Christopher M. Spring

Plasma fibronectin depletion enhances platelet aggregation and thrombus formation in mice lacking fibrinogen and von Willebrand factor

We previously showed that platelet aggregation and thrombus formation occurred in mice lacking both fibrinogen (Fg) and von Willebrand factor (VWF) and that plasma fibronectin (pFn) promoted thrombus growth and stability in injured arterioles in wild-type mice. To examine whether pFn is required for Fg/VWF-independent thrombosis, we generated Fg/VWF/conditional pFn triple-deficient (TKO; Cre(+), Fn(flox/flox), Fg/VWF(-/-)) mice and littermate control (Cre(-), Fn(flox/flox), Fg/VWF(-/-)) mice. Surprisingly, TKO platelet aggregation was not abolished, but instead was enhanced in both heparinized platelet-rich plasma and gel-filtered platelets. This enhancement was diminished when TKO platelets were aggregated in pFn-positive control platelet-poor plasma (PPP), whereas aggregation was enhanced when control platelets were aggregated in pFn-depleted TKO PPP. The TKO platelet aggregation can be completely inhibited by our newly developed mouse anti-mouse beta(3) integrin antibodies but was not affected by anti-mouse GPIbalpha antibodies. Enhanced platelet aggregation was also observed when heparinized TKO blood was perfused in collagen-coated perfusion chambers. Using intravital microscopy, we further showed that thrombogenesis in TKO mice was enhanced in both FeCl(3)-injured mesenteric arterioles and laser-injured cremaster arterioles. Our data indicate that pFn is not essential for Fg/VWF-independent thrombosis and that soluble pFn is probably an important inhibitory factor for platelet aggregation.

Fibrinogen is required for maintenance of platelet intracellular and cell-surface P-selectin expression

Platelet P-selectin plays important roles in inflammation and contributes to thrombosis and hemostasis. Although it has been reported that von Willebrand factor (VWF) affects P-selectin expression on endothelial cells, little information is available regarding regulation of platelet P-selectin expression. Here, we first observed that P-selectin expression was significantly decreased on platelets of fibrinogen and VWF double-deficient mice. Subsequently, we identified this was due to fibrinogen deficiency. Impaired P-selectin expression on fibrinogen-deficient platelets was further confirmed in human hypofibrinogenemic patients. We demonstrated that this impairment is unlikely due to excessive P-selectin shedding, deficient fibrinogen-mediated cell surface P-selectin binding, or impaired platelet granule release, but rather is due to decreased platelet P-selectin content. Fibrinogen transfusion completely recovered this impairment in fibrinogen-deficient (Fg(-/-)) mice, and engagement of the C-terminus of the fibrinogen gamma chain with beta3 integrin was required for this process. Furthermore, Fg(-/-) platelets significantly increased P-selectin expression following transfusion into beta3 integrin-deficient mice and when cultured with fibrinogen. These data suggest fibrinogen may play important roles in inflammation, thrombosis, and hemostasis via enhancement of platelet P-selectin expression. Since human fibrinogen levels vary significantly in normal and diseased populations, P-selectin as an activation marker on platelets should be used with caution.

Plasma fibronectin supports hemostasis and regulates thrombosis

Plasma fibronectin (pFn) has long been suspected to be involved in hemostasis; however, direct evidence has been lacking. Here, we demonstrated that pFn is vital to control bleeding in fibrinogen-deficient mice and in WT mice given anticoagulants. At the site of vessel injury, pFn was rapidly deposited and initiated hemostasis, even before platelet accumulation, which is considered the first wave of hemostasis. This pFn deposition was independent of fibrinogen, von Willebrand factor, β3 integrin, and platelets. Confocal and scanning electron microscopy revealed pFn integration into fibrin, which increased fibrin fiber diameter and enhanced the mechanical strength of clots, as determined by thromboelastography. Interestingly, pFn promoted platelet aggregation when linked with fibrin but inhibited this process when fibrin was absent. Therefore, pFn may gradually switch from supporting hemostasis to inhibiting thrombosis and vessel occlusion following the fibrin gradient that decreases farther from the injured endothelium. Our data indicate that pFn is a supportive factor in hemostasis, which is vital under both genetic and therapeutic conditions of coagulation deficiency. By interacting with fibrin and platelet β3 integrin, pFn plays a self-limiting regulatory role in thrombosis, suggesting pFn transfusion may be a potential therapy for bleeding disorders, particularly in association with anticoagulant therapy.

Animal model of fetal and neonatal immune thrombocytopenia: role of neonatal Fc receptor in the pathogenesis and therapy

Fetal and neonatal immune thrombocytopenia (FNIT) is a severe bleeding disorder in which maternal antibodies cross the placenta and destroy fetal/neonatal platelets. It has been demonstrated that the neonatal Fc receptor (FcRn) regulates immunoglobulin G (IgG) homeostasis and plays an important role in transplacental IgG transport. However, the role of FcRn in the pathogenesis and therapy of FNIT has not been studied. Here, we developed an animal model of FNIT using combined β3 integrin-deficient and FcRn-deficient (β3(-/-)FcRn(-/-)) mice. We found that β3(-/-)FcRn(-/-) mice are immunoresponsive to β3(+/+)FcRn(-/-) platelets. The generated antibodies were β3 integrin specific and were maintained at levels that efficiently induced thrombocytopenia in adult β3(+/+)FcRn(-/-) mice. FNIT was observed when immunized β3(-/-)FcRn(+/+) females were bred with β3(+/+)FcRn(+/+) males, while no FNIT occurred in β3(-/-)FcRn(-/-) females bred with β3(+/+)FcRn(-/-) males, suggesting that FcRn is indispensable for the induction of FNIT. We further demonstrated that fetal FcRn was responsible for the transplacental transport of various IgG isotypes. We found that anti-FcRn antibody and intravenous IgG prevented FNIT, and that intravenous IgG ameliorated FNIT through both FcRn-dependent and -independent pathways. Our data suggest that targeting FcRn may be a potential therapy for human FNIT as well as other maternal pathogenic antibody-mediated diseases.

Relative efficacy of steroid therapy in immune thrombocytopenia mediated by anti-platelet GPIIbIIIa versus GPIbα antibodies.

Immune thrombocytopenia (ITP) is characterized by platelet clearance mediated primarily by autoantibodies against the platelet GPIIbIIIa and/or GPIbα. Steroid therapy is a first‐line treatment for ITP. However, some patients are refractory to this therapy and currently no method can predict which patients will respond. To evaluate whether steroids are equally efficacious in treating patients with ITP caused by anti‐GPIIbIIIa versus anti‐GPIbα antibodies, we performed a retrospective study on 176 newly diagnosed patients with acute ITP who had severe bleeding symptoms and were admitted as resident patients to the hospital. The patients were treated first with intravenous administration of high‐dose dexamethasone (DXM), followed by oral administration of prednisone. Response to therapy was observed in a majority of patients with antibodies specific for GPIIbIIIa (31/43) or without detectable antibodies against either GPIIbIIIa or GPIbα (36/45). In contrast, the steroid response was significantly lower in patients with anti‐GPIbα antibodies (9/34) or with antibodies against both GPIbα and GPIIbIIIa (16/54). The preliminary findings of this study suggest that in future prospective clinical trials including corticosteroids, the anti‐GPIbα, and ‐GPIIbIIIa status should be assessed in order to test its potential relevance in deciding future treatments.

Cadherin 6 Has a Functional Role in Platelet Aggregation and Thrombus Formation

Objective—Thrombosis occurs at sites of vascular injury when platelets adhere to subendothelial matrix proteins and to each other. Platelets express many surface receptor proteins, the function of several of these remains poorly characterized. Cadherin 6 is expressed on the platelet surface and contains an arginine-glycine-aspartic acid motif, suggesting that it might have a supportive role in thrombus formation. The aim of this study was to characterize the role of cadherin 6 in platelet function. Methods and Results—Platelet aggregation was inhibited by both antibodies and exogenous soluble cadherin 6. Platelet adhesion to immobilized cadherin 6 was inhibited by arginine-glycine-aspartic acid-serine tetrapeptides. Antibodies to &agr;IIb&bgr;3 inhibited platelet adhesion to cadherin 6. Because platelet aggregation occurs in fibrinogen and von Willebrand factor double-deficient mice, we investigated whether cadherin 6 is an alternative ligand for the integrin &agr;IIb&bgr;3. Platelet aggregation in fibrinogen and von Willebrand factor double-deficient mice was significantly inhibited by an antibody to cadherin 6. In flow-based assays, inhibition of cadherin 6 caused a marked reduction in thrombus formation in both human and mouse blood. Conclusion—This study demonstrates the role of cadherin 6 as a novel ligand for &agr;IIb&bgr;3 and highlights its function in thrombus formation.

Angiogenic Dysfunction in Bone Marrow-Derived Early Outgrowth Cells from Diabetic Animals Is Attenuated by SIRT1 Activation

Impaired endothelial repair is a key contributor to microvascular rarefaction and consequent end‐organ dysfunction in diabetes. Recent studies suggest an important role for bone marrow‐derived early outgrowth cells (EOCs) in mediating endothelial repair, but the function of these cells is impaired in diabetes, as in advanced age. We sought to determine whether diabetes‐associated EOC dysfunction might be attenuated by pharmacological activation of silent information regulator protein 1 (SIRT1), a lysine deacetylase implicated in nutrient‐dependent life span extension in mammals. Despite being cultured in normal (5.5 mM) glucose for 7 days, EOCs from diabetic rats expressed less SIRT1 mRNA, induced less endothelial tube formation in vitro and neovascularization in vivo, and secreted less of the proangiogenic ELR+ CXC chemokines CXCL1, CXCL3, and CXCL5. Ex vivo SIRT1 activation restored EOC chemokine secretion and increased the in vitro and in vivo angiogenic activity of EOC conditioned medium derived from diabetic animals to levels similar to that derived from control animals. These findings suggest a pivotal role for SIRT1 in diabetes‐induced EOC dysfunction and that its pharmacologic activation may provide a new strategy for the restoration of EOC‐mediated repair mechanisms.

Early outgrowth cells release soluble endocrine antifibrotic factors that reduce progressive organ fibrosis

Adult bone marrow‐derived cells can improve organ function in chronic disease models, ostensibly by the release of paracrine factors. It has, however, been difficult to reconcile this prevailing paradigm with the lack of cell retention within injured organs and their rapid migration to the reticuloendothelial system. Here, we provide evidence that the salutary antifibrotic effects of bone marrow‐derived early outgrowth cells (EOCs) are more consistent with an endocrine mode of action, demonstrating not only the presence of antifibrotic factors in the plasma of EOC‐treated rats but also that EOC conditioned medium (EOC‐CM) potently attenuates both TGF‐β‐ and angiotensin II‐induced fibroblast collagen production in vitro. To examine the therapeutic relevance of these findings in vivo, 5/6 subtotally nephrectomized rats, a model of chronic kidney and heart failure characterized by progressive fibrosis of both organs, were randomized to receive i.v. injections of EOC‐CM, unconditioned medium, or 106 EOCs. Rats that received unconditioned medium developed severe kidney injury with cardiac diastolic dysfunction. In comparison, EOC‐CM‐treated rats demonstrated substantially improved renal and cardiac function and structure, mimicking the changes found in EOC‐treated animals. Mass spectrometric analysis of EOC‐CM identified proteins that regulate cellular functions implicated in fibrosis. These results indicate that EOCs secrete soluble factor(s) with highly potent antifibrotic activity, that when injected intravenously replicate the salutary effects of the cells themselves. Together, these findings suggest that an endocrine mode of action may underlie the effectiveness of cell therapy in certain settings and portend the possibility for systemic delivery of cell‐free therapy. Stem Cells 2013;31:2408–2419

Novel nuclear hENT2 isoforms regulate cell cycle progression via controlling nucleoside transport and nuclear reservoir

Nucleosides participate in many cellular processes and are the fundamental building blocks of nucleic acids. Nucleoside transporters translocate nucleosides across plasma membranes although the mechanism by which nucleos(t)ides are translocated into the nucleus during DNA replication is unknown. Here, we identify two novel functional splice variants of equilibrative nucleoside transporter 2 (ENT2), which are present at the nuclear envelope. Under proliferative conditions, these splice variants are up-regulated and recruit wild-type ENT2 to the nuclear envelope to translocate nucleosides into the nucleus for incorporation into DNA during replication. Reduced presence of hENT2 splice variants resulted in a dramatic decrease in cell proliferation and dysregulation of cell cycle due to a lower incorporation of nucleotides into DNA. Our findings support a novel model of nucleoside compartmentalisation at the nuclear envelope and translocation into the nucleus through hENT2 and its variants, which are essential for effective DNA synthesis and cell proliferation.

Improved resolution in extracellular vesicle populations using 405 instead of 488 nm side scatter

ABSTRACT Improvements in identification and assessment of extracellular vesicles (EVs) have fuelled a recent surge in EV publications investigating their roles as biomarkers and mediators of disease. Meaningful scientific comparisons are, however, hampered by difficulties in accurate, reproducible enumeration and characterization of EVs in biological fluids. High-sensitivity flow cytometry (FCM) is presently the most commonly applied strategy to assess EVs, yet its utility is limited by variant ability to resolve smaller EVs. Here, we propose the use of 405 nm (violet) wavelength lasers in place of 488 nm (blue) for side scatter (SSC) detection to obtain greater resolution of EVs using high-sensitivity FCM. To test this hypothesis, we modelled EV resolution by violet versus blue SSC in silico and compared resolution of reference beads and biological EVs from plasma and bronchoalveolar lavage (BAL) fluid using either violet or blue wavelength SSC EV detection. Mie scatter modelling predicted that violet as compared to blue SSC increases resolution of small (100–500 nm) spherical particles with refractive indices (1.34–1.46) similar to EVs by approximately twofold in terms of light intensity and by nearly 20% in SSC signal quantum efficiency. Resolution of reference beads was improved by violet instead of blue SSC with two- and fivefold decreases in coefficients of variation for particles of 300–500 nm and 180–240 nm size, respectively. Resolution was similarly improved for detection of EVs from plasma or BAL fluid. Violet SSC detection for high-sensitivity FCM allows for significantly greater resolution of EVs in plasma and BAL compared to conventional blue SSC and particularly improves resolution of smaller EVs. Notably, the proposed strategy is readily implementable and inexpensive for machines already equipped with 405 nm SSC or the ability to accommodate 405/10 nm bandpass filters in their violet detector arrays.