Hamster platelets and frog thrombocytes: a comparative study of the ultrastructure before and during exposure to thrombogenic agents
McKinnon, Elizabeth Louise
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The fine structure of normal hamster platelets and frog thrombocytes was studied and compared. Ultrast ructure of the latter was described for the first time. Frog thrombocytes are characterized by a high nucleo-plasmic index and by deep furrows extending 1 /4 to 3/4 through the nucleus. The presence of alpha, beta (mitochondrion), gamma (microvesicles), and delta (vacuoles) granulomeres (Schulz et al, 1959) in the cytoplasm of thrombocytes and platelets lends further support to the morphological and functional analogy between these blood elements. When osmium fixed platelets and thrombocytes were suspended in agar and carried through the dehydration and embedding procedures without centrifugation, cells were separated from each other by at least 300A. Cells recovered in pellet form by centrifugation after each step in the above procedures were separated by free spaces of 50A or less. Plasma membrane distortion, seen in pellet preparations, did not occur when platelets and thrombocytes were prepared in agar blocks. Disintegration of alpha granules, one source of platelet factor 3 in mammals (Johnson, et al, 1959), was a common occurrence in both platelets and thrombocytes and paralleled the distortion of plasma membranes, another source of platelet factor 3 (Marcus and Zucker-Franklin, 1964). The functional analogy between mammalian platelets and amphibian thrombocytes was also extended by comparison of their responses to topically applied bovine thrombin (25 N.I.H. units/ml.). Occluding thrombi developed from mural aggregates in less than sixty seconds in blood vessels of frog retrolingual membrane and hamster cheek pouch preparations. Fine structural changes in platelets and thrombocytes were similar to those described for human platelets during the same stages of aggregation and thrombocytorrhexis (Rodman, et L, 1963). Plasma membrane deformation was usually accompanied by the appearance of electron lucent areas in normally homogeneous electron dense alpha granules, while a substructural lattice in the granules remained intact. Marcus and Zucker-Franklin (1965) have shown that the phospholipids in plasma membranes and in alpha granules of platelets are correlated with clotting activity. Persistence of mitochondria even when discharged from aggregating cells indicates that these organelles are less affected by platelet viscous metamorphosis than either the plasma membrane or alpha granules. Adenosine diphosphate (4.0 ug/ml.) and serotonin (100 mgms.%) displayed thrombogenic action on blood vessels of the hamster cheek pouch, but not in those of the frog retrolingual membrane. Whether the difference in response was due to basic biochemical differences in the blood of these two species was not determined. When adenosine diphosphate was applied topically to exposed blood vessels in each of three hamster cheek pouches, occluding thrombi did not appear to develop from mural thrombi in any vessels under observation. Venules were occluded only when a circulating embolus reached a critical size and became lodged in the vessel. This seems to indicate that adenosine diphosphate acts by directly altering the state of the plasma membrane of circulating platelets. Alterations in the vascular endothelium could not be demonstrated at the ultrastructural level after exposure to any of the thrombogenic agents. Formation of white thrombi in hamster blood vessels exposed to serotonin occurred in the same manner as that described for adenosine diphosphate. However, the thrombus consisted of aggregated polymorphonuclear leukocytes and not of platelets. Any platelets seen in the vicinity appeared as isolated and intact structures. Fibrin was absent in the artificial thrombus osmium-fixed ninety seconds after formation. Absence of fibrin in biochemically and electrically induced thrombi fixed in osmium five minutes after formation of occluding thrombi indicates that fibrin formation occurs in a later stage of the hemostatic process.
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