A study of intracellular transport of secretory glycoproteins in rat liver.

To study the transport of secretory glycoproteins in the endoplasmic reticulum of rat liver, the distribution of nascent glycoproteins in the membrane and luminal fraction of rough and smooth microsomes has been examined after a short-time incorporation of radioactive glucosamine in vivo. 50--60% of the radioactivity was associated with the membranes of rough and smooth microsomes, whereas about 10% of the serum albumin was found in the same fractions. The relative amount of radioactivity in the membranes was the same whether the luminal content of the microsomal vesicles was released by sonication, French press, Triton X-100, Brij 35 or sodium deoxycholate. The distribution of labeled glycoproteins between the membrane and luminal fraction of rough and smooth microsomes did not change during the time interval of 15--120 min after administration of the isotope. The similarity of the labeling patterns obtained after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis indicated that the same set of glycoproteins were located in the lumen and the membrane of rough and smooth microsomes. A specific precipitation of nascent glycoproteins from both the membrane and luminal fractions of rough and smooth microsomes were obtained with rabbit antiserum against rat serum. The nascent glycoproteins associated with the membranes were not released by high ionic strength or treatment with mercaptoethanol. A slow exchange between [14C]glucosamine-labeled glycoproteins in the lumen and membrane fraction was, however, found.

Comparison of protein and lipid composition of the human platelet alpha-granule membranes and glycerol lysis membranes.

Platelet glycerol lysis membranes and alpha-granule membranes were compared with respect to protein and lipid composition. Crossed immunoelectrophoresis using antibodies against whole platelets, and sodium dodecyl sulphate polyacrylamide gel electrophoresis, revealed the presence of the glycoproteins IIb and IIIa, myosin and an antigen termed G4 in both membrane fractions. The glycoproteins Ia, Ib and IIIb, in addition to beta 2-microglobulin and actin, appeared specific for the glycerol lysis membranes, whereas two antigens, termed G8 and G18, were observed only in the alpha-granule membranes. The localization of glycoprotein IIa was inconclusive. Comparison with the surface-located proteins revealed that the glycerol lysis membranes represented a reasonable approximation to a plasma membrane preparation. Radioactively labelled immunoprecipitates obtained after crossed immunoelectrophoresis of 125I-labelled platelets were cut out and applied to sodium dodecyl sulphate electrophoresis on polyacrylamide slab gels. Autoradiography of the dried gels revealed that antigen G4 represented a protein with an average molecular weight of 146 000 in its unreduced state and 132 000 in its reduced state. Antigen G18 represented a protein of molecular weight 130 000-135 000 in the reduced as well as unreduced state. Quantitation of protein and lipids showed that the alpha-granule membranes contained about one-third as much cholesterol and 2-times as much protein in relation to phospholipids as compared to the glycerol lysis membranes. No significant difference between the two membrane preparations was found as regards the composition of their phospholipids.

Characterization of the proteins of isolated human platelet alpha-granules. Evidence for a separate alpha-granule-pool of the glycoproteins IIb and IIIa.

The protein composition of a well-defined alpha-granule preparation isolated from human platelets has been studied. Crossed immunoelectrophoresis against polyspecific platelet antibodies revealed more than 20 immunoprecipitates. The glycoprotein IIb-IIIa complex represented a major antigen in the Triton X-100-solubilized alpha-granule preparation and cross-reacted with the corresponding platelet membrane antigen. Furthermore, after lactoperoxidase-catalyzed 125I-iodination of whole platelets it was not labelled, in contrast to its membrane-located counterpart. This indicates an intracellular location of glycoproteins IIb and IIIa, probably as constituents of the alpha-granules. Fibrinogen, platelet factor 4, albumin, factor VIII-related antigen and the main granule glycoprotein (thrombinsensitive protein, thrombospondin) were identified in the alpha-granule preparation by the crossed immunoelectrophoresis technique. Crossed affinity immunoelectrophoresis using lectins revealed the presence of at least seven glycoproteins, and six sialoglycoproteins were identified by their altered electrophoretic mobility after neuraminidase treatment. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of reduced samples of the alpha-granules revealed at least 15 Coomassie Brilliant Blue-staining polypeptide bands, one of which comigrated with myosin heavy chain. No prominent band was observed in the actin region. Five glycopolypeptide bands were observed after periodic acid-Schiff staining. The dominant three represented the main granule glycoprotein, glycoprotein IIb and glycoprotein IIIa, respectively. More glycoproteins seem to be present in the alpha-granules than was previously recognized.

Demonstration of 125I-labelled thrombin binding platelet proteins by use of crossed immunoelectrophoresis and autoradiography.

A possible receptor for thrombin on the platelet membrane has been identified. Whole platelets were treated with 125I-labelled thrombin followed by washing of the platelets, solubilization in Triton X-100, crossed immunoelectrophoresis and autoradiography. A heavily labelled antigen which migrated slightly more slowly than albumin was observed. No corresponding arc was seen on the same immunoplate when stained with Coomassie brilliant blue, indicating that the antigen possessed weak antigenic properties and/or was present in very small amounts. When 125I-labelled thrombin that had been inactivated by phenylmethylsulphonyl fluoride was used, no such labelled arc was seen. The radiolabelled immunoprecipitate does not represent any of the antigens identified hitherto in the immunoelectrophoretic patterns obtained with platelets or platelet material. The electrophoretic mobility of the antigen was influenced neither by neuraminidase treatment of the platelets prior to the 125I-labelled thrombin exposure nor by inclusion of concanavalin A, wheat-germ lectin or lentil lectin in the gel during the first-dimension electrophoresis. This suggests that the antigen does not represent a glycoprotein. Upon subcellular fractionation the radioactively labelled arc was observed in the cytosol fraction following crossed immunoelectrophoresis and autoradiography. Analysis of the secreted proteins after induction of the release reaction with 125I-labelled thrombin revealed labelling of immunoprecipitates representing thrombospondin, albumin and the 'line' form of platelet factor 4. This confirms that stable complexes of 125I-labelled thrombin and platelet proteins can exist in the presence of Triton X-100 and during electrophoresis.

Evidence for release of soluble, but not of membrane-integrated, proteins from human platelet alpha-granules.

Proteins released from stimulated platelets were compared to those of a well-defined preparation of alpha-granules and the soluble cytoplasm by crossed immunoelectrophoresis. Nearly all releasable proteins were detected in the alpha-granule, whereas the true proteins of the soluble cytoplasm were not released. The released glycoproteins interacted with lectins similarly to their alpha-granula-located counterparts. The alpha-granules were divided into soluble contents and membranes by ultrasonication followed by ultracentrifugation. The proteins of the soluble content corresponded to those released from the stimulated platelets. This observation was also supported by SDS-polyacrylamide gel electrophoresis. The results indicate that the bulk of the proteins released from stimulated platelets originate from the soluble content of the alpha-granules. Two major alpha-granule antigens as well as the myosin heavy chain were not released and recovered in the alpha-granule membrane. These results support the hypothetical exocytosis mechanism for the release of alpha-granule proteins from platelets.

Involvement of divalent cations in the complex between the platelet glycoproteins IIb and IIIa.

The major immunoprecipitate (No. 16) seen on crossed immunoelectrophoresis of Triton X-100-solubilized platelet proteins against whole platelet antibodies represents a complex containing the membrane glycoproteins IIb and IIIa. When EDTA is present during the solubilization, immunoprecipitate 16 as such is not observed, and two new arcs, termed 16a and 16b, appear. As with 16 these immunoprecipitates become radioactively labelled on lactoperoxidase-catalyzed iodination of platelets. Immunoprecipitate 16a showed partial immunochemical identity with 16, and was precipitated by an antibody raised against immunoprecipitate 16. The areas covered by immunoprecipitates 16, 16a and 16b were strongly reduced compared to normal with platelets from a patient with Glanzmann's thrombasthenia type II. Such platelets are known to contain reduced amounts of glycoproteins IIb and IIIa. The new arcs appearing when divalent cations are chelated by EDTA thus represent proteins derived from the immunoprecipitate 16 proteins, and divalent cations seem to be necessary to preserve the protein complex containing glycoprotein IIb and IIIa. The different complex formations between the components of immunoprecipitate 16 may reflect biochemical alterations of functional importance.

Dissociation of the glycoprotein IIb-IIIa complex in isolated human platelet membranes. Dependence of pH divalent cations.

The platelet membrane glycoproteins IIb and IIIa normally exist as a complex which forms a predominant immunoprecipitate after crossed immunoelectrophoresis of Triton-X-100-solubilized platelets. Dissociation of the complex occurs by solubilization in the presence of EDTA or EGTA at pH 8.7 and is readily verified by crossed immunoelectrophoresis. Incubations of isolated membranes with EDTA or EGTA at various pH levels were performed. Removal of the chelators and solubilization showed no dissociation of the glycoprotein IIb-IIIa complex in membranes incubated at pH below 8.0. At pH above 8.0 a dissociation which increased with increasing pH was seen. Under these conditions, dissociation appears to take place already in the intact membranes. The tendency of the glycoprotein IIb-IIIa complex to become dissociated with EDTA or EGTA at increasing pH seems to be due to increased chelating capacity of the chelators concomitant with a decreased chelating capacity of glycoprotein IIb and IIIa. The divalent cations Ca2+ and Mg2+, but not Cu2+, Zn2+, Mn2+ or Sr2+, in molar concentrations below that of EGTA were able to prevent the dissociation of the glycoprotein IIb-IIIa complex by the chelator at pH 9.0, indicating that Ca2+ as well as Mg2+ can be used to keep the complex together. In some experiments it was possible to reverse the dissociation in the membranes after removal of EDTA. At pH 7.5 reassociation occurred within 15 min whether divalent cations were added or not. At pH 9.0. reassociation occurred within 2 h provided Ca2+ was present. The tendency of glycoprotein IIb and IIIa to form a complex thus appeared to be most pronounced over the physiological pH range and to be a rapid process in platelet membranes under such conditions.

Demonstration of a new glycoprotein Ib-related component in platelet extracts prepared in the presence of leupeptin.

The water-soluble protein glycocalicin is generated during platelet lysis by a proteolytic attack on the integral membrane glycoprotein GP Ib. However, only small amounts of glycocalicin are formed when platelets are solubilized by 1% Triton X-100. Crossed immunoelectrophoresis of such extracts using an antiserum to glycocalicin, shows a continuous immunoprecipitate consisting of two peaks, one representing glycocalicin and the other GP Ib. When leupeptin was present during solubilization, subsequent immunoelectrophoresis revealed yet another GP Ib-related component represented by a third, slow-migrating peak of the immunoprecipitate. During incubation of platelets with dibucaine followed by solubilization in the presence of leupeptin, a gradual transformation of this new form of GP Ib into the previously defined one took place prior to the formation of glycocalicin. An increase followed by a decrease in the agglutination response of the platelets to bovine von Willebrand factor occurred concomitant with these transformations. SDS-polyacrylamide gel electrophoresis of Triton X-100 extracts of platelets did not reveal any difference in the size of GP Ib whether or not leupeptin had been present during the solubilization.

Utility of a modified calibration model for reliable conversion of thromboplastin times to international normalized ratios.

The combined thromboplastin reagent, Normotest, has been calibrated against the secondary international reference preparation for bovine thromboplastin, OBT/79. Three expert laboratories measured up to 62 patients on stabilized oral anticoagulant therapy and up to 20 normals in order to establish an INR-scale for Normotest. It was found that the model recommended by the WHO was less suited for the calibration of this thromboplastin. This is the first study in which three independent laboratories demonstrate a similar bias of the WHO calibration model. A modified model in which a correction factor is introduced was applied to the problem and proved to give a reliable calculation method for INR on Normotest. The mean coefficient of variation of INR calculated between measurements with Normotest and OBT/79 (scatter of data around calibration line) was 4.2-5.0% as compared to 5.1-5.7% for the WHO-method. A conversion scale for percent activities between Normotest and Thrombotest was established showing that the recommended therapeutic range of 5-10% Thrombotest (INR = 4.8-2.8) corresponds to 10-20% Normotest.

Diagnosis of heterozygotes in Glanzmann's thrombasthenia.

A study of a family with a propositus suffering from classical thrombasthenia type I has shown that the new immunochemical methods detect heterozygotes with high reliability. There was no overlapping between heterozygotes and normals, and the concentration of the glycoproteins IIb-IIIa-complex is remarkable constant around 50-60% in the heterozygotes. Furthermore, heterozygotes as a group show an increased bleeding tendency.

Comparison of three D-dimer assays for the diagnosis of DVT:ELISA, latex and an immunofiltration assay (NycoCard D-Dimer).

Ninety-two consecutive patients referred for suspicion of deep venous thrombosis (DVT) were analyzed for D-dimer using ELISA, latex test, and a new immunofiltration method (NycoCard D-Dimer). Contrast venography verified the diagnosis in 40, and excluded the diagnosis in 52 patients. The sensitivity, negative predictive values, specificity and positive predictive values were, for ELISA 98%, 95%, 38% and 54, for NycoCard D-Dimer 100%, 100%, 42% and 57% and for the latex test 73%, 78%, 75%, and 69%, respectively. Sensitivity and specificity were inversely related with increasing pathological cut-off value. Comparison of test results by concentration category revealed a good agreement between ELISA and NycoCard D-Dimer, but to less extent between latex and the two other tests. It is concluded that NycoCard D-Dimer and D-dimer ELISA are well-suited as exclusion tests for DVT. A plasma sample is tested with NycoCard D-Dimer in less than 2 min. Thus, this test combines advantageous analytical properties comparable to the ELISA-test, with rapidity and simplicity comparable to the latex test.

Complex-formation between the fibrin-derived plasmic fragments DD and E demonstrated by crossed immunoelectrophoresis.

The formation of a complex between the fibrin fragments DD and E was studied by crossed immunoelectrophoresis using antibodies against human fibrinogen. The complex formation was seen by a common electrophoretic migration of the DD-fragment and part of the E-fragments. This effect was abolished by a further incubation with plasmin of the preparation containing the (DD) E-complex. This also led to an anodal shift in migration of the E-fragment indicating a transfer from E1 to E3.

Platelet factor XIII is an active enzyme after solubilization and crossed immunoelectrophoresis.

Crossed immunoelectrophoresis of platelets against antiplatelet antibodies has proved to be a valuable tool in the study of platelet proteins (1-8). The advantage of this separation system is that the proteins are separated under nondenaturating conditions and thus to some extent would be expected to maintain their functional properties. Previously, the binding of several proteins to immobilized thrombin (5) and immobilized heparin (9) during crossed immunoelectrophoresis of platelet proteins solubilized in a Triton X-loo-containing buffer has been described. Furthermore, it has been demonstrated that fibrinogen is able to bind to immunoprecipitates containing the glycoprotein IIb-IIIa-complex (7). These studies indicate that the proteins contained in the immunoprecipitates represent biologically active entities. In the present study we provide direct evidence for this by demonstrating enzymatic activity associated with the immunoprecipitate containing Factor XIII in immunoplates obtained after crossed immunoelectrophoresis of solubilized platelets against anti-platelet antibodies.

A new bleeding disorder: lack of platelet aggregatory response to adrenaline and lack of secondary aggregation to ADP and platelet activating factor (PAF).

A bleeding disorder, probably familial, with absent adrenaline-induced platelet aggregation and lack of secondary aggregation response to ADP and platelet activating factor (PAF), is described. The laboratory findings do not fit any hitherto recognized hemorrhagic disease. The disorder was not caused by alpha-adrenergic receptor deficiency, but the ultimate defect has not yet been unraveled. This patient illustrates that a normal response to more than one aggregating stimulus is necessary for normal hemostasis, and indicates a physiopathological role for adrenaline not hitherto recognized. Whether this also applies to PAF remains to be proven.

Glycoprotein IIb-IIIa complex in platelets of patients and heterozygotes of Glanzmann's thrombasthenia.

The concentration of the glycoprotein (GP) IIb-IIIa complex in thrombasthenic platelets of 8 patients of 6 families has been estimated. In the thrombasthenic platelets of 3 patients this complex is absent (thrombasthenia type I and subtype I). In 2 patients only traces are detectable and in 3 patients GP IIb-IIIa complex is strongly reduced (less than 5%). On the basis of the haemostatic data as well as the content of GP IIb-IIIa complex and platelet fibrinogen the classification of these types as subtypes of thrombasthenia type II is discussed. The diagnostic applicability of GP IIb-IIIa complex determination for heterozygote detection in types of thrombasthenia with absent or extremely reduced GP IIb-IIIa complex is shown.

The N-DSK gamma-chain binds to immunoprecipitated GP IIb-IIIa.

The CNBr-split N-terminal disulphide knot of the fibrinogen molecule (N-DSK) binds to ADP-stimulated gel-filtered platelets and immunoprecipitated fibrinogen receptor. To investigate which part of the N-DSK molecule that is involved in this binding, the glycoprotein IIb-IIIa complex (the fibrinogen receptor) was immunoprecipitated in crossed immunoelectrophoresis of Triton X-100 extracts of platelets against rabbit antibodies to whole platelet proteins. The immunoelectrophoresis plates were incubated with solubilized, carboxymethylated 125I-labelled A alpha -, B beta - or gamma-chains of N-DSK, and investigated for binding by autoradiography. The N-DSK gamma-chain, but not the A alpha - or B beta -chains demonstrated binding to the GP IIb-IIIa complex. These results show that the fibrinogen molecule contains a third sequence of amino acids, in addition to the two previously reported ones that can be involved in binding of fibrinogen to the fibrinogen receptor on the platelets.

Competitions between fibrinogen with its degradation products for interactions with the platelet-fibrinogen receptor.

Direct binding of 125-I-labelled plasmic and CNBr-derived fibrin (ogen) fragments (pre-X, X, Y, D, Degta, Efg, E1, N-DSK, N-dsk) to gel-filtered platelets was compared to their ability to support or inhibit ADP-induced aggregation, and to compete with fibrinogen for binding to ADP-stimulated platelets. Pre-X was the only fragment that supported aggregation. All fragments tested except for E derived from fibrinogen (Efg) and Degta bound specifically to the platelets and inhibited ADP-induced aggregation in the presence of fibrinogen. Competitive binding studies with fibrinogen and fragments labelled with different isotopes of iodine, or inhibition of binding of labelled fibrinogen with unlabelled fragments showed that all of the fragments except Efg and Degta were able to compete with fibrinogen for binding. When simultaneous binding of N-dsk and fibrinogen was studied, an increased binding of both ligands was observed probably due to complex formation. The results fully agree with previous findings of binding to immunoprecipitated glycoprotein IIb-IIIa after crossed immunoelectrophoresis. We conclude that the fibrinogen molecule contains at least six sequences responsible for platelet interaction, two in the E domain and two in each of the C-terminal parts of the fibrinogen molecule.

Further studies on the interaction between thrombin and GP Ib using crossed immunoelectrophoresis. Effect of thrombin inhibitors.

The platelet surface protein GP Ib (glycocalicin-related protein) has been shown to be retarded by thrombin-Sepharose 4B in a crossed immunoelectrophoresis system. The interaction between GP Ib and thrombin was abolished when thrombin was blocked either at the active serine site with tosyl-lysine-chloromethyl-ketone (TLCK) or phenylmethylsulfonylfluoride (PMSF) or at the fibrinogen binding site (macromolecular binding site) with N-bromosuccinimide (NBS) or heparin, indicating that both sites have to be freely accessible for the retention of the glycocalicin-related protein by thrombin.

Demonstration of variable forms of the platelet factor 4 immunoprecipitate using crossed immunoelectrophoresis.

Proteins with different electrophoretic properties were precipitated by a monospecific antiserum to platelet factor 4 either as a "line" or as a "peak" precipitate. The "line" form seen on crossed immunoelectrophoresis of whole platelets was retained when immobilized thrombin was included in the intermediate gel. The retention was partially abolished when thrombin had been blocked at the active serine site or at the fibrinogen binding site. The "peak" form seen on analysis of material secreted from platelets passed unaffected through thrombin-Sepharose. It is suggested that platelet factor 4 exists in the platelets in a state different from that observed extracellularly after platelet secretion.

Binding of 125I-labelled fibrin(ogen) fragments to platelets and to immunoprecipitated glycoprotein IIb-IIIa complex.

To further investigate which parts of the fibrinogen molecule that are responsible for its binding to the fibrinogen receptor on human platelets, the following approaches were made: The glycoprotein IIb-IIIa complex (the putative fibrinogen receptor) was immunoprecipitated in crossed immunoelectrophoresis of Triton X-100-extracts of platelets against antibodies to whole platelet proteins. Subsequently, the immunoplates were incubated with 125I-labelled, plasmin- or CNBr-cleaved fibrinogen fragments (pre-X,X,Y,D,Degta,Efg,N-DSK) or fibrin fragments (E1,N-dsk), characterized by partial sequenation. The immunoplates were exposed to X-ray films, and binding of the fragments to the glycoprotein IIb-IIIa complex was examined. The findings were compared to the results obtained from studies on binding of the same fragments to intact gel-filtered platelets after ADP-stimulation. The following conclusions were made: All fragments except Efg and Degta bound to the immunoprecipitated GPIIb-IIIa complex as well as to ADP-stimulated platelets suggesting that at least two sequences in the E domain and one in each of the D domains of fibrinogen are involved in binding to the platelet receptor. The GPIIb-IIIa complex is the only surface-located platelet antigen that binds fibrinogen and the aforementioned fragments. The binding of the fragments to the receptor is dependent on divalent cations.