I. Field of the Invention
The present invention relates to the fields of vascular biology and inflammation. More particularly, it relates to assays to detect soluble endothelial protein C receptor as a measure of thrombin generation.
II. Related Art
In the U.S., more than 55 million people currently suffer from conditions associated with thrombosis. The American Heart Association has estimated that the annual direct expense to the U.S. healthcare system for the treatment of thrombotic disease exceeds $200 billion. On an annual basis worldwide, nearly 2 million patients will undergo either cardiopulmonary bypass surgery or coronary angioplasty to treat such conditions. Thrombosis and associated diseases affect millions of patients and their manifestations are a source of significant morbidity and mortality.
Perturbations of hemostasis are central to the pathogenesis of a hypercoagulable state and may be triggered by multiple, overlapping influences. These include, but are not limited to environmental effects (surgery, diet, smoking, childbirth, trauma), manifestations of a primary disease (heart disease, diabetes, sepsis, hypertension, autoimmune diseases, malignancy) or inheritable defects in the hemostatic factors (protein C, protein S, factor V Leiden, prothrombin, antithrombin III) (Kearon et al., 2000; Carey et al., 1998; Lane et al., 2000). The clinical settings include disseminated intravascular coagulation (DIC), deep vein thrombosis (DVT), or perioperative DVT prevention and heart conditions such as acute myocardial infarction, mechanical prosthetic heart valves, mitral valve prolapse, atrial fibrillation, rheumatic mitral valve disease, and dilated cardiomyopathy. In the U.S. alone, more than 1.5 million patients with atrial fibrillation or mechanical heart valves are currently undergoing preventative oral anticoagulant therapy to help prevent stroke and thrombus formation.
The current therapeutic approach to a patient with thrombotic disease typically starts with an intravenous course of unfractionated heparin or low-molecular weight heparin, followed by a course of oral anticoagulation. Both therapeutic regimes seek to reduce the activity of thrombin, a potent enzyme responsible for activation of the cells and creation of fibrin that form the basis of a clot. In North America, Warfarin (4-hydroxycoumarin) is the most widely used oral anticoagulant, prescribed for preventing and treating venous or arterial thrombosis and embolism.
However, Warfarin is a potentially hazardous drug because the therapeutic window is relatively narrow and the dose-response differs significantly between patients. Warfarin therapy causes major bleeding in about 1–8% of people treated, and intracranial bleeding in about 0.1–0.5% of patients during each year of therapy (van der Meer et al., 1993; Connegieter et al., 1995; Levine et al., NEED YEAR). Patients with transient ischemic attack or minor stroke appear to be particularly at risk for bleeding from Warfarin therapy (Laupacis et al., 1994; Ann. Nuerol., 1997; N.E. Jour. Med., 1995). A small population of patients (0.01–0.1%) may exhibit Warfarin-induced skin necrosis, due to thrombosis of veins and capillaries of the subcutaneous fat (Chan et al., 2000). This can be associated with deficiencies of anti-coagulant proteins, or with unbalanced hemostatic control due to the rapid Warfarin-induced loss of the anticoagulants protein S and/or protein C (hours) compared with the procoagulant protein prothrombin (days). Thus, continual monitoring of coagulation status is required.
Direct measurement of thrombin is virtually impossible, due to its 30-second half-life in the circulation. While this appears to be a short time, thrombin is an extraordinarily potent enzyme and 30 seconds is more than enough time for it to act on its protein substrates and cellular receptors. Thus, the challenge to coagulation test designers is to create an assay that reflects thrombin levels without interference from other proteins or in vitro parameters, an assay that is reproducible and can be standardized, and ideally an assay that reports a history of thrombin levels. This latter challenge is equivalent to that provided by hemoglobin Alc (glycohemoglobin) levels in diabetic patients. Patient compliance and therapeutic efficacy is measured in these patients by the glycosylation levels of their hemoglobin Alc, in essence a mirror of their plasma glucose levels over a 2–4 month period (Goldstein et al., 1994). For patients on oral anticoagulant therapy, none of these challenge parameters are met by the currently available anticoagulation tests, and improved methods are needed.