Marstacimab, a tissue factor pathway inhibitor neutralizing antibody, improves coagulation parameters of ex vivo dosed haemophilic blood and plasmas.

INTRODUCTION: Tissue factor pathway inhibitor (TFPI) is an endogenous inhibitor of the extrinsic pathway that negatively regulates thrombin production during coagulation. Under haemophilic conditions, where the intrinsic coagulation pathway is impaired, inhibition of TFPI may improve clotting. AIM: We investigated the ex vivo effects of a human TFPI neutralizing antibody, marstacimab (previously PF-06741086), in coagulation assays including rotational thromboelastometry (ROTEM), thrombin generation assay (TGA) and the dilute prothrombin time (dPT) assay, performed in haemophilic whole blood and plasmas. We compared the effects of marstacimab to the effects of recombinant coagulation factors and investigated the reproducibility of marstacimab in restoring haemostasis by comparing its effect in whole blood collected from the same study participants on differing days. METHODS: Citrated whole blood and plasmas obtained from haemophilia participants were supplemented ex vivo with vehicle, marstacimab, recombinant FVIII (rFVIII) or recombinant factor IX (rFIX) and analysed in ROTEM, TGA and the dPT assay using low tissue factor concentrations to trigger coagulation. RESULTS: Marstacimab induced pro-coagulant responses in ROTEM parameters including reduction in clotting times and increases in angle. Similarly, participant plasmas supplemented with marstacimab exhibited improvements in TGA parameters, including reduced lag times, increased peak thrombin concentrations and reductions in dPT clotting time. Concentrations of marstacimab tested showed activity comparable to addition of rFVIII or rFIX and were reproducible. CONCLUSIONS: These studies show the ex vivo potency of marstacimab in restoring haemostasis in whole blood and plasmas from haemophilia participants and comparability to ex vivo reconstitution with recombination coagulation factors.

The origin and function of platelet glycosyltransferases.

Platelets are megakaryocyte subfragments that participate in hemostatic and host defense reactions and deliver pro- and antiangiogenic factors throughout the vascular system. Although they are anucleated cells that lack a complex secretory apparatus with distinct Golgi/endoplasmic reticulum compartments, past studies have shown that platelets have glycosyltransferase activities. In the present study, we show that members of 3 distinct glycosyltransferase families are found within and on the surface of platelets. Immunocytology and flow cytometry results indicated that megakaryocytes package these Golgi-derived glycosyltransferases into vesicles that are sent via proplatelets to nascent platelets, where they accumulate. These glycosyltransferases are active, and intact platelets glycosylate large exogenous substrates. Furthermore, we show that activation of platelets results in the release of soluble glycosyltransferase activities and that platelets contain sufficient levels of sugar nucleotides for detection of glycosylation of exogenously added substrates. Therefore, the results of the present study show that blood platelets are a rich source of both glycosyltransferases and donor sugar substrates that can be released to function in the extracellular space. This platelet-glycosylation machinery offers a pathway to a simple glycoengineering strategy improving storage of platelets and may serve hitherto unknown biologic functions.

The spectrin-based membrane skeleton stabilizes mouse megakaryocyte membrane systems and is essential for proplatelet and platelet formation.

Megakaryocytes generate platelets by remodeling their cytoplasm first into proplatelets and then into preplatelets, which undergo fission to generate platelets. Although the functions of microtubules and actin during platelet biogenesis have been defined, the role of the spectrin cytoskeleton is unknown. We investigated the function of the spectrin-based membrane skeleton in proplatelet and platelet production in murine megakaryocytes. Electron microscopy revealed that, like circulating platelets, proplatelets have a dense membrane skeleton, the main fibrous component of which is spectrin. Unlike other cells, megakaryocytes and their progeny express both erythroid and nonerythroid spectrins. Assembly of spectrin into tetramers is required for invaginated membrane system maturation and proplatelet extension, because expression of a spectrin tetramer-disrupting construct in megakaryocytes inhibits both processes. Incorporation of this spectrin-disrupting fragment into a novel permeabilized proplatelet system rapidly destabilizes proplatelets, causing blebbing and swelling. Spectrin tetramers also stabilize the "barbell shapes" of the penultimate stage in platelet production, because addition of the tetramer-disrupting construct converts these barbell shapes to spheres, demonstrating that membrane skeletal continuity maintains the elongated, pre-fission shape. The results of this study provide evidence for a role for spectrin in different steps of megakaryocyte development through its participation in the formation of invaginated membranes and in the maintenance of proplatelet structure.

Megakaryocyte-derived microparticles: direct visualization and distinction from platelet-derived microparticles.

Platelet microparticles are a normal constituent of circulating blood. Several studies have demonstrated positive correlations between thrombotic states and platelet microparticle levels. Yet little is known about the processes by which platelet microparticles are generated in vivo. We now characterize microparticles derived directly from megakaryocytes. Video microscopy of live mouse megakaryocytes demonstrated that microparticles form as submicron beads along the lengths of slender, unbranched micropodia. These microparticles are CD41(+), CD42b(+), and express surface phosphatidylserine. Megakaryocyte microparticle generation is resistant to inhibition of microtubule assembly, which is critical to platelet formation, and augmented by inhibition of actin polymerization. To determine whether circulating microparticles are derived primarily from activated platelets or megakaryocytes, we identified markers that distinguish between these 2 populations. CD62P and LAMP-1 were found only on mouse microparticles from activated platelets. In contrast, full-length filamin A was found in megakaryocyte-derived microparticles, but not microparticles from activated platelets. Circulating microparticles isolated from mice were CD62P(-), LAMP-1(-) and expressed full-length filamin A, indicating a megakaryocytic origin. Similarly, circulating microparticles isolated from healthy volunteers were CD62P(-) and expressed full-length filamin A. Cultured human megakaryocytes elaborated microparticles that were CD41(+), CD42b(+), and express surface phosphatidylserine. These results indicate that direct production by megakaryocytes represents a physiologic means to generate circulating platelet microparticles.

Visualization of microtubule growth in living platelets reveals a dynamic marginal band with multiple microtubules.

The marginal band of microtubules maintains the discoid shape of resting blood platelets. Although studies of platelet microtubule coil structure conclude that it is composed of a single microtubule, no investigations of its dynamics exist. In contrast to previous studies, permeabilized platelets incubated with GTP-rhodamine-tubulin revealed tubulin incorporation at 7.9 (+/- 1.9) points throughout the coil, and anti-EB1 antibodies stained 8.7 (+/- 2.0) sites, indicative of multiple free microtubules. To pursue this result, we expressed the microtubule plus-end marker EB3-GFP in megakaryocytes and examined its behavior in living platelets released from these cells. Time-lapse microscopy of EB3-GFP in resting platelets revealed multiple assembly sites within the coil and a bidirectional pattern of assembly. Consistent with these findings, tyrosinated tubulin, a marker of newly assembled microtubules, localized to resting platelet microtubule coils. These results suggest that the resting platelet marginal band contains multiple highly dynamic microtubules of mixed polarity. Analysis of microtubule coil diameters in newly formed resting platelets indicates that microtubule coil shrinkage occurs with aging. In addition, activated EB3-GFP-expressing platelets exhibited a dramatic increase in polymerizing microtubules, which travel outward and into filopodia. Thus, the dynamic microtubules associated with the marginal band likely function during both resting and activated platelet states.

Dynamin 3 participates in the growth and development of megakaryocytes.

High-density oligonucleotide microarrays were used to compare gene expression profiles from uncultured CD34+/CD38lo cells and culture-derived megakaryocytes (MKs). As previously published, three replicate microarray data sets from three different sources of organ donor marrow were analyzed using the software program Rosetta Resolver. After setting a stringent p value of

Translational Pharmacokinetic/Pharmacodynamic Characterization and Target-Mediated Drug Disposition Modeling of an Anti-Tissue Factor Pathway Inhibitor Antibody, PF-06741086.

Tissue factor pathway inhibitor (TFPI) exhibits multiple isoforms, which are known to present in multiple locations such as plasma, endothelium, and platelets. TFPI is an endogenous negative modulator of the coagulation pathway, and therefore, neutralization of TFPI function can potentially increase coagulation activity. A human monoclonal antibody, PF-06741086, which interacts with all isoforms of TFPI is currently being tested in clinic for treating hemophilia patients with and without inhibitors. To support clinical development of PF-06741086, pharmacokinetics (PK) and pharmacodynamics of PF-06741086 were characterized in monkeys. In addition, a mechanistic model approach was used to estimate PK parameters in monkeys and simulate PK profiles in human. The results show that PF-06741086 exhibited target-mediated drug disposition and had specific effects on various hemostatic markers including diluted prothrombin time, thrombin generation, and thrombin-antithrombin complex in monkeys after administration. The model-predicted and observed human exposures were compared retrospectively, and the result indicates that the exposure prediction was reasonable within less than 2-fold deviation. This study demonstrated in vivo efficacy of PF-06741086 in monkeys and the utility of a rational mechanistic approach to describe PK for a monoclonal antibody with complex target binding.

Abnormal clotting of the intrinsic/contact pathway in Alzheimer disease patients is related to cognitive ability.

Alzheimer disease (AD) is a neurodegenerative disorder characterized by extracellular ß-amyloid (Aß) deposition. Although peripheral inflammation and cerebrovascular pathology are reported in AD, there is a lack of plasma biomarkers in this field. Because the contact system is triggered in patient plasma, we hypothesized that the hemostasis profile could be a novel biomarker in AD. Here, we assessed the clotting profile in plasma from AD patients and age-matched controls. Utilizing clinically relevant assays, thromboelastography and activated partial thromboplastin time, we found impaired clot initiation and formation rate in AD patient plasma. These coagulation end points correlated with cerebrospinal fluid neurofilament-light levels and cognition and were more profound in younger AD patients. Ex vivo intrinsic clotting of plasma from AD mice expressing human amyloid precursor protein (APP) was also delayed in an age-dependent manner, suggesting that this phenotype is related to APP, the parent protein of Aß. Further analysis of coagulation factors in human plasma indicated that endogenous inhibitor(s) of factors XII and XI in AD plasma contribute to this delayed clotting. Together, these data suggest that delayed clotting in young AD patients is a novel biomarker and that therapies aimed to correct this phenotype might be beneficial in this patient population. Follow-up studies in additional AD patient cohorts are warranted to further evaluate these findings.

Endomucin prevents leukocyte-endothelial cell adhesion and has a critical role under resting and inflammatory conditions.

Endomucin is a membrane-bound glycoprotein expressed luminally by endothelial cells that line postcapillary venules, a primary site of leukocyte recruitment during inflammation. Here we show that endomucin abrogation on quiescent endothelial cells enables neutrophils to adhere firmly, via LFA-1-mediated binding to ICAM-1 constitutively expressed by endothelial cells. Moreover, TNF-α stimulation downregulates cell surface expression of endomucin concurrent with increased expression of adhesion molecules. Adenovirus-mediated expression of endomucin under inflammatory conditions prevents neutrophil adhesion in vitro and reduces the infiltration of CD45(+) and NIMP-R14(+) cells in vivo. These results indicate that endomucin prevents leukocyte contact with adhesion molecules in non-inflamed tissues and that downregulation of endomucin is critical to facilitate adhesion of leukocytes into inflamed tissues.

Differential roles of cAMP and cGMP in megakaryocyte maturation and platelet biogenesis.

The cyclic nucleotides cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) regulate the activity of protein kinase A (PKA) and protein kinase G (PKG), respectively. This process helps maintain circulating platelets in a resting state. Here we studied the role of cAMP and cGMP in the regulation of megakaryocyte (MK) differentiation and platelet formation. Cultured, platelet-producing MKs were differentiated from fetal livers harvested from 13.5 days postcoital mouse embryos. MK development was accompanied by a dramatic increase in cAMP production and expression of soluble guanylate cyclase, PKG, and PKA as well as their downstream targets vasodilator-stimulated phosphoprotein (VASP) and MENA. Stimulation of prostaglandin E(1) receptor/adenylyl cyclase or soluble guanylate cyclase/PKG in cultured MKs increased VASP phosphorylation, indicating that these components share a common signaling pathway. To dissect out the role of cyclic nucleotides in MK differentiation, cAMP/PKA and cGMP/PKG signaling were alternately blocked in cultured MKs. Down-regulation of cAMP pathway effectors decreased MK numbers and ploidy. Notably, cGMP levels increased at the beginning of MK development and returned to basal levels in parallel with MK maturation. However, inhibition of cGMP pathway effectors had no effect on MK development. In addition, platelet release from mature MKs was enhanced by cGMP and inhibited by cAMP. Our data suggest that cAMP plays an important role in MK differentiation, while cAMP and cGMP have opposite effects on platelet production. Identifying the signaling pathways that underpin MK development and proplatelet formation will provide greater insights into thrombopoiesis and may potentially yield useful therapeutic targets.

Signal transduction in vasculogenesis and developmental angiogenesis.

The vasculature is a highly specialized organ that functions in a number of key physiological tasks including the transport of oxygen and nutrients to tissues. Formation of the vascular system is an essential and rate-limiting step in development and occurs primarily through two main mechanisms, vasculogenesis and angiogenesis. Both vasculogenesis, the de novo formation of vessels, and angiogenesis, the growth of new vessels from pre-existing vessels by sprouting, are complex processes that are mediated by the precise coordination of multiple cell types to form and remodel the vascular system. A host of signaling molecules and their interaction with specific receptors are central to activating and modulating vessel formation. This review article summarizes the current state of research involving signaling molecules that have been demonstrated to function in the regulation of vasculogenesis and angiogenesis, as well as molecules known to play a role in vessel maturation, hypoxia-driven angiogenesis and arterial-venous specification.

Cytoskeletal mechanics of proplatelet maturation and platelet release.

Megakaryocytes generate platelets by remodeling their cytoplasm into long proplatelet extensions, which serve as assembly lines for platelet production. Although the mechanics of proplatelet elongation have been studied, the terminal steps of proplatelet maturation and platelet release remain poorly understood. To elucidate this process, released proplatelets were isolated, and their conversion into individual platelets was assessed. This enabled us to (a) define and quantify the different stages in platelet maturation, (b) identify a new intermediate stage in platelet production, the preplatelet, (c) delineate the cytoskeletal mechanics involved in preplatelet/proplatelet interconversion, and (d) model proplatelet fission and platelet release. Preplatelets are anucleate discoid particles 2-10 µm across that have the capacity to convert reversibly into elongated proplatelets by twisting microtubule-based forces that can be visualized in proplatelets expressing GFP-ß1-tubulin. The release of platelets from the ends of proplatelets occurs at an increasing rate in time during culture, as larger proplatelets undergo successive fission, and is potentiated by shear.

Direct visualization of the endomitotic cell cycle in living megakaryocytes: differential patterns in low and high ploidy cells.

Endomitosis in megakaryocytes (MKs) involves repeated DNA replication in the absence of cytokinesis and is a crucial part of MK development. However, chromosomal dynamics have never been observed in living MKs. We developed a new transgenic mouse model in which the expression of human histone H2B fused in-frame to green fluorescent protein is targeted to MKs. Ex vivo time-lapse microscopy analysis indicated that chromosomal condensation occurs at early mitosis in all MKs. In high ploidy MKs (>or=8N), late anaphase was marked by a ring-type alignment of chromosomes with multiple territories formed between them. By contrast, in low ploidy MKs mitotic chromosomes segregated to form two groups separated by a clear space before re-joining to one cluster. This is the first study to document chromosomal segregation patterns during endomitosis ex vivo and to indicate their potential differential regulation in low and high ploidy cells.

Angiogenesis is regulated by a novel mechanism: pro- and antiangiogenic proteins are organized into separate platelet alpha granules and differentially released.

Platelets, in addition to their function in hemostasis, play an important role in wound healing and tumor growth. Because platelets contain angiogenesis stimulators and inhibitors, the mechanisms by which platelets regulate angiogenesis remain unclear. As platelets adhere to activated endothelium, their action can enhance or inhibit local angiogenesis. We therefore suspected a higher organization of angiogenesis regulators in platelets. Using double immunofluorescence and immunoelectron microscopy, we show that pro- and antiangiogenic proteins are separated in distinct subpopulations of alpha-granules in platelets and megakaryocytes. Double immunofluorescence labeling of vascular endothelial growth factor (VEGF) (an angiogenesis stimulator) and endostatin (an angiogenesis inhibitor), or for thrombospondin-1 and basic fibroblast growth factor, confirms the segregation of stimulators and inhibitors into separate and distinct alpha-granules. These observations motivated the hypothesis that distinct populations of alpha-granules could undergo selective release. The treatment of human platelets with a selective PAR4 agonist (AYPGKF-NH(2)) resulted in release of endostatin-containing granules, but not VEGF-containing granules, whereas the selective PAR1 agonist (TFLLR-NH(2)) liberated VEGF, but not endostatin-containing granules. In conclusion, the separate packaging of angiogenesis regulators into pharmacologically and morphologically distinct populations of alpha-granules in megakaryocytes and platelets may provide a mechanism by which platelets can locally stimulate or inhibit angiogenesis.