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The expressions of platelet-specific glycoprotein(GP)s Ib, IIb and IIIa were analyzed in 2 megakaryoblastic cell lines: CMK and UT-7. Phorbol-12 myristate 13-acetate (PMA) treatment of CMK induced expressions of GPs IIb and IIIa that peaked on the 4th day post-treatment, while treated UT-7 cells showed maximal levels of these GPs during the 6-8th days. Antibody staining to detect the formation of GPIb alpha after PMA induction showed the presence of the intact GP in UT-7 and the degraded form in CMK. In UT-7, synthesis of GPIIb mRNA increased on days 4-6 after PMA treatment, in parallel with the increase of GPIIb. PMA increased the cytoskeletal protein (actin and myosin) contents in both lines, but in contrast to the two GPs, the increase in these proteins started immediately after PMA addition to the cells. Cell surface proteins of CMK and UT-7 cells were rapidly modified after PMA induction. Especially notable were the degradations of 93-kDa and 140-kDa proteins that occurred on days 1-2 after PMA treatment. These studies demonstrate that the expression of platelet-specific proteins shows a different time dependency than the increment of cytoskeletal proteins, indicating that the syntheses of these two classes of proteins are most likely induced through different mechanisms.

We investigated the effects of host species and resource (carbon dioxide, nitrate) availability on activity of detoxication enzymes in the gypsy moth,Lymantria dispar. Larvae were fed foliage from quaking aspen or sugar maple grown under ambient or elevated atmospheric CO2, with low or high soil NO 3 (-) availability. Enzyme solutions were prepared from larval midguts and assayed for activity of cytochrome P-450 monooxygenase, esterase, glutathione transferase, and carbonyl reductase enzymes. Activity of each enzyme system was influenced by larval host species, CO2 or NO 3 (-) availability, or an interaction of factors. Activity of all but glutathione transferases was highest in larvae reared on aspen. Elevated atmospheric CO2 promoted all but transferase activity in larvae reared on aspen, but had little if any impact on enzyme activities of larvae reared on maple. High NO 3 (-) availability enhanced activity of most enzyme systems in gypsy moths fed high CO2 foliage, but the effect was less consistent for insects fed ambient CO2 foliage. This research shows that gypsy moths respond biochemically not only to interspecific differences in host chemistry, but also to resource-mediated, intraspecific changes in host chemistry. Such responses are likely to be important for the dynamics of plantinsect interactions as they occur now and as they will be altered by global atmospheric changes in the future.

The mechanisms of platelet adhesion to collagen type III-coated wells and Matrigel-coated wells were analyzed. The adhesion of 51Cr-labeled platelets to collagen-coated wells showed a biphasic pattern. The early stage of adhesion was inhibited by antibodies against platelet glycoprotein(GP)s Ia/IIa and VI. The later stage of platelet adhesion was inhibited by an antibody against the GPIIb/IIIa complex and a concomitant release of 14C-labeled serotonin was observed. The percentage of adhered platelets was increased when a higher platelet concentration was added in the reaction medium. These results indicated that the adhesion assay of platelets to collagen-coated wells was composed of two reactions: the first one is the platelet-collagen interaction that depends on GPIa/IIa and GPVI on the platelet surface; and the second reaction is the platelet-platelet interaction, platelet aggregation, which depends on GPIIb/IIIa. The adhesion of platelets to Matrigel-coated wells was indicated to involve platelet-Matrigel interactions that were partly dependent on the laminin in the Matrigel solution.

The genetic basis for Glanzmann's thrombasthenia (GT) was elucidated on a compound heterozygote with glycoprotein (GP)IIb gene: an opal mutation at the end of exon 17 (CGA----TGA) results in only a trace amount of GPIIb mRNA, and a splicing mutation at the acceptor site of exon 26 (CAG----GAG) causes an in-frame, exon skipping process from exon 25 to 27. This aberrant transcript encodes a single-chain polypeptide characterized by a 42-amino acid deletion, which includes the proteolytic cleavage site(s) and a unique, proline-rich region at the location corresponding to the carboxyl-terminal of the normal GPIIb alpha-chain. These characteristics are shared by a previously reported defective GPIIb molecule, which is neither assembled with GPIIIa nor transported to the cellular surface. Despite its normal transcription level, expression of the present defective GPIIb molecule was significantly decreased (approximately 6% of the control level). Because the precursor GPIIb molecule is assembled with GPIIIa in the endoplasmic reticulum (ER) and its processing, as well as stability, is dependent on the GPIIIa subunit, the defective GPIIb molecule may be rapidly degraded by the intrinsic quality control system of the ER due to its inability to form a stable heterodimer complex as a consequence of its misfolded structure. Although we did not confirm that the GPIIIa genes of this individual were normal, GPIIIa may be secondarily decreased (approximately 11% of control), because a large part of it could not be complexed, making it vulnerable to proteolysis. To elucidate the molecular basis for GT, we propose here a classification of GT based on the biosynthetic pathway of the GPIIb-IIIa complex.

Glycoproteins, present on the outer surface of platelets, function in the aggregating and releasing reactions of these cells. Some of the hereditary diseases of platelet dysfunction, such as, thrombasthenia and Bernard-Soulier syndrome, have been shown to result from defects in some of these glycoproteins. Recent advances in molecular biology have enabled us to analyze the genes which encode the defective glycoproteins in these patients. The information from these studies also helps us to understand in detail the function of the glycoproteins. At present, the major glycoproteins, GP II b/IIIa and GP I b/IX, have been studied extensively and we know the details of the mechanisms through which they function. However, as to the minor glycoproteins, GP I a/II a, GP IV and GP VI, details remain to be elucidated.

In 1988, we reported a thrombasthenic patient whose platelets contained an abnormal molecular weight GPIIb (Blood, 71, 915, 1988). Here we describe another patient whose platelets contain a variant GPIIb with properties similar to the previously reported abnormal GPIIb. This patient has a small amount of the abnormal GPIIb and was suggested to be a heterozygote characterized by deficient and abnormal GPIIb genes. The abnormal GPIIb was suggested to be related to the precursor form of GPIIb because the molecular weights of both proteins are the same. The abnormal GPIIb may lack a peptide region around the cleavage site of the two chains, as suggested from its lack of reactivity towards antibodies against these epitopes.

Platelet glycoprotein (GP) Ib from 131 healthy Japanese was analyzed using SDS-polyacrylamide gel electrophoresis and specific staining with peroxidase-coupled wheat germ agglutinin after it was transferred to nitrocellulose membranes. Four slightly different species of GPIb were observed and designated as A, B, C, and D for glycoproteins with molecular weights of 168,000, 162,000, 159,000, and 153,000 daltons, respectively. The respective gene frequencies were calculated to be .073, .011, .561, and .355 for A-, B-, C-, D-type GPIb. Portions from each type of GPIb molecule (alpha-chain and glycocalicin) showed heterogeneity with the same molecular weight difference, indicating that the variance would be derived from the polypeptide portion that is exposed to the outer medium. The different types of GPIb were the same with respect to their accessibility to lactoperoxidase, reactivity to lectins, and affinity to TLCK-thrombin. Although Bolin et al reported patients with a bleeding tendency whose platelets have double GPIb bands, here we found that platelets with different GPIb phenotypes showed no significant differences in aggregating activity and platelet retention. Analysis of GPIb phenotype should be important for structural and physiologic studies on GPIb and glycocalicin.

Platelet proteins (0.5-5 micrograms) were electrophoresed in a one-dimensional or an unreduced-reduced, two-dimensional sodium dodecyl sulfate gel system. The separated proteins were then transferred electrophoretically to nitrocellulose and reacted with peroxidase-conjugated lectins. Visualization of specific glycoproteins which bound the lectins was made by the chromogenic reaction catalyzed by peroxidase utilizing 3,3'-diaminobenzidine as the substrate. Wheat germ agglutinin specifically reacted with and allowed the visualization of glycoprotein Ib. Peanut agglutinin also specifically stained glycoprotein Ib after treatment of the nitrocellulose transferred proteins with neuraminidase. Ricinus communis agglutinin I stained thrombospondin, a 260 kDa protein, and factor VIII. Concanavalin A stained mainly glycoproteins IIb, III, IV, and V. Glycoproteins Ia, Ic, IIa, and other minor glycoproteins could be separated by unreduced-reduced, two-dimensional gel electrophoresis and were stained weakly with wheat germ agglutinin conjugates. These techniques were found to be reproducible as well as easily applied to the analysis and identification of platelet glycoproteins, particularly when dealing with a limited amount of platelets.

Actin filament formation during early stages of platelet activation by stimulants was analyzed by determining the distributions of cytoskeletal proteins to a low speed centrifuge pellet (TP), ultracentrifuge pellet (UP), and ultracentrifuge supernatant (US) after lysing platelets with 1% Triton X-100. TP contained actin binding protein, myosin, alpha-actinin and actin, and UP contained these cytoskeletal proteins and other proteins. During thrombin activation, total protein and actin in TP increased with time, while they decreased in UP. Aggregating agents (ADP, PMA, thrombin) and, to a small extent, colchicine and nocodazole, microtubule inhibitors, increased cytoskeletal proteins and total protein in TP, while cytochalsin B, an inhibitor of actin polymerization, had the opposite effect. When the amounts of the respective proteins in TP and UP were summed, these values were not affected by agonists and inhibitors, except in the case of thrombin stimulation. These data suggest that the actin in UP is a form intermediate between the actin filaments in TP and actin monomer in the soluble fraction, and there may be a dynamic conversion between these forms upon platelet activation. UP was also characterized by immunostaining with antibodies against fibrinogen, tubulin, and glycoprotein Ib after SDS-PAGE/electrophoretic transfer to nitrocellulose sheets.

Platelet glycoprotein (GP) Ib from healthy individuals from the United States was analyzed using SDS-polyacrylamide gel electrophoresis and staining with peanut agglutinin-coupled peroxidase or wheat germ agglutinin-coupled peroxidase after transfer of electrophoresed proteins to nitrocellulose. The GPIb from American Caucasians (109 individuals) was found to be polymorphic, showing types A, B, C, and D of GPIb, similar to what was observed in Japanese. The phenotype distribution pattern, however, was very different from that observed in the Japanese population. GPIb heterogeneity was also observed in American Blacks, Asian Americans, and Americans of Hispanic origin. Thus, it appears that the polymorphism of GPIb is a general phenomenon which should be regarded as an important factor when doing studies on this glycoprotein.

We describe an individual with abnormal platelet glycoprotein (GP) IIb of different molecular weight (mol wt), a defect that distinguishes this patient from previously reported thrombasthenics. The patient, a 21-year-old female, has a mild bleeding tendency; her platelets lack adenosine diphosphate (ADP) aggregation and have severely suppressed collagen aggregation but a normal response to ristocetin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of her platelets indicates that they contain two types of GPIIb molecules: one with an abnormal mol wt (122 kd, unreduced; 128 kd, reduced) and one with a normal mol wt (128 kd, unreduced; 118 kd, reduced). Relative to the amount of GPIIb in normal platelets, her platelets contain approximately 35% abnormal GPIIb and 20% normal GPIIb. Fibrinogen binding assays on the patient's platelets indicated that they contained 25% of the normal amount of fibrinogen receptors. Crossed immunoelectrophoresis of the patient's platelets demonstrated the formation of a GPIIb/IIIa complex that was mainly composed of normal mol wt GPIIb and GPIIIa. The patient's father has decreased ADP aggregability, and his platelets also contained both abnormal and normal GPIIb (about 50% of the normal level and about 50% of the normal number of fibrinogen receptors); her mother has only normal GPIIb. These results indicate that the patient has heterozygous GPIIb molecules with an abnormality of GPIIb at the molecular level. Studies on this abnormal GPIIb should provide information about the function of GPIIb and the mechanism of its biosynthesis.

A fully automatic instrument for the determination of electrophoretic mobility of colloidal particles was applied to human platelets. A significant increase in platelet electrophoretic mobility was observed one day after a laparotomy. This suggests that a selective consumption of platelets with smaller surface negative charge may occur during postoperative hemostatic plug formation or under surgical stress. In addition, the difference in electrophoretic mobility observed between males and females suggests an effect of estrogen on platelets.

Several lectins have been studied for their effects on the interaction of thrombin with human platelets. Wheat germ agglutinin, concanavalin A and Ricinus communis lectin increased the number of high affinity sites for diisopropylphosphothrombin on washed platelets from 3000 to about 12 000 but the binding affinities were unchanged (Kd approx 4 nM). Two other lectins, Lens culinaris and Bandieria simplicifolia, were without effect. (2) Using formalinized platelets to avoid possible complications of the platelet release reaction, wheat germ agglutinin showed a marked increase (5-fold) in the binding of active thrombin, peanut agglutinin had no effect while Ricinus communis and :Bandieria simplicifolia showed marginal increases (2-fold). Thrombin binding was decreased to about one quarter with Lens culinaris, Phaseolus vulgaris and concanavalin A. (3) Wheat germ agglutinin caused a synergistic increase of platelet aggregation at low concentrations of thrombin (12.5 mU/ml) and ADP (1 microM), both in the absence and presence of added fibrinogen, but had no effect on ristocetin-induced aggregation.

The relationship between platelet surface negative charge and hyperfunction was examined by determining electrophoretic mobility (EPM), aggregability, and sialic acid of platelets in prostatic cancer, prostatic cancer with estrogen, prostatic cancer with estrogen and aspirin, prostatic hypertrophy, and healthy aged males. Estrogen treated prostatic cancer patients had significantly higher platelet EPM. A good linear correlation was found between sialic acid and EPM (r = 0.97, p less than 0.001). EPM was negatively correlated with primary aggregations by adrenaline and ADP but not with secondary or maximum aggregations, suggesting increased surface negative charge may inhibit primary aggregation. Estrogen and platelet population changes influenced surface negative charge. Neuraminidase removal of platelet surface sialic acid resulted in dose-dependent decreases of EPM which paralleled decreases in sialic acid. Aspirin treated patients and platelets incubated with aspirin in vitro both showed increased platelet EPM. These results suggest that platelet surface negative charge may directly affect platelet function.

To examine the relationship between glycoprotein Ib and other proteins in the platelet membrane and the interaction of this protein with thrombin, platelets were crosslinked by two cleavable reagents, SADP (N-succinimidyl(4-azidophenyldithio)propionate) and DTSSP (3,3'-dithiobis(sulfosuccinimidyl propionate]. Two-dimensional, unreduced-reduced sodium dodecyl sulphate (SDS)-polyacrylamide electrophoresis and staining by silver or wheat germ agglutinin-conjugated peroxidase, after protein transfer to nitrocellulose, demonstrated that SADP intramolecularly crosslinked glycoprotein Ib and formed intermolecular complexes of glycorprotein IIb and some high molecular weight proteins. DTSSP intermolecularly crosslinked glycoprotein Ib, glycoprotein IIb, and other high molecular weight proteins. With a low concentration of 125I-labeled TLCK-thrombin (6 nM), crosslinking with SADP yielded a 200 000 Da complex containing radioactive-labeled thrombin, and high TLCK-thrombin concentration (0.1 microM) gave the complex and a 167 000 band. alpha- and TLCK-thrombin crosslinking with DTSSP also yielded the 200 000 complex formed by reaction with SADP or DTSSP was markedly reduced by preincubation of platelets with excess unlabeled TLCK-thrombin and had a pI similar to glycoprotein Ib. These results suggest that glycoprotein Ib is one of the proteins composing the high affinity receptor for thrombin.

Human platelets were separated into density subpopulations by using a step-wise gradient of Percoll in Tris-NaCl buffer. The absolute value of the electrophoretic mobility (EPM) of the density subpopulations was found to be a linear function of the density of the platelets, with EPM becoming less negative with increasing platelet density. Platelet volume distributions, mode volume, and sialic acid and protein contents were found to increase with platelet density, while no differences were found in GPII, GPIII, and GPIV contents among the subpopulations. An estimate of charge density was made from the ratio between the PAS-staining material (membrane GP's) and platelet surface area. The ratio was found to decrease as platelet density increased, consistent with the less negative EPM values observed for the higher density platelets. This lower surface charge of heavier platelets, which would lower charge repulsion between cells, agrees with the premise that heavier platelets are more active.