James J. Hathcock

Alternatively spliced human tissue factor: a circulating, soluble, thrombogenic protein

Tissue factor (TF) is an essential enzyme activator that forms a catalytic complex with FVIIa 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.

Role of Risk Factors in the Modulation of Tissue Factor Activity and Blood Thrombogenicity

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 (n=36), smokers (n=10), and untreated hyperlipidemic subjects (n=10) 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. Patients with improvement in glycemic control showed a reduction in circulating TF (362±135 versus 243±74 pmol/L per min FXa, P =0.0001). A similar effect was observed in BT (15 445±1130 versus 12 072±596 &mgr;m/mm2, P =0.01). Two hours after smoking 2 cigarettes, TF was increased (217±72 versus 283±106 pmol/L per min FXa, P =0.003). Hyperlipidemic subjects showed higher TF (237±63 versus 195±44 pmol/L per min FXa, P =0.035) than healthy volunteers. Conclusions—These findings suggest that high levels of circulating TF may be the mechanism of action responsible for the increased thrombotic complications associated with the presence of these cardiovascular risk factors. These observations strongly emphasize the usefulness of the management of the patients based on their global risk assessment.

Flow Effects on Coagulation and Thrombosis

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.

Hydroxychloroquine protects the annexin A5 anticoagulant shield from disruption by antiphospholipid antibodies: evidence for a novel effect for an old antimalarial drug

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.

Identification and characterization of murine alternatively spliced tissue factor.

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.