Although tissue factor (TF), the principial initiator of physiological coagulation and pathological thrombosis, has recently been proposed to be present in human blood, the functional significance and location of the intravascular TF is unknown. In the plasma portion of blood, we found TF to be mainly associated with circulating microvesicles. By cell sorting with the specific marker CD42b, platelet-derived microvesicles were identified as a major location of the plasma TF. This was confirmed by the presence of full-length TF in microvesicles acutely shedded from the activated platelets. TF was observed to be stored in the α-granules and the open canalicular system of resting platelets and to be exposed on the cell surface after platelet activation. Functional competence of the blood-based TF was enabled when the microvesicles and platelets adhered to neutrophils, as mediated by P-selectin and neutrophil counterreceptor (PSGL-1, CD18 integrins) interactions. Moreover, neutrophil-secreted oxygen radical species supported the intravascular TF activity. The pools of platelet and microvesicle TF contributed additively and to a comparable extent to the overall blood TF activity, indicating a substantial participation of the microvesicle TF. Our results introduce a new concept of TF-mediated coagulation crucially dependent on TF associated with microvesicles and activated platelets, which principally enables the entire coagulation system to proceed on a restricted cell surface.Key words: lipopolysaccharide • platelet rich plasma • superoxide dismutase • catalase • microparticles T wo principal events that are initiated after disrupture of the endothelial barrier are thought to mark the initiation of hemostasis. Blood platelets adhere to subendothelial collagen providing a provisional, mechanically unstable closure of the vessel perforation. Concomitantly, the coagulation process is started. This is mainly due to the formation of an initiator complex between tissue factor (TF), an integral cell membrane protein predominantly present in the adventitial layer of the vessel wall, and the blood-based factor VII/VIIa (1). The TF/factor VIIa complex proteolytically activates factor X, which, in turn, elicits the formation of thrombin. The TF/factor VIIa complex is likely to play a central role in the genesis of arterial and venous thrombosis (2, 3), leading causes of mortality in many countries. TF is present in the lipid rich core of unstable atherosclerotic plaques and may be a major determinant of the thrombogenicity of the plaques (4-6). Indeed, on rupture of the plaque, the interaction of TF with factor VIIa substantially contributes to the rapid formation of the occluding thrombus, the principal final step in the genesis of coronary ischemic disease.Apart from its presence in the vascular wall, TF has also been detected in the blood (intravascular TF). So far, no clear conclusion has been reached about the localization and the functional meaning of the blood-based TF. In the plasma compartment, TF is present un...

Blood cell progenitors were scanned for the presence of the coagulation starter protein tissue factor (TF) by immunoelectron microscopy. Thereby, substantial TF expression was observed in the precursor cells of eosinophils. TF levels were lower in basophil precursors and barely detectable in neutrophil progenitors. In peripheral blood immediately processed to avoid activation of the TF gene, mature eosinophils were found to considerably express TF, unique among the granulocyte and monocyte fractions. TF was preferentially located in the specific granules in resting eosinophils. Platelet-activating factor (PAF), and more pronounced, granulocyte-macrophage colony-stimulating factor (GM-CSF) plus PAF, caused translocation of preformed TF to the eosinophil cell membrane. GM-CSF/PAF also increased the TF transcript levels. The activated eosinophils exhibited procoagulant activity that was abrogated by TF inhibition. Targeting the extracellular domain of TF with specific antibodies markedly suppressed the initial phase of the eosinophil passage across the IL-4-activated endothelium. Eosinophil rolling and firm adhesion remained unaffected. This suggests that TF specifically facilitates the early transendothelial migration of the eosinophils. In summary, eosinophils maintain a high TF expression during maturation, providing a main source of preformed TF in blood, which might be relevant for the thrombogenesis promoted by hypereosinophilic conditions. IntroductionTissue factor (TF), the crucial starter protein of hemostasis and a major determinant of its pathologic sequelae, 1 is basically expressed in the plasma membrane of cells located in the adventitial and medial layers of the vascular wall. TF binds the serine protease factor VIIa with high affinity, thereby enhancing its proteolytic activity by several orders of magnitude, whereby coagulation is initiated. A series of new findings suggests that preformed TF is present in intravascular compartments in humans, at variance with its designation. For a deeper understanding of the start process of intravascular coagulation, it is essential to know where TF is located and how its expression is regulated. Recently, circulating microvesicles (or microparticles) were revealed as source for preexisting TF in blood, which are apparently derived from leukocytes and platelets. 2-5 They most likely represent the earlier described TF pool in acellular plasma 6-9 because the selective removal of microvesicles strongly decreases the plasma TF contents. 3 Microvesicles are rapidly recruited to the site of vascular injury in vivo, where they elicit the coagulation start in a TF-dependent way. 10 Moreover, TF has been reported to appear on the cell surface and on microvesicles secreted from activated platelets. [11][12][13] In other work, TF was not detectable on platelets. 14 In addition, TF has been proposed to be present in neutrophils (summarized by Nakamura et al 15 ), although this has been called into question. 16 Notably, the functional competence of TF is not restricted to ...

Transplantation of pancreatic islets reconstitutes glucose homeostasis in diabetes mellitus. Before transplantation, islets are disrupted from the surrounding blood vessels by the isolation procedure, with the grafted tissue being subject to ischemic damage. The survival of transplanted islets is assumed to depend on effective revascularization. Perfusion studies suggest that newly formed microvessels supplying the graft with nutrients are exclusively rebuilt by the host. It is generally not known whether isolated islets contain endothelial cells (EC), which potentially participate in the revascularization process. Therefore, we tried to detect immature EC in isolated islets by transformation with polyoma middle T antigen. Endothelioma cells were generated, implicating the presence of de-differentiated EC within isolated islets. When embedded in a fibrin gel, the islets developed cellular cords consisting of EC, whereas FGF-2 and VEGF stimulated the formation of cord-like structures. Furthermore, we studied the presence of donor EC in islet grafts by using transgenic mice with an EC lineage-specific promotor-LacZ reporter construct (Tie-2LacZ). Following islet transplantation, Tie-2LacZ-positive EC of both donor and recipient were identified in the vicinity of or within the graft up to 3 wk after transplantation. In conclusion, EC and/or their progenitors with angiogenic capacity reside within isolated islets of different species, and their proliferative potential can be stimulated by various inducers. These graft-related endothelia persist after islet transplantation and are integrated within newly formed microvessels.

Recently, the homolog of yeast protein Sec63p was identified in dog pancreas microsomes. This pancreatic DnaJ-like protein was shown to be an abundant protein, interacting with both the Sec61p complex and lumenal DnaK-like proteins, such as BiP. The pancreatic endoplasmic reticulum contains a second DnaJ-like membrane protein, which had been termed Mtj1p in mouse. Mtj1p is present in pancreatic microsomes at a lower concentration than Sec63p but has a higher affinity for BiP. In addition to a lumenal J-domain, Mtj1p contains a single transmembrane domain and a cytosolic domain which is in close contact with translating ribosomes and appears to have the ability to modulate translation. The interaction with ribosomes involves a highly charged region within the cytosolic domain of Mtj1p. We propose that Mtj1p represents a novel type of co-chaperone, mediating transmembrane recruitment of DnaK-like chaperones to ribosomes and, possibly, transmembrane signaling between ribosomes and DnaK-like chaperones of the endoplasmic reticulum.

While the adenosine 5-diphosphate (ADP) pathway is known to enhance thrombus formation by recruiting platelets and leukocytes to the primary layer of collagenadhering platelets, its role for the initiation of coagulation has not been revealed. Ex vivo inhibition of the P2Y 12 ADP receptor by clopidogrel administration diminished the rapid exposure of tissue factor (TF), the major initiator of coagulation, in conjugates of platelets with leukocytes established by the contact of whole blood with fibrillar collagen. Under in vitro conditions, the P2Y 12 and P2Y 1 ADP receptors were both found to be implicated in the exposure of TF in collagen-activated whole blood. Immunoelectronmicroscopy revealed that collagen elicited the release of TF from its storage pools within the platelets. Functional activation of the intravascular TF was reduced by inhibition of the ADP receptors, partially due to the disruption of the platelet-neutrophil adhesions. Injection of collagen into the venous system of mice increased the number of thrombin-antithrombin complexes, indicative for the formation of thrombin in vivo. In P2Y 1 -deficient mice, the ability of collagen to enhance the generation of thrombin was impaired. In conclusion, the platelet ADP pathway supports the initiation of intravascular coagulation, which is likely to contribute to the concomitant formation of fibrin at the site of the growing thrombus. (Blood.