Tissue factor (TF) is an essential enzyme activator that forms a catalytic complex with FVII(a) and initiates coagulation by activating FIX and FX, ultimately resulting in thrombin formation. TF is found in adventitia of blood vessels and the lipid core of atherosclerotic plaques. In unstable coronary syndromes, plaque rupture initiates coagulation by exposing TF to blood. Biologically active TF has been detected in vessel walls and circulating blood. Elevated intravascular TF has been reported in diverse pro-thrombotic syndromes such as myocardial infarction, sepsis, anti-phospholipid syndrome and sickle-cell disease. It is unclear how TF circulates, although it may be present in pro-coagulant microparticles. We now report identification of a form of human TF generated by alternative splicing. Our studies indicate that alternatively spliced human tissue factor (asHTF) contains most of the extracellular domain of TF but lacks a transmembrane domain and terminates with a unique peptide sequence. asHTF is soluble, circulates in blood, exhibits pro-coagulant activity when exposed to phospholipids, and is incorporated into thrombi. We propose that binding of asHTF to the edge of thrombi contributes to thrombus growth by creating a surface that both initiates and propagates coagulation.
Although it is generally accepted that the initial event in coagulation and intravascular thrombus formation is the exposure of tissue factor (TF) to blood, there is still little agreement about the mechanisms of thrombus propagation and the identities of the molecular species participating in this process. In this study, we characterized the thrombotic process in real-time and under defined flow conditions to determine the relative contribution and spatial distribution of 3 components of the thrombi: circulating or blood-borne TF (cTF), fibrin, and platelets. For this purpose, we used high-sensitivity, multicolor immunofluorescence microscopy coupled with a laminar flow chamber. Freshly drawn blood, labeled with mepacrine (marker for platelets and white cells), anti-hTF1 Alexa.568 (marker for tissue factor), and anti-T 2 G 1 Cy-5 (marker for fibrin) was perfused over collagen-coated glass slides at wall shear rates of 100 and 650 s ؊1 . A motorized filter cube selector facilitated imaging every 5 seconds at 1 of 3 different wavelengths, corresponding to optimal wavelengths for the 3 markers above. Real-time video recordings obtained during each of 10 discrete experiments show rapid deposition of platelets and fibrin onto collagen-coated glass. Overlay images of fluorescent markers corresponding to platelets, fibrin, and cTF clearly demonstrate colocalization of these 3 components in growing thrombi. These data further support our earlier observations that, in addition to TF present in the vessel wall, there is a pool of TF in circulating blood that contributes to the propagation of thrombosis at a site of vascular injury. IntroductionTissue factor (TF) is an integral membrane protein that is found primarily on the surface of certain cell types that are located outside the vasculature. 1,2 Because of its ideal physical location in the vasculature, vessel wall TF remains separated from the various coagulation proteins that circulate in the blood, thus preventing thrombus formation in intact vessels. The most widely accepted view of coagulation and thrombosis is that following vascular injury, vessel wall TF is exposed to flowing blood, whereupon it forms a complex with factor VII/VII a (FVII/VII a ), thus initiating the coagulation cascade and eventually resulting in clot or thrombus formation. Therefore, in this current view of arterial thrombosis, propagation of thrombi requires that various coagulation reactions as well as platelet deposition occur on the luminal thrombus surface. However, thrombi are relatively large structures extending 1 to 3 mm above the vessel wall. Vessel wall-derived activated coagulation factors would have to diffuse from the injured vessel wall site through the deposited thrombus to the luminal surface to be present at the site of the growing thrombus. Even in an ideal situation of free diffusion, a protein of molecular weight 50 000 would require hours to reach the apex of a 1-mm thrombus. Circulating or blood-borne TF (cTF), carried by unidentified cells or vesicles in flowing bloo...
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