ALBERT

Description: 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.

Description: Key Points The novel FVIII variant (FVIII-RH) has enhanced stability and procoagulant activity in both in vitro and in vivo models. FVIII-RH is efficacious and safe; thus, it is an attractive molecule for protein replacement and as a transgene in gene-therapy strategies.

Description: HIT is an iatrogenic complication of heparin therapy caused by antibodies that recognize the platelet chemokine, platelet factor 4 (PF4), complexed to heparin or to cellular glycosaminoglycans (GAG). Unlike most other immune thrombocytopenias, HIT is markedly prothrombotic. We have proposed that this prothrombotic tendency is due to binding of pathogenic antibodies to PF4 complexes attached to the surface GAGs expressed by all intravascular cells, including platelets with their relatively low affinity surface GAGs, chondroitin sulfates, and monocytes with their higher affinity membrane GAGs, heparan and dermatan sulfates. Using isolated monocytes from healthy volunteers, we show by scanning electron microscopy that the addition of 10-50 µg/ml of recombinant human PF4 causes the appearance of ∼200 nm “knobs” on the cell surface. Subsequent addition of a HIT-like monoclonal antibody KKO at 50 µg/ml to the PF4-coated cells markedly alters their surface with the appearance of larger, up to 1-2 µm, membrane “blebs”. These blebs increase in size over time (15-60 minutes) and are shed from the cells. After shedding of these blebs, the monocytes lose their typical ruffled surface and appear spherical. These surface changes in the presence of KKO and PF4 are not seen in the presence of PF4 and 50 µg/ml of the anti-PF4 monoclonal antibody RTO, which does not induce the prothrombotic state of HIT. Platelets in suspension exposed to PF4 and KKO show by scanning electron microscopy similar knobs on their surface, but only minimally form blebs or microvesiculate. Platelets spread on fibrinogen in culture medium stimulated with PF4 and KKO and observed by hopping probe ion conductance microscopy progressively developed surface protrusions over a period of an hour, becoming more spherical. This morphological change was also observed in platelets exposed to IgG purified from 5 patients with HIT, but not when the KKO Fab fragment was tested. Neither PF4 alone nor with RTO antibody induced this morphological transition. Exposure to KKO plus PF4 for an hour induced minimal microvesiculation of platelets as measured by flow cytometry. Platelets adherent to fibrinogen underwent a similar morphological transition and did not microvesiculate after adding 50 mM of a thrombin-receptor activating peptide, whereas ADP-stimulated platelets rapidly microvesiculated during the same period of time. We believe that the “knobs” observed on monocytes and platelets represent aggregates of PF4-GAG complexes that are the targets of HIT antibodies. Bleb formation on monocytes and morphological transition of platelets result from clustering of knobs caused by bivalent HIT antibodies which cross-link Fc receptors. These blebs are released from monocytes, potentially becoming the microparticles found in the plasma of patients with HIT. In contrast, platelets treated with KKO plus PF4 showed minimal microvesiculation. This finding differs from reported high platelet microparticle counts in the plasma of HIT patients, suggesting that additional factors may be required to induce platelets to microvesiculate. Our images represent the first visualization of surface events when platelets and monocytes assume an active prothrombotic state. Whether these are unique to HIT or have wider applicability to the changes that occur in other prothrombotic, proinflammatory states needs to be addressed. Disclosures: No relevant conflicts of interest to declare.

Description: Two major pathways contribute to Ras-proximate-1–mediated integrin activation in stimulated platelets. Calcium and diacyglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI, RasGRP2) mediates the rapid but reversible activation of integrin αIIbβ3, while the adenosine diphosphate receptor P2Y12, the target for antiplatelet drugs like clopidogrel, facilitates delayed but sustained integrin activation. To establish CalDAG-GEFI as a target for antiplatelet therapy, we compared how each pathway contributes to thrombosis and hemostasis in mice. Ex vivo, thrombus formation at arterial or venous shear rates was markedly reduced in CalDAG-GEFI−/− blood, even in the presence of exogenous adenosine diphosphate and thromboxane A2. In vivo, thrombosis was virtually abolished in arterioles and arteries of CalDAG-GEFI−/− mice, while small, hemostatically active thrombi formed in venules. Specific deletion of the C1-like domain of CalDAG-GEFI in circulating platelets also led to protection from thrombus formation at arterial flow conditions, while it only marginally increased blood loss in mice. In comparison, thrombi in the micro- and macrovasculature of clopidogrel-treated wild-type mice grew rapidly and frequently embolized but were hemostatically inactive. Together, these data suggest that inhibition of the catalytic or the C1 regulatory domain in CalDAG-GEFI will provide strong protection from athero-thrombotic complications while maintaining a better safety profile than P2Y12 inhibitors like clopidogrel.

Description: 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.

Description: 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.

Description: Thrombosis is the most striking complication of heparin-induced thrombocytopenia (HIT). We have shown that monocytes are preferentially targeted by HIT antibodies because of the higher affinity of monocyte surface glycosaminoglycans (GAGs) for PF4 than the chondroitin sulfate GAGs on platelets. The contribution and mechanism by which monocytes promote thrombosis in HIT have not been fully elucidated. It has been reported that HIT antibodies activate monocytes through FcγRI and then by the MEK1-ERK1/2 intracellular pathway leading to the expression of tissue factor (TF) (Kasthuri et al., Blood 2012 119:5285). While activation of platelets by HIT antibodies through FcγRIIA is well established, the role of this receptor in monocyte activation in HIT is not clear. We examined the role of monocytes and their family of Fcγ receptors in HIT using several in vitro models, including a novel microfluidic system that allowed us to examine the prothrombotic pathways using human- and murine-based systems. Our studies showed that monocytes were key to the prothrombotic state; simply adding monocytes coated with PF4 and pre-activated with KKO, a HIT-like monoclonal antibody, was sufficient to form platelet-fibrin clots in reconstituted blood samples combining isolated red cells, platelets and mononuclear cells. Using three separate approaches, we also found that HIT antibodies bound to surface PF4/GAG complexes activate monocytes via the same Fc receptor that mediates platelet activation, i.e., FcγRIIA. First, using a strategy of blocking individual classes of Fcγ receptors known to be present on monocytes - FcγRI, FcγRIIA and FcγRIII - by monoclonal antibodies, we found that only anti-FcγRIIA decreased fibrin deposition (by 52 ± 8%; p

Description: Sepsis is a high-risk clinical setting often resulting in multi-organ failure and death. Release of chromatin NETS (neutrophil extracellular traps) from neutrophils and the toxic role of highly-positively charged histones in late sepsis have been noted previously. Also, for NET formation to occur, peptidylarginine deiminase 4 activity must be present in the neutrophils, leading to citrullinated (cit) histones formation and loss of a portion of the positive charge. The four histones (H2A, H2B, H3 and H4) alone and as octamers of the four units tightly bind DNA. H3 and H4 histones as well as mixed octameric histones can induce a sepsis-like state in mice. One feature previously noted was that histones could inhibit activated protein C (aPC) production in the presence of thrombomodulin (TM). Since aPC generation is felt to protect against vascular damage, it was felt that this might - in part - account for the deleterious effects of histones in sepsis. We have shown that another highly-positive, small molecule, platelet factor 4 (PF4, CXCL4), which exists as a tetramer and which is stored in high concentrations in platelet alpha granules to be released in large amounts post-platelet activation, binds to the chondroitin sulfate (CS) side-chain of TM (TMCS) and enhanced aPC production along a bell-shaped curve with a peak effect around 25 µg/ml. Non-modified mixed histones had a similar bell-shaped effect on aPC generation and [histones + PF4] are additive on affecting aPC generation via TMCS. We wondered, because of this overlapping biology and the fact that significant levels of free PF4 are available in late sepsis, whether PF4 might affect other histone pathobiological pathways in late sepsis focusing on PF4’s interactions with non-modified and cit-histones. We first asked whether released PF4 might affect the binding of histones to DNA within NETS. We found that PF4 binds to DNA with greater affinity than histones in a competitive binding assay and that this effect was more marked for cit-histone consistent with its decreased positive charge. We then studied PF4 biology in three known targets of histone in sepsis. (1) In aPC generation, we examined cit-histones (either mixed, H3 or H4) relative to non-modified histones in stimulating aPC generation and found that they had a more limited effect on aPC generation with TMCS, but that again, PF4 cooperated in inducing aPC generation along a bell-shaped curve. (2) Histones are known to activate platelets (known to involve the toll-like receptor 4), likely contributing to the observed thrombocytopenia in late sepsis. We affirmed this affect with mixed histones and H4. Cit-mixed histones and cit-H4 also activated platelets in a platelet aggregation system, but much more weakly. PF4 had no effect on platelet activation by non-modified histones, but enhanced platelet activation by both cit-mixed histones and cit-H4. This was especially true for platelet activation studies with cit-H4 which on its own had nearly no affect on platelet activation though in the presence of moderate levels of PF4 (25 µg/ml), cit-H4 activated platelets as well as non-modified histones. (3) Finally, both non-modified and cit-histones activate endothelial cells (EC) by binding to their cell surfaces and likely contribute to the vascular damage of late sepsis. Using a microfluidic system involving controlled photochemical injury of the EC lining we found that PF4 enhanced the observed damage after cit-H4 exposure, but not notably after a comparable H4 exposure so that peak damage (as detected by propidium iodide staining) after cit-H4 approached that seen after H4 alone. In conclusion, NET formation involves citrullination of histones, and these modified histones likely contribute significantly to pathobiology in late sepsis. We now propose that in late sepsis, free histones, especially cit-histones, are mobilized out of NETs by PF4 because the PF4 binds DNA with higher affinity. After the histones and cit-histones are released from DNA, PF4 modifies the biology of these histones, especially the cit-histones enhancing their effects on aPC generation, platelet activation and EC injury. These studies provide additional insights of how histones achieve their pathobiological effects in sepsis. Such new insights may be critical for both understanding and monitoring clinical outcome and may lead to new therapeutic targets in sepsis. Disclosures No relevant conflicts of interest to declare.

Description: 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.