The neurorepellent, Slit2, prevents macrophage lipid loading by inhibiting CD36-dependent binding and internalization of oxidized low-density lipoprotein.

Atherosclerosis is characterized by retention of modified lipoproteins, especially oxidized low density lipoprotein (oxLDL) within the sub-endothelial space of affected blood vessels. Recruited monocyte-derived and tissue-resident macrophages subsequently ingest oxLDL by binding and internalizing oxLDL via scavenger receptors, particularly CD36. The secreted neurorepellent, Slit2, acting through its transmembrane receptor, Roundabout-1 (Robo-1), was previously shown to inhibit recruitment of monocytes into nascent atherosclerotic lesions. The effects of Slit2 on oxLDL uptake by macrophages have not been explored. We report here that Slit2 inhibits uptake of oxLDL by human and murine macrophages, and the resulting formation of foam cells, in a Rac1-dependent and CD36-dependent manner. Exposure of macrophages to Slit2 prevented binding of oxLDL to the surface of cells. Using super-resolution microscopy, we observed that exposure of macrophages to Slit2 induced profound cytoskeletal remodeling with formation of a thick ring of cortical actin within which clusters of CD36 could not aggregate, thereby attenuating binding of oxLDL to the surface of cells. By inhibiting recruitment of monocytes into early atherosclerotic lesions, and the subsequent binding and internalization of oxLDL by macrophages, Slit2 could represent a potent new tool to combat individual steps that collectively result in progression of atherosclerosis.

Procoagulant Phosphatidylserine-Exposing Platelets in vitro and in vivo.

The physiological heterogeneity of platelets leads to diverse responses and the formation of discrete subpopulations upon platelet stimulation. Procoagulant platelets are an example of such subpopulations, a key characteristic of which is exposure either of the anionic aminophospholipid phosphatidylserine (PS) or of tissue factor on the activated platelet surface. This review focuses on the former, in which PS exposure on a subpopulation of platelets facilitates assembly of the intrinsic tenase and prothrombinase complexes, thereby accelerating thrombin generation on the activated platelet surface, contributing importantly to the hemostatic process. Mechanisms involved in platelet PS exposure, and accompanying events, induced by physiologically relevant agonists are considered then contrasted with PS exposure resulting from intrinsic pathway-mediated apoptosis in platelets. Pathologies of PS exposure, both inherited and acquired, are described. A consideration of platelet PS exposure as an antithrombotic target concludes the review.

Analysis of procoagulant phosphatidylserine-exposing platelets by imaging flow cytometry.

BACKGROUND: Upon platelet activation, a subpopulation of procoagulant platelets is formed, characterized by the exposure of the anionic aminophospholipid phosphatidylserine (PS) on the surface membrane. OBJECTIVE: To evaluate procoagulant PS-exposing platelets by imaging flow cytometry. METHODS: Platelet ultrastructure was examined by transmission electron microscopy, and a comprehensive analysis of procoagulant platelets was performed using imaging flow cytometry; platelets were fluorescently labeled for the markers glycoprotein (GP)IX, activated integrin αIIbß3, CD62P, and PS exposure. RESULTS: A subpopulation of platelets stimulated in suspension by the physiological agonists thrombin+collagen, and all platelets stimulated by the calcium ionophore A23187, had a distinct round morphology. These platelets were PS-exposing, larger in size, had an increased circularity index, and had reduced internal complexity compared with non-PS-exposing platelets. They expressed CD62P and αIIbß3 in an inactive conformation on the surface, and demonstrated depolarized inner mitochondrial membranes. For the first time, using imaging flow cytometry, a large proportion of PS-exposing platelets possessing platelet-associated extracellular vesicles (EVs) was observed, which demonstrated heterogeneous platelet marker expression that was different from free released EVs. CONCLUSIONS: Innovative imaging flow cytometry allowed detailed fluorescence-based, quantitative morphometric analysis of PS-exposing platelets; in becoming procoagulant, platelets undergo remarkable morphological changes, transforming into spherical "balloons," almost devoid of their normal internal architecture. Almost all PS-exposing platelets have associated EVs that are not detectable by traditional flow cytometry. While their functions have yet to be fully elucidated, the heterogeneity of platelet-associated and released EVs suggests that they may contribute to different aspects of hemostasis and of thrombosis.

Laboratory diagnosis of inherited platelet function disorders.

Platelets respond to vessel wall injury by forming a primary hemostatic plug to arrest blood loss. Hemostatic plug formation is complex, and involves platelet adhesion to the subendothelium that results in platelet activation and ultimately, aggregation. If any of these processes are deficient, primary hemostasis is impaired. Inherited platelet function disorders (IPFDs) are a heterogeneous group of defects in these processes, with patients experiencing mainly mucocutaneous bleeding symptoms that can range from very mild to life threatening, depending on the specific disorder. Here, we review the approach to an initial patient assessment required to inform laboratory testing, and the frequently used clinical laboratory assays for diagnostic evaluation of IPFDs. Newer testing approaches that may improve laboratory diagnosis in the near future are described.

Young steady-state rabbit platelets do not have an enhanced capacity to expose procoagulant phosphatidylserine.

Platelets are recognized to be physiologically and functionally heterogeneous. An example of the diversity in reactivity is the formation of a distinct subpopulation of procoagulant phosphatidylserine (PS)-exposing platelets upon activation. Platelet age has been proposed as a determinant of platelet function, and it has been reported that young platelets are more reactive in exposing PS; using the same methodology of thiazole orange (TO) staining to distinguish young and old platelets, the percentages of procoagulant platelets produced by thrombin plus collagen activation of platelets from healthy controls were examined by flow cytometry. The procoagulant subpopulation formed by TO-positive platelets (with high TO fluorescence), purported to be young reticulated platelets, was observed to be significantly larger than that formed by TO-negative platelets (with low TO fluorescence), purported to be older platelets. However, it was noted that TO fluorescence in the total platelet population was unimodal and increased with platelet size, assessed by forward scatter. This observation raised the concern that TO-positive platelets are not necessarily the youngest platelets in the condition of steady-state platelet production. Thus, to unequivocally determine whether platelet age is a factor in procoagulant platelet formation, a different approach to identify young, steady-state platelets was employed. Rabbits were injected with biotin to label >95% of circulating platelets in vivo; 24 hours post-biotinylation, the non-biotinylated platelets in the circulation, detected flow cytometrically, are the youngest, newly-formed platelets. It was demonstrated that these youngest platelets were not larger in size than older, biotinylated platelets, and that they did not have an enhanced capacity to expose PS.

The integrin PSI domain has an endogenous thiol isomerase function and is a novel target for antiplatelet therapy.

Integrins are a large family of heterodimeric transmembrane receptors differentially expressed on almost all metazoan cells. Integrin ß subunits contain a highly conserved plexin-semaphorin-integrin (PSI) domain. The CXXC motif, the active site of the protein-disulfide-isomerase (PDI) family, is expressed twice in this domain of all integrins across species. However, the role of the PSI domain in integrins and whether it contains thiol-isomerase activity have not been explored. Here, recombinant PSI domains of murine ß3, and human ß1 and ß2 integrins were generated and their PDI-like activity was demonstrated by refolding of reduced/denatured RNase. We identified that both CXXC motifs of ß3 integrin PSI domain are required to maintain its optimal PDI-like activity. Cysteine substitutions (C13A and C26A) of the CXXC motifs also significantly decreased the PDI-like activity of full-length human recombinant ß3 subunit. We further developed mouse anti-mouse ß3 PSI domain monoclonal antibodies (mAbs) that cross-react with human and other species. These mAbs inhibited αIIbß3 PDI-like activity and its fibrinogen binding. Using single-molecular Biomembrane-Force-Probe assays, we demonstrated that inhibition of αIIbß3 endogenous PDI-like activity reduced αIIbß3-fibrinogen interaction, and these anti-PSI mAbs inhibited fibrinogen binding via different levels of both PDI-like activity-dependent and -independent mechanisms. Importantly, these mAbs inhibited murine/human platelet aggregation in vitro and ex vivo, and murine thrombus formation in vivo, without significantly affecting bleeding time or platelet count. Thus, the PSI domain is a potential regulator of integrin activation and a novel target for antithrombotic therapies. These findings may have broad implications for all integrin functions, and cell-cell and cell-matrix interactions.

Regulation of platelet activity in a changing redox environment.

SIGNIFICANCE: The regulation of platelet function is finely tuned by a balance between the vasculature's redox environment and the oxidative processes that occur in it. The activation of platelets at sites of vascular damage is essential for the maintenance of normal hemostasis. In the extracellular milieu, a normal redox environment is maintained by thiol/disulfide redox couples, which include reduced and oxidized glutathione (GSH/GSSG) and cysteine (Cys/CySS). Oxidative changes in either of the plasma redox potentials are directly linked with risk factors for cardiovascular disease. RECENT ADVANCES: Many proteins found on the surface of platelets contain cysteine residues that are targets for oxidation. These include platelet-specific integrins and thiol isomerase enzymes that respond to changes in the extracellular redox environment, thus influencing normal platelet responses. CRITICAL ISSUES: The post-translational modification of critical cysteine thiol groups is linked to alterations in redox potentials and occurs both intracellularly and extracellularly in normal platelet activation. Platelet integrins, in particular, are prime targets for redox modification due to their high cysteine content. Although the role of thiol/disulfide bond exchange in platelet activation is established, the effects of a changing redox environment on platelet reactivity are unclear. FUTURE DIRECTIONS: A thorough understanding of these mechanisms and how they interact with other platelet signaling events is of the utmost importance for the development of novel therapeutic targets so that we can protect against inappropriate thrombus formation.