TLR9 localizes to a novel intracellular compartment called the T granule to promote immune signaling by platelets.

Nanoparticles are currently being investigated in a number of human clinical trials. As information on how nanoparticles function in humans is difficult to obtain, animal studies that can be correlative to human behavior are needed to provide guidance for human clinical trials. Here, we report correlative studies on animals and humans for CRLX101, a 20-to 30-nm-diameter, multifunctional, polymeric nanoparticle containing camptothecin (CPT). CRLX101 is currently in phase 2 clinical trials, and human data from several of the clinical investigations are compared with results from multispecies animal studies. The pharmacokinetics of polymer-conjugated CPT (indicative of the CRLX101 nanoparticles) in mice, rats, dogs, and humans reveal that the area under the curve scales linearly with milligrams of CPT per square meter for all species. Plasma concentrations of unconjugated CPT released from CRLX101 in animals and humans are consistent with each other after accounting for differences in serum albumin binding of CPT. Urinary excretion of polymer-conjugated CPT occurs primarily within the initial 24 h after dosing in animals and humans. The urinary excretion dynamics of polymer-conjugated and unconjugated CPT appear similar between animals and humans. CRLX101 accumulates into solid tumors and releases CPT over a period of several days to give inhibition of its target in animal xenograft models of cancer and in the tumors of humans. Taken in total, the evidence provided from animal models on the CRLX101 mechanism of action suggests that the behavior of CRLX101 in animals is translatable to humans.nanomedicine | clinical translation | interspecies scaling | pharmacodynamics | Nanoparticles

There has been recent controversy as to whether platelet ␣-granules represent a single granule population or are composed of different subpopulations that serve discrete functions. To address this question, we evaluated the localization of vesicle-associated membrane proteins (VAMPs) in spread platelets to determine whether platelets actively sort a specific subpopulation of ␣-granules to the periphery during spreading. Immunofluorescence microscopy demonstrated that granules expressing VAMP-3 and VAMP-8 localized to the central granulomere of spread platelets along with the granule cargos von Willebrand factor and serotonin. In contrast, ␣-granules expressing VAMP-7 translocated to the periphery of spread platelets along with the granule cargos TIMP2 and VEFG. Time-lapse microscopy demonstrated that ␣-granules expressing VAMP-7 actively moved from the granulomere to the periphery during spreading. Platelets from a patient with gray platelet syndrome lacked ␣-granules and demonstrated only minimal spreading. Similarly, spreading was impaired in platelets obtained from Unc13d Jinx mice, which are deficient in Munc13-4 and have an exocytosis defect. These studies identify a new ␣-granule subtype expressing VAMP-7 that moves to the periphery during spreading, supporting the premise that ␣-granules are heterogeneous and demonstrating that granule exocytosis is required for platelet spreading. IntroductionPlatelets are replete with granules containing cargo that is required for platelet function in hemostasis, thrombosis, inflammation, angiogenesis, and malignancy. [1][2][3][4] Platelet granule types include ␣-granules, dense granules, and lysosomes. Of these granule types, the ␣-granule is by far the most abundant with 50 to 80 granules/ platelet, compared with 3 to 6 dense granules/platelet and 0 to 3 lysosomes/platelet. Recent studies indicate that ␣-granules may not constitute a homogenous population. There is evidence that ␣-granule subpopulations can be distinguished on the basis of morphology, 5 cargo type, 6-8 and response to agonists. 7-10 However, experiments using high resolution immunofluorescence microscopy have raised the possibility that the distribution of cargo among ␣-granules is largely stochastic and that the apparent segregation observed by standard immunofluorescence microscopy could result from segregation within granules. 11,12 Although the study of ␣-granule heterogeneity has focused primarily on the localization and release of granule cargo, granules also serve an essential role in membrane remodeling. In nucleated cells, granules provide an internal reservoir of membrane to expand and reshape the plasma membrane during cell movement, 13 membrane resealing, 14,15 neurite outgrowth, [16][17][18] and development of the phagocytotic cup in macrophages. 19,20 Different subpopulations of granules demonstrate different behaviors during membrane remodeling. Recent studies demonstrate that these different granule types can be distinguished by the vesicle-associated membrane proteins (VAMPs) that th...

Key Points Parmodulins are a new class of PAR1 inhibitors that target the cytosolic face of PAR1 to block signaling through Gαq, but not Gα12/13. Unlike vorapaxar, which causes endothelial injury, parmodulins selectively block proinflammatory, but not cytoprotective, signaling.

Key Points VAMP-7 functions in platelet granule exocytosis and spreading. VAMP-7 associates with VARP and Arp2/3, thereby linking granule exocytosis and actin reorganization.

Following platelet activation, platelets undergo a dramatic shape change mediated by the actin cytoskeleton and accompanied by secretion of granule contents. While the actin cytoskeleton is thought to influence platelet granule secretion, the mechanism for this putative regulation is not known. We found that disruption of the actin cytoskeleton by latrunculin A inhibited α-granule secretion induced by several different platelet agonists without significantly affecting activationinduced platelet aggregation. In a cell-free secretory system, platelet cytosol was required for α-granule secretion. Inhibition of actin polymerization prevented α-granule secretion in this system and purified platelet actin could substitute for platelet cytosol to support α-granule secretion. To determine whether SNAREs physically associate with the actin cytoskeleton, we isolated the Triton X-100 insoluble actin cytoskeleton from platelets. VAMP-8 and syntaxin-2 associated only with actin cytoskeletons of activated platelets. Syntaxin-4 and SNAP-23 associated with cytoskeletons isolated from either resting or activated platelets. When syntaxin-4 and SNAP-23 were tested for actin binding in a purified protein system, only syntaxin-4 associated directly with polymerized platelet actin. These data show that the platelet cytoskeleton interacts with select SNAREs and that actin polymerization facilitates α-granule release.The role of the actin cytoskeleton in granule exocytosis is enigmatic. It has been demonstrated to act both as a physical barrier that limits granule secretion and as a positive regulator of membrane fusion and cargo release. The ability of the resting actin cytoskeleton to serve as a barrier to granule secretion has been demonstrated in neutrophils, neurons, chromaffin cells, melanotrophs, pancreatic beta cells, and acinar cells (1)(2)(3)(4)(5)(6). We have previously demonstrated that platelet granules are coated with actin and that the actin cytoskeleton impedes platelet dense granule and α-granule release (7). Partial disruption of this barrier results in augmented and more rapid release of granule contents from platelets. This actin cytoskeletal barrier may help prevent unregulated release of thrombogenic substances into the circulation (7).Yet accumulating evidence indicates that actin polymerization can promote membrane fusion. Actin polymerization contributes to homotypic fusion of yeast vacuoles (8), fusion of phagosomes with endocytotic organelles (9) as well as secretion of granules from neuroendocrine cells (6,10,11), and mast cells (12). In some cells, actomyosin contraction and/ † Supported by NIH grants HL63250 and HL87203 (R.F.) and T32 HL07917 (K.W., L.D., P.S.B., C.G.P. (23,25,(27)(28)(29)(30).The dramatic morphologic change and granule centralization that occurs upon exposure to a strong agonist has lead to speculation that the cytoskeleton provides a contractile force that facilitates granule release (31)(32)(33). Yet this hypothesis remains unproven. An additional or alternative possibility is ...

Purpose: Increased tumor hypoxia and hence elevated hypoxia-inducible factor-1a (HIF1a) is thought to limit the efficacy of vascular endothelial growth factor (VEGF) pathway-targeting drugs by upregulating adaptive resistance genes. One strategy to counteract this is to combine antiangiogenic drugs with agents able to suppress HIF1a. One such possibility is the investigational drug CRLX101, a nanoparticle-drug conjugate (NDC) containing the payload camptothecin, a known topoisomerase-I poison.Experimental Design: CRLX101 was evaluated both as a monotherapy and combination with bevacizumab in a preclinical mouse model of advanced metastatic ovarian cancer. These preclinical studies contributed to the rationale for undertaking a phase II clinical study to evaluate CRLX101 monotherapy in patients with advanced platinum-resistant ovarian cancer.

Stimulation of protease-activated receptor 1 (PAR1) on endothelium by activated protein C (APC) is protective in several animal models of disease, and APC has been used clinically in severe sepsis and wound healing. Clinical use of APC, however, is limited by its immunogenicity and its anticoagulant activity. We show that a class of small molecules termed "parmodulins" that act at the cytosolic face of PAR1 stimulates APC-like cytoprotective signaling in endothelium. Parmodulins block thrombin generation in response to inflammatory mediators and inhibit platelet accumulation on endothelium cultured under flow. Evaluation of the antithrombotic mechanism showed that parmodulins induce cytoprotective signaling through Gβγ, activating a PI3K/Akt pathway and eliciting a genetic program that includes suppression of NF-κB-mediated transcriptional activation and up-regulation of select cytoprotective transcripts. is among the up-regulated transcripts, and knockdown of stanniocalin-1 blocks the protective effects of both parmodulins and APC. Induction of this signaling pathway in vivo protects against thromboinflammatory injury in blood vessels. Small-molecule activation of endothelial cytoprotection through PAR1 represents an approach for treatment of thromboinflammatory disease and provides proof-of-principle for the strategy of targeting the cytoplasmic surface of GPCRs to achieve pathway selective signaling.