Severe Aortic Valve Stenosis.

Aortic valve stenosis (AVS) is the most common valve disease in adults. Severe forms are associated with acquired von Willebrand syndrome (aVWS) with loss of the largest von Willebrand factor (VWF) multimers. Diagnostic gold standard is the VWF multimer analysis. Valve replacement rapidly restores the VWF structure. Uncertainty exists if this effect is permanent and how functional VWF assays perform compared with multimer analysis. We studied 21 consecutive patients with severe AVS before and 6 to 18 months after valve surgery and compared them with 14 controls without valve disease referred for coronary angiography. The VWF multimers, VWF antigen (VWF:Ag), VWF collagen binding capacity (VWF:CB), VWF:CB/VWF:Ag ratio, in vitro bleeding time (PFA-100), factor VIII coagulation activity (FVIII:C), and VWF ristocetin cofactor activity (VWF:RCo) were determined. In all patients with AVS, the large VWF multimers were strongly reduced (56 ± 13% of normal plasma); all controls had normal multimers. The PFA-100 collagen/ADP closure times (coll/ADP CT) were prolonged in patients with AVS compared with the controls (175 ± 56 seconds vs 86 ± 14 seconds, P < .001). The VWF:CB/VWF:Ag ratio was pathological in 20 of the 21 patients but normal in controls. After surgery, the multimers normalized in all patients and coll/ADP CT shortened (pre 184 ± 65 seconds vs post 102 ± 22 seconds; P < .001). The VWF:CB/VWF:Ag ratio strongly improved ( P < .001) and normalized in 14 of 17 patients. In conclusion, all consecutive patients with severe AVS had an aVWS. The combination of coll/ADP CT and VWF:CB/VWF:Ag ratio detected the aVWS in all patients. More than 6 months after valve replacement, the VWF multimers were still normalized in all patients indicating a permanent cure of the aVWS.

Albumin is recycled from the primary urine by tubular transcytosis.

Under physiologic conditions, significant amounts of plasma protein pass the renal filter and are reabsorbed by proximal tubular cells, but it is not clear whether the endocytosed protein, particularly albumin, is degraded in lysosomes or returned to the circulatory system intact. To resolve this question, a transgenic mouse with podocyte-specific expression of doxycycline-inducible tagged murine albumin was developed. To assess potential glomerular backfiltration, two types of albumin with different charges were expressed. On administration of doxycycline, podocytes expressed either of the two types of transgenic albumin, which were secreted into the primary filtrate and reabsorbed by proximal tubular cells, resulting in serum accumulation. Renal transplantation experiments confirmed that extrarenal transcription of transgenic albumin was unlikely to account for these results. Genetic deletion of the neonatal Fc receptor (FcRn), which rescues albumin and IgG from lysosomal degradation, abolished transcytosis of both types of transgenic albumin and IgG in proximal tubular cells. In summary, we provide evidence of a transcytosis within the kidney tubular system that protects albumin and IgG from lysosomal degradation, allowing these proteins to be recycled intact.

Factor XII activation markers do not reflect FXII dependence of thrombin generation induced by polyvinylchloride.

The role of factor XII (FXII) as the main trigger of the coagulation cascade during haemodialysis has been recently challenged. Polyvinylchloride (PVC) is the standard polymer for haemodialysis circuit tubings, but its interaction with FXII has not been extensively characterized. In a modified Chandler tubing loop model using heparinized fresh human whole blood we selectively inhibited coagulation factors VII, X or XII with monospecific antibodies. Contact of whole blood with PVC induced a strong thrombin generation [thrombin-antithrombin complexes (TAT) 64 ± 24 µg/l, before <1 µg/l]. Despite this, levels of FXII coagulation activity, free FXIIa or FXIIa-C1 inhibitor complexes remained unchanged. The anti-FXII antibody abolished thrombin generation (TAT 8 ± 5 µg/l, P < 0.05) and made the free FXIIa undetectable. Inhibition of FVII did not affect coagulation activation (TAT 68 ± 26 µg/l). Our data provide definitive evidence that PVC triggers the coagulation system via FXII. However, all FXII activation markers in plasma failed to detect contact activation.

Anticoagulant-free Genius haemodialysis using low molecular weight heparin-coated circuits.

BACKGROUND: Regional citrate anticoagulation or saline flushes are often used in haemodialysis patients at high risk of bleeding. In an alternative approach we evaluated the effects of covalent circuit coating with low molecular weight heparin (LMWH) for intermittent haemodialysis. METHODS: In vitro, we compared the thrombogenicity of an uncoated polyvinylchloride (PVC) tubing set with LMWH-coated tubing (AOThel) and a reference tubing with end-point attached heparin coating (Carmeda Bioactive surface) under dynamic blood contact. In vivo, five chronic haemodialysis patients were studied using the Genius dialysis system and F60S filters. Each patient underwent three dialysis sessions separated by a standard haemodialysis each: (1) standard dialysis (uncoated circuit and regular dalteparin dosage), (2) dialysis with LMWH-coated circuit and regular dalteparin dosage and (3) dialysis with a completely LMWH-coated circuit without anticoagulant use. RESULTS: In vitro, both coated tubings showed significantly reduced thrombin-antithrombin (TAT) complex levels compared with PVC. The reference coating (Carmeda) released substantial antifactor Xa (antiXa) activity into the plasma. The LMWH coating (AOThel) released low antiXa activity only during the initial rinsing. In vivo, all dialysis sessions were well tolerated and completed without major clotting. Antithrombin levels and platelet counts were similar in all groups. P-selectin and D-dimer levels increased similarly in all groups. TAT levels were comparable in all groups during the first 3 h and significantly increased in the anticoagulant-free group after the fourth hour. CONCLUSIONS: LMWH surface coating reduces thrombogenicity in vitro without releasing significant amounts of heparin from the surface. In vivo, anticoagulant-free haemodialysis using a completely LMWH-coated circuit is feasible and safe in stable chronic dialysis patients with normal coagulation.

Factor Xa-activated whole blood clotting time (Xa-ACT) for bedside monitoring of dalteparin anticoagulation during haemodialysis.

BACKGROUND: Low molecular weight heparins (LMWH) like dalteparin are increasingly used for anticoagulation during haemodialysis (HD). The available laboratory tests for monitoring LMWH anticoagulation are time-consuming and expensive, and the suitability of the conventional activated clotting time (ACT) is controversial. A simple and cheap bedside test would be useful. METHODS: We studied the factor Xa-activated whole blood clotting time (Xa-ACT) in vitro and in vivo in nine patients undergoing chronic HD with i.v. dalteparin bolus anticoagulation and compared it with the conventional ACT. Plasma anti-factor Xa (antiXa) activity was determined with a chromogenic assay. Thrombin-antithrombin complexes were measured to detect coagulation activation. RESULTS: Xa-ACT and ACT were prolonged with rising dalteparin concentration. In vitro, both clotting times were strongly correlated with the antiXa levels (r = 0.94 and 0.89, respectively). Nevertheless, compared with the ACT, the Xa-ACT was considerably more sensitive to the LMWH in vitro (healthy blood: Xa-ACT 90 s/U vs ACT 26 s/U; uraemic blood: Xa-ACT 96 s/U vs ACT 31 s/U) as well as in vivo (Xa-ACT 81 s/U vs ACT 22 s/U) and reflected different intensities of anticoagulation. An initial dalteparin bolus of 80+/-11 U/kg body weight was able to prevent coagulation activation for up to 4 h of HD. CONCLUSION: For monitoring LMWH anticoagulation the Xa-ACT was superior to the conventional ACT in vitro as well as in vivo during HD. The Xa-ACT can be useful as a LMWH bedside test. The ACT was not sensitive enough to serve as a LMWH monitoring tool.

In vitro studies on hirudin elimination by haemofiltration: comparison of three high-flux membranes.

BACKGROUND: Recombinant hirudin (r-hirudin) is a highly selective thrombin inhibitor used for anticoagulation in heparin-induced thrombocytopenia type II. R-hirudin is increasingly applied to patients with renal failure and on renal replacement therapy. Since kidney function impairment strongly prolongs r-hirudin elimination half-life, severe accumulation and bleeding complications may occur. Data on the r-hirudin permeability and elimination capacity of different haemofilters are limited. METHODS: Three haemofilter types were investigated: high-flux polysulphone (Fresenius), AN69 (Hospal), and polyamide (Gambro). We used two in vitro haemofiltration models: (i) an open post-dilution haemofiltration model with ultrafiltration and fluid substitution (model 1) simulating hirudin intoxication, and (ii) a closed model with ultrafiltrate reinfusion (model 2) to determine steady-state sieving coefficients (SC). Fresh human heparinized blood (2 IU unfractionated heparin/ml blood) was used. In model 2, SC obtained with human whole blood were compared with isotonic saline. RESULTS: In model 1, r-hirudin levels decreased significantly faster with polysulphone than with AN69 or polyamide (P<0.05). In accordance with this, in model 2 the observed SC in whole blood were 1.11+/-0.28 (polysulphone), 0.61+/-0.15 (AN69) and 0.33+/-0.13 (polyamide), and clearances were 28+/-7 (polysulphone), 15+/-4 (AN69) and 8+/-3 ml/min (polyamide) (P<0.001 for all comparisons). The SC in saline were slightly but significantly lower for polysulphone (0.88+/-0.12), similar for AN69 (0.59+/-0.1), and significantly improved for polyamide (0.83+/-0.1). CONCLUSIONS: Elimination of r-hirudin by haemofiltration strongly depended on the membrane material. Using human blood, we observed large differences between the three high-flux membranes. The saline experiments suggest a membrane-dependent impact of plasma proteins and pH on hirudin sieving. Our findings have implications for r-hirudin dosage in haemofiltration, for treatment of overdosage, and for future in vitro haemofiltration studies.