Search

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.

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.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.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.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.

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% (p0.05) 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.

Intravenous recombinant (r)-hirudin has a potent antithrombotic effect in aspirin- and heparin-resistant platelet-dependent thrombus formation in baboon models. However, these thrombi reform when therapy is stopped after 60 minutes. To determine if 4 hours of therapy can produce a lasting antithrombotic effect, we investigated the extent of deposition of 111In-labeled platelets onto 0.5-cm2 segments of Dacron vascular grafts for 53 hours. These grafts had been incorporated into exteriorized permanent femoral arteriovenous shunts in baboons. Platelet deposition in eight untreated animals was generally sigmoidal. Maximum platelet deposition, 1.7% +/- 0.9% of injected labeled platelets, was reached after approximately 4 hours. Deposition then gradually decreased to 0.4% +/- 0.2% of injected labeled platelets after 53 hours. After a thrombus was allowed to form for 15 minutes in six animals, intravenous treatment with r-hirudin at a dose of 20 nmol (0.14 mg)/kg-min-1 (aPTT300 seconds) was started and maintained for 4 hours. Platelet deposition was interrupted during treatment. After infusion was stopped, platelets accumulated again, but not as much as in the untreated animals. Maximum platelet deposition, 0.7% +/- 0.2% of injected labeled platelets, was significantly less (P.01), and was reached after approximately 23 hours. Thereafter, deposition decreased to 0.4% +/- 0.2% at 53 hours. The shunts in all of the untreated animals occluded at some stage during the study, while only one shunt occluded in the treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)

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, p0.001), 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, p0.001) after 0.79 (median, range 0.00 to 6.70) nmol sialic acid/10(8) platelets was removed (p0.001). 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)

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)

The relative sensitivities for the various in vivo and in vitro tests for platelet activation are unknown. This was studied in a baboon model where limited and more substantial injury to the vascular endothelium was inflicted. The endothelium of a segment of the right carotid artery was removed with a balloon catheter on day 0 (limited de-endothelialisation), and that of the left carotid artery, abdominal aorta and left femoral artery on day 7 (substantial de-endothelialisation). Eight baboons (Papio ursinus) were used. Baseline tests for platelet activation (platelet volume, platelet density, platelet aggregate ratio, and platelet and plasma levels of platelet factor 4 [PF4] and beta-thromboglobulin [beta-TG]) were performed 7 days before de-endothelialisation and repeated on days 1, 9 and 16. The kinetics of indium-111-labelled platelets were measured after substantial de-endothelialisation. Sham operations were done on 3 animals exactly as in the test, except that the balloon injuries were not inflicted. No influence on the results of the platelet function tests was found. The only test capable of detecting limited injury to the endothelium was the measurement of plasma PF4. The mean platelet life-span (MPLS) shortened, mean platelet density decreased, the circulating platelet aggregate ratio decreased, and plasma levels of PF4 and beta-TG increased (P less than 0.05 in all instances) after the substantial endothelial injury. The mean platelet volume, intraplatelet PF4 and beta-TG, and the in vivo distribution and sites of sequestration of labelled platelets were poor tests for in vivo platelet activation.(ABSTRACT TRUNCATED AT 250 WORDS)

We have compared in baboons the capacity of two types of synthetic vascular grafts to accumulate thrombus, activate circulating platelets, and generate occlusive platelet microemboli. Grafts were incorporated into femoral arterial-arterial shunts placed unilaterally in 10 baboons; the unoperated contralateral limbs served as controls. The accumulation of indium 111 (111In)-labeled platelets onto the grafts (expanded polytetrafluoroethylene [ePTFE] or knitted Dacron, 4 mm inner diameter) and the appearance of 111In radioactivity in distal microcirculatory beds (calf and foot) were quantified by dynamic scintillation camera imaging. After 1 hour total platelet deposition per graft was higher with Dacron (49.0 +/- 8.0 x 10(9) platelets) than with ePTFE (3.7 +/- 0.6 x 10(9) platelets, p less than 0.01). Platelet counts decreased and beta-thromboglobulin levels increased with Dacron graft placement but were unaffected by ePTFE graft placement (p less than 0.05 and p less than 0.01, respectively). Emboli shed from Dacron grafts were detected as multifocal, irregular, and changing deposits in the calves and feet. Indium 111 platelet activity in the feet distal to the Dacron grafts increased 81.1% +/- 21.4% from baseline values over 1 hour, whereas the activities in the feet distal to the ePTFE grafts were unchanged (p less than 0.05). The increase 111In-platelet radioactivity above the control limb values (excess radioactivity) was higher for the Dacron graft group than for the ePTFE group in both the feet (139.6% +/- 46.9% vs 6.2%, p less than 0.05) and the calves (86.7% +/- 21.7% vs 7.3% +/- 3.6%, p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.