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.

Antithrombotic effect of platelet glycoprotein Ib-blocking monoclonal antibody Fab fragments in nonhuman primates.

Platelet adhesion in arterial blood flow is mainly supported by the platelet receptor glycoprotein (GP) Ib, which interacts with von Willebrand factor (vWF) that is bound to collagen at the site of vessel wall injury. Antibody 6B4 is a monoclonal antibody (MoAb) raised against purified human GPIb. MoAb 6B4 inhibits both ristocetin- and botrocetin-induced, vWF-dependent human platelet agglutination. MoAb 6B4 furthermore blocks shear-induced adhesion of human platelets to collagen I. We studied the antithrombotic effect of this inhibitory murine MoAb 6B4 in a baboon model of arterial thrombosis. When injected into baboons, intact IgG and its F(ab')(2) fragments caused almost immediate thrombocytopenia, whereas injection of the Fab fragments alone did not. Fab fragments were subsequently used to investigate their in vivo effect on platelet deposition onto a thrombogenic device, consisting of collagen-rich, glutaraldehyde-fixed bovine pericardium (0.6 cm(2)), at a wall shear rate ranging from 700 to 1000 s(-1). Baboons were either pretreated with Fabs to study the effect of inhibition on platelet adhesion or treated 6 minutes after placement of the thrombogenic device to investigate the effect on interplatelet cohesion. Pretreatment of the animals with bolus doses ranging from 80 to 640 microgram/kg Fab fragments significantly reduced (111)In-labeled platelet deposition onto the collagen surface by approximately 43% to 65%. Only the highest dose caused a significant prolongation (doubling) of the bleeding time. Ex vivo ristocetin-induced platelet agglutination was equally reduced. Treatment with a bolus of 110 microgram/kg Fab fragments after a thrombus was allowed to form for 6 minutes had no effect on further platelet deposition. We therefore conclude that Fab fragments or derivatives of inhibitory anti-GPIb antibodies may be useful compounds to prevent thrombosis.

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.

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.

Antithrombotic activity of Bay u3405, a thromboxane A2-antagonist, in patients with Dacron aortic grafts: a random controlled clinical trial.

Twelve patients with Dacron aortic grafts participated in a placebo controlled, crossover trial to investigate the effect of Bay u3405, a thromboxane A2 receptor antagonist, on graft thrombogenicity. During each treatment period (seven days, Bay u3405 or placebo), 111In-platelet survival and platelet deposition on the grafts were measured daily by gamma-camera imaging and blood radioactivity analysis. Bay u3405 substantially reduced the deposition of platelets and the thrombogenic index, while platelet survival remained unchanged. The ex vivo platelet aggregation response to ADP and epinephrine was significantly inhibited. The bleeding time increased slightly but not to any clinically relevant extent, and no adverse side effects were recorded. Bay u3405 seems to be a safe and effective drug for the inhibition of platelet deposition on aortic Dacron grafts. The use of quantitative imaging techniques is also more sensitive than the measurement of platelet survival for the assessment of antiplatelet drug efficacy in patients with aortic grafts.

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)

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)

The influence of platelet-graft interaction on platelet survival in patients with aortobifemoral Dacron grafts.

In patients with arterial grafts, platelet consumption may be due either to platelet interaction with the graft, and/or concomitant platelet consumption in the rest of the arterial tree. This hypothesis was tested by quantifying the kinetics and platelet-graft interaction of indium-111-labelled platelets with double velour Dacron grafts in 13 patients with arterial insufficiency ascribed to atherosclerosis. Mean platelet lifespan (MPLS), 149 +/- 46 hours, was significantly shorter (P = 0.001) than normal. Labelled platelets were transiently deposited onto the graft surfaces. Peak 111In deposition on the grafts, 1.33 +/- 1.02% of injected labelled platelets, was reached at 70 +/- 33 hours. Thereafter the graft-platelet radioactivity decreased in parallel with platelet radioactivity in the circulation. There was no statistical correlation between MPLS and the factors known to be associated with graft platelet deposition: graft size; peak graft radioactivity; and the time to attain peak graft radioactivity. It is therefore concluded that in patients with arterial disease requiring graft implantation, the observed increased platelet consumption cannot only be ascribed to the interaction of platelets with the graft. Concomitant atherosclerosis is probably the important modifier of platelet consumption. The significant contribution of this factor to the shortening of the MPLS should be taken into account when assessing, by measuring platelet lifespan, the thrombogenicity of grafts.

Blood platelet kinetics in normal subjects modelled by compartmental analysis.

The purpose of this study was to describe the function of platelets throughout their life span by expressing their in vivo distribution and kinetic behaviour in mathematical terms by using multicompartmental analysis. The distribution of indium-111 labelled platelets in five normal subjects was imaged and quantified with a scintillation camera image processing system. Serial blood samples were also obtained. The data were modelled using the SAAM (Simulation Analysis and Modelling) compartmental computer program. Five models were entertained to evaluate the role of platelets that were either functional or injured during collection and their interaction with the liver, spleen and vascular endothelium. Models were evaluated by comparing F values calculated from the least squares estimate obtained from each model. The Dornhorst function was used to describe the sequestration of platelets in the compartmental model. Results indicated that the data could not be satisfactorily simulated when compartments were included that simulated only functional and sequestered platelets (model 1). It was necessary to include compartments that simulated the kinetics of collection-injured platelets in the liver (model 2) and spleen (model 3). The model that simulated the interaction with the vascular endothelium (model 5) showed a visual but not significant improvement in the fitting of the observed data compared to model 3. The mean organ uptake and range indicated in parentheses were calculated at equilibrium. There were 20% (15%-27%) of the injected platelets in the spleen, 10% (8%-11%) in the liver and 70% (64%-75%) in the circulation.(ABSTRACT TRUNCATED AT 250 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.

Radionuclide cholescintigraphic imaging: an evaluation of several 99mTc labelled hepatobiliary radiopharmaceuticals.

Recently, much interest has been shown in the development of 99mTc labelled cholescintigraphic agents for imaging the hepatobiliary tract. In this study six cholescintigraphic agents were compared in rabbits with respect to transit efficiency through the liver and the halftime on the washout portion of the liver time-activity curve. The agents compared were p-butyl-IDA (PBIDA), diisopropyl-IDA (DISIDA), two mebrofenin (MBF) agents and two pyridoxylaminates (PDA). Best transit efficiencies were obtained with MBF (34.1 and 31.2%) followed by PDA (27.7 and 24.9%) while DISIDA (23%) and PBIDA (19.3%) were the lowest. The same phenomenon was observed regarding the washout halftime, with MBF the most rapid (6.3 and 5.9 min), PDA more prolonged (10.1 and 12.0 min) and DISIDA and PBIDA the slowest (23.0 and 23.2 min). This study confirms the difference in physiological behaviour of the various cholescintigraphic agents and shows identical flow patterns for locally produced and imported compounds.

Evaluation of (o)-[77Br]bromohippuran as renal tubular function agent.

(o)-[77Br]bromohippuran (BHIP) was developed as renal tubular function agent due to its favourable chemical and physical properties and compared to (o)-[131I]iodohippuran (IHIP). Renograms obtained from baboons were compared and absorbed radiation dose calculations performed. Although BHIP showed a delayed kidney uptake and washout pattern, good kidney clearance of the radionuclide was obtained after 30 min. Radiation dose values for BHIP were markedly lower than for IHIP indicating that larger activities of BHIP could be administered to increase counting statistics. BHIP imaging in normal volunteers did however not substantiate the favourable behaviour obtained in the primate.