GTPase activating protein Rap1GAP2 and synaptotagmin-like protein 1 interact and are involved in platelet dense granule secretion

Platelets are anucleate cells that play a major role in hemostasis and thrombosis in the vasculature. During primary hemostasis platelets adhere to sites of vascular damage and the initial platelet coat is reinforced by additional platelets forming a stable aggregate. At the same time platelets secrete their intracellular granules containing substances that further activate platelets in an autocrine and paracrine fashion and affect local coagulation and endothelial smooth muscle cell function. The small guanine nucleotide binding protein Rap1 regulates the activity of the platelet integrin alphaIIbbeta3 and thus platelet aggregation. Rap1 activity is controlled by guanine nucleotide exchange factors and GTPase activating proteins. In platelets, Rap1GAP2 is the only GTPase activating protein of Rap1. In order to identify Rap1GAP2-associated proteins, a genetic two-hybrid screening in yeast was performed and synaptotagmin-like protein 1 (Slp1, also called JFC1) was found as a new putative binding partner of Rap1GAP2. Slp1 is a tandem C2 domain containing protein and is known to bind to Rab27, a small GTPase involved in platelet dense granule secretion. The direct interaction between Rap1GAP2 and Slp1 was confirmed in yeast and in transfected cells. More importantly, Slp1 is expressed in platelets and binding of endogenous Rap1GAP2 and Slp1 was verified in these cells. The Rap1GAP2 and Slp1 interaction sites were mapped by mutational analysis. Rap1GAP2 binds through the -TKXT- motif within its C-terminus to the C2A domain of Slp1. Moreover, the Slp1 binding -TKXT- motif of Rap1GAP2 was confirmed by complementary approaches using short synthetic Rap1GAP2 peptides. The C2A domain of Slp1 is a phospholipid binding domain and thus mediates binding of Slp1 to the plasma membrane. Phospholipid overlay assays revealed that simultaneous binding of Slp1 via its C2A domain to Rap1GAP2 and to phospholipids can occur. In addition, the interaction between Rap1GAP2 and Slp1 is regulated by cAMP-dependent protein kinase (cAK or PKA), and kinase activation in platelets enhanced binding of endogenous Rap1GAP2 to Slp1. In-vitro phosphorylation assays revealed that Slp1 is a substrate of PKA, and serine 111 was identified as phosphorylation site. Since Slp1 is a Rab27 binding protein, a trimeric complex of Slp1, Rab27 and Rap1GAP2 is conceivable. The association of Slp1, Rab27 and Rap1GAP2 was investigated by immunofluorescence and co-immuno-precipitation experiments in both, transfected cells and platelets. By Slp1 affinity chromatography and subsequent mass spectrometric analysis additional Slp1 binding proteins were identified in platelets, and binding of Slp1 to Rab8 was confirmed in pull-down assays. To investigate the functional significance of the interaction between Rap1GAP2 and Slp1, an assay system was established to determine serotonin secretion of streptolysin-O permeabilized platelets. Addition of recombinant Slp1 protein to permeabilized platelets strongly inhibited platelet dense granule secretion, whereas addition of recombinant Rap1GAP2 protein or synthetic Rap1GAP2 peptide enhanced secretion. Deleting the Slp1 binding -TKXT- motif abolished the stimulatory effect of Rap1GAP2 on secretion. Addition of Rap1 to permeabilized platelets had no effect on secretion. These findings indicate that the Rap1GAP2 effect on platelet secretion does not depend on the GTPase activating function of Rap1GAP2, but is rather dependent on the -TKXT- mediated interaction of Rap1GAP2 with Slp1. In addition, in-vitro GAP assays revealed that Slp1 binding to Rap1GAP2 does not affect the Rap1GAP activity of Rap1GAP2, and adhesion assays excluded a role for the Rap1GAP2/Slp1 interaction in cell adhesion. Altogether, the results of the present study demonstrate that besides its function in platelet aggregation by controlling the activity of the small guanine nucleotide binding protein Rap1, Rap1GAP2 is involved in platelet dense granule secretion by the new -TKXT- mediated interaction with the Rab27 and membrane binding protein Slp1. In addition, the interaction between Rap1GAP2 and Slp1 is embedded into an elaborate network of protein-protein interactions in platelets which appear to be regulated by phosphorylation. Future studies will in particular aim to dissect the molecular details of Rap1GAP2 and Slp1 action in platelet secretion and investigate the potential biochemical and pharmacological value of the unique protein binding -TKXT- motif of Rap1GAP2.Blutplättchen, auch Thrombozyten genannt, sind kernlose Zellen, die unter physiologischen Umständen eine wichtige Rolle in der primären Hämostase spielen. Nach einer Gefäßverletzung adhärieren Thrombozyten an Strukturen der freigelegten subendothelialen Matrix. Bei dieser primären Adhäsion werden Thrombozyten aktiviert und bilden durch Wechselwirkung untereinander ein zunächst fragiles Thrombozytenaggregat. Dieser initial gebildete Thrombus wird in der sekundären Hämostase durch Fibrin verfestigt und ermöglicht dann die stabile Abdichtung der Gefäßwandläsion gegenüber dem Blutstrom (Kehrel 2003). Die Aktivierung von Thrombozyten durch Adhäsion oder durch lösliche Plättchenaktivatoren bewirkt eine Formveränderung der Thrombozyten. Gleichzeitig werden intrazelluläre Speichergranula von Thrombozyten ausgeschüttet. Thrombozyten enthalten drei verschiedene Arten von Granula: alpha-Granula, dichte Granula und Lysosomen. Die Granula in Thrombozyten dienen als Speicherorte für Proteine und Substanzen, welche sowohl autokrin die Thrombozytenaktivierung verstärken als auch parakrin noch ruhende Thrombozyten aus der Blutzirkulation rekrutieren. Die dichten Granula in Thrombozyten enthalten niedermolekulare Substanzen wie zum Beispiel ADP, ATP, Ca2+ und Serotonin. In den alpha-Granula dagegen befinden sich vor allem Proteine, die für die Adhäsion, Aggregation und die Blutgerinnung wichtig sind. Zusätzlich enthalten alpha-Granula Zytokine und Wachtumsfaktoren. Die lysosomalen Granula in Thrombozyten enthalten hydrolytische Enzyme wie beispielsweise beta-Hexosaminidase und Heparitinase (Gawaz 1999). ... Zielsetzung dieser Arbeit: Um weitere Einblicke in die Funktion und Regulation von Rap1GAP2 in Thrombozyten zu gewinnen, wurde ein Hefe-zwei-Hybrid-Screening durchgeführt (O. Danielewski und A. Smolenski, IBCII, Frankfurt). Neben 14-3-3-Proteinen (Hoffmeister et al. 2008), wurde das Rab27-bindende Protein synaptotagmin-like protein 1 (Slp1, auch JFC1 genannt) als neuer putativer Interaktionspartner von Rap1GAP2 gefunden. Ausgehend von den Ergebnissen dieses Hefe-zwei-Hybrid-Screenings waren die konkreten Ziele der vorliegenden Arbeit (1) die Bindung von Slp1 an Rap1GAP2 zu verifizieren, (2) die für die Bindung verantwortlichen Bindebereiche zu identifizieren, (3) die mögliche Komplexbildung von Rap1GAP2, Slp1, Rab27 und anderen Proteinen zu untersuchen sowie (4) die funktionale Bedeutung der Interaktion zwischen Rap1GAP2 und Slp1 zu bestimmen. ... Fazit Die Ergebnisse der vorliegenden Arbeit zeigen, dass das Rab27-bindende Protein Slp1 ein neuer direkter Interaktionspartner des GTPase-aktivierenden Proteins Rap1GAP2 in Thrombozyten ist. Die Expression von Slp1 wurde in Thrombozyten nachgewiesen. Damit ist Slp1 neben Munc13-4 das zweite bisher bekannte Rab27-bindende Protein in diesen Zellen. Der Nachweis der Interaktion von Slp1 mit GTP-gebundenen Rab27 legte eine Funktion von Slp1 als Rab27-Effektorprotein nahe und es konnte in dieser Arbeit gezeigt werden, dass Slp1 die Sekretion der dichten Granula in Thrombozyten beeinflusst. Darüber hinaus zeigen die Ergebnisse der vorliegenden Arbeit, dass Rap1GAP2, zusätzlich zu seiner bereits bekannten Funktion bei der Thrombozytenaggregation durch die Regulation des kleinen G-Proteins Rap1, auch an der Sekretion der dichten Granula in Thrombozyten beteiligt ist. Der Rap1GAP2 Effekt auf die Sekretion in Thrombozyten beruht auf der -TKXT- 111 vermittelten Wechselwirkung mit Slp1, welche Teil eines komplexen Netzwerks von Protein-Protein-Interaktionen in Thrombozyten ist. Rap1GAP2 könnte somit eine zentrale Rolle bei der Koordination der wichtigen Funktionen Aggregation und Sekretion in Thrombozyten spielen

FUNCTIONAL ROLES FOR POST-TRANSLATIONAL MODIFICATIONS OF t-SNARES IN PLATELETS

Platelets affect vascular integrity by secreting a host of molecules that promote hemostasis and its sequela. Given its importance, it is critical to understand how platelet exocytosis is controlled. Post-translational modifications, such as phosphorylation and acylation, have been shown to affect signaling pathways and platelet function. In this dissertation, I focus on how these modifications affect the t-SNARE proteins, SNAP-23 and syntaxin-11, which are both required for platelet secretion. SNAP-23 is regulated by phosphorylation. Using a proteoliposome fusion assay, I demonstrate that purified IκB Kinase (IKK) phosphorylated SNAP-23, which increased the initial rates of SNARE-mediated liposome fusion. SNAP-23 mutants containing phosphomimetics showed enhanced initial fusion rates. These results, combined with previous work in vivo, confirm that SNAP-23 phosphorylation is involved in regulating membrane fusion, and that IKK-mediated signaling contributes to platelet exocytosis. To address the role(s) of acylation, I sought to determine how syntaxin-11 and SNAP-23 are associated with plasma membrane. Using metabolic labeling, I showed that both proteins contain thioester-linked acyl groups which turn over in resting cells. Mass spectrometry mapping showed that syntaxin-11 is modified on C275, 279, 280, 282, 283 and 285, while SNAP-23 is modified on C79, 80, 83, 85, and 87. To probe the effects of acylation, I measured ADP/ATP release from platelets treated with the acyl-transferase inhibitor, cerulenin, or the thioesterase inhibitor, palmostatin B. Cerulenin pretreatment inhibited t-SNARE acylation and platelet function while palmostatin B had no effect. Interestingly, pretreatment with palmostatin B blocked the inhibitory effects of cerulenin suggesting that maintaining the acylation state of platelet proteins is important for their function. Thus my work indicates that the enzymes controlling protein acylation could be valuable targets for modulating platelet exocytosis in vivo

CONTROLLING PLATELET SECRETION TO MODULATE HEMOSTASIS AND THROMBOSIS

Upon vascular injury, activated blood platelets fuse their granules to the plasma membrane and release cargo to regulate the vascular microenvironment, a dynamic process central to platelet function in many critical processes including hemostasis, thrombosis, immunity, wound healing, angiogenesis etc. This granule- plasma membrane fusion is mediated by a family of membrane proteins- Soluble N-ethyl maleimide Attachment Receptor Proteins(SNAREs). SNAREs that reside on vesicle (v-SNAREs) /Vesicle-Associated Membrane Proteins(VAMPs) interact with target/t-SNAREs forming a trans-bilayer complex that facilitates granule fusion. Though many components of exocytic machinery are identified, it is still not clear how it could be manipulated to prevent occlusive thrombosis without triggering bleeding. My work addresses this question by showing how the rates and extents of granule secretion could be regulated by various v-SNAREs. We also show that the granule cargo decondensation is an intermediate to secretion that also contributes to rates of cargo release. Platelets contain four major VAMP isoforms (-2, -3, -7, and -8), however, VAMP-8 and -7 play a primary role while VAMP-2 and -3 are ancillary in secretion. To exploit this heterogeneity in VAMP usage, platelet-specific V-2/3-/- and V-2/3/8-/- mouse models were generated and characterized to understand how secretion influences hemostasis. We found that each VAMP isoform differentially contributes by altering the rates and extents of cargo release. The loss of VAMP-2 and -3 had a minimal impact while the loss of VAMP-2, -3 and -8 significantly reduced the granule secretion. Platelet activation and aggregation were not affected though the spreading was reduced in V-2/3/8-/- platelets indicating the importance of secretion in spreading. Though coagulation pathways were unaltered, PS exposure was reduced in both V-2/3-/- and V-2/3/8-/- platelets suggesting diminished procoagulant activity. In vivo experiments showed that V-2/3/8-/- animals bled profusely upon tail transaction and failed to form occlusive thrombus upon arterial injury while V-2/3-/- animals did not display any hemostatic deficiency. These data suggest that about 40-50% reduction in secretion provides protection against thrombosis without compromising hemostasis and beyond 50% secretion deficiency, the animals fail to form functional thrombi and exhibit severe bleeding. Additionally, detailed structural analysis of activated platelets suggests that the post-stimulation cargo dissolution depends on an agonist concentration and stimulation duration. This process is VAMP-dependent and represents intermediate steps leading to a full exodus of cargo. Moreover, we also show that VAMP-8 is important for compound fusion events and regulates fusion pore size. This is a first comprehensive report that shows how manipulation of the exocytic machinery have an impact on secretion and ultimately on hemostasis. These animals will be instrumental in future investigations of platelet secretion in many other vascular processes

PLATELET ENDOCYTOSIS: ROLES IN HEMOSTASIS AND INNATE IMMUNITY

Endocytosis is key to fibrinogen (Fg) uptake, receptor trafficking of integrins (αIIbβ3, αvβ3) and purinergic receptors (P2Y1, P2Y12), and thereby for normal platelet function. However, platelet endocytosis could potentially be critical for actively sensing changes in vascular micro-environments and responding accordingly to what is being taken up. This is a more dynamic view of platelets as active surveyors of the vasculature; extending the importance of platelet endocytosis beyond granule biogenesis and perhaps even hemostasis. The mechanistic underpinnings of endocytosis, its importance in platelets, and the molecular machinery required and possible trafficking routes are however understudied, in part due to a lack of experimental tools. The work presented here, puts forth new players that regulate platelet endocytosis and mediate cargo loading and hemostasis as well as provides a novel mechanistic understanding of how endocytosis allows platelets to act as immune cells and become the first responders to pathogens in the vasculature. Previously we showed the importance of ADP-ribosylation factor 6 (Arf6), which regulates αIIbβ3-mediated Fg uptake/storage and affects acute platelet functions e.g., clot retraction and spreading. To further identify elements of this endocytic machinery, we examined the role of a vesicle-residing Soluble N-ethylmaleimide Factor Attachment Protein Receptor (v-SNARE) called Cellubrevin/Vesicle Associated Membrane Protein-3 (VAMP-3) in platelet function. VAMP-3 KO mice had less platelet-associated Fg, indicating a defect in Fg uptake/storage. Loss of VAMP-3 led to defective uptake of fluorescently-tagged Fg and low-molecular dextran in platelets though it had a greater negative effect on receptor-mediated Fg uptake than on the fluid-phase marker uptake. Additionally, we followed the time-dependent trafficking of Fg and dextran into platelets using 3D-Structured Illumination Microscopy. Wild-type platelets endocytosed both cargo but quickly sorted them into distinct compartments with partial overlap occurring only at early time points. Sorting was unaffected in VAMP-3KO platelets. VAMP-3 loss did affect some acute platelet functions leading to enhanced spreading on Fg and faster clot retraction compared to wild-type. Additionally, the rate of JAK2 phosphorylation, initiated through the thrombopoietin receptor (TPOR/Mpl) activation, was affected in VAMP-3 KO platelets. The idea that platelets can act as immune cells and contribute to innate immunity has been increasingly gaining ground. Groups have correlated thrombocytopenia with clinical outcomes of viremia and bacteremia. Chronic viral infections, e.g., HIV-1, severely increase the risk of acute myocardial infarction (MI), possibly via some level of platelet activation, contributing to increased thrombotic potential. Platelets do endocytose viruses and bacteria, but the molecular machinery is ill-defined. In nucleated cells, responses to HIV-1 are mediated by virus phagocytosis/endocytosis, degradation to release Toll-like Receptor ligands, and subsequent receptor activation. Is this process recapitulated in platelets? Here we show that platelets indeed use VAMP-3 and Arf6-dependent pathways to endocytose HIV-1 virions, degrade retroviral particles to release TLR ligands, which initiate platelet activation and secretion. HIV-1 uptake and subsequent activation is abolished in VAMP-3 and Arf6 KO mice. Collectively, our studies shed light on how platelets act at the early stage of pathogen recognition and are able to process them to initiate an immune response

Master of Science

thesisChediak-Higashi Syndrome (CHS) is a rare, autosomal recessive genetic disorder caused by mutations in the lysosomal trafficking regulator gene (CHS1) that affects vesicle morphology. The CHS1 gene encodes a protein, Lyst, which belongs to a family of proteins containing a conserved BEACH (BEige And Chediak-Higashi) domain and WD40-repeat domain. The beigej mouse has been the best-studied animal model for CHS. Factor associated with neutral sphingomyelinase activation or FAN is the smallest member of the BEACH family. FAN interacts with the Tumor Necrosis Factor Receptor (TNFR1) to activate Neutral SphingoMyelinase2 (NSMase2). The absence of Lyst or FAN gives rise to enlarged lysosomes. Loss of both proteins results in an additive effect, as demonstrated by increased lysosome size in FAN-/- /beigej mouse. An additive phenotype suggests that there are at least two pathways to regulate lysosome size. Overexpression of Lyst or FAN results in smaller than normal lysosomes. Overexpression of FAN in the absence of Lyst does not give rise to small lysosomes. NSMases are present on lysosomes and inhibition of NSMases, using GW4869 or 3-OMethylsphingomyelin, results in enlarged lysosomes. Further, the decrease in lysosome size seen in FAN-overexpressing cells is blocked by GW4869. These results suggest that FAN activation of NSMase at the lysosome is a crucial step in regulating lysosome size

INFLAMMATORY INTERACTIONS AND SECRETION IN CARDIAC REMODELING

Heart failure contributes to nearly 60,000 deaths per year in the USA and is often caused by hypertension and preceded by the development of left ventricular hypertrophy (LVH). LVH is usually accompanied by intensive interstitial and perivascular fibrosis which may contribute to arrhythmogenic sudden cardiac death. Emerging evidence indicates that LV dysfunction in patients and animal models of cardiac hypertrophy is closely associated with perivascular inflammation. To investigate the role of perivascular inflammation in coronary artery remodeling and cardiac fibrosis during hypertrophic ventricular remodeling, we used a well-established mouse model of pressure-overload-induced LVH: transverse aortic constriction (TAC). Early perivascular inflammation was indicated by accumulation of macrophages and T lymphocytes 24 hours post-TAC and which peaked at day 7. Coronary luminal platelet deposition was observed along with macrophages and lymphocytes at day 3. Also, LV protein levels of VEGF and MCP-1 were significantly increased. Consistent with lymphocyte accumulation, cardiac expression of IL-10 mRNA was elevated. Furthermore, circulating platelet-leukocyte aggregates tended to be higher after TAC, compared to sham controls. Platelets have been shown to modulate perivascular inflammation and may facilitate leukocyte recruitment at sites of inflamed endothelium. Therefore, we investigated the impact of thrombocytopenia in the response to TAC. Immunodepletion of platelets decreased early perivascular accumulation of T lymphocytes and IL-10 mRNA expression, and altered subsequent coronary artery remodeling. The contribution of lymphocytes was examined in Rag1-/- mice, which displayed significantly more intimal hyperplasia and perivascular fibrosis compared to wild-type mice following TAC. Collectively, our studies support a role of early perivascular accumulation of platelets and T lymphocytes in pressure overload-induced inflammation which will contribute to long-term LV remodeling. One potential mechanism for inflammatory cells to modulate their environment and affect surrounding cells is through release of cargo stored in granules. To determine the contribution of granule release from inflammatory cells in the development of LVH, we used Unc13dJinx (Jinx) mice, which contain a single point mutation in Unc13d gene resulting in defects in Munc13-4. Munc13-4 is a limiting factor in vesicular priming and fusion during granule secretion. Therefore, Jinx mice have defects in degranulation of platelets, NK cells, cytotoxic T lymphocytes, neutrophils, mast and other cells. With the use of bone marrow transplantation, Jinx chimeric mice were created to determine whether the ability of hematopoietic cells to secrete granule contents affects the development of LVH. Wild-type mice (WT) that were transplanted with WT bone marrow (WT\u3eWT) and WT mice that received Jinx bone marrow (Jinx\u3eWT) developed LVH and a classic fetal reprogramming response early after TAC (7 days), but at later times (5 weeks), Jinx\u3eWT mice failed to sustain the cardiac hypertrophic response observed in WT\u3eWT mice. No difference in cardiac fibrosis was observed at early or late times. Repetitive injection of WT platelets or platelet releasate restored cardiac hypertrophy in Jinx\u3eWT mice. These results suggest that sustained LVH in the setting of pressure overload depends on factor(s) secreted, likely from platelets. In conclusion, our studies demonstrate that platelets and lymphocytes are involved in early perivascular inflammation post-TAC, which may contribute to later remodeling in the setting of LVH. Factors released from hematopoietic cells, including platelets, in a Munc13-4-dependent manner are required to promote cardiac hypertrophy. These findings focus attention on modulating perivascular inflammation and targeting granule cargo release to prevent the development and consequences of LVH

ADP-RIBOSYLATION FACTOR 6 (ARF6) REGULATES INTEGRIN αIIbβ3 TRAFFICKING, PLATELET SPREADING, AND CLOT RETRACTION

Endocytic trafficking of platelet surface receptors plays a role in the accumulation of granule cargo (i.e. fibrinogen and VEGF) and thus could contribute to hemostasis, angiogenesis, or inflammation. However, the mechanisms of platelet endocytosis are poorly understood. The small GTP-binding protein, ADP-ribosylation factor 6 (Arf6), regulates integrin trafficking in nucleated cells; therefore, we posited that Arf6 functions similarly in platelets. To address this, we generated platelet-specific, Arf6 knockout mice. Arf6-/- platelets had a storage defect for fibrinogen but not other cargo, implying Arf6’s role in integrin αIIbβ3 trafficking. Additionally, platelets from Arf6-/- mice injected with biotinylated-fibrinogen, showed lower accumulation of the modified protein than did WT mice. Resting and activated αIIbβ3 levels, measured by FACS, were unchanged in Arf6-/- platelets. Arf6-/- platelets had normal agonist-induced aggregation and ATP release; however, they showed faster clot retraction and enhanced spreading, which appears due to altered αIIbβ3 trafficking since myosin light chain phosphorylation and Rac1 activation, in response to thrombin, were unaffected. Arf6-/- mice showed no hemostasis defect in tail-bleeding or FeCl3–induced carotid injury assays. These data suggest a role for Arf6 in integrin αIIbβ3 trafficking in platelets. Additionally, the regulation of Arf6 in platelets was also investigated, focusing on integrin αIIbβ3 outside-in signaling which was suggested to be responsible for the second wave of Arf6-GTP loss. G protein-coupled receptor kinase-interacting protein 1 (GIT1), a GTPase-activating protein (GAP) toward Arf6, is suggested to be involved in αIIbβ3 downstream signaling. I found that GIT1, complex with β-PIX, was translocated to the detergent-insoluble pellet upon human platelet activation, a process that is blocked by RGDS and myrArf6 peptide treatment. Moreover, tyrosine-phosphorylation of GIT1 was impaired by treatment with both peptides or with actin polymerization inhibitors. GIT1’s role in platelets was further studied using platelet-specific, GIT1 knockout mice. GIT1-/- platelets failed to show any defect, including clot retraction or fibrinogen storage. Unlike human platelets, GIT1 expression levels were much lower in mouse platelets, suggesting that GIT2 may be the functionally relevant Arf6-GAP in mouse platelets. The data in this dissertation identify that Arf6 mediates fibrinogen storage, implying its role in integrin αIIbβ3 trafficking in platelets

Role of Pikfyve in Platelet Lysosomal Homeostasis

PIKfyve is a lipid kinase that is essential for the synthesis of phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2], and for the regulation of membrane dynamics within the endolysosomal system in mammals. Depletion of intracellular pools of PtdIns(3,5)P2 in humans and in mice is associated with neurodegeneration and early lethality. However, the biological role of PtdIns(3,5)P2 in non-neural tissues is not well understood. Platelets are hematopoietic cells that function in a variety of physiological responses. Essential to many of these functions is the activation-dependent release of effectors from distinct storage granules - alpha granules, dense granules, and lysosomes - that derive from the endolysosomal system. In this work, we show that platelet-specific ablation of the PIKfyve gene in mice results in accelerated arterial thrombosis, but also unexpectedly to multiorgan defects that impair development, body mass, fertility, and survival by inducing inappropriate inflammatory responses characterized by macrophage accumulation in multiple tissues. Platelet depletion in vivo significantly impairs the progression of multiorgan defects in these mice, confirming that these defects reflect a platelet-specific process. Although PIKfyve-null platelets generate and release normal amounts of alpha granule and dense granule contents, they develop defective maturation and excessive storage of lysosomal enzymes, which are released upon platelet activation. Remarkably, impairing the secretion of lysosomes from PIKfyve-deficient platelets in vivo significantly attenuates the multiorgan defects in mice, suggesting that platelet lysosome secretion contributes to pathogenesis. Together, these results demonstrate that PIKfyve is an essential regulator for the biogenesis of platelet lysosomes, and highlight the previously unrecognized and important pathological contributions of platelet lysosomes in inflammation, arterial thrombosis, and macrophage biology