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N O Solum

Publications and source records attributed to N O Solum.

At least 37 records · Page 2Linked to original sources
PubMed1995

Platelet-derived microvesicles and activated platelets express factor Xa activity.

Activated platelets and platelet-derived microvesicles demonstrate procoagulant properties. It is known that following stimulation, negatively charged phospholipids and factor Va become located on their surfaces. The aim of this study was to see whether activated platelets and platelet-derived microvesicles also expressed some factor Xa activity on their surfaces in a system where factor Xa did not come from external sources. In order to study this question, flow cytometry, as well as the use of a chromogenic substrate to factor Xa and a clotting assay in a factor X depleted plasma, were applied. A prothrombinase assay was also applied using prothrombin, CaCl2 and a chromogenic substrate to thrombin. The platelets were gel-filtered or washed, suspended in Tris-buffered saline, and activated by calcium ionophore A23187 or the thrombin receptor agonist peptide SFLLRN. Microvesicles and activated platelets were separated by centrifugation. Flow cytometry using a monoclonal antibody against factor Xa demonstrated the presence of factor Xa on the surface of the activated platelets. In addition, platelet-derived microvesicles and activated platelets demonstrated factor Xa activity on their surfaces detected directly by splitting of the chromogenic substrate to factor Xa, or by the prothrombinase assay. The thrombin generation in the last assay could be inhibited by a selective factor Xa inhibitor (recombinant tick anticoagulant peptide (rTAP)), soybean trypsin inhibitor, and antithrombin III plus LMW-heparin, all inhibiting at the factor Xa level, as well as by leupeptin which also inhibited the thrombin-chromogenic substrate interaction as such.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Coagulation↗
PubMed1995

Stimulated Glanzmann's thrombasthenia platelets produced microvesicles. Microvesiculation correlates better to exposure of procoagulant surface than to activation of GPIIb-IIIa.

The mechanism of formation of platelet-derived microvesicles remains controversial. The aim of the present work was to study the formation of microvesicles in view of a possible involvement of the GPIIb-IIIa complex, and of exposure of negatively charged phospholipids as procoagulant material on the platelet surface. This was studied in blood from three Glanzmann's thrombasthenia patients lacking GPIIb-IIIa and healthy blood donors. MAb FN52 against CD9 which activates the complement system and produces microvesicles due to a membrane permeabilization, ADP (9.37 microM), and the thrombin receptor agonist peptide SFLLRN (100 microM) that activates platelets via G-proteins were used as inducers. In a series of experiments platelets were also preincubated with PGE1 (20 microM). The number of liberated microvesicles, as per cent of the total number of particles (including platelets), was measured using flow cytometry with FITC conjugated antibodies against GPIIIa or GPIb. Activation of GPIIb-IIIa was detected as binding of PAC-1, and exposure of aminophospholipids as binding of annexin V. With normal donors, activation of the complement system induced a reversible PAC-1 binding during shape change. A massive binding of annexin V was seen during shape change as an irreversible process, as well as formation of large numbers of microvesicles (60.6 +/- 2.7%) which continued after reversal of the PAC-1 binding. Preincubation with PGE1 did not prevent binding of annexin V, nor formation of microvesicles (49.5 +/- 2.7%), but abolished shape change and PAC-1 binding after complement activation. Thrombasthenic platelets behaved like normal platelets after activation of complement except for lack of PAC-1 binding (also with regard to the effect of PGE1 and microvesicle formation). Stimulation of normal platelets with 100 microM SFLLRN gave 16.3 +/- 1.2% microvesicles, and strong PAC-1 and annexin V binding. After preincubation with PGE1 neither PAC-1 nor annexin V binding, nor any significant amount of microvesicles could be detected. SFLLRN activation of the thrombasthenic platelets produced a small but significant number of microvesicles (6.4 +/- 0.8%). Incubation of thrombasthenic platelets with SFLLRN after preincubation with PGE1, gave results identical to those of normal platelets. ADP activation of normal platelets gave PAC-1 binding, but no significant annexin V labelling, nor production of microvesicles. Thus, different inducers of the shedding of microvesicles seem to act by different mechanisms. For all inducers there was a strong correlation between the exposure of procoagulant surface and formation of microvesicles, suggesting that the mechanism of microvesicle formation is linked to the exposure of aminophospholipids. The results also show that the GPIIb-IIIa complex is not required for formation of microvesicles after activation of the complement system, but seems to be of importance, but not absolutely required, after stimulation with SFLLRN.

Adult↗
PubMed1994

Partial expression of GP Ib measured by flow cytometry in two patients with Bernard-Soulier syndrome.

Bernard-Soulier syndrome is a rare, congenital bleeding disorder caused by absent or defective GP Ib platelet membrane receptor for the von Willebrand factor (vWF). We studied two brothers with moderate bleeding symptoms. Bleeding time was prolonged and ristocetin-induced platelet aggregation was absent. Flow cytometric analysis showed that both boys had a subnormal expression of GP Ib. One antibody used (AN51) was bound only to 30% of the platelets and at a subnormal density. A second antibody (SZ2) also bound at a subnormal density but a normal fraction of the platelets were immunoreactive. Ristocetin stimulation of the patients' platelets in the presence of plasma resulted in a low binding of vWF, about 30% of healthy controls. On the other hand the expression of GP IIb/IIIa on the platelet membrane appeared to be supernormal even when the increased platelet size was taken into account as shown by the ratio between the density of GP IIIa and CD 9 structures. We conclude that these brothers have a variant of the Bernard-Soulier syndrome with a low expression of a GP Ib receptor.

Bernard-Soulier Syndrome↗
PubMed1994

Complement-mediated permeabilization of platelets by monoclonal antibodies to CD9: inhibition by leupeptin, and effects on the GP Ib-actin-binding protein system.

Two monoclonal antibodies to CD9 of the IgM and IgG2a categories (FN 52 and FN 99), reproducibly induced platelet alterations in platelet-rich plasma by activation of the complement system with membrane incorporation of the pore-forming C5b-9 complex. The permeabilization could be monitored by measurements of extracellular ATP and observed as a shape change followed by an increase in light transmission in the aggregometer, and was associated with formation of tiny platelet aggregates. This could be accomplished by only minor lysis observed as extracellular lactate dehydrogenase (LDH). When leupeptin was added prior to, or immediately after the antibody, a total inhibition of the platelet alterations could be obtained. When added soon after the shape change, leupeptin had little effect on the liberation of ATP. However, whereas the ability of the platelets to become agglutinated by ristocetin was lost during the complement-mediated platelet alterations, addition of leupeptin immediately after the shape change, prevented this loss. The lost ability of the permeabilized platelets to undergo ristocetin-induced agglutination is not ascribed to degradation of GP Ib as this was relatively little affected in these studies as compared to the actin-binding protein (ABP) which was profoundly degraded. This protein represents a link between GP Ib and the submembraneous cytoskeleton, and the inhibition of its degradation by leupeptin, was clearly demonstrated. Experiments with digitonin-induced permeabilization showed that leupeptin did not inhibit permeabilization as such, but it did prevent the loss of ristocetin-induced agglutination even with this inducer.

Actins↗
PubMed1994

Demonstration of platelet-derived microvesicles in blood from patients with activated coagulation and fibrinolysis using a filtration technique and western blotting.

Platelet vesiculation in vitro is correlated to platelet activation. It was therefore considered of interest to see if microvesicles (MV) are present in blood in clinical situations associated with platelet activation in vivo. Patients with both activated coagulation and fibrinolysis, implying that thrombin has been generated, suit such a purpose. Thus, the aim of this study was to investigate whether microvesicles could be detected in patients with activated coagulation and fibrinolysis, as diagnosed by the presence of soluble fibrin (positive ethanol gelation tests) and positive tests for fibrin degradation products (FDP). Platelet-rich plasma was prepared from citrated blood from patients (n = 22) and healthy controls (n = 32) matched as to age and sex. The intact platelets were removed from plasma by centrifugation. Any MV present were isolated from the platelet-free plasma by a filtration procedure, washed, solubilized in Triton X-100 and subjected to SDS-PAGE with Western blotting using a MAb against GPIIb alpha as an indicator of the presence of microvesicles. All of the 22 patients showed the presence of microvesicles detectable by the content of GPIIb alpha, whereas this could be observed in only 4 out of the 32 normal controls and then in small or trace amounts only. The presence of microvesicles among cell-derived material in the plasma of two of the patients was also confirmed by electron microscopy. To the best of our knowledge this is the first report on the presence of microvesicles in plasma from patients with both activated coagulation and fibrinolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

The difference between platelet and plasma FXIII used to study the mechanism of platelet microvesicle formation.

The formation of microvesicles from platelets was induced either by activation of the complement system by a monoclonal antibody to CD9, or by incubation of platelets with the calcium ionophore A23187. A filter technique to isolate the microvesicles without plasma contamination is described. The microvesicles contained FXIIIa2 from the platelet cytoplasm which shows that these particles contain significant amounts of intracellular material. This was shown by the use of crossed immunoelectrophoresis with rabbit antibodies to total human platelet proteins in the second dimension gel and polyclonal antibodies against the a- and b-subunit of FXIII in the intermediate gel. The FXIIIa2 in the microvesicle was found to be functional as an enzyme. To prove this, it was shown that FXIII in its immunoprecipitate arc could catalyze the incorporation of monodansylcadaverine into casein as identified by fluorescence of this arc in ultraviolet light. The observation that the plasma form of FXIII (FXIIIa2b2) was absent from the microvesicles collected by the filtration technique, whereas it was present in platelet fragments obtained by mechanical disruption by ultrasonication, indicates that the activation-dependent microvesicles are formed by a true budding process with the inclusion of intracellular, but not extracellular material.

Antibodies, Monoclonal↗
PubMed1993

Correlation of light transmission changes to changes of platelet glycoprotein Ib during storage of platelet concentrates.

The present study compares some properties of platelet concentrates, i.e. their ability to transmit light and the degree of platelet lysis and the deterioration of the platelet membrane glycoprotein Ib (GPIb). Extracellular levels of glycocalicin and lactate dehydrogenase (LDH) served as indicators for the proteolytic cleavage of GPIb and platelet lysis, respectively. The light transmission of 16 platelet apheresis concentrates was monitored continuously during storage using a specially designed apparatus. Determination of platelet counts and extracellular glycocalicin levels was carried out on day 1. In addition, after 7 days of storage extracellular concentrations of LDH and glycocalicin were determined. After 7 days highly significant correlations were found both when comparing light transmission changes and extracellular glycocalicin levels (r = 0.78; p < 0.001) and when comparing light transmission changes and extracellular concentrations of LDH (r = 0.83; p < 0.001).

Animals↗
PubMed1992

Binding of human platelet glycoprotein Ib and actin to fragments of actin-binding protein.

Actin-binding protein (ABP) is degraded into fragments of 190 and 90 kDa by calpain. A monoclonal antibody (MAb TI10) against the 90 kDa fragment of ABP coprecipitated with the glycoprotein Ib (GP Ib) peak observed on crossed immunoelectrophoresis of Triton X-100 extracts of platelets prepared without calpain inhibitors. MAb PM6/317 against the 190 kDa fragment was not coprecipitated with the GP Ib peak under such conditions. The 90 kDa fragment was adsorbed on protein A agarose from extracts that had been preincubated with antibodies to GP Ib. This supports the idea that the GP Ib-ABP interaction resides in the 90 kDa region of ABP. GP Ib was sedimented with the Triton-insoluble actin filaments in trace amounts only, and only after high speed centrifugation (100,000 x g, 3 h). Both the 190 kDa and the 90 kDa fragments of ABP were sedimented with the Triton-insoluble actin filaments.

Actins↗
PubMed1992

Platelets and vitronectin: immunocytochemical localization and platelet interaction with exogenously added vitronectin.

Vitronectin stores were localized in human platelets by immunofluorescence, confocal laser microscopy, and immunoelectron microscopy. In ultrathin cryosections, the immunogold labelling was mainly observed in the alpha-granules, although occasional immunolabelling of the platelet surface was present. The interaction between thrombin-stimulated platelets and exogenously added vitronectin was also explored. This vitronectin had been purified by heparin-affinity chromatography. Binding of vitronectin to thrombin-stimulated platelets was demonstrated by flow cytometry. The immunoelectron microscopical studies revealed that this binding was mainly restricted to clumps of released alpha-granular material on the platelet surface. The possible significance of the interaction between exogenous vitronectin and released platelet proteins is discussed.

Amino Acid Sequence↗
PubMed1992

Membrane fluidity and platelet aggregation: dibucaine permits ristocetin-induced platelet aggregation with low-molecular-weight von Willebrand multimers.

The effects of brief incubation of platelets with dibucaine were studied. Membrane fluidity was increased after incubation with 1 mM dibucaine for 1 min as determined by fluorescence polarization. Platelet aggregation was increased when ristocetin was employed as modulator and normal plasma used as a source of von Willebrand factor (vWF). In contrast, aggregation was decreased with botrocetin. After cryoprecipitation the supernatant, which contained predominantly low-molecular-weight vWF multimers, was effective with ristocetin only after prior incubation of the platelets with dibucaine. This indicates that low-molecular-weight vWF multimers can also support ristocetin-induced aggregation when the membrane fluidity is increased. This idea was supported by the use of plasma from a patient with von Willebrand disease type IIa. Being a multimeric protein, von Willebrand factor contains multiple binding sites for glycoprotein Ib (GP Ib). GP Ib-dependent aggregation by agents that do not contain multiple binding sites, i.e. wheat germ agglutinin or polyclonal antibodies to GP Ib, was decreased after brief incubation with dibucaine. These observations are discussed in relation to the hypothesis that the increased membrane fluidity allows an increased number of GP Ib molecules to bind to each vWF multimer.

Crotalid Venoms↗
PubMed1991

Effects of toluene on platelet membrane glycoprotein Ib and actin-binding protein.

Effects of the organic solvent toluene on the platelet membrane receptor glycoprotein Ib (GP Ib) and the cytoskeletal protein, actin-binding protein (ABP), were studied and related to the effects of the local anesthetic dibucaine. The glycocalicin-region of GP Ib contains the binding site for von Willebrand factor; intracellularly GP Ib is linked to the cytoskeleton via ABP. Both GP Ib and ABP are substrates for a calcium-dependent protease, calpain. Washed platelets were incubated with toluene or dibucaine. The toluene concentration in the platelet suspension was analysed by gas chromatography. Using 1.5-2.8 mmol/L toluene, calpain was activated, leading to degradation of ABP and release of glycocalicin from GP Ib. The latter phenomenon was paralleled by a reduced von Willebrand factor-induced platelet agglutination. At lower toluene concentrations (0.3-1.4 mmol/L), degradation of ABP was not detected but an initial increased agglutination that declined to the control level with time was observed. These effects of toluene on the GP Ib-ABP complex are similar to those observed with 1 mmol/L dibucaine. The lowest toluene concentrations used correspond to those that have been found in blood from toluene abusers ("sniffers").

Blotting, Western↗
PubMed1990

Studies on a patient with thrombocytopenia, giant platelets and a platelet membrane glycoprotein Ib with reduced amount of sialic acid.

A 70-year-old patient with a life-long bleeding tendency, giant platelets and thrombocytopenia (10-40 x 10(9) platelets/l) has been studied. This is a condition often associated with lack of platelet membrane glycoprotein Ib (GP Ib). Electron microscopy of fixed platelets incubated with monoclonal antibodies to GP Ib (AN 51, AP 1) and gold-labelled goat anti-mouse IgG, showed a distinct distribution of GP Ib on the patient's platelets, however. Crossed immunoelectrophoresis and SDS-PAGE demonstrated a reduced mobility of the patient's GP Ib which could be explained by absence of sialic acid. Blotting with peroxidase-conjugated peanut agglutinin confirmed this conclusion. This lectin binds to galactose-N-acetyl-galactosamine residues exposed terminally when sialic acid is absent from the carbohydrate side-chains. Such binding could be seen with normal GP Ib only after neuraminidase treatment. Fluorescence studies with FITC-conjugated peanut agglutinin showed binding of the lectin to intact patient platelets, indicating that lack of sialic acid was not introduced during the platelet isolation procedure. Neither could the lack of sialic acid be attributed to increased neuraminidase activity as studied in vitro. Platelets treated with neuraminidase in vivo or in vitro are rapidly cleared from the circulation. Therefore the patient's thrombocytopenia may be associated with the reduced amount of GP Ib sialic acid. As far as we know, similar cases have not been described previously.

Aged↗
PubMed1989

The PhastSystem equipment used for crossed immunoelectrophoresis combined with immunoblotting of coprecipitated monoclonal antibodies as studied with platelet membrane receptor proteins.

The Pharmacia PhastSystem equipment has been used for crossed immunoelectrophoresis combined with a technique for immunoblotting with monoclonal antibodies. This miniaturized gel system is compared to the conventional approach using platelet membrane receptor proteins as a model. Whereas in the conventional system the electrophoretic procedure takes place within 20 h, 3 h are adequate for the small gel system. Because of the short second-dimensional electrophoresis, and only one overnight incubation, the total electrophoretic and blotting procedure could be reduced from about 48 h to 24 h. The amount of antiserum used during the second-dimensional electrophoresis could be reduced roughly by a factor of 5. The examples with electrophoresis and immunoblotting using platelet extracts in 1% Triton X-100 demonstrate that membrane receptor proteins can be studied even when present as noncovalent complexes. The immunoblotting can be used with monoclonal antibodies that do not function in Western blotting.

Animals↗
PubMed1989

The use of PhastSystem crossed immunoelectrophoresis with immunoblotting to demonstrate a complex between glycoprotein Ib and the actin-binding protein (ABP) of human platelets.

The study shows how a technique described in an accompanying paper can be applied to solve a biological problem. The technique makes use of the observation that a monoclonal antibody that has been coprecipitated with its antigen during crossed immunoelectrophoresis can be transferred to a nitrocellulose membrane and visualized. Previous studies using crossed immunoelectrophoresis of Triton X-100 extracts of platelets have indicated that a particular immunoprecipitate (peak III) of the membrane receptor glycoprotein Ib (GP Ib) might contain a complex between the receptor and the actin-binding protein (filamin). When a monoclonal antibody (PM6/317) directed towards the actin-binding protein was added to a platelet extract prior to immunoelectrophoresis and blotting, this was visualized on the blot as a replica of the peak III immunoprecipitate. This demonstrates a colocalization of GP Ib and the actin-binding protein in the precipitate, and thus the existence of a complex between the membrane receptor and the cytoskeletal protein.

Antibodies, Monoclonal↗
PubMed1988

Characterization of a monoclonal antibody ITI-Pl 1 directed against human platelet membrane glycoprotein IIb using extracts of whole platelets and platelet surface and intracellular membranes.

A monoclonal antibody (mAb) termed ITI-Pl 1 has been prepared by the hybridoma procedure. Using immuno-absorption and crossed immunoelectrophoresis of Triton X-100 extracts of untreated and EDTA-treated human platelets it was shown to be directed against the surface membrane glycoprotein IIb (GP IIb). This mAb binds to whole platelets independently of ADP-stimulation and the presence of Ca2+-ions. It saturates at around 870 ng/10(8) cells corresponding to approximately 35,800 molecules/platelet. ITI-Pl 1 did not significantly inhibit GP IIb-IIIa dependent functions such as platelet aggregation or fibrinogen binding. Immunofluorescence could be demonstrated using ITI-Pl 1 and intact normal platelets, but not with platelets from a Glanzmann's thrombasthenia patient. Crossed immuno-electrophoresis with platelet extracts from four different thrombasthenic patients gave a line precipitate in the intermediate gel with 125I-labelled ITI-Pl 1 and autoradiography indicating trace amounts of free GP IIb or the GP IIb-IIIa complex. The epitope on GP IIb detected by ITI-Pl 1 is not destroyed by neuraminidase treatment. Thus the mAb also interacts with neuraminidase-treated GP IIb-IIIa complex in highly purified platelet surface membrane fractions as well as with GP IIb-IIIa from untreated internal membranes isolated by continuous flow electrophoresis.

Animals↗
PubMed1987

Interactions of human blood platelets with three circulating plasma fibrinogens of different molecular weights.

The ability of three naturally occurring human fibrinogen species, HMW, LMW and LMW', to support ADP-induced platelet aggregation was investigated and compared to their ability to bind to gel-filtered platelets. Whereas HMW had intact subunit chains, LMW and LMW' had defined lesions in the C-terminal part of one (LMW) or both (LMW') of the A alpha-chains. The ADP-induced aggregation of gel-filtered platelets in the presence of LMW was about 75 per cent of that obtained with HMW (0.2 mumol/l of fibrinogen and 10 mumol/l of ADP). A mixture of equal amounts of LMW and LMW' showed around 50% decrease in aggregation. Compared to these difference in aggregation co-factor function, direct binding to gel-filtered platelets was less affected by the A alpha-chain degradation. However, a difference between LMW, LMW' and HMW binding was significant when the fibrinogens labelled with two different isotopes of iodine were present simultaneously. These results demonstrate that naturally occurring plasma fibrinogens differ in their interactions with platelets. As the HMW/LMW ratio changes during the acute phase, this may be of physiological significance.

Adenosine Diphosphate↗