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.

BackgroundUpon platelet activation, a subpopulation of procoagulant platelets is formed, characterized by the exposure of the anionic aminophospholipid phosphatidylserine (PS) on the surface membrane.ObjectiveTo evaluate procoagulant PS‐exposing platelets by imaging flow cytometry.MethodsPlatelet 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 αIIbβ3, CD62P, and PS exposure.ResultsA 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 αIIbβ3 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.ConclusionsInnovative 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.

Integrins are a large family of heterodimeric transmembrane receptors differentially expressed on almost all metazoan cells. Integrin β 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 β3, and human β1 and β2 integrins were generated and their PDI-like activity was demonstrated by refolding of reduced/denatured RNase. We identified that both CXXC motifs of β3 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 β3 subunit. We further developed mouse anti-mouse β3 PSI domain monoclonal antibodies (mAbs) that cross-react with human and other species. These mAbs inhibited αIIbβ3 PDI-like activity and its fibrinogen binding. Using single-molecular Biomembrane-Force-Probe assays, we demonstrated that inhibition of αIIbβ3 endogenous PDI-like activity reduced αIIbβ3-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.

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.

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Platelet adhesion and aggregation at sites of vascular injury are key events in thrombosis and hemostasis. Platelet β3 integrins and their ligands are essential in mediating these processes. Therefore the understanding of β3 integrin-ligand interactions is crucial in elucidating mechanisms of thrombosis and hemostasis. In an effort to identify unknown ligands for β3 integrin, we used immobilized human platelet β3 integrin to capture proteins from human plasma. The isolated proteins were further analysed by 2D electrophoresis and mass spectrometry, and apolipoprotein A-IV (apoA-IV) was identified. ApoA-IV is an abundant plasma lipid binding protein secreted by the small intestine during dietary lipid absorption. Several studies in different ethnic populations have suggested that the level of apoA-IV is inversely correlated with cardiovascular diseases. However, the roles of apoA-IV in platelets and thrombosis are completely unknown. A single-molecule technique, biomembrane force probe (BFP), was employed to detect direct interactions between apoA-IV and platelet αIIbβ3 integrin. The BFP adhesion frequency assay demonstrated that apoA-IV bound to αIIbβ3 integrin on ADP treated platelets. ApoA-IV also bound to purified activated αIIbβ3 integrin or the integrin expressed on Chinese hamster ovary (CHO) cells. In comparison, apoA-IV did not significantly bind to αIIbβ3 integrin on resting platelets, GPIb-complex expressed on CHO cells, αMβ2 integrin expressed on K562 cells, nor purified α5β1 and αvβ3 integrins. Importantly, apoA-IV-αIIbβ3 interactions in these experiments could be completely inhibited by a blocking monoclonal antibody (M1) against β3 integrin. These data clearly demonstrated the specificity of apoA-IV for αIIbβ3 integrin. Furthermore, 2D kinetics measurements revealed that the effective 2D affinity of apoA-IV-αIIbβ3 is 43% of that between fibrinogen and αIIbβ3. The BFP competition assay showed that apoA-IV competitively inhibited fibrinogen-αIIbβ3 interactions at its physiological concentration. Platelet functional studies in vitro showed that recombinant apoA-IV significantly inhibited both mouse and human platelet aggregation following stimulation with various agonists. Consistently, platelet aggregation in platelet rich plasma of apoA-IV deficient mice (apoA-IV-/-) was enhanced. Depletion of human plasma apoA-IV also enhanced ADP-induced human platelet aggregation. In ex vivo perfusion chambers, recombinant apoA-IV inhibited human and mouse thrombus growth and dissolved pre-formed thrombi, while absence of apoA-IV in blood enhanced ex vivo thrombus growth under both low and high shear stresses. Using two in vivo intravital microscopy thrombosis models and a carotid artery thrombosis model, we demonstrated that FeCl3- and laser-induced thrombosis were enhanced in apoA-IV-/-mice, while transfusion of recombinant apoA-IV markedly attenuated this process. In addition, we found recombinant apoA-IV significantly decreased platelet P-selectin expression, and consistently more P-selectin expression was observed on ADP treated platelets from apoA-IV-/- mice, suggesting that apoA-IV occupancy may inhibit fibrinogen or other prothrombotic ligands mediated αIIbβ3 outside-in signaling. We further found that the N-terminus of apoA-IV plays a key role in its inhibitory function and the exposure of N-terminus is negatively regulated by its C-terminus. Furthermore, mutation of the two aspartic acid (D) residues at apoA-IV N-terminal 5 and 13 abolished its binding for αIIbβ3 integrin as demonstrated by BFP adhesion frequency assay, resulting in the loss of these inhibitory effects.These findings suggest that D5 and D13 of apoA-IV are the potential binding sites for αIIbβ3 integrin. Thus, apoA-IV is identified as a novel endogenous inhibitor of thrombosis and represents a new link between lipoprotein metabolism and platelet function, both of which play critical roles in cardiovascular diseases. These findings may also contribute to hemostasis, P-selectin mediated postprandial platelet activation and inflammation. Disclosures No relevant conflicts of interest to declare.

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.