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The use of fibrinolytic agents to prevent new thrombus formation is limited by an increased risk of bleeding due to lysis of hemostatic clots that prevent hemorrhage in damaged blood vessels. We sought to develop an agent that provides thromboprophylaxis without carrying a significant risk of causing systemic fibrinolysis or disrupting hemostatic clots. We previously showed that platelet (PLT) α granule-delivered urokinase plasminogen activator (uPA) is highly effective in preventing thrombosis, while being associated with little systemic fibrinolysis or bleeding. Here, we generated a chimeric prodrug composed of a single-chain version of the variable region of an anti-αIIbβ3 mAb fused to a thrombin-activatable, low-molecular-weight pro-uPA (PLT/uPA-T). PLT/uPA-T recognizes human αIIbβ3 on both quiescent and activated platelets and is enzymatically activated specifically by thrombin. We found that this prodrug binds tightly to human platelets even after gel filtration, has a prolonged half-life in mice transgenic for human αIIb compared with that of uPA-T, and prevents clot formation in a microfluidic system. Importantly, in two murine injury models, PLT/uPA-T did not lyse preexisting clots, even when administration was delayed by as little as 10 minutes, while it concurrently prevented the development of nascent thrombi. Thus, PLT/uPA-T represents the prototype of a platelet-targeted thromboprophylactic agent that selectively targets nascent over preexisting thrombi.

Introduction Cancer patients have a 4- to 7- fold increased risk of venous thromboembolism (VTE) compared with general population. Most tumor cells express tissue factor (TF) and constitutively release small membrane microvesicles called tumor microvesicles (TMVs). Clinical studies have shown that circulating MP-TF activity is associated with VTE in pancreatic cancer but not in other types of cancer. Thrombin is a potent platelet agonist and activates platelets via protease activated receptors (PARs). Aim To determine the contribution of the TF + TMV-thrombin-platelet pathway to cancer-associated thrombosis. Materials and Methods A human pancreatic adenocarcinoma cell line expressing high levels of TF (BxPc-3) was selected to study the effect of TF + TMVs on platelet activation and thrombosis. Results TF + TMVs induced platelet activation in vitro in a thrombin-dependent manner. The presence of orthotopically grown BxPc-3 tumors in mice was associated with increased levels of thrombin-antithrombin III complexes (TATc) and larger thrombi in an inferior vena cava stenosis model compared with control mice. Furthermore, injection of BxPc-3 TF + TMVs into mice triggered platelet activation and enhanced venous thrombosis in a TF-dependent manner. Importantly, BxPc-3 TF + TMV-enhanced thrombosis was reduced in Par4-deficient mice and wild-type mice treated with the platelet inhibitor clopidogrel, suggesting that platelet activation was required for the enhanced thrombosis. Conclusions These studies suggest that platelet inhibitors may reduce thrombosis in cancer patients with elevated levels of TF + TMVs.

Essentials Cancer patients have a high rate of venous thrombosis (VT) but the underlying mechanisms are unknown. Tumor-derived, tissue factor-positive microvesicles in platelet activation in vitro and in vivo were studied. Tumor-derived, tissue factor-positive microvesicles enhanced VT in mice. Platelets may contribute to VT in some cancer patients, and this could be prevented with antiplatelet drugs. SummaryBackground Cancer patients have an approximately 4-fold increased risk of venous thromboembolism (VTE) compared with the general population, and cancer patients with VTE have reduced survival. Tumor cells constitutively release small membrane vesicles called microvesicles (MVs) that may contribute to thrombosis in cancer patients. Clinical studies have shown that levels of circulating tumor–derived, tissue factor–positive (TF+) MVs in pancreatic cancer patients are associated with VTE. Objectives We tested the hypothesis that TF+ tumor–derived MVs (TMVs) activate platelets in vitro and in mice. Materials and methods We selected two human pancreatic adenocarcinoma cell lines expressing high (BxPc-3) and low (L3.6pl) levels of TF as models to study the effect of TF+ TMVs on platelets and thrombosis. Results and conclusions We found that both types of TF+ TMVs activated human platelets and induced aggregation in vitro in a TF and thrombin-dependent manner. Further, injection of BxPc-3 TF+ TMVs triggered platelet activation in vivo and enhanced thrombosis in two mouse models of venous thrombosis in a TF-dependent manner. Importantly, BxPc-3 TF+ TMV-enhanced thrombosis was reduced in Par4-deficient mice and in wild-type mice treated with clopidogrel, suggesting that platelet activation was required for enhanced thrombosis. These studies suggest that TF+ TMV-induced platelet activation contributes to thrombosis in cancer patients.

Understanding platelet biology has been aided by studies of mice with mutations in key megakaryocytic transcription factors. We have shown that point mutations in the GATA1 cofactor FOG1 that disrupt binding to the nucleosome remodeling and deacetylase (NuRD) complex have erythroid and megakaryocyte lineages defects. Mice that are homozygous for a FOG1 point mutation (ki/ki), which ablates FOG1-NuRD interactions, have platelets that display a gray platelet syndrome (GPS)–like macrothrombocytopenia. These platelets have few α-granules and an increased number of lysosomal-like vacuoles on electron microscopy, reminiscent of the platelet in patients with GATA1-related X-linked GPS. Here we further characterized the platelet defect in ki/ki mice. We found markedly deficient levels of P-selectin protein limited to megakaryocytes and platelets. Other α-granule proteins were expressed at normal levels and were appropriately localized to α-granule–like structures. Treatment of ki/ki platelets with thrombin failed to stimulate Akt phosphorylation, resulting in poor granule secretion and platelet aggregation. These studies show that disruption of the GATA1/FOG1/NuRD transcriptional system results in a complex, pleiotropic platelet defect beyond GPS-like macrothrombocytopenia and suggest that this transcriptional complex regulates not only megakaryopoiesis but also α-granule generation and signaling pathways required for granule secretion.

Platelet transfusions are often a life-saving intervention, and the use of platelet transfusions has been increasing. Donor-derived platelet availability can be challenging. Compounding this concern are additional limitations of donor-derived platelets, including variability in product unit quality and quantity, limited shelf life and the risks of product bacterial contamination, other transfusion-transmitted infections, and immunologic reactions. Because of these issues, there has been an effort to develop strategies to generate platelets from exogenously generated precursor cells. If successful, such platelets have the potential to be a safer, more consistent platelet product, while reducing the necessity for human donations. Moreover, ex vivo–generated autologous platelets or precursors may be beneficial for patients who are refractory to allogeneic platelets. For patients with inherited platelet disorders, ex vivo–generated platelets offer the promise of a treatment via the generation of autologous gene-corrected platelets. Theoretically, ex vivo–generated platelets also offer targeted delivery of ectopic proteins to sites of vascular injury. This review summarizes the current, state-of-the-art methodologies in delivering a clinically relevant ex vivo–derived platelet product, and it discusses significant challenges that must be overcome for this approach to become a clinical reality.

We previously reported on a novel compound (Compound 1; RUC-1) identified by high-throughput screening that inhibits human αIIbβ3. RUC-1 did not inhibit αVβ3, suggesting that it interacts with αIIb, and flexible ligand/rigid protein molecular docking studies supported this speculation. We have now studied RUC-1's effects on murine and rat platelets, which are less sensitive than human to inhibition by Arg-Gly-Asp (RGD) peptides due to differences in the αIIb sequences contributing to the binding pocket. We found that RUC-1 was much less potent in inhibiting aggregation of murine and rat platelets. Moreover, RUC-1 potently inhibited fibrinogen binding to murine platelets expressing a hybrid αIIbβ3 receptor composed of human αIIb and murine β3, but not a hybrid receptor composed of murine αIIb and human β3. Molecular docking studies of RUC-1 were consistent with the functional data. In vivo studies of RUC-1 administered intraperitoneally at a dose of 26.5 mg/kg demonstrated antithrombotic effects in both ferric chloride carotid artery and laser-induced microvascular injury models in mice with hybrid hαIIb/mβ3 receptors. Collectively, these data support RUC-1's specificity for αIIb, provide new insights into the αIIb binding pocket, and establish RUC-1's antithrombotic effects in vivo.

Two major clinical limitations to the use of thrombolytic agents are their short half-life and lack of targeting specificity. We aimed to circumvent these limitations by targeting a platelet-bound, thrombin-activatable, low molecular weight urokinase, termed PLT/uPA-T, to nascent thrombi by a combination of two strategies: (1) Attaching the drug through its N-terminal scFv portion to human αIIb/β3 on the surface of platelets with nM affinity. (2) Protecting the uPA from rapid activation/inactivation in the circulation while simultaneously requiring its activation by thrombin through a two amino acid deletion at the plasmin cleavage site that concurrently creates a thrombin cleavage/activation site. These two properties constrain the activity of PLT/uPA-T on mature clots, which express low levels of thrombin and transiently recruit only a few new platelets to the shell rather than the core of the thrombus. These two properties of PLT/uPA-T also enhance the drug’s lifespan by attaching it to the platelet cell surface and preventing its inactivation before reaching its intended target. PLT/uPA-T binds specifically to human platelets and to hαIIb mouse platelets that transgenically expressed human (h) αIIb on its surface. In hαIIb mice, the half-life of retro-orbitally infused PLT/uPA-T was ~2 hours, ~100-fold longer than similarly infused uPA-T and did not cause any spontaneous bleeding or lower platelet counts 4 to 24 hours later. We now report two in vivo models to test the efficacy of the PLT/uPA-T as a thromboprophylactic agent versus uPA-T, taking into account the much shorter circulating half-life of the latter. In the hαIIb mice model, a tail-vein clipping model was done to represent the “mature clot” as follows: following clipping, blood was collected into 37°C water for 10 minutes. The tail was then removed from the water and a bolus of PLT/uPA-T (0.5 µg/g mouse) injected retro-orbitally followed by a continuous infusion of the same dose over the next 30 minutes. uPA-T was similarly infused but both bolus and infusion were given at 10-fold higher doses. A no-drug treatment control was also included. After the drug infusion was started, the tail was placed into fresh 37°C water and bleeding was documented from these “mature clots” over the ensuing 30 minutes. To study “nascent thrombi”, mice were bolused/infused with same drug regimens, and a FeCl3 carotid artery injury study was performed contemporaneously. PLT/uPA-T was as effective at 1/10th the dose as uPA-T at preventing these “nascent thrombi” (FeCl3 injuries), but did not cause bleeding from “mature clots” (tail clippings) relative to the no-treatment control, while the uPA-T treatment lead to ~5-fold greater rebleeding compared to the no-treatment control (p 10 animals per arm. The second in vivo model targeted human platelets infused into immunocompromized NOD-SCID γ-interferon-deficient (NSG) male mice to generate a calculated 10% of all the circulating platelets being human at the initiation of the studies. The pre-drug “mature clots” and the post-drug “nascent thrombi” were both arteriolar laser cremaster injuries. We enumerated mouse platelets incorporated into these thrombi over time. There was an ~50% decrease in the size of laser-induced post-drug “nascent thrombi” after PLT/uPA-T or a 10-fold higher dose of uPA-T relative to no drug treatment. However, PLT/uPA-T did not affect the size of the laser-induced pre-drug “mature clots” relative to no drug-treatment, while there was a decrease in size of the mature clots after treatment with uPA-T. These studies describe two preclinical models of comparative thromboprophylactic efficacy and safety that are independent of drug half-life. Our studies demonstrate that a combination of platelet-targeting and need for thrombin-activation makes PLT/uPA-T a very potent and targeted thrombolytic agent to prevent new thrombus formation, while leaving older, hemostatic clots intact. Disclosures No relevant conflicts of interest to declare.

Through an extremely complicated equilibrium called homeostasis, all living organisms maintain their survival in the face of both externally and internally generated "stimuli". This apparent harmony is constantly challenged. Survival through successful adaptation is maintained as close to steady state as possible by adaptive responses, which may also be called perturbation responses since they have a constitutively defined dynamic capacity, i.e., an immediate limit, in a series of balancing and feedback activities reflecting an astounding array of biological, psychological and sociological behaviors. The broad spectrum of stimuli capable of engaging this protective response is remarkable. We define stress as a type of stimulation that is stronger and lasts for a longer duration, upsetting a typical perturbation response given its dynamic parameters. The stress response, which evolves out of the perturbation response, involves inducible signal molecules, i.e., cytokines. We surmise that the ability to exist in an ever-changing environment was a requirement for all life forms, including invertebrates and single celled organisms. It would be expected that these organisms exhibit both perturbation and stress responses. In this regard, we demonstrate that these organisms have mammalian-like signal molecule systems, i.e., opioid, and corresponding behaviors that are similar to those found in mammals with regard to both perturbation and stress responses. Thus, it would appear that these responses evolved first in simpler organisms and were then maintained and enhanced during evolution.

Abstract 3450 Our goal is to generate sufficient PLTs from ex vivo-generated MKs for clinical utility in PLT transfusions. A critical step in this process begins with ex vivo-generated hMKs and deriving clinically relevant PLTs. We reported that infused mature, high-ploidy murine (m) MKs derived from fetal liver (FL) cells increased PLT counts in recipient mice in a clinically relevant fashion, thus avoiding the need to generate ex vivo functional PLTs. We examined whether this strategy applies to hMKs derived from FL cells (gestational age, 17–21 weeks) and bone marrow (BM) cells in a xeno-transfusion model using immunodeficient NOD/SCID/IL-2Rγcnull (NSG) mice. Infused hPLTs isolated from blood had a half-life (T1/2) of 10 hours (hrs), compared to 24 hrs for infused murine PLTs. The hPLTs were functional in NSG mice as demonstrated by their incorporation into growing thrombi in situ. Both hFL hematopoietic mononuclear cells and hBM-CD34+ cells were cultured in serum-free media supplemented with optimized cytokine cocktails to generate hMKs. In contrast to the murine studies where the FL cell-derived mMKs were the most efficient source of derived mPLTs, FL cell-derived hMKs had low ploidy (0% ≥ 8N ploidy), gave rise to ∼16 PLTs/infused hMK, and had a short T1/2 (6 hrs). In contrast, 17% of hBM cell-derived MKs had a ploidy of ≥ 8N, and after infusion into NSG mice, resulted in a wave of MKs transiently entrapped in the pulmonary microvasculature and then over ∼0.5–3 hrs released PLTs with a T1/2 of 10 hrs, comparable to infused hPLTs. Maximally, we achieved a level of 5% of circulating total PLTs being derived from human cells with ∼32 PLTs/infused hMK. These hPLTs were normal in size, displayed normal levels of surface markers, were functional, and incorporated into growing thrombi. One strategy to increase hPLT yield is to expose developing MKs to drugs reported to increase MK maturation, thrombopoiesis, and/or facilitate hematopoietic progenitor cell expansion. Such drugs include dimethylfasudil (diMF) (an inhibitor of several kinases involved in polyploidization), UNC0638 (a G9a histone methyltransferase inhibitor), SR1 (an AhR antagonist), and nicotinamide (a sirtuin histone/protein deacetylases inhibitor). Although diMF promoted size and polyploidization of hMKs, diMF markedly worsened yield of PLTs/infused hMK and decreased PLTs T1/2 in vivo. UNC0638 led to significant cell expansion, but lowered hMKs ploidy and PLTs/infused hMK yield. Nicotinamide increased maturation, size and polyploidization of hMKs, but PLT release following MK infusion needs further study. Of note, SR1 that has been reported to promote the expansion of human HSC, not only increased size and ploidy of hMKs, but also hPLT release in vitro and in vivo. SR1-treated hMKs resulted in a 3-fold increased yield of normal size, T1/2 and functional PLTs/infused hMK compared to a DMSO-treated control. In summary, like mMKs, infused hMKs into mice release PLTs in the pulmonary vasculature though at a lower efficiency. Released hPLTs were functional and T1/2 was as expected. diMF enhanced MK ploidy, but worsened PLT yield and T1/2, while an AhR antagonist SR1 that also improved MK ploidy appears to markedly enhance yield of PLT/infused hMK, while maintaining T1/2. The ability of SR1 to enhance PLT release from induced pluripotent stem cells (iPSCs)-derived MKs remains to be tested, but this drug appears to be a strong candidate for a therapeutic strategy to take ex vivo-grown hMKs and generate PLTs in clinical relevant numbers. Disclosures: No relevant conflicts of interest to declare.

Abstract 1171 Thrombolytic therapies are limited in their use to acute life- or limb-threatening thrombosis due to the high risk of significant bleeding. To overcome this limitation, we propose a novel strategy incorporating two targeting features: 1) an N-terminal single-chain antibody variable region (scFv) chain that binds with high affinity to human platelet alphaIIb, and 2) a C-terminal thrombin-specific activatable low molecular weight urokinase (uPA-T) that has its plasmin-activation site replaced by a thrombin cleavage site. We anticipate that such pro-drugs would preferentially bind and become activated at sites of active clot propagation. Two distinct anti-alphaIIb scFvs were studied: one (312.8) derived from a monoclonal antibody that bound human activated or quiescent alphaIIb whether complexed to human or mouse beta3, and one (LIBS) that bound preferentially to activated human or mouse alphaIIb bound to either human or mouse beta3. We anticipated that the 312.8/uPA-T chimera would have a more prolonged half-life, but that the LIBS version would be more effective at the nascent clot where activated platelets predominate. All proteins studied (uPA-T, 312.8/uPA-T and LIBS/uPA-T) were expressed and isolated from Drosophila S2 insect cells. Flow cytometric studies confirmed the expected binding of the chimera proteins to resting and activated human platelets, wildtype (WT) mouse platelets and mouse platelets that expressed only human alphaIIb/mouse beta3 (haIIb/mb3) on their surface. Neither chimeric protein interfered with platelet aggregation stimulated by adenosine diphosphate (10 μM). Further studies using diverse serine proteases confirmed that induction of fibrinolytic activities of both chimeras was thrombin-specific, and both were activated by thrombin to the same extent as isolated uPA-T. In haIIb/mb3 mice, both flow cytometric studies and tracking of 125I-labeled chimeric proteins showed 312.8/uPA-T had a longer half-life than uPA-T or LIBS/uPA-T. Ex vivo studies of thromboprophylactic efficacy of the chimeric proteins relative to uPA-T were performed using a microfluidic system with the surfaces coated with species-specific von Willebrand Factor. Platelets were isolated from whole human or haIIb/mb3 murine blood, exposed to a fibrinolytic agent or control, gel filtered to remove unbound drug, and added back to reconstitute whole blood. 312.8/uPA-T decreased platelet aggregation and virtually eliminated fibrin formation at drug concentrations Disclosures: Cines: Amgen: Consultancy; Genzyme: Consultancy; Sanofi: Consultancy; Eisai: Consultancy.

Abstract 635 HIT is an immune mediated prothrombotic disorder often associated with life- and limb-threatening thrombosis caused by antibodies to a complex between platelet factor 4 (PF4) and heparin. Platelet activation and clearance are considered key to the pathogenesis. Direct thrombin inhibitors, the most commonly used anticoagulant therapy in the treatment of HIT, provide incomplete prevention against development of new thrombi and little effect on the incidence of loss of limb and life. Thus, there is a need for a better understanding of pathogenesis of HIT and new approaches to therapy. We and others have shown that HIT not only is associated with platelet activation, but also involves activation of monocytes and endothelial cells, which together increase thrombin generation that may affect both the amount and structural properties of the resultant fibrin clot. However, this proposed increase in fibrin formation though suspected, has never been directly investigated. Previously, we have shown in the cremaster muscle laser-injury model of thrombosis induced by the HIT-like murine monoclonal anti-hPF4/heparin antibody KKO that transgenic mice expressing both human PF4 (hPF4+) and hFcγRIIA developed larger, more fibrin-rich occlusive thrombi than in control mice expressing hPF4 or hFcγRIIA alone. To quantify fibrin formation in a more controlled setting, we simulated HIT in a BioFlux microfluidic channel system coated with von Willebrand factor by perfusing whole blood at a venule shear stress of 20 dyne/cm2 at 37°C for 10 min. Platelets were labeled by adding Calcein-AM (3 μM) and fibrin was visualized by adding Alexa 647 labeled fibrinogen (1.5 μg/ml) to the whole blood prior to the perfusion. Using NaCitrate-anticoagulated human blood, we observed that recalcified human blood samples exposed to KKO plus hPF4 formed large platelet thrombi and an extensive fibrin network, with fibers radiating from the platelet aggregates and often organized along the direction of flow. In contrast, control samples exposed to a combination of the non-pathogenic anti-hPF4 monoclonal antibody RTO plus hPF4 showed little fibrin and less organization. Quantitative fluorescence analysis showed nine times more fibrin formed after stimulation with KKO plus PF4 than RTO plus PF4. Inhibition of KKO-mediated platelet activation by blocking FcgRIIA with Fab fragments of monoclonal antibody IV.3 in whole blood suppressed platelet adhesion by > 80%, but decreased fibrin formation by only ∼40%. On the other hand, addition of a selective inhibitor of the Syk tyrosine kinase PRT-060318 (Reilly et al., Blood 2011:117:2241–6; kindly provided by Dr. Uma Sinha, Portola Pharmaceuticals) to whole blood at a concentration of 3 μM suppressed both platelet adhesion and fibrin formation by 80% and 70%, respectively. IV.3 inhibits platelet activation alone, while we have shown that PRT-060318 inhibits both platelet activation and monocyte activation with the subsequent release of tissue factor-rich microparticles. These results provide a mechanistic basis for the use of novel therapies in HIT such as fibrinolytic agents. To do so, we studied a novel chimeric pro-fibrinolytic composed of a C-terminal thrombin-specific activatable low molecular weight urokinase (uPA-T) that has its plasmin-activation site replaced by a thrombin cleavage site and linked at its N-terminus to a single-chain variable region (scFv) that binds with high affinity to human platelet aIIb, designed to deliver the agent to sites of incipient thrombosis. Preliminary results show that uPA-T profoundly suppressed fibrin accumulation in both in vitro and in an in vivo model of HIT. This novel approach to therapy takes advantage of our growing understanding of the pathogenesis of the prothrombotic nature of HIT including monocyte activation and formation of fibrin-rich clots. Such therapeutics may be especially effective as targeted therapy in HIT. Disclosures: Cines: Amgen Inc.: Consultancy; GlaxoSmithKline: Consultancy; Eisai: Consultancy.

Abstract 3339 Use of plasminogen activators (PAs) is restricted to life-threatening thrombotic conditions because high concentrations are required to diffuse into clots, overcome PA inhibitors and compensate for rapid clearance, thereby predisposing to bleeding. We hypothesized that targeting pro-PAs to platelets would circumvent these obstacles and preferentially lyse nascent, pathological clots that are actively recruiting platelets, while sparing preformed hemostatic clots. To test this concept, we expressed a chimeric protein (anti-PLT scFv/uPA-T) composed of an N-terminal scFv generated from a monoclonal antibody MoAb 312.8 directed to human αIIb integrin chain linked via a serine-rich linker peptide to human low molecular weight thrombin activatable pro-urokinase (uPA-T) wherein the plasmin cleavage site was replaced with a thrombin cleavage site, which we reasoned would be activated preferentially at sites of active clot propagation. Anti-PLT scFv/uPA-T expressed in Drosophila S2 insect cells bound specifically to human platelets and to transgenic mouse platelets that contain only human αIIb/mouse β3 on their cell surface (HαIIb+ Tg), but not to wild type mouse platelets. The anti-PLT scFv/uPA-T bound specifically to immobilized human αIIbβ3 with a Kd of ∼80 nM. The anti-PLT scFv/uPA-T did not interfere with either ADP-induced platelet aggregation or activation as demonstrated by expression of P-selectin by flow cytometry, and retained its zymogenic properties until activated specifically by thrombin. The fibrinolytic activity of anti-PLT scFv/uPA-T and uPA-T were compared using the FeCl3 carotid artery injury model in HαIIb+ Tg mice. The mice were protected from forming occlusive thrombi for at least 10 hrs post injection of anti-PLT scFv/uPA-T whereas even five-fold higher concentrations of uPA-T were effective for only 2–5 min. These studies support a novel approach for prophylactic targeting drug delivery by combining a pro-drug that requires activation by thrombin with platelet delivery to sites of incipient thrombotic vascular occlusion. Disclosures: No relevant conflicts of interest to declare.

Abstract SCI-47 The clinical need for platelets transfusion continues to grow. Platelet units have all the usual concerns with other donor-derived blood products and in addition have concerns over the need to store the platelets at room temperature and of the resulting increased risk of bacterial contamination. Several groups have been able to develop platelets from embryonic stem cells, but achieving significant numbers of pro-platelets and/or platelets from in vitro grown megakaryocytes (Megs) remains elusive. Three sites of thrombopoiesis in vivo have been described: 1) intramedullary, 2) at the medullary/vascular junction, or 3) intrapulmonary from circulating Megs. We investigated whether infusing murine Megs into mice can release a large number of functional platelets both to better understand thrombopoiesis and also to see if such an approach may be an alternative to generating platelets for clinical usage. We show that infused fetal-liver-derived Megs results in 100–200 platelets/Meg. Levels as high as 12% of the total platelets in the recipient mouse were from the infused Megs. These platelets had a delayed presentation, peaking at 90 mins. In spite of these being new platelets, their half-life was only ∼75% of infused platelets. The shortened half-life was not due to the fact that these platelets were fetal in nature in that infused adult-liver-derived Megs had a similar temporal profile. These derived platelets had normal-size distribution, were not activated, and had not been affected by ADAM17 during in vitro culture as determined by surface glycoprotein 1b alpha. Platelet functionality was shown by their incorporation into clots in arterioles and venules in a cremaster laser injury model and in a ferric chloride carotid artery injury model. Using BrdU-labeled Megs we show that most of the Megs are trapped in the lungs with a few also appearing in the splenic red pulp. None were seen in other tissues. Staining with species-specific CD41 antibodies showed that detectable Meg cytoplasm peaked in the pulmonary vasculature at 30 min, and like the nuclei, was mostly gone by 5 hrs. We estimate that only ∼1% of the available pulmonary capillary bed is affected by the infused Megs, and most studied animals survived the infusion of the Megs. These studies support the lungs as a potential site for thrombopoiesis and that in vitro differentiated Megs can be infused directly avoiding the challenge of developing ex vivo technology to release large number of non-activated, but functional platelets. Disclosures: No relevant conflicts of interest to declare.

Abstract 225 Thrombopoiesis, the process by which circulating platelets (plts) arise from megakaryocytes (Megs) remains incompletely understood. Two models had been proposed: (1) release of plts produced from Megs within the marrow space and (2) release of plts by circulating Megs within the pulmonary vasculature. Recently, murine two-photon electron microscopy suggested that the actual process may involve an intermediate model, wherein Megs adjacent to vascular spaces within the marrow shed large cytoplasmic fragments into the circulation, which presumably go on to form plts in the vascular space. We have now tested whether infused cultured Megs can form circulating, functional plts. Cultured Megs from the fetal livers of Day 14 wildtype (WT) C57Bl6 murine embryos grown in the presence of thrombopoietin for 10 days were separated using a bovine serum albumin gradient into two cell fractions: (1) large, mature CD41+/CD42+ Megs (average ploidy = 16–32N) and (2) an admixture of proplatelets and small CD41+/CD42+ Megs (average ploidy = 2-4N). These fractions were separately infused into hαIIb mice that expressed human, but not mouse, αIIb on their platelets. Donor-derived circulating plts were detected using species-specific antibodies to distinguish them from recipient hαIIb+ plts. Infusions of isolated WT plts as a positive control resulted in immediately detectable donor plts, and these plts lasted 24–36 hrs. Infused large Megs produced plts with delayed kinetics. Few donor plts were detectable at 5 mins, followed by a peak at 30–60 min. These plts were normal in size as determined by side-scatter and lasted for ∼24 hrs. The peak yield was ∼50-100 plts/infused Meg. In these studies, these derived plt levels reached as high as 10% of the total circulating plts in these mice who had plt counts of >8×105/μl. Maximal tolerated dose of infused large Megs was not tested. Infusion of the proplatelets/small Megs fraction generated an immediate peak of circulating plts and these lasted Disclosures: Poncz: Diagnostica Stago: Patents & Royalties.

The search for novel inhibitors of the platelet αIIbβ3 receptor continues with the dual goals of better defining structure-function relationships and developing second generation oral agents. We previously reported on a novel small compound (Compound 1; RUC-1) identified by high throughput screening that inhibits human αIIbβ3. RUC-1 did not inhibit αVβ3, suggesting that it interacts with αIIb, and molecular docking studies supported this speculation. RUC-1 also induced less extensive changes in αIIbβ3 conformation than existing small molecule inhibitors, which may be therapeutically desirable. We have now studied RUC-1’s effects on murine and rat platelets, which are less sensitive than human to inhibition by RGD peptides due to differences in the αIIb sequences contributing to the binding pocket. We found that RUC-1 (100 μM) was much less potent in inhibiting platelet aggregation of murine and rat platelets than human platelets or the platelets of a mouse expressing a hybrid receptor composed of human αIIb and murine β3 (hαIIb/mβ3) (mouse, 6±6%, n=4; rat, 0±15%, n=3; human, 97±2% n=3; hαIIb/mβ3 99±1%, n=4). RUC-1 also inhibited fibrinogen binding to murine platelets expressing the hybrid hαIIb/mβ3 receptor (94± 2%, n=4), but not a hybrid receptor composed of murine αIIb and human β3 (mαIIb/hβ3; 0%, n=4). Molecular docking studies of RUC-1 were consistent with the functional data with RUC-1 binding entirely within the β-propeller. αIIb In vivo studies of RUC-1 administered intraperitoneally (IP) at a dose of 26.5 mg/kg demonstrated antithrombotic effects in the FeCl3 carotid artery model in mice expressing hαIIb/mβ3 (Figure 1A), but did not protect WT mice from thrombotic occlusion at the same dose (Figure 1B). Collectively, these data support RUC-1’s specificity for αIIb, provide new insights into the αIIb ligand-binding pocket, and establish RUC-1’s anti-thrombotic effects in vivo. In addition, the hαIIb/mβ3 mice provide a convenient model for testing low molecular weight αIIbβ3 antagonist drugs such as RUC-1 for toxicity and therapeutic potential. Figure 1. RUC-1 protects hαIIb/mβ3 mice, but not WT mice from FeCl3-induced carotid artery thrombotic occlusion. A. Mice expressing hαIIb/mβ3 receptor were injected IP with vehicle control (n=4), an inactive congener of RUC-1 (RUC-1-piperidine; n=4; 26.5 mg/kg), or RUC-1 (n=7; 26.5 mg/kg) 25 min before carotid artery injury with 20% FeCl3 for 3 min. Blood flow through the carotid artery was monitored for 30 min with a Doppler flow probe. Kaplan-Meier analysis was calculated from the time the FeCl3 was applied to the artery until complete occlusion. The control curve contains the combined data from the mice treated with vehicle and RUC-1-piperidine. B. WT mice (n=4) were given RUC-1 (26.5 mg/kg) and their data are compared to those reported in panel A for mice expressing hαIIb/mβ3. Figure 1. RUC-1 protects hαIIb/mβ3 mice, but not WT mice from FeCl3-induced carotid artery thrombotic occlusion. A. Mice expressing hαIIb/mβ3 receptor were injected IP with vehicle control (n=4), an inactive congener of RUC-1 (RUC-1-piperidine; n=4; 26.5 mg/kg), or RUC-1 (n=7; 26.5 mg/kg) 25 min before carotid artery injury with 20% FeCl3 for 3 min. Blood flow through the carotid artery was monitored for 30 min with a Doppler flow probe. Kaplan-Meier analysis was calculated from the time the FeCl3 was applied to the artery until complete occlusion. The control curve contains the combined data from the mice treated with vehicle and RUC-1-piperidine. B. WT mice (n=4) were given RUC-1 (26.5 mg/kg) and their data are compared to those reported in panel A for mice expressing hαIIb/mβ3.