H. F. Kotze

Quantification of the distribution of 111In-labelled platelets in organs

A simple and practical approach to the in vivo quantification of 111indium-oxine labelled blood platelets with a scintillation camera and computer assisted imaging system was evaluated. Radioactivity of the 172 and 247 keV energies was measured in a phantom at various source distances from the collimator and the accuracy of anterior and posterior mode measurements compared with that of the geometrical mean (GM) method, with and without correction for Compton scatter (CS). Organ radioactivity, expressed as a percentage of whole body radioactivity, was determined in vivo in five baboons and the accuracy of the methods verified by post mortem quantification in the animals. Measurement in the anterior mode significantly overestimates hepatic and underestimates splenic radioactivity; posterior mode quantification reverses these results. Correction with the GM method made the accuracy and reproducibility very acceptable. Further correction for anterior-posterior attenuation and/or CS did not improve results materially. The GM method could readily be applied in five human subjects. This study shows that the GM method is an accurate and practical method for the in vivo quantification of organ and regional distribution of 111In-labelled platelets.

A computer program in compiled basic for the IBM personal computer to calculate the mean platelet survival time with the multiple-hit and weighted mean methods

We developed an easy-to-operate computer program for the IBM personal computer to calculate, display and store in a database platelet kinetic data determined by analysis of the rate of clearance of radiolabeled blood platelets from the circulation. This was done by curve fitting using the weighted mean method and multiple-hit model. These models are complementary and calculating the mean platelet survival time with both is recommended. Improvement of the weighted mean method was investigated. The optimized weighting and fitting the exponential function with the Marquardt non-linear least squares method improved the weighted mean method. The weighted mean and multiple-hit models fit the survival curve data equally well. The calculation of the mean platelet survival time with the weighted mean method was very fast. The duration of calculation with the multiple-hit model could take up to 2 minutes. Calculation of the mean platelet survival time using both models has the advantage that conditions when calculation of the mean platelet survival time would be invalid, can be detected. The computer program will promote the valid comparison of results obtained at different institutions.

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)

Thromboembolic potential of synthetic vascular grafts in baboons

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)

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.

Kinetics of indium- 111-labelled platelets in HIV-infected patients with and without associated thrombocytopaenia

Abstract:  Seven to 12% of HIV‐infected patients have thrombocytopaenia. The pathophysiology of the thrombocytopaenia is not clear. It has been variously suggested that it may be caused by an increased peripheral platelet destruction, a defect in platelet production, or by a combination of these. The aim of the study was to elucidate the pathogenesis of HIV‐associated thrombocytopaenia. We determined the mean platelet life span (MPLS) and calculated the turnover of autologous indium‐111‐labelled platelets in 17 HIV‐positive patients, seven with thrombocytopaenia. The sites of sequestration of labelled platelets were quantified. The thrombocytopaenic patients had a very short MPLS (3.0±3.8 h) and a marked increase in platelet production (18.2±12.6× 109/l/h). The majority of these patients (5 of 7) had excessive sequestration of platelets in the spleen. Five of the patients with a normal blood platelet count had a shortened MPLS (109±23 h) and increased platelet turnover (3.8±1.2×109/l/h), i.e. the increased peripheral platelet destruction was compensated for by increased platelet production. The other five patients with a normal platelet count had normal MPLS (195±11 h) and slightly increased platelet production (2.5±0.6× 109/l/h). We conclude that patients with HIV‐associated thrombocytopaenia have increased peripheral platelet destruction. Platelet production is elevated but is insufficient to maintain a normal peripheral platelet count. In these patients platelets are predominantly sequestrated in the spleen. Patients with HIV infection and a normal blood platelet count may also have increased platelet production. This may be an early subclinical phase in the development of full‐blown HIV‐associated thrombocytopaenia.

The influence of the ‘collection injury’ on the survival and distribution of Indium- 111 -labelled canine platelets

Summary. The extent of the ‘collection injury’ sustained by platelets during labelling with In‐lll‐oxine was investigated in three matched pairs of beagle dogs. The influence of the procedure on the survival, kinetics, in vivo distribution and fate of the labelled platelets was determined by serial blood sampling and quantitative computerized scintillation camera studies. Injured labelled platelets were removed in the matched dog acting, as a biological filter. The survival, distribution and fate of the ‘filtered’ and ‘unfiltered’ platelets were compared. The mean platelet lifespan of the ‘filtered’ and ‘unfiltered’ platelets did not differ significantly, but the shape of the survival curve of the filtered platelets fitted a linear function more closely than that of the unfiltered platelets. Radioactivity in the different organs and regions was serially quantitated and expressed as a percentage of whole body radioactivity. Splenic and hepatic radioactivity of filtered and unfiltered platelets did not differ significantly at equilibrium or at the end of platelet lifespan. It is concluded that the currently employed isolation and labelling techniques for platelets are suitable for quantitative in vivo studies with a computerized scintillation camera system. The shape of platelet survival curves should, however, be interpreted with some caution as it may be influenced by these procedures.

In vitro effect of a thrombin inhibition peptide selected by phage display technology

A repeated selection of phages from a cyclic heptapeptide phage display library resulted in the enrichment of phages that bind to human alpha-thrombin. One clone of the binding phages that competed with PPACK for binding to thrombin and that had the best binding characteristics was chosen. The amino acid sequence of the peptide displayed on this phage was determined and a peptide with the sequence, Cys-Asn-Arg-Pro-Phe-Ile-Pro-Thr-Cys was synthesised. This peptide, thrombin-inhibiting peptide (TIP), is a full competitive inhibitor of thrombin with an inhibition constant (K(i)) of 0.4974 mM. It lengthened the thrombin time and inhibited thrombin-induced platelet activation and the platelet release reaction, both in a dose-dependent manner. It also reduced platelet adhesion onto a human microvascular endothelial matrix in the parallel plate flow chamber under both arterial and venous shear conditions. Thus, we have selected and synthesised a cyclic heptapeptide that competes with PPACK to bind to thrombin and that can be developed as a direct antithrombin.

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 plateles 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. The relatively high accumulation of activity in the spleen was as a result of the slow transit time of the functional platelets through the spleen of 5.1 (3.5–6.0) min compared with the transit time through the liver of 0.33 (0.19–0.50) min. The 9% (5%–12%) collection-injured platelets in the spleen and 10% (5%–16%) in the liver had longer transit times than functional platelets. Platelet sequestration was well simulated with the compartmental model. The mean platelet survival time estimated by the compartmental model and standard curve fitting techniques did not differ significantly. A multicompartmental model and reference range for platelet kinetics have been established and may prove to be clinically useful in platelet disease.

The effect of blood ozonation on mitochondrial function and apoptosis of peripheral blood mononuclear cells in the presence and absence of plasma antioxidants

Ozone-autohemotherapy (O3-AHT) has recently gained interest as a form of alternative and complementary medicine. There is, however, some concern regarding its toxicity and effectiveness. Ozone is a powerful oxidant and when introduced into biological fluids react with most cellular components including proteins, lipids and DNA. We assessed the effect of O3-AHT on peripheral blood mononuclear cells (PBMC) viability, apoptosis and mitochondrial function in the presence and absence of plasma antioxidants. Exposure to ozone increased lactate dehydrogenase (LDH) release and caspase 3/7 activity in PBMC. A decrease in mitochondrial function was measured as a decrease in ATP levels and an increase in NADH/ NAD+ ratio. Complex IV (cytochrome c oxidase) of the respiratory chain was almost completely inhibited by ozone. These results indicated that the death of PBMC was probably through apoptosis. These effects were more evident in the absence of plasma antioxidants. Therefore, high concentrations of ozone were damaging to the cells, but this effect was diminished by antioxidants present in plasma. It is not certain if the in vitro damage will be propagated when ozonated blood is injected back into individuals. One must bear in mind that only a fraction of the total blood volume is ozonated.