine) abolished [Ca2+]i elevation in human platelets, while in rat and human macrophages the maximal inhibition was 76% and 85%, respectively. On the contrary, the antagonist WEB 2086 (3-[4-(2-chlorophenyl)-9-methyl-6Hthieno[3,2-f] [1,2,4]triazolo-[4,3-a] [1,4]-diazepin-2-yl]-1-(4-morpholiny)-1-propanon, apafant) totally inhibited the effect of PAF in both platelets and macrophages. The WEB 2086 concentration-response curves had a slope <1 in the three cell types, indicating interaction with heterogeneous receptors. Accordingly, 3H-WEB 2086 bound to two different classes of sites. Both phases of [Ca2+]i elevation (influx or release) were equally affected by the antagonists. These data support the notions that: 1) PAF receptors are heterogeneous; 2) the two antagonists have a different selectivity toward the receptor subtypes: WEB 2086 recognizes two different receptors both in platelets and in macrophages, while SCH 37370 does not discriminate between receptor subtypes in platelets, and only interacts with one subtype in macrophages; and 3) both SCH 37370 and WEB 2086 display different potencies in rat and human macrophages.

Platelet alpha 2-adrenergic receptors in hypercholesterolemia: relationship between binding studies and epinephrine-induced platelet aggregation.

BACKGROUND: Platelets isolated from patients with hypercholesterolemia are more sensitive in vitro to various aggregating agents, including epinephrine, than those isolated from normocholesterolemic subjects. Increased platelet reactivity is one mechanism that may explain the enhanced risk of thromboembolism in hypercholesterolemia. This study assessed whether platelet hyperreactivity to epinephrine in hypercholesterolemia is associated with higher alpha 2-adrenergic receptor density or affinity for epinephrine. METHODS: Platelet aggregation and binding studies, with use of [3H]yohimbine as ligand, were performed on platelets isolated from 30 patients with type IIa hypercholesterolemia and 23 control subjects. RESULTS: Platelet aggregation in response to epinephrine was significantly higher in patients with hypercholesterolemia than in control subjects. A statistically significantly higher alpha 2-adrenergic receptor density was observed in a subgroup of 13 patients with hypercholesterolemia than in 13 sex- and age-matched control subjects (280 +/- 61 and 230 +/- 49 fmol/mg protein respectively; p < 0.03), but no difference was observed in receptor affinity for the ligand. In these subgroups plasma total and levels of low-density lipoprotein (LDL) cholesterol were inversely correlated with platelet aggregation but directly correlated with platelet receptor density. CONCLUSION: Platelet alpha 2-adrenergic receptor density is increased in hypercholesterolemia and directly correlates with plasma total and levels of LDL cholesterol, providing at least a partial explanation for the enhanced platelet response to epinephrine that is observed in hypercholesterolemia.