Intrahepatic kinetics of indium-111-labelled platelets

The intrahepatic kinetics of 111indium-labelled platelets have been studied using dynamic gamma camera scintigraphy immediately following injection. Platelets labelled in saline with 111In-oxine or 111In-acetylacetonate underwent rapidly reversible hepatic sequestration, indicating that they were "activated". Platelets labelled in plasma with 111In-tropolonate, however, did not display this phenomenon. On the assumption that plasma-labelled platelets display a normal initial bio-distribution, mean intrahepatic platelet transit time, as a factor of the transit time of 99m-Tc labelled red cells, was 1.45 +/- SE 0.12 (n = 6), implying the normal presence of a small intrahepatic platelet pool. Unlike the liver, transit through the spleen was not sensitive to the labelling medium; thus the mean intrasplenic transit time of plasma-labelled platelets was 9.3 +/- SE 0.7 min (n = 10), and of saline-labelled platelets 9.5 +/- SE 0.3 min (n = 8).     

Indium-111 labeled platelets: Studies on preparation and evaluation of in vitro and in vivo functions

Platelets have been labeled with a lipid soluble complex of the radionuclide indium-111. The complex is formed with 8-hydroxyquinoline and extracted in chloroform which is then evaporated to dryness. The residue is dissolved in 50 μl of ethanol and diluted to 200 μl with normal saline. Platelets are separated by differential centrifugation, washed and suspended either in Tyrodes-albumin solution or in normal saline. The solution of ¹¹¹In-complex is added to separated platelets and more than 95% of the radioactivity is incorporated with the platelets. The function of the labeled platelets has been studied by their aggregability using adenosine diphosphate and collagen as the stimulating agents. No adverse effects have been observed. The survival of the indium-labeled platelets was similar to that observed with chromium-51 labeled platelets. Labeled canine platelets have been administered to dogs in whom venous thrombi had been induced by alteration of the intima by an electric current. The thrombi were detected by imaging 50 minutes after administration of the labeled platelets. Twenty-four hours later the thrombi were removed and had 20–50 times the radioactivity of an equal weight of blood. Accumulation of large amounts of radioactivity in surgical wounds has also been observed. Damage of the intima of carotid arteries in animals by a balloon catheter demonstrated 8 to 20 times more activity in the damaged artery than in the normal artery. Results indicate that In-111 labeled platelets have potential both for platelet survival studies and for evaluation of vascular damage.     

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.     

Indium-111-labeled platelet scintigraphy in carotid atherosclerosis

We evaluated platelet accumulation in carotid arteries by means of a dual-radiotracer method, using indium-111-labeled platelets and technetium-99m-labeled human serum albumin, in 123 patients (92 men, 31 women; median age 60 years). Sixty patients had symptoms of transient ischemic carotid artery disease, and 63 patients with peripheral arterial occlusive disease served as controls. Antiplatelet treatment with acetylsalicylic acid was taken by 53 of the 123 patients. In 36 of the 60 symptomatic patients, platelet scintigraphy was repeated 3-4 days after carotid endarterectomy. Comparison of different scintigraphic parameters (platelet accumulation index and percent of the injected dose of labeled platelets at the carotid bifurcation) showed no significant differences between symptomatic and asymptomatic patients, and the severity of stenosis and the presence of plaque ulceration also had no influence on the parameters. There was no difference between patients with a short (less than 4 weeks) or long (greater than 4 weeks) interval from the last transient ischemic attack to scintigraphy and no difference between patients with or without antiplatelet treatment. Classifying the patients according to plaque morphology judged by high-resolution real-time ultrasonography also demonstrated no differences. No significant correlation was found between any scintigraphic parameter and other platelet function parameters such as platelet survival time, platelet turnover rate, and concentration of platelet-specific proteins. Quantification of platelet deposition after carotid endarterectomy in 36 patients demonstrated a significant increase of the median platelet accumulation index and the percent injected dose index. There were no significant differences between patients receiving high-dose (1.0 g/day) or low-dose (1.0 g/day) acetylsalicylic acid in scintigraphic results.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characteristics of thrombin-degranulated human platelets: development of a method that does not use proteolytic enzymes for deaggregation

A method has been developed for preparing suspensions of washed human platelets that have lost as much as 90% of their dense granule and alpha granule contents as a result of stimulation by thrombin (0.9 U/ml for 3 min at 37 degrees C), and recovering the platelets without using a proteolytic enzyme. Glycyl-L-prolyl-L-arginyl-L-proline (GPRP) was used to prevent polymerization of released fibrinogen and arginyl-glycyl-aspartyl-serine (RGDS) to block the interaction of released fibrinogen, vWf or fibronectin with the glycoprotein IIb/IIIa complex. The thrombin used to degranulate the platelets was neutralized with D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (FPRCH2Cl) and prostaglandin E1 was added to return the platelets towards a disc shape. The degranulated platelets aggregated in response to ADP, platelet activating factor, arachidonate and the thromboxane A2 mimetic, U46619 in the presence of added fibrinogen; the platelets changed shape but did not aggregate in response to collagen. Thrombin and the calcium ionophore, A23187, caused aggregation without added fibrinogen. Synergism between pairs of aggregating agents at low concentrations was observed. Little TXB2 was formed when the platelets were reaggregated by thrombin. RGDS and F(ab')2 fragments of an antibody to fibrinogen inhibited reaggregation induced by thrombin and A23187 indicating that small amounts of fibrinogen at the platelet surface may support aggregation by strong agonists. Adherence of thrombin-degranulated platelets to a collagen-coated surface was less than for controls, but spreading was more extensive. Electron-microscopic immunogold cytochemistry with anti-human fibrinogen IgG showed numerous gold particles in platelet vacuoles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Improvement of the periodate-borohydride surface-labeling method for human blood platelets

The periodate/sodium boro[3H]hydride ([3H]-NaBH4) method is extensively used for the specific labeling of cell surface glycoproteins. Reduction with tritiated borohydride is also used in other surface-labeling techniques, the neuraminidase/galactose oxidase/[3H]-NaBH4 method (specific for terminal galactose and N-acetyl-galactosamine residues) and the pyridoxal phosphate/[3H]-NaBH4 method (specific for protein). By modification of the reaction conditions during the periodate-oxidation and borohydride-reduction, the ratio of the incorporated to the total added radioactivity could be increased by a factor of 50, while the specific activity of the labeled material was twice as high as in the original method. Alternatively, by another modification, the specific activity of the labeled material could be increased about 10-fold. The influence of the most important parameters was investigated in detail. Sodium dodecyl sulfate gel electrophoresis and fluorography demonstrate that the labeling pattern of the membrane glycoproteins is the same as with the conventional method.     

Chloroquine: a multipotent inhibitor of human platelets in vitro

Chloroquine inhibited human platelet aggregation in vitro both at receptor- and nonreceptor-operated stimuli. The inhibition was dose-dependent, recorded on isolated platelets as well as in platelet-rich plasma, and followed the rank order of stimuli: adrenaline (second phase)>phorbol 12-myristate 13 acetate>adenosine diphosphate>adrenaline (first phase)>thrombin>calcium ionophore A23187. In thrombin-activated platelets, chloroquine decreased in a dose-dependent manner phospholipase A(2)-induced arachidonic acid liberation from membrane phospholipids, malondialdehyde formation (a marker of membrane phospholipid peroxidation), and thromboxane generation, considered the most potent autoaggregatory agent. Chloroquine only slightly altered the arachidonic acid cascade of platelets stimulated with A23187 and phorbol 12-myristate 13 acetate. Histamine formation and liberation induced with thrombin and A23187 were not affected by chloroquine. On the other hand, thrombin-stimulated serotonin secretion was significantly decreased with chloroquine in the concentration of 10 micromol/L. This indicated that chloroquine might interfere with stimulated secretion from platelets. The results suggest that chloroquine inhibited activated platelets: first, intracellularly; second, in a close relationship to the intraplatelet Ca(2+) mobile pool; and third, most probably at the site of platelet phospholipase A(2) activation.     

Sheep platelets as a model for human platelets: evidence for specific PAF (platelet activating factor) receptors

In a number of animals, the platelet response to Platelet Activating Factor (PAF) has been shown to differ considerably from that in humans. However, aggregation, release and particularly shape change were quite similar for human and sheep platelets. In this study, aggregation and shape change analysis were used to measure the response of sheep platelets to various synthetic analogs of PAF. Response is greatly reduced with no alkyl group in position 2 of PAF and decreases progressively as the number of carbons of the alkyl gets larger than three. A reduction of activity is also seen as the ether linkage at position 1 of PAF is replaced by an ester linkage. These changes are indicative of a specific membrane receptor for PAF in sheep platelets and confirm the usefulness of sheep platelets as a model for PAF-platelet interaction in humans.     

Immunological evidence for prothrombin in human platelets

An attempt was made to isolate from plasma the platelet surface substrate for thrombin, glycoprotein V (GPV), because a GPV antigen was reported to be present in plasma. Plasma fractionation based on procedures for purification of GPV from platelets revealed a thrombin-sensitive protein with appropriate electrophoretic mobility. The protein was purified; an antiserum against it reacted with detergent-solubilized platelet proteins or secreted proteins in a double diffusion assay, adsorbed a protein from the supernatant solution of activated platelets, and inhibited thrombin-induced platelet activation, but the antiserum did not adsorb labeled GPV. The purified protein was immunochemically related to prothrombin rather than to GPV. Other antibodies against prothrombin were also able to adsorb a protein from platelets. It is concluded that plasma does not contain appreciable amounts of GPV, and platelets contain prothrombin or an immunochemically similar protein.     

Autoradiographic detections of 111indium-labeled platelets in brain tissue sections

Various techniques for detection of blood platelets in tissue section were evaluated in an incremental air embolism model of ischemia in order to further investigate the accumulation of platelets in brains subjected to cell-damaging ischemia. The experimental animals were dogs anesthetized with alpha-chloralose. A new autoradiographic technique was devised that allows precise localization of 111Indium-labeled platelets in brain sections. The technique is described in detail along with some effects of the label on platelet function and behavior. The technique can be performed in conjunction with 14C-iodoantipyrine autoradiographic measurement of blood flow without causing mutual interference or other forms of interaction. This permits the simultaneous investigation of local blood flow and deposition of platelets.     

3H-imipramine binding in human platelets: a study in normal twins

3H-imipramine binding was determined in freshly prepared intact platelets from 17 monozygotic (MZ) and 15 dizygotic (DZ) twin pairs, all of them male, adult, drug-free, and healthy volunteers. Sixteen males served as controls for determination of intraindividual variation of binding parameters. Both MZ and DZ twin pairs exhibited high intraclass correlations of Bmax values, but DZ twins were nearly as similar as MZ pairs. Interindividual variation of binding parameters is not large enough to reveal a significant genetic control.     

The megakaryocyte in thrombocytopenia: a microscopic study which supports the theory that platelets are produced in the pulmonary circulation

Thrombocytopenia can increase platelet production and thereby facilitate investigations into the mechanism. Megakaryocytes in the bone marrow, lung, spleen and liver have been studied by light and transmission electron microscopy after 6 days of experimentally induced thrombocytopenia in the rabbit. The findings support the theory that platelets are produced in the pulmonary circulation by the physical fragmentation of megakaryocyte cytoplasm.     

Successful transsphenoidal removal of sphenoidal sinus tumor in a patient with severe thrombocytopenia

The paper describes a clinical case of successful transsphenoidal removal of sphenoidal sinus tumor in a patient with severe thrombocytopenia. It also discusses the problems of infusion-transfusion therapy policy in such patients during surgery.     

Leucopenia and thrombocytopenia possibly associated with lamotrigine use in a patient.

Haematological side effects are rather exceptional with lamotrigine. We report the case of a 25-year-old woman with epilepsy who developed combined leucopenia and thrombocytopenia eight weeks after starting lamotrigine. Within weeks after lamotrigine was discontinued, all of the haematopoietic abnormalities had disappeared. To our knowledge, this is the first report of combined leucopenia and thrombocytopenia associated with lamotrigine treatment suggesting, in our patient, a causal reaction.     

A method for the isolation of α-granules from human platelets

A method for the isolation of α-granules is described. A two-step French pressure cell homogenization procedure which directly produced an organelle concentrate suited for loading on density gradients was developed. The procedure was optimalized with respect to recovery of intact α-granules. The organelle homogenate was loaded to 17.5 – 27.5% metrizamide gradients and centrifuged. Organelle aggregate formation was minimized by controlling the ionic conditions and the shape of the gradient. The α-granules were separated from lysosomes and dense bodies, but overlapped with the mitochondria. The α-granules were recovered from the gradient in order to omit the major amount of mitochondria from the final preparation. A washing procedure which introduced a minimum of α-granule adhesivity and fragility is presented.