Characterization of the platelet membrane glycoprotein abnormalities in Bernard-Soulier syndrome and comparison with normal by surface-labeling techniques and high-resolution two-dimensional gel electrophoresis.

The platelets from three patients with Bernard-Soulier syndrome have been analyzed by surface-labeling coupled with two-dimensional gel electrophoresis and compared with normals. As well as the previously described absence or deficiency in glycoprotein (GP) Ib(alpha) it could be shown that GP Ib beta and an additional low molecular weight glycoprotein GP17 were not detectable using carbohydrate-labeling methods or deficient to the same extent as the GPIb alpha subunit. In addition, the thrombin cleavable glycoprotein could not be detected using carbohydrate-labeling methods in two patients and was deficient in a third. This finding was confirmed in a fourth patient by one-dimensional gel electrophoresis. Thus, the changes in the membrane of Bernard-Soulier platelets are more complex than previously thought.

Platelet membrane glycoproteins implicated in ristocetin-induced aggregation. Studies of the proteins on platelets from patients with Bernard-Soulier syndrome and von Willebrand's disease.

The antibiotic ristocetin only aggregates platelets in the presence of plasma von Willebrand factor. Platelets from patients with Bernard-Soulier syndrome do not aggregate upon addition of ristocetin although, in contrast to von Willebrand's disease, plasma levels of factor VIII complex (factor VIII clotting activity, von Willebrand factor activity, and von Willebrand antigen) are normal. The membrane surface of normal platelets was modified and compared to the surface of platelets from a patient with Bernard-Soulier syndrome in an attempt to identify the receptor involved in von Willebrand factor-ristocetin-induced aggregation. After the incubation of washed normal platelets with a preparation of ristocetin previously shown to contain a proteolytic contaminant, the aggregation response is significantly decreased on addition or normal plasma. Analaysis by gel electrophoresis of such platelets when stained for carbohydrate revealed a decrease in the relative amounts of membrane glycopro-eins. Chymotrypsin-treated normal platelets had less membrane glycoproteins in addition to giving a reduced aggregation response in ristocetin-induced aggregation. Staining of gels for protein and carbohydrate indicated that there was an extensive change in the surface of Bernard-Soulier platelets, whereas those from patients with von Willebrand's disease appeared the same as normal. Platelets from patients were labeled by the lactoperoxidase iodination technique. Not only was the relative intensity of staining of platelet-specific proteins and glycoproteins changed in Bernard-Soulier platelets, but the iodination of the glycoproteins on the membrane surface relative to other membrane constituents was lower. In contrast, platelets from patients with von Willebrand's disease showed a normal exposure of membrane components. These data suggest therefore that membrane glycoproteins may play a functional role in ristocetin-induced aggregation.

Evidence that the platelet plasma membrane does not contain a (Ca2+ + Mg2+)-dependent ATPase.

The present study was designed to determine the subcellular distribution of the platelet (Ca2+ + Mg2+)-ATPase. Human platelets were surface labeled by the periodate-boro[3H]hydride method. Plasma membrane vesicles were then isolated to a purity of approx. 90% by a procedure utilizing wheat germ agglutinin affinity chromatography. These membranes were found to be 2.6-fold enriched in surface glycoproteins compared to an unfractionated vesicle fraction and almost 7-fold enriched compared to intact platelets. In contrast, the isolated plasma membranes showed a decreased specific activity of the (Ca2+ + Mg2+)-ATPase compared to the unfractionated vesicle fraction. This decrease in specific activity was found to be similar to that of an endoplasmic reticulum marker, glucose-6-phosphatase, and to that of a platelet inner membrane marker, phospholipase A2. We conclude, therefore, that the (Ca2+ + Mg2+)-ATPase is not located in the platelet plasma membrane but is restricted to membranes of intracellular origin.

On the significance of the influx of calcium ions into stimulated human blood platelets.

Blood platelets, upon stimulation with various substances, take up calcium ions from the suspending medium. This influx occurs simultaneously with the release reaction, i.e. the specific secretion of a variety of substances from storage organelles and the second wave of aggregation. Various inhibitors of the release reaction inhibit this Ca2+ influx. Platelets previously loaded with 45Ca show an increased efflux of the cation upon stimulation by thrombin. These results suggest that the plasma membrane acquires an increased permeability to Ca2+ only in a later phase of platelet activation, in most cases after the earlier release of Ca2+ into the cytoplasm from Castoring organelles. Rapid shape change and release proceed independently of external calcium, whereas clot retraction depends upon a prolonged increased permeability of the plasma membrane to this cation.

Platelet activation studied by fluorescence polarization.

Platelet activation was elevated by changes in the fluorescence anisotropy of the sulfhydryl-reactive fluorescent probe, (5-[2-(iodoacetyl) aminacetyl]aminonaphthalene-1-sulfonic acid. The membrane-permeable fluorophore was shown to bind to a multitude of cytoplasmic and membrane proteins. Platelets were stimulated by addition of thrombin, arachidonic acid or ADP under conditions that did not induce aggregation. A sudden increase in the fluorescence anisotropy, r of moderate degree (25-33%) occurred during the first 60 s after exposure of platelets to the aggregating agents and was sustained during the entire period of observation (15-18 min). Phenylmethylsulfonyl thrombin was unable to produce these changes in fluorescence anisotropy. Preincubation of platelets with colchicine reduced r within 30-60 s after platelets were exposed to thrombin. These findings are interpreted as an indication of a general decrease in the 'motional freedom' of the fluorophores and indirectly their ligand molecules.

High resolution two-dimensional gel electrophoresis of the proteins and glycoproteins of human blood platelets and platelet membranes.

The proteins and glycoproteins of human blood platelets and platelet membranes in both the reduced and the unreduced states have been analysed by isoelectric focusing and sodium dodecyl sulphate-discontinuosus polyacrylamide gel electrophoresis in a two-dimensional technique. Gels which had been stained with periodic acid-Schiff's reagent could be counter-stained with Coomassie Brilliant Blue, simplifying the recognition of components which stain with both reagents. The major glycoproteins and some of the proteins have been identified and the characteristics of the membrance and of the whole platelet components established in this system.

Regulation of the intracellular calcium level in human blood platelets: cyclic adenosine 3',5'-monophosphate dependent phosphorylation of a 22,000 dalton component in isolated Ca2+-accumulating vesicles.

Two protein kinase activities have been separated from the supernatants of homogenized human blood platelets by DEAE cellulose chromatography. One of them (peak I enzyme) is an efficient stimulator of the uptake of Ca2+ into isolated membrane vesicles in the presence of cyclic AMP and ATP. The second (peak II enzyme), although equally active towards histone, exerts only about one third of the activity of the peak I enzyme. The stimulation of Ca2+ uptake is accompanied by the phosphorylation of a membrane protein with an apparent molecular weight of 22 000, which appears to play an essential role in the regulation of the intracellular Ca2+ level and hence of platelet activity.

Further characterization of calcium-accumulating vesicles from human blood platelets.

Human blood platelets are capable of removing Ca2+ from the cytoplasm by means of an active, ATP-dependent and cyclic AMP-stimulated transport system. Calcium-accumulating vesicles are obtained by sonicating platelets. On density gradient centrifugation, this activity is found in the heavier of two membrane fractions. Concentrated in this fraction are also the Ca2+-stimulated Mg2+-ATPase and glucose-6-phosphatase, believed to be a marker for internal membrane systems. When the isolated vesicles are loaded with Ca2+, a third band separates from the two vesicular fractions in the density gradient. This band C contains virtually all the Ca2+-accumulating activity. Evidence that this activity is due to an active uptake and not to surface binding or adsorption is presented. Whereas electron microscopy does not reveal striking differences between active and inactive fractions, differences in protein composition are revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Furthermore, this band contains an enzyme system which converts arachidonic acid to malondialdehyde and therefore this fraction must be the site of prostaglandin synthesis. Membranes prepared by loading platelets with glycerol, followed by osmotic lysis are unable to accumulate calcium. In sodium dodecyl sulphate-polyacrylamide gel electrophoresis such membranes show significant differences in their protein pattern as compared to the actively Ca2+-accumulating vesicular membranes of band C. All preparations with Ca2+-accumulating activity also contain markers for plasma membranes and the question whether this activity is due exclusively to an intracellular structural element equivalent to the sarcoplasmic reticulum of muscle or whether an "extrusion pump" expelling Ca2+ to the outside of the cell is also involved, cannot yet be ;nswered.

A comparative study of the effect of modification of the surface of human platelets on the receptors for aggregated immunoglobulins and for ristocentin-von Willebrand factor.

The receptors for aggregated immunoglobulin G (IgG) (an Fc receptor) and for ristocetin-von Willebrand factor on human platelets were studied by means of various modifications of the platelet surface. The expression of these receptors was measured by the agglutination of platelets to ristocetin in the presence of von Willebrand factor, which is part of the factor VIII complex, and by the binding of aggregated IgG coupled to 3H-labelled diazobenzene. Treatment of platelets with chymotrypsin, trypsin, papain and pronase which removed protein and glycoprotein from the platelet under conditions where the release reaction was inhibited caused loss of the expression of the receptor for ristocetin-von Willebrand factor and an enhancement of that for aggregated IgG. Induction of membrane changes with ADP and of the release reaction with the ionophore A23187 abolished agglutination to ristocentin-von Willebrand factor but did not alter the receptor for aggregated IgC. Possible contributions of unspecific membrane changes, produced by protease treatment of platelets, to the modification of receptor expression were eliminated by the use of formaldehyde-treated platelets. Trypsin, papain and pronase destroyed the ability of these platelets to agglutinate to ristocetin-von Willebrand factor but produced no change in the binding of aggregated IgC. Therefore, the receptor for ristocetin-von Willebrand factor is truly sensitive to proteolysis while the Fc receptor is not, but is partially masked by protease-sensitive material.

Isolation of the membrane glycoproteins of human blood platelets by lectin affinity chromatography.

The major platelet membrane glycoproteins have been solubilized in 1.0% sodium deoxycholate and subjected to affinity chromatography on the lectins from Lens culinaris, wheat germ and Abrus precatorius. Polyacrylamide gel electrophoresis in the presence and absence of a reducing agent together with the differential binding of the lectins to the glycoproteins permitted the distinction of at least seven separate glycoprotein entities. A new nomenclature for the glycoproteins is proposed to accomodate the additional data. Using combinations of lectin columns, glycoproteins Ia and Ib could be prepared in a pure state and IIb and IIIa could be greatly purified. The binding of lectins to glycoprotein Ib has been strongly implicated as a necessary step in the aggregation response of platelets to lectins.

Stimulation of calcium uptake in platelet membrane vesicles by adenosine 3',5'-cyclic monophosphate and protein kinase.

The events involved in platelet shape change, aggregation, the release reaction and contraction are thought to be mediated by the availability of Ca2+. Increased cytoplasmic calcium, released from intracellular stores, triggers platelet activity, and increased concentration of adenosine 3',5'-cyclic monophosphate (cyclic AMP) inhibits platelet alterations. We have studied the hypothesis that cyclic AMP may regulate the level of platelet cytoplasmic calcium by stimulating calcium removal by a membrane system. Such a hypothesis would be consistent with the reversibility of most manifestations of platelet activation. Human platelets were sonicated and unlysed platelets, mitochondria and granules were removed by centrifugation at 19 000 X g. Electron microscopy shows that the sediment, after centrifugation of the supernatant at 40 000 X g consists to a large extent of membrane vesicles. Such preparations actively concentrate calcium, as measured by the uptake of 45Ca, and also have the maximal calcium-stimulated ATPase activity. Optimal calcium uptake requires ATP and oxalate, and release of calcium from loaded vesicles was stimulated by the calcium ionophore A23187 and inhibited by LaCl3. These data indicate that calcium was being actively concentrated within membrane vesicles. After washing of such preparations in the absence of ATP, their capacity to take up Ca2+ is reduced to an initial value of 2.8 nmol/mg protein per min. In the presence of 2 - 10(6) M cyclic AMP to which was added a protein kinase preparation from human platelets, up to a 3-fold increase of this rate of uptake was observed. These results suggest that in platelets, as in muscle, cyclic AMP is a regulatory factor in the control of cytoplasmic calcium. Although the cyclic nucleotide may have still other functions, it appears likely that the well-known inhibition of many platelet activities by high intracellular cyclic AMP concentrations is directly linked to the stimulation of the removal of Ca2+ from the cytoplasm.

Distribution of platelet glycoproteins and phosphoproteins in hydrophobic and hydrophilic phases in Triton X-114 phase partition.

Platelets, either unlabelled, surface-labelled by the periodate NaB3H4 method or metabolically labelled with 32P were solubilized in Triton X-114 and partitioned into aqueous and detergent phases. The phases were analysed by two-dimensional polyacrylamide gel electrophoresis followed by silver-staining, fluorography or indirect autoradiography. Each of the phases contains a distinct set of proteins. The surface-labelled glycoproteins partition into the hydrophobic phase with the notable exceptions of glycoproteins Ib and GP17(5.8-6.5) and minor amounts of a few others. The phosphoproteins which undergo increased phosphorylation on platelet activation in general separate in the hydrophobic phase, while higher molecular weight phosphoproteins were principally in the hydrophilic phase. This method might be used as a first step in purifying many platelet components.

Characterization of human blood platelet membrane proteins and glycorproteins by their isoelectric point (pI) and apparent molecular weight using two-dimensional electrophoresis and surface-labelling techniques.

Intact human blood platelets were radioactively labelled at the surface by techniques specific for proteins or glycoproteins. Labelled platelet samples were analyzed by a high-resolution two-demensional separation system involving isoelectric focusing in the first dimension and discontinuous sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the second. The major platelet membrane glycoprotein (GP) bands (Ib, IIb, IIIa and IIIb) were found to be highly heterogeneous even after removal of terminal sialic acid residues. Lactoperoxidase-catalyzed iodination of platelets showed that the major labelled proteins (Ib, IIb, IIIa and IIIb) had altered isoelectric points (pI) and molecular weights after neuraminidase treatment. A number of membrane glycoproteins previously undetected by one-dimensional gel electrophoresis were demonstrated and good evidence provided that the major platelet surface proteins are glycosylated.

Tryptic peptide map analysis of the major human blood platelet membrane glycoproteins separated by two-dimensional polyacrylamide gel electrophoresis.

Washed platelets were surface-labelled by lactoperoxidase catalyzed iodination and either the platelets or membranes were solubilized in detergent and applied to a wheat germ agglutinin-Sepharose column and a Lens culinaris lectin Sepharose column coupled sequentially. The glycoproteins eluted from the lectin columns were separated by two-dimensional gel electrophoresis. Alternatively, labelled whole platelets or membranes were solubilized and then directly separated by two-dimensional polyacrylamide gel electrophoresis. Spots corresponding to specific glycoproteins identified by apparent isoelectric point (pI), apparent molecular weight (Mr), staining and labelling characteristics were cut from the gels and analyzed by tryptic peptide mapping. The maps of the individual glycoproteins(GP) Ia, Ib, IIa, IIb, GP4-4.5 132-135, IIIa, IIIb and IIIc were all different. Glycoproteins with the same Mr but different pI were distinct with the exception of regions of GP Ib. There were minor differences in the maps of glycoproteins separated in the reduced or non-reduced state. Tryptic peptide maps provide a valuable additional parameter for the identification and characterization of platelet glycoproteins.

The formation of the haemostatic plug--a special case of platelet aggregation. An experiment and a survey of the literature.

The formation of the haemostatic plug is an extremely fast process. This excludes, at least in its first phase, the involvement of soluble activating agents released from or produced by the platelets. An experiment with ADP-activated, formaldehyde-fixed platelets shows that platelets with activated fibrinogen receptors will bind inactive platelets in the presence of fibrinogen and Ca(2+)-ions. A survey of the literature shows that platelet activation is accompanied by the clustering of the fibrinogen receptors. The surface of an activated platelet, which makes part of the growing haemostatic plug therefore is covered with patches of tightly packed fibrinogen. This allows the multisite combination with the statistically distributed low affinity receptors of the newly arriving platelets. Tightly packed fibrinogen, as present on clusters of the activated GP IIb/IIIa receptors as well as when absorbed to artificial surfaces acts as an activator of platelets. Thus, the propagation of the activation process is possible without a requirement for other, external activators. Such agents, which are released from platelets and, finally, thrombin formation, are nonetheless of vital importance, not for the formation but for the consolidation of the haemostatic plug.

Movement of calcium ions and their role in the activation of platelets.

The increase of the cytoplasmic Ca-concentration plays a central role in the initiation of platelet activation. Four kinds of movements of Ca-ions are presumed to occur during this process: a) Ca-ions liberated from membranes induce the rapid shape change. b) Vesicular organelles release Ca-ions into the cytoplasm which initiate the release reaction. c) The storage organelles called dense bodies, secrete their contents including Ca-ions to the outside during the release reaction. d) At the same time a rearrangement of the plasma membrane occurs, resulting in an increase in its permeability for Ca-ions as well as in an increase in the number of Ca-binding sites. Since most processes occurring during platelet activation are reversible, the platelet must be equipped with a mechanism which removes Ca-ions from the cytoplasm. A vesicular fraction obtained from homogenized platelets indeed accumulates Ca actively. This Ca-pump is stimulated by cyclic AMP and protein kinase; it may be involved in the recovery of platelets after activation.

Surface labeling of human platelets by reductive methylation.

A simple, reproducible method for the specific labeling of the surface proteins of human platelets with tritium is described. The labeled platelets were analyzed by two-dimensional gel electrophoresis (isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis) followed by fluorography and the results compared with those obtained by conventional methods. Reductive methylation gave an intense labeling of membrane glycoproteins. There was no cross-linking of the labeled membrane proteins by formaldehyde nor could clear evidence be found that inner proteins were labeled by permeation of the reagents through the plasma membrane. As well as previously described platelet membrane glycoproteins, several others could be detected that were not invariable seen using labeling techniques.

Binding of anti-actin autoantibodies to platelets.

Normal platelets incubated with anti-actin autoantibodies (AAA) (from the serum of patients with chronic aggressive hepatitis) do not show binding of these antibodies as seen by indirect immunofluorescence. AAA serum does not inhibit thrombin-induced clot retraction, despite the binding of the antibodies to platelets in the clot. Similarly, AAA serum does not affect "reversible" or "irreversible" aggregation (induced by ADP, collagen or epinephrine), despite the binding of the antibodies to platelet actin under such circumstances. AAA also bind to platelets when aggregation is inhibited by EDTA. The incubation of "reversibly" aggregated platelet with AAA results in a small but definite binding of AAA to platelets. These findings suggest that during "irreversible" and/or "reversible" aggregation, changes take place at the surface of platelets which expose the antigen at the surface of the cell.

Improvement of the periodate-borohydride surface-labeling method for human blood platelets.

The periodate/sodium boro[3H]hydride ([3H]-NaBH4) method is extensively used for the specific labeling of cell surface glycoproteins. Reduction with tritiated borohydride is also used in other surface-labeling techniques, the neuraminidase/galactose oxidase/[3H]-NaBH4 method (specific for terminal galactose and N-acetyl-galactosamine residues) and the pyridoxal phosphate/[3H]-NaBH4 method (specific for protein). By modification of the reaction conditions during the periodate-oxidation and borohydride-reduction, the ratio of the incorporated to the total added radioactivity could be increased by a factor of 50, while the specific activity of the labeled material was twice as high as in the original method. Alternatively, by another modification, the specific activity of the labeled material could be increased about 10-fold. The influence of the most important parameters was investigated in detail. Sodium dodecyl sulfate gel electrophoresis and fluorography demonstrate that the labeling pattern of the membrane glycoproteins is the same as with the conventional method.

Morphology of the interaction of collagen fibrils with normal human platelets and thrombasthenic platelets.

The ultrastructure of the platelet contacts with collagen fibrils (CF) as well as the course taken by CF on the platelet surface were studied on ultrathin sections of platelets and CF. Platelets from normal donors and from a patient with thrombasthenia were incubated in citrated plasma with collagen. For electron microscopy a protein-stabilizing fixation procedure was applied. Platelet-collagen contacts (PCC) are characterized by a distance of 7 +/- 3 nm between the platelet membrane and the CF; the gap contains electron-dense bridges. The PCC of normal and thrombasthenic platelets are morphologically identical. Hence, it is unlikely that the glycoproteins IIb/IIIa-complex, which is absent in thrombasthenic platelets, plays a significant role in the platelet collagen interaction. The CF induce random movements of the platelets and their pseudopods, whereby the fibrils, which often show multisite attachment, coil up around the platelet surface; they become bent and often display drastic directional changes. CF are found inside invaginations of the platelet membrane as well as in depressions of the platelet surface. These processes require an involvement of the platelet's contractile system, which appears to interact reversibly with the platelet plasma membrane.