Vitamin D, besides having an essential role in calcium and bone metabolism, also acts as a mediator of many non-calcemic effects through modulations of several biological responses. Vitamin D exists in its two major forms, vitamin D 2 , or commonly known as ergocalciferol, and vitamin D 3 , or commonly known as cholecalciferol. Both of these forms bind to vitamin D-binding protein to get transported to all vital target organs, where it serves as a natural ligand to vitamin D receptors for enabling their biological actions. Clinical reports corroborating vitamin D deficiency with an increase in thrombotic episodes implicate the role of vitamin D and its associated molecule in the regulation of thrombosis-related pathways. Thrombosis is the formation and propagation of a blood clot, known as thrombus. It can occur either in the arterial or the venous system resulting in many severe complications, including myocardial infarction, stroke, ischemia, and venous thromboembolism. Vitamin D, directly or indirectly, controls the expression of several genes responsible for the regulation of cellular proliferation, differentiation, apoptosis, and angiogenesis. All of these are the processes of potential relevance to thrombotic disorders. This review, thus, discussed the effects of vitamin D on pathways involved in thrombosis, such as hemostatic process, inflammatory pathway, and endothelial cell activation, with a focus on the molecular mechanisms associated with them.

There are several genetic and acquired risk factors for venous thromboembolism. Exposure to high altitude (HA), either during air travel, ascension of mountains, or while engaging in sports activities, has been observed to result in a hypercoagulable state, thus predisposing to thromboembolic events. Although several previous studies have suggested that conditions present at HAs contribute to establish a prothrombotic milieu, published reports are contradictory and the exact underlying mechanism remains poorly understood. Results from HA studies also show that environmental conditions at HA such as hypoxia, dehydration, hemoconcentration, low temperature, use of constrictive clothing as well as enforced stasis due to severe weather, would support the occurrence of thrombotic disorders. The three leading factors of Virchow triad, that is, venous stasis, hypercoagulability, and vessel-wall injury, all appear to be present at HA. In synthesis, the large list of environmental variables suggests that a single cause of HA-induced thromboembolic disorders (TED) may not exist, so that this peculiar phenomenon should be seen as a complex or multifactorial trait. Further investigation is needed to understand the risk of TED at HA as well as the possible underlying mechanisms.

Key Points• Hypoxia induces altered platelet proteome/reactivity, which correlates with a prothrombotic phenotype.• CAPNS1-dependent calpain activity in platelet activation cascade is associated with hypoxia-induced thrombogenesis.Oxygen-compromised environments, such as high altitude, air travel, and sports, and pathological conditions, such as solid tumors, have been suggested to be prothrombotic. Despite the indispensable role of platelets in thrombus formation, the studies linking hypoxia, platelet reactivity, and thrombus formation are limited. In the present study, platelet proteome/reactivity was analyzed to elucidate the acute hypoxia-induced prothrombotic phenotype. Rats exposed to acute simulated hypoxia (282 torr/8% oxygen) demonstrated a decreased bleeding propensity and increased platelet reactivity. Proteomic analysis of hypoxic platelets revealed 27 differentially expressed proteins, including those involved in coagulation. Among these proteins, calpain small subunit 1, a 28-kDa regulatory component for calpain function, was significantly upregulated under hypoxic conditions. Moreover, intraplatelet Ca 21 level and platelet calpain activity were also found to be in accordance with calpain small subunit 1 expression. The inhibition of calpain activity demonstrated reversal of hypoxia-induced platelet hyperreactivity. The prothrombotic role for calpain was further confirmed by an in vivo model of hypoxia-induced thrombosis. Interestingly, patients who developed thrombosis while at extreme altitude had elevated plasma calpain activities and increased soluble P-selectin level. In summary, this study suggests that augmented calpain activity is associated with increased incidence of thrombosis under hypoxic environments. (Blood. 2014;123(8):1250-1260

Numerous studies have reported the presence of oxidatively modified high-density lipoprotein (OxHDL) within the intima of atheromatous plaques as well as in plasma; however, its role in the pathogenesis of thrombotic disease is not established. We now report that OxHDL, but not native HDL, is a potent inhibitor of platelet activation and aggregation induced by physiologic agonists. This antithrombotic effect was concentration and time dependent and positively correlated with the degree of lipoprotein oxidation. Oxidized lipoproteins are known ligands for scavenger receptors type B, CD36 and scavenger receptor B type I (SR-BI), both of which are expressed on platelets. Studies using murine CD36(-/-) or SR-BI(-/-) platelets demonstrated that the antithrombotic activity of OxHDL depends on platelet SR-BI but not CD36. Binding to SR-BI was required since preincubation of human and murine platelets with anti-SR-BI blocking antibody abrogated the inhibitory effect of OxHDL. Agonist-induced aggregation of platelets from endothelial nitric oxide synthase (eNOS)(-/-), Akt-1(-/-), and Akt-2(-/-) mice was inhibited by OxHDL to the same degree as platelets from wild-type (WT) mice, indicating that the OxHDL effect is mediated by a pathway different from the eNOS/Akt pathway. These novel findings suggest that contrary to the prothrombotic activity of oxidized low-density lipoprotein (OxLDL), HDL upon oxidation acquires antithrombotic activity that depends on platelet SR-BI.

Recent studies have identified a novel family of oxidized phosphatidylcholines (oxPC CD36 ) that serve as highly specific ligands for scavenger receptor CD36. oxPC CD36 accumulate in vivo and mediate macrophage foam cell formation as well as promote platelet hyper-reactivity in hyperlipidemia via CD36. The structural basis of oxPC CD36 binding to CD36 has not been elucidated. We used liquid-phase binding to glutathione S-transferase fusion proteins containing various regions of CD36 to initially identify the region spanning CD36 amino acids 157-171 to contain a major binding site for oxPC CD36 CD36 is a 472-amino-acid, 88-kDa heavily glycosylated transmembrane protein that is expressed in various cell types including macrophages, platelets, microvascular endothelial cells, and adipocytes (1, 2). CD36 has been shown to play a significant role in a number of physiological and pathological processes in vivo including atherogenesis, lipid sensing and metabolism, innate immune responses, angiogenesis, uptake of apoptotic cells, and diabetes (2-4). CD36 involvement in such a variety of processes can be partially explained by its capacity to recognize a number of various distinct ligands. Examples of CD36 ligands include thrombospondin-1 (5), oxidized low density lipoproteins (oxLDL) 2 (6), oxidized phospholipids (7-9), fatty acids (10), microbial diacylglycerides (4), hexarelin (3), collagen (11), and malarial parasite-infected erythrocytes (12).That CD36 can function as a multiligand receptor is conceivable assuming that it has multiple ligand binding domains. Several studies suggest, for example, that the binding sites of thrombospondin-1 and oxLDL on CD36 are different (13,14). Two distinct binding sites are proposed for oxLDL on CD36. Studies using a monoclonal antibody have shown that domain 155-183 of CD36 plays a critical role in the binding of LDL oxidized by copper (15). Solid phase binding assays using recombinant fusion proteins spanning various regions of CD36, however, implicate the domain 28 -93 as the major binding site for oxLDL (16).We have recently identified a novel family of oxidized choline glycerophospholipids (oxPC CD36 ) that mediate CD36-dependent recognition of LDL oxidized by various pathways. The structural aspect of oxPC CD36 essential for high affinity binding to CD36 is an sn-2 acyl group that incorporates a terminal ␥-hydroxy(or oxo)-␣,␤-unsaturated carbonyl. A characteristic feature of oxPC CD36 conformation is a negatively charged distal end of the sn-2 acyl chain residue that partitions into the aqueous phase (17). oxPC CD36 are formed during the oxidation of LDL by multiple distinct pathways, serve as specific high affinity ligands for CD36 (9), and are present in vivo at sites of enhanced oxidative stress (18 -20). OxPC CD36 mediate foam cell formation induced by oxidized LDL via macrophage CD36 and induce a prothrombotic phenotype in hyperlipidemia via platelet CD36 (18,20).In this current study, we investigated the structural basis for the recognition of oxPC CD36 by CD36 using...

Hypercholesterolemia is associated with increased platelet sensitivity to agonists and a prothrombotic phenotype. Mechanisms of platelet hypersensitivity are poorly understood; however, increased platelet cholesterol levels associated with hypercholesterolemia were proposed as leading to hypersensitivity. Scavenger receptor class B type I (SR-BI) in the liver controls plasma high-density lipoprotein (HDL) levels, and SR-BI-deficient mice display a profound dyslipoproteinemia. SR-BI is also expressed on platelets, and recent studies have suggested a role for SR-BI in platelet function; however, its role in hemostasis is unknown. Our present studies demonstrated that nonbone marrow-derived SR-BI deficiency and the dyslipidemia associated with it lead to platelet hyperreactivity that was mechanistically linked to increased platelet cholesterol content. Platelet-specific deficiency of SR-BI, on the other hand, was associated with resistance to hyper- IntroductionDyslipidemia is frequently associated with increased platelet reactivity and thrombogenic potential. [1][2][3][4][5] Although subjects with increased measures of platelet reactivity are at increased prospective risk for coronary events and death, 3,6-9 the mechanisms modulating platelet reactivity in vivo during dyslipidemia are still poorly understood. A mechanistic link between oxidative stress associated with dyslipidemia and a prothrombotic phenotype have been recently established by us. 10 Our studies demonstrated that specific oxidized phospholipids accumulate in plasma in dyslipidemia and interact with platelet CD36, leading to enhanced platelet reactivity, activation, and thrombosis. 10 Dyslipidemia is associated with changes in cellular cholesterol balance leading, in some cases, to increases in platelet cholesterol content or cholesterol/phospholipids ratio. A direct role of excessive platelet cholesterol in induction of platelet hyperreactivity has been shown. 1,2,11,12 However, the molecular mechanisms and pathways linking increased platelet reactivity and cholesterol levels in platelets are not well understood. One critical player in cholesterol metabolism is scavenger receptor class B type I (SR-BI), a multiligand receptor of the CD36 superfamily. 13 Its major physiologic function is selective uptake of cholesteryl esters from high-density lipoprotein (HDL) in steroidogenic tissues and liver. 13 SR-BI also stimulates the bidirectional flux of free cholesterol between cells and lipoproteins, modifies membrane cholesterol distribution, and induces signaling events. 14 SR-BI-deficient mice are hypercholesterolemic with abnormally large circulating HDL particles. 15 Platelets of SR-BI-deficient mice exhibit abnormally high unesterified cholesterol, abnormal morphologies, and elevated rates of clearance from the circulation. 16 Surprisingly, despite high platelet cholesterol content, platelets of SR-BIdeficient mice exhibited in vitro either normal or blunted aggregation responses to agonist. 16 Recent studies have shown that SR-BI is exposed on re...

Specific oxidized phospholipids (oxPC CD36 ) accumulate in vivo at sites of oxidative stress and serve as high affinity ligands for scavenger receptors class B (CD36 and SR-BI). Recognition of oxPC CD36 by scavenger receptors plays a role in several pathophysiological processes. The structural basis for the recognition of oxPC CD36 by CD36 and SR-BI is poorly understood. A characteristic feature of oxPC CD36 is an sn-2 acyl group that incorporates a terminal ␥-hydroxy (or oxo)-␣,␤-unsaturated carbonyl. In the present study, a series of model oxidized phospholipids were designed, synthesized, and tested for their ability to serve as ligands for CD36 and SR-BI. We demonstrated that intact the sn-1 hydrophobic chain, the sn-3 hydrophilic phosphocholine or phosphatidic acid group, and the polar sn-2 tail are absolutely essential for high affinity binding. We further found that a terminal negatively charged carboxylate at the sn-2 position suffices to generate high binding affinity to class B scavenger receptors. In addition, factors such as polarity, rigidity, optimal chain length of sn-2, and sn-3 positions and negative charge at the sn-3 position of phospholipids further modulate the binding affinity. We conclude that all three positions of oxidized phospholipids are essential for the effective recognition by scavenger receptors class B. Furthermore, the structure of residues in these positions controls the affinity of the binding. The present studies suggest that, in addition to oxPC CD36 , other oxidized phospholipids observed in vivo may represent novel ligands for scavenger receptors class B. Specific oxidized phospholipids (oxPC CD36 )3 accumulate at sites of oxidative stress in vivo such as within atherosclerotic lesions and plasma in dyslipidemia (1, 2). They serve as high affinity ligands for scavenger receptors class B: CD36 and SR-BI (3, 4). Recognition of oxPC CD36 on the surface of cell membranes and lipoprotein particles by scavenger receptors class B plays an important role in several pathophysiological processes, including atherosclerosis and thrombosis. oxPC CD36 phospholipids mediate uptake of oxidized low density lipoprotein (oxLDL) by macrophages via CD36 and promote a pro-thrombotic state via platelet scavenger receptor CD36 (1, 2). oxPC CD36 phospholipids also prevent binding of high density lipoprotein by SR-BI because of the close proximity of the binding sites for these two ligands on SR-BI. Furthermore, oxPC CD36 interfere with SR-BI-mediated selective uptake of cholesteryl esters in hepatocytes (4). These data demonstrate that oxidative stress and accumulation of specific oxidized phospholipids may have a detrimental effect due to specific interaction with scavenger receptors class B. However, the exact molecular mechanism of the recognition of oxPC CD36 by scavenger receptors class B is poorly understood.Initial studies have demonstrated that the sn-2 acyl group of oxPC CD36 that incorporates a terminal ␥-hydroxy (or oxo)-␣,␤-unsaturated carbonyl is essential for high affinity binding to...

We have recently demonstrated that specific oxidized phospholipids (oxPC CD36 ) accumulate at sites of oxidative stress in vivo such as within atherosclerotic lesions, hyperlipidemic plasma, and plasma with low high-density lipoprotein levels. oxPC CD36 serve as high affinity ligands for the scavenger receptor CD36, mediate uptake of oxidized low density lipoprotein by macrophages, and promote a pro-thrombotic state via platelet scavenger receptor CD36. We now report that oxPC CD36 represent ligands for another member of the scavenger receptor class B, type I (SR-BI). oxPC CD36 prevent binding to SR-BI of its physiological ligand, high density lipoprotein, because of the close proximity of the binding sites for these two ligands on SR-BI. Furthermore, oxPC CD36 interfere with SR-BI-mediated selective uptake of cholesteryl esters in hepatocytes. Thus, oxidative stress and accumulation of specific oxidized phospholipids in plasma may have an inhibitory effect on reverse cholesterol transport.Atherosclerosis is a chronic inflammatory disease in which macrophage accumulation of cholesterol and subsequent foam cell formation are critical events. Accumulation of cholesterol in macrophages is a result of failure to adequately adjust cellular cholesterol efflux in conditions of dramatically increased cholesterol acquisition. Efflux of excess of cholesterol from macrophages to HDL 2 and to free apolipoproteins and subsequent delivery to the liver for excretion, a process called reverse cholesterol transport, are critical for the maintenance of cholesterol balance. One of the key steps in reverse cholesterol transport is binding of HDL in the liver to hepatocyte SR-BI followed by selective uptake of cholesteryl esters (CE) from HDL. Multiple studies employing various murine models of atherosclerosis demonstrated that hepatic SR-BI is atheroprotective (1-4). Processes that interfere with SR-BI-mediated selective uptake of CE are of particular interest because they are potentially proatherogenic and may represent novel mechanisms contributing to the development of atherosclerosis.SR-BI belongs to the evolutionarily conserved CD36 family of proteins, sharing 30% sequence identity with CD36 (5). SR-BI is an 82-kDa membrane glycoprotein containing a large extracellular domain and two transmembrane domains with short cytoplasmic amino-and carboxyl-terminal tails (6). Similar to CD36 it is a multifunctional protein; however, its major function is selective uptake of CE from HDL in steroidogenic tissues. SR-BI-mediated selective uptake of HDL CE is a two-step process. The first step involves lipoprotein binding to the extracellular domain of SR-BI, and the second step is the selective transfer of lipid from HDL to the plasma membrane. SR-BI shares with CD36 an affinity for a wide array of ligands, including native and modified lipoproteins, advanced glycation end products, and anionic phospholipids (7,8).Atherosclerosis is associated with oxidative stress and the generation of biologically active oxidized lipids. It has been prev...