Publications (9) View all
-
Article: C560Rβ3 caused platelet integrin αIIbβ3 to Bind Fibrinogen Continuously yet Resulted in a Severe Bleeding Syndrome and Increased Murine Mortality.
[show abstract] [hide abstract]
ABSTRACT: BACKGROUND AND OBJECTIVES: β3-deficient(-/-) megakaryocytes were modified by human β3-lentivirus transduction and transplantation to express sufficient levels of a C560Rβ3 amino acid substitution to investigate how an activated αIIbβ3 conformation affects platelets in vivo in mice. PATIENT/METHODS: Identical to our previous report of a R560β3 mutation in a patient with Glanzmann thrombasthenia, R560β3 murine platelets spontaneously bound antibody that only recognizes activated αIIbβ3 bound to its ligand, fibrinogen. RESULTS: With this murine model, we now show that αIIb-R560β3 mutation mediated continuous binding of fibrinogen occurs in the absence of P-selectin surface expression indicating that the integrin was in an active conformation, although the platelets circulated in a quiescent manner. Remarkably, only 35% of R560β3 "mutant" mice survived six months after transplant while 87% of C560β3 "wildtype" mice remained alive. Pathological examination revealed that R560β3 mice had enlarged spleens with extramedullary hematopoiesis and increased hemosiderin indicating haemorrhage. R560β3 megakaryocytes and platelets displayed abnormal morphology and irregular granule distribution. Interestingly, R560β3 washed platelets could aggregate upon simultaneous addition of fibrinogen and physiological agonists, but aggregation failed when platelets were exposed to fibrinogen before activation in vitro and in vivo. CONCLUSIONS: Thus, the results demonstrate that continuous occupancy of αIIbβ3 with fibrinogen disrupts platelet structure and function leading to haemorrhagic death consistent with Glanzmann thrombasthenia rather than a thrombotic state. © 2013 International Society on Thrombosis and Haemostasis.Journal of Thrombosis and Haemostasis 04/2013; · 5.73 Impact Factor -
Article: Arachidonic Acid-Induced Dilation in Human Coronary Arterioles: Convergence of Signaling Mechanisms on Endothelial TRPV4-Mediated Ca2+ Entry.
[show abstract] [hide abstract]
ABSTRACT: Arachidonic acid (AA) and/or its enzymatic metabolites are important lipid mediators contributing to endothelium-derived hyperpolarizing factor (EDHF)-mediated dilation in multiple vascular beds, including human coronary arterioles (HCAs). However, the mechanisms of action of these lipid mediators in endothelial cells (ECs) remain incompletely defined. In this study, we investigated the role of the transient receptor potential vanilloid 4 (TRPV4) channel in AA-induced endothelial Ca(2+) response and dilation of HCAs. AA induced concentration-dependent dilation in isolated HCAs. The dilation was largely abolished by the TRPV4 antagonist RN-1734 and by inhibition of endothelial Ca(2+)-activated K(+) channels. In native and TRPV4-overexpressing human coronary artery ECs (HCAECs), AA increased intracellular Ca(2+) concentration ([Ca(2+)]i), which was mediated by TRPV4-dependent Ca(2+) entry. The AA-induced [Ca(2+)]i increase was inhibited by cytochrome P450 (CYP) inhibitors. Surprisingly, the CYP metabolites of AA, epoxyeicosatrienoic acids (EETs), were much less potent activators of TRPV4, and CYP inhibitors did not affect EET production in HCAECs. Apart from its effect on [Ca(2+)]i, AA induced endothelial hyperpolarization, and this effect was required for Ca(2+) entry through TRPV4. AA-induced and TRPV4-mediated Ca(2+) entry was also inhibited by the protein kinase A inhibitor PKI. TRPV4 exhibited a basal level of phosphorylation, which was inhibited by PKI. Patch-clamp studies indicated that AA activated TRPV4 single-channel currents in cell-attached and inside-out patches of HCAECs. AA dilates HCAs through a novel mechanism involving endothelial TRPV4 channel-dependent Ca(2+) entry that requires endothelial hyperpolarization, PKA-mediated basal phosphorylation of TRPV4, and direct activation of TRPV4 channels by AA.Journal of the American Heart Association. 01/2013; 2(3):e000080. -
Article: Platelet gene therapy improves hemostatic function for integrin alphaIIbbeta3-deficient dogs.
[show abstract] [hide abstract]
ABSTRACT: Activated blood platelets mediate the primary response to vascular injury. Although molecular abnormalities of platelet proteins occur infrequently, taken collectively, an inherited platelet defect accounts for a bleeding diathesis in ≈1:20,000 individuals. One rare example of a platelet disorder, Glanzmann thrombasthenia (GT), is characterized by life-long morbidity and mortality due to molecular abnormalities in a major platelet adhesion receptor, integrin αIIbβ3. Transfusion therapy is frequently inadequate because patients often generate antibodies to αIIbβ3, leading to immune-mediated destruction of healthy platelets. In the most severe cases allogeneic bone marrow transplantation has been used, yet because of the risk of the procedure it has been limited to few patients. Thus, hematopoietic stem cell gene transfer was explored as a strategy to improve platelet function within a canine model for GT. Bleeding complications necessitated the use of a mild pretransplant conditioning regimen; therefore, in vivo drug selection was used to improve engraftment of autologously transplanted cells. Approximately 5,000 αIIbβ3 receptors formed on 10% of platelets. These modest levels allowed platelets to adhere to αIIbβ3's major ligand (fibrinogen), form aggregates, and mediate retraction of a fibrin clot. Remarkably, improved hemostatic function was evident, with ≤135-fold reduced blood loss, and improved buccal bleeding times decreased to 4 min for up to 5 y after transplant. One of four transplanted dogs developed a significant antibody response to αIIbβ3 that was attenuated effectively with transient immune suppression. These results indicate that gene therapy could become a practical approach for treating inherited platelet defects.Proceedings of the National Academy of Sciences 06/2011; 108(23):9583-8. · 9.68 Impact Factor -
Article: Heparin promotes platelet responsiveness by potentiating αIIbβ3-mediated outside-in signaling.
[show abstract] [hide abstract]
ABSTRACT: Unfractionated heparin (UFH) is a widely used anticoagulant that has long been known to potentiate platelet responses to subthreshold doses of platelet agonists. UFH has been reported to bind and induce modest conformational changes in the major platelet integrin, αIIbβ3, and induce minor changes in platelet morphology. The mechanism by which UFH elicits these platelet-activating effects, however, is not well understood. We found that both human and murine platelets exposed to UFH, either in solution or immobilized onto artificial surfaces, underwent biochemical and morphologic changes indicative of a potentiated state, including phosphorylation of key cytosolic signaling molecules and cytoskeletal changes leading to cell spreading. Low molecular weight heparin and the synthetic pentasaccharide, fondaparinux, had similar platelet-potentiating effects. Human or mouse platelets lacking functional integrin αIIbβ3 complexes and human platelets pretreated with the fibrinogen receptor antagonists eptifibatide or abciximab failed to become potentiated by heparin, demonstrating that heparin promotes platelet responsiveness via its ability to initiate αIIbβ3-mediated outside-in signaling. Taken together, these data provide novel insights into the mechanism by which platelets become activated after exposure to heparin and heparin-coated surfaces, and suggest that currently used glycoprotein IIb-IIIa inhibitors may be effective inhibitors of nonimmune forms of heparin-induced platelet activation.Blood 03/2011; 117(18):4946-52. · 9.90 Impact Factor -
Article: TRPV4-mediated endothelial Ca2+ influx and vasodilation in response to shear stress.
[show abstract] [hide abstract]
ABSTRACT: The transient receptor potential vallinoid type 4 (TRPV4) channel has been implicated in the endothelial shear response and flow-mediated dilation, although the precise functions of this channel remain poorly understood. In the present study, we investigated the role of TRPV4 in shear stress-induced endothelial Ca(2+) entry and the potential link between this signaling response and relaxation of small resistance arteries. Using immunohistochemical analysis and RT-PCR, we detected strong expression of TRPV4 protein and mRNA in the endothelium in situ and endothelial cells freshly isolated from mouse small mesenteric arteries. The selective TRPV4 agonist GSK1016790A increased endothelial Ca(2+) and induced potent relaxation of small mesenteric arteries from wild-type (WT) but not TRPV4(-/-) mice. Luminal flow elicited endothelium-dependent relaxations that involved both nitric oxide and EDHFs. Both nitric oxide and EDHF components of flow-mediated relaxation were markedly reduced in TRPV4(-/-) mice compared with WT controls. Using a fura-2/Mn(2+) quenching assay, shear was observed to produce rapid Ca(2+) influx in endothelial cells, which was markedly inhibited by the TRPV4 channel blocker ruthenium red and TRPV4-specific short interfering RNA. Flow elicited a similar TRPV4-mediated Ca(2+) entry in HEK-293 cells transfected with TRPV4 channels but not in nontransfected cells. Collectively, these data indicate that TRPV4 may be a potential candidate of mechanosensitive channels in endothelial cells through which the shear stimulus is transduced into Ca(2+) signaling, leading to the release of endothelial relaxing factors and flow-mediated dilation of small resistance arteries.AJP Heart and Circulatory Physiology 12/2009; 298(2):H466-76. · 3.71 Impact Factor
