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Background Antiplatelet therapy is the mainstay of secondary prevention of cardiovascular events. Studies suggest that women do not obtain equal therapeutic benefit from antiplatelet therapy compared with men. The link between sex differences in platelet biology and response to antiplatelet therapies is unclear. We therefore investigated the role of sex differences in platelet reactivity in a cohort of outpatients with chest pain, in response to treatment with antiplatelet agents. Methods Platelet reactivity was measured in 382 randomly selected patients participating in the Myocardial Ischemia Detection by Circulating Biomarkers (MYOMARKER) study, an observational cohort study of outpatients suspected of myocardial ischemia. In all patients, blood was collected during diagnostic workup, and platelet reactivity was assessed with a flow cytometry–based platelet activation test that quantifies both platelet degranulation (P‐selectin expression) and platelet aggregation (fibrinogen binding to integrin αIIbβ3) in whole blood. Results Platelet reactivity was higher in women compared with men when activated with protease activating receptor 1–activating peptide SFLLRN (PAR1‐AP) and adenosine 5′‐phosphate (ADP), independent of age, basal activation status, estimated glomerular filtration rate

The genetic disorder Down syndrome is associated with a decreased susceptibility for atherosclerotic cardiovascular disease. Hematological and immune abnormalities occur frequently in Down syndrome patients. We evaluated, in a preclinical setting, the impact of a Down syndrome-like hematological/immune phenotype on atherosclerosis susceptibility. Hereto, hypercholesterolemic low-density lipoprotein receptor knockout mice were transplanted with bone marrow from either a trisomic Ts65Dn mouse or euploid wild-type control and subsequently fed a Western-type diet to induce the development of atherosclerotic lesions. T and B cell concentrations were markedly reduced in blood of Ts65Dn bone marrow recipients (p < 0.001). Expression levels of the pro-atherogenic scavenger receptor CD36 were respectively 37% and 59% lower (p < 0.001) in trisomic monocytes and macrophages. However, these combined effects did not translate into an altered atherosclerosis susceptibility. Notably, blood platelet numbers were elevated in Ts65Dn bone marrow recipients (+57%; p < 0.001), which was paralleled by higher platelet GPVI protein expression (+35%; p < 0.001) and an enhanced collagen-induced platelet activation (p < 0.001). In conclusion, we have shown that providing mice with a Down syndrome-like hematological profile does not change the susceptibility to atherosclerosis. Furthermore, our studies have uncovered a novel effect of the trisomy on platelet functionality that may be relevant in human clinical settings.

Staphylococcus aureus virulence has been associated with the production of phenol-soluble modulins (PSMs). These PSMs have distinct virulence functions and are known to activate, attract and lyse neutrophils. These PSM-associated biological functions are inhibited by lipoproteins in vitro. We set out to address whether lipoproteins neutralize staphylococcal PSM-associated virulence in experimental animal models. Serum from both LCAT an ABCA1 knockout mice strains which are characterised by near absence of high-density lipoprotein (HDL) levels, was shown to fail to protect against PSM-induced neutrophil activation and lysis in vitro. Importantly, PSM-induced peritonitis in LCAT−/− mice resulted in increased lysis of resident peritoneal macrophages and enhanced neutrophil recruitment into the peritoneal cavity. Notably, LCAT−/− mice were more likely to succumb to staphylococcal bloodstream infections in a PSM-dependent manner. Plasma from homozygous carriers of ABCA1 variants characterized by very low HDL-cholesterol levels, was found to be less protective against PSM-mediated biological functions compared to healthy humans. Therefore, we conclude that lipoproteins present in blood can protect against staphylococcal PSMs, the key virulence factor of community-associated methicillin resistant S. aureus.

Background delta-storage pool disease (delta-SPD) is a bleeding disorder characterized by a reduced number of platelet-dense granules. The diagnosis of delta-SPD depends on the measurement of platelet ADP content, but this test is time consuming and requires a relatively large blood volume. Flow cytometric analysis of platelet mepacrine uptake is a potential alternative, but this approach lacks validation, which precludes its use in a diagnostic setting.Objectives To evaluate the performance of platelet mepacrine uptake as a diagnostic test for delta-SPD.Patients/Methods Mepacrine fluorescence was determined with flow cytometry before and after platelet activation in 156 patients with a suspected platelet function disorder and compared with platelet ADP content as a reference test. Performance was analyzed with a receiver operating characteristic (ROC) curve.Results Eleven of 156 patients had delta-SPD based on platelet ADP content. Mepacrine fluorescence was inferior to platelet ADP content in identifying patients with delta-SPD, but both mepacrine uptake (area under the ROC curve [AUC] 0.87) and mepacrine release after platelet activation (AUC 0.80) had good discriminative ability. In our tertiary reference center, mepacrine uptake showed high negative predicitive value (97%) with low positive predictive value (35%). Combined with a negative likelihood ratio of 0.1, these data indicate that mepacrine uptake can be used to exclude delta-SPD in patients with a bleeding tendency.Conclusion Mepacrine fluorescence can be used as a screening tool to exclude delta-SPD in a large number of patients with a suspected platelet function disorder.

Introduction At the clinical introduction of antiangiogenic agents as anticancer agents, no major toxicities were expected as merely just endothelial cells (ECs) in tumors would be affected. However, several (serious) toxicities became apparent, of which underlying mechanisms are largely unknown. We investigated to what extent sunitinib (multitargeted antiangiogenic tyrosine kinase inhibitor (TKI)), sorafenib (TKI) and bevacizumab [specific antibody against vascular endothelial growth factor (VEGF)] may impair platelet function, which might explain treatment-related bleedings. Materials and methods In vitro, the influence of sunitinib, sorafenib, and bevacizumab on platelet aggregation, P-selectin expression and fibrinogen binding, platelet–EC interaction, and tyrosine phosphorylation of c-Src was studied by optical aggregation, flow cytometry, real-time perfusion, and western blotting. Ex vivo, platelet aggregation was analyzed in 25 patients upon sunitinib or bevacizumab treatment. Concentrations of sunitinib, VEGF, and platelet and EC activation markers were measured by LC–MS/MS and ELISA. Results In vitro, sunitinib and sorafenib significantly inhibited platelet aggregation (20 μM sunitinib: 71.3%, p

Background: Membrane-exposed sulfatides are proposed to contribute to P-selectin-dependent platelet aggregation. Here, we demonstrated that P-selectin-mediated platelet aggregation on a collagen-coated surface under flow indeed depended on sulfatides and that this interaction differed considerably from the interaction of P-selectin with P-selectin Glycoprotein Ligand-1 (PSGL-1), which underlies leukocyte-endothelium adhesion. Methods and Results: Upon platelet activation, sulfatides were translocated to the platelet surface to form focal hot-spots. Interestingly, P-selectin was observed to exclusively interact with liposomes with a sulfatide density higher than 21% (w/w), indicating that the binding profile of P-selectin for sulfatide-rich liposomes was dependent on sulfatide density. Sulfatide-liposome binding to P-selectin and sulfatide/P-selectin-dependent platelet aggregation was blunted by peptide antagonists, carrying the EWVDV motif within N-terminal extensions, such as CDVEWVDVSC (half maximal inhibitory concentration IC50 = 0.2 &mu, M), but not by the EWVDV core motif itself (IC50 &gt, 1000 &mu, M), albeit both being equally potent inhibitors of PSGL-1/P-selectin interaction (IC50= 7&ndash, 12 &mu, M). Conclusions: Our data suggest that the sulfatide/P-selectin interaction implicates multiple binding pockets, which only partly overlap with that of PSGL-1. These observations open ways to selectively interfere with sulfatide/P-selectin-dependent platelet aggregation without affecting PSGL-1-dependent cell adhesion.

Tissue factor, coagulation factor XII, platelets, and neutrophils are implicated as important players in the pathophysiology of (experimental) venous thrombosis (VT). Their role became evident in mouse models in which surgical handlings were required to provoke VT. Combined inhibition of the natural anticoagulants antithrombin (Serpinc1) and protein C (Proc) using small interfering RNA without additional triggers also results in a venous thrombotic phenotype in mice, most notably with vessel occlusion in large veins of the head. VT is fatal but is fully rescued by thrombin inhibition. In the present study, we used this VT mouse model to investigate the involvement of tissue factor, coagulation factor XII, platelets, and neutrophils. Antibody-mediated inhibition of tissue factor reduced the clinical features of VT, the coagulopathy in the head, and fibrin deposition in the liver. In contrast, genetic deficiency in, and small interfering RNA–mediated depletion of, coagulation factor XII did not alter VT onset, severity, or thrombus morphology. Antibody-mediated depletion of platelets fully abrogated coagulopathy in the head and liver fibrin deposition. Although neutrophils were abundant in thrombotic lesions, depletion of circulating Ly6G-positive neutrophils did not affect onset, severity, thrombus morphology, or liver fibrin deposition. In conclusion, VT after inhibition of antithrombin and protein C is dependent on the presence of tissue factor and platelets but not on coagulation factor XII and circulating neutrophils. This study shows that distinct procoagulant pathways operate in mouse VT, dependent on the triggering stimulus.

Heart failure with preserved ejection fraction (HFpEF) is a syndrome involving microvascular dysfunction. No treatment is available yet and as the HFpEF patient group is expanding due to the aging population, more knowledge on dysfunction of the cardiac microvasculature is required. Endothelial dysfunction, impaired angiogenesis, (perivascular) fibrosis and the pruning of capillaries (rarefaction) may all contribute to microvascular dysfunction in the heart and other organs, e.g., the kidneys. The HFpEF patient group consists mainly of post-menopausal women and female sex itself is a risk factor for this syndrome. This may point toward a role of estrogen depletion after menopause in the development of HFpEF. Estrogens favor the ratio of vasodilating over vasoconstricting factors, which results in an overall lower blood pressure in women than in men. Furthermore, estrogens improve angiogenic capacity and attenuate (perivascular) fibrosis formation. Therefore, we hypothesize that the drop of estrogen levels after menopause contributes to myocardial microvascular dysfunction and renders post-menopausal women more vulnerable for heart diseases that involve the microvasculature. This review provides a detailed summary of molecular targets of estrogen, which might guide future research and treatment options.

BACKGROUND: Platelet (PLT) concentrates are prophylactically given to prevent major bleeding complications. The corrected count increment (CCI) is currently the only tool to monitor PLT transfusion efficacy. PLT function tests cannot be performed in patients with thrombocytopenia. Therefore, an optimized agonist-induced assay was used to determine PLT function, in patients with severe thrombocytopenia before and after transfusion. STUDY DESIGN AND METHODS: PLT reactivity toward adenosine diphosphate (ADP), thrombin receptor-activating peptide SFLLRN (TRAP), and convulxin (CVX) was assessed by flow cytometry. P-selectin expression was measured on PLTs from 11 patients with thrombocytopenia before and 1 hour after transfusion, on stored PLTs, and on stored PLTs incubated for 1 hour in whole blood from patients ex vivo. RESULTS: The mean (±SEM) CCI after 1 hour was 11.4 (±1.5). After transfusion, maximal agonist-induced PLT P-selectin expression was on average 29% higher for ADP (p = 0.02), 25% higher for TRAP (p = 0.007), and 24% higher for CVX (p = 0.0008). ADP-induced reactivity of stored PLTs increased with 46% after ex vivo incubation (p = 0.007). These PLTs also showed an overall higher P-selectin expression compared to PLTs 1 hour after transfusion (p = 0.005). After normalization for this background expression, a similar responsiveness was observed. CONCLUSIONS: Our study shows recovery of PLT function after transfusion in patients with thrombocytopenia. The majority of functional PLTs measured after transfusion most likely represents stored transfused PLTs that regained functionality in vivo. The difference in baseline P-selectin expression in vivo versus ex vivo suggests a rapid clearance from circulation of PLTs with increased P-selectin expression.

Background and aims: Thrombocytopenia in scavenger receptor BI (SR-BI) knockout mice is suggested to result from augmented platelet clearance induced by elevated intracellular unesterified cholesterol (UC) levels. We hypothesize that SR-BI deficiency may also influence platelet production at the level of its precursor cell in the bone marrow, the megakaryocyte. Methods: In this study, we compared megakaryopoiesis and platelet production in SR-BI knockout and wild-type mice. Results: In line with our hypothesis, megakaryocytes from SR-BI knockout mice exhibited UC accumulation while no accumulation of UC was detectable in wild-type megakaryocytes. Bone marrow expression of transcription factors involved in megakaryocyte maturation was induced, but megakaryocyte counts were unchanged in bone marrow of SR-BI knockout mice. Interestingly, we did find a striking 62% decrease (p < 0.01) in proplatelet production by SR-BI knockout megakaryocytes. SR-BI knockout mice displayed an impaired increase in circulating platelet concentrations and bone marrow megakaryocyte numbers upon thrombopoietin challenge. Importantly, megakaryocytes from normolipidemic bone marrow-specific SR-BI knockout mice exhibited a normal ability to produce proplatelets. Moreover, bone marrow-specific deletion of SR-BI did not impair the thrombopoietin response or induce thrombocytopenia, confirming that absence of megakaryocyte SR-BI does not underlie the thrombocytopenic phenotype in total body SR-BI knockout mice. Conclusions: In conclusion, the elevation of plasma unesterified cholesterol levels impairs megakaryopoiesis and platelet production in SR-BI knockout mice. Our findings suggest that, in addition to an increased platelet clearance, a decrease in platelet production may also, in part, explain the thrombocytopenic phenotype associated with SR-BI deficiency in mice.

Objective: Familial Hypercholesterolemia (FH) is associated with an increased risk of cardiovascular disease (CVD). However, whether an individual heterozygous FH patient will develop CVD depends on other genetic-and environmental risk factors as well. LDL receptor-related protein with 11 ligand binding repeat (LR11) and its soluble form (sLR11) play a role in the progression of atherosclerosis. We investigated the involvement of LR11 and sLR11 in CVD development in FH patients and in LDLR deficient (Ldlr(-/-)) mice. Approach and results: In statin-treated asymptomatic male heterozygous FH subjects, plasma sLR11 levels correlated with carotid intima-media thickness. Increased plasma sLR11 levels were found in Ldlr(-/-) and also in wild-type mice exclusively after high-fat feeding. Hepatic LR11 mRNA levels, however, were higher in chow-fed Ldlr(-/-) in comparison with wild-type mice and were further increased after a high fat diet. Similar results were obtained with Apoe(-/-) mice, but not with wild-type mice. LR11 mRNA and protein levels and release of sLR11 from cultured HepG2 and aortic smooth muscle cells were upregulated by postprandial triglyceride-rich lipoproteins (TGRL). Overexpression of human LR11 in CHO cells resulted in increased binding and association of 12I-labeled TGRL, but not of 12I-labeled LDL. Conclusion: Our data strongly suggest an involvement of LR11 in mediating the harmful effects of a high-fat diet on CVD progression. Elevated sLR11 levels may increase the CVD risk especially in subjects with delayed clearance of triglyceride-rich remnants, such as in FH patients. (C) 2015 Elsevier Ireland Ltd. All rights reserved.

Background and objective Prophylactic platelet transfusions are administered to prevent bleeding in haemato-oncological patients. However, bleeding still occurs, despite these transfusions. This practice is costly and not without risk. Better predictors of bleeding are needed, and flow cytometric evaluation of platelet function might aid the clinician in identifying patients at risk of bleeding. This evaluation can be performed within the hour and is not hampered by low platelet count. Our objective was to assess a possible correlation between bleeding and platelet function in thrombocytopenic haemato-oncological patients. Materials and Methods Inclusion was possible for admitted haemato-oncology patients aged 18 years and above. Furthermore, an expected need for platelet transfusions was necessary. Bleeding was graded according to the WHO bleeding scale. Platelet reactivity to stimulation by either adenosine diphosphate (ADP), cross-linked collagen-related peptide (CRP-xL), PAR1- or PAR4-activating peptide (AP) was measured using flow cytometry. Results A total of 114 evaluations were available from 21 consecutive patients. Platelet reactivity in response to stimulation by all four studied agonists was inversely correlated with significant bleeding. Odds ratios (OR) for bleeding were 0·28 for every unit increase in median fluorescence intensity (MFI) [95% confidence interval (CI) 0·11–0·73] for ADP; 0·59 [0·40–0·87] for CRP-xL; 0·59 [0·37–0·94] for PAR1-AP; and 0·43 [0·23–0·79] for PAR4-AP. The platelet count was not correlated with bleeding (OR 0·99 [0·96–1·02]). Conclusion Agonist-induced platelet reactivity was significantly correlated to bleeding. Platelet function testing could provide a basis for a personalized transfusion regimen, in which platelet transfusions are limited to those at risk of bleeding.

Patients on P2Y12 inhibitors may still develop thrombosis or bleeding complications. Tailored antiplatelet therapy, based on platelet reactivity testing, might reduce these complications. Several tests have been used, but failed to show a benefit of tailored antiplatelet therapy. This could be due to the narrowness of current platelet reactivity tests, which are limited to analysis of platelet aggregation after stimulation of the adenosine diphosphate (ADP)-pathway. However, the response to ADP does not necessarily reflect the effect of P2Y12 inhibition on platelet function in vivo. Therefore, we investigated whether measuring platelet reactivity toward other physiologically relevant agonists could provide more insight in the efficacy of P2Y12 inhibitors. The effect of in vitro and in vivo P2Y12 inhibition on αIIbβ3-activation, P-selectin and CD63-expression, aggregate formation, release of alpha, and dense granules content was assessed after stimulation of different platelet activation pathways. Platelet reactivity measured with flow cytometry in 72 patients on P2Y12 inhibitors was compared to VerifyNow results. P2Y12 inhibitors caused strongly attenuated platelet fibrinogen binding after stimulation with peptide agonists for protease activated receptor (PAR)-1 and -4, or glycoprotein VI ligand crosslinked collagen-related peptide (CRP-xl), while aggregation was normal at high agonist concentration. P2Y12 inhibitors decreased PAR-agonist and CRP-induced dense granule secretion, but not alpha granule secretion. A proportion of P2Y12-inhibitor responsive patients according to VerifyNow, displayed normal fibrinogen binding assessed with flow cytometry after stimulation with PAR-agonists or CRP despite full inhibition of the response to ADP, indicating suboptimal platelet inhibition. Concluding, measurement of platelet fibrinogen binding with flow cytometry after stimulation of thrombin- or collagen receptors in addition to ADP response identifies different patients as nonresponders to P2Y12 inhibitors, compared to only ADP-induced aggregation-based assays. Future studies should investigate the value of both assays for monitoring on-treatment platelet reactivity.

Platelets are key players in hemostasis, the process that is essential in the prevention of blood loss in response to injury of a blood vessel. When platelets encounter breaches in the vascular wall, they rapidly adhere to the site of injury and aggregate to form a platelet plug. Subsequently coagulation will be initiated resulting in a fibrin network that reinforces the plug. Failure to form an adequate plug leads to a bleeding tendency. On the other hand, excessive platelet reactivity leads to an increased risk of vascular occlusion and thrombosis. In order to better understand how platelets function, it is essential to have insight into their overall morphology and (ultra)structure. This chapter will contribute to this and present our current view of the platelet structure and physiology in health and disease and the recent techniques available to visualize this.

Objective— Platelets abundantly express the membrane receptor CD36 and store its ligand thrombospondin-1 (TSP1) in the α-granules. We investigated whether released TSP1 can support platelet adhesion and thrombus formation via interaction with CD36. Approach and Results— Mouse platelets deficient in CD36 showed reduced adhesion to TSP1 and subsequent phosphatidylserine expression. Deficiency in either CD36 or TSP1 resulted in markedly increased dissolution of thrombi formed on collagen, although thrombus buildup was unchanged. In mesenteric vessels in vivo, deficiency in CD36 prolonged the time to occlusion and enhanced embolization, which was in agreement with earlier observations in TSP1-deficient mice. Thrombi formed using wild-type blood stained positively for secreted TSP1. Releasate from wild-type but not from TSP1-deficient platelets enhanced platelet activation, phosphatidylserine expression, and thrombus formation on collagen. The enhancement was dependent on CD36 because it was without effect on thrombus formation by CD36-deficient platelets. Conclusions— These results demonstrate an anchoring role of platelet-released TSP1 via CD36 in platelet adhesion and collagen-dependent thrombus stabilization. Thus, the TSP1–CD36 tandem is another platelet ligand–receptor axis contributing to the maintenance of a stable thrombus.

Background and aims Women who develop preeclampsia during pregnancy are at a higher risk for developing cardiovascular disease. As platelets are affected by preeclampsia, we set out to identify whether platelets carry information in their transcriptome on cardiovascular risk in women with former preeclampsia. Methods Platelets were isolated from asymptomatic women with previous preeclampsia, who underwent screening with coronary computed tomography angiography. Platelet RNA was isolated and used to construct gene networks using an unbiased approach. Platelet gene modules assembled from the network were related to risk factors and clinical traits of these women, including coronary artery calcium scores (CACS). Results We found multiple gene modules which correlated with CACS (correlation coefficients: 0.44 to 0.59, p = 0.05 to 0.007). The genes from two clinically relevant modules were expressed at a higher level in the group with calcifications (p = 3.9 × 10−10 and 0.02) and enriched for platelet-related gene-sets such as platelet activation. The first of these modules was also enriched (ppermutation = 0.0546) for genes mapped to known coronary artery disease susceptibility loci. Additional unbiased network analyses in platelet RNA of patients with overt cardiovascular disease underlined the importance of the identified modules for disease by high preservation. (p = 1.6 × 10−9 to 1.7 × 10−47). Conclusions We found platelet RNA modules that correlated with CACS in asymptomatic women with previous preeclampsia. Whether or not platelets directly contribute to this disease trajectory, or reflect the underlying plaque substrate remains to be determined, but enrichment for coronary artery disease susceptibility genes emphasizes the importance for the disease.

containing lipoproteins can provide cholesterol for synthesis of glucocorticoids. Here we assessed adrenal glucocorticoid function in LCAT knockout (KO) mice to determine the specific contribution of HDL-cholesteryl esters to adrenal glucocorticoid output in vivo. LCAT KO mice exhibit an 8-fold higher plasma free cholesterol-to-cholesteryl ester ratio (P

Recently, platelets, neutrophils, and factor XII (FXII) have been implicated as important players in the pathophysiology of venous thrombosis. Their role became evident in mouse models in which surgical handling was used to provoke thrombosis. Inhibiting anticoagulation in mice by using small interfering RNA (siRNA) targeting Serpinc1 and Proc also results in a thrombotic phenotype, which is spontaneous (no additional triggers) and reproducibly results in clots in the large veins of the head and fibrin deposition in the liver. This thrombotic phenotype is fatal but can be fully rescued by thrombin inhibition. The mouse model was used in this study to investigate the role of platelets, neutrophils, and FXII. After administration of siRNAs targeting Serpinc1 and Proc, antibody-mediated depletion of platelets fully abrogated the clinical features as well as microscopic aspects in the head. This was corroborated by strongly reduced fibrin deposition in the liver. Whereas neutrophils were abundant in siRNA-triggered thrombotic lesions, antibody-mediated depletion of circulating Ly6G-positive neutrophils did not affect onset, severity, or thrombus morphology. In addition, absence of circulating neutrophils did not affect quantitative liver fibrin deposition. Remarkably, siRNA-mediated depletion of plasma FXII accelerated the onset of the clinical phenotype; mice were affected with more severe thrombotic lesions. To summarize, in this study, onset and severity of the thrombotic phenotype are dependent on the presence of platelets but not circulating neutrophils. Unexpectedly, FXII has a protective effect. This study challenges the proposed roles of neutrophils and FXII in venous thrombosis pathophysiology.

Many patients are treated with platelet inhibitors such as aspirin and clopidogrel for prevention of thrombotic cardiovascular events. However, the inhibitory effect of antiplatelet therapy is variable between patients; in some, the platelets are hardly inhibited, while in others, the platelets are excessively inhibited. The newer and more potent platelet inhibitors, prasugrel and ticagrelor, often lead to low platelet reactivity, which potentially leads to bleeding events. Preoperative measurement of platelet reactivity in patients receiving platelet inhibitors who undergo cardiac surgery, could be useful to identify those with low platelet reactivity and thus have an increased risk of bleeding during or after surgery. In this review, we discuss the most commonly used platelet inhibitors and platelet function tests. Furthermore, we will provide an overview of the evidence for the prediction of post-operative bleeding at the operation site with preoperative platelet reactivity testing in patients undergoing cardiac surgery.

Objective— The A 2B adenosine receptor (A 2B R) is highly expressed in macrophages and vascular smooth muscle cells and has been established as an important regulator of inflammation and vascular adhesion. Recently, it has been demonstrated that A 2B R deficiency enhances neointimal lesion formation after vascular injury. Therefore, we hypothesize that A 2B R agonism protects against injury-induced intimal hyperplasia. Methods and Results— Apolipoprotein E–deficient mice were fed a Western-type diet for 1 week, after which the left common carotid artery was denuded. Mice were treated with the A 2B receptor agonist BAY60-6583 or vehicle control for 18 days. Interestingly, lumen stenosis as defined by the neointima/lumen ratio was inhibited by treatment with the A 2B receptor agonist, caused by reduced smooth muscle cell proliferation. Collagen content was significantly increased in the BAY60-6583–treated mice, whereas macrophage content remained unchanged. In vitro, vascular smooth muscle cell proliferation decreased dose dependently whereas collagen content of cultured smooth muscle cells was increased by BAY60-6583. Conclusion— Our data show that activation of the adenosine A 2B receptor protects against vascular injury, while it also enhances plaque stability as indicated by increased collagen content. These outcomes thus point to A 2B receptor agonism as a new therapeutic approach in the prevention of restenosis.

BACKGROUND: In mice, the scavenger receptor class B type I (SR-BI) is essential for the delivery of high-density lipoprotein (HDL) cholesterol to the liver and steroidogenic organs. Paradoxically, elevated HDL cholesterol levels are associated with increased atherosclerosis in SR-BI-knockout mice. It is unclear what role SR-BI plays in human metabolism.METHODS: We sequenced the gene encoding SR-BI in persons with elevated HDL cholesterol levels and identified a family with a new missense mutation (P297S). The functional effects of the P297S mutation on HDL binding, cellular cholesterol uptake and efflux, atherosclerosis, platelet function, and adrenal function were studied.RESULTS: Cholesterol uptake from HDL by primary murine hepatocytes that expressed mutant SR-BI was reduced to half of that of hepatocytes expressing wild-type SR-BI. Carriers of the P297S mutation had increased HDL cholesterol levels (70.4 mg per deciliter [1.8 mmol per liter], vs. 53.4 mg per deciliter [1.4 mmol per liter] in noncarriers; PCONCLUSIONS: We identified a family with a functional mutation in SR-BI. The mutation carriers had increased HDL cholesterol levels and a reduction in cholesterol efflux from macrophages but no significant increase in atherosclerosis. Reduced SR-BI function was associated with altered platelet function and decreased adrenal steroidogenesis. (Funded by the European Community and others.).

Lipoprotein-associated cholesterol has been suggested to make a significant contribution to adrenal steroidogenesis in vivo. To determine whether lipoproteins indeed contribute to optimal adrenal steroidogenesis in mice, in the current study we have determined the effect of relative lipoprotein deficiency on adrenal steroidogenesis in C57BL/6 wild-type mice. Feeding C57BL/6 mice the lipid-lowering drug probucol (0.25% wt/wt) for 2 wk induced a 90% decrease in plasma high-density lipoprotein (HDL) cholesterol levels and a 77% reduction in low-density lipoprotein (LDL) cholesterol levels. Neutral lipid stores were depleted upon probucol treatment specifically in the glucocorticoid-producing zona fasciculata of the adrenal, leading to a 44% decreased plasma corticosterone level under basal conditions. Exposure to lipopolysaccharide (LPS) induced a 37% increase in the adrenal uptake of HDL cholesteryl esters. Probucol-treated mice could induce only a relatively minor corticosterone response upon a LPS challenge compared with controls, which coincided with an approximately twofold increased hepatic expression level of interleukin-6 and tumor necrosis factor (TNF)α and an 89% higher TNFα response in plasma. Furthermore, a compensatory two- to fivefold upregulation of LDL receptor (cholesterol uptake) and HMG-CoA reductase (cholesterol synthesis) expression was noticed in the adrenals of probucol-treated mice. In conclusion, we have shown that lipoprotein deficiency in mice as a result of probucol feeding is associated with decreased adrenal cortex cholesterol levels, a lower basal and stress-induced plasma glucocorticoid level, and an increased susceptibility to LPS-induced inflammation. Therefore, it is suggested that plasma lipoproteins are required for optimal adrenal steroidogenesis and protection against endotoxemia in mice.

Essentials The diagnosis of mild platelet function disorders (PFDs) is challenging. Validation of flow cytometric testing in patients with suspected PFDs is required. Flow cytometry has added value to light transmission aggregometry (LTA) in diagnosis of PFDs. There is fair agreement in diagnosing PFDs between LTA and flow cytometry. Summary: Background Light transmission aggregometry (LTA) is the most commonly used test for the diagnosis of platelet function disorders (PFDs), but has moderate sensitivity for mild PFDs. Flow cytometry has been recommended for additional diagnostics of PFDs but is not yet standardized as a diagnostic test. We developed a standardized protocol for flow cytometric analysis of platelet function that measures fibrinogen binding and P-selectin expression as platelet activation markers in response to agonist stimulation. Objectives To determine the additional value of flow cytometric platelet function testing to standard LTA screening in a cross-sectional cohort of patients with a suspected PFD. Methods Platelet function was assessed with flow cytometry and LTA in 107 patients suspected of a PFD in whom von Willebrand disease and coagulation factor deficiencies were excluded. Both tests were compared in terms of agreement and discriminative ability for diagnosing patients with PFDs. Results Out of 107 patients, 51 patients had an elevated bleeding score; 62.7% of the patients had abnormal platelet function measured with flow cytometry and 54.2% of the patients were abnormal based on LTA. There was fair agreement between LTA and flow cytometry (κ = 0.32). The discriminative ability of flow cytometric analysis in patients with an elevated bleeding score was good (AUC 0.82, 0.74–0.90), but moderate for LTA (AUC 0.70, 0.60–0.80). Both tests combined had a better discriminative ability (AUC 0.87, 0.80–0.94). Conclusion Flow cytometric analysis of platelet function has added value in diagnostics of PFDs in patients with unexplained bleeding tendency.

Item does not contain fulltext The serotonin transporter is implicated in the uptake of the vasoconstrictor serotonin from the circulation into the platelets, where 95% of all blood serotonin is stored and released in response to vascular injury. In vivo studies indicated that platelet-derived serotonin mediates liver regeneration after partial hepatectomy. We have recently generated serotonin transporter knockout rats and demonstrated that their platelets were almost completely depleted of serotonin. Here we show that these rats exhibit impaired hemostasis and contain about 1-6% of wild-type serotonin levels in the blood. Despite the marked reduction of serotonin levels in blood and platelets, efficient liver regeneration and collagen-induced platelet aggregation occur in rats lacking the serotonin transporter. These results provide evidence that liver regeneration is not dependent on the release of serotonin from platelets. Our findings indicate that very low levels of serotonin in blood are sufficient for liver regeneration.

Objective— The interaction of platelets with low density lipoprotein (LDL) contributes to the development of cardiovascular disease. Platelets are activated by native LDL (nLDL) through apoE Receptor 2′ (apoER2′)-mediated signaling to p38 MAPK and by oxidized LDL (oxLDL) through lysophosphatidic acid (LPA) signaling to Rho A and Ca 2+ . Here we report a new mechanism for platelet activation by oxLDL. Methods and Results— Oxidation of nLDL increases p38 MAPK activation through a mechanism that is (1) independent of LPA, and (2) unlike nLDL-signaling not desensitized by prolonged platelet-LDL contact or inhibited by receptor-associated protein or chondroitinase ABC. Antibodies against scavenger receptors CD36 and SR-A alone fail to block p38 MAPK activation by oxLDL but combined blockade inhibits p38 MAPK by >40% and platelet adhesion to fibrinogen under flow by >60%. Mouse platelets deficient in either CD36 or SR-A show normal p38 MAPK activation by oxLDL but combined deficiency of CD36 and SR-A disrupts oxLDL-induced activation of p38 MAPK by >70%. Conclusion— These findings reveal a novel platelet-activating pathway stimulated by oxLDL that is initiated by the combined action of CD36 and SR-A.

Introduction Although the role of platelets in rheumatoid arthritis (RA) is relatively unexplored, recent studies point towards a contribution of platelets in arthritis. We set out to determine platelet phenotype in RA and studied whether this could be influenced by the presence of anti-citrullinated protein antibodies (ACPA). Methods Platelets from healthy controls were incubated in the presence of plasma of patients with RA or age- and sex-matched healthy controls and plasma from ACPAneg or ACPApos patients or in the presence of plate-bound ACPA. Characteristics of platelets isolated from patients with RA were correlated to disease activity. Results Platelets isolated from healthy controls displayed markers of platelet activation in the presence of plasma derived from RA patients, as determined by P-selectin expression, formation of aggregates and secretion of soluble CD40 ligand (sCD40L). Furthermore, levels of P-selectin expression and sCD40L release correlated with high ACPA titres. In accordance with these findings, enhanced platelet activation was observed after incubation with ACPApos plasma versus ACPAneg plasma. Pre-incubation of platelets with blocking antibodies directed against low-affinity immunoglobulin G receptor (FcγRIIa) completely inhibited the ACPA-mediated activation. In addition, expression of P-selectin measured as number of platelets correlated with Disease Activity Score in 44 joints, C-reactive protein level, ACPA status and ACPA level. Conclusions We show for the first time that ACPA can mediate an FcγRIIa-dependent activation of platelets. As ACPA can be detected several years before RA disease onset and activated platelets contribute to vascular permeability, these data implicate a possible role for ACPA-mediated activation of platelets in arthritis onset. Electronic supplementary material The online version of this article (doi:10.1186/s13075-015-0665-7) contains supplementary material, which is available to authorized users.

Summary: Background: The autonomic nervous system attenuates inflammation through activation of the α7 nicotinic acetylcholine receptor (α7nAChR), a pathway termed the cholinergic anti-inflammatory reflex. Interestingly, α7nAChR is expressed on immune cells and platelets, both of which play a crucial role in the development of atherosclerosis. Objective: To investigate the role of hematopoietic α7nAChR in inflammation and platelet function in atherosclerotic ldlr-/- mice and to identify its consequences for atherosclerotic lesion development. Methods: Bone marrow from α7nAChR-/- mice or wild-type littermates was transplanted into irradiated ldlr-/- mice. After a recovery period of 8 weeks, the mice were fed an atherogenic Western-type diet for 7 weeks. Results: Hematopoietic α7nAChR deficiency clearly increased the number of leukocytes in the peritoneum (2.6-fold, P < 0.001), blood (2.9-fold; P < 0.01), mesenteric lymph nodes (2.0-fold; P < 0.001) and spleen (2.2-fold; P < 0.01), indicative of an increased inflammatory status. Additionally, expression of inflammatory mediators was increased in peritoneal leukocytes (TNFα, 1.6-fold, P < 0.01; CRP, 1.8-fold, P < 0.01) as well as in the spleen (TNFα, 1.6-fold, P < 0.01). The lack of α7nAChR on platelets from these mice increased the expression of active integrin αIIbβ3 upon stimulation by ADP (1.9-fold, P < 0.01), indicating increased activation status, while incubation of human platelets with an α7nAChR agonist decreased aggregation (-35%, P < 0.05). Despite the large effects of hematopoietic α7nAChR deficiency on inflammatory status and platelet function, it did not affect atherosclerosis development or composition of lesions. Conclusions: Hematopoietic α7nAChR is important for attenuation of inflammatory responses and maintaining normal platelet reactivity, but loss of hematopoietic α7nAChR does not aggravate development of atherosclerosis.

Cardiovascular disease is the main cause of death and disability in the Western society. Lipoproteins are important in the development of cardiovascular disease since they change the properties of different cells involved in atherosclerosis and thrombosis. The interaction of platelets with lipoproteins has been under intense investigation. Particularly the initiation of platelet signaling pathways by low density lipoprotein (LDL) has been studied thoroughly, since platelets of hypercholesterolemic patients, whose plasma contains elevated LDL levels due to absent or defective LDL receptors, show hyperaggregability in vitro and enhanced activity in vivo. These observations suggest that LDL enhances platelet responsiveness. Several signaling pathways induced by LDL have been revealed in vitro, such as signaling via p38 mitogen-activated protein kinase and p125 focal adhesion kinase. High density lipoprotein (HDL) consists of two subtypes, HDL2 and HDL3, which have opposing effects on platelet activation. This review provides a summary of the activation of signaling pathways after platelet-LDL and platelet-HDL interaction, with special emphasis on their role in the development of thrombosis and atherosclerosis.

Platelets and lipoproteins are both key elements in the development of atherosclerosis and thrombosis. Based on their density, five classes of lipoproteins have been identified which all influence platelets via distinct mechanisms. The activation of platelets starts with binding of apolipoproteins to different platelet receptors and is followed by the activation of signaling pathways resulting in activation or inhibition of platelet functions like aggregation or secretion. In addition to apolipoproteins, lipoproteins are also associated to a large amount of proteins, enzymes and lipids that also can induce platelet activation or inhibition. This review provides a summary of the activation of signaling pathways after platelet–lipoprotein interactions initiated by lipoprotein-associated proteins and lipids.

Platelets and coagulation factors are involved in the process of haemostasis, which ensures undisturbed blood flow upon vessel wall damage. However, excessive platelet aggregation and/or coagulation may lead to arterial or venous thrombosis. Pro-atherogenic lipoproteins, including native and oxidized low-density lipoprotein (LDL), are associated with an increased susceptibility to thrombosis. In contrast, numerous epidemiological studies have established an inverse correlation between high-density lipoprotein (HDL) levels and the risk for thrombosis. In addition to its role in reverse cholesterol transport, HDL also interacts with platelets, the coagulation cascade, and the vascular endothelium. Native HDL prevents platelet hyperreactivity by limiting intraplatelet cholesterol overload, as well as by modulating platelet signalling pathways after binding platelet HDL receptors such as scavenger receptor class B type I (SR-BI) and apoER2'. The antithrombotic properties of native HDL are also related to the suppression of the coagulation cascade and stimulation of clot fibrinolysis. Furthermore, HDL stimulates the endothelial production of nitric oxide and prostacyclin, which are potent inhibitors of platelet activation. Thus, HDL's antithrombotic actions are multiple and therefore, raising HDL may be an important therapeutic strategy to reduce the risk of arterial and venous thrombosis.

We investigated the mechanism of b -very low density lipoprotein ( b -VLDL)-induced foam cell forma- tion derived from peritoneal macrophages from control mice and low density lipoprotein (LDL) receptor-deficient mice to elucidate the role of the LDL receptor in this process. The LDL receptor appeared to be of major im- portance for b -VLDL metabolism. Consequently, the accu- mulation of cholesteryl esters in LDL receptor 2 / 2 mac- rophages is 2.5-fold lower than in LDL receptor 1 / 1 macrophages. In the absence of the LDL receptor, how- ever, b -VLDL was still able to induce cholesteryl ester accumulation and subsequently we characterized the prop- erties of this residual b -VLDL recognition site(s) of LDL receptor 2 / 2 macrophages. Although the LDL receptor- related protein is expressed on LDL receptor 2 / 2 mac- rophages, the cell association of b -VLDL is not influenced by the receptor-associated protein, and treatment of the macrophages with heparinase and chondroitinase was also ineffective. In contrast, both oxidized LDL (OxLDL) and anionic liposomes were able to inhibit the cell association of 125 I-labeled b -VLDL in LDL receptor 2 / 2 macrophages by 65%. These properties suggest a role for scavenger re- ceptor class B (SR-B), and indeed, in the LDL receptor 2 / 2 macrophages the selective uptake of cholesteryl esters from b -VLDL was 2.2-fold higher than that of apolipopro- teins, a process that could be inhibited by OxLDL, high density lipoprotein (HDL), and b -VLDL. In conclusion, the LDL receptor on peritoneal macrophages is directly in- volved in the metabolism of b -VLDL and the subsequent foam cell formation. When the LDL receptor is absent, SR-B appears to mediate the remaining metabolism of cholesteryl esters from b -VLDL. —Herijgers, N., M. Van Eck, S. J. A. Korporaal, P. M. Hoogerbrugge, and T. J.C. Van Berkel. Relative importance of the LDL receptor and scavenger receptor class B in the b -VLDL-induced uptake and accumulation of cholesteryl esters by peritoneal mac- rophages. J. Lipid Res . 2000. 41: 1163-1171.

While numerous studies have aimed to develop strategies to inhibit the development and progression of atherosclerosis, recent attention has focussed on the regression of pre-existing atherosclerotic plaques. As important regulator of total body cholesterol homeostasis, the liver X receptor (LXR) could possibly be an important target to induce regression. Here, we describe the effect of LXR activation by the synthetic agonist T0901317 on lesion regression in different mouse models with early fatty streak lesions or advanced collagen-rich lesions. Although T0901317 caused a dramatic increase in plasma (V)LDL levels in low-density lipoprotein (LDL) receptor knockout mice, no further increase in lesion size was observed, which points to beneficial LXR activity in the vascular wall. In normolipidemic C57BL/6 mice with cholate diet-induced atherosclerotic lesions, T0901317 treatment improved plasma lipoprotein levels and induced lesion regression (-43%, p0.05). Apolipoprotein E (APOE) reconstitution in APOE knockout mice by means of bone marrow transplantation dramatically improved plasma lipoprotein profiles and resulted in a marked regression of initial (-45%, p0.001) and advanced lesions (-23%, p0.01). Atherosclerosis regression was associated with a decrease in the absolute macrophage content (-84%, p0.001). T0901317 supplementation further decreased the size of early (-71%, p0.001 vs baseline; -48%, p0.01 vs chow diet alone) and more advanced atherosclerotic lesions (-36%, p0.001 and -17%, p=0.06 respectively). In conclusion, our study highlights the potential of LXR agonist T0901317 to stimulate removal of macrophages from atherosclerotic lesions ultimately leading to a highly significant plaque regression of both early and advanced atherosclerotic lesions.

Objective— Despite common disbelief that neutrophils are involved in atherosclerosis, evidence is accumulating for a causal role of neutrophils in atherosclerosis. CC chemokine ligand (CCL)3 is an inflammatory chemokine and its expression is significantly increased during atherosclerotic lesion formation in mice. It has recently been shown that under conditions of inflammation neutrophils can migrate along a CCL3 gradient. In this study, we aimed to elucidate the role of leukocyte-derived CCL3 in atherogenesis. Methods and Results— Irradiated low density lipoprotein receptor –/– mice, reconstituted with CCL3 –/– or littermate bone marrow showed markedly reduced CCL3 response to lipopolysaccharide treatment, establishing the critical relevance of leukocytes as source of CCL3. Hematopoietic deficiency of CCL3 significantly reduced aortic sinus lesion formation by 31% after 12 weeks of western-type diet. Interestingly, whereas plaque macrophage, collagen, and vascular smooth muscle cell content were unchanged, neutrophil adhesion to and presence in plaques was significantly attenuated in CCL3 –/– chimeras. These mice had reduced circulating neutrophil numbers, which could be ascribed to an increased neutrophil turnover and CCL3 –/– neutrophils were shown to be less responsive toward the neutrophil chemoattractant CXC chemokine ligand 1. Conclusion— Our data indicate that under conditions of acute inflammation leukocyte-derived CCL3 can induce neutrophil chemotaxis toward the atherosclerotic plaque, thereby accelerating lesion formation.

Early ischemic ventricular fibrillation (VF) in the setting of an acute myocardial infarction (AMI) due to thrombotic coronary occlusion remains a major health problem. Several animal studies have shown that platelet-dense granule contents released during thrombus formation can induce arrhythmias. We hypothesize that the platelet release reaction is involved in the predisposition to early ischemic VF. A case-control study was performed in patients who survived VF during a first AMI ("cases," n = 26) and in patients with one previous AMI without arrhythmias ("controls," n = 24). All patients were on aspirin 100 mg OD. Baseline platelet activation was assessed with flow cytometry. Response to activation was assessed with aggregometry, flow cytometry and PFA-100 analysis. Differences in platelet contents and content release were assessed by labeling platelet-dense granules with mepacrine and by measuring serotonin and ADP/ATP content. Patient and infarct characteristics and baseline platelet function tests were similar between groups. The mean time from event was 4.9 (±3.2) years among cases and 4.7 (±2.7) years among controls. Dense granule release was similar in cases versus controls. Platelet serotonin content in cases was higher than in controls (611 ± 118 ng/10E(9) platelets vs. 536 ± 141 ng/10(9), p = 0.048). Even years after the event, elevations in the platelet dense granule contents between VF survivors and controls may be detected. These preliminary findings shed new light on the pathophysiological mechanisms underlying ischemic VF, as platelet-dense granules may contain mediators of early ischemic VF risk.

International audience; ABCA7, a close relative of ABCA1 which facilitates cholesterol efflux to lipid-poor apoproteins, has been implicated in macrophage lipid efflux and clearance of apoptotic cells in in vitro studies. In the current study, we investigated the in vivo effects of macrophage ABCA7 deficiency on lipid metabolism and atherosclerosis. Chimeras with dysfunctional ABCA7 in macrophages and other blood cells were generated by transplantation of bone marrow from ABCA7 knockout (KO) mice into irradiated low-density lipoprotein receptor (LDLr) KO mice. Unexpectedly, macrophage ABCA7 deficiency did not significantly affect atherosclerosis susceptibility of LDLr KO mice after 10 weeks Western-type diet feeding. However, ABCA7 deficiency was associated with 2-fold (p0.05) as compared to wild type transplanted mice. However, single deletion of ABCA1 had a similar effect (1.8-fold, p

Purpose of review Scavenger receptor class B type I (BI) is primarily known for its role in the selective uptake of cholesteryl esters from HDL, the final step in reverse cholesterol transport. Here, we will discuss findings that highlight the recently established novel link between scavenger receptor BI and adrenal and platelet function. Recent findings Human heterozygote carriers of a functional P297S mutation in the scavenger receptor BI gene show an attenuated adrenal glucocorticoid output and an altered platelet function. Scavenger receptor BI knockout mice lack adrenal cholesteryl ester stores and suffer from primary adrenal glucocorticoid insufficiency, indicating that adrenal HDL cholesteryl ester uptake by scavenger receptor BI is needed for generating the cholesterol pool used for steroidogenesis. Scavenger receptor BI knockout mice exhibit thrombocytopenia, an impaired platelet aggregation response, and higher susceptibility for arterial thrombosis. Bone marrow-specific deletion of scavenger receptor BI in mice indicates that scavenger receptor BI indirectly modulates platelet function through regulation of plasma cholesterol levels. Summary Scavenger receptor BI is not merely a crucial mediator of reverse cholesterol transport, but rather acts as a multipurpose player in cholesterol and steroid metabolism. Further understanding of the contribution of scavenger receptor BI's roles in adrenal steroidogenesis and platelet function to the pathogenesis of atherosclerosis and atherothrombosis will hopefully show its potential as a therapeutic target for cardiovascular benefit.

Objective— Disruption of scavenger receptor class B type I (SR-BI) in mice impairs high-density lipoprotein (HDL)–cholesterol (HDL-C) delivery to the liver and induces susceptibility to atherosclerosis. In this study, it was investigated whether introduction of cholesteryl ester transfer protein (CETP) can normalize HDL-C transport to the liver and reduce atherosclerosis in SR-BI knockout (KO) mice. Methods and Results— Expression of human CETP in SR-BI KO mice resulted in decreased plasma HDL-C levels, both on chow diet (1.8-fold, P P KO mice. Unexpectedly, expression of CETP in SR-BI KO mice did not reduce atherosclerotic lesion development, probably because of consequences of SR-BI deficiency, including the persistence of higher VLDL-cholesterol (VLDL-C) levels, unchanged elevated free cholesterol/total cholesterol ratio, and the increased oxidative status of the animals. In addition, CETP expression did not normalize other characteristics of SR-BI deficiency, including female infertility, reticulocytosis, thrombocytopenia, and impaired platelet aggregation. Conclusion— CETP restores HDL-C levels in SR-BI KO mice, but it does not change the susceptibility to atherosclerosis and other typical characteristics that are associated with SR-BI disruption. This may indicate that the pathophysiology of SR-BI deficiency is not a direct consequence of changes in the HDL pool.

Objective—The sensitivity of platelets to aggregating agents increases when low-density lipoprotein (LDL) binds to apolipoprotein E receptor 2′ (apoER2′), triggering activation of p38MAPKand formation of thromboxane A2. LDL signaling is terminated by PECAM-1 through recruitment and activation of the Ser/Thr protein phosphatase PP2A, but platelets remain unresponsive to LDL when PECAM-1 activation disappears. We report a second mechanism that halts LDL signaling and in addition lowers platelet responsiveness to aggregating agents.Methods and Results—After a first stimulation with LDL, platelets remain unresponsive to LDL for 60 minutes, despite normal apoER2′ activation by a second dose of LDL. A possible cause is persistent activation of the tyrosine phosphatases SHP-1 and SHP-2, which may not only block a second activation of p38MAPK, PECAM-1, and PP2A by LDL but also seem to reduce aggregation by TRAP, collagen, and ADP.Conclusion—These findings reveal that p38MAPKphosphorylation and platelet activation by LDL are suppressed by two mechanisms: (1) short activation of PECAM-1/PP2A, and (2) prolonged activation of SHP-1 and SHP-2. Activation of SHP-1 and SHP-2 is accompanied by reduced responsiveness to aggregating agents, which—if present in vivo—would make LDL an aggregation inhibitor during prolonged contact with platelets.

Binding of low density lipoprotein (LDL) to platelets enhances platelet responsiveness to various aggregation-inducing agents. However, the identity of the platelet surface receptor for LDL is unknown. We have previously reported that binding of the LDL component apolipoprotein B100 to platelets induces rapid phosphorylation of p38 mitogen-activated protein kinase (p38MAPK). Here, we show that LDL-dependent activation of this kinase is inhibited by receptor-associated protein (RAP), an inhibitor of members of the LDL receptor family. Confocal microscopy revealed a high degree of co-localization of LDL and a splice variant of the LDL receptor family member apolipoprotein E receptor-2 (apoER2') at the platelet surface, suggesting that apoER2' may contribute to LDL-induced platelet signaling. Indeed, LDL was unable to induce p38MAPK activation in platelets of apoER2-deficient mice. Furthermore, LDL bound efficiently to soluble apoER2', and the transient LDL-induced activation of p38MAPK was mimicked by an anti-apoER2 antibody. Association of LDL to platelets resulted in tyrosine phosphorylation of apoER2', a process that was inhibited in the presence of PP1, an inhibitor of Src-like tyrosine kinases. Moreover, phosphorylated but not native apoER2' co-precipitated with the Src family member Fgr. This suggests that exposure of platelets to LDL induces association of apoER2' to Fgr, a kinase that is able to activate p38MAPK. In conclusion, our data indicate that apoER2' contributes to LDL-dependent sensitization of platelets.

Mildly oxidized low-density lipoprotein activates platelets through lysophosphatidic acid (LPA). Hence, the platelet-activating properties attributed to native low-density lipoprotein (nLDL) might be caused by LPA contamination. We show that nLDL enhances thrombin receptor-activating peptide (TRAP)-induced fibrinogen binding to KIIbL L3. The LPA receptor blocker N-palmitoyl-L-serine-phosphoric acid did not affect nLDL-enhanced fibrinogen binding induced by TRAP, but reduced TRAP-induced binding. cAMP and inhibitors of protein kinase C and Ca 2+ rises completely blocked ligand binding by TRAP and nLDL/TRAP. Inhibitors of p38 MAPK and ADP secretion interfered only partially. Blockade of Rho-kinase increased ligand binding 2^3-fold. We conclude that nLDL enhances TRAP-induced fibrinogen binding independent of LPA. fl 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Objective— Scavenger receptor BI (SR-BI) is a cell surface receptor that promotes the selective uptake of cholesteryl esters from high-density lipoprotein (HDL) by the liver. In mice, SR-BI deficiency results in increased plasma HDL cholesterol levels and enhanced susceptibility to atherosclerosis. The aim of this study was to investigate the role of SR-BI deficiency on platelet function. Methods and Results— SR-BI-deficient mice were thrombocytopenic, and their platelets were abnormally large, probably because of an increased cholesterol content. The FeCl 3 acute injury model to study arterial thrombosis susceptibility showed that SR-BI wild-type mice developed total arterial occlusion after 24±2 minutes. In SR-BI-deficient mice, however, the time to occlusion was reduced to 13±1 minutes ( P =0.02). Correspondingly, in SR-BI-deficient mice, platelets circulated in an activated state and showed increased adherence to immobilized fibrinogen. In contrast, platelet-specific disruption of SR-BI by bone marrow transplantation in wild-type mice did not alter plasma cholesterol levels or affect platelet count, size, cholesterol content, or reactivity, suggesting that changes in plasma cholesterol levels were responsible for the altered responsiveness of platelets in SR-BI-deficient mice. Conclusion— The function of SR-BI in HDL cholesterol homeostasis and prevention of atherosclerosis is indirectly also essential for maintaining normal platelet function and prevention of thrombosis.

The interaction of platelets with low-density lipoprotein (LDL) plays an important role in the pathogenesis of atherosclerosis and thrombosis. Previously, we have shown that native LDL (nLDL) is a mild activator of platelets increasing their sensitivity to aggregation-inducing agents. Binding of nLDL to platelets was saturable, reversible and initiated signal transduction to p38MAPK suggesting the involvement of a receptor. A peptide mimic of the B-site in apoB100 resembled nLDL in its platelet-activating properties, suggesting that the receptor is a member of the LDL-receptor family. Platelets from familial hypercholesterolemia patients, who lack or have a defective apoB/E receptor, responded normally to nLDL and an antibody against this receptor left nLDL-induced activation of normal platelets undisturbed, excluding the involvement of the classical LDL (apoB/E)-receptor. In this study, we provide evidence that nLDL initiates platelet signaling to p38MAPK via a splice variant of the LDL-receptor family member Apolipoprotein E Receptor 2 (ApoER2′). This conclusion is based on (i) blockade of nLDL-induced p38MAPK activation by receptor-associated protein (RAP), an inhibitor of ligand binding to members of the LDL-receptor family, (ii) confocal microscopy showing a high degree of co-localization of nLDL and ApoER2′ at the platelet surface, (iii) binding of both nLDL and the B-site peptide to soluble ApoER2′, (iv) activation of p38MAPK by an anti-ApoER2 antibody with similar kinetics as nLDL, and (v) tyrosine phosphorylation of ApoER2′ upon binding of nLDL. The nLDL-induced phosphorylation of ApoER2′ could be abolished by PP1, an inhibitor of Src-like tyrosine kinases. In the absence of PP1, ApoER2′ phosphorylation was accompanied by co-association with the Src-family member Fgr. We conclude that binding of nLDL to platelets involves ApoER2′. Upon nLDL binding, the receptor is phosphorylated which induces the recruitment of Fgr, a kinase known to activate p38MAPK. The ApoER2′-Fgr complex subsequently activates p38MAPK, an upstream element in the formation of thromboxane A2 that primes the platelets to further stimulation by aggregation-inducing agents.