Prevention effect of orally active heparin conjugate on cancer-associated thrombosis.

Thrombogenesis is a major cause of morbidity and mortality in cancer patients. Prophylaxis with low-molecular-weight heparin (LMWH) is recommended for cancer patients, but requires non-parenteral delivery methods for long-term treatments. In this study, we sought to generate a new oligomeric-bile acid conjugate of LMWH that can be used for oral delivery. We first synthesized a tetramer of deoxycholic acid (tetraDOCA), which was site-specifically conjugated at the end saccharide of LMWH. When LMWH-tetraDOCA conjugate (LHe-tetraD) was orally administered at a dose of 5 mg/kg in ICR mice, the maximum anti-factor Xa level was increased up to 0.62±0.05 IU/mL without any evidence of liver toxicity, gastrointestinal damage, or thrombocytopenia. The cancer-associated thrombosis was induced in tumor-bearing mice by local heat application, and the fibrin deposition in tumors was evaluated. The oral administration of LHe-tetraD (either a single dose or multiple daily doses for up to 10 days) in mice substantially abolished the coagulation-dependent tropism of fibrinogen in the heated tumors and significantly decreased hemorrhage, compared to the mice treated with saline or subcutaneous injection of LMWH. Thus, the anticoagulation effect of oral LHe-tetraD invokes the benefits of oral delivery and promises to provide an effective and convenient treatment for cancer patients at risk of thrombosis.

Development of idraparinux and idrabiotaparinux for anticoagulant therapy.

Idraparinux is an analogue of fondaparinux binding with high affinity to antithrombin. It was designed for weekly, rather than daily, administration, with an exceptionally long half-life. One potential problem with small heparin-like fragments of this type is the difficulty of neutralising excessive activity in the case of side-effects or overdose. The efficacy of idraparinux was was proven in clinical studies with patients suffering from venous thromboembolism (VTE) or atrial fibrillation. Due to major bleeding events during treatment for more than six months the development of idraparinux was stopped. Idrabiotaparinux has an attached biotin moiety at the non-reducing end unit, which allows its neutralisation with avidin, an egg-derived protein with low antigenicity. This compound is currently investigated in clinical trials for prevention of recurrent VTE in patients with acute pulmonary embolism. The future of idrabiotaparinux depends also on the safety and efficacy of avidin.

Thrombin generation assays are superior to traditional tests in assessing anticoagulation reversal in vitro.

Even though new anticoagulants are being devised with the notion that they do not require regular monitoring, when bleeding occurs, it is important to have an antidote and a reliable test to confirm whether the anticoagulant effects are persisting. We examined the effects of five heparinoids, unfractionated heparin (UFH), tinzaparin, enoxaparin, danaparoid and fondaparinux on the traditional APTT and anti-Xa assays as well as on the calibrated automated thrombogram (CAT). We also studied the ability of protamine sulphate (PS), NovoSeven, FEIBA and FFP to reverse maximum anticoagulation induced by the different heparinoids. The CAT was the only test to detect the coagulopathy of all the anticoagulants. PS produced complete reversal of UFH, and this could be monitored with all three tests. Tinzaparin can also be completely neutralised in vitro with high doses of PS, but the maximum enoxaparin reversal achieved with PS is only approximately 60%. Fondaparinux does not significantly affect the APTT and PS has no significant effect on its reversal. Only NovoSeven was able to correct the fondaparinux induced CAT abnormalities whilst having no effect on the anti-Xa level. None of the reversal agents was very effective in danaparoid spiked plasma but NovoSeven, at high dose, increased the ETP by 40% and reduced the anti-Xa level from 0.93 to 0.78 IU/ml. We conclude that the CAT is superior to the traditional coagulation tests in that it not only detects the coagulopathy of all the heparinoids but can be also be used to monitor their reversal.

Autologous blood transfusion for cardiopulmonary bypass: effects of storage conditions on platelet function.

OBJECTIVES: Cardiopulmonary bypass impairs formation of large stable platelet aggregates (macroaggregation), although formation of small aggregates (microaggregation) is preserved. A factor in the uncertain benefits of intraoperative autologous blood transfusion may be the effects of storage on platelet function. The effects of citrate preservative and heparinization before storage on platelet function was therefore assessed. METHODS: Twenty-seven patients undergoing elective coronary artery bypass grafting were randomly allocated to have 450 to 1,000 mL of blood taken into CPDA anticoagulant bags either before (n = 14) or after heparinization (n = 13). Samples from the patients and stored blood were anticoagulated with rhirudin, 200 U/mL. The macroaggregatory response to submaximal collagen was measured by impedance aggregometry and microaggregation by single platelet counting. RESULTS: During macroaggregation, before cardiopulmonary bypass, the ex vivo median (interquartile range) response was 16.3 (12.4-18.7) Omega. This decreased 10 minutes after heparin to 8.9 (3.3-11.0) Omega (p < 0.0001). In the blood bags (in vitro), the initial response for nonheparinized blood was 4.8 (0.1-7.5) Omega (p < 0.002 v ex vivo) and at end-cardiopulmonary bypass was 2.4 (1.6-8.2) Omega. During microaggregation, in vivo heparinization decreased microaggregation both ex vivo and in vitro in CPDA blood; the in vitro response of nonheparinized blood at end-cardiopulmonary bypass was greater than that seen after in vivo heparinization (p < 0.007). No difference in bleeding or transfusion requirements was seen. CONCLUSIONS: Collecting blood into CPDA anticoagulant caused a marked deterioration in platelet function. This was worse after in vivo heparinization and included depression of microaggregation.

Enhancement of the leukocyte-endothelial cell interaction in collecting venules of skeletal muscle by protamine.

OBJECTIVE: A transient but severe systemic leukopenia regularly occurs after the antagonization of heparin by protamine in patients and in animals. The aim of the present study was to investigate the site and mechanisms of white blood cell retention during this transient leukopenia by studying the leukocyte-endothelial cell interaction in skeletal muscle venules. METHODS: Syrian golden hamsters were equipped with a dorsal skinfold chamber for intravital fluorescence microscopy and arterial and venous catheters for drug infusion, blood pressure measurement, and blood sampling. Microhemodynamic parameters and leukocyte-endothelial cell interactions were observed in one single collecting venule per animal after intravenous infusion of saline solution (control, n = 10), of protamine (n = 9), and after infusion of heparin followed by either intravenous protamine (n = 9) or intraarterial protamine (n = 9). RESULTS: All parameters remained unchanged in the control group. Whereas venular diameters remained unchanged, protamine transiently increased arterial blood pressure and venular erythrocyte velocity in all groups. Systemic leukocyte counts and the venular leukocyte discharge concentration decreased concurrently after protamine administration by about 60% to 70% at 2 minutes while the fraction of rolling leukocytes and the number of adherent leukocytes remained unchanged. Two and one-half minutes later, systemic leukocyte counts and venular discharge concentrations normalized while the fraction of leukocytes rolling slowly along or adhering firmly to the venular endothelial wall increased considerably and similarly in all groups receiving protamine. Myeloperoxidase (an indicator of polymorphonuclear leukocytes) determination in 20 separate hamsters 2 minutes after protamine infusion revealed increased myeloperoxidase activity exclusively in the lungs. CONCLUSION: The response of leukocytes to protamine infusion with or without prior heparinization is biphasic: initial retention of leukocytes in the lungs is followed by enhanced leukocyte-endothelial cell interaction in the systemic circulation.