6 research outputs found
An advocate for minorities in science
No full textDr. Teshell Greene, who grew up on the Caribbean island of St. Kitts, moved to New York at 14 to pursue her dream of being a science researcher. Today she is an instructor in the VCU Department of Biology and a staunch advocate for underrepresented minorities in the field
Understanding and improving platelet-delivered Factor (F) VIII hemostatic efficacy in hemophilia A mice
No full textHemophilia A is the most common, inherited severe bleeding diathesis and is due to a deficiency of functional Factor (F) VIII. Two challenges to current FVIII replacement therapy are maintaining sustained FVIII corrections and treating the 30% population of Hemophilia A patients who develop FVIII inhibitors. Gene therapy promises to be an attractive treatment for hemophilia A as there is a wide therapeutic window for FVIII corrective plasma levels. Since platelets target to hemostatic injuries, ectopically expressed platelet (p) FVIII is an attractive alternative FVIII delivery strategy. We have already shown the effectiveness of platelet human B-domainless factor VIII (phBF8) in the presence of circulating inhibitors in FVIIInull mice. However, the clots formed are unstable and lead to increased embolization with phBF8. This dissertation describes platelet-specific gene therapy using three FVIII variants with enhanced activity: inactivation resistant FVIII (IR8), canine (c) B-domainless FVIII (cBF8), and hBF8R1645 H. Each improved clotting efficacy in FVIIInull mice and decreased embolic risk though to different degrees. For example, we found that despite pcBF8 having ~30% antigenic levels of phBF8, it corrected clot instability seen in a cremaster injury model in FVIIInull mice. Since IR8 does not bind FVIII carrier protein, Von Willebrand Factor (vWF), efficiently, we tested the effectiveness of this variant in the presence of circulating FVIII inhibitors, and found that vWF binding was not needed for IR8 to be effective. To understand the cellular mechanism for decreased cBF8 relative to hBF8 expression level in megakaryocytes, we studied mRNA levels and the effect of pcBF8 on megakaryopoiesis and found that the level of cBF8 is related to increased cellular apoptosis. Finally to understand the mechanistic basis of pcBF8 increased hemostatic effectiveness, we examined whether mutation of the cleavage site for the Golgi apparatus enzyme PACE/furin in hBF8 to that of cBF8, which increases the FVIII’s activity, would also enhance hBF8R1645H pFVIII activity and affirm that this occurs. These studies provide new insights into pFVIII during megakaryopoiesis and how to optimize pFVIII hemostatic efficacy that should be useful in translating this therapeutic model to a large animal hemophilia A model and future clinical trials
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The kinetics of αIIbβ3 activation determines the size and stability of thrombi in mice: implications for antiplatelet therapy.
No full textTwo 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 A(2). 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
CXCL5 Regulates Chemokine Scavenging and Pulmonary Host Defense to Bacterial Infection
No full textSummaryThe chemokine sink hypothesis pertaining to erythrocyte Duffy Antigen Receptor for Chemokines (DARC) during inflammation has received considerable attention, but lacks direct in vivo evidence. Here we demonstrate, using mice with a targeted deletion in CXCL5, that CXCL5 bound erythrocyte DARC and impaired its chemokine scavenging in blood. CXCL5 increased the plasma concentrations of CXCL1 and CXCL2 in part through inhibiting chemokine scavenging, impairing chemokine gradients and desensitizing CXCR2, which led to decreased neutrophil influx to the lung, increased lung bacterial burden and mortality in an Escherichia coli pneumonia model. In contrast, CXCL5 exerted a predominant role in mediating neutrophil influx to the lung during inflammation after LPS inhalation. Platelets and lung resident cells were the sources of homeostatic CXCL5 in blood and inflammatory CXCL5 in the lung respectively. This study presents a paradigm whereby platelets and red cells alter chemokine scavenging and neutrophil-chemokine interaction during inflammation
Monocyte-bound PF4 in the pathogenesis of heparin-induced thrombocytopenia
No full textAbstractHeparin-induced thrombocytopenia (HIT) is a life- and limb-threatening thrombotic disorder that develops after exposure to heparin, often in the setting of inflammation. We have shown previously that HIT is associated with antibodies to complexes that form between platelet factor 4 and glycosaminoglycan (GAG) side chains on the surface of platelets. However, thrombosis can occur in the absence of thrombocytopenia. We now show that platelet factor 4 binds to monocytes and forms antigenic complexes with their surface GAG side chains more efficiently than on platelets likely due to differences in GAG composition. Binding to monocytes is enhanced when the cells are activated by endotoxin. Monocyte accumulation within developing arteriolar thrombi was visualized by situ microscopy. Monocyte depletion or inactivation in vivo attenuates thrombus formation induced by photochemical injury of the carotid artery in a modified murine model of HIT while paradoxically exacerbating thrombocytopenia. These studies demonstrate a previously unappreciated role for monocytes in the pathogenesis of arterial thrombosis in HIT and suggest that therapies targeting these cells might provide an alternative approach to help limit thrombosis in this and possibly other thrombotic disorders that occur in the setting of inflammation.</jats:p
In vivo efficacy of platelet-delivered, high specific activity factor VIII variants
No full textAbstractEctopically expressed, human B-domainless (hB) factor 8 (F8) in platelets improves hemostasis in hemophilia A mice in several injury models. However, in both a cuticular bleeding model and a cremaster laser arteriole/venule injury model, there were limitations to platelet-derived (p) hBF8 efficacy, including increased clot embolization. We now address whether variants of F8 with enhanced activity, inactivation resistant F8 (IR8) and canine (c) BF8, would improve clotting efficacy. In both transgenic and lentiviral murine model approaches, pIR8 expressed at comparable levels to phBF8, but pcBF8 expressed at only approximately 30%. Both variants were more effective than hBF8 in cuticular bleeding and FeCl3 carotid artery models. However, in the cremaster injury model, only pcBF8 was more effective, markedly decreasing clot embolization. Because inhibitors of F8 are stored in platelet granules and IR8 is not protected by binding to von Willebrand factor, we also tested whether pIR8 was effective in the face of inhibitors and found that pIR8 is protected from the inhibitors. In summary, pF8 variants with high specific activity are more effective in controlling bleeding, but this improved efficacy was inconsistent between bleeding models, perhaps reflecting the underlying mechanism(s) for the increased specific activity of the studied F8 variants.</jats:p
