Arterial thrombosis is considered to arise from the interaction of tissue factor (TF) in the vascular wall with platelets and coagulation factors in circulating blood. According to this paradigm, coagulation is initiated after a vessel is damaged and blood is exposed to vessel-wall TF. We have examined thrombus formation on pig arterial media (which contains no stainable TF) and on collagen-coated glass slides (which are devoid of TF) exposed to f lowing native human blood. In both systems the thrombi that formed during a 5-min perfusion stained intensely for TF, much of which was not associated with cells. Antibodies against TF caused Ϸ70% reduction in the amount of thrombus formed on the pig arterial media and also reduced thrombi on the collagencoated glass slides. TF deposited on the slides was active, as there was abundant fibrin in the thrombi. Factor VII ai , a potent inhibitor of TF, essentially abolished fibrin production and markedly reduced the mass of the thrombi. Immunoelectron microscopy revealed TF-positive membrane vesicles that we frequently observed in large clusters near the surface of platelets. TF, measured by factor X a formation, was extracted from whole blood and plasma of healthy subjects. By using immunostaining, TF-containing neutrophils and monocytes were identified in peripheral blood; our data raise the possibility that leukocytes are the main source of blood TF. We suggest that blood-borne TF is inherently thrombogenic and may be involved in thrombus propagation at the site of vascular injury.Tissue factor (TF) present in the arterial wall has been considered to be responsible for the initiation of the coagulation cascade and thrombus formation (1). The role of vascular TF in acute thrombosis and atherosclerosis has been proposed based on our previous studies (2-5). To investigate the role of circulating TF in thrombogenesis, we have used a system in which pig aortic media or collagen-coated slides were mounted in a laminar flow chamber and perfused with native human blood. We noted that when stained either with derivatized factor VII a (FVII a ) or with specific anti-TF antibodies, the thrombi contained large amounts of TF staining, whereas the media and collagen-coated slides were essentially negative. Thus, we surmised that the TF came from the blood; accordingly, we examined whole blood and plasma for TF activity that we have extracted and assayed. We conclude that there is circulating, potentially active TF in normal subjects. We present evidence that this pool is thrombogenic in model flow systems. We also present evidence suggesting the TF comes from leukocytes and hypothesize that the cell-surface TF is completely encrypted (6-8) but becomes available during thrombosis. METHODSReagents. Human recombinant FVII a was a gift from NovoNordisk, Copenhagen. Factor X was purified from human plasma (9). Affigel-15 was purchased from Bio-Rad. The phospholipids used for relipidation of TF consisted of 30% 1,2-dioleoyl-sn-glycero-3-phosphatidylserine and 70% 1,2-dioleoyl-sn-gl...

Rab proteins represent a large family of ras‐like GTPases that regulate distinct vesicular transport events at the level of membrane targeting and/or fusion. We report here the primary sequence, subcellular localization and functional activity of a new member of the rab protein family, rab9. The majority of rab9 appears to be located on the surface of late endosomes. Rab9, purified from Escherichia coli strains expressing this protein, could be prenylated in vitro in the presence of cytosolic proteins and geranylgeranyl diphosphate. In vitro‐prenylated rab9 protein, but not C‐terminally truncated rab9, stimulated the transport of mannose 6‐phosphate receptors from late endosomes to the trans Golgi network in a cell‐free system that reconstitutes this transport step. Rab7, a related rab protein that is also localized to late endosomes, was inactive in the in vitro transport assay, despite its efficient prenylation and capacity to bind and hydrolyze GTP. These results strongly suggest that rab9 functions in the transport of mannose 6‐phosphate receptors between late endosomes and the trans Golgi network. Moreover, our results confirm the observation that a given organelle may bear multiple rab proteins with different biological functions.

Abstract. Newly synthesized lysosomal enzymes bind to mannose 6-phosphate receptors (MPRs) in the TGN, and are carded to prelysosomes, where they are released. MPRs then return to the TGN for another round of transport. Rab9 is a ms-like GTPase which facilitates MPR recycling to the TGN in vitro. We show here that a dominant negative form of rab9, rab9 S21N, strongly inhibited MPR recycling in living cells. The block was specific in that the rates of biosynthetic protein transport, fluid phase endocytosis and receptor-mediated endocytosis were unchanged.Expression of rab9 S21N was accompanied by a decrease in the efficiency of lysosomal enzyme sorting. Cells compensated for the presence of the mutant protein by inducing the synthesis of both soluble and membrane-associated lysosomal enzymes, and by internalizing lysosomal enzymes that were secreted by default. These data show that MPRs are limiting in the secretory pathway of cells expressing rab9 S21N and document the importance of MPR recycling and the rab9 GTPase for efficient lysosomal enzyme delivery.M ANNOSE 6-phosphate receptors (MPRs)t deliver newly synthesized, soluble lysosomal enzymes from the TGN to pre-lysosomes CKornfeld and Mellman, 1989;Kornfeld, 1992). Two types of MPRs have been identified to date. One is a 300-kD transmembrane glycoprotein which also binds insulin-like growth factor II; the second is a dimer or tetramer of 45-kD subunits and requires divalent cations for ligand binding in vitro. Both types of MPRs release their ligands upon encountering the low pH within pre-lysosomes, and then return to the Golgi complex to reinitiate another cycle of biosynthetic enzyme transport. MPRs are also present at the cell surface. These receptors are in rapid equilibrium with their intracellular counterparts, and at least the 300-kD MPR is capable of endocytosing extracellular lysosomal hydrolases and delivering them to endocytic compatmtents.Work from a number of laboratories has suggested that the rab family of ras-like GTPases plays a key role in regulating receptor trafficking (Zerial and Stenmark, 1993;Pfeffer, 1992). For example, rab5 has been shown to regulate early endosome fusion both in vitro (Gorvel et al., 1991) and in vivo (Bucci et al., 1992). Rab4 functions in receptor recycling between early endosomes and the cell surface (van der Sluijs et al., 1992). In addition, rabl and its yeast homolog, YPT1, play a key role in the transport of proteins between the ER and the Golgi (Plutner et al., 1991;Tisdale et al., Address all correspondence to S. Pfeffer, Department of Biochemistry, B400, Stanford University Medical School, Stanford, CA 94305-5307.L Abbreviations used in this paper: ECL, enhanced chemiluminescence, man6P, mannose 6-phosphate; MPR, mannose 6-phosphate receptor.1992; Rexach and Schekman, 1991;Segev, 1991). Rab proteins are thought to function in transport vesicle targeting and/or fusion events, because SEC4 mutant yeast strains accumulate secretory vesicles (Novick et al., 1980), anti-FPT/ antibodies inhibit ER-derived tran...

We describe thrombogenic tissue factor (TF) on leukocyte-derived microparticles and their incorporation into spontaneous human thrombi. Polymorphonuclear leukocytes and monocytes transfer TF+particles to platelets, thereby making them capable of triggering and propagating thrombosis. This phenomenon calls into question the original dogma that vessel wall injury and exposure of TF within the vasculature to blood is sufficient for the occurrence of arterial thrombosis. The transfer of TF+ leukocyte-derived particles is dependent on the interaction of CD15 and TF with platelets. Both the inhibition of TF transfer to platelets by antagonizing the interaction CD15 with P-selectin and the direct interaction of TF itself suggest a novel therapeutic approach to prevent thrombosis.