Inhibition of Thrombin-Induced Aggregation of Human Platelets by Heparin and Antithrombin III

Heparin aggregated washed platelets in the presence of divalent cations. The aggregation was not significantly influenced by antithrombin III, albumin, or by the presence of serum. The significance of this finding is discussed. Heparin, together with antithrombin III, inhibited the release of platelet adenine nucleotides and also the platelet aggregation induced by thrombin. The inhibition was rapid and progressive. Much higher thrombin concentrations were inhibited than with antithrombin III alone. The inhibition was more pronounced at 37° C than at 19° C, and could not be reversed by addition of polybrene.     

Biochemical Characterisation of A Blood Group Activity on Human Platelets

A butanol extraction procedure has been used to isolate the blood group A antigen determinants from platelet membranes and to determine their biochemical structure-glycolipid and/or glycoprotein. The blood group A serological activity was found to reside entirely in the butanol (organic) fraction. When this fraction was analyzed by thin-layer chromatography, the A-inhibitory activity was found to comigrate with extracts of erythrocyte type Aa, Ab and Ac variant structures. The results of this study indicate that the ABO blood group A determinants on human platelets are glycolipid, similar in structure to glycolipid A determinants on erythrocytes.     

Fine Structural Localization of Adenosine Triphosphatase in Human Platelets and Other Blood Cells

Adenosine triphosphatase activity has been localized in human blood cellsby combined histochemical and electron microscopic technics. Deposits oflead phosphate, indicative of enzymic activity, were observed on the surfacemembranes of erythrocytes, lymphocytes and platelets. Intracellular ATPaseactivity was found in lymphocyte mitochondria and nucleoli. Blood plateletscontained organelles in their hyaloplasm with dense accumulations of theenzyme reaction product. The distribution of lead phosphate in platelet organelles suggested that they were mitochondria, but some platelet granules mayalso contain ATPase activity.

Submitted on October 26, 1964 Accepted on March 2, 1965     

Relation between the Inhibition of Aggregation and the Concentration of cAMP in Human and Rat Platelets

Adenosine inhibits the aggregation of human but not of rat platelets whereas both are inhibited by prostaglandin E1 or by the pyrimido-pyrimidine compound RA233. In human platelets all three agents increase adenosine-3'-5'-cyclic monophosphate (cAMP). If the inhibition of aggregation depended on this increase, adenosine might be expected not to increase cAMP in rat platelets. Under conditions in which adenosine inhibited aggregation and increased cAMP in human platelets, adenosine caused a similar increase in cAMP in rat platelets without inhibiting their aggregation. The aggregation of rat platelets was inhibited as effectively as that of human platelets by PGE1 or RA233 at concentrations which caused greater increases in cAMP than did the highest concentrations (2.8 X 10(-4) M) of adenosine it was possible to use. When the increase of cAMP in rat platelets by PGE1 was limited to that produced by adenosine, PGE1 like adenosine failed to inhibit aggregation. Therefore, the difference in the inhibitory effectiveness of adenosine on rat and human platelets was quantitative rather than qualitative and apparently depended on the inability of adenosine to increase cAMP sufficiently in rat platelets. When cAMP had been increased by adenosine, PGE1 or RA233, the addition of ADP caused cAMP to decrease rapidly in both human and rat platelets to between +22 and -18% of control values, except that the decrease in rat platelets was to +40% after RA233 had been present for 0.5 min before ADP. The increase in cAMP produced in rat platelets by adenosine at 5 X 10(-6) to 2.8 X 10(-4) M for 3 min was associated with a small increase in aggregation velocity. It is suggested that the comparative ineffectiveness of adenosine as an inhibitor of platelet aggregation, particularly with rat but less so also with human platelets, is because, unlike PGE1 or RA233, adenosine has two opposing actions on aggregation; one being inhibition by activating adenylate cyclase and increasing cAMP, and the other being potentiation by uptake. This hypothesis accounts for the present results as well as for the earlier observation that dipyridamole which prevents the uptake of adenosine potentiates its inhibitory effect on the aggregation of human platelets.     

The Effect of Phospholipase C on Rat Blood Platelets in Vivo

Rat blood platelets were treated with phospholipase C in vitro or phospholipase C was injected i.v. to rats. In both cases its effect on the functions of the platelets in vivo has been studied. No change was found in primary bleeding time or in platelet survival. Treatment with phospholipase C gave a moderate reduction of ADP-induced platelet aggregation in the pulmonary circulation whereas the aggregation induced by thrombin was unchanged. I.v. injection of phospholipase C caused a rapid, very moderate and transient increase of ⁵¹Cr-activity in the lungs without concomitant overt respiratory distress. A moderate increase in ⁵¹Cr-activity was noted in liver and kidney 24 and 48 h after injection of phospholipase C. This may be caused by a slightly increased leakage of ⁵¹Cr-labelled material from the platelets during exposure to phospholipase C.     

Competitive Inhibition of Beef Heart Cyclic AMP Phosphodiesterase by Cytokinins and Related Compounds

Two cytokinins and four related analogs, none of which is a cyclic ribonucleotide, have been shown to act as competitive inhibitors of the high K(m) cyclic-AMP phosphodiesterase (3':5'-cyclic-AMP 5'-nucleotidohydrolase, EC 3.1.4.17) activity from beef heart. Weak inhibition of the low K(m) cyclic AMP phosphodiesterase activity was also observed, suggesting a possible mechanism for regulation of intracellular cyclic AMP levels by the exogenously added compounds. In addition to the kinetic data, obtained on the six inhibitors in four different heterocyclic series, 15 other cytokinins and related compounds have been shown to inhibit the high K(m) cyclic AMP phosphodiesterase activity at single concentrations of substrate and inhibitor. Heterocycles such as adenosine and 7-amino-3-methylpyrazolo[4,3-d]pyrimidine, which lack the N-substituent, were inactive as cyclic AMP phosphodiesterase inhibitors. The observed inhibition of cyclic AMP phophodiesterase supports prior observations which implicate exogenously added cytokinins in cyclic AMP metabolism.     

The Effect of Synthetic Phosphatidyl Serines on Platelet Aggregation, Blood Coagulation and Haemostasis

The effects of synthetic phosphatidyl serines on blood coagulation, platelet aggregation, the formation of haemostatic plugs, and deposit formation in extracorporeal shunts were examined. In vitro , phosphatidyl serines with C₁₆ saturated fatty acids inhibited blood coagulation but did not inhibit platelet aggregation induced by collagen, thrombin or antigen-antibody complexes. C₁₀ phosphatidyl serine inhibited platelet aggregation induced by these stimuli but did not inhibit blood coagulation. This effect of C₁₀ phosphatidyl serine appeared to be due to inhibition of the release of platelet constituents. C₁₂ and C₁₄ phosphatidyl serines had intermediate effects; phosphatidyl serines with C₈, C₆ and C₂ fatty acids were relatively inert. In dogs, the intravenous administration of C₁₆ phosphatidyl serine inhibited blood coagulation and prolonged the bleeding time; C₁₀ phosphatidyl serine did not inhibit blood coagulation, but did inhibit platelet aggregation and prolonged the bleeding time. This compound had similar effects in guinea-pigs and rabbits. C₁₀ phosphatidyl serine inhibited thrombus formation in extracorporeal shunts. It appears that the fatty acid composition of phosphatidyl serines determines their effects on blood coagulation and platelet aggregation.     

Effects of Epinephrine, Adrenocorticotrophic Hormone, and Theophylline on Adenosine 3′,5′-Monophosphate Phosphodiesterase Activity in Fat Cells

When rat fat cells were incubated with ACTH, epinephrine, or theophylline for 2 to 10 min, cyclic AMP phosphodiesterase (3':5'-cyclic-AMP 5'-nucleotidohydrolase, E.C. 3.1.4.17) activity (K(m) about 0.39 muM) in the 100,000 x g sediment fraction of homogenates was increased 35 to 50%. The effects of epinephrine and ACTH were concentration dependent and maximal increases were produced with concentrations similar to those that maximally stimulate lipolysis. Theophylline (0.5 mM) similarly increased phosphodiesterase activity but did not enhance the effects of maximally effective concentrations of the hormones. The changes in phosphodiesterase activity following addition of ACTH or theophylline paralleled changes in cell cyclic AMP content; both reached a maximum within 5 min and then declined, approaching basal levels after 20 or 30 min. The increased phosphodiesterase activity in cells incubated for 5 min with epinephrine reverted to basal levels within 2.5 min after the addition of propranolol. Our data are consistent with the view that there is a component of the fat-cell phosphodiesterase, perhaps localized in the plasma membrane, whose activity can be acutely modified by the concentration of its substrate, cyclic AMP.As previously reported (J. Biol. Chem.248, 7164-7170, 1973), exposure of fat cells to insulin increases the activity of a low K(m) phosphodiesterase also localized in the 100,000 x g sediment fraction of fat cell homogenates. In the presence of insulin, however, phosphodiesterase remained elevated for at least 40 min and there was no significant change in fat-cell cyclic AMP content. When phosphodiesterase activity was elevated and cyclic AMP content maintained at a high level by incubation of cells with ACTH plus theophylline, insulin produced a further increase in enzyme activity. Whether or not insulin and ACTH (or epinephrine or theophylline) affect the same phosphodiesterase, there seems little doubt that the underlying mechanisms are different.     

ADP-Induced Aggregation of Washed Platelets Effect of Platelet and Plasma Fibrinogen

ADP-induced aggregation of washed human and porcine platelets has been studied. Plasma from a patient genetically deficient in fibrinogen lacked ADP-cofactor activity. Apyrase totally inhibited the small platelet aggregation observed after addition of fibrinogen to washed platelets. Lysates of washed porcine platelets contained 1.34 mg/10⁹ platelets of protein in the soluble form and 0.44 mg/10⁹ platelets as insoluble protein. Platelet fibrinogen in soluble fraction was 0.16 mg/10⁹ platelets. Partly purified porcine platelet fibrinogen showed cofactor activity for ADP-induced aggregation of washed porcine platelets, but compared to plasma fibrinogen a higher concentration of the platelet fibrinogen was needed to obtain the same effect.     

Changes in Platelet Aggregation in Whole Blood,Plasma and Washed Platelets in Streptozotocin-induced Diabetic Rats: Time-dependent Change in the Antiaggregatory Activity of Diabetic Rat Plasma

Time-dependent changes in platelet aggregation in whole blood, platelet-rich plasma (PRP) and washed platelets were studied in streptozotocin-induced diabetic rats. Collagen-induced aggregation of whole blood and PRP from diabetic rats were significantly reduced within 8 weeks after induction of diabetes, although that in washed platelets were increased from 8 weeks. Plasma from diabetic rats within 8 weeks attenuated platelet aggregation, whereas diabetic plasma at 12 weeks showed no inhibitory effect. Insulin treatment normalized aggregation in whole blood and PRP and abolished the antiaggregatory activity of diabetic plasma. These results suggest the plasma antiaggregating activity appears in the early stage of diabetes, which may contribute to the hypoaggregation in whole blood and PRP. The inhibitory activity disappeared in the later stage. Plasma factor(s) accounting for the antiaggregatory effect of diabetic plasma has not yet characterized.     

Changes in Platelet Aggregation in Whole Blood, Plasma and Washed Platelets in Streptozotocin-induced Diabetic Rats: Time-dependent Change in the Antiaggregatory Activity of Diabetic Rat Plasma

Time-dependent changes in platelet aggregation in whole blood, platelet-rich plasma (PRP) and washed platelets were studied in streptozotocin-induced diabetic rats. Collagen-induced aggregation of whole blood and PRP from diabetic rats were significantly reduced within 8 weeks after induction of diabetes, although that in washed platelets were increased from 8 weeks. Plasma from diabetic rats within 8 weeks attenuated platelet aggregation, whereas diabetic plasma at 12 weeks showed no inhibitory effect. Insulin treatment normalized aggregation in whole blood and PRP and abolished the antiaggregatory activity of diabetic plasma. These results suggest the plasma antiaggregating activity appears in the early stage of diabetes, which may contribute to the hypoaggregation in whole blood and PRP. The inhibitory activity disappeared in the later stage. Plasma factor(s) accounting for the antiaggregatory effect of diabetic plasma has not yet characterized.     

Positive Interaction between Agonists in the Aggregation Response of Human Blood Platelets: Interaction between ADP, Adrenaline and Vasopressin

ADP, adrenaline and vasopressin interact positively as agonists in aggregating human blood platelets in vitro. This interaction is maximal if the addition of two of the agonists is separated by 10--20 s but decreases rapidly at longer intervals especially at low agonist concentrations. The agonist concentrations at which positive interaction gives full aggregation are significantly less than those required for such a response to each agonist alone. The lowest concentrations at which adrenaline and vasopressin interact positively are at least two orders of magnitude greater than the normal blood concentrations of these hormones, and at least an order of magnitude greater than the concentrations achieved in pathological states. Specifically antagonizing the adrenaline and ADP receptors showed that the response was to the second agonist added to the system. An inhibitor of intracellular Ca2+ movement (tetracaine) is equally effective in blocking the responses generated by a single agonist or by interaction of two agonists. Inhibitors which increase cyclic-3',5'-AMP concentration (adenosine, prostaglandin E1, dipyridamole) are more effective against the response to a single agonist than that to agonist interaction. These data suggest that positive agonist interaction results from effects on the concentrations of second messengers within the platelet rather than from a direct interaction on the membrane receptors or the transmembrane coupling mechanisms.     

The Abilities of Human Blood Platelets to Bind Extracellular Calcium and to be Aggregated by Adenosine Diphosphate are Related

S ummary . A technique is described for preparing platelet rich plasma containing a very low level of extracellular calcium. Using this Ca-depleted PRY it has been possible to demonstrate that extraplatelet calcium is in equilibrium with a small amount of calcium that is bound to the platelet surface. When Ca-depleted PRP is incubated at 37°C the platelets lose their ability to bind calcium. At the same time they lose their ability to aggregate. It is likely that the binding of calcium to the platelet surface is a prerequisite for ADP-induced aggregation.     

Induction of Aggregation of Human Blood Platelets by Ultraviolet Light: Action Spectrum and Structural Changes

Exposure of washed human platelets toultraviolet light is followed by platelet aggregation. The effect occurs at wavelengths between 302 and 225 mµ with amaximal response at 248 mµ, and the effectis greatly enhanced by the addition offibrinogen. Platelets exposed to ultravioletlight show ultrastructural changes whichcan be induced independently of aggregation if the addition of fibrinogen is omitted.

Submitted on January 30, 1973 Revised on April 19, 1973 Accepted on April 20, 1973     

The Potentiating Effect of Serotonin and Epinephrine on Collagen-induced Whole Blood Aggregation and Secretion

An unusual mechanism has been demonstrated for the in vitro proaggregating interaction between human platelets and human epidermoid carcinoma A431 cells. A431 cells induce platelet aggregation in a dodependent manner, depending on the rate of ADP release from tumour cells, which occurs in the presence not only of platelet rich plasma (PRP) but also of platelet poor plasma (PPP) or serum. This ADP release appears to be correlated to C₃ cleavage and binding of C_3c to the A431 cell membrane. The interaction between A431 cells and PRP is characterized by typical morphological changes of A431 cells, leading to formation of mixed aggregates showing long projections of tumour cells deeply penetrating into the aggregate. These features, lacking in the presence of gel-filtered platelets (GFP), and reduced in the presence of thrombin degranulated platelets (TDP), are inhibited by cytochalasin and RGDS. The same activation of A431 cell cytoskeleton is induced by PDGF, but not by ADP or thromboxane receptor agonist U46619 or TGFP. These findings suggest a cooperative mechanism of tumour cell platelet interaction, in which a complementdependent ADP release from A431 cells induces platelet degranulation, PDGF release and aggregation. PDGF may induce in A431 cells Ca²⁺ influx, cytoskeleton activation and changes in exposition of surface adhesion molecules, while fibrinogen binding causes mixed tumour cell-platelet aggregates to form.     

The Haemostatic Effect of 51Cr-Labelled Blood Platelets An Experimental Study in the Rabbit

The haemostatic effect of ⁵¹Cr-labelled platelets was studied in 5 rabbits made thrombocytopenic (35,000/μl blood) by whole body ionizing irradiation. Bleeding times were recorded after standardized cuts on the inner side of the rabbit's ear, a method with an acceptable reproducibility. The animals were then each transfused with concentrates of labelled platelets from 2 healthy donor rabbits. This increased the platelet counts to about 2 times 10⁵/μl blood. Bleeding time values were markably prolonged before transfusion and became normalized when tested 1 and 4 h after transfusion. In 3 control experiments, where unlabelled platelet rich plasma was transfused to thrombocytopenic recipients, a similar shortening of the bleeding time was observed. It is concluded that ⁵¹Cr-labelled platelets retain haemostatic ability comparable to non-labelled platelets, when circulating in a recipient animal.