Membrane Changes in Human Platelets Induced by Lipopolysaccharide Endotoxin

The unexplained occurrence of thrombocytopenia in cases of Gramnegative sepsis in man led us, in the light of animal experiments indicating the blood platelet as the target cell for endotoxin, to examine the effect of Salmonella enteritidis lipopolysaccharide B on human platelets. Human platelets were separated from a coat of plasma proteins by Sepharose 2B filtration or by a combined procedure of albumin gradient and Sepharose 2B filtration. The action of endotoxin on human platelets resulted in membrane changes manifested by dose-dependent release of [3H]serotonin and adenine nucleotides. Cytoplasmic marker, lactic dehydrogenase, and lysosomal marker, beta glucuronidase, were retained indicating that the release reaction was selective. Release of [3H]serotonin was specific for endotoxin since other particulates, zymosan and erythrocyte stroma, were without effect. Endotoxin, added to gel-filtered human platelets, induced a significant evolution of platelet factor 3 procoagulant activity. Preincubation of endotoxin with a membrane-rich homogenate of human platelets inhibited its labilizing effect on human platelets thus suggesting an interaction between endotoxin and the platelet membrane itself. Other plausible factors in this interaction [fibrinogen, adenine nucleotides, thrombin, sialic acid residues, and IgG] were eliminated on the basis of a series of control experiments. From the negligible effect of aspirin and indomethacin, we may infer that the interaction of endotoxin with platelets does not depend on the platelet prostaglandin synthesis pathway. The direct interaction of endotoxin with the human platelet membrane comprises a new mechanism which may help to clarify the pathogenesis of vascular and haemostatic disorders accompanying bloodstream infections due to Gram-negative bacteria.     

Ultrastructural Changes in Human Platelets During Arachidonic Acid-induced Aggregation

This study was undertaken to visualizethe sequence of ultrastructural changesoccurring in human platelets during aggregation induced by arachidonic acid.Aggregometer recordings were obtainedup to the time of adding glutaraldehydeto begin fixation of platelets for electronmicroscopy. A correlation was shown between the degree of aggregation andchanges in the fine structure of humanplatelets which had been allowed to aggregate for 15, 30, 90, or 180 sec. Thepattern of ultrastructural changes foundin the platelets aggregating in response toarachidonic acid was similar to that reported previously for other aggregatingagents such as collagen or ADP. Amorphous material was observed within thedilated canalicular system of platelets,supporting the thesis that its channelsserve as a pathway for the products released during aggregation. The dilatationof the canalicular system of platelets induced by arachidonic acid could bemediated by an intermediate in prostaglandin synthesis.

Submitted on January 4, 1974 Revised on February 14, 1974 Accepted on February 26, 1974     

Coronary Artery Shape and Flow Changes Induced by Myocardial Bridging

Changes in coronary shape and blood flow induced by myocardial bridging were analyzed in a 56-year-old patient with symptoms of unstable angina after the exclusion of other heart disease. Coronary angiography demonstrated a 1.8-cm long myocardial bridge in the middle part of the left anterior descending coronary artery. In systole, an eccentric compression of the artery occurred, resulting in a stenosis that occupied 86% of the diameter and 96% of the area. Intraluminal ultrasound was performed with a 20-MHz transducer in a 4.8-Fr catheter sheath (Boston Scientific Corp.) connected to an ultrasound console (Diasonics Inc.). A side saddle catheter was introduced into the left anterior descending coronary artery via a giant guiding catheter. A circular shape with typical systolic pulsation was seen in the proximal part of the artery (maximal and minimal diameters 3.6 mm and 3.5 mm, respectively). Distally an eccentric compression of the coronary artery was visualized, decreasing one diameter from 3.0 to 2.6 mm, whereas the orthogonal diameter remained constant at 3.3 mm. The myocardial bridge compressed 160°–180° of the circumference of the artery, leading to a change from a circular to an elliptical arterial shape. A delayed relaxation of the bridging was demonstrated. Only the proximal part of the vessel in the muscle bridge could be passed. Coronary flow was measured using a Doppler 3-Fr 20-MHz catheter (Millar Instruments Inc.) using a pulse repetition rate of 62.5 kHz. Coronary flow velocity was calculated in the proximal part of the left anterior descending coronary artery before and after intracoronary injection of 10 mg papaverine. Phasic coronary flow velocity increased from 14 to 21 cm/sec and mean flow from 6 to 13.5 cm/sec, yielding an estimated flow reserve of 1.5 and 2.2, respectively (normal > 3.0). Thus, intravascular and Doppler ultrasound are useful techniques for analyzing the effect of myocardial bridging on changes in coronary shape and blood flow. An eccentric compression of the coronary artery was visualized with delayed relaxation. Coronary flow reserve was reduced. Further studies in larger patient populations are necessary to demonstrate whether reduction of coronary flow reserve is, in general, related to delayed relaxation in diastole.     

ADP Induced Surface Charge Changes of Adult and Newborn Platelets

In experiments reported here electrophoretic mobility of washed platelets and platelets suspended in diluted plasma obtained from adults and newborns was practically the same. No significant difference was observed in the pH—mobility relationship of the two types of platelets. These comparative studies indicate that the actual charge density, i.e. the number and sign of the charged groups at the newborn and adult platelet surfaces are essentially identical.
However, a significant difference was found between the two platelet populations in the mobility changes induced by ADP. On the basis of cross over experiments between the platelets and plasma of adults and newborns it seems likely that the different behaviour of neonatal platelets arises from a difference between adult and newborn plasma. Preliminary results indicate that this may be due to the presence in adult plasma of a component with molecular weight about 10 000.     

Fine Structural Alterations Induced in Platelets by Adenosine Diphosphate

Blood platelets exposed to the nucleotide adenosine diphosphate (ADP)lose their discoid appearance and adhere to one another. The basis for theADP induced shape change has remained obscure. In the present study thestructural transformations of platelets under the influence of ADP have beenexamined in the electron microscope. Loss of lentiform appearance is closelyrelated to a marked reorganization of the platelet hyaloplasm. The marginalbundle of microtubules decreases in circumference and is moved into theinterior of the platelet. Granules and other organelles are transported to thecell center where they are enveloped within a web of microtubules and microfilaments. These changes are completely reversed after the influence of ADPdisappears. The movement of the circumferential bundle of microtubulesfrom its position under the cell wall appears to be the principle cause forthe loss of discoid shape. Constriction of hyaloplasmic elements followed bycomplete recovery of unaltered appearance are indicative of a reversible waveof contraction occurring in the platelet hyaloplasm. Thus the change in plateletsurface contour after exposure to ADP is not due to a direct modification ofthe cell wall by ADP, but to a contractile wave which ADP indirectly triggers in the substance of the cells.

Submitted on March 28, 1967 Accepted on November 1, 1967     

Localization of 5-Hydroxytryptamine in Blood Platelets: An Autoradiographic and Ultrastructural Study

5-Hydroxytryptamine (serotonin, 5-HT) was found by electronmicroscope autoradiography to be localized in dense granules in platelets. Evidence of radioactivity from tritium-labelled 5-HT was also found at other sites within platelets, but without evidence of concentration at those sites. Studies on human platelets showed that the dense granules were present in different forms, and structural evidence suggested that the dense granules containing 5-HT may be derived from alpha granules. After clotting of platelet-rich plasma, and before the structural integrity of the platelets was lost, radioactivity was present in relation to dense granules, but had been lost from other sites, suggesting firmer binding of 5-HT to the granules.     

Cycles of Agglutination-Disagglutination Induced by Ristocetin in Thrombasthenic Platelets

An oscillatory pattern of platelet agglutination-disagglutination in response to Ristocetin (R) at narrow concentration ranges was observed in citrated platelet rich plasma (PRP) of 10 patients with Glanzmann's thrombasthenia. The cyclic pattern decreased in intensity over time, was reproducible, and was not pH dependent. Formalin-fixed thrombasthenic platelets agglutinated with R but did not show a cyclic pattern. Incubation with 2.5 microM ADP inhibited R oscillation response, but small increases in R dose overcame this inhibition. The addition of ATP or creatine phosphate/creatine phosphokinase to thrombasthenic platelets inhibited by ADP restored the R oscillation response. In the platelets of a single patient, intracellular levels of ADP and ATP were shown to diminish during an oscillation response to R. There was an increase in AMP levels during the same period of time. The changes in these three intracellular nucleotides were gradual over time and did not vary with phases of the oscillation. Acetyl salicylic acid (ASA), at concentrations shown to block cyclooxygenase activity in control platelets, enabled thrombasthenic platelets to respond to R with full agglutination without oscillations. Lower concentrations of ASA in the PRP gave a return of the oscillation response. Our data suggest that the disagglutination phase of the R response of thrombasthenic platelets is not a function of the known glycoprotein membrane defect, but depends on materials originating in the platelet whose release is blocked by ASA.     

An Electron Microscopic Study of the Changes in Platelets during Viscous Metamorphosis

Viscous metamorphosis of platelets has been studied with the light microscope, and ultra-thin sections have been prepared at progressive stages forexamination in the electron microscope.

The phase contrast light microscope reveals rapid aggregation and distortionof platelets and gives the impression of their fusion into structureless aggregates during viscous metamorphosis.

Sectioned material collected during viscous metamorphosis of plateletsand examined in the electron microscope reveals a remarkable degree of retention of structure in a majority of the platelets. All become deficient ingranules and devoid of vesicular spaces, but most retain intact cell membranes,and the structureless masses seen with the light microscope are found to consist of densely aggregated platelets. Fusion and complete loss of identityoccurs in the minority.

The retracted clot was found to contain densely aggregated, distorted andelongated platelets, empty of granules and intimately related to fibrin particles.

Submitted on March 26, 1962 Accepted on July 12, 1962     

Platelets in the Carcinoid Syndrome: A Chemical and Ultrastructural Investigation

A combined chemical and electron microscopic study of normal platelets and platelets from patients with the carcinoid syndrome has revealed an association between their serotonin content and certain alterations in their cytoplasmic structure. In addition to a fibrillary structure of possible cytoskeletal significance, platelets also contain a system of microtubules and vesicles. When the platelet serotonin content is high this tubular structure is prominent and communicates with large vacuoles which may contain granular material. Normal platelets incubated with serotonin show similar alterations in structure. In addition, serial investigations of platelets from two carcinoid patients suggest that in addition to the normal active transport process for absorbing serotonin a diffusion process can operate when the cell is exposed to high environmental concentrations. The significance of these storage mechanisms in relation to normal and elevated platelet serotonin concentration is discussed.     

Experimental Colitis Induced by Trinitrobenzenesulfonic Acid (An Ultrastructural and Histochemical Study)

Inflammatory bowel disease (IBD) of humans is achronic and devastating disease of unknown etiology.Models of acute colitis in animals have been achieved byintrarectal administration of agents such as 2,4,6-trinitrobenzenesulfonic acid (TNBS) intorat colon. This agent induces focal inflammation andalterations in the colon with features similar to thosefound in chronic inflammatory diseases in humans. The aim of this study was to assess the effectof TNBS administration on histological andultrastructural features of the rat colon, especially inareas not affected by transmural inflammation. Also in areas without transmural inflammation, weobserved a significant increase in crypt diameter and inthe number and area of the goblet cells, as well asalterations in the contents of mucin in goblet cells. We conclude that TNBS treatment in rats led tosevere changes in normal architecture of the colon andalso in damaged areas where no direct inflammation wasproduced.     

Lymphocyte Transformation Induced by Autologous Platelets in a Case of Thrombocytopenic Purpura

In a case of chronic thrombocytopenicpurpura, significant lymphocyte transformation could be induced with autologous platelets as a stimulant. The reaction was not due to serum factors since itoccurred when culture medium was supplemented either with autologous orpooled human serum. Controls excludednonspecific lymphocyte transformation.Modulation of normal platelet antigensby viral or chemical factors or adsorption of such factors on the platelet membrane could account for the observedreactions. However, since other auto-antibodies were demonstrated, the hypothesis of altered cellular "self-recognition" mechanisms is tentatively proposedas an alternative explanation for thisobservation.

Submitted on April 8, 1970 Accepted on May 11, 1970     

东亚钳蝎毒诱发大肠癌细胞凋亡的电镜观察

东亚钳蝎毒素是东亚钳蝎毒腺细胞分泌的一种具有多种生物活性的小分子多肽类物质。我们应用体外细胞培养的方法,已证实蝎毒素对大肠癌细胞具有显著的抑杀作用。为进一步探讨蝎毒素抑杀肿瘤细胞的机理,我们用东亚钳蝎毒素灌胃治疗大鼠实验性大肠癌,详细研究了用药后大肠肿瘤细胞超微结构的变化,结果表明,蝎毒素能诱发大肠癌细胞程序化死亡,本实验详尽地观察了大肠癌肿瘤细胞程序化死亡的基本过程,这种作用可能是蝎毒抗肿瘤的重要途径之一。     

Inhibition of Thrombin Induced Aggregation of Human Platelets by Antithrombin III

The influence of antithrombin III (At-III) on the thrombin induced aggregation of human platelets was studied by the aggregometer technique according to Born. When At-III was mixed with platelet suspension, subsequent addition of thrombin resulted in decreased aggregation as compared to the control. The inhibition was dependent on the At-III: thrombin ratio. Above 0.2 NIH U/ml of thrombin, no inhibitory effect was observed with physiological At-III concentrations. The effect was more pronounced at 37° C than at 20° C.     

Function ex vivo of 111 In-Labelled Human Platelets Simultaneous Aggregation of Labelled and Unlabelled Platelets Induced by Collagen

The function of ¹¹¹In-labelled platelets has been assessed by collagen-induced aggregation of platelets in samples of whole blood. The blood samples were drawn after injection of autologous ¹¹¹In-labelled platelets in 19 subjects undergoing platelet kinetic studies. It was thus possible to measure the aggregability of labelled and unmanipulated platelets simultaneously. ¹¹¹In-labelled platelets aggregated to the same extent as unmanipulated platelets when tested from 10 min to 24 h after injection of the labelled platelets. The results confirm the assumption that minimal damage is inflicted on the platelets during the isolation and labelling procedures, and support the concept that platelets manipulated in vitro may recover in vivo within a few minutes after reinjection.     

Platelets Stored in a Glucose-Free Additive Solution or in Autologous Plasma – An Ultrastructural and Morphometric Evaluation

We evaluated a new glucose-free citrate-acetate-NaCl platelet additive solution (PAS 2). In series I, platelet concentrates (PC) were prepared by apheresis and subsequently stored either in plasma (n =16) or in PAS 2 (n =15). In series II, PCs were prepared from pools of four buffy coats (BC) and stored in plasma (n =12) or in PAS 2 (n =11). By means of ultrastructural morphometry, the volume fractions of α-granules, the open canalicular system (OCS) and the fraction of storage granules secreted into the OCS were analyzed during storage for up to 8 days. Additionally, we determined pH, glucose, lactate, pCO₂, HCO₃^–, lactate dehydrogenase and platelet factor 4. Apheresis platelets stored in plasma showed no changes in their contents of α-granules and in the fractions of the OCS. In contrast, apheresis platelets stored in PAS 2 displayed a decrease of their relative volume fraction of α-granules from 9.1±1% on day 1 to 3.7±0.9% on day 5. The fraction of the OCS increased from 7.4±0.8% on day 1 to 17.1±1.4% on day 3. On day 8, 93±9% of all platelets were lysed. Levels of glucose were significantly lower in these preparations and after day 3 glucose consumption decreased to zero. Among PC derived from pooled BC, differences between storage in PAS 2 or plasma were less striking. Only the fraction of α-granules secreted into the OCS was significantly greater in BC derived PC stored in PAS 2 on all days. These PCs stored in PAS 2 had a higher plasma carryover (30%) in comparison to apheresis PC stored in PAS 2 (10%). We conclude that plasma is superior to PAS 2 for storage of both apheresis and buffy coat platelets. For preservation of the structural integrity of platelets, the use of PAS 2 requires a minimum of 30% plasma carryover.     

Changes in Phosphoinositides in Rabbit Platelets during Clot Formation. Comparison of Platelets Stimulated by ADP or by Thrombin in the Presence of Polymerising Fibrin

Platelet phosphoinositide metabolism was examined during platelet-fibrin clot formation stimulated by ADP (10 μM) plus reptilase, or by thrombin (1 U/ml), for 120 s in the presence of fibrinogen, to determine which changes are specifically associated with this process. Stirring at 200 rpm was used to minimise the contribution of aggregation to the platelet changes. Under these conditions, thrombin caused extensive release of the contents of platelet granules; ADP plus reptilase did not. The presence of fibrinogen decreased the amount of extractable phosphatidylinositol 4,5-bisphosphate (PIP(2)) by 46.4±5.5% when thrombin was the stimulus, and by 47.4±5.5% when the platelets were stimulated by ADP plus reptilase. Fibrinogen did not decrease the extraction of other phospholipids. The amount of phosphatidylinositol 4-phosphate (PIP) increased when platelets were stimulated in either the presence or absence of fibrinogen. These increases were greater in the presence of fibrinogen and the thrombin-induced increase was smaller than the increase induced by ADP plus reptilase; with ADP plus reptilase, the increase in PIP more than accounted for the loss of extractable PIP(2). In platelets prelabelled with [(3)H]inositol, the decrease in PIP(2) labelling induced by fibrinogen with ADP plus reptilase as the stimulus was accounted for by the increase in PIP labelling; the decrease induced by fibrinogen with thrombin as the stimulus was not. With thrombin, 46.5% of the decrease in PIP(2) labelling, caused by fibrinogen, was accounted for by label that remained with the interfacial protein after lipid extraction; with ADP plus reptilase, the amount of label with this protein was the same with or without fibrinogen. Only thrombin increased the amount of label in inositol trisphosphate (IP(3)) and the amount of phosphatidic acid (PA); these changes were not increased by fibrinogen. Thus, the results with ADP plus reptilase indicate that clot formation is not dependent on release of granule contents, formation of detectable IP(3) or PA (and hence does not require activation of phospholipase C) or association of [(3)H]inositol-labelled compounds with protein. Clot formation is associated with a shift in the PIP(2)-PIP equilibrium toward PIP.     

Changes in 32P-Content of Phosphatidic Acid and the Phosphoinositides of Rabbit Platelets during Aggregation Induced by Collagen or Thrombin

S ummary . During the ADP-induced shape change and aggregation of washed platelets that have been labelled with ³²PO₄ there is increased incorporation of ³²P into phosphatidic acid (PA) and the phosphoinositides of the platelets. When added to suspensions of washed platelets, collagen and thrombin cause not only a change in shape and aggregation, but also the release reaction. The present experiments were carried out to study the effect of collagen and thrombin on the ³²P-content of PA and the phosphoinositides of washed platelets from rabbits. Both collagen and thrombin caused an increase in the labelling of PA that was apparent when the platelets had changed shape and before aggregation of platelets was detected. Unlike the change in labelling of PA that occurs during ADP-induced aggregation, there was a marked increase in labelling of PA after the commencement of platelet aggregation. Comparison with the maximum response caused by ADP showed that both collagen and thrombin caused a greater increase in the ³²P-content of PA than can be obtained with ADP. Like ADP, collagen and thrombin also caused an increase in labelling of mono-phosphatidylinositol (MPI). Collagen caused an increase in labelling of diphosphatidylinositol (DPI) but not triphosphatidylinositol (TPI). As with ADP in previously reported experiments, the increase in labelling of DPI occurred after the increase in labelling of PA but preceded the increase in labelling of MPI. An increase in the ³²P-content of DPI has not been demonstrated previously in similar in vitro experiments with other cells using other stimuli. The changes in extent of incorporation of ³²P into PA and phosphoinositides may reflect changes in these phospholipids which are important in the mechanism of the change in platelet shape and the release reaction.     

Sexual Morphogenesis in Achlya: Ultrastructural Basis for the Hormonal Induction of Antheridial Hyphae

Male strains of the water mold Achlya ambisexualis produce antheridial hyphae in response to the steroid hormone antheridiol. The antheridial hypha is postulated to be initiated through a localized wall softening with the enzyme cellulase. Freeze-etch studies of hormone-treated hyphae were conducted to determine if aggregates of vesicles are induced at the location of new antheridial hyphae. Localized aggregates of vesicles were found in conjunction with areas of wall thinning. These data suggest that the processes of vesiculation and secretion provide a mechanism for concentration of cellulase at the site of initiation of antheridial hyphae.     

Long-Lived Conformation Changes Induced by Electric Impulses in Biopolymers

Electric impulses are capable of inducing long-lived conformational changes in (metastable) biopolymers. Results of experiments with poly(A).2 poly(U) and ribosomal RNA, which are known to develop metastabilities, are reported. A polarization mechanism is proposed to explain the structural transitions observed in the biopolymers exposed to the impulses. In accordance with this idea, the applied electric field (of about 20 kV/cm and decaying exponentially, with a decay time of about 10 musec) induces large dipole moments by shifting the ionic atmosphere of multistranded polynucleotide helices. This shift, in turn, causes strand repulsion and partial unwinding. The fields used in our experiments are of the same order of magnitude as those in nerve impulses. The significance of the impulse experiments with regard to the question of biological memory recording is briefly discussed.