Showing papers by "Barbara C. Furie published in 2005"

Leukocyte-versus microparticle-mediated tissue factor transfer during arteriolar thrombus development

Abstract: Circulating tissue factor accumulates in the developing thrombus and contributes to fibrin clot formation. To determine whether tissue factor derived from hematopoietic cells is delivered to the thrombus via tissue factor-bearing microparticles or circulating leukocytes expressing tissue factor on the plasma membrane, we compared the kinetics of tissue factor accumulation in the developing arteriolar thrombus with the time course of leukocyte-thrombus interaction and microparticle-thrombus interaction in the microcirculation of a living mouse using intravital high-speed widefield and confocal microscopy. Tissue factor rapidly accumulated in the developing thrombus, appearing immediately following vessel wall injury, reaching a first peak in approximately 100 s. In contrast, leukocyte-thrombus interaction was not observed until after 2-3 min following vessel wall injury. Maximal leukocyte rolling and firm leukocyte adherence on thrombi in wild-type mice were observed after approximately 8 min and were dependent on P-selectin and P-selectin glycoprotein ligand-1. This delay in P-selectin-dependent leukocyte rolling is a result of time-dependent platelet activation and P-selectin expression on the luminal surface of the thrombus. In contrast, microparticle accumulation in the developing arteriolar thrombus was rapid, and peak accumulation was within 60 s. The accumulation of hematopoietic cell-derived tissue factor in the developing thrombus correlates to the kinetics of microparticle accumulation and does not correlate temporally with leukocyte-thrombus interaction. These results indicate that tissue factor derived from hematopoietic cells is delivered by microparticles during the initial phase of thrombus development in vivo.

PTP-1B is an essential positive regulator of platelet integrin signaling

Abstract: Outside-in integrin αIIbβ3 signaling is required for normal platelet thrombus formation and is triggered by c-Src activation through an unknown mechanism. In this study, we demonstrate an essential role for protein–tyrosine phosphatase (PTP)–1B in this process. In resting platelets, c-Src forms a complex with αIIbβ3 and Csk, which phosphorylates c-Src tyrosine 529 to maintain c-Src autoinhibition. Fibrinogen binding to αIIbβ3 triggers PTP-1B recruitment to the αIIbβ3–c-Src–Csk complex in a manner that is dependent on c-Src and specific tyrosine (tyrosine 152 and 153) and proline (proline 309 and 310) residues in PTP-1B. Studies of PTP-1B–deficient mouse platelets indicate that PTP-1B is required for fibrinogen-dependent Csk dissociation from αIIbβ3, dephosphorylation of c-Src tyrosine 529, and c-Src activation. Furthermore, PTP-1B–deficient platelets are defective in outside-in αIIbβ3 signaling in vitro as manifested by poor spreading on fibrinogen and decreased clot retraction, and they exhibit ineffective Ca2+ signaling and thrombus formation in vivo. Thus, PTP-1B is an essential positive regulator of the initiation of outside-in αIIbβ3 signaling in platelets.

Interactions of platelets, blood-borne tissue factor, and fibrin during arteriolar thrombus formation in vivo.

Abstract: Thrombus formation following vascular injury is an essential component of both hemostasis and pathologic vessel occlusion. This process occurs in a closed, pressurized environment in which blood flows rapidly over the injury site. Thrombus formation must occur quickly to reduce blood loss, but is carefully modulated to limit vessel occlusion. Circulating cells, plasma proteins, vessel wall components, and physical forces such as shear all influence thrombus formation. Historically, thrombus formation has been studied by isolating the separate components of blood involved in clot formation. With improved optical techniques, investigators have increasingly studied thrombus formation under conditions of flow in vitro and in live animals in vivo. Using multichannel fluorescence intravital videomicroscopy, the authors have studied the changes in the kinetics and deposition of platelets, fibrin, and tissue factor at the injury site during thrombosis in transgenic mice, bone marrow transplanted mice, and mice treated with pharmacological agents that modulate thrombosis. The differences in the kinetics of accumulation of the various components of thrombus in these mice have provided new insights about thrombus formation in arterioles. This review discusses the role of platelet intracellular signaling, P-selectin expression on platelets, and tissue factor-bearing microparticles in thrombus formation.

Precursors of Novel Gla-Containing Conotoxins Contain a Carboxy-Terminal Recognition Site That Directs gamma-Carboxylation(,).

Abstract: Vitamin K-dependent γ-glutamyl carboxylase catalyzes the conversion of glutamyl residues to γ-carboxyglutamate. Its substrates include vertebrate proteins involved in blood coagulation, bone mineralization, and signal transduction and invertebrate ion channel blockers known as conotoxins. Substrate recognition involves a recognition element, the γ-carboxylation recognition site, typically located within a cleavable propeptide preceding the targeted glutamyl residues. We have purified two novel γ-carboxyglutamate-containing conotoxins, Gla-TxX and Gla-TxXI, from the venom of Conus textile. Their cDNA-deduced precursors have a signal peptide but no apparent propeptide. Instead, they contain a C-terminal extension that directs γ-carboxylation but is not found on the mature conotoxin. A synthetic 13-residue “postpeptide” from the Gla-TxXI precursor reduced the Km for the reaction of the Conus γ-carboxylase with peptide substrates, including FLEEL and conantokin-G, by up to 440-fold, regardless of whether it w...