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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 2, 2003 as doi:10.1096/fj.02-0574fje. |
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* Physiologisches Institut and
|| Vaskuläre Biologie und Hämostase, Institut für Klinische Chemie, Ludwig-Maximilians-Universität München, 81377 München, Germany;
Institut für Pathologie, Technische Universität Dresden, 01307 Dresden Germany;
Medizinische Biologie, Universität des Saarlandes, Campus Homburg, 66421 Homburg, Germany; and
Institut für Biochemie, Universität Giessen, 35392 Giessen, Germany
2Correspondence: Institut für Klinische Chemie, Vaskuläre Biologie und Hämostase, Klinikum der Universität München, Marchioninistr. 15, 81377 München, Germany. E-mail: Bernd.Engelmann{at}klch.med.uni-muenchen.de
SPECIFIC AIMS
Tissue factor (TF), an integral membrane protein of the vessel wall, is the principal initiator of physiological coagulation and a major trigger of arterial and venous thrombosis. Recently, several intravascular TF locations have been proposed. The aim of our study was to identify the major localizations of functional competent TF in human blood.
PRINCIPAL FINDINGS
1. Plasma TF is present on circulating microvesicles originating from platelets
In human blood stimulated with collagen, TF was detected on the surface of microvesicles with diameters of 300–600 nm. Selective removal of circulating microvesicles reduced the plasma TF contents by more than two thirds. In platelet derived microvesicles isolated from the total plasma microvesicles by cell sorting, full length TF was detected. Thus, the plasma associated TF is mainly present on circulating microvesicles.
2. Circulating microvesicles and platelets support TF dependent fibrin formation to a comparable extent
Depletion of circulating microvesicles and selective removal of platelets from whole blood reduced the TF dependent fibrin formation to a similar degree. Also in platelet rich plasma, removal of the microvesicles substantially delayed the formation of fibrin (Fig. 1
a). The TF mediated generation of factor Xa in the presence of the isolated microvesicles was enhanced by addition of the isolated platelets (Fig. 1b
). Increased numbers of microvesicles, similar to those found in conditions associated with high risk for arterial thrombosis, resulted in excessive factor Xa generation (Fig. 1c
). At an hematocrit of 50% and a platelet count of 3 x 108/mL blood, it could be calculated that in 1 mL of human blood, 30 pg and 110 pg of TF will be associated with the platelets and the plasma compartment, respectively. Together, the findings suggest that the microvesicle TF substantially contributes to thrombogenesis in vivo.
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3. Localization of TF in resting and activated platelets
In resting platelets, TF was identified in the membrane and matrix of 
-granules, and in the open canalicular system by immunoelectron microscopy (Fig. 2
a–d). Upon activation of platelets with collagen and thrombin, TF was exposed on the platelet surface (Fig. 2e
). Stimulation with ADP was barely effective. Moreover, TF was secreted from the activated platelets in association with shed microvesicles. Thus, the preformed TF stored in the platelets is exposed on the surface and secreted in association with microvesicles after stimulation with strong platelet agonists.
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4. Functional activity of blood based TF is promoted by adhesion to neutrophils and reactive oxygen species
Disrupting the adhesion of the TF-bearing blood components to the neutrophils diminished the ability of the intravascular TF to initiate coagulation. The TF activity and subsequent fibrin formation elicited by TF on microvesicles and activated platelets was promoted by reactive oxygen species, as evidenced from the inhibitory effects of catalase and superoxide dismutase. Thus, neutrophil adhesion molecules and secretion products substantially promoted the intravascular TF activity.
CONCLUSIONS AND SIGNIFICANCE
In the present study, circulating microvesicles and activated platelets are identified as the main sites of functionally active TF in human blood. Circulating microvesicles were found to play a major role for the TF dependent fibrin formation in blood. Indeed, their contribution is comparable to the one elicited by the activated platelets. We demonstrate for the first time that TF is present within resting platelets, by providing evidence for its location in the -granules and in the open canalicular system. Subsequent to platelet activation, the preformed TF stored in the platelets is exposed on the cell surface and on microvesicles shed from the plasma membrane. The activity of the intravascular TF is promoted by adhesion of the TF-bearing components to the neutrophils, and by reactive oxygen species released from the leukocytes. Our findings imply that the whole coagulation process can take place on the same cell membrane.
Platelets occupy a central role as mediator of the primary occlusion of vessel perforations. Moreover, platelets directly promote coagulation by enabling the assembly of several protein complexes, including, among others, the prothrombinase complex catalyzing the formation of thrombin. The results from the present study add a substantial new element to the role of platelets in coagulation. Since the platelet surface is able to present TF upon activation and assemble the extrinsic tenase complex, the factor Xa generated by the TF/factor VIIa system can be directly introduced into the prothrombinase complex leading to the formation of thrombin. Why should there be a necessity for an intravascular TF pathway in addition to the one located in the vessel wall? We assume that the blood-based TF will be of particular relevance for the stabilization of the developing clot, which is formed after the vessel wall perforation. In most cases, the clot is growing in the direction of the vessel lumen. Under those conditions, the distance between the growing clot and the activated coagulation factors generated by the adventitial TF might be too long to allow sufficient fibrin to reach the thrombus. Moreover, the platelet layer that is formed on the collagen fibers immediately after the vessel rupture will provide a barrier for the diffusion of the coagulation factors previously activated by the vessel wall TF. Thus, there is substantial evidence that the local intravascular TF pathway is needed for the stabilization of the interaction of newly recruited platelets with the preexisting thrombus.
Neutrophils appear to play a major role for the activation of the intravascular TF, since the firm adhesion of the TF-bearing microvesicles and platelets to these cells as well as the neutrophil secretion products substantially enhanced the functional competence of the blood based TF. Whereas the exact mechanism will need to be evaluated in future studies, apart from the reactive oxygen species, also serine proteases, a further group of secretion products, contribute to the activation of the blood based TF. Since the reactive oxygen species might stimulate the secretion of the proteases, a cooperation of both secretion products might be anticipated. Adhesion of platelets to the neutrophils has been proposed to result in the formation of a restricted microenvironment containing higher concentrations of the neutrophil secretion products as compared to the bulk plasma compartment. In this intercellular space, the platelet and microvesicle TF might be particularly effective. In line with the thrombogenic influence of the platelet-neutrophil interactions, statistical associations of high numbers of platelet-neutrophil aggregates with an increased risk for arterial thrombosis have been well documented. Our results extend earlier work on the presence of TF in plasma. Whereas plasma TF was found to be partially associated with circulating microvesicles derived from platelets, further plasma locations of unknown origin exist. Circulating microvesicles have previously been detected in plasma under physiological conditions, and their concentration is increased in patients with acute coronary disease and other prothrombotic states. Under the same pathological conditions, elevated numbers of microvesicles have been observed. The high procoagulant activities found in our experiments with increased concentrations of microvesicles suggest that the microvesicle TF represents an even more important prothrombotic trigger under pathological conditions.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0574fje; to cite this article, use FASEB J. (January 2, 2003) 10.1096/fj.02-0574fje 
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, microvesicles plus neutrophils; 
, microvesicles plus neutrophils and platelets. The values given on y axis refer to the increase in microvesicle number compared to the basal number of microvesicles (3x104, set as "1"). Representative experiment of a total of 5 experiments. Neutrophils and platelets were present in similar amounts as in panel b).
