Vesicle Associated Membrane Protein 8 (VAMP8)-mediated Zymogen Granule Exocytosis Is Dependent on Endosomal Trafficking via the Constitutive-Like Secretory Pathway

Acinar cell zymogen granules (ZG) express 2 isoforms of the vesicle-associated membrane protein family (VAMP2 and -8) thought to regulate exocytosis. Expression of tetanus toxin to cleave VAMP2 in VAMP8 knock-out (−/−) acini confirmed that VAMP2 and -8 are the primary VAMPs for regulated exocytosis, each contributing ∼50% of the response. Analysis of VAMP8^−/− acini indicated that although stimulated secretion was significantly reduced, a compensatory increase in constitutive secretion maintained total secretion equivalent to wild type (WT). Using a perifusion system to follow secretion over time revealed VAMP2 mediates an early rapid phase peaking and falling within 2–3 min, whereas VAMP8 controls a second prolonged phase that peaks at 4 min and slowly declines over 20 min to support the protracted secretory response. VAMP8^−/− acini show increased expression of the endosomal proteins Ti-VAMP7 (2-fold) and Rab11a (4-fold) and their redistribution from endosomes to ZGs. Expression of GDP-trapped Rab11a-S25N inhibited secretion exclusively from the VAMP8 but not the VAMP2 pathway. VAMP8^−/− acini also showed a >90% decrease in the early endosomal proteins Rab5/D52/EEA1, which control anterograde trafficking in the constitutive-like secretory pathway. In WT acini, short term (14–16 h) culture also results in a >90% decrease in Rab5/D52/EEA1 and a complete loss of the VAMP8 pathway, whereas VAMP2-secretion remains intact. Remarkably, rescue of Rab5/D52/EEA1 expression restored the VAMP8 pathway. Expressed D52 shows extensive colocalization with Rab11a and VAMP8 and partially copurifies with ZG fractions. These results indicate that robust trafficking within the constitutive-like secretory pathway is required for VAMP8- but not VAMP2-mediated ZG exocytosis.     

Vesicle-associated membrane protein 8 (VAMP8) is a SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) selectively required for sequential granule-to-granule fusion

Compound exocytosis is found in many cell types and is the major form of regulated secretion in acinar and mast cells. Its key characteristic is the homotypic fusion of secretory granules. These then secrete their combined output through a single fusion pore to the outside. The control of compound exocytosis remains poorly understood. Although soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) such as syntaxin 2, SNAP23 (synaptosome-associated protein of 23 kDa), and SNAP25 have been suggested to play a role, none has been proven. Vesicle-associated membrane protein 8 (VAMP8) is a SNARE first associated with endocytic processes but more recently has been suggested as an R-SNARE in regulated exocytosis. Secretion in acinar cells is reduced when VAMP8 function is inhibited and is less in VAMP8 knock-out mice. Based on electron microscopy experiments, it was suggested that VAMP8 may be involved in compound exocytosis. Here we have tested the hypothesis that VAMP8 controls homotypic granule-to-granule fusion during sequential compound exocytosis. We use a new assay to distinguish primary fusion events (fusion with the cell membrane) from secondary fusion events (granule-granule fusion). Our data show the pancreatic acinar cells from VAMP8 knock-out animals have a specific reduction in secondary granule fusion but that primary granule fusion is unaffected. Furthermore, immunoprecipitation experiments show syntaxin 2 association with VAMP2, whereas syntaxin 3 associates with VAMP8. Taken together our data indicate that granule-to-granule fusion is regulated by VAMP8 containing SNARE complexes distinct from those that regulate primary granule fusion.     

EMILIN2 Regulates Platelet Activation,Thrombus Formation,and Clot Retraction

Thrombosis, like other cardiovascular diseases, has a strong genetic component, with largely unknown determinants. EMILIN2, Elastin Microfibril Interface Located Protein2, was identified as a candidate gene for thrombosis in mouse and human quantitative trait loci studies. EMILIN2 is expressed during cardiovascular development, on cardiac stem cells, and in heart tissue in animal models of heart disease. In humans, the EMILIN2 gene is located on the short arm of Chromosome 18, and patients with partial and complete deletion of this chromosome region have cardiac malformations. To understand the basis for the thrombotic risk associated with EMILIN2, EMILIN2 deficient mice were generated. The findings of this study indicate that EMILIN2 influences platelet aggregation induced by adenosine diphosphate, collagen, and thrombin with both EMILIN2-deficient platelets and EMILIN2-deficient plasma contributing to the impaired aggregation response. Purified EMILIN2 added to platelets accelerated platelet aggregation and reduced clotting time when added to EMILIN2-deficient mouse and human plasma. Carotid occlusion time was 2-fold longer in mice with platelet-specific EMILIN2 deficiency, but stability of the clot was reduced in mice with both global EMILIN2 deficiency and with platelet-specific EMILIN2 deficiency. In vitro clot retraction was markedly decreased in EMILIN2 deficient mice, indicating that platelet outside-in signaling was dependent on EMILIN2. EMILIN1 deficient mice and EMILIN2:EMILIN1 double deficient mice had suppressed platelet aggregation and delayed clot retraction similar to EMILIN2 mice, but EMILIN2 and EMILIN1 had opposing affects on clot retraction, suggesting that EMILIN1 may attenuate the effects of EMILIN2 on platelet aggregation and thrombosis. In conclusion, these studies identify multiple influences of EMILIN2 in pathophysiology and suggest that its role as a prothrombotic risk factor may arise from its effects on platelet aggregation and platelet mediated clot retraction.     

Functional Divergence of Platelet Protein Kinase C (PKC) Isoforms in Thrombus Formation on Collagen

Arterial thrombosis, a major cause of myocardial infarction and stroke, is initiated by activation of blood platelets by subendothelial collagen. The protein kinase C (PKC) family centrally regulates platelet activation, and it is becoming clear that the individual PKC isoforms play distinct roles, some of which oppose each other. Here, for the first time, we address all four of the major platelet-expressed PKC isoforms, determining their comparative roles in regulating platelet adhesion to collagen and their subsequent activation under physiological flow conditions. Using mouse gene knock-out and pharmacological approaches in human platelets, we show that collagen-dependent α-granule secretion and thrombus formation are mediated by the conventional PKC isoforms, PKCα and PKCβ, whereas the novel isoform, PKCθ, negatively regulates these events. PKCδ also negatively regulates thrombus formation but not α-granule secretion. In addition, we demonstrate for the first time that individual PKC isoforms differentially regulate platelet calcium signaling and exposure of phosphatidylserine under flow. Although platelet deficient in PKCα or PKCβ showed reduced calcium signaling and phosphatidylserine exposure, these responses were enhanced in the absence of PKCθ. In summary therefore, this direct comparison between individual subtypes of PKC, by standardized methodology under flow conditions, reveals that the four major PKCs expressed in platelets play distinct non-redundant roles, where conventional PKCs promote and novel PKCs inhibit thrombus formation on collagen.     

Structure,Sulfatide Binding Properties,and Inhibition of Platelet Aggregation by a Disabled-2 Protein-derived Peptide

Disabled-2 (Dab2) targets membranes and triggers a wide range of biological events, including endocytosis and platelet aggregation. Dab2, through its phosphotyrosine-binding (PTB) domain, inhibits platelet aggregation by competing with fibrinogen for α_IIbβ₃ integrin receptor binding. We have recently shown that the N-terminal region, including the PTB domain (N-PTB), drives Dab2 to the platelet membrane surface by binding to sulfatides through two sulfatide-binding motifs, modulating the extent of platelet aggregation. The three-dimensional structure of a Dab2-derived peptide encompassing the sulfatide-binding motifs has been determined in dodecylphosphocholine micelles using NMR spectroscopy. Dab2 sulfatide-binding motif contains two helices when embedded in micelles, reversibly binds to sulfatides with moderate affinity, lies parallel to the micelle surface, and when added to a platelet mixture, reduces the number and size of sulfatide-induced aggregates. Overall, our findings identify and structurally characterize a minimal region in Dab2 that modulates platelet homotypic interactions, all of which provide the foundation for rational design of a new generation of anti-aggregatory low-molecular mass molecules for therapeutic purposes.     

PRIP (Phospholipase C-related but Catalytically Inactive Protein) Inhibits Exocytosis by Direct Interactions with Syntaxin 1 and SNAP-25 through Its C2 Domain

Membrane fusion for exocytosis is mediated by SNAREs, forming trans-ternary complexes to bridge vesicle and target membranes. There is an array of accessory proteins that directly interact with and regulate SNARE proteins. PRIP (phospholipase C-related but catalytically inactive protein) is likely one of these proteins; PRIP, consisting of multiple functional modules including pleckstrin homology and C2 domains, inhibited exocytosis, probably via the binding to membrane phosphoinositides through the pleckstrin homology domain. However, the roles of the C2 domain have not yet been investigated. In this study, we found that the C2 domain of PRIP directly interacts with syntaxin 1 and SNAP-25 but not with VAMP2. The C2 domain promoted PRIP to co-localize with syntaxin 1 and SNAP-25 in PC12 cells. The binding profile of the C2 domain to SNAP-25 was comparable with that of synaptotagmin I, and PRIP inhibited synaptotagmin I in binding to SNAP-25 and syntaxin 1. It was also shown that the C2 domain was required for PRIP to suppress SDS-resistant ternary SNARE complex formation and inhibit high K⁺-induced noradrenalin release from PC12 cells. These results suggest that PRIP inhibits regulated exocytosis through the interaction of its C2 domain with syntaxin 1 and SNAP-25, potentially competing with other SNARE-binding, C2 domain-containing accessory proteins such as synaptotagmin I and by directly inhibiting trans-SNARE complex formation.     

Point Mutation in Syntaxin-1A Causes Abnormal Vesicle Recycling,Behaviors, and Short Term Plasticity

Syntaxin-1A is a t-SNARE that is involved in vesicle docking and vesicle fusion; it is important in presynaptic exocytosis in neurons because it interacts with many regulatory proteins. Previously, we found the following: 1) that autophosphorylated Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), an important modulator of neural plasticity, interacts with syntaxin-1A to regulate exocytosis, and 2) that a syntaxin missense mutation (R151G) attenuated this interaction. To determine more precisely the physiological importance of this interaction between CaMKII and syntaxin, we generated mice with a knock-in (KI) syntaxin-1A (R151G) mutation. Complexin is a molecular clamp involved in exocytosis, and in the KI mice, recruitment of complexin to the SNARE complex was reduced because of an abnormal CaMKII/syntaxin interaction. Nevertheless, SNARE complex formation was not inhibited, and consequently, basal neurotransmission was normal. However, the KI mice did exhibit more enhanced presynaptic plasticity than wild-type littermates; this enhanced plasticity could be associated with synaptic response than did wild-type littermates; this pronounced response included several behavioral abnormalities. Notably, the R151G phenotypes were generally similar to previously reported CaMKII mutant phenotypes. Additionally, synaptic recycling in these KI mice was delayed, and the density of synaptic vesicles was reduced. Taken together, our results indicated that this single point mutation in syntaxin-1A causes abnormal regulation of neuronal plasticity and vesicle recycling and that the affected syntaxin-1A/CaMKII interaction is essential for normal brain and synaptic functions in vivo.     

Aspirin,Platelet Aggregation,and the Circadian Variation of Acute Thrombotic Events

The onset of several acute cardiovascular diseases occurs in a circadian pattern, with a peak incidence in the hours soon after awakening. This finding, coupled with laboratory data that confirm a surge in platelet activation during the early morning hours, suggests that acute changes in platelet aggregability may be an important trigger of thrombosis. Therefore, the efficacy of antiplatelet agents, such as aspirin, in reducing risks of vascular occlusion may result, at least in part, from a blunting of these short-term changes in platelet aggregability. In this review, clinical and laboratory evidence describing these cyclical changes is discussed, as is current evidence of the effects of aspirin on platelet function and the circadian variation of acute thrombosis.     

Aspirin, Platelet Aggregation, and the Circadian Variation of Acute Thrombotic Events

The onset of several acute cardiovascular diseases occurs in a circadian pattern, with a peak incidence in the hours soon after awakening. This finding, coupled with laboratory data that confirm a surge in platelet activation during the early morning hours, suggests that acute changes in platelet aggregability may be an important trigger of thrombosis. Therefore, the efficacy of antiplatelet agents, such as aspirin, in reducing risks of vascular occlusion may result, at least in part, from a blunting of these short-term changes in platelet aggregability. In this review, clinical and laboratory evidence describing these cyclical changes is discussed, as is current evidence of the effects of aspirin on platelet function and the circadian variation of acute thrombosis.     

Sec22 Regulates Endoplasmic Reticulum Morphology but Not Autophagy and Is Required for Eye Development in Drosophila

The endoplasmic reticulum (ER) is a highly dynamic organelle that plays a critical role in many cellular processes. Abnormal ER morphology is associated with some human diseases, although little is known regarding how ER morphology is regulated. Using a forward genetic screen to identify genes that regulated ER morphology in Drosophila, we identified a mutant of Sec22, the orthologs of which in yeast, plants, and humans are required for ER to Golgi trafficking. However, the physiological function of Sec22 has not been previously investigated in animal development. A loss of Sec22 resulted in ER proliferation and expansion, enlargement of late endosomes, and abnormal Golgi morphology in mutant larvae fat body cells. However, starvation-induced autophagy was not affected by a loss of Sec22. Mosaic analysis of the eye revealed that Sec22 was required for photoreceptor morphogenesis. In Sec22 mutant photoreceptor cells, the ER was highly expanded and gradually lost normal morphology with aging. The rhabdomeres in mutants were small and sometimes fused with each other. The morphology of Sec22 mutant eyes resembled the eye morphology of flies with overexpressed eyc (eyes closed). eyc encodes for a Drosophila p47 protein that is required for membrane fusion. A loss of Syntaxin5 (Syx5), encoding for a t-SNARE on Golgi, also phenocopied the Sec22 mutant. Sec22 formed complexes with Syx5 and Eyc. Thus, we propose that appropriate trafficking between the ER and Golgi is required for maintaining ER morphology and for Drosophila eye morphogenesis.     

Identification of SNARE and cell trafficking regulatory proteins in the salivary glands of the lone star tick,Amblyomma americanum (L.)

Prostaglandin E(2) (PGE(2)) stimulates secretion of tick salivary gland proteins via a phosphoinositide signaling pathway and mobilization of intracellular Ca(2+) (). Highly conserved intracellular SNARE (soluble NSF attachment protein receptors) complex proteins are associated with the mechanism of protein secretion in vertebrate and invertebrate neuronal and non-neuronal cells. Proteins in the salivary glands of partially fed female lone star ticks cross-react individually with antibodies to synaptobrevin-2 (vesicle (v)-SNARE), syntaxin-1A, syntaxin-2 and SNAP-25 (target (t)-SNAREs), cytosolic alpha/beta SNAP and NSF (N-ethylmaleimide-sensitive fusion protein), Ca(2+) sensitive synaptotagmin, vesicle associated synaptophysin, and regulatory cell trafficking GTPases Rab3A and nSec1. V-SNARE and t-SNARE proteins form an SDS-resistant, boiling sensitive core complex in the salivary glands. Antibodies to SNARE complex proteins inhibit PGE(2)-stimulated secretion of anticoagulant protein in permeabilized tick salivary glands. We conclude that SNARE and cell trafficking regulatory proteins are present and functioning in the process of PGE(2)-stimulated Ca(2+) regulated protein secretion in tick salivary glands.     

竹叶青蛇毒血小板聚集剂的分离纯化及生化特性

经ＳｅｐｈａｄｅｘＧ－７５凝胶过滤和ＦＰＬＣＭｏｎｏＱ阴离子交换柱层析及Ｓｕｐｅｒｏｓｅ－１２凝胶过滤,从竹叶青蛇的蛇毒中纯化到一个能诱导人血小板聚集的均一组分．经ＳＤＳ－聚丙烯酰胺凝胶电泳测定其分子量为６８０００左右．等电点为４．３．测定了它的氨基酸组成,对它活化血小板的作用机理进行了初步研究,结果表明它是一种强的血小板激动剂．     

Loss of Granuphilin and Loss of Syntaxin-1A Cause Differential Effects on Insulin Granule Docking and Fusion

The Rab27 effector granuphilin/Slp4 is essential for the stable attachment (docking) of secretory granules to the plasma membrane, and it also inhibits subsequent fusion. Granuphilin is thought to mediate these processes through interactions with Rab27 on the granule membrane and with syntaxin-1a on the plasma membrane and its binding partner Munc18-1. Consistent with this hypothesis, both syntaxin-1a- and Munc18-1-deficient secretory cells, as well as granuphilin null cells, have been observed to have a deficit of docked granules. However, to date there has been no direct comparative analysis of the docking defects in those mutant cells. In this study, we morphometrically compared granule-docking states between granuphilin null and syntaxin-1a null pancreatic β cells derived from mice having the same genetic background. We found that loss of syntaxin-1a does not cause a significant granule-docking defect, in contrast to granuphilin deficiency. Furthermore, we newly generated granuphilin/syntaxin-1a double knock-out mice, characterized their phenotypes, and found that the double mutant mice represent a phenocopy of granuphilin null mice and do not represent phenotypes of syntaxin-1a null mice, including their granule-docking behavior. Because granuphilin binds to syntaxin-2 and syntaxin-3 as well as syntaxin-1a, it likely mediates granule docking through interactions with those multiple syntaxins on the plasma membrane.     

Munc18a Does Not Alter Fusion Rates Mediated by Neuronal SNAREs,Synaptotagmin, and Complexin

Sec1/Munc18 (SM) proteins are essential for membrane trafficking, but their molecular mechanism remains unclear. Using a single vesicle-vesicle content-mixing assay with reconstituted neuronal SNAREs, synaptotagmin-1, and complexin-1, we show that the neuronal SM protein Munc18a/nSec1 has no effect on the intrinsic kinetics of both spontaneous fusion and Ca²⁺-triggered fusion between vesicles that mimic synaptic vesicles and the plasma membrane. However, wild type Munc18a reduced vesicle association ∼50% when the vesicles bearing the t-SNAREs syntaxin-1A and SNAP-25 were preincubated with Munc18 for 30 min. Single molecule experiments with labeled SNAP-25 indicate that the reduction of vesicle association is a consequence of sequestration of syntaxin-1A by Munc18a and subsequent release of SNAP-25 (i.e. Munc18a captures syntaxin-1A via its high affinity interaction). Moreover, a phosphorylation mimic mutant of Munc18a with reduced affinity to syntaxin-1A results in less reduction of vesicle association. In summary, Munc18a does not directly affect fusion, although it has an effect on the t-SNARE complex, depending on the presence of other factors and experimental conditions. Our results suggest that Munc18a primarily acts at the prefusion stage.     

The Carboxyl-terminal Tail of Noxa Protein Regulates the Stability of Noxa and Mcl-1

The BH3-only protein Noxa is a critical mediator of apoptosis and functions primarily by sequestering/inactivating the antiapoptotic Bcl-2 family protein Mcl-1. Although Noxa is a highly labile protein, recent studies suggested that it is degraded by the proteasome in a ubiquitylation-independent manner. In the present study, we investigated the mechanism of Noxa degradation and its ability to regulate the stability of Mcl-1. We found that the ubiquitylation-independent degradation of Noxa does not require a physical association with Mcl-1. A short stretch of amino acid residues in the C-terminal tail was found to mediate the proteasome-dependent degradation of Noxa. Ectopic placement of this degron was able to render other proteins unstable. Surprisingly, mutation of this sequence not only attenuated the rapid degradation of Noxa, but also stabilized endogenous Mcl-1 through the BH3-mediated direct interaction. Together, these results suggest that the C-terminal tail of Noxa regulates the stability of both Noxa and Mcl-1.     

Importance of the Rab3a-GTP Binding Domain for the Intracellular Stability and Function of Rabphilin3a in Secretion

Abstract: We had previously demonstrated that Rab3a-GTP inhibits and the Rab3a-binding protein Rabphilin3a enhances secretion in bovine chromaffin cells. In this study, we investigated the role of Rab3a-GTP binding in the intracellular expression and the function of Rabphilin3a in regulated exocytosis in bovine chromaffin cells. Using transient transfections, we found that a minimal domain, Rp(51–190), that inhibits secretion coincides with a minimal domain that effectively binds Rab3a-GTP and allows intracellular stability of the construct. This domain includes a cysteine-rich, Zn²⁺-binding domain whose integrity is also required for Rab3a-GTP binding and the ability to inhibit secretion. A Rabphilin3a mutant, containing both C2 domains but defective in Rab3a-GTP, and wild-type Rabphilin3a both localized to chromaffin granules and stimulated secretion similarly despite lessened intracellular expression of the mutant protein. The data are consistent with a sequence of events in which a Rab3a-GTP · Rabphilin3a complex forms on the secretory granule as a precursor in a pathway that enhances secretion. The complex dissociates (perhaps because of GTP hydrolysis) to permit the enhancement of secretion by Rabphilin3a.     

Roles of Platelet STIM1 and Orai1 in Glycoprotein VI- and Thrombin-dependent Procoagulant Activity and Thrombus Formation

In platelets, STIM1 has been recognized as the key regulatory protein in store-operated Ca²⁺ entry (SOCE) with Orai1 as principal Ca²⁺ entry channel. Both proteins contribute to collagen-dependent arterial thrombosis in mice in vivo. It is unclear whether STIM2 is involved. A key platelet response relying on Ca²⁺ entry is the surface exposure of phosphatidylserine (PS), which accomplishes platelet procoagulant activity. We studied this response in mouse platelets deficient in STIM1, STIM2, or Orai1. Upon high shear flow of blood over collagen, Stim1^−/− and Orai1^−/− platelets had greatly impaired glycoprotein (GP) VI-dependent Ca²⁺ signals, and they were deficient in PS exposure and thrombus formation. In contrast, Stim2^−/− platelets reacted normally. Upon blood flow in the presence of thrombin generation and coagulation, Ca²⁺ signals of Stim1^−/− and Orai1^−/− platelets were partly reduced, whereas the PS exposure and formation of fibrin-rich thrombi were normalized. Washed Stim1^−/− and Orai1^−/− platelets were deficient in GPVI-induced PS exposure and prothrombinase activity, but not when thrombin was present as co-agonist. Markedly, {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365, a blocker of (receptor-operated) Ca²⁺ entry, inhibited Ca²⁺ and procoagulant responses even in Stim1^−/− and Orai1^−/− platelets. These data show for the first time that: (i) STIM1 and Orai1 jointly contribute to GPVI-induced SOCE, procoagulant activity, and thrombus formation; (ii) a compensating Ca²⁺ entry pathway is effective in the additional presence of thrombin; (iii) platelets contain two mechanisms of Ca²⁺ entry and PS exposure, only one relying on STIM1-Orai1 interaction.     

Effects of monovalent cations and anions on ADP-induced aggregation of bovine platelets, and mechanism thereof

The effects of monovalent cations - inorganic alakali metal cations and organic quanternary ammonium cations - and monovalent inorganic anions on ADP-induced aggregation of bovine platelets were investigated. In the presence of K⁺, Rb⁺, Cs⁺, choline or tetramethylammonium, aggeregation proceeded. However, aggregation was markedly restricted in media containing Li⁺, Na⁺, tetrabutylammonium or dimethyldibenzylammonium. With anions, aggregation proceeded in the order Cl⁻ > Br⁻ > I⁻ > Clo₄⁻ > SCN⁻. The effects of cations significantly depended on Ca²⁺ concentration, whereas those of the anions depended little of Ca²⁺. Anions such as SCN⁻ and ClO₄⁻ markedly decreased the fluorescence of the surface charge probe 2-p-tuluidinylnaphthalene-6-sulfonate, whereas cations had less pronouced effects. The relative effects of the anions on the fluorescence were consistent with their relative inhibitory effects on aggregation. These results suggest that inhibition of platelet aggregation by the anions is due to a change in the surface change of the platelet plasma membrane. On the other hand, kinetic analysis suggests that the effects of monovalent cations on platelet aggregation are due to their competition with Ca²⁺ during the process of aggregation.     

Heparanase Regulates Secretion,Composition, and Function of Tumor Cell-derived Exosomes

Emerging evidence indicates that exosomes play a key role in tumor-host cross-talk and that exosome secretion, composition, and functional capacity are altered as tumors progress to an aggressive phenotype. However, little is known regarding the mechanisms that regulate these changes. Heparanase is an enzyme whose expression is up-regulated as tumors become more aggressive and is associated with enhanced tumor growth, angiogenesis, and metastasis. We have discovered that in human cancer cells (myeloma, lymphoblastoid, and breast cancer), when expression of heparanase is enhanced or when tumor cells are exposed to exogenous heparanase, exosome secretion is dramatically increased. Heparanase enzyme activity is required for robust enhancement of exosome secretion because enzymatically inactive forms of heparanase, even when present in high amounts, do not dramatically increase exosome secretion. Heparanase also impacts exosome protein cargo as reflected by higher levels of syndecan-1, VEGF, and hepatocyte growth factor in exosomes secreted by heparanase-high expressing cells as compared with heparanase-low expressing cells. In functional assays, exosomes from heparanase-high cells stimulated spreading of tumor cells on fibronectin and invasion of endothelial cells through extracellular matrix better than did exosomes secreted by heparanase-low cells. These studies reveal that heparanase helps drive exosome secretion, alters exosome composition, and facilitates production of exosomes that impact both tumor and host cell behavior, thereby promoting tumor progression.