Chronic inflammation is a major cause of morbidity and mortality in end-stage renal disease. The associated anemia in these patients due to renal cortical atrophy and erythropoietin deficiency is treated with recombinant erythropoietin. Recent reports suggest a growing incidence of symptomatic venous thrombosis in cancer patients treated with recombinant erythropoietin. Several investigators have reported on different mechanisms of thrombosis in these patients. We hypothesize that thrombosis in patients with end-stage renal disease due to increased expression of C-reactive protein (CRP) as a result of chronic inflammation promotes the release of thrombin activatable fibrinolytic inhibitor causing fibrinolytic deficit and eventually thrombosis. Furthermore, because endothelial nitric oxide is responsible for the maintenance of the normal vascular function, the decreased levels of nitric oxide in chronic inflammation cause endothelial damage and result in thrombosis. To test this hypothesis, blood samples were collected from 106 patients (49 male and 57 female, aged 59.8+/-15.7 years) with end-stage renal disease undergoing hemodialysis and treated with recombinant erythropoietin at a mean dose of 201.8 U/kg/week. Blood samples were drawn in 5-mL tubes containing 3.2% sodium citrate just before the hemodialysis procedure. These blood samples were immediately centrifuged to obtain platelet-poor plasma, which was aliquoted and frozen at -70 degrees C until further analysis. Erytropoietin antibodies were measured using an anti-EPO enzyme-linked immunosorbent assay (ELISA) method developed in our laboratory. Nitric oxide was measured using a NO analyzer (Sievers 280I, Ionics, Boulder, CO). Plasma CRP levels were measured with a highly sensitive ELISA method IMUNOCLONE CRP ELISA (American Diagnostica, Greenwich, CT). TAFI antigen levels in plasma were analyzed with an IMUCLONE TAFI ELISA kit (American Diagnostica, Greenwich, CT). TAFI functional activity was assayed with an ACTICHROME TAFI activity kit. The measured levels of nitric oxide, CRP, TAFI antigen, and TAFI functional were 37.36+/-36.8 (normal value, 37.49+/-18.96; range, 19.3-102 microM), 12.27+/-10.6 (normal value, < 1 microg/mL), 146.9+/-28.4% NHP (normal, 100% NHP), and 102.55+/-37% NHP (normal range, 22.3-165.7; mean, 89.5% NHP), respectively. The erythropoietin antibody was detected in 9.4% of the patient group. While 20% of the erythropoietin antibody-positive and 27.1% of the erythropoietin antibody-negative patients experienced chest pain, thrombotic events developed in 9.4% of the erythropoietin antibody-negative patients. These data provide the rationale for a novel mechanism of thrombosis through increased activity of CRP, nitric oxide, and TAFI, leading to fibrinolytic deficit and thrombosis in patients treated with erythropoietin.

Several of the newly developed anti-Xa and anti-IIa agents have been shown to influence the International Normalized Ratio (INR) values. During phase I trials with normal healthy volunteers and phase II study patients who were given warfarin and concomitant anti-IIa or anti-Xa agents, it has been reported that INR values were falsely elevated. It is of critical importance to know of the effects of these agents on INR to avoid dosage errors. To study the influence of these agents on INR, we used several anti-IIa agents (argatroban, recombinant hirudin, efegatran, and PEG-hirudin) and anti-Xa drugs (pentasaccharides such as fondaparinux and idraparinux, DX-9065a and JTV-803). The anti-IIa drugs were supplemented in citrated plasma at a concentration of 0 to 1 microg/mL level and anti-Xa drugs in the range of 0 to 25 microg/mL. The IC(50) values for each of these agents were calculated. Four different commercially available prothrombin time (PT) reagents were used to perform the PT assays and to calculate the relative INR values. Direct synthetic factor IIa and Xa inhibitors exhibited a concentration-dependent increase in the INR values. Hirudin, efegatran, and PEG-hirudin showed a weaker effect, whereas argatroban showed a much higher elevation of the INR values. Synthetic indirect anti-Xa agents such as the pentasaccharide did not show any effect on the INR values. Furthermore, prothrombin time reagents with high ISI values exhibited disproportionally higher INR values for both the direct anti-Xa and anti-IIa agents. Elevation of INR values has therapeutic implications when non-oral anticoagulant drugs are used in combination with drugs such as warfarin. Because of the false elevation of INR values with some of the non-oral anticoagulant drugs, patients who are on concomitant warfarin therapy should be carefully evaluated for their corresponding INR values for proper dosing. To avoid dosing errors it is best not to use the INR values in the therapeutic monitoring of anti-Xa and anti-IIa agents either in the monotherapeutic or polytherapeutic modalities. These data also warrant the development clinically relevant methods for the monitoring of the concomitant use of newly developed anti-Xa and anti-IIa drugs with oral anticoagulants.

Heparin and low molecular weight heparins exert their vascular effects by mobilizing tissue factor pathway inhibitor (TFPI) from the vascular endothelium into the blood circulation. We compared the influence of molecular weight on the TFPI release by heparin and its fractions in a non-human primate model. Primates were treated with unfractionated heparin, a low molecular weight heparin (gammaparin), or a heparin-derived oligosaccharide mixture (C3). Endothelial TFPI release was determined using both immunologic and functional assays. After intravenous administration, all agents significantly increased TFPI levels ( p < 0.05) in a dose dependent manner. The increase produced by unfractionated heparin and gammaparin was greater than that by C3 at an equal dosage (p < 0.05). With subcutaneous injection, all agents produced less TFPI release. Repeated administration of heparin-derived oligosaccharides gradually increased TFPI release. A 1.89 fold increase in TFPI levels was observed 4 days after C3 treatment (2.5 mg/ kg). Our findings indicated that TFPI release is dependent on the molecular weight of heparin and its derivatives. Heparin oligosaccharides exert their vascular effects through increased TFPI release after longterm repeated administration.

Long-distance air travel is increasing and cases of venous thromboembolism (VTE) following air travel have attracted both considerable public attention and legal claims against airlines. VTE is a common disorder worldwide with a notably high incidence in older individuals. Many biochemical factors that lead to, or accentuate, thrombus formation are associated with increased risk of VTE. These factors include thrombophilia, activated protein C resistance and factor V Leiden, prothrombin gene mutation, antiphospholipid antibodies, protein S and protein C deficiencies, and methylene tetrahydrofolate reductase polymorphism and homocysteinemia. Individual physical characteristics including age, weight and height are significant for personal risk of VTE as are other factors such as use of oral contraceptives in women. In the case of air travel-related venous thrombosis, superimposed upon these individual factors are the environmental factors directly related to air travel. Travel-related factors include stasis associated with prolonged periods of immobility, physiological stresses resulting from exposure to the cabin environment (low humidity and hypoxia) in long-haul flight and other in-flight factors. It is suggested that passenger behavior (movement, avoidance of dehydration and of alcohol) and appropriate pharmacological prophylaxis for high-risk travelers can reduce the likelihood of VTE. Physical prophylaxis (use of compression stockings or in-flight exercise devices) may also be of general benefit to passengers. It is recommended that airlines become more proactive in educating passengers concerning the dangers of VTE and in promoting passenger actions that can reduce risk. Airlines should also work to avoid cramped seating conditions (seat size and pitch) that contribute to prolonged immobility. Governments and regulatory authorities should mandate the provision of adequate seating conditions and a good cabin environment and should support studies that will define risks and determine the efficacy of protocols to minimize dangers of VTE. Increased long-haul air traffic and an aging population suggest that travel-related VTE may present a growing healthcare threat and has highlighted a need for additional biomedical research into the causes and potential solutions to this problem.

Burn and septic injuries induce profound changes in coagulation status. This study examined the changes in plasma tissue factor pathway inhibitor (TFPI) and thrombin activatable fibrinolytic inhibitor (TAFI) levels in a rat model of burn and septic injuries. Rats underwent 30% TBSA cutaneous scald burn injury and septic insult was induced by caecal ligation and puncture (CLP). CLP was superimposed on burn injury to mimic the clinical model of sepsis complicating burn injury. Rats were pretreated with Cprofloxacin orally to colonize their gut with Enterococcus faecalis. TFPI and TAFI plasma levels were measured using functional activity assay kit with a chromogenic method at 24 and 72 hours following the injuries. TFPI levels decreased significantly at 24 hours in burn, CLP, and burn+CLP groups, followed by incomplete rebound recovery at 72 hours in all three groups. On the other hand, TAFI levels increased significantly at 24- and 72-hour time points in all three groups. These results suggest that burn, septic, and their combined injuries perturb coagulation cascade and thrombotic process toward the procoagulant pathway by impairing fibrinolysis.

Synthetic direct inhibitors of factor Xa are capable of prolonging the global anticoagulant assay times in a concentration-dependent fashion. The relative degree of thrombin generation inhibition at an equivalent prolongation is not similar to the results observed with heparins and oral anticoagulant drugs. In addition, the direct factor Xa inhibitors prolong the Russell's viper venom test (RWT) and Heptest clotting times. Ecarin clotting time (ECT) and thrombin time (TT) remain unaffected. The kinetics of factor Xa inhibition are markedly different than those observed with pentasaccharide and heparins. Therefore, the methods developed for heparins and pentasaccharides may not be applicable for the monitoring of factor Xa inhibitors. To test the feasibility of using the prothrombin time (PT), International Normalized Ratio (INR), activated partial thromboplastin time (aPTT), Heptest, thrombin time, RVVT, ECT, and a modified anti-Xa amidolytic assay, to monitor a synthetic factor Xa inhibitor, normal human pool plasma samples were spiked with a synthetic factor Xa inhibitor in the concentration range of 0 to 1 microg/mL and 0 to 25 microg/mL. Different laboratory tests were performed and INR and other ratios were calculated. The anticoagulant effects on whole blood were measured using the activated clotting time (ACT). Further studies on the effect of factor Xa inhibitor on platelet aggregation; factor II, VII, and X functional levels; and fibrinopeptide A (FPA) generation were carried out at equivalent INR levels in comparison to oral anticoagulant and antithrombin agents. FPA generation at equivalent anticoagulant level in comparison to heparin (twice the baseline) was also carried out. Factor Xa inhibitor produced a concentration-dependent prolongation of the ACT. ACT was doubled at a concentration of 4 to 5 microg/mL. There was a marked difference in the prolongation of the PT by a synthetic factor Xa inhibitor dependent on the ISI of the PT reagent used. When the results were calculated to determine INR, marked variations were noted between the recombinant thromboplastin and rabbit brain thromboplastin. The rabbit brain thromboplastin reagent gave markedly high INR values. Similar results were observed when different aPTT reagents were studied. In the anti-Xa assay, modification of the incubation time was employed to extend the proper sensitivity range. These studies warrant further investigation to understand the mechanism of action of factor Xa inhibitors.

No abstract

No abstract