Shaojing Ye

The Platelet Release Reaction: Just when you thought platelet secretion was simple

Purpose of reviewIn response to agonists produced at vascular lesions, platelets release a host of components from their three granules: dense core, alpha, and lysosome. This releasate activates other platelets, promotes wound repair, and initiates inflammatory responses. Although widely accepted, the specific mechanisms underlying platelet secretion are only now coming to light. This review focuses on the core machinery required for platelet secretion. Recent findingsProteomic analyses have provided a catalog of the components released from activated platelets. Experiments using a combination of in-vitro secretion assays and knockout mice have led to assignments of both vesicle-soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (v-SNARE) and target membrane SNARE to each of the three secretion events. SNARE knockout mice are also proving to be useful models for probing the role of platelet exocytosis in vivo. Other studies are beginning to identify SNARE regulators, which control when and where SNAREs interact during platelet activation. SummaryA complex set of protein–protein interactions control the membrane fusion events required for the platelet release reaction. SNARE proteins are the core elements but the proteins that control SNARE interactions represent key points at which platelet signaling cascades could affect secretion and thrombosis.

Distinct Roles for Rap1b Protein in Platelet Secretion and Integrin αIIbβ3 Outside-in Signaling*

Background: Rap1b is a small G protein that is a key regulator for platelet activation. Results: Agonist-induced Rap1b activation plays a role in platelet secretion, and integrin outside-in signaling-mediated Rap1b activation is important in platelet spreading on fibrinogen and clot retraction. Conclusion: There are dual activation mechanisms of Rap1 that play distinct roles in platelet function. Significance: Learning two novel functions of Rap1b in platelets. Rap1b is activated by platelet agonists and plays a critical role in integrin αIIbβ3 inside-out signaling and platelet aggregation. Here we show that agonist-induced Rap1b activation plays an important role in stimulating secretion of platelet granules. We also show that αIIbβ3 outside-in signaling can activate Rap1b, and integrin outside-in signaling-mediated Rap1b activation is important in facilitating platelet spreading on fibrinogen and clot retraction. Rap1b-deficient platelets had diminished ATP secretion and P-selectin expression induced by thrombin or collagen. Importantly, addition of low doses of ADP and/or fibrinogen restored aggregation of Rap1b-deficient platelets. Furthermore, we found that Rap1b was activated by platelet spreading on immobilized fibrinogen, a process that was not affected by P2Y12 or TXA2 receptor deficiency, but was inhibited by the selective Src inhibitor PP2, the PKC inhibitor Ro-31-8220, or the calcium chelator demethyl-1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis. Clot retraction was abolished, and platelet spreading on fibrinogen was diminished in Rap1b-deficient platelets compared with wild-type controls. The defects in clot retraction and spreading on fibrinogen of Rap1b-deficient platelets were not rescued by addition of MnCl2, which elicits αIIbβ3 outside-in signaling in the absence of inside-out signaling. Thus, our results reveal two different activation mechanisms of Rap1b as well as novel functions of Rap1b in platelet secretion and in integrin αIIbβ3 outside-in signaling.

Platelet secretion and hemostasis require syntaxin-binding protein STXBP5

Genome-wide association studies (GWAS) have linked genes encoding several soluble NSF attachment protein receptor (SNARE) regulators to cardiovascular disease risk factors. Because these regulatory proteins may directly affect platelet secretion, we used SNARE-containing complexes to affinity purify potential regulators from human platelet extracts. Syntaxin-binding protein 5 (STXBP5; also known as tomosyn-1) was identified by mass spectrometry, and its expression in isolated platelets was confirmed by RT-PCR analysis. Coimmunoprecipitation studies showed that STXBP5 interacts with core secretion machinery complexes, such as syntaxin-11/SNAP23 heterodimers, and fractionation studies suggested that STXBP5 also interacts with the platelet cytoskeleton. Platelets from Stxbp5 KO mice had normal expression of other key secretory components; however, stimulation-dependent secretion from each of the 3 granule types was markedly defective. Secretion defects in STXBP5-deficient platelets were confirmed via lumi-aggregometry and FACS analysis for P-selectin and LAMP-1 exposure. Interestingly, STXBP5-deficient platelets had altered granule cargo levels, despite having normal morphology and granule numbers. Consistent with secretion and cargo deficiencies, Stxbp5 KO mice showed dramatic bleeding in the tail transection model and defective hemostasis in the FeCl3-induced carotid injury model. Transplantation experiments indicated that these defects were due to loss of STXBP5 in BM-derived cells. Our data demonstrate that STXBP5 is required for normal arterial hemostasis, due to its contributions to platelet granule cargo packaging and secretion.

Characterization of a Novel Integrin Binding Protein, VPS33B, Which is Important for Platelet Activation and in vivo Thrombosis and Hemostasis

Background— Integrins are heterodimeric (&agr;/&bgr;) membrane proteins that play fundamental roles in many biological processes, for example, cell adhesion and spreading, which are important for platelet function and hemostasis. The molecular mechanism that regulates integrin activation is not completely understood. Methods and Results— Here, we show that VPS33B, a member of the Sec1/Munc18 family, binds directly to the integrin &bgr; subunit. Overexpression of VPS33B in Chinese hamster ovary cells potentiated &agr;IIb&bgr;3 outside-in signaling but not inside-out signaling. Platelets, from megakaryocyte- and platelet-specific VPS33B conditional knockout mice, had normal morphology, yet their spreading on fibrinogen was impaired and they failed to support clot retraction. Platelet aggregation and ATP secretion in response to low-dose agonists were reduced in the VPS33B knockout mice. &agr;IIb&bgr;3-mediated endocytosis of fibrinogen was also defective. Tail bleeding times and times to occlusion in an FeCl3-induced thrombosis model were prolonged in the VPS33B knockout mice. Furthermore, VPS33B acted upstream of the RhoA-ROCK-MLC and Rac1-dependent pathways that lead to clot retraction and cell spreading, respectively. Conclusions— Our work demonstrates that vesicular trafficking complexes, containing VPS33B, are a novel class of modifiers of integrin function. Our data also provide insights into the molecular mechanism and treatment of arthrogryposis, renal dysfunction, and cholestasis syndrome.

Advanced cardiac chemical exchange saturation transfer (cardioCEST) MRI for in vivo cell tracking and metabolic imaging.

An improved pre‐clinical cardiac chemical exchange saturation transfer (CEST) pulse sequence (cardioCEST) was used to selectively visualize paramagnetic CEST (paraCEST)‐labeled cells following intramyocardial implantation. In addition, cardioCEST was used to examine the effect of diet‐induced obesity upon myocardial creatine CEST contrast. CEST pulse sequences were designed from standard turbo‐spin‐echo and gradient‐echo sequences, and a cardiorespiratory‐gated steady‐state cine gradient‐echo sequence. In vitro validation studies performed in phantoms composed of 20 mM Eu‐HPDO3A, 20 mM Yb‐HPDO3A, or saline demonstrated similar CEST contrast by spin‐echo and gradient‐echo pulse sequences. Skeletal myoblast cells (C2C12) were labeled with either Eu‐HPDO3A or saline using a hypotonic swelling procedure and implanted into the myocardium of C57B6/J mice. Inductively coupled plasma mass spectrometry confirmed cellular levels of Eu of 2.1 × 10−3 ng/cell in Eu‐HPDO3A‐labeled cells and 2.3 × 10−5 ng/cell in saline‐labeled cells. In vivo cardioCEST imaging of labeled cells at ±15 ppm was performed 24 h after implantation and revealed significantly elevated asymmetric magnetization transfer ratio values in regions of Eu‐HPDO3A‐labeled cells when compared with surrounding myocardium or saline‐labeled cells. We further utilized the cardioCEST pulse sequence to examine changes in myocardial creatine in response to diet‐induced obesity by acquiring pairs of cardioCEST images at ±1.8 ppm. While ventricular geometry and function were unchanged between mice fed either a high‐fat diet or a corresponding control low‐fat diet for 14 weeks, myocardial creatine CEST contrast was significantly reduced in mice fed the high‐fat diet. The selective visualization of paraCEST‐labeled cells using cardioCEST imaging can enable investigation of cell fate processes in cardioregenerative medicine, or multiplex imaging of cell survival with imaging of cardiac structure and function and additional imaging of myocardial creatine. Copyright

Lysophospholipid mediators in the vasculature

Acting through cell surface receptors, “extracellular” lysophosphatidic acid (LPA) influences cell growth, differentiation, apoptosis and development in a wide spectrum of settings [1–5]. Within the vasculature, smooth muscle cells [6, 7], endothelial cells [8] and platelets [9, 10] display notable responses to LPA [11, 12], which likely regulate blood vessel development and contribute to vascular pathology. The bioactive effects of LPA are mediated by a family of G-protein coupled receptors with at least six members (termed LPA1-6 that are encoded by the LPAR genes in humans and Lpar in mice) [1–3]. LPA may also serve as a ligand for the receptor for advanced glycation end products (RAGE) [13]. This review summarizes evidence to support a role for LPA signaling in vascular biology based on studies of LPA receptors and enzymes that produce or metabolize the lipid (Figure 1). Figure 1 Autotaxin (ATX) and LPA actions in blood and vascular cells. LPA receptors The receptors for LPA are widely distributed on blood and vascular cells. In preclinical animal models, targeting the LPA receptors genetically and pharmacologically suggests that they may contribute to vascular injury and inflammatory responses, as well as endothelial barrier function and vascular stability. Single and multiple deletions of LPA receptors in mice produce differing vascular phenotypes. Deficiency of Lpar1, which results in 50% neonatal lethality, gives rise to the development of spontaneous frontal hematomas [14]. This suggests a role for LPA1 in stabilization of vessels, as no defect in hemostasis has been observed in these animals. In experimental arterial injury models, LPA1 regulates the development of intimal hyperplasia, a complex response involving inflammation and smooth muscle cell proliferation and migration. LPA1 may influence the vascular remodeling response via the Gα12/Gα13 pathway that couples to RhoGEF to activate RhoA, given the similarities in development on intimal hyperplasia after injury in the Lpar1−/− mice [6] and those lacking the Gα12/Gα13 and Rho pathways [15] in smooth muscle cells. The lack of LPA1 disrupts the endothelial barrier and results in increased vascular permeability in response to inflammatory stimuli in the lung [16] and the skin [17]. Conversely, LPA1 antagonists prevent inflammation in response to peritoneal injection of lipopolysaccharide [18]. Whether either a defect in smooth muscle or endothelial cell function accounts for the bleeding observed in the Lpar1−/− mice remains unknown. Knockout of both Lpa1 and Lpa2 increases the incidence of prefrontal hematomas [19], impairs the response to vascular injury [6], and results in the development of pulmonary hypertension with age [20]; the latter phenotype in not observed in mice with deficiency of either of the receptors alone. Together, these results suggest some redundancy or overlap between the 2 receptor systems. Likewise, LPA1 and LPA3 antagonists reduce arterial remodeling elicited by denudation injury [7] in mice, perhaps due to attenuated signaling through both G12/G13 and Gq/G11 signaling pathways, which appear to regulate vascular remodeling antagonistically. Lpar4-deficient mice display a genetic background-dependent defect in formation of vasculature. On the C57Bl/6 background, the mice develop hemorrhage and edema due to a maturation defect from lack of smooth muscle cell and pericytes recruitment vessels [21]. As described in more detail below, studies in zebrafish also support a role for several of the canonical LPA receptors in blood vessel formation. Additionally, LPA signaling through RAGE may also affect SMC function [13].

Pharmacological Elevation of Circulating Bioactive Phosphosphingolipids Enhances Myocardial Recovery After Acute Infarction

Acute myocardial infarction (AMI) triggers mobilization of bone marrow (BM)‐derived stem/progenitor cells (BMSPCs) through poorly understood processes. Recently, we postulated a major role for bioactive lipids such as sphingosine‐1 phosphate (S1P) in mobilization of BMSPCs into the peripheral blood (PB). We hypothesized that elevating S1P levels after AMI could augment BMSPC mobilization and enhance cardiac recovery after AMI. After AMI, elevating bioactive lipid levels was achieved by treating mice with the S1P lyase inhibitor tetrahydroxybutylimidazole (THI) for 3 days (starting at day 4 after AMI) to differentiate between stem cell mobilization and the known effects of S1P on myocardial ischemic pre‐ and postconditioning. Cardiac function was assessed using echocardiography, and myocardial scar size evolution was examined using cardiac magnetic resonance imaging. PB S1P and BMSPCs peaked at 5 days after AMI and returned to baseline levels within 10 days (p < .05 for 5 days vs. baseline). Elevated S1P paralleled a significant increase in circulating BMSPCs (p < .05 vs. controls). We observed a greater than twofold increase in plasma S1P and circulating BMSPCs after THI treatment. Mechanistically, enhanced BMSPC mobilization was associated with significant increases in angiogenesis, BM cell homing, cardiomyocytes, and c‐Kit cell proliferation in THI‐treated mice. Mice treated with THI demonstrated better recovery of cardiac functional parameters and a reduction in scar size. Pharmacological elevation of plasma bioactive lipids after AMI could contribute to BMSPC mobilization and could represent an attractive strategy for enhancing myocardial recovery and improving BMSC targeting.

Cardiac Chemical Exchange Saturation Transfer MR Imaging Tracking of Cell Survival or Rejection in Mouse Models of Cell Therapy

Purpose To examine whether cardiac chemical exchange saturation transfer (CEST) imaging can be serially and noninvasively used to probe cell survival or rejection after intramyocardial implantation in mice. Materials and Methods Experiments were compliant with the National Institutes of Health Guidelines on the Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee. One million C2C12 cells labeled with either europium (Eu) 10-(2-hydroxypropyl)-1,4,7-tetraazacyclododecane-1,4,7-triacetic acid (HP-DO3A) or saline via the hypotonic swelling technique were implanted into the anterior-lateral left ventricular wall in C57BL/6J (allogeneic model, n = 17) and C3H (syngeneic model, n = 13) mice. Imaging (frequency offsets of ±15 parts per million) was performed 1, 10, and 20 days after implantation, with the asymmetrical magnetization transfer ratio (MTRasym) calculated from image pairs. Histologic examination was performed at the conclusion of imaging. Changes in MTRasym over time and between mice were assessed by using two-way repeated-measures analysis of variance. Results MTRasym was significantly higher in C3H and C57BL/6J mice in grafts of Eu-HP-DO3A-labeled cells (40.2% ± 5.0 vs 37.8% ± 7.0, respectively) compared with surrounding tissue (-0.67% ± 1.7 vs -1.8% ± 5.3, respectively; P < .001) and saline-labeled grafts (-0.4% ± 6.0 vs -1.2% ± 3.6, respectively; P < .001) at day 1. In C3H mice, MTRasym remained increased (31.3% ± 9.2 on day 10, 28.7% ± 5.2 on day 20; P < .001 vs septum) in areas of in Eu-HP-DO3A-labeled cell grafts. In C57BL/6J mice, corresponding MTRasym values (11.3% ± 8.1 on day 10, 5.1% ± 9.4 on day 20; P < .001 vs day 1) were similar to surrounding myocardium by day 20 (P = .409). Histologic findings confirmed cell rejection in C57BL/6J mice. Estimation of graft area was similar with cardiac CEST imaging and histologic examination (R2 = 0.89). Conclusion Cardiac CEST imaging can be used to image cell survival and rejection in preclinical models of cell therapy.