Immunocytochemical evidence for the translocation of alpha-granule membrane glycoprotein IIb/IIIa (integrin alpha IIb beta 3) of human platelets to the surface membrane during the release reaction.

The localization of glycoprotein (GP) IIb/IIIa (integrin alpha IIb beta 3) in both resting and thrombin-activated platelets was studied immunocytochemically. By the preembedding method where only the GP IIb/IIIa molecules on the surface of platelets were immunostained, the distribution of protein A-colloidal gold label was randomly distributed along the surface membrane of resting platelets at a density of 18.0 +/- 2.7 gold particles/microns of membrane. At 15 s after stimulation by 0.1 U/ml of thrombin in an unstirred platelet suspension, the spheroid-shaped platelets with pseudopodia still had normal numbers of alpha-granules, and the density of gold particles was 19.7 +/- 3.6 particles/microns. At 5 min, the alpha-granules were no longer present because of the release reaction, and the density of gold particles significantly increased (27.0 +/- 3.7 particles/microns; p less than 0.01). In immuno-stained ultra-thin frozen sections, the gold particles were detected not only on the surface membrane, including the open canalicular system (OCS), but also on the alpha-granule membranes of resting platelets. At 30 s after thrombin stimulation the alpha-granules fused with the OCS, resulting in the formation of a swollen OCS, which still had gold particles on its membrane. At 5 min, the gold particles were detected on the membrane of the swollen OCS located near the surface membrane, while very few gold particles were present on the membrane of the OCS in the central part of the platelets. These results demonstrate that alpha-granule membrane GPIIb/IIIa translocates to the surface membrane through the membrane of the OCS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fibrinogen distribution on surfaces and in organelles of ADP stimulated human blood platelets

The fibrinogen distribution in platelet organelles after ADP-stimulation was investigated with anti-human fibrinogen using protein A-gold applied to serial sections. Fibrinogen was detected in the so-called alpha-granules of platelets and also in granule protrusions which were observed after ADP-stimulation. The ends of these protrusions were formed as coated membranes and the tips were often in apposition to the surface connected membranes or the plasmalemma. At such places fusion events and hence signs of an exocytosis could be demonstrated by means of cryofixation and cryosubstitution. Examination of serial sections revealed fibrinogen on all these granule profiles. Surface connected membranes, free surfaces and the characteristic structure of the contact zones of aggregated platelets were also labelled by gold particles but less than anticipated. On the platelet surfaces and surface connected membranes fibrinogen was rarely demonstrable with ferritin-labelled anti-human fibrinogen on washed or thrombin-stimulated, almost fibrinogen free platelets. After addition of human fibrinogen to the thrombin stimulated and disaggregated platelets a part of the platelets aggregated spontaneously and formed characteristic contact zones. Anti-human fibrinogen was observed on the free surfaces, in filamentous bridges between the contact spaces and in a tubular surface connected membrane system with involvement of coated membranes at the central ends of these structures. The results indicate the following: all alpha-granules contain fibrinogen; after ADP-stimulation secretion takes place with involvement of coated membranes; during aggregation fibrinogen binds to platelet surfaces and forms contact spaces; fibrinogen is taken up by the surface connected system with involvement of coated membranes.     

Internalization of cationized ferritin by isolated pancreatic acinar cells

The internalization of cationized ferritin (CF) was studied in isolated pancreatic acinar cells in vitro. Horseradish peroxidase (HRP) was used in conjunction with CF to compare internalization of soluble-phase and membrane-bound tracers. The mode of internalization of CF was dependent upon tracer concentration and origin of the plasma membrane (apical vs. lateral-basal). At the lower tracer concentrations (0.19 and 0.38 mg/ml), internalization from the apical cell surface occurred via small vesicles. The tracer then appeared in multivesicular bodies, in tubules, and in irregular membrane-bound structures. After 15 min, CF particles were seen in many small vesicles near the Golgi apparatus, but not in the Golgi saccules. In contrast, at the lateral-basal cell surface the CF particles tended to form clusters. These clusters were more pronounced at higher CF concentrations (0.76 and 1.5 mg/ml) and were associated with elongated cellular processes, which seemed to engulf CF accumulations in a phagocytic manner. Once internalized, CF was found primarily in large irregular structures which appeared to migrate slowly toward the nucleus, reaching a juxtanuclear position after approximately 30 min. CF was observed in lysosomes after 30-45 min and by 90 min most of the CF was confined to large vacuoles and to trimetaphosphatase-positive lysosomes. Similar routes were observed when cells were double-labeled with CF and HRP, where endocytic structures showed co-localization of both tracers. The results of this study indicate the importance of the Golgi region in the intracellular sorting of internalized apical membrane. Furthermore, this work confirms the presence of distinct endocytic pathways at the apical and lateral-basal cell surfaces.     

Correlated measurement of platelet release and aggregation in whole blood

We have used a technique for the simultaneous measurement of platelet activation and aggregation in whole blood using two-color immunofluorescence and flow cytometry to study the relationship between the release reaction and aggregation. A monoclonal antibody specific for the alpha granule membrane protein GMP-140 was used to measure the release reaction, and a monoclonal antibody specific for platelet membrane glycoprotein Ib (GPIb) was used to identify platelets and platelet aggregates. Aggregates were identified as particles expressing both levels of GPIb and size larger than that of resting single platelets. Anticoagulated whole blood was incubated with platelet agonists. At various times samples of the blood were removed and immediately fixed with paraformaldehyde. Blood that had been anticoagulated with ethylenediamine tetraacetic acid showed progressive release of platelets but little or no aggregation. However, blood anticoagulated with citrate or heparin showed correlated release and aggregation. The degree of aggregation was greater in heparin than in citrate. The expression of GPIb and GMP-140 increased in direct proportion to the size of the aggregates. Aggregates were observed varying in apparent diameter up to approximately 20 microns. During prolonged incubation there was progressive disaggregation of adenosine diphosphate (ADP)-induced aggregates. After disaggregation the proportion of GMP-140 negative single platelets increased, indicating that both released and nonreleased platelets participated in the aggregation. There was little or no disaggregation of phorbol myristate acetate (PMA)-induced aggregates. The relatively small size and reversibility of platelet aggregates that we have observed in whole blood may be relevant to phenomena occurring in vivo and in extracorporeal circulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructural changes of platelets during ADP- stimulated aggregation (in the presence of fibrinogen)

The ultrastructure of resting and stimulated human blood platelets (P) was studied by the transmission electron microscopy. The cells were chemically fixed (using tannic acid and OsFeCN mixture) 1, 3, 5 and 15 min after the addition of ADP and fibrinogen (F). Early changes in P ultrastructure consist in drastic reduction of the electron-dense layer of glycocalyx and in an increase of the plasma membrane permeability. At the early stages of P aggregation the cells contact with each other due to rapidly arising pseudopodia. Later, the extracellular network containing an exogenous F participates in the aggregation process.     

Cationized ferritin as a platelet-stimulating surface probe. Binding to platelets and effects on platelet function

Polycationic derivatives of ferritin containing primary amino groups (CFah) or tertiary amino groups (CFdmp) were potent platelet agonists inducing shape change, aggregation and secretion, but also agglutination in the presence of EDTA. Pretreatment of platelets with neuraminidase, PGE1, indomethacin, or creatine kinase/creatine phosphate inhibited CF-induced activation. In contrast, neuraminidase and PGE1 increased the agglutination by CF, indicating an inverse relationship between activation and CF-induced agglutination. At pH 7.4, the cationic charges of CFdmp exceeded those of CFah by a factor of 1.5 and the platelets bound approximately 1.5 times more CFah than CFdmp, suggesting the same number of anionic surface sites for both CF preparations. The capacity of the platelets to bind CF was diminished by 55% at 0 degree C or by 62% after aldehyde fixation and by 13% with PGE1. This suggests that the binding capacity depends on the mobility of the binding sites in the plane of the membrane but is only slightly increased by platelet activation. Binding to fixed or cold platelets approached equilibrium within a few seconds whereas saturation required several minutes at 37 degrees C. Neuraminidase preferentially reduced the slow binding and much less the rapid binding. Since activation by CF developed during seconds, suppressible by a brief treatment with neuraminidase 25 mU/ml, a small portion of neuraminidase-sensitive sites appears to be necessary for CF-induced platelet activation. Full activation and agglutination occurred at CF concentrations far below saturating concentrations. The results show that neither CF-induced activation nor agglutination depend on a simple neutralization of the negative surface charge.     

Localization of fibrinogen during ADP- or thrombin-induced aggregation of washed rabbit platelets

In contrast to human platelets, which aggregate poorly in response to ADP unless fibrinogen is present in the external medium, washed rabbit platelets form large aggregates in response to ADP without fibrinogen in the suspending medium. Addition of fibrinogen to the suspending medium of rabbit platelets frequently has little or no effect on the extent of ADP-induced platelet aggregation. We examined washed rabbit platelets by immunocytochemistry during ADP-induced aggregation and deaggregation and during thrombin-induced aggregation when the external medium did not contain added fibrinogen to determine if (a) fibrinogen was expressed on the surface of rabbit platelets that could support aggregation when the platelets were stimulated, or (b) fibrinogen secreted from the alpha granules supports platelet aggregation. Glutaraldehyde-fixed samples were prepared at different times after addition of ADP or thrombin, embedded in Lowicryl K4M, sectioned, incubated with sheep anti-rabbit fibrinogen, washed, reacted with gold-labeled anti-sheep IgG, and prepared for electron microscopy. The alpha granules of rabbit platelets were heavily labeled with immunogold; the platelet membrane was not labeled. During platelet aggregation and deaggregation in response to ADP, fibrinogen was not detectable on the platelet surface. In response to thrombin, large aggregates formed before fibrinogen was secreted from the alpha granules; fibrinogen was detectable focally at sites of granule discharge by 30-60 sec and fibrin formed by 3 min. Therefore, stimulated washed rabbit platelets can adhere to each other without large amounts of fibrinogen taking part in the close platelet-to-platelet contact, since aggregation occurs before detectable secretion, and large areas where the platelets are in contact are devoid of fibrinogen between the adherent membranes. Adhesion mechanisms not involving fibrinogen may support the aggregation of washed rabbit platelets.     

Rearrangement of the open-canalicular system of the human blood platelet after incorporation of surface-bound ligands

Summary The three-dimensional configuration of the membrane system in human blood platelets following administration of cationized ferritin (CF) was reconstructed by means of thick serial sectioning in combination with high-voltage electron microscopy (HVEM).Surface-bound CF was rapidly internalized at 37 ° C by the platelets, and a conglomerate of vacuolar and tubular structures containing CF was formed in their interior. Three-dimensional reconstruction of these incorporated membranous structures revealed that most of the internalized structures were interconnected with each other and that the number of sites at which they opened onto the cell surface was significantly reduced compared with the number found in intact platelets. In some cases, the openings completely disappeared. This may indicate that most of the internalized membranous structures originate from the open canalicular system (OCS), and that, following the incorporation of the ligand, the OCS gradually loses its connections with the surface membrane forming a conglomerate of the ligand-containing membranous structures in the interior of the platelet.     

Membrane structure of nonactivated and activated human blood platelets as revealed by freeze-fracture: evidence for particle redistribution during platelet contraction

The distribution of intramembrane particles of nonactivated and activated human blood platelets was studied by freeze-fracture under various experimental conditions to see whether morphological evidence for a structural coupling between the platelet actomyosin system and the fibrin network in a retracting clot could be established. Membrane particles were evenly distributed in nonactivated platelets; the total number (E + P faces) was approximately 1,500/micrometers 2 of membrane, and there were two to three times more particles present on the E face than on the P face. Transformation of discoid platelets to "spiny spheres" by cooling did not change the particle distribution. Platelet activation and aggregation by serum or ADP caused no change in membrane particle density or distribution. Particle distribution was not changed in Ca2+-activated platelets fixed immediately before fibrin formation, but after fibrin formation and during clot retraction, particles were sometimes most frequent on the P face and tended to form distinct clusters, and aggregates of E face pits were observed. Blood platelets contain contractile proteins that are distinct as filaments in platelets in retracting clots. We suggest that the redistribution of particles seen in activated platelets during clot retraction reflects the esablishment of mechanical transmembrane links between the platelet actomyosin system and the fibrin net. The P-face particle clusters may represent sites of force transmission between actin filaments bonded to the inside of the membrane and the fibrin network at the outside. Thus, whereas membrane particles may not be directly involved in the attachment of actin filaments to membranes, the transmission of the force of the contractile system to an exterior substrate apparently involves the intramembrane particles.     

The origin of the membrane convolute in degranulating platelets. A comparative study of normal and "gray" platelets

Thrombin-stimulated normal platelets contain a membrane system of dilated channels with openings to the exterior. Whether these membranes originate from the surface connected system (SCS), the alpha-granules or internalized portions of the plasmalemma has not yet been defined. The present study traces in series of ultrathin sections the rearrangement of these membranes during shape change, degranulation and internalization of surface membranes in washed normal and "gray" platelets upon the stimulation with thrombin (1 IU/ml). Cationized ferritin (CF) was used as a surface marker in order to recognize internalized portions of the plasmalemma. Within the first seconds after stimulation, both normal and gray platelets changed their shape by extrusion of the SCS membranes. Simultaneously they started to internalize surface membrane and formed surface membrane invaginations closely attached to the outer rim of the cytoskeletal sphere which developed during the internal contraction. CF was internalized in these invaginations. CF was not observed within the system of dilated channels of stimulated platelets, however. Thrombin-stimulated gray platelets showed a markedly reduced number of dilated channels or none at all. This observation may be due to the fact "gray" platelets are deficient in alpha-granules. It is concluded that the dilated system of membranes in degranulated normal platelets originates from membranes of the alpha-granules which have performed compound exocytosis.     

Long-range interactions in mammalian platelet aggregation. I. Evidence from kinetic studies in brownian diffusion.

The Smoluchowski theory describing aggregation in suspensions of spherical colloidal particles due to Brownian diffusion-controlled two-body collisions, was used to obtain collision efficiencies, alpha B, for adenosine diphosphate (ADP)-induced platelet aggregation in citrated platelet-rich plasma (PRP) from humans, dogs, and rabbits. For these diffusion studies, PRP was stirred with 10 microM ADP for 0.5 s, then kept nonstirred at 37 degrees C for varying times before fixation; the percent aggregation was computed from the decrease in particle concentration with time measured with a resistive particle counter. Up to 20% of rabbit platelets formed microaggregates within 60 s of ADP addition to such nonstirred suspensions, corresponding to mean alpha B values of approximately 0.9. However, human and dog platelets aggregated approximately 10 times and 2-3 times faster than rabbit platelets within the first 60 s of ADP addition, corresponding to alpha B approximately 8 and 2, respectively. These high alpha B (much greater than 1) for human platelets were independent of initial platelet count and were equally observed with the calcium ionophore A23187 as activator. In about one-third of human, dog, or rabbit PRP, comparable and lower values of alpha B (less than 0.5) were obtained for a slower second phase of aggregation seen for the nonstirred PRP over 60-300 s post ADP-addition. Platelet aggregability in continually stirred PRP was distinct from that observed in Brownian diffusion (nonstirred) because comparable aggregation was observed for all three species' stirred PRP, whereas greater than 3-8 times more ADP is required to yield 50% of maximal rates of aggregation for nonstirred than for stirred PRP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective translocation of protein kinase c isozymes by PAF in rabbit platelets

The action of platelet activating factor (PAF) on subcellular distribution and activity of protein kinase C (PKC) isoforms in rabbit platelets was analyzed. The results showed an increase of PKC alpha in membrane fraction, concomitantly with a decrease in cytosolic fraction after 5 min PAF treatment, indicating that a translocation of PKC alpha occurred. In addition, PKC zeta was redistributed in a "reverse" form, from the membrane to cytosolic fraction after PAF treatment. PAF induced an increase of PKC alpha activity, whereas a decrease rather than increase in PKC zeta was observed by using immunoprecipitation assays. In addition, some results indicated that PI3 kinase activation was not involved in PAF-induced PKC zeta translocation as occur in several cells and with other agonists. These actions were time- and concentration-dependent, and were inhibited by the treatment with a PAF antagonist. No translocation was observed when the platelets were incubated with lysoPAF, a PAF related compound.The redistribution of PKC isoforms take place through the activation of high specificity PAF binding sites. The pretreatment of the rabbit platelets with staurosporine, a putative inhibitor of PKC, completely blocked the PAF-evoked aggregation without affecting to PAF-evoked shape change and serotonin release. All together, these data could suggest that the specific translocation of PKC isoforms play an important role in the activation of rabbit platelets.     

PLATELET PHAGOCYTOSIS AND AGGREGATION

The addition of latex particles to native (no anticoagulant) or citrated human platelet-rich plasma (PRP), or to a once-washed platelet suspension causes platelet aggregation. This aggregation is associated with phagocytosis of the latex particles by the platelets and appears to be due to release of adenosine diphosphate (ADP) from the platelets. Adenosine and adenosine monophosphate, which are known to inhibit platelet aggregation induced by ADP, also block that induced by latex. These compounds do not prevent the phagocytosis of latex particles by the platelet. The addition of iodoacetate and 2,4-dinitrophenol in appropriate concentrations to the PRP, prior to the addition of the latex, blocks platelet aggregation and phagocytosis. This is also true for the chelating agent ethylenediaminetetraacetate (EDTA). Platelets left in contact with latex for a sufficient period of time show loss of their granules. Leucocytes phagocytose both latex and platelets that had themselves phagocytosed latex. It is concluded that phagocytosis of latex particles by platelets resembles that by white cells, and that in both processes metabolic changes appear to be involved.     

Apolipoprotein A-I mimetic peptide 4F blocks sphingomyelinase-induced LDL aggregation

Lipolytic modification of LDL particles by SMase generates LDL aggregates with a strong affinity for human arterial proteoglycans and may so enhance LDL retention in the arterial wall. Here, we evaluated the effects of apoA-I mimetic peptide 4F on structural and functional properties of the SMase-modified LDL particles. LDL particles with and without 4F were incubated with SMase, after which their aggregation, structure, and proteoglycan binding were analyzed. At a molar ratio of L-4F to apoB-100 of 2.5 to 20:1, 4F dose-dependently inhibited SMase-induced LDL aggregation. At a molar ratio of 20:1, SMase-induced aggregation was fully blocked. Binding of 4F to LDL particles inhibited SMase-induced hydrolysis of LDL by 10% and prevented SMase-induced LDL aggregation. In addition, the binding of the SMase-modified LDL particles to human aortic proteoglycans was dose-dependently inhibited by pretreating LDL with 4F. The 4F stabilized apoB-100 conformation and inhibited SMase-induced conformational changes of apoB-100. Molecular dynamic simulations showed that upon binding to protein-free LDL surface, 4F locally alters membrane order and fluidity and induces structural changes to the lipid layer. Collectively, 4F stabilizes LDL particles by preventing the SMase-induced conformational changes in apoB-100 and so blocks SMase-induced LDL aggregation and the resulting increase in LDL retention.     

Adenine nucleotide metabolism of blood platelets. X. Formaldehyde stops centrifugation-induced secretion after A23187-stimulation and causes breakdown of metabolic ATP.

A23187 induced shape change, aggregation and secretion of platelets in plasma. When rapid cooling was used to stop secretion and centrifugation to separate the cells from the medium, maximal amounts of storage ATP plus ADP and preadsorbed [14C]serotonin were found in the supernatant immediately (less than 5 s) after A23187 addition. These results suggested that A23187 could cause shape change and aggregation through secreted ADP and not directly. When secretion was stopped with chilling and formaldehyde treatment before centrifugation, the secreted substances appeared after a lag of 60-120 s, i.e. after shape change was terminated and aggregation was well on its way. These two platelet responses thus seemed to be independent of secretion and induced directly by A23187. The absence of a lag period when secretion was stopped by chilling alone was thought to be due to centrifugation-induced secretion of platelets conditioned by A23187. Formaldehyde completely inhibited centrifugation-induced secretion. At 37 degrees C, formaldehyde caused rapid breakdown of metabolic ATP in platelets with a pattern dependent on the formaldehyde concentration: Below 50 mM, ATP was converted to inosine plus hypoxanthine via ADP, AMP and IMP and the adenylate energy charge was preserved. Above 100 mM, AMP was the end product with a drastic reduction in the adenylate energy charge. These changes were not due to lysis of the platelets, but were apparently caused by an formaldehyde-induced increase in cellular ATP consumption. Platelet secretion is usually associated with a conversion of metabolic ATP to hypoxanthine. Formaldehyde had to be used to stop secretion and since it caused breakdown of ATP, additional smaples were taken out for nucleotide determination during stirring of platelet-rich plasma with A23187. It was found that metabolic ATP was converted to inosine plus hypoxanthine only during the secretion step.     

Interaction of human platelet membrane glycoproteins with collagen and lectins

The binding of platelets to collagen is the first step in hemostasis. We attempted three approaches for elucidation of the chemical nature of receptors of human platelets for collagen. First, we examined the effect of platelet surface alteration by chymotrypsin treatment. On increasing the concentration of chymotrypsin, collagen-induced platelet aggregation and the release reaction decreased, and in parallel with this change, remarkable decrease of membrane glycoproteins IIb and V, as well as 400 kDa and 300 kDa membrane proteins, was observed. Secondly, effects of several lectins on the platelet-collagen interaction were examined. Lens culinaris agglutinin was found to specifically inhibit the platelet aggregation and release reaction induced by collagen. This inhibition appeared to be caused mainly by blocking of the collagen receptors on platelets by Lens culinaris agglutinin. Furthermore, Lens culinaris agglutinin was found to bind preferentially to glycoprotein IIb as identified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of platelet membranes followed by staining with ¹²⁵I-Lens culinaris agglutinin. In addition, a polymerized preparation of Lens culinaris agglutinin induced platelet aggregation. Thirdly, the membrane component which could bind to collagen-Sepharose 4B was determined. Analysis by SDS-polyacrylamide gel electrophoresis combined with autoradiography or fluorography revealed that glycoprotein IIb was most enriched in the bound fraction to collagen. From these results, glycoprotein IIb is most likely a receptor for collagen on human platelet membranes.     

Internalization of cationized ferritin receptors by rat hepatoma ascites cells.

Cationized ferritin (CF) was used to label the cell surface anionic sites of Chang rat hepatoma ascites cells. If the hepatoma cells were fixed with glutaraldehyde and treated with CF, the label was distributed evenly over the external surface of the plasma membrane. Treatment of unfixed ascites cells with CF yielded clusters of ferritin particles separated by label-free areas of the plasma membrane. Some unfixed ascites cells were treated firstly with CF, then incubated in veronal buffered saline at 37 °C for 10, 20, 30 and 45 min, subsequently fixed in glutaraldehyde and re-exposed to CF. After 10 min of incubation, the label was arranged into large clusters with the remaining areas of the plasma membrane lightly labelled with CF. At 20 min, only clusters of ferritin were present on the plasma membrane; the remaining area of the cell surface was totally free of label. The ability of the plasma membrane to bind additional CF was completely restored after 45 min of incubation. These results suggest that for some period of time after internalization of CF label on cell surface the plasma membrane is devoid of any detectable negative charge.     

Biphasic effects of phospholipase A2 on platelet aggregation. Effect of prostaglandin synthesis inhibitors and essential fatty acid deficiency

Phospholipase A₂ has a biphasic action upon the aggregation of rat platelets. In the first phase, occurring after shorter incubation periods with the enzyme, aggregation is enhanced. Longer incubation periods lead to an inhibition of the aggregation. The first phase disappears after the addition of indomethacin whereas the second phase persists. Incubation of platelets with phospholipase A₂ leads to serotonin release. Prostaglandins are formed without platelet aggregation. Whereas the same effects occurred at the high dose of phospholipase A₂ when platelets of essential fatty acid deficient rats were used, a difference was seen at the lower dose.It is concluded that in the first phase, arachidonic acid is liberated and transformed into aggregation inducing intermediates which are formed in the prostaglandin synthesis. In the second phase, changes may occur in the outer membrane which lead to a diminished sensitivity to aggregating agents.     

An inhibitor selective for collagen-stimulated platelet aggregation from the salivary glands of hard tick Haemaphysalis longicornis and its mechanism of action

Soluble materials of salivary glands from Haemaphysalis longicornis were found to inhibit collagen, ADP, and thrombin-stimulated platelet aggregation. One inhibitory component was purified to salivary gland homogeneity by a combination of gel filtration, ion-exchange, and C_8 reverse phase HPLC. The purified activity, named longieornin, is a protein of moleeular weight 16 000 on SDS-PAGE under both reduced and nonredueed conditions. Collagen-mediated aggregation of platelets in plasma and of washed platelets (IC_(50) was approximately 60 nmol/L) was inhibited with the same efficacy. No inhibition of aggregation stimulated by other effeetors, including ADP, arachidonic acid, thrombin, ristocetin, calcium ionophore A23187, thromboxane A2 mimetic U46619 and 12-O-phorbol-13-myristate acetate, was observed. Longieonin had no effect on platelet adhension to collagen. Not only platelet aggregation but also release reaction, and increase of intraeellar Ca~(2 ) level of platelets in response to collagen were com     

Alterations of wheat-germ agglutinin binding pattern on cell surface of blood platelets after thrombin stimulation

An attempt was made to demonstrate wheat-germ agglutinin (WGA) binding sites on platelet surfaces after thrombin stimulation, by means of a post-embedding cytochemical technique using colloidal gold as marker at an ultrastructural level. In unstimulated platelets washed with EDTA, an intense uniform labeling of WGA-gold complexes was found on the surface membrane. When washed platelets were stimulated by thrombin in the absence of Ca2+, only a release reaction was induced. WGA labeling on the surface membranes of these platelets decreased dramatically. However, the labeling intensity of WGA-gold complexes on the surface membrane of aggregated platelets induced by thrombin in the presence of Ca2+ increased significantly compared to that of thrombin-stimulated platelets in the absence of Ca2+. In contrast to the uniform labeling on the surface membranes of unstimulated platelets, clusters of gold label were often found on the surface membrane of the aggregated platelets, although there was no significant quantitative difference in the labeling intensity between these two groups. Thus, we present direct morphological evidence demonstrating qualitative and quantitative alterations of WGA labeling on the surface membrane of platelets after thrombin stimulation. The possibility is considered that WGA-binding glycoproteins in the surface membrane are involved in the aggregation response after thrombin stimulation.