Internalization of tissue factor by platelets.

Tissue factor (TF) has been found associated with platelets. Mechanisms responsible for TF-platelet interactions and transport are not fully understood. We explored the response of isolated washed platelets to preparations of recombinant (rTF) or placental (pTF) human TF, exposed on lipid microvesicles (MVs). Sequential ultrastructural and immunocytochemical studies revealed trafficking of these TF preparations, being endocytosed by platelets into channels of the open canalicular system (OCS) and accumulating in the cytoplasm and occasionally in alpha-granules. The process of internalization of TF-MVs was accomplished in less than 30 min, being faster for the placental (pTF) than for the recombinant (rTF) preparation. Signs of mild platelet activation with pseudopodia formation were observed at early stages of internalization. Platelets returned to an apparent resting state after 5-10 min. All of these observations paralleled with modifications on patterns of tyrosine phosphorylation for several signaling proteins. Our studies demonstrate that platelets possess mechanisms to capture and incorporate TF-rich vesicles. These processes were accelerated by the presence of other contaminating cellular antigens in the vesicles (pTF). TF carried by platelets could play a potential role in platelet thrombus formation and by extension in the development of ischemic complications.

Tissue factor-dependent coagulation is preferentially up-regulated within arterial branching areas in a baboon model of Escherichia coli sepsis.

Endothelium plays a critical role in the pathobiology of sepsis by integrating systemic host responses and local rheological stimuli. We studied the differential expression and activation of tissue factor (TF)-dependent coagulation on linear versus branched arterial segments in a baboon sepsis model. Animals were injected intravenously with lethal doses of Escherichia coli or saline and sacrificed after 2 to 8 hours. Whole-mount arterial segments were stained for TF, TF-pathway inhibitor (TFPI), factor VII (FVII), and markers for endothelial cells (ECs), leukocytes, and platelets, followed by confocal microscopy and image analysis. In septic animals, TF localized preferentially at branches, EC surface, leukocytes, and platelet aggregates and accumulated in large amounts in the subendothelial space. FVII strongly co-localized with TF on ECs and leukocytes but less so with subendothelial TF. TFPI co-localized with TF and FVII on endothelium and leukocytes but not in the subendothelial space. Focal TF increases correlated with fibrin deposition and increased endothelial permeability to plasma proteins. Biochemical analysis confirmed that aortas of septic baboons expressed more TF mRNA and protein than controls. Branched segments contained higher TF protein levels and coagulant activity than equivalent linear areas. These data suggest that site-dependent endothelial heterogeneity and rheological factors contribute to focal procoagulant responses to E. coli.

Activity, folding, misfolding, and aggregation in vitro of the naturally occurring human tissue factor mutant R200W.

Tissue factor (TF), a small transmembrane receptor, binds factor VIIa (FVIIa), and the formed complex initiates blood coagulation by proteolytic activation of substrate factors IX and X. A naturally occurring mutation in the human TF gene was recently reported, where a single-base substitution results in an R200W mutation in the TF extracellular domain [Zawadzki, C., Preudhomme, C., Gaveriaux, V., Amouyel, P., and Jude, B. (2002) Thromb. Haemost. 87, 540-541]. This mutation appears to be associated with low monocyte TF expression and may protect against thrombosis but has not been associated with any pathological condition, and individuals who present the heterozygous trait appear healthy. Here, we report the activity, folding, and aggregation behavior of the R200W mutant of the 219-residue soluble extracellular domain of TF (sTF(R200W)) compared to that of the wild-type protein (sTF(wt)). No differences in stability or FVIIa cofactor activity but an impaired ability to promote FX activation at physiological conditions between the sTF(R200W) mutant and sTF(wt) were evident. Increased binding of 1-anilino-8-naphthalene-sulfonic acid (ANS) to sTF(R200W) indicated a population of partially folded intermediates during denaturation. sTF(R200W) showed a dramatically increased propensity for aggregate formation compared to sTF(wt) at mildly acidic pHs, with an increased rate of aggregation during conditions, promoting the intermediate state. The lowered pH resistance could explain the loss of sTF(R200W) in vivo because of aggregation of the mutant. The intrinsic structure of the sTF aggregates appears reminiscent of amyloid fibrils, as revealed by thioflavin T fluorescence, atomic force microscopy, and transmission electron microscopy. We conclude that the lowered activity for FX activation and the propensity of the mutant protein to misfold and aggregate will both contribute to decreased coagulation activity in TF(R200W) carriers, which could protect from thrombotic disease.

Crystal structure of a humanized Fab fragment of anti-tissue-factor antibody in complex with tissue factor.

Tissue factor (TF) is a membrane-anchored protein that initiates the extrinsic cascade of blood coagulation. TF forms a complex with serine protease Factor VIIa, and then activates Factor X zymogen to Factor Xa, leading to the blood coagulation. Humanized anti-TF antibody hATR-5 strongly inhibits TF-initiated blood coagulation, and is of potential use for various thrombotic diseases. The Fab fragment of antibody hATR-5 is obtained for crystallization. The crystal structure of the complex of the Fab with extracellular domains of human TF was determined with the molecular replacement method, and refined to an R factor of 0.196 at 2.1 A resolution. All the complementarity-determining regions (CDRs) of the Fab are involved in interaction with the C-terminal-side extracellular domain of TF through 19 hydrogen bonds. The interface between the Fab and TF molecules contains 15 water molecules, and yields buried surface areas as wide as 2000 A2. The TF surface in the interface is possibly involved in the activation of Factor X, by forming a transient ternary complex of Factor X-TF-Factor VIIa. Electrostatic interactions are predominantly observed between the heavy-chain CDRs and TF. These hydrogen-bonding and electrostatic interactions together with the wide buried areas contribute to the high affinity of the antibody toward TF, leading to the effective inhibition of the TF-initiated blood coagulation.

Crystallization and preliminary X-ray analysis of human tissue factor extracellular domain.

The extracellular domain (residues 1 to 220) of human tissue factor has been cloned and expressed in Escherichia coli and purified to isoelectric homogeneity. Single crystals suitable for X-ray analysis have been obtained by vapour diffusion. They belong to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2 with a = b = 45.2 A, c = 231.5 A, contain one molecule per asymmetric unit and diffract to 2.6 A resolution. Native and derivative data sets have been collected to 3.6 and 3.9 A, respectively.

[Tissue thromboplastin in the human brain]. / Tkanevoi tromboplastin golovnogo mozga cheloveka.

The studies by the scanning electron microscopy have shown that there are small amounts of membrane-like structures in the dry preparation of tissues thromboplastin obtained from the human brain. In water phospholipids of thromboplastin form hexagonal (H11) cylinders. Protein moiety of the tissue factor essentially influences on the phase state of phospholipids and on the dynamic properties of their fatty acid chains' radicals. The interaction of Ca2+ with the tissue thromboplastin does not change the phase state of phospholipids and at the same time rises rigidity of polar parts of phospholipids and their hydrophobic segments in the internal structures.