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.
Abstract-Thrombosis occurs in a dynamic rheological field that constantly changes as the thrombus grows to occlusive dimensions. In the initiation of thrombosis, flow conditions near the vessel wall regulate how quickly reactive components are delivered to the injured site and how rapidly the reaction products are disseminated. Whereas the delivery and removal of soluble coagulation factors to the vessel is thought to occur via classic convection-diffusion phenomena, the movement of cells and platelets to the injured wall is strongly augmented by flow-dependent cell-cell collisions that enhance their ability to interact with the wall. In addition, increased shear conditions have been shown to activate platelets, alter the cellular localization of proteins such as tissue factor (TF) and TF pathway inhibitor, and regulate gene production. In the absence of high shearing forces, red cells, leukocytes, and platelets can form stable aggregates with each other or cells lining the vessel wall, which, in addition to altering the biochemical makeup of the aggregate or vessel wall, effectively increases the local blood viscosity. Thus, hemodynamic forces not only regulate the predilection of specific anatomic sites to thrombosis, but they strongly influence the biochemical makeup of thrombi and the reaction pathways involved in thrombus formation. T hrombosis is thought to begin with an event such as plaque rupture, vessel damage, or dysfunctioning endothelium, resulting in the exposure of active tissue factor (TF) on the vessel wall. 1 The exposed TF binds circulating factor VII/VIIa (fVII/fVIIa) with high-affinity (Kd Ͻ10 pM), forming a reactive vessel surface that proteolytically cleaves fIX and fX. The rate of this surface-bound reaction depends not only on biochemical factors (number and surface density of TF:VIIa complexes, intrinsic kinetic activity, and local phospholipid composition 2 ) but on the rate at which the substrates fIX and fX are transported by the flowing medium to the reactive surface 3 and the rate at which product is removed. 4 Moreover, the local accumulation of reaction product may be critical in overpowering endogenous inhibitors and successfully initiating coagulation.Although convective flow forces dominate the axial movement of blood components (coagulation factors, platelets, red cells, leukocytes, and inhibitors) throughout most of the body, in the few microns approaching the vessel wall where thrombosis is thought to initiate, the frictional drag of the vessel wall causes the axial flow velocity to slow to 0, and the convective and diffusive motion of blood components becomes comparable. 5 In this initial coagulation reaction, blood flowing parallel to the wall convectively transports coagulation factors (fIX and fX) from upstream to a region close to the reactive site, and Brownian motion mediates the radial movement toward the reactive wall. In the case of platelets adhering to the wall, a similarly defined adhesion rate is often inferred. 6 The bound proteins fIX and fX become proteolyt...
Background-Several studies suggest a role for an increased circulating pool of tissue factor (TF) in atherothrombotic diseases. Furthermore, certain cardiovascular risk factors, such as diabetes, hyperlipemia, and smoking, are associated with a higher incidence of thrombotic complications. We hypothesized that the observed increased blood thrombogenicity (BT) observed in patients with type 2 diabetes mellitus may be mediated via an increased circulating tissue factor activity. We have extended our study to smokers and hyperlipidemic subjects. Methods and Results-Poorly controlled patients with type 2 diabetes mellitus (nϭ36), smokers (nϭ10), and untreated hyperlipidemic subjects (nϭ10) were studied. Circulating TF was immunocaptured from plasma, relipidated, and quantified by factor Xa (FXa) generation in the presence of factor VIIa. BT was assessed as thrombus formation on the Badimon perfusion chamber.
Annexin A5 (AnxA5) is a potent anticoagulant protein that crystallizes over phospholipid bilayers (PLBs), blocking their availability for coagulation reactions. Antiphospholipid antibodies disrupt AnxA5 binding, thereby accelerating coagulation reactions. This disruption may contribute to thrombosis and miscarriages in the antiphospholipid syndrome (APS). We investigated whether the antimalarial drug, hydroxychloroquine (HCQ), might affect this prothrombotic mechanism. Binding of AnxA5 to PLBs was measured with labeled AnxA5 and also imaged with atomic force microscopy. Immunoglobulin G levels, AnxA5, and plasma coagulation times were measured on cultured human umbilical vein endothelial cells and a syncytialized trophoblast cell line. AnxA5 anticoagulant activities of APS patient plasmas were also determined. HCQ reversed the effect of antiphospholipid antibodies on AnxA5 and restored AnxA5 binding to PLBs, an effect corroborated by atomic force microscopy. Similar reversals of antiphospholipid-induced abnormalities were measured on the surfaces of human umbilical vein endothelial cells and syncytialized trophoblast cell lines, wherein HCQ reduced the binding of antiphospholipid antibodies, increased cell-surface AnxA5 concentrations, and prolonged plasma coagulation to control levels. In addition, HCQ increased the AnxA5 anticoagulant activities of APS patient plasmas. In conclusion, HCQ reversed antiphospholipid-mediated disruptions of AnxA5 on PLBs and cultured cells, and in APS patient plasmas. These results support the concept of novel therapeutic approaches that address specific APS disease mechanisms.
Treatment with the antimalarial drug hydroxychloroquine (HCQ) has been associated with reduced risk of thrombosis in the antiphospholipid (aPL) syndrome (APS) and, in an animal model of APS, with reduction of experimentally induced thrombosis. Recognition of 2-glycoprotein I (2GPI) by aPL antibodies appears to play a major role in the disease process. We therefore used the techniques of ellipsometry and atomic force microscopy (AFM) to investigate whether HCQ directly affects the formation of aPL IgG-2GPI complexes on phospholipid bilayers. HCQ, at concentrations of 1 g/mL and greater, significantly reduced the binding of aPL-2GPI complexes to phospholipid surfaces and THP-1 (human acute monocytic leukemia cell line) monocytes. The drug also reduced the binding of the individual proteins to bilayers. This HCQmediated reduction of binding was completely reversed when the HCQ-protein solutions were dialyzed against buffer. HCQ also caused modest, but statistically significant, reductions of clinical antiphospholipid assays. In conclusion, HCQ reduces the formation of aPL-2GPI complexes to phospholipid bilayers and cells. This effect appears to be due to reversible interactions between HCQ and the proteins and may contribute to the observed reduction of thrombosis in human and experimental APS. These results support the possibility that HCQ, or analogous molecules, may offer novel nonanticoagulant therapeutic strategies for treating APS. (Blood. 2008;112:1687-1695) IntroductionThe antiphospholipid (aPL) syndrome (APS) is a thrombophilic disorder that is defined by the presence of autoantibodies against phospholipid-binding cofactor proteins in patients with vascular thrombosis and/or pregnancy complications. 1 Of the various phospholipid-binding proteins, aPL antibody recognition of the phospholipid-binding protein, 2-glycoprotein I (2GPI), appears to particularly correlate with thrombosis 2 and is associated with significantly increased risk of thrombosis. 3 Antiphospholipid antibodies have been demonstrated to play a causal role in the development of thrombosis in animal models (reviewed in Rand 4 ). Long-term anticoagulation with warfarin, a medication that carries a significant risk of bleeding complications, 5 is the standard treatment for APS-associated thrombosis. 6 Hydroxychloroquine (HCQ), an amphiphilic antimalarial compound, has proven to be an effective immunosuppressive treatment of systemic lupus erythematosus (SLE). [7][8][9][10][11] The Hopkins Lupus Cohort reported that the presence of aPL antibodies is an independent predictor of thrombosis in SLE, and that treatment of SLE patients with HCQ was associated with a reduced risk of thrombosis. 12 A cross-sectional study that compared aPL antibody-positive patients with thrombosis to a group of patients having the antibodies but who did not have thrombotic histories indicated that HCQ may be protective against thrombosis. 13 HCQ significantly reduced the extent of thrombosis in an animal model of injuryinduced thrombosis in APS, 14 and, in a si...
Upon plaque rupture or vascular injury, tissue factor (TF) protein in the vessel wall becomes exposed to flowing blood, initiating a cascade of reactions resulting in the deposition of fibrin and platelets on the injured site. Paradoxically, the growing thrombus may act as a barrier, restricting the convective and diffusive exchange of substrates and coagulation products between the blood and reactive vessel wall, thus limiting the role TF plays in thrombus growth. In this study, various in vitro, platelet-fibrin clots were prepared on TF:VIIa-coated surfaces and the rate at which factor (F) X in the well-mixed clot supernatant permeates the clot and is converted to X a was monitored over several hours. The apparent diffusion coefficients of FX (a) in fibrin and plateletfibrin clots at 37°C was 2.3 ؋ 10 ؊7 and 5.3 ؋ 10 ؊10 cm 2 /second, respectively, indicating that the mean time required for FX (a) , and likely FIX (a) , to diffuse 1 mm in a fibrin clot is 4 hours, and in the presence of platelets, 3.6 months. As complete human thrombotic occlusion has been observed within 10 minutes, an alternative source of procoagulant activity that can localize to the outer surface of growing thrombi, such as platelet factor XI or blood-borne TF, appears essential for rapid thrombus growth. IntroductionIn the generally accepted view of thrombosis, rupture or erosion of an atherosclerotic plaque exposes procoagulant regions on the vascular wall that bind platelets and initiate thrombus formation. The exposed material thought responsible for initiating coagulation is tissue factor (TF), which complexes with VII (a) to activate factor (F) IX and FX. Immediately following injury, FIX and FX are rapidly delivered to the vessel wall by flowing blood resulting in localized thrombin generation. However, as fibrin and platelets accrue to the injured surface, flow to the underlying TF region becomes restricted and substrates must percolate through an overlying platelet-fibrin mesh in order to become activated ( Figure 1). Furthermore, to achieve effective prothrombinase activity (X a :V a ) near the outer surface of a growing thrombus, the products (IX a or X a ) must diffuse a substantial distance back through the overlying mass. Through its interaction with VIIIa, factor IX (a) plays a crucial role in amplifying the amount of X a generated; However, since the only accepted source of Ixa in most coagulation models is its generation by TF:VII a , IX a would have to percolate a substantial distance through the overlying platelets and fibrin in order to generate tenase activity near the surface of the growing thrombus. (Factor XI-mediated activation of IX through the intrinsic pathway is generally not regarded as an essential step in thrombosis and is addressed later.) Hence, to obtain occlusive thrombi, the substrates (IX and X) and their respective enzymes, either individually or working in concert, must travel several millimeters through a growing thrombus without the aid of convection. As diffusion of proteins the size of fact...
Summary. Tissue factor (TF) is a transmembrane glycoprotein that initiates coagulation and plays a critical role in regulating hemostasis and thrombosis. We have recently reported a naturally occurring, soluble form of human tissue factor (asTF) generated by alternative splicing. This splice variant has a novel C-terminus with no homology to that of the full-length TF (flTF), lacks a transmembrane domain, and is active in the presence of phospholipids. Mouse models offer unique opportunities to examine the relative importance of flTF and asTF in mediating thrombosis, the response to arterial injury, and ischemic damage. To that end, we have identified and characterized murine asTF (masTF). Like the human splice variant, masTF lacks a transmembrane domain and has a unique C-terminus. We have generated antibodies specific to masTF and murine flTF (mflTF) to examine the expression of both forms of TF. masTF antigen is widely and abundantly expressed, with a pattern similar to that of mflTF, in adult tissues, in experimentally induced thrombi, and during development. These studies demonstrate that masTF contributes to the pool of total TF and may thus play an important role in mediating TF-dependent processes.
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