Geir O. Gogstad

Measurement of protein in cell suspensions using the Commassie brilliant blue dye-binding assay

Abstract The direct measurement of protein in cell suspensions using the Coomassie brilliant blue dye-binding assay demonstrated markedly lower values compared to those obtained with the Lowry assay. It is shown that the addition of small amounts of Triton X-100 or NaOH to the cell suspensions prior to addition of the dye reagent corrected this discrepancy. Standards of soluble proteins may be used for the quantitation of protein in cell suspensions with the dyebinding assay provided that the same amounts of Triton X-100 or NaOH are added to both the standards and the samples.

A radioimmunoassay for thrombospondin, used in a comparative study of thrombospondin, β-thromboglobulin and platelet factor 4 in healthy volunteers

A radioimmunoassay was developed for the platelet alpha-granule protein thrombospondin; concentrations of thrombospondin as low as 3 ng ml-1 could be measured. There was no interference from other components of human biological fluids and no crossreactivity with beta-thromboglobulin (beta-TG) or platelet factor 4 (PF4). Plasma samples were stable when stored at -20 degrees C. Normal human plasma contained 105.0 +/- 31.0 ng thrombospondin ml-1 compared with beta-TG concentrations of 37.2 +/- 10.9 ng ml-1 and PF4 concentrations of 14.7 +/- 10.1 ng ml-1 when samples were carefully taken into a platelet inhibitor cocktail and processed at 0-4 degrees C. Release of thrombospondin during clotting of blood occurred at the same time as that of beta-TG and PF4 and resulted in a serum concentration of 17.5 +/- 5.5 micrograms ml-1. Assay of whole blood gave a platelet thrombospondin content of 89.1 +/- 28.3 ng/10(6) platelets. The concentration in normal urine fluctuated widely from 3 to 22.5 ng ml-1, and was unrelated to urine flow. The half-life of thrombospondin in vivo was about 9 h, much longer than that of either beta-TG or PF4. Unlike PF4, it was not released into the blood following an intravenous heparin injection. Bovine, ovine, canine and porcine sera contained thrombospondin which crossreacted immunologically with the human molecule; these species would be suitable animal models for the study of thrombospondin and its value as a platelet release marker.

Characterization of the proteins of isolated human platelet α-granules: Evidence for a separate α-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 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.

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

Heparin‐binding platelet proteins demonstrated by crossed affinity immunoelectrophoresis

Summary. Platelet proteins that interact with heparin were studied using crossed affinity immunoelectrophoresis. Platelet proteins solubilized in Triton X‐100 were applied to crossed immunoelectrophoresis against anti‐platelet antibodies, and an intermediate gel containing heparin covalently linked to Sepharose 4B was inserted. Six immunoprecipitates were absent or showed an altered position compared to control immunoplates, indicating that the corresponding antigens were bound to the immobilized heparin. These precipitates represented platelet factor 4, thrombospondin, glycoprotein lb, and three antigens termed G4,17 and 25. The subcellular location of the heparin‐binding proteins was either in the surface membrane (glycoprotein lb and the antigens 17 and 25), or in the a‐granules (platelet factor 4, thrombospondin and G4). Both forms of platelet factor 4 appearing after crossed immunoelectrophoresis, i.e. a line‐form and a peak‐form, bound strongly to the heparin. Glycoprotein lb showed a weak binding whereas its proteolytic split product glycocalicin did not significantly bind to the heparin in the present system. It is concluded that the platelets contain at least six heparin‐binding proteins which are present on the cellular surface or are able to be exposed to the extracellular medium after the release‐reaction has occurred.

A method for the isolation of α-granules from human platelets

Abstract A method for the isolation of α-granules is described. A two-step French pressure cell homogenization procedure which directly produced an organelle concentrate suited for loading on density gradients was developed. The procedure was optimalized with respect to recovery of intact α-granules. The organelle homogenate was loaded to 17.5 – 27.5% metrizamide gradients and centrifuged. Organelle aggregate formation was minimized by controlling the ionic conditions and the shape of the gradient. The α-granules were separated from lysosomes and dense bodies, but overlapped with the mitochondria. The α-granules were recovered from the gradient in order to omit the major amount of mitochondria from the final preparation. A washing procedure which introduced a minimum of α-granule adhesivity and fragility is presented.

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 Glanzmanns 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.

Evidence for release of soluble, but not of membrane-integrated. Proteins from human platelet α-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.

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.