Activation of monocytes and platelets by monoclonal antibodies or malaria-infected erythrocytes binding to the CD36 surface receptor in vitro.

The CD36 leukocyte differentiation antigen, recognized by MAbs OKM5 and OKM8 and found on human monocytes and endothelial cells, has been implicated as a sequestration receptor for erythrocytes infected with the human malaria parasite Plasmodium falciparum (IRBC). CD36 is also expressed on platelets and appears to be identical to platelet glycoprotein IV. We investigated receptor activation of monocytes and platelets by anti-CD36 MAbs and by IRBC. Incubation of human monocytes with anti-CD36 MAbs or IRBC resulted in stimulation of the respiratory burst as measured by reduction of nitroblue tetrazolium and generation of chemiluminescence. Incubation of human platelets with anti-CD36 MAbs resulted in platelet activation as measured by aggregation or ATP secretion. Activation of monocytes and platelets required appropriate intracellular transmembrane signaling and was inhibited by calcium antagonists or by specific inhibitors of protein kinase C or guanine nucleotide binding proteins. Soluble CD36 inhibited binding of IRBC to both monocytes and platelets, suggesting that these interactions are mediated by the CD36 receptor. Using a cytochemical electron microscopic technique, the presence of reactive oxygen intermediates was identified at the interface between human monocytes and IRBC. These data provide support for the hypothesis that reactive oxygen intermediates produced by monocytes when IRBC ligands interact with cell surface receptors may play a role in the pathophysiology of falciparum malaria.     

Signaling role of CD36 in platelet activation and thrombus formation on immobilized thrombospondin or oxidized low‐density lipoprotein

Summary. Background and Objective: Platelets abundantly express glycoprotein CD36 with thrombospondin‐1 (TSP1) and oxidized low‐density lipoprotein (oxLDL) as proposed ligands. How these agents promote platelet activation is still poorly understood. Methods and Results: Both TSP1 and oxLDL caused limited activation of platelets in suspension. However, immobilized TSP1 and oxLDL, but not LDL, strongly supported platelet adhesion and spreading with a major role of CD36. Platelet spreading was accompanied by potent Ca²⁺ rises, and resulted in exposure of P‐selectin and integrin activation, all in a CD36‐dependent manner with additional contributions of α_IIbβ₃ and ADP receptor stimulation. Signaling responses via CD36 involved activation of the protein tyrosine kinase Syk. In whole blood perfusion, co‐coating of TSP1 or oxLDL with collagen enhanced thrombus formation at high‐shear flow conditions, with increased expression on platelets of activated α_IIbβ₃, P‐selectin and phosphatidylserine, again in a CD36‐dependent way. Conclusions: Immobilized TSP1 and oxLDL activate platelets partly via CD36 through a Syk kinase‐dependent Ca²⁺ signaling mechanism, which enhances collagen‐dependent thrombus formation under flow. These findings provide novel insight into the role of CD36 in hemostasis.     

Platelet CD36 mediates interactions with endothelial cell-derived microparticles and contributes to thrombosis in mice

CD36 is a scavenger receptor that binds multiple ligands, including phosphatidyl serine (PS). Although CD36(-) mice do not have a bleeding diathesis, we show here that they do have significantly prolonged thrombotic occlusion times in response to FeCl(3)-induced vascular injury. Because cell-derived microparticles (MPs) are generated in response to vascular injury and circulate in patients with prothrombotic diseases, we hypothesized that PS exposed on their surfaces could be an endogenous CD36 ligand that transmits an activating signal to platelets. We found that MPs prepared from human ECs, monocytes, or platelets or isolated from blood of normal subjects bound to platelets. Binding was not observed with platelets from CD36(-) donors and was inhibited by an anti-CD36 antibody or by blockade of exposed PS by annexin V or anti-PS IgM. Preincubation of platelets with MPs led to CD36-dependent augmentation of platelet activation in response to low doses of ADP, as assessed by measuring alpha(2b)beta(3) activation, P-selectin expression, and aggregation. Immunofluorescence confocal microscopy of murine carotid thrombi from CD36(-) mice showed a significant decrement in endothelial antigen accumulation, which suggests that CD36 plays a role in MP recruitment into thrombi. These results provide what we believe to be a novel role for CD36 in thrombosis.     

Complement-dependent Clearance of Apoptotic Cells by Human Macrophages

Apoptotic cells are rapidly engulfed by phagocytes, but the receptors and ligands responsible for this phenomenon are incompletely characterized. Previously described receptors on blood- derived macrophages have been characterized in the absence of serum and show a relatively low uptake of apoptotic cells. Addition of serum to the phagocytosis assays increased the uptake of apoptotic cells by more than threefold. The serum factors responsible for enhanced uptake were identified as complement components that required activation of both the classical pathway and alternative pathway amplification loop. Exposure of phosphatidylserine on the apoptotic cell surface was partially responsible for complement activation and resulted in coating the apoptotic cell surface with C3bi. In the presence of serum, the macrophage receptors for C3bi, CR3 (CD11b/CD18) and CR4 (CD11c/CD18), were significantly more efficient in the uptake of apoptotic cells compared with previously described receptors implicated in clearance. Complement activation is likely to be required for efficient uptake of apoptotic cells within the systemic circulation, and early component deficiencies could predispose to systemic autoimmunity by enhanced exposure to and/or aberrant deposition of apoptotic cells.     

The Immune Nature of Platelets Revisited

Platelets are the primary cellular mediators of hemostasis and this function firmly acquaints them with a variety of inflammatory processes. For example, platelets can act as circulating sentinels by expressing Toll-like receptors (TLR) that bind pathogens and this allows platelets to effectively kill them or present them to cells of the immune system. Furthermore, activated platelets secrete and express many pro- and anti-inflammatory molecules that attract and capture circulating leukocytes and direct them to inflamed tissues. In addition, platelets can directly influence adaptive immune responses via secretion of, for example, CD40 and CD40L molecules. Platelets are also the source of most of the microvesicles in the circulation and these miniscule elements further enhance the platelet’s ability to communicate with the immune system. More recently, it has been demonstrated that platelets and their parent cells, the megakaryocytes (MK), can also uptake, process and present both foreign and self-antigens to CD8+ T-cells conferring on them the ability to directly alter adaptive immune responses. This review will highlight several of the non-hemostatic attributes of platelets that clearly and rightfully place them as integral players in immune reactions.     

The Effect of Psychoactive Drugs on in vitro Platelet Function

BACKGROUND: Neuro-hormonal and hemostatic mechanisms are important in a wide range of psychological and cardiovascular diseases. The use of psychoactive drugs in mental illnesses is often involved with hematologic side effects including impaired platelet function. Subsequently, the risk for the development of cardiovascular diseases may be higher in these patients. Interestingly, platelets that play a key role in cardiovascular complications contain quite a number of neuronal receptors which are involved in psychotic disorders. It has been widely discussed whether psychoactive drugs used in the therapy of psychotic disorders have a direct effect on platelet function and whether the effects are transmitted through the corresponding receptors on the platelet surface. MATERIAL AND METHODS: In this study, we tested several psychoactive drugs regarding their impact on whole blood platelet aggregation. RESULTS: Antidopaminergics preferentially inhibited ADP-induced aggregation whereas anticholinergics mainly inhibited U46619-induced aggregation. Because platelets respond selectively to different psychoactive drugs we assume that corresponding receptors have a functional aspect on platelets and that receptor blockade affects platelet aggregation through different mechanisms. CONCLUSION: The knowledge about the effects of psychoactive drugs on platelet function may help to characterize neuronal receptors on platelets and may contribute to a better understanding of altered platelet function during therapy with psychoactive drugs.     

Scavenger receptors and oxidized low density lipoproteins.

Oxidized LDL has been shown to exhibit a number of potentially proatherogenic actions and properties, including receptor-mediated uptake and lipid accumulation within macrophages. It has been postulated that rapid, unregulated uptake of oxidatively modified LDL could account for the transformation of monocyte-derived macrophages to foam cells in atherosclerotic lesions. In support of this hypothesis, oxidized LDL and lipid peroxidation products have been shown to exist in atheromas in vivo. Furthermore, a number of cell membrane proteins that can bind oxidized LDL with high affinity have been identified on the surface of macrophages, endothelial cells and smooth muscle cells. One characteristic that almost all of these 'scavenger receptors' share is the ability to bind with high affinity to a broad spectrum of structurally unrelated ligands. Of all of the different classes of scavenger receptors that have been identified, the scavenger receptor class A type I/II (SR-AI/II) has received the most attention. Studies with macrophages from mice deficient in the gene for SR-AI/II provide direct evidence that a receptor other than the SR-AI/II is responsible for most of the uptake of oxidized LDL in murine macrophages. This article provides an overview of the characterization and functions of the scavenger receptors that have been shown to interact with oxidized LDL, including SR-AI/II, CD36, SR-BI, macrosialin/CD68, LOX-1, and SREC. Isolation and characterization of these and other scavenger receptors has increased our understanding of their role in the uptake of oxidized LDL and the pathogenesis of atherosclerosis.     

Isoimmunization against CD36 (glycoprotein IV): description of four cases of neonatal isoimmune thrombocytopenia and brief review of the literature

BACKGROUND: Platelet CD36 (glycoprotein [GP] IV) deficiency occurs in 3 to 5 percent of persons of Asian or African ancestry. A subset of these individuals is at risk for immunization against CD36, but the magnitude of this problem and its significance in transfusion medicine have not yet been clarified. STUDY DESIGN AND METHODS: Clinical and laboratory aspects of neonatal thrombocytopenia involving five infants born to four CD36- mothers were characterized. The CD36 gene was sequenced in three mothers. The literature concerning isoimmunization against CD36 was reviewed and summarized. RESULTS: Isoantibodies reactive with CD36 on normal platelets and platelets from the fathers were identified in each of the four mothers. Two African-American mothers were homozygous for a 1264TG mutation in the CD36 gene. A mother of Italian ancestry was homozygous for a previously unidentified deletion of exons 1 through 3. Previously reported cases of isoimmunization against CD36 were reviewed and summarized. CONCLUSION: Isoimmunization against CD36 can cause neonatal isoimmune thrombocytopenia (NITP), refractoriness to platelet transfusions, and post-transfusion purpura. Immunization against this glycoprotein (GP) should be considered in patients with apparent alloimmune platelet disorders not explained by immunization against recognized platelet-specific alloantigens, especially in persons of African, Asian, and, possibly, Mediterranean ancestry.     

Platelet‐based coagulation: different populations,different functions

Summary. Platelets in a thrombus interact with (anti)coagulation factors and support blood coagulation. In the concept of cell‐based control of coagulation, three different roles of platelets can be distinguished: control of thrombin generation, support of fibrin formation, and regulation of fibrin clot retraction. Here, we postulate that different populations of platelets with distinct surface properties are involved in these coagulant functions. Platelets with elevated Ca²⁺ and exposed phosphatidylserine control thrombin and fibrin generation, while platelets with activated α_IIbβ₃ regulate clot retraction. We review how coagulation factor binding depends on the platelet activation state. Furthermore, we discuss the ligands, platelet receptors and downstream intracellular signaling pathways implicated in these coagulant functions. These insights lead to an adapted model of platelet‐based coagulation.     

A functional integrin ligand on the surface of platelets: intercellular adhesion molecule-2.

Activated platelets express P-selectin and release leukocyte chemoattractants; however, they have not been known to express integrin ligands important in the stabilization of leukocyte interactions with the vasculature. We now demonstrate the presence of intercellular adhesion molecular-2 (ICAM-2) (CD102), and lack of expression of other beta 2-integrin ligands, ICAM-1 (CD54) and ICAM-3 (CD50), on the surface of resting and stimulated platelets. ICAM-2 isolated from platelets migrates as a band of 59,000 M(r) in reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Staining of bone marrow aspirates with anti-ICAM-2 mAb demonstrates strong reactivity to megakaryocytes. Using frozen thin sections and immunogold labeling, the antigen was shown to be present on the plasma membrane and surface-connected canalicular system of resting platelets. The average number of ICAM-2 molecules per platelet is 3,000 +/- 230 and does not change after activation. In adhesion assays, resting and stimulated platelets were capable of binding through ICAM-2 to purified leukocyte function-associated antigen-1. Activation of T lymphocytes with PMA stimulated binding to platelets that was Mg2+ dependent and could be specifically inhibited by mAbs to either ICAM-2 or leukocyte function-associated antigen-1. ICAM-2 is the only known beta 2-integrin ligand present on platelets, suggesting that it may play an important role in leukocyte-platelet interactions in inflammation and thrombosis.     

Analysis of the kinetics of TPO uptake during platelet transfusion

BACKGROUND: It has been shown in several studies that platelets play a role in the removal of TPO from the circulation. For instance, in vitro studies have shown that platelets can bind and internalize TPO, and transfusion studies have shown that the concentration of circulating TPO decreased after platelet transfusion. In the current study, the in vivo kinetics of plasma TPO levels and TPO uptake by transfused platelets is analyzed in more detail. STUDY DESIGN AND METHODS: Serial blood samples from patients who received a platelet transfusion were analyzed with respect to platelet count, plasma TPO concentration, and TPO content per platelet. In addition, the capacity of transfused platelets to bind TPO in vitro was assessed. RESULTS: Platelet counts increased immediately after transfusion, but subsequently started to decrease. Conversely, TPO levels decreased significantly but then returned to baseline level by 44 hours after transfusion. Platelet count and plasma TPO concentration were inversely correlated (r(p) = -0.9; p<0.05). The decrease in TPO concentration upon transfusion was accompanied by a significant increase in the platelet-associated TPO concentration. After transfusion, platelets isolated from the patient still displayed functional TPO receptors, as indicated by their intact capacity to bind TPO in vitro. CONCLUSION: The decrease in plasma TPO followed by the increase in platelet TPO provides evidence that platelets are responsible for the clearance of TPO in circulation. In vivo, platelets can bind and may degrade TPO upon platelet transfusion.     

人血小板表面CD36糖蛋白检测方法的建立及初步应用

个体血小板表面CD36抗原缺乏在随机输注时有产生抗-CD36免疫反应的风险,是血小板输注无效的原因之一。本研究应用流式细胞术检测杭州地区单采血小板供者的血小板表面CD36抗原表达情况,并分析个体血小板上CD36缺失表型的频率。留取献血者新鲜抗凝血样,经离心获取富血小板血浆,洗涤并调整血小板计数至1×106。采用CD36-FITC、CD41-PE单克隆抗体和血小板孵育反应,然后用流式细胞仪检测和分析血小板表面糖蛋白CD36抗原表达情况。对于血小板表面CD36抗原阴性的标本,进一步筛查其单核细胞表面CD36的表达情况。结果表明:192例无偿献血者筛查出7例血小板表面CD36抗原阴性,CD36缺失型频率为3.6%,均为Ⅱ型缺失。人群中个体CD36抗原表达强度存在差异,参照CD36几何平均荧光强度数值大小,59例为低表达,126例为高表达。结论:人群中存在CD36Ⅱ型缺失表型,这些数据将为研究CD36抗原分布提供参考,有助于解决血小板输注无效问题。     

The SLAM family member CD84 is regulated by ADAM10 and calpain in platelets

Hofmann S, Vögtle T, Bender M, Rose‐John S, Nieswandt B. The SLAM family member CD84 is regulated by ADAM10 and calpain in platelets. J Thromb Haemost 2012; 10: 2581–92. Summary. Background and objective: Ectodomain shedding is a major mechanism to modulate platelet receptor signaling and to downregulate platelet reactivity. Proteins of the a disintegrin and metalloproteinase (ADAM) family are implicated in the shedding of various platelet receptors. The signaling lymphocyte activation molecule (SLAM) family receptor CD84 is highly expressed in platelets and immune cells, but its role in platelet physiology is not well explored. Because of its ability to form homodimers, CD84 has been suggested to mediate contact‐dependent signaling and contribute to thrombus stability. However, nothing is known about the cellular regulation of CD84. Methods: We studied the regulation of CD84 in murine platelets by biochemical approaches and use of three different genetically modified mouse lines. Regulation of CD84 in human platelets was studied using inhibitors and biochemical approaches. Results: We show that CD84 is cleaved from the surface of human and murine platelets in response to different shedding inducing agents and platelet receptor agonists. CD84 downregulation occurs through ectodomain‐shedding and intracellular cleavage. Studies in transgenic mice identified ADAM10 as the principal sheddase responsible for CD84 cleavage, whereas ADAM17 was dispensable. Western blot analyses revealed calpain‐mediated intracellular cleavage of the CD84 C‐terminus, occurring simultaneously with, but independently of, ectodomain shedding. Furthermore, analysis of plasma and serum samples from transgenic mice demonstrated that CD84 is constitutively shed from the platelet surface by ADAM10 in vivo.Conclusions: These results reveal a dual regulation mechanism for platelet CD84 by simultaneous extra‐ and intracellular cleavage that may modulate platelet‐platelet and platelet‐immune cell interactions.     

Automated plateletpheresis does not cause an increase in platelet activation in volunteer donors.

Activation of platelets during collection and storage has been implicated as a major cause of the platelet storage lesion. In this study, we investigated the effect of an automated plateletpheresis procedure on the in vivo platelet activation in 20 volunteer donors. Peripheral blood samples were collected immediately before and after plateletpheresis on the Haemonetics V50 Blood Cell Separator. Activation of platelets was determined by quantitating the amount of platelet P-selectin (CD62) expression using a whole blood method on flow cytometry. Adenosine diphosphate (ADP), collagen, and ristocetin induced platelet aggregations were also measured on a whole blood impedance aggregometer. Plateletpheresis caused a significant decrease in the CD62-positive platelet percentage and aggregation responses to 3 agonists. We concluded that the plateletpheresis procedure did not cause an increase in platelet activation in donors. Further studies are required to elucidate whether activated platelets are collected during the procedure or removed from the circulation of the donor and replaced by resting platelets, activated platelets bind to leukocytes or endothelial cells, and the plateletpheresis procedure is a powerful stimulus for platelet activation.     

Identification of a new class of inducible receptors on platelets. Thrombospondin interacts with platelets via a GPIIb-IIIa-independent mechanism

Thrombospondin with fibrinogen, fibronectin, and von Willebrand factor binds to platelets stimulated with agonists and support platelet adhesive functions. The receptors for the latter three proteins are associated with membrane glycoprotein GPIIb-IIIa. Thrombasthenic platelets deficient in GPIIb-IIIa have been utilized to examine the role of this membrane protein in the interactions of thrombospondin with platelets. Radioiodinated thrombospondin bound to thrombin-stimulated platelets from normal and thrombasthenic donors with a similar affinity and capacity. As monitored with a monoclonal antibody to thrombospondin, the divalent ion-dependent and -independent pathways for the expression of the endogenous pool of thrombospondin on the surface of thrombin-stimulated platelets from normal and thrombasthenic donors were also qualitatively and quantitatively similar. GPIIb-IIIa or ligands associated with GPIIb-IIIa thus are not essential for the binding of thrombospondin to platelets. Therefore, thrombospondin interacts with unique receptors on platelets.     

Platelet chemokines and their receptors: what is their relevance to platelet storage and transfusion practice?

The role of platelets as inflammatory cells is demonstrated by the fact that they can release many growth factors and inflammatory mediators, including chemokines, when they are activated. The best known platelet chemokine family members are platelet factor 4 (PF4) and beta-thromboglobulin (beta-TG), which are synthesized in megakaryocytes, stored as preformed proteins in alpha-granules and released from activated platelets. However, platelets also contain many other chemokines such as interleukin-8 (IL-8), growth-regulating oncogene-alpha(GRO-alpha), epithelial neutrophil-activating protein 78 (ENA-78), regulated on activation normal T expressed and secreted (RANTES), macrophage inflammatory protein-1alpha (MIP-1alpha), and monocyte chemotactic protein-3 (MCP-3). They also express chemokine receptors such as CCR4, CXCR4, CCR1 and CCR3. Platelet activation is a feature of many inflammatory diseases such as heparin-induced thrombocytopenia, acquired immunodeficiency syndrome, and congestive heart failure. Substantial amounts of PF4, beta-TG and RANTES are released from platelets on activation, which may occur during storage. Although very few data are available on the in vivo effects of transfused chemokines, it has been suggested that the high incidence of adverse reactions often observed after platelet transfusions may be attributed to the chemokines present in the plasma of stored platelet concentrates.     

Identification of a functional receptor for granulocyte colony-stimulating factor on platelets.

Since granulocyte colony-stimulating factor (G-CSF) is thought to be a granulocyte lineage-specific cytokine, G-CSF receptors on blood cells other than those of granulocyte or monocyte lineage have not been well investigated. We now report that G-CSF receptors are present on platelets. The expression of G-CSF receptors on platelets was demonstrated by flow cytometry and radioreceptor assay. The mean number of G-CSF-binding sites per cell was 41 and the binding affinity was high (Kd 300 pM), similar to the affinity observed on granulocytes. Cross-linking assay revealed that G-CSF receptors were present on a single subunit protein of approximately 150 kD on the platelets. To clarify whether or not G-CSF might produce some direct functional influence on platelet response, the effects on platelet aggregation were studied. Although G-CSF itself did not affect platelet aggregation in vitro, preincubation with G-CSF augmented a secondary aggregation of platelets induced by low concentrations of adenosine diphosphate (ADP). There was a dose-response relationship for this G-CSF activity at concentrations of up to 10 ng/ml. Furthermore, the augmented ADP-induced secondary aggregation of platelets on G-CSF receptors was completely abrogated in the presence of anti-G-CSF polyclonal antibodies. These results indicate that platelets possess functional G-CSF receptors.     

Efficient phagocytosis of periodontopathogens by neutrophils requires plasma factors,platelets and TLR2

Summary. Background: Periodontitis represents a chronic infection of supportive dental tissues by distinct gram‐negative bacteria. It is characterized by chronic and local inflammation as well as transient bacteremia with frequently occurring infections at distant sites. Objectives: The present work aimed to clarify the role of platelets and plasma factors in neutrophil interactions with the periodontopathogens A. actinomycetemcomitans and P. gingivalis. Methods: Phagocytosis, cell–cell interactions and activation of platelets and neutrophils in response to periodontopathogens were analyzed by flow cytometry, confocal microscopy and bacteria survival assay. Plasma factors, platelet signaling pathways and receptors involved in platelet‐neutrophil‐bacteria interactions were determined. The role of platelet and neutrophil TLR2 in phagocytosis was further evaluated in a murine TLR2 knockout model. Results: In the presence of plasma neutrophil‐mediated clearance of periodontopathogens is doubled due to opsonisation of bacteria. Platelets, which become activated by periodontopathogens, further enhance clearance of bacteria by 20%, via direct interaction with neutrophils. Plasma factors (e.g. CD14) are required for platelet activation, which is mainly TLR2 dependent and results in PI3K/Akt activation. In a murine TLR2 knockout model we prove that platelet TLR2 is important for formation of platelet–neutrophil aggregates and enhanced phagocytosis of periodontopathogens. In contrast, neutrophil TLR2 is not involved in platelet–neutrophil aggregate formation but is required for efficient phagocytosis. Conclusions: These data indicate that efficient elimination of periodontopathogens by neutrophils involves a complex interplay of plasma factors as well as platelets and requires functional TLR2. By enhancing neutrophil activation platelets contribute to immune defense but may also foster inflammation.