Mitigation of coagulation by removing clotting factors part 1: in vitro feasibility study.

Heparin is associated with adverse effects in some patients during extracorporeal circulation. A potential alternate anticoagulation strategy explored in this investigation involved mitigation of coagulation by removing clotting factors from blood by adsorption on a protamine-immobilized Sepharose matrix (PSM). Human or porcine plasmas treated with PSM in vitro were tested for clotting factors I (fibrinogen), II (prothrombin), VIII, and X, and proteins C and S, and for prothrombin time (PT), activated partial thromboplastin time (APTT), and total protein concentration. Bovine blood treated with PSM was also perfused through a hollow-fiber cartridge to assess thrombogenic potential in a shear flow system. PT increased with increasing protamine-Sepharose-to-plasma ratios and with increasing mixing time. When the PT and APTT of treated plasma were prolonged three to six times the baseline, Factors II and X were significantly removed (>90%), Factors I and VIII were partly removed (<35%), and total protein concentration remained >80% of the initial value. When blood depleted of clotting factors was perfused through hollow-fiber cartridges without an anticoagulant, cartridge patency was prolonged compared with cartridges perfused with untreated blood. This investigation demonstrated that inhibition of blood coagulation by removal of key clotting proteins is feasible.

Mitigation of coagulation by removing clotting factors part 2: heparin-free extracorporeal circulation in a porcine model.

A subpopulation of patients would benefit from an anticoagulation strategy during extracorporeal circulation (ECC) that does not involve systemic administration of heparin and protamine. Inhibition of coagulation by adsorption of plasma clotting factors using protamine immobilized on a Sepharose matrix (PSM) has been explored. This investigation extends previous in vitro studies and demonstrates the feasibility of heparin-free ECC. In a porcine ex vivo circuit, plasma was separated from blood via plasmapheresis, passed through a column containing PSM beads, and returned to the animal. Hemodialyzers and stents were placed in the circuit before, during, and after ECC and examined for device thrombosis. After 90 minutes, prothrombin time (PT) was prolonged >10 times the baseline, and blood clotting Factors I, II, VIII, and X were decreased significantly (>90%); this state was maintained for 2.5 hours without detectable adverse consequences. After cessation of ECC, PT approached normal levels within 60 minutes. Examination of hemodialyzers and coronary stents placed in the circuit revealed that the removal of clotting factors significantly reduced device thrombosis and that transfusion of homologous blood ( approximately 10% V/V) resulted in immediate restoration of hemostasis. It is possible to remove clotting factors from circulating blood to allow extracorporeal circulation of blood without the use of heparin.

Elevated fibrinogen fragment levels in uremic plasma inhibit platelet function and expression of glycoprotein IIb-IIIa.

Hemostatic dysfunction is frequently noted in uremia, but the mechanisms responsible for it are poorly understood and are assumed to be multifactorial. Preliminary findings from our laboratory suggest that elevated levels of circulating fibrinogen fragments (FF) might contribute to the hemostatic defect in uremic patients. Defibrinated plasma obtained from chronic hemodialysis (HD) patients as well as normal subjects were examined by SDS-PAGE and immunoblotting and quantified by an immunoassay. In addition, endogenous FF isolated from normal and uremic plasma using affinity chromatography were examined by flow cytometry for their effect on glycoprotein (GP) IIb-IIIa receptor expression and tested for their ability to inhibit platelet aggregation. The mean FF concentration in uremic plasma (1.14 +/- 0.85 mg/ml) was noted to be eight times greater than in normal plasma (0.15 +/- 0.01 mg/ml) (P < 0.05). Moreover, the mean FF level decreased by 48.25% following HD (from 1.14 +/- 0.85 mg/ml to 0.59 +/- 0.33 mg/ml; P < 0.05). SDS-PAGE and immunoblotting experiments showed that the decrease was observed in both medium-sized (20-60 kDa) as well as large (>100 kDa) FF. Further, FF isolated from uremic plasma inhibited platelet aggregation by (46.8 +/- 18.1)% (P < 0.05) and the GP IIb-IIIa receptor expression by (28.0 +/- 7.6)% (P < 0.05 vs. control). The results show that (1) FF levels are elevated in uremic plasma, (2) HD results in significant decrease in FF and (3) endogenous FF inhibit platelet function, presumably via competitive binding to the fibrinogen receptor GP IIb-IIIa. The decrease in plasma levels of FF > 100 kDa following HD suggests that adsorption to the dialysis membrane contributes to their removal.

Different responses by cultured aortic and venous smooth muscle cells to gamma radiation.

BACKGROUND: Stenosis of hemodialysis arteriovenous grafts is usually focal and caused by the proliferation of vascular smooth muscle cells (SMCs). External radiation of the graft is a potential strategy to prevent stenosis; however, the relative responsiveness of arterial and venous SMCs to radiation is unknown. METHODS: Human aortic and saphenous vein SMCs were cultured in a medium containing growth factors and serum and treated with 0 to 50 Gy in a gamma irradiator. At 2 to 20 days post-irradiation, cell counting, methylthiazoletetrazolium dye reduction, [(3)H]-thymidine uptake, and bromodeoxyuridine (BrdU) incorporation assays were performed. RESULTS: All assays showed that 1 to 50 Gy inhibited the proliferation of both aortic and venous SMCs in a dose-dependent manner. Importantly, venous cells were less susceptible to radiation in all assays, compared to aortic cells. At day 10, 1 to 50 Gy of radiation inhibited the increase in the number of aortic cells by 24% to 66% and venous cells by 8% to 25% (P < 0.01) (aortic vs. venous). The differences between aortic and venous cells varied among different assays and were most pronounced in the BrdU assay. CONCLUSION: Inasmuch as myointimal hyperplasia occurs at both arterial and venous anastomoses, future strategies using radiation to prevent hemodialysis vascular access stenosis should take these differences into consideration.

In vitro pharmacological inhibition of human vascular smooth muscle cell proliferation for the prevention of hemodialysis vascular access stenosis.

BACKGROUND: Vascular access for chronic hemodialysis often fails as a result of stenosis caused primarily by the proliferation of vascular smooth muscle cells (VSMC). Various drugs have been shown to inhibit the proliferation of VSMC under different conditions. METHODS: In this study, we compared the inhibitory effect of ten drugs on the proliferation of human aortic smooth muscle cells (SMC) in culture. Quiescent cells were cultured in the presence of growth factors, fetal bovine serum and incremental concentrations of the test drug. Cell proliferation was assessed by the MTT reduction assay. RESULTS: Aspirin, enalaprilat, heparin, hydroxyurea, indomethacin and tirofiban were ineffective. While dipyridamole, paclitaxel, tranilast and verapamil inhibited cell proliferation, the concentrations required were significantly higher than the clinical plasma levels achieved after systemic administration. CONCLUSION: Local delivery of these drugs to the target site may therefore be a more effective and appropriate strategy for the prevention of hemodialysis vascular access stenosis.

Inhibition of neointimal hyperplasia in vascular grafts by sustained perivascular delivery of paclitaxel.

BACKGROUND: Neointimal hyperplasia occurs commonly at the anastomoses of arteriovenous grafts for chronic hemodialysis, causing stenosis and occlusion. Antiproliferative drugs may be effective in inhibiting hyperplasia, but local drug delivery would be required to minimize systemic side effects. We examined the feasibility of local drug delivery to inhibit neointimal hyperplasia at dialysis grafts in a canine model. METHODS: Bilateral polytetrafluoroethylene loop grafts (10-cm length and 6-mm internal diameter) were placed between the femoral artery and ipsilateral femoral vein of five mongrel dogs. At the time of surgery or 1 to 5 weeks later, 2 mL of a thermosensitive biodegradable copolymer (ReGel) mixed with 0.26 mg or 0.65 mg paclitaxel were applied to the external surface of one graft around the anastomoses to provide a depot for sustained release of the drug. ReGel alone without paclitaxel was applied to the contralateral graft as a control. The grafts and the connecting vessels were explanted at eight or nine weeks, and the cross-sections were examined histologically. The degree of hyperplasia at the anastomoses was graded by five blinded independent reviewers, with scores ranging from 0 to 5. RESULTS: The median (25th-75th percentile) hyperplasia score of both arterial and venous anastomoses was 1.80 (0.90-3.05) in the grafts treated with ReGel alone, and 0.95 (0.70-1.50) in the grafts treated with ReGel/paclitaxel (N= 8; P < 0.05 by Wilcoxon signed rank test). There were no noticeable localized or systemic complications attributed to the treatments in these animals. Paclitaxel levels in the plasma obtained from forelimb veins were undetectable (<10 ng/mL). CONCLUSION: These results suggest that the local delivery of antiproliferative agents using a thermosensitive, injectable biodegradable copolymer (ReGel) for sustained delivery is a promising strategy to inhibit neointimal hyperplasia of arteriovenous hemodialysis grafts.