Suzanne J.A. Korporaal

Genetic variant of the scavenger receptor BI in humans

BACKGROUNDnIn 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.nnnMETHODSnWe 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.nnnRESULTSnCholesterol 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; P<0.001) and a reduced capacity for efflux of cholesterol from macrophages, but the carotid artery intima-media thickness was similar in carriers and in family noncarriers. Platelets from carriers had increased unesterified cholesterol content and impaired function. In carriers, adrenal steroidogenesis was attenuated, as evidenced by decreased urinary excretion of sterol metabolites, a decreased response to corticotropin stimulation, and symptoms of diminished adrenal function.nnnCONCLUSIONSnWe 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.).

Platelet Activation by Oxidized Low Density Lipoprotein Is Mediated by Cd36 and Scavenger Receptor-A

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 p38MAPK and by oxidized LDL (oxLDL) through lysophosphatidic acid (LPA) signaling to Rho A and Ca2+. Here we report a new mechanism for platelet activation by oxLDL. Methods and Results—Oxidation of nLDL increases p38MAPK 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 p38MAPK activation by oxLDL but combined blockade inhibits p38MAPK by >40% and platelet adhesion to fibrinogen under flow by >60%. Mouse platelets deficient in either CD36 or SR-A show normal p38MAPK activation by oxLDL but combined deficiency of CD36 and SR-A disrupts oxLDL-induced activation of p38MAPK 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.

Activation of Human Platelets by Misfolded Proteins

Objective—Protein misfolding diseases result from the deposition of insoluble protein aggregates that often contain fibrils called amyloid. Amyloids are found in Alzheimer disease, atherosclerosis, diabetes mellitus, and systemic amyloidosis, which are diseases where platelet activation might be implicated. Methods and Results—We induced amyloid properties in 6 unrelated proteins and found that all induced platelet aggregation in contrast to fresh controls. Amyloid-induced platelet aggregation was independent of thromboxane A2 formation and ADP secretion but enhanced by feedback stimulation through these pathways. Treatments that raised cAMP (iloprost), sequestered Ca2+ (BAPTA-AM) or prevented amyloid-platelet interaction (sRAGE, tissue-type plasminogen activator [tPA]) induced almost complete inhibition. Modulation of the function of CD36 (CD36−/− mice), p38MAPK (SB203580), COX-1 (indomethacin), and glycoprotein Ib&agr; (Nk-protease, 6D1 antibody) induced ≈50% inhibition. Interference with fibrinogen binding (RGDS) revealed a major contribution of &agr;IIb&bgr;3-independent aggregation (agglutination). Conclusions—Protein misfolding resulting in the appearance of amyloid induces platelet aggregation. Amyloid activates platelets through 2 pathways: one is through CD36, p38MAPK, thromboxane A2–mediated induction of aggregation; the other is through glycoprotein Ib&agr;–mediated aggregation and agglutination. The platelet stimulating properties of amyloid might explain the enhanced platelet activation observed in many diseases accompanied by the appearance of misfolded proteins with amyloid.

Platelet Activation by Low Density Lipoprotein and High Density Lipoprotein

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.

Native high-density lipoproteins inhibit platelet activation via scavenger receptor BI Role of negatively charged phospholipids

OBJECTIVESnHIGH-density lipoproteins (HDL) are a negative predictor of platelet-dependent thrombus formation and reduced platelet activation has been observed in vitro in the presence of HDL3, a major HDL fraction. However, mechanisms underlying the anti-thrombotic effects of HDL3 are poorly understood. Scavenger receptors class B represent possible HDL3 binding partners on platelets. We here investigated the role of scavenger receptor class B type I (SR-BI) and CD36 in mediating inhibitory effects of native HDL3 on thrombin-induced platelet activation.nnnMETHODS AND RESULTSnRhodamine isothiocyanate-labeled HDL3 bound specifically to platelets and HDL3 binding was inhibited by scavenger receptor class B ligands such as phosphatidylserine (PS)- or phosphatidylinositol (PI)-containing liposomes or maleylated albumin (mBSA). By contrast, scavenger receptor class A ligands failed to displace HDL3 from platelets. HDL3, PS- and PI-liposomes, and mBSA inhibited thrombin-induced platelet aggregation, fibrinogen binding, P-selectin expression and mobilization of intracellular Ca(2+). In addition, PS- and PI-liposomes emulated HDL3-induced intracellular signaling cascades including diacylglycerol production and protein kinase C activation. The reduction of platelet activation by liposomes was related to their PS or PI content. Moreover, inhibitory effects of native HDL3 were enhanced after enriching lipoproteins with PS, while PS- and PI-poor HDL2 failed to inhibit platelet aggregation and Ca(2+) mobilization. Both, HDL3 and PS-containing liposomes failed to inhibit thrombin-induced activation of platelets obtained from SR-BI-deficient mice but not CD36-deficient mice.nnnCONCLUSIONnWe suggest that SR-BI is a functional receptor for native HDL3 on platelets that generates an inhibitory signal for platelet activation. The content of negatively charged phospholipids (PS, PI) in HDL may be an important determinant of their anti-thrombotic potential.

Deletion of the High-Density Lipoprotein Receptor Scavenger Receptor BI in Mice Modulates Thrombosis Susceptibility and Indirectly Affects Platelet Function by Elevation of Plasma Free Cholesterol

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 FeCl3 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.

Restoration of High-Density Lipoprotein Levels by Cholesteryl Ester Transfer Protein Expression in Scavenger Receptor Class B Type I (SR-BI) Knockout Mice Does Not Normalize Pathologies Associated With SR-BI Deficiency

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-BIKO mice resulted in decreased plasma HDL-C levels, both on chow diet (1.8-fold, P<0.001) and on challenge with Western-type diet (1.6-fold, P<0.01). Furthermore, the presence of CETP partially normalized the abnormally large HDL particles observed in SR-BIKO mice. Unexpectedly, expression of CETP in SR-BIKO 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-BIKO 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.

Leukocyte-Specific CCL3 Deficiency Inhibits Atherosclerotic Lesion Development by Affecting Neutrophil Accumulation

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.

Deletion of the serotonin transporter in rats disturbs serotonin homeostasis without impairing liver regeneration.

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.

Effect of Oxidation on the Platelet-Activating Properties of Low-Density Lipoprotein

Objective—Because of the large variation in oxidizing procedures and susceptibility to oxidation of low-density lipoprotein (LDL) and the lack in quantification of LDL oxidation, the role of oxidation in LDL–platelet contact has remained elusive. This study aims to compare platelet activation by native LDL (nLDL) and oxidized LDL (oxLDL). Methods and Results—After isolation, nLDL was dialyzed against FeSO4 to obtain LDL oxidized to well-defined extents varying between 0% and >60%. The oxLDL preparations were characterized with respect to their platelet-activating properties. An increase in LDL oxidation enhances platelet activation via 2 independent pathways, 1 signaling via p38MAPK phosphorylation and 1 via Ca2+ mobilization. Between 0% and 15% oxidation, the p38MAPK route enhances fibrinogen binding induced by thrombin receptor (PAR-1)-activating peptide (TRAP), and signaling via Ca2+ is absent. At >30% oxidation, p38MAPK signaling increases further and is accompanied by Ca2+ mobilization and platelet aggregation in the absence of a second agonist. Despite the increase in p38MAPK signaling, synergism with TRAP disappears and oxLDL becomes an inhibitor of fibrinogen binding. Inhibition is accompanied by binding of oxLDL to the scavenger receptor CD36, which is associated with the fibrinogen receptor, &agr;IIb&bgr;3. Conclusion—At >30% oxidation, LDL interferes with ligand binding to integrin &agr;IIb&bgr;3, thereby attenuating platelet functions.