A radiological method to evaluate alveolar bone regeneration in the Chacma baboon (Papio ursinus).

INTRODUCTION: The evaluation of alveolar bone healing may have a role in dental implantology, in prosthodontics in the post-extraction phase and in monitoring fracture repair. There are several radiological techniques described to evaluate alveolar bone regeneration. However, most are expensive and time consuming. OBJECTIVES: To develop and evaluate a radiological method utilising readily available equipment to measure alveolar bone regeneration. MATERIALS AND METHODS: An apparatus was designed to enable the acquisition of standardized x-ray images, consisting of a disposable impression tray, digital positioning system, aluminum step wedge, digital x-ray sensor, Rinn apparatus and laboratory putty. Bone biopsies were collected from each oral quadrant in each of five Chacma baboons (Papio ursinus). Accurately standardised x-ray images of the biopsy sites were taken pre-operatively, directly post-operatively and again after three and six week intervals. These images were analysed using a graded histogram provided in a computer software program. RESULTS: The average gray-scale value on the histogram of the selected biopsy area was determined on the series of standardised images. The average values for the three biopsied sites per quadrant were expressed as percentages of pre-operative density. The results ndicated a mean ncrease of 6.3% (+/- 1.4%) (mean +/- 1 SEM) in bone density after three weeks and 12.6% (+/- 1.7%) six weeks post-operatively. CONCLUSION: A standardised radiologica examination method was developed which, together with a computer ised evaluation technique, could be applied to accurately determine relative bone density. This method was shown to provide comparative bone density values during the regeneration process of alveolar bone over a six week period.

Outcome determinants of urethroplasty in the management of inflammatory anterior urethral strictures.

BACKGROUND: Limited data are available on outcomes of the surgical management of inflammatory urethral strictures secondary to infection, a major cause of stricture. Several shortcomings that need to be addressed have been identified in the past. OBJECTIVE: To determine the impact of stricture length, position and degree of obliterative urethral lumen on the surgical outcomes of corrective procedures for inflammatory anterior urethral strictures. METHODS: This retrospective analysis used the records of patients who presented with proven infective anterior urethral strictures at an academic hospital from 2007 to 2010. All patients were followed up after 48 months. Urethroplasty outcomes were analysed according to stricture location and length and effect of urethral obliteration. RESULTS: The median age of the 174 patients in the study was 47 (range 21 - 86) years. Anastomotic urethroplasty was successful in 59/99 (59.6%) patients. Augmented anastomotic urethroplasty was successful in 11/15 (73.3%) patients. Dorsal onlay buccal mucosa graft urethroplasty was successful in 23/32 (71.9%) patients, significantly higher than in 2/9 (22.2%) patients who underwent ventral onlay buccal mucosa graft urethroplasty (p=0.017; hazard ratio 3.4; 95% confidence interval 1.29 - 9.40). The one-stage circular pedicled penile skin-flap urethroplasty was successful in 1/12 (8.3%) patients. Two-stage urethroplasty was successful in 5/7 (71.4%) patients. A primary component analysis of the 73 failed procedures showed that stricture length was the main contributor to failure (eigenvalue 1.79; 45%). CONCLUSIONS: Urethroplasty remains a challenge in inflammatory urethral strictures, where stricture length was the main reason for treatment failure.

Evaluation of mathematic models to assess platelet kinetics.

Twelve mathematic methods used to calculate the mean platelet survival time were compared by determining the "goodness of fit" of the models to the platelet survival curves of 15 reference subjects and 54 patients. Platelets were labeled with [111In]oxine. The linear (LN), exponential, weighted mean, multiple hit (MH), Dornhorst (DH), Meuleman (ML), alpha order (AO), and polynomial (PO) mathematic models were investigated. The goodness of fit for the exponential model was determined by the nonlinear least squares method (EP), and also by the linear least squares method on logarithmically transformed data (EX) as is recommended. The modified weighted mean (MWM) and the usual weighted mean method (WM) obtained with these exponential models were tested. The Dornhorst (DH10) and Meuleman (ML10) models, where the potential age-dependent platelet survival times were kept constant at 10 days, were also evaluated. The goodness of fit results, expressed as % s.d. indicated that the LN (5.2%), EX (5.0%), EP (4.4%), WM (3.7%), DH10 (3.7%), and ML10 (3.7%) models all fitted the data significantly worse than the MWM, MH, DH, ML, AO, and PO models (range 3.2-3.3%). The mean platelet survival time determined with the MH model differed significantly from the results with the DH, ML, and AO models. The results of mean platelet survival time calculated with different mathematic models cannot, therefore, be compared directly. The models that fitted the platelet survival curve well varied slightly in sensitivity to noise as is indicated by the coefficient of variation of the mean platelet survival time estimates for the reference subjects (range 7.9-12.0%). Fitting data to at least two mathematic models has definite advantages. Data on which the calculations are based are probably invalid if the following are true: (a) if the mean platelet survival time estimated with the alpha order model is shorter than that estimated with the EP, MWM, or MH models, or (b) the mean platelet survival time estimated with either the DH, ML, AO, or PO models, is longer than the LN, MWM, or MH estimate of the mean platelet survival time. We conclude that the mean platelet survival time can be reliably estimated by fitting the data to either the MWM method (if limited computing facilities are available) or the MH model. Confidence in the result will be increased if considered in conjunction with the finding obtained with one other model; in those cases where the platelet survival time is very short, the alpha order model is recommended.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparison of oxine and tropolone methods for labeling human platelets with indium-111.

The effect of the chelates oxine and tropolone, used to label platelets, on the kinetics of indium-111-(111In) labeled platelets was studied in twelve normal human subjects. Autologous platelets were labeled either in saline with 111In-oxine or in plasma with 111In-tropolone. Mean platelet lifespan was estimated by fitting the disappearance curve of platelets from the circulation to the multiple hit and other mathematical models. The in vivo distribution of platelets was quantitatively imaged with a scintillation camera. The in vivo recovery of 111In-oxine and 111In-tropolone did not differ, and the mean platelet lifespan was also similar (111In-oxine: 230 +/- 29 hr; 111In-tropolone: 226 +/- 13 hr). At equilibrium (90 min after reinjection of labeled platelets) and at the end of platelet lifespan, 111In-oxine and 111In-tropolone radioactivities in the spleen and liver were similar. These results demonstrate that the results of kinetics measured with 111In-oxine or 111In-tropolone do not differ significantly.

Kinetics of In-111-platelets in the baboon: I. Isolation and labelling of a viable and representative platelet population.

A fully representative and viable platelet population was isolated from the blood of 15 baboons by a multiwash procedure, and labelled with In-111-oxine. The recovery of the total platelet population in the circulation was 85% +/- 9. Mean platelet life span was 146 hr +/- 13. Correcting for plasma radioactivity (always less than 3.5%) did not significantly affect the estimate of platelet life span (145 hr +/- 16) or recovery (85% +/- 12). Platelet survival estimates, repeated at different times, were reproducible. In 5 baboons, platelets were also harvested by a single step differential centrifugation. The mean life span of a representative platelet population was significantly longer than that of platelets harvested by a single step. Recovery values of the representative and non-representative population were similar. We conclude that it may be important to harvest and label a fully representative platelet population for kinetic studies. The proposed method is simple and reproducible, and may be applied in studies in humans.

Kinetics of In-111-platelets in the baboon: II. In vitro distribution and sites of sequestration.

The kinetics and sites of sequestration of a fully representative population of In-111-platelets were determined in 11 baboons. The in vivo method of quantification with computer assisted scintillation camera image analysis was validated by sacrificing 5 baboons and measuring and comparing the distribution of organ radioactivity. Recovery of platelets in the circulation was 87% +/- 7, and their mean survival time was 147 hr +/- 15. The mean splenic platelet pool was 16.0 +/- 1.9. At equilibrium 15.8% +/- 2.9 of the In-111-platelets were in the hepatic blood pool. Senescent platelets were destroyed in the reticulo-endothelial system. The major sites of sequestration were: liver (37.6% +/- 6.0), and the spleen (23.3% +/- 4.6). The bone marrow sequestrated 14.4% +/- 1.7 of the labelled platelets, and 15.5% +/- 4.0 were present in various other tissues. We conclude that the in vivo method of In-111-quantification is accurate. Senescent platelets are mainly sequestrated in the reticuloendothelial tissue, with the liver, spleen and the bone marrow important sites of sequestration.

Transient interruption of arterial thrombosis by inhibition of factor Xa results in long-term antithrombotic effects in baboons.

Recombinant tick anticoagulant peptide (r-TAP) is a potent and specific inhibitor of activated coagulation factor X which effectively interrupts in vivo arterial thrombosis during treatment. It is, however, uncertain if it also affects thrombosis after treatment is stopped. This was tested in a baboon model of arterial thrombosis where platelet deposition onto Dacron vascular graft segments, inserted as extensions into permanent femoral arteriovenous shunts, was measured. The baboons were intravenously treated with 10 micrograms/kg/min (low dose, aPTT = 39 +/- 1 s) and 25 micrograms/kg/min (high dose, aPTT = 58 +/- 2 s) r-TAP for two hours. During treatment the r-TAP inhibited thrombin formation and dose-dependently interrupted platelet deposition onto the graft segment. This effect lasted for up to two hours after treatment with the low dose. Following treatment with the high dose, the graft segments were kept in place for 53 h. After treatment was stopped, platelets again deposited, but at a much lower rate than in control studies. Maximum deposition was approximately 38% lower than in the control studies. Total platelet deposition over 55 h, calculated as the area under the deposition curve, was approximately 40% (p < 0.05) less than in the control studies. A significant shortening in the mean platelet life span and an approximately 15-fold increase in thrombin-antithrombin III complexes during the first 31 h indicated that the thrombus surface remained thrombogenic and that the effect of r-TAP was transient. We have shown that 2 h of treatment with a full antithrombotic dose of r-TAP markedly reduced both the rate of platelet deposition after treatment was stopped and the total number of platelets deposited over 55 h. This was in spite of the finding that the antithrombotic effect of r-TAP was transient.

In vivo inhibition of acute platelet-dependent thrombosis in a baboon model by Bay U3405, a thromboxane A2-receptor antagonist.

Bay U3405 is a thromboxane A2 (TxA2)-receptor antagonist that inhibits the binding of TxA2 to its target cells. The aim of this study was to determine if Bay U3405 could be used to inhibit arterial thrombosis. A thrombogenic device, consisting of uncrimped Dacron vascular graft material (0.5 cm2) built into the wall of silicone rubber tubing with 4 mm inside diameter, was exposed to native flowing blood under arterial blood flow conditions (100-140 ml/min) by interposing the devices as extension segments into permanent femoral arteriovenous shunts implanted in baboons. Thrombus formation was quantified in vivo by measuring the deposition of 111In-labelled platelets onto the graft material with a scintillation camera. In six baboons, a bolus injection of Bay U3405, calculated to attain an initial plasma concentration of 300 ng/ml, reduced the maximum thrombus formation measured over a 2 h study period. Platelet deposition was reduced by 33 +/- 14% (SD) at 2 h as compared to control studies done in the same baboons. The accumulation of additional platelets onto a thrombus that was allowed to form for 1 h, was reduced by 58 +/- 28% at 2 h. Ex vivo platelet aggregation in response to ADP, activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) were not affected by the treatment. Ex vivo platelet aggregation in response to collagen was markedly inhibited for 2 h after treatment. The results demonstrated that selective blocking of the TxA2-receptor on platelets reduced platelet-dependent thrombus formation and the accumulation of additional platelets in a freshly formed thrombus.(ABSTRACT TRUNCATED AT 250 WORDS)

Indium-111-labelled human platelets: a method for use in severe thrombocytopenia.

We describe and evaluate a simple method for labelling autologous human platelets with Indium-111-oxine in patients with severe thrombocytopenia. Twenty patients with immune thrombocytopenia and platelet counts ranging from 5 to 119 X 10(9)/1 were investigated. Platelets were isolated from blood by differential centrifugation, residual platelets were repeatedly washed from the red cell layer and buffy coat and labelled with In 111 in saline. A mean of 55% +/- 21 of platelets were harvested from the blood, labelled with 49% +/- 24 efficiency and 15.8 X 10(8) labelled platelets reinjected to the patients. Contamination of the platelets with red cells and plasma was low. The labelled platelets were viable as assessed by in vitro aggregation, recovery in the circulation and mean survival time. This method permits quantitative platelet imaging with autologous labelled platelets in patients with severe thrombocytopenia.

Prolonged inhibition of acute arterial thrombosis by high dosing of a monoclonal anti-platelet glycoprotein IIb/IIIa antibody in a baboon model.

The in vivo activity of MA-16N7C2, the first monoclonal antibody that contains an echistatin-like RGD-sequence and inhibits platelet glycoprotein (GP)IIb/IIIa function, was determined in baboons. A dose-finding study assessing haemostatic variables such as bleeding time and ex vivo platelet aggregation showed that doses of as low as 0.2-0.3 mg/kg resulted in a pronounced effect. The effects were dose-dependent and lasted for several days, implying that MA-16N7C2 is a potent and long-acting GPIIb/IIIa inhibitor. Following the initial studies, the antithrombotic effect of 0.1 and 0.3 mg/kg of the antibody, given as a bolus, was determined in a baboon model of platelet-dependent, arterial-type thrombus formation. In these studies, a thrombogenic device consisting of Dacron vascular graft material was inserted as extension segments into a permanent arteriovenous shunt. The results confirmed the potent and long-lasting antithrombotic effect of MA-16N7C2. Surprisingly, the antithrombotic effect was stronger 48 h after a dose of 0.3 mg/kg administration than on the day of treatment with 0.1 mg/kg, despite the fact that comparable numbers of GPIIb/IIIa receptors were occupied on resting platelets. We postulate that with the high dose of MA-16N7C2 and after an extended period, occupied GPIIb/IIIa may be internalised by the platelets. Upon platelet activation, these receptors become reexposed but are unable to participate in thrombus formation. This is in contrast to unoccupied internal GPIIb/IIIa receptors early after a low dose of MA-16N7C2.

Influence of platelet membrane sialic acid and platelet-associated IgG on ageing and sequestration of blood platelets in baboons.

Platelets were isolated from blood of baboons and treated with neuraminidase to remove platelet membrane sialic acid, a process which artificially ages the platelets. The platelets were then labelled with 111In and their mean life span, in vivo distribution and sites of sequestration were measured. The effect of removal of sialic acid on the attachment of immunoglobulin to platelets were investigated and related to the sequestration of the platelets by the spleen, liver, and bone marrow. Removal of sialic acid by neuraminidase did not affect the aggregation of platelets by agonists in vitro, nor their sites of sequestration. The removal of 0.51 (median, range 0.01 to 2.10) nmol sialic acid/10(8) platelets shortened their life span by 75 h (median, range 0 to 132) h (n = 19, p < 0.001), and there was an exponential correlation between the shortening of the mean platelet life span and the amount of sialic acid removed. The increase in platelet-associated IgG was 0.112 (median, range 0.007 to 0.309) fg/platelet (n = 25, p < 0.001) after 0.79 (median, range 0.00 to 6.70) nmol sialic acid/10(8) platelets was removed (p < 0.001). There was an exponential correlation between the shortening of mean platelet life span after the removal of sialic acid and the increase in platelet-associated IgG. The results suggest that platelet membrane sialic acid influences ageing of circulating platelets, and that the loss of sialic acid may have exposed a senescent cell antigen that binds IgG on the platelet membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics and in vivo distribution of in-111-labelled platelets and platelet function in familial hypercholesterolaemia.

The kinetics, in vivo distribution and sites of sequestration of autologous In-111-labelled platelets and other platelet function parameters were studied in ten patients with type IIa or IIb familial hypercholesterolaemia and thrombotic complications of atherosclerosis. The in vitro platelet aggregation response to ADP (P = 0.50) and collagen (P = 0.46); binding of fibrinogen to platelets (P = 0.61); and plasma beta-thromboglobulin levels (P = 0.42) of the patients and normal reference subjects did not differ significantly. The in vivo distribution of In-111-labelled platelets at equilibrium was within normal limits, and at the end of platelet life-span the sequestration pattern of labelled platelets in the reticuloendothelial system was also normal (spleen P = 0.31; liver P = 0.54). There was minimal evidence of in vivo platelet activation: only mean platelet lifespan (MPLS), 195 +/- 57 hours (difference between mean MPLS of patients and controls was 25 hours, with a 95% confidence interval from 23 to 31 hours; P = 0.02); mean platelet platelet turnover, 2298 +/- 824 platelets/microliter/hour (P = 0.005); plasma platelet factor 4 (P = 0.02); and the mean circulating platelet aggregate ratio, 0.8 +/- 0.1 (P = 0.02); differed significantly from normal. These results suggest that abnormalities of platelet function and kinetics observed in type II hyperlipoproteinaemia cannot be ascribed wholly to the hyperlipidaemia, but may be induced by the associated atherosclerosis.

Kinetics, distribution, and sites of destruction of indium-111 oxine labelled red cells in haemolytic anaemia.

The survival of red cells labelled with indium-111 oxine in the circulation was determined. In vivo distribution at equilibrium and sites of deposition at the T50In--that is, the half life of labelled red cells--were quantitated with a scintillation camera and computer assisted image analysis. Although the rate of elution. Of 111In from the red cells was higher than that of chromium-51-disodium chromate, estimates of T50In and T50Cr corresponded reasonably well and were shortened in haemolytic anaemia. In normal subjects red cells were sequestered mainly in the liver and spleen. In five patients with different types of haemolytic anaemia two distinct patterns of red cell sequestration could be recognised: mainly splenic sequestration, and destruction of red cells in the liver, spleen, and the bone marrow. These patterns were expected for the particular disease studied.

Production of a recombinant antithrombotic and fibrinolytic protein, PLATSAK, in Escherichia coli.

The three main components involved in thrombosis and haemostasis are thrombin, platelets, and plasmin. Almost all inhibitors of thrombosis are focused either on the inhibition of thrombin or on the inhibition of platelets. We designed a construct using the fibrinolytic activity of staphylokinase, fused via a cleavable linker to an antithrombotic peptide of 29 amino acids. The peptide was designed to include three inhibitory regions: (1) the Arg-Gly-Asp (RGD) amino acid sequence to prevent fibrinogen binding to platelets; (2) a part of fibrinopeptide A, an inhibitor of thrombin; and (3) the tail of hirudin, a potent direct antithrombin. The amino acid sequence of the 29 amino acid peptide was reverse translated, and the gene was chemically synthesised and cloned into an expression vector as a 3' fusion to the staphylokinase gene. Gene expression was induced in E. coli Top 10 cells and the fusion protein, designated PLATSAK, was purified using metal affinity chromatography. The purified fusion protein significantly lengthened the activated partial thromboplastin time and thrombin time and inhibited the amidolytic activity of thrombin. The fibrinolytic activity was almost equal to that of recombinant staphylokinase as measured with a thrombelastograph. Platelet aggregation was not markedly inhibited by PLATSAK, probably due to the unfavourable three dimensional structure, with the Arg-Gly-Asp sequence buried inside. Our results confirm that it is feasible to design and produce a hybrid multifunctional protein that targets various components of the haemostatic process.

PLATSAK, a potent antithrombotic and fibrinolytic protein, inhibits arterial and venous thrombosis in a baboon model.

Antiplatelet-antithrombin-staphylokinase (PLATSAK) is a chimeric protein that was recombinantly produced in Escherichia coli cells. The protein was designed to target haemostasis at three different levels. It consists of staphylokinase for activation of fibrinolyis, the Arg-Gly-Asp sequence for the prevention of platelet aggregation, and an antithrombotic peptide for the inhibition of thrombin. The in vivo activity of PLATSAK was evaluated by assessing its effect on platelet deposition in a baboon model of arterial and venous thrombosis. Dacron vascular graft segments and expansion chambers, inserted as extensions into permanent femoral arteriovenous shunts, were used to simulate arterial and venous thrombosis, respectively. PLATSAK (3.68 mg/kg) was administered as a bolus 10 minutes before placement of the thrombogenic devices. Platelet deposition onto the graft surface and in the expansion chamber was imaged in real time with a scintillation camera as the deposition of 111In-labeled platelets. After 2 hours, platelet deposition in the graft segments and expansion chambers was inhibited by 50% and 85%, respectively, when compared to control studies. The activated partial thromboplastin time was lengthened to greater than 120 seconds. Interestingly, the level of fibrinogen degradation products in plasma did not increase after administration of PLATSAK. These results demonstrate that PLATSAK effectively inhibited platelet deposition in both arterial- and venous-type thrombosis in an animal model.

A formula for correcting for the in vitro release of platelet beta-thromboglobulin.

The interpretation of platelet beta-thromboglobulin (BTG) and platelet factor 4 (PF4) levels as indicators of in vivo platelet activation is complicated by the artefactual release of these proteins in vitro. A formula was devised to correct for in vitro platelet activation and release of BTG. Blood was collected from normal volunteers by an ideal method and BTG and PF4 levels determined by radioimmunoassay; these were the reference values. Blood from normal volunteers was activated in vitro by standing at room temperature. The BTG and PF4 released was measured at different time intervals. The relationship between BTG and PF4 released was measured at different time intervals. The relationship between BTG and PF4 was measured mathematically best described by a second degree polynomial function. The true plasma BTG value was then calculated by correcting for in vitro release by the general formula: BTG corrected = BTG measured - BtG for PF4 measured + BtG for PF4 reference The validity of the correction formula was tested in 10 normal subjects and in patients with either recent myocardial infarction(n = 10), familial hypercholesterolaemia(n = 10) or arterial prostheses(n = 14). Correction was adequate in normal subjects if the plasma BTG levels did not exceed 260ng/ml. In patients with a thrombotic tendency, the formula overcorrected for in vitro release. This could be ascribed to increased in vivo PF4 levels in these patients, especially those with prostheses. The reference values for PF4 in these patients, and especially those with vascular prostheses, were also higher than normal. The PF4 measured in their plasma thus reflects both in vivo and in vitro released protein. The hypothesis on which the correction formula was based, is therefore not always applicable. It may be possible to improve the correction by establishing formulae for specific disease groups.

Determination of the dosage of recombinant hirudin to inhibit arterial thrombosis in baboons.

Recombinant hirudin, a potent and direct inhibitor of thrombin, effectively inhibits platelet-dependent thrombosis. Our aim was to establish the plasma concentration at which r-hirudin expresses its optimal antithrombotic effect. We measured the extent of inhibition of (111)In-labeled platelet deposition onto 0.6 cm(2) segments of Dacron vascular grafts. These grafts were incorporated as extension segments into exteriorized permanent femoral arteriovenous shunts in baboons. In six control studies a mean of 1.99 +/- 0.26 x 10(9) platelets were deposited at the end of 120 min. In the treatment studies, a thrombus was allowed to form for 10 min in six animals. Treatment for 30 min with r-hirudin at dosages of 140, 70, and 35 microgram/kg/min, but not 14 microgram/kg/min, dose dependently interrupted platelet deposition. The relationship between the percent inhibition of platelet deposition caused by r-hirudin and the plasma concentration of hirudin was exponential (i.e., % Inhibition = 95(1-e(0.23 x [r-hirudin])) (R(2) = 0.76). From this, we estimated that 50% inhibition of platelet deposition will occur at a plasma concentration of approximately 3.3 microgram r-hirudin/mL and 80% at 8.1 microgram/mL. The relationship between the inhibition of platelet deposition and the plasma concentration of hirudin makes it possible to estimate the dose of hirudin that will result in a given level of inhibition of platelet deposition.

Sites of elimination and pharmacokinetics of recombinant [131I]lepirudin in baboons.

Lepirudin has a short half-life, and only 50-60% of the intravenously administered dose is excreted by the kidneys. The fate of the remainder is unknown. We designed a study to determine the fate of this lepirudin. In each of six baboons, [131I]lepirudin was given intravenously as a bolus or infused over 30 min, 24 h apart. The in vivo redistribution of [131I]lepirudin was determined and quantified by scintillation camera imaging. In all studies, the half-life of [131I]lepirudin, as determined from the disappearance of radioactivity, was 21 +/- 3 min. The half-life determined from the disappearance of lepirudin, measured by the Ecarin Clotting Time (ECT) method, was similar at 23 +/- 8 min. Results obtained with the labeled lepirudin are therefore comparable with those obtained using the plasma concentration of lepirudin. When lepirudin was administered as a bolus, the half-life was 18 +/- 4 min, and lepirudin was cleared from the plasma at a rate of 42 +/- 12 mL/min and by the kidneys at 23 +/- 2 mL/min. Following infusion over 30 min, the half-life and total and renal clearances were not significantly different. In both studies, between 50 and 60% of the administered lepirudin was excreted by the kidney. Studies on sacrificed baboons showed that appreciable amounts of lepirudin were present in the bile, indicating the liver as a contributor to the elimination of lepirudin.

The effect of r-hirudin vs. heparin on blood-membrane interactions during hemodialysis.

UNLABELLED: The aim of this study was to determine if recombinant (r-)hirudin, used as anticoagulant during hemodialysis, has favourable effects on blood-membrane interactions. The results were compared with that obtained when standard unfractionated heparin, the anticoagulant of choice, was used. MATERIAL AND METHODS: Eleven patients with chronic renal failure and on maintenance hemodialysis were included in this open cross-over study. Heparin was administered according to the existing protocol in use at the Dialysis Unit during the first dialysis of the study (5,000 to 10,000 IU). r-Hirudin, 0.15 mg/kg, was given as a bolus at the start of the second dialysis, two days later. The effect of the anticoagulant on leukocyte and complement activation, thrombogenesis, release of platelet activating factor and pulmonary gas exchange was studied. RESULTS: In most cases after dialysis with heparin (8 of 11), but not with r-hirudin, macroscopically visible thrombi formed at the inlet of the artificial kidneys. Irrespective of the anticoagulant used, a transient leukopenia (neutropenia) developed ten minutes after dialysis was started. Heparin anticoagulation resulted in a significant increase in plasma levels of complement C3a at all time points, whereas with r-hirudin the increase was significant after only 30 and 240 min. The O2 saturation decreased significantly during the first two hours of dialysis with heparin. The partial O2 pressure decreased significantly during the first two hours of dialysis, irrespective of the anticoagulant used. CONCLUSIONS: We conclude that r-hirudin is superior to heparin with regard to inhibition of thrombus formation in the dialyzer during hemodialysis. Slight, but favourable effects on complement activation. O2 saturation and lung CO diffusing capacity were also found with r-hirudin.

r-Hirudin inhibits platelet-dependent thrombosis during cardiopulmonary bypass in baboons.

BACKGROUND: Systemic anticoagulation is required during cardiopulmonary bypass (CPB) to inhibit the activation of platelets, the coagulation system and ultimately thrombus formation. Unfractionated heparin is most commonly used, but it is neither entirely safe nor completely effective. The use of protamine sulphate to reverse the anticoagulant effect further complicates the use of heparin. The clinical need for a heparin substitute is therefore obvious. We evaluated the efficacy of r-Hirudin, a potent and specific inhibitor of thrombin, as anticoagulant in a baboon model of cardiopulmonary bypass. METHODS: Ten baboons, divided into two groups of five each, were used. The one group received 0.7 mg/kg r-Hirudin as a bolus before CPB was started, followed by a constant infusion of 1.4 mg/kg/hr for the 90 min of CPB. The other group received a bolus of 2.5 mg/kg heparin before the start of CPB, followed by maintenance dosages to maintain the activated clotting time (ACT) >400 sec. RESULTS: Adequate anticoagulation was obtained with both anticoagulants. Haemodilution due to priming the extracorporeal system with Ringer's lactate and appropriately anticoagulated donor blood, was equivalent in both groups. During CPB with heparin, but not with hirudin, there was a significant increase in the number of circulating platelet aggregates, thrombin-antithrombin (TAT) complexes and 111In-labelled platelet accumulation in the oxygenator. After the initial decrease in platelet count due to haemodilution, it further decreased significantly during CPB with heparin but remained relatively constant when r-Hirudin was used. CONCLUSIONS: Our results strongly suggest that r-Hirudin is superior to heparin especially with respect to its inhibitory effect on platelet dependent thrombogenesis caused by the biomembranes of the oxygenator.