1. The specificity of platelet receptor sites for 5-HT uptake and for the rapid morphological change and aggregation was investigated with 5-hydroxy-tryptamine (5-HT) and seventeen analogues as well as with some antagonists of 5-HT.2. The analogues, with the exception of 5-hydroxy-N'N'-dibutyltryptamine, caused the rapid morphological change in platelets. In concentrations below those needed to produce the agonistic action (viz. 0.05-2.0 muM), these analogues themselves inhibited competitively the shape change caused by 5-HT.3. The velocity of change in shape caused by 5-HT was reduced in low Na media.4. Ten analogues produced platelet aggregation; three of these, viz. 5-methoxy-alpha-methyltryptamine, 5-hydroxy-alpha-methyltryptamine and 5-hydroxy-N'N'-diisopropyltryptamine), were approximately equipotent with 5-HT. Six analogues did not induce platelet aggregation.5. All the analogues which prevented the initial change in shape of platelets caused by 5-HT also inhibited its aggregating effect, apparently competitively with low K(i) values (0.02-1.6 muM).6. As with the inhibition of shape change, the inhibition of aggregation shows relatively low structural specificity of the receptor site.7. Methysergide was a potent inhibitor of shape change and aggregation (K(i) approximately 0.03 muM); imipramine was much less inhibitory (K(i) approximately 5-10 muM).8. Only one analogue (5-hydroxy-alpha-methyltryptamine) was taken up like 5-HT by platelets. All the other analogues inhibited the uptake of 5-HT by platelets (K(i)=0.2-2.7 muM).9. Methysergide was a weak inhibitor of 5-HT uptake (K(i) approximately 125 muM) whereas imipramine was very effective (K(i) approximately 0.3 muM).10. Our results show that the initial change in shape of platelets is required for and precedes aggregation. The structural specificity of the platelet receptor concerned with shape change and aggregation caused by 5-HT appears low whereas the uptake mechanism is a highly specific one. The uptake probably proceeds through more than one step, the relationship between the steps is not yet clear.

Blood Coagulation↗

PubMed1972

Quantitative investigations of the adhesiveness of circulating polymorphonuclear leucocytes to blood vessel walls.

1. A new simple method is described for quantitating the adhesiveness of circulating polymorphonuclear leucocytes, or granulocytes, to the walls of blood vessels. The cheek pouch of anaesthetized hamsters or a small part of the mesentery of anaesthetized mice were prepared for continuous microscopic observation of selected venules. Those granulocytes which moved sufficiently slowly to be individually visible were counted for 1 or 2 min periods as they rolled past a selected point on one side of a vessel. The velocity distribution of these cells was determined by analysing films. Films were used also to measure mean blood flow velocity in the venules by observing embolizing platelet thrombi induced by the iontophoretic application of adenosine diphosphate. Emigration of granulocytes into the tissues was quantitated by enumerating them in standard areas of stained histological sections.2. In control experiments with hamster cheek pouch venules, the rolling granulocyte count usually passed through a maximum shortly after the preparation was set up and then fell to a low constant value. In mouse mesentery venules the count remained at a low approximately constant value from the beginning for at least 3 hr.3. The mean velocity of blood flow in the venules was between 900 and 200 mu/sec. All rolling granulocytes moved much more slowly; in hamster cheek pouch venules the mean velocity was about 20 mu/sec and in mouse mesentery venules about 10 mu/sec. Around these means the velocity distribution of individual cells was narrow.4. Rolling of granulocytes was abolished by superfusing ethylenediamine tetra-acetate (EDTA, 0.1 M) suggesting that the phenomenon depends on calcium or magnesium ions.5. Agents were applied locally to the observed venules. Human serum albumin, trypsin or histamine in high concentrations did not affect the rolling granulocyte count.6. The rolling granulocyte count was increased during the application of Hammarsten casein or Escherichia coli culture filtrate which are chemotactic to granulocytes in vitro. These agents did not cause alterations in mean blood flow velocity in the observed venules which might have accounted for the effect on the rolling granulocyte counts. When E. coli culture filtrate was perfused through mouse intestine the increase in rolling granulocyte count in the draining venous blood was proportional to the logarithm of the concentration of filtrate.7. The rolling granulocyte count was also increased by the local application of plasma globulin permeability factor or lymph node permeability factor.8. Granulocyte counts in standard histological sections showed no significant increases in control preparations but considerable increases following the application of Hammarsten casein.

Animals↗

PubMed1972

The initiation of platelet thrombi in normal venules and its acceleration by histamine.

1. In cheek pouch preparations of anaesthetized hamsters, platelet thrombi or ;white bodies' were produced in venules by the micro-ionto-phoretic application of adenosine diphosphate (ADP). Currents of 10-400nA were passed through micropipettes containing 10 mM-ADP, the tips of which were less than 5 mu from the outer wall of the venule. The effect was quantitated by determining the time between starting the currents and the first appearance of platelets adhering inside the venule opposite the tip of the micropipette.2. Repeated applications of ADP to the same site on a venule caused the appearance of white bodies after intervals which were almost constant for up to 3 hr.3. The time to first appearance of a white body was inversely related to the iontophoretic current between about 10 and 200 nA. Currents smaller than 10 nA had no effect on the platelets. With currents of 200 nA or more the time remained at a minimum of less than 20 sec.4. With currents of about 300 nA the minimum time increased little as the pipette tip was withdrawn up to 20 mu from the venule; with greater distances the time increased progressively.5. Histamine caused gaps to appear between endothelial cells in cheek pouch venules. Histamine at a concentration of 10 mM in micropipettes was applied iontophoretically by currents of 300 nA to venules at the same sites as ADP. Histamine alone had no effect on circulating platelets. When applied before and together with ADP, histamine decreased the time to first appearance of white bodies by up to 40% below that determined with ADP alone. Iontophoretically applied histamine did not alter the mean blood flow velocity in the venules.6. After stopping the application of histamine, the time to first appearance of white bodies produced by ADP increased again in about 5 min to the control values.7. Bradykinin, which does not cause endothelial gaps in cheek pouch venules, did not accelerate the induction of white bodies like histamine.8. There were no microscopic abnormalities in venules in which white bodies had formed. Venules exposed to histamine accumulated circulating carbon particles in discrete wall areas.9. It is concluded that adhering white bodies can be induced repeatedly in normal venules by direct action of externally applied ADP on circulating platelets and that the accelerating effect of histamine on white body formation is due to the separation of endothelial cells which accelerates the inward diffusion of ADP.

Adenine Nucleotides↗

PubMed1972