Presently, thrombin-specific inhibitors are in clinical trials as anticoagulant drugs for the treatment of arterial (after coronary artery angioplasty) and deep venous thrombosis. The specificity of these drugs is reflected in the dissociation constants ranging from 2 picomoles/L - 2 nanomoles/L. The thrombin specific inhibitors have demonstrated efficacy in animal models for both treatment and prevention of arterial and venous thrombosis. Recombinant hirudin, derived from the medicinal leech Hirudo medicinalis, is the thrombin-specific inhibitor most studied. Effective doses and their range for therapeutic plasma levels in humans have been determined. The low and high ends of the range are used for venous and arterial thrombosis, respectively.
Several U.S. patents have been issued on anti-thrombin agents for use as medicinals or bound to implantable materials. U.S. Pat. No. 4,944,943 to Eschenfelder, et al. teaches use of hirudin and t-PA for treatment of thrombosis. U.S. Pat. No. 4,952,562 to Klein, et al, discloses anti-thrombotic peptides and pseudopeptides. U.S. Pat. No. 5,019,393 to Ito, et al. teaches use of implantable materials having a thrombogenesis inhibitor immobilized thereon. U.S. Pat. No. 5,053,453 to Ku discloses hirudin or hirudin derivatives covalently linked to support materials to avoid formation of thrombi. U.S. Pat. No. 5,640,814 to Klein, et al. discloses an anti-thrombotic peptide for administration as a medicinal. U.S. Pat. No. 5,087,613 to Courtney, et al. discloses hirudin variants for use as inhibitors of thrombin activity. U.S. Pat. No. 5,095,092 discloses a process for isolation and purification of hirudin. None of the cited patents disclose a quantitative thrombin-time test as taught herein.
Several methods are presently used to assess whether the patient taking the new thrombin-specific inhibitors is therapeutically anticoagulated. It is possible to measure plasma thrombin-specific inhibitors levels by high performance liquid chromatography (HPLC). Though accurate, HPLC is generally available only in large referral hospitals and commercial labs and requires a high level of staff expertise. Even when available in large referral hospitals, the technique is time consuming. Hence, there is delay in obtaining results. This delay is unacceptable when a clinical practitioner is attempting to closely monitor and control the progress of the patient.
Another approach is to monitor the prolongation of the activated partial thromboplastin time (APTT). The APTT is a standard screening test of the coagulation system. This test is commonly used to follow the degree of anticoagulation in patients receiving the anticoagulant heparin. However, in the absence of specific thrombin inhibitors a variety of coagulopathies [e.g. isolated or multiple factor deficiency, abnormal fibrinogen, decreased fibrinogen concentration or antiphospholipid antibodies] tend to prolong the APTT results. Some patients with coagulopathies associated with the nephrotic syndrome or the lupus anticoagulant also develop thrombosis requiring treatment with anticoagulants. In these cases, the APTT is already prolonged and cannot be used to monitor anticoagulation with thrombin specific inhibitors.
Another clotting assay, the standard thrombin time, is routinely used in a qualitative fashion in clinical laboratories to determine the presence of heparin and abnormal or low levels of fibrinogen. The standard thrombin time is not effective and has not been used to quantitate the concentrations of thrombin-specific inhibitors in the blood because of the inherent sensitivity of the standard thrombin time, as presently developed, to factors other than these inhibitors (Walenga J. M. et al. Seminars in Thrombosis Hemostasis 17:103, 1991). In addition, plasmas obtained from patients with liver disease, dysfibrinogenemia, hypofibrinogenemia, or who have received fibrinolytic therapy are likely to produce a prolonged standard thrombin time.
Besides HPLC and the APTT, further methods including amidolytic assays for thrombin and immunologic assays for the hirudinthrombin complex have been suggested. However, the techniques are not easily applied to the clinical laboratory. Because of the potential therapeutic efficacy of the thrombin-specific inhibitors, a new and improved laboratory test for determining plasma levels that can be performed in clinical laboratories with presently available instrumentation is needed. Furthermore, such a new process capable of providing results that are easily interpreted by clinical practitioners would be of great utility.