ALBERT

Description: End Stage Renal Disease (ESRD) manifests hematologic, cardiovascular, inflammatory, and immunologic dysfunctions. Erythropoietin deficiency is present in ESRD leading to anemic states. ESRD patients receive regular/repeated administration of commercially available recombinant erythropoietin (r-E) (Epogen® Procrit® Patients treated with r-E have been known to develop thrombotic complications during r-E therapy. Antibodies to erythropoietin are generated in some patients and lead to erythropoietic aplasia. The mechanisms of the anti-erythropoietin antibody induced aplasia and reported hypercoagulability state in patients treated with r-E are not fully understood. Anticoagulant drugs such as heparin are used to reduce the incidence of thrombotic complications during r-E therapy. To understand pathogenesis leading to hypercoagulable state and its potential link with r-E treatment, blood samples taken from a group of 60 patients with ESRD were drawn prior to initiation of antithrombotic therapy. These patients on periodic hemodialysis were administered r-E 6–8 weeks prior to blood drawn. Anti-erythropoietin antibody titer, anti-phospholipid antibody (APA) titer, CRP levels, fibrinopeptide-A (FPA), nitric oxide (NO) levels, asymmetric 1,3-dimethylarginine (ADMA) levels, and thrombin activatable fibrinolytic inhibitor (TAFI) levels were measured. Five of 60 patients (8.3%) showed higher titers of anti-erythropoietin antibodies. Three of 60 patients (5%) exhibited positive titers of anti-phospholipid antibodies. Marked elevation of the CRP, FPA, NO, ADMA, and TAFI levels were noted in comparison to match controls. Patients with positive levels of anti-erythropoietin antibody titer (3 to 5 folds) exhibited a simultaneous elevation of FPA, CRP and TAFI suggesting the potential role of anti-erythropoietin antibody in mediating these responses. These observations indicate that ESRD patients treated with r-E are at a higher risk of developing thrombotic complications. The upregulation of NO and ADMA in these patients is suggestive of ongoing inflammatory processes. Anticoagulants such as low molecular weight heparins may be useful in the management of these patients.

Description: Background: Because of the relative insensitivity of the global clot based assays such as the activated partial thromboplastin time (APTT), the low molecular weight heparins (LMWHs) are potency evaluated/optimized in the anti-Xa (AXa) and heptest. A new clot based assay namely, prothrombin induced clotting time (PiCT) is sensitive to the anticoagulant effects of LMWHs and related drugs. As the LMWHs are standardized using the anti-Xa methods, using the International Standards, this study was designed to cross validate the 2nd International Standard for LMWHs(NIBSC 01/608) in various assay methods. Methods: Commercially available LMWHs, Dalteparin (D), Enoxaparin (E), Tinzaparin (T) and the 1st International Standard (85/600) were crossed referenced against the 2nd International Standard (NIBSC 01/608) using an amidolytic AXa method. Each of these LMWHs were compared in the AXa, adjusted concentration range of 0–1.0 U/ml using the Heptest, AXa, AIIa and PiCT. In addition plasma samples from patients receiving a LMWH, E for therapeutic and interventional purposes were measured using various tests. Results: The AXa potency adjusted LMWHs (D, E, and T) and 1st International Standard provided superimposable concentration curves in the amidolytic AXa assays. However marked differences in the heptest and PiCT were noted. Major differences were noted in the AIIA levels, even between the two International standards. When patients samples (n=75) from a therapeutic trial (1.0 mg/kg BID/SC) were evaluated, assay based differences were further amplified. The amidolytic AXa assay consistently measured higher AXa levels. When the two standards were cross-referenced with one another in different assays, major differences were noted in the clot-based assays. Even in the AXa assay at equivalent AXa levels, differences were obvious. Conclusions: These results suggest that both of the International Standards of LMWH are valid for only the cross standardization of the AXa activities of LMWHs. If any of the other methods were used, significantly different results were obtained with each of the individual LMWHs. Thus, the 2nd International Standard should only be used for amidolytic AXa assay for potency referencing purposes. Moreover, the stated potency of the 2nd Internation Standard may need to be readjusted against the 1st Standard to obtain valid results. These standards are of limited value in the clinical monitoring of LMWHs. It is therefore proposed that each of the LMWHs should be cross referenced by its own standard and the clinical monitoring of these drugs should only be carried out utilizing the specific drug used in a given patient. The PiCT test offers a global test which is capable of monitoring the effects of all components of heparins regardless of their affinity to serpines. Moreover, the effect of TFPI released on clotting processes is also measured. Thus, the PiCT test provides a physiologically relevant anticoagulant index.

Description: ADAMTS13 alternatively known as von Willebrand Factor (vWF) cleaving protease is a Zinc metalloprotease which cleaves macromolecular M-vWF multimers at the Tyr (1605)-Met (1606) bond located in the AT domain of vWF. Down regulation of ADAMTS13 is known to be associated with thrombotic thrombocytopenic purpura (TTP). ADAMTS13 forms stable complex with factor XI and factor XIa. Measurement of ADAMTS13/FXI complexes in plasma may provide additional information on the pathogenesis of ESRD associated thrombotic complications. ADAMTS13/FXI can be measured using an ELISA based immunoassay. A polyclonal antibody reacts with a peptide in C-terminal region of the ADAMTS13 molecule is quoted on the micro titer plate. ADAMTS13 present in these test samples bind the antibody. After washing the bound ADAMTS13/FXI complexes were quantified. To test the hypothesis that ADAMTS13 levels are altered in ESRD, blood samples from 62 patients with ESRD prior to dialysis session were collected. Control group comprised of 34 normal healthy age match controls. The vWF antigen levels were also measured in both the groups using an ELISA method. The ESRD group exhibited a marked variation in the ADAMTS13/FXI complex levels with a range of 0 to 1100% (mean = 161±315) of the 62 samples. Eleven patients exhibited a greater than 250% where as the 51 patients exhibited less than 100 % complex. The distribution of ADMTS13/FXI complex was bi-modal. In the normals, the ADAMTS13/FXI complex activity ranges from 147–201% with a mean of 169±16. Unlike the ESRD population the normal group exhibited guassian distribution. The vWF antigen levels relatively higher in the ESRD patients (175±30) vs the normal (88±19). These results suggest that ADAMTS13/FXI is dysregulated in ESRD patients. While most of the patients exhibit a marked decrease in ADAMTS13 level, some of these patients exhibit markedly higher levels. Such fluctuation in the ADAMTS13/FXI level may contribute to the potential thrombotic and bleeding complications in ESRD pathogenesis.