Viscous Metamorphosis Research Articles

The role of platelets in the initial stages of atherosclerosis.

The role of platelets in the initial stages of atherosclerosis.

Platelet membrane interactions

The term 'viscous metamorphosis' was coined in early studies to describe the fusion and disintegration of platelets into an amorphous mass sealing sites of vascular injury. Improvements in morphological techniques revealed that disintegration was not involved in the platelet hemostatic reaction, and that part of the definition was eliminated. Fusion was retained, however, even though platelet fusion was never observed. The present study has examined the different forms of platelet membrane interaction to determine if fusion is an appropriate term for any aspect of platelet physiology or pathology. Fusion was not observed during platelet-platelet, platelet-surface or platelet-fibrin interaction at any stage in the development. Only fusion of secretory organelles with channels of the open canalicular system (OCS) during the release reaction represents involvement of the phenomenon. Pathological fusion occurs during long-term storage when alpha granules bind together to form giant organelles. The only examples of surface membrane fusion develops when platelets are incubated with the cholinergic agent, carbachol (carbamyl choline chloride). Ethylenediamine tetraacetic acid also causes intimate associations of OCS channels and interacting surface membranes, but not the fusion of lipid bilayers that carbachol induces. Thus, fusion does not appear to be an appropriate term to describe platelet-membrane interactions, except for the process of secretion.

Subcellular changes in platelets in acute and subacute african swine fever

The morphological changes in platelets in acute and subacute African swine fever (ASF) and their relationship to pathogenesis were studied. Eight pigs were inoculated with a highly virulent strain of African swine fever (Malawi '83) and 14 with a moderately virulent strain (Dominican Republic '78) for ultrastructural study of platelets, monocyte/macrophages and vascular structures in the liver, spleen, lymph node, bone marrow, lung and kidney. Both viruses produced activation and degranulation of platelets from day 3 after inoculation onwards, coinciding with activation of the mononuclear phagocyte system and virus replication in monocyte/macrophages. Platelet aggregation and viscous metamorphosis of platelets were observed at 5 and 7 days after inoculation with the highly virulent strain, coinciding with endothelial alterations, but platelet aggregation was less prevalent and there was no sign of viscous metamorphosis in animals inoculated with the moderately virulent strain. Virions within platelets were observed at the final stage of acute ASF and at 5-7 days after inoculation in subacute ASF. This suggests that platelets assist in disseminating ASF virus within the body, especially in subacute infections.

Heparin Ablates Force Development during Platelet Mediated Clot Retraction

Although snake venom enzymes such as reptilase do not cause viscous metamorphosis, platelet secretion or clot retraction; when batroxobin and calcium are added to citrated blood significant platelet force development occurs. When this batroxobin-calcium system was applied to the study of platelet function during cardiopulmonary bypass (CPB), force development was found to be completely inhibited. After heparin reversal by protamine sulfate, significant recovery of force occurred. The present investigation was performed to evaluate the role of heparin in reducing force development during CPB. At concentrations above 0.10 U/ml, heparin totally suppressed force development in normal plasma. Addition of protamine sulfate to heparinized plasma caused complete recovery of force development. These concentrations of heparin had little effect on platelet aggregation by ADP or collagen. Possible direct effects of heparin on fibrin assembly and structure were studied by adding varying amounts of heparin to plasma and then inducing clot formation with batroxobin. At 1 U/ml, heparin reduced the size of fibrin fibers by 33%. Higher heparin concentrations had no additional effect. These results indicate that heparin may be responsible for a significant component of the decreased platelet force noted during cardiopulmonary bypass. To test whether heparin's effect could be due to suppression of thrombin activity, the effects of the antithrombin hirudin on force development were measured. Hirudin also inhibited force development in a concentration dependent manner. Thus, heparin's reduction of platelet force development may be due, at least in part, to suppression of thrombin activity.

Restudying of viscous metamorphosis after addition of calcium to PRP

Scanning electron microscopic observation of morphological change of platelets when calcium (Ca) was added to platelet-rich plasma (PRP) was carried out in healthy, patients with hemophilia and thrombocythemia.1) In PRP, many platelets showed pseudopods or aggregation more or less. This may be attributed to stimulation on platelet at blood collection and centrifugation. But platelets with severe abnormality in thrombocythemia showed on pseudopods at all. 2) When fluid was moved after addition of Ca to PRP, platelets was transformed physiologicaly and showed “cherry blossom-like” in generaly. 3) In the case of hemophilia, fibrin-nets were produced independently from the platelets or products of transformed platelets. 4) Morphological change of platelets when Ca was added to PRP of thrombocythemia were multifariousness. When Ca was added to PRP of thrombocythemia, moved and fixed at a proper good time, a few fibrin-like markedly long pseudopods were seen in platelets.Even morphological changes that could not be recognized in normal could be inferred by using PRP of hemophilia and thrombocythemia.

A study of the relationship between the platelet shape and it's function

It is widely accepted that the platelet which is originally discoid is deformed into spiny spheres by ADP, thrombin or collagen (viscous metamorphosis) and forms thrombus by its function-adhesiveness and aggregation. But we have hardly achieved the quantative analysis of the morphological changes and disclosed the relationship between the deformed platelets and their functions.In order to clarify this relationship, we examined the influences of the following procedures on human platelets.1) preservation (non-anticoagulated whole blood, citrated whole blood), 2) stirring (the same blood samples), 3) preservation after stirring (citrated whole blood) and 4) surgical operation with laparotomy on 10 patients with various diseases.After fixing the platelets by 1% glutaraldehyde, the platelets were classified into disc, hemisphere, sphere and the other form, and checked for pseudopod formation under a light microscope. As a platelet function, platelet retention rate was measured by a modified Hellem-II method.Platelet shape was easily changed and pseudopods issued by preservation and stirring. These morphological changes were brought about more strikingly by stirring than by preservation. The retension rate decreased when spherification and pseudopod formation had prominently advanced. However, it increased when the incidences of non-discoids and the pseudopods increased to some degree, e. g. when citrated blood was preserved up to 60min. at room temperature and when it was stirred for 1min. These phenomena were similarly observed in vivo condition (during operation).These results suggested that some relationship existed between the platelet shape and its function and that halfly deformed platelets were in an activated phase and prominently deformed platelets were rather in a refractory phase. This relationship was probably derived fromnot only extra-platelet factors such as activation of plasma coagulation factors (contact factor and prothrombin) but also from internal factors (or character) of platelets. The latter factors were considered to be more important because both shape change and the increase of retention rate were demonstrated in some experiments to occur before the silicon PTT became shortened.

Mechanism of the hypercoagulant effect of hyperoxia

It was shown in man and rabbits that a 30-minute inhalation of a 100% O2 under normal atmospheric pressure was accompanied by an elevation of the blood coagulation capacity and a sharp reduction of the count of platelets with the change of their structure. The trigger mechanism of the described hypercoagulation effect is possibly the viscous metamorphosis of platelets developing under the effect of oxygen activation of Hageman's factor.

Clot retraction and contractile protein

Clot retraction was ihanced only by a living platelet, not by a destroyed or a denatured platelet. The phenomenon was inhibited by G-EDTA or cytochalasin B (Cy-B), and accelerated by colchicine. The inhibition by Cy-B was observed above 10mcg/ml, but at the concentration of 30 or 50mcg/ml slight retraction was observed at the bottom of some test tubes. These results indicated that natural actomyosin purified by us from platelets played a main role on the retraction. But the inhibitory concentration on clot ratraction by Cy-B was higher than that for free cell migration. Then it may be impossible to explain that the clot retraction was responsible only for the contractile protein.Ultastructure of platelet in clot incubated with Cy-B exhibited irdiriclul fibrin fiber that showed no adhesion and no bend, and that the cell shape was round or partially protrudent. No viscous metamorphosis or no pseudopod formation could be observed that was ordinartly observed in a normal retracted clot. Intracellular granules were expelled, vacuolized, or put aside in the cytoplasm. Microtubules were preserved and became clear, because the number of granules were diminished. But microfilament that was observed in the normal platelet in retracted or colchicine induced clots could not be found in the Cy-B treated platelet. These results indicated that microtubules were fundamentally different from microfilaments, and that microfilaments were not made from microfilaments, judging from the attitude of Cy-B or colchicine induced clots.When H-merromyosin were applied to the glycerinated clot that had been treated with Cy-B, according to the method of Ishikawa, no arrowhead structural filament was formed in the platelet. In the cytoplasma only some thin filaments could be observed.The above findings may suggest that the contractile protein plays an important roleon the clot retraction, but the phenomenon might not be explained only by the action of the protein.

Electrochemical features of platelet interactions

Platelets participate in hemostasis and thrombus formation. The physicochemical basis at the molecular level for platelet adhesion, cohesion to form aggregates and the resultant viscous metamorphosis is not well understood. Under physiological conditions, blood platelets possess a net negative charge and decreases or changes in this charge have been implicated in both their adhesion and aggregation. The peripheral zone of the platelet contains glycoproteins and glycolipids. A variety of charged groups are present including positively charged amino groups and at least three types of negatively charged groups. The carboxyl group of terminal sialic acid residues is considered to be a major contributor to the negative charge of platelets. Removal of as little as 10 percent of the peripheral zone sialic acid by neuraminidase results in a shortened platelet lifespan in vivo . The electrokinetic properties of blood platelets are changed by interaction with a great variety of agents. In those cases where specific adsorption occurs it is possible to study the relationship between adsorption, change in surface charge, and the process of aggregation. It should be noted that in physiological media only those charge groups within about 8 Å of the electrophoretic surface of the platelet will contribute to a significant extent to the electrophoretic properties. The electrophoretic mobility of platelets has been determined for a variety of animal species and in humans for a wide spectrum of disease situations. The measurement of platelet electrophoretic mobilities before and after treatment with adenosine diphosphate and norepinephrine has been introduced as a test of platelet function. However, so many variables may influence the surface properties of blood platelets including method of preparation, time, temperature, pH, contact phenomena, shear conditions, ionic strength and composition of suspending media, conditions for mobility measurements, etc., that considerable apparent disagreement exists in the literature regarding platelet electrokinetics.

The spurting of erythrocytes through junctions of the vascular endothelium treated with snake venom

The vascular endothelium treated with the venom of Trimeresurus flavoviridis, a crotalid, was investigated by electron microscopy. The results demonstrated that the erythrocytes, changing their shape as does the amoeba, spurt through opened junctions of the endothelial cell lining, the adjacent basement membrane being disrupted to permit eventual extravasation of the erythrocytes. A small number of platelets gathers at the site of hemorrhage but they show no sign of viscous metamorphosis. These results, together with our recent findings that highly purified venom hemorrhagic principles can disrupt the isolated basement membrane in an enzymatic mode of action and also can inhibit the in vitro aggregation of platelets, do allow us to visualize the physiological mechanisms involved in hemorrhage.

ChemInform Abstract: BINDING OF THROMBIN TO HUMAN PLATELETS

MUCH work has been carried out aimed at elucidating the mode of action of thrombin on platelets1–5. These studies were based on the assumption that hydrolysis of a protein by thrombin constitutes the primary event culminating in aggregation of platelets. Results of recent studies suggest that the primary step in this process might be binding of thrombin to a receptor site, triggering viscous metamorphosis of the platelets6,7. The processes of binding and proteolysis need not be mutually exclusive, however, binding may be followed by proteolysis. Here I present direct evidence of thrombin binding by human platelets and discuss some of its implications.

Ultrastructural study of experimental thrombosis in pulmonary muscular arteries of rabbits.

In order to clarify the ultrastructural change of initial phase of thrombosis, an autologous clot Injection or one shot of thrombin was performed via an auricular vein of each rabbit.In the groups of clot injection, platelets located between an inserted thrombus and endothelial cells show viscous metamorphosis. Degeneration and disappearance of the endothelial cells are followed by invasion of platelets and dense liquid materials into the subendothelial space and the media of the muscular arteries in both experimental groups. There is no detectable morphological difference between thrombosis in rivo induced by an injection of thrombin and thromboembolism induced by an injection of autologous whole blood clots coagulated in vitro.Phagocytosis in the course of thrombosis is mainly derived from the activity of subendothelial cells and partly from that of endothelial cells. Ultrastructural features of the subendothelial edema are based on the detachment of endothelial cells from the elastic lamina or on the edematous swelling of subendothelial cell cytoplasm. The initial stage of the organization of thrombus, showing smooth muscle cells arranged in the vertical position towards the thrombus, is noted three days after a clot or thrombin administration. Myofilamentous structures with dotted density are detected in the endothelial cells of muscular artery, not only of experimental groups but also of control ones.

Binding of thrombin to human platelets.

MUCH work has been carried out aimed at elucidating the mode of action of thrombin on platelets1–5. These studies were based on the assumption that hydrolysis of a protein by thrombin constitutes the primary event culminating in aggregation of platelets. Results of recent studies suggest that the primary step in this process might be binding of thrombin to a receptor site, triggering viscous metamorphosis of the platelets6,7. The processes of binding and proteolysis need not be mutually exclusive, however, binding may be followed by proteolysis. Here I present direct evidence of thrombin binding by human platelets and discuss some of its implications.

The Function of the Platelet Surface in Coagulation

SummaryPlatelets washed by the albumen density gradient technique were used as the sole source of thromboplastin in a new test, and also as the sole source of factor X in a modification of the one-stage factor X assay test of Denson. These platelets were shown to be capable of generating considerable amounts of thrombin during periods of preincubation of up to 8 minutes. For at least 3 minutes of preincubation their size and shape remained invariant, as measured by counting and by sizing on a Coulter channelyser. It is suggested that this test measures a function of the platelet surface, rather than of the platelet after viscous metamorphosis. This function is tentatively named platelet catalytic activity (PCA). Support for this concept was given by the fact that aspirin and phenylbutazone did not affect the test. The factor X assay system confirmed the presence of a small amount of mostly inactive factor X on the surface of the platelet. This factor X could be activated by Russell viper venom.

Physical and biochemical characteristics of platelets in severely malnourished children with purpura.

Platelets from a group of 24 malnourished children with purpura, and a group of 28 clinically healthy adults and 5 normal children, were studied with determination of platelet mean diameter, soluble protein, lactate concentration and platelet viscous metamorphosis.The data obtained from platelet rich plasma in malnourished children showed an increase in lactate concentration and an inability to increase its lactate when thrombin was added; an abnormal viscous metamorphosis was found also in some patients, due to either a plasma or a platelet abnormality and also in a third group of patients to the presence of both defects.

Viscous metamorphosis of platelets and functional changes in the blood clotting system under the influence of alimentary lipemia

Viscous metamorphosis of platelets and functional changes in the blood clotting system under the influence of alimentary lipemia

The Induction of the Release Reaction in Human Blood Platelets by Close Cell Contact

SummaryWhen blood platelets are centrifuged at elevated pH they release serotonin. This reaction is similar to the release reaction which makes part of the complex called viscous metamorphosis. The experiments support the hypothesis that contact between platelets forms the stimulus which initiates the release reaction.

Qualitative description of factors involved in the retraction and lysis of dilute whole blood clots and in the aggregation and retraction of platelets.

Dilute whole blood clots were prepared by addition of thrombin to blood diluted 1:10 in phosphate buffer. The pH of this buffer was 7.4 and the ionic strength was 0.084. Though the ionic strength was low, there was no hemolysis of red corpuscles due to the contribution to the osmotic gradient by plasma salts and proteins. In the standard assay the clot was formed by addition of thrombin at 4 degrees C then incubated at 37 degrees C. Retraction and lysis of these clots were inhibited by removal of platelets and by increasing concentrations of purified thrombin. Retraction and lysis were also inhibited by inactivation of any one of the following factors: gammaM globulin, complement components C4 and 3, and (in the case of lysis) plasminogen. Evidence that some of the above serum factors were adsorbed to the platelet membrane was obtained by aggregation of washed platelets by antisera to these factors (i.e. fibrinogen, gammaM, and C4 or C3). These platelets were not aggregated by antisera to other serum proteins (by albumin, transferrin, gammaG globulin). These and other studies suggested that platelets, thrombin, fibrinogen, gammaM globulin (cold agglutinin), complement components, and plasminogen influenced and facilitated retraction and lysis of clots. These studies also suggested that platelets and some of these factors were physically associated. Because of this physical association, and because of the fact that clot retraction is associated with aggregation and retraction of platelets, we extended the above observations to include a study of the effect of these same serum factors on serum-induced aggregation and retraction of washed platelets. (Other terms which have been in use in the past to describe serum-induced platelet aggregation and retraction have included those such as platelet "fusion" and "viscous metamorphosis," neither of which fully described the phenomena.)Platelet aggregation and retraction induced by serum was markedly accelerated by addition of increasing concentrations of thrombin and (or) cold agglutinin. Hirudin and antisera to gammaM globulin inhibited seruminduced aggregation and retraction of platelets. Reconstitution of inactivated serum with purified C4, 3, and 5 and thrombin restored its capacity to induce aggregation and retraction of platelets.Therefore, we postulated that platelet aggregation and retraction were necessary for clot retraction and that platelet aggregation and clot retraction facilitated clot lysis. More specifically we postulated that thrombin, in addition to catalyzing clot formation, also modified the platelet membrane such that gammaM globulin (cold agglutinin) and complement components can act on the platelet membrane leading to (a) aggregation and retraction of the platelets, (b) retraction of the clot, and (c) to the activation of plasminogen either on the surface of the platelet by C8i and (or) by release of platelet activators of plasminogen.

Release of elastolytic activity from blood platelets.

THE degradation of elastin early in atherosclerosis1 has been attributed to elastolytic enzymes of the blood or vessel wall2, but no enzyme other than pancreatic elastase (EG 3.4.4.7) (ref. 2) has yet been demonstrated conclusively. We have recently reported the presence of an elastolytic enzyme in blood platelets3 and now present evidence that it can be released from blood platelets by certain factors which produce aggregation and viscous metamorphosis of platelets. The elastolytic enzyme(s) can be separated from acid cathepsins and partially purified4.

Platelet Changes Induced by Elatericin A

SummaryElatericin A at a concentration of 3 μg/ml causes spicule formation on platelets and inhibits spreading on glass surfaces, viscous metamorphosis and clot retraction. Elatericin A has no effect whatsoever on the activity of isolated platelet factors. However platelet factor 3-availability is decreased by elatericin A. Platelets treated with elatericin A up to a limiting concentration of 30 μg/ml regain their clot retracting activity if washed and reincubated in normal plasma.Elatericin A does not affect the ATP content nor the ATPase activity of platelets. A normal ADP-release is obtained upon addition of thrombin to elatericin A treated platelets. Addition of ATP and Mg ions does not restore the clot retracting activity of elatericin A treated platelets.