Biomedical subjects

N O Solum

Publications and source records attributed to N O Solum.

At least 19 recordsLinked to original sources

PubMed2000

CXC-chemokines, a new group of cytokines in congestive heart failure--possible role of platelets and monocytes.

OBJECTIVES: The purpose of the present study was to examine the circulating levels of CXC-chemokines in patients with various degree of congestive heart failure (CHF). BACKGROUND: CXC-chemokines may be important mediators in the persistent immune activation observed in CHF patients by activation of circulating neutrophils, T-cells and monocytes and possibly by the recruitment of these cells into the failing myocardium. METHODS: Levels of interleukin (IL)-8, growth-regulated oncogene (GRO) alpha and epithelial neutrophil activating peptide (ENA)-78 were measured both in serum and in platelet-free plasma by enzyme immunoassay in 47 patients with CHF and in 20 healthy controls. RESULTS: (i) CHF patients had significantly elevated levels of all the three CXC-chemokines with IL-8 and GRO alpha showing a gradual increase along with increasing NYHA class. (ii) There was an inverse correlation between IL-8 and left ventricular ejection fraction (EF) and cardiac index (CI). (iii) Both unstimulated and lipopolysaccharide (LPS)-stimulated monocytes from CHF patients released markedly elevated amounts of all three CXC-chemokines. (iv) Platelets from patients with severe CHF were characterised by decreased content of GRO alpha and ENA-78 as well as decreased release of these chemokines upon thrombin receptor stimulation. (v) Activated platelets stimulated peripheral blood mononuclear cells in vitro to enhanced release of IL-8, and neutralising antibodies against ENA-78 inhibited this interaction. CONCLUSIONS: This study demonstrates for the first time elevated levels of CXC-chemokines in CHF, which may be of importance for progression of heart failure. Our findings further suggest that activated monocytes and platelets may contribute to enhanced CXC-chemokine levels in CHF.

Analysis of Variance↗

PubMed1999

Studies on the dual effects on platelets of a monoclonal antibody to CD9, and on the properties of platelet CD9.

The article describes effects on human platelets of a murine monoclonal antibody of the IgG2a subtype (clone FN99) directed against the membrane glycoprotein CD9. This antibody exerts a dual action on human platelets in plasma depending on whether the complement system can be activated or not, resulting either in membrane permeabilization or a true platelet aggregation. Secretion from the alpha-granules during permeabilisation was not observed in the sense that the granule-located protein thrombospondin was retained in the platelets, as opposed to what was seen with platelets that had undergone an antibody-induced aggregation. Only a small fraction of P-selectin was found on the surface of the permeabilised platelets. The cytoskeletal protein actin-binding protein (filamin) was profoundly degraded during membrane permeabilisation, however, and scanning electron microscopy showed platelets that were swollen with only a few pseudopodia. Preincubation of platelets with three different antibodies to CD9 showed strong inhibition of a subsequent binding of FITC-labelled Fab fragment of FN99 indicating that antibodies tend to bind in the same area of the CD9 molecule. No association of CD9 to the platelet actin-based cytoskeleton was observed. CD9 was present on the surface of microvesicles derived from calcium ionophore-treated platelets.

Antibodies, Monoclonal↗

PubMed1999

Enhanced levels of soluble and membrane-bound CD40 ligand in patients with unstable angina. Possible reflection of T lymphocyte and platelet involvement in the pathogenesis of acute coronary syndromes.

BACKGROUND: The CD40 ligand (CD40L) on activated T cells and platelets may be activating matrix metalloproteinases, inducing procoagulant activity, and be involved in the pathogenesis of acute coronary syndromes by promoting plaque rupture in atheroma. METHODS AND RESULTS: To study the role of CD40L-CD40 interaction in coronary disease, we analyzed levels of soluble (s) and membrane-bound CD40L in the peripheral blood from 29 patients with stable angina, 26 with unstable angina, and 19 controls. Our main findings follow. (1) Patients with unstable angina had significantly raised serum levels of sCD40L when compared with patients with stable angina and controls. (2) Platelets could release large amounts of sCD40L when stimulated ex vivo with the thrombin receptor-agonist peptide SFLLRN in both patients and controls. (3) Platelets in patients with unstable angina were characterized ex vivo by decreased intracellular levels and decreased SFLLRN-stimulated release of sCD40L, which may possibly represent a higher percentage of degranulated platelets in these patients. (4) T cells in patients with unstable angina had enhanced surface expression of CD40L and increased release of sCD40L on anti-CD3/anti-CD28 stimulation in vitro when compared with patients with stable angina and controls. (5) Recombinant CD40L and serum from patients with unstable angina who had high sCD40L levels induced enhanced release of monocyte chemoattractant peptide-1 from mononuclear cells, a CC-chemokine involved in the pathogenesis of atherosclerosis. CONCLUSIONS: This first demonstration of enhanced levels of soluble and membrane-bound forms of CD40L in angina patients, with particularly high levels in patients with unstable angina, suggests that CD40L-CD40 interaction may play a pathogenic role in both the long-term atherosclerotic process and in the triggering and propagation of acute coronary syndromes.

Acute Disease↗

PubMed1999

Procoagulant expression in platelets and defects leading to clinical disorders.

Hemostasis is a result of interactions between fibrillar structures in the damaged vessel wall, soluble components in plasma, and cellular elements in blood represented mainly by platelets and platelet-derived material. During formation of a platelet plug at the damaged vessel wall, factors IXa and VIIIa form the "tenase" complex, leading to activation of factor X on the surface of activated platelets. Subsequently, factors Xa and Va form the "prothrombinase" complex, which catalyzes the formation of thrombin from prothrombin, leading to fibrin formation. An enhanced expression of negatively charged phosphatidylserine in the outer membrane leaflet resulting from a breakdown of the phospholipid asymmetry is essential for the formation of the procoagulant surface. An ATP-driven and inward-acting aminophospholipid "translocase" and a "floppase" counterbalancing this have been postulated to maintain the dynamic state of phospholipid asymmetry. A phospholipid-nonspecific "scramblase," believed to be responsible for the fast breakdown of the asymmetry during cell activation, has recently been isolated from erythrocytes, cloned, and characterized. An intracellular calcium-binding segment and one or more thioesterified fatty acids are probably of importance for calcium-induced activation of this transporter protein. Cytosolic calcium ions also activate the calcium-dependent protease calpain associated with shedding of microvesicles from the transformed platelet membrane. These are shed with a procoagulant surface and with surface-exposed P-selectin from the alpha-granules. Theoretically, therefore, microvesicles can be involved in both coagulation and inflammation. Scott syndrome is probably caused by a defect in the activation of an otherwise normal scramblase, resulting in a relatively severe bleeding tendency. In Stormorken syndrome, the patients demonstrate a spontaneous surface expression of aminophospholipids. Activated platelets and the presence of procoagulant microvesicles have been demonstrated in several clinical conditions, such as thrombotic and idiopathic thrombocytopenia, disseminated intravascular coagulation, and HIV-1 infection, and have been found to be associated with fibrin in thrombosis. Procoagulant microvesicles may also be formed from other cells as a result of apoptosis.

Blood Coagulation Disorders↗

PubMed1998

Shear-induced platelet activation and platelet microparticle formation in native human blood.

Shear-induced platelet activation and platelet microparticle formation are triggered in native human blood by high arterial shear or by a sudden increase in shear as introduced by a stenosis with potential consequences for collagen-induced platelet thrombus formation. Blood was drawn from healthy volunteers and directly perfused ex vivo over various well-defined eccentric stenoses. Shear-induced platelet activation was determined by using flow cytometry to assess: 1) GPIIb-IIIa activation by fluorescein isothiocyanate (FITC)-labeled Mab PAC-1; and 2) translocation of membrane aminophospholipids (procoagulant activity) by FITC-labeled Annexin V. Microparticle formation was measured by flow cytometry and FITC-labeled Mab Y2/51 directed against GPIIIa. Significant platelet activation and platelet microparticle formation were elicited when the wall shear rate reached 10,500 sec-1 for a period of 0.075 sec. Prolonged exposure to or a rapid increase in shear further enhanced activation and microparticle formation. Shear-induced platelet activation was associated with significantly increased collagen-induced platelet thrombus formation that was insensitive to aspirin ingestion. Exposure of native blood to very high shear thus activates platelets to express GPIIb-IIIa, renders the platelet membrane procoagulant and stimulates microparticle formation. These responses are associated with enhanced collagen-induced thrombus formation by prostaglandin-independent mechanisms.

Adult↗

PubMed1998

Enhanced activation of platelets with abnormal release of RANTES in human immunodeficiency virus type 1 infection.

Besides their role in hemostasis, platelets are involved in inflammatory and immunological processes, and we hypothesize that platelet activation may play an immunopathogenetic role in HIV-1 infection. Blood was drawn from 15 controls and 20 HIV-1-infected patients with normal platelet counts, classified into groups of non-AIDS and AIDS. Platelet activation was detected using flow cytometry with mAbs against the release markers P-selectin and CD63, mAb against GPIb, and the probe annexin V detecting surface exposure of aminophospholipids. The amount of microvesicles was measured using mAb against GPIIIa. Compared to controls, blood samples from HIV-1-infected patients showed significantly enhanced levels of microvesicles and activated platelets as detected by their exposure of P-selectin, CD63, and aminophospholipids, as well as reduction in GPIb expression. Increased expression of P-selectin and amounts of microvesicles were most pronounced in advanced clinical and immunological disease. When studying the effect of HIV-1 protease inhibitor therapy (indinavir) on platelet activation, we found that concomitant with a profound decrease in plasma viral load, there was a near normalization of several of the parameters reflecting enhanced platelet activation. Finally, we demonstrated that platelets may be an important source of the chemokine RANTES in HIV-1-infected patients. Although both unstimulated and SFLLRN-stimulated platelets from asymptomatic patients had enhanced release of RANTES, platelets from AIDS patients were characterized by markedly enhanced spontaneous, but decreased SFLLRN-stimulated release of this chemokine. Taken together, these results, which demonstrate for the first time increased platelet activation in HIV-1-infected patients with normal platelet counts, may represent a previously unrecognized immunopathogenic factor in HIV-1 infection.

Anti-HIV Agents↗

PubMed1998

Microvesicles bind soluble fibrinogen, adhere to immobilized fibrinogen and coaggregate with platelets.

In the present study we have investigated whether platelet derived microvesicles can bind soluble fibrinogen, bind to immobilized fibrinogen, and coaggregate with platelets. Flow cytometry was used for studies on binding of soluble fibrinogen and coaggregation, whereas ELISA wells were used to study binding of microvesicles to immobilized fibrinogen. Biotinylated microvesicles produced by stimulation with A23187, thrombin or SFLLRN of platelets which had been surface-labelled with biotin, were used both for the coaggregation experiments and for the binding studies with immobilized fibrinogen. Unlabelled microvesicles and biotinylated fibrinogen were employed when studying binding of soluble fibrinogen to the microvesicles. For the flow cytometry, the biotinylated proteins were reacted with avidin or streptavidin which was PE-conjugated, whereas the same substances were conjugated with alkaline phosphatase for the ELISA studies. The microvesicles formed after stimulation of platelets by SFLLRN or A23187 clearly bound the soluble, biotinylated fibrinogen. Moreover, isolated biotinylated microvesicles added to washed platelets prior to activation, were associated to the microaggregates that formed after stimulation. A significant binding of biotinylated microvesicles to immobilized fibrinogen could also be detected. The binding of microvesicles to soluble and immobilized fibrinogen and association to platelets was clearly specific and at least partly dependent on the GPIIb-IIIa complex, as all of these phenomena could be prevented or reduced by addition of the c7E3 Fab which blocks the activated form of this receptor complex. From these in vitro results it is clear that microvesicles can bind to immobilized fibrinogen, bind soluble fibrinogen and are able to coaggregate with platelets. It may be speculated that these results also reflect a haemostatic role of microvesicles in vivo.

Blood Platelets↗

PubMed1997

Shear-induced platelet activation and platelet microparticle formation at blood flow conditions as in arteries with a severe stenosis.

In the present study, we investigated whether high arterial shear stresses at various exposure times or a sudden increase in shear stress introduced by a stenosis affect platelet activation and platelet microparticle formation in native human blood. We used a parallel-plate perfusion chamber device through which nonanticoagulated human blood was drawn (10 mL/min) by a pump directly from an antecubital vein through the flow channel of a perfusion chamber at wall shear rates of 420, 2600, and 10500 s-1. In another set of experiments, an eccentric stenosis was introduced into the flow channel. Wall shear rates of 2600 or 10500 s-1 at the stenosis apex were maintained at the same flow rate. The wall shear rate upstream and downstream of these stenoses was 420 s-1. A shear rate of 420 s-1 is within the range of those encountered in healthy small coronary arteries, whereas those of 2600 and 10500 s-1 are representative for vessels with various degrees of stenotic lesions. The blood was exposed to these shear rates for periods varying from 0.075 to 3.045 seconds. Platelet activation was assessed as activated glycoprotein (GP) IIb/IIIa by FITC-labeled monoclonal antibody (MAb) PAC-1 and aminophospholipid translocation by FITC-labeled annexin V. Microparticle formation was quantified by FITC-labeled MAb Y2/51 directed against GP IIIa. Significant platelet activation and formation of microparticles were observed at 10500 s-1 only (P < .008). This shear-induced platelet activation and microparticle formation were enhanced by introduction of a thrombus-promoting surface consisting of type III human collagen fibrils. Introduction of the most severe stenosis at 10500 s-1 further increased platelet activation (P < .017). The collagen-induced thrombus formation increased the platelet thrombus volume at 10500 s-1 from 16.5 to 33.8 microns3/microns2 (P < .003) on the stenosis apex when the most severe stenosis was used. A correlation (P < .0001) between platelet thrombus volume and platelet microparticle formation was observed in the presence of the eccentric stenoses. Apparently, high shear stress (315 dynes/cm2 at 10500 s-1), as encountered in severe atherosclerotic arteries, activated platelets and triggered platelet microparticle formation. In contrast, no significant platelet activation or formation of platelet microparticles was observed at physiological shear (420 s-1) or at the shear condition simulating shear in arteries with a less severe stenosis (2600 s-1). The data imply that platelets are activated and form microparticles in native blood at very high shear stresses. These events are potentiated by prolonged exposure to the high shear or by a sudden change of increasing shear due to the stenosis. The latter situation apparently enhances platelet thrombus formation at the stenosis.

Annexin A5↗

PubMed1995

Detection of biotinylated proteins in crossed immunoelectrophoresis gels: studies on platelet membrane receptors and microparticles.

Biotinylation can be used as an alternative for surface labeling of cell membrane proteins. The use of the water soluble N-hydroxysulfosuccinimide (NHSS)-biotin or the more lipophilic N-hydroxysuccinimide (NHS)-biotin reagent has been investigated in the present study labeling two central receptor complexes on the platelet surface, i.e. the glycoprotein (GP) Ib-IX and the GP IIb-IIIa complexes involved in platelet adhesion and aggregation. Lack of labeling of the intracellularly located albumin was used as a negative control. The labeling has been studied using crossed immunoelectrophoresis in the PhastSystem format after extraction of the labeled cells in Triton X-100, and it is shown that, using enzyme-conjugated avidin and chromogenic substrates, the biotinylated proteins can be visualized directly in the dried electrophoresis gel without the need for a transfer to a blotting membrane as is used after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Suitable conditions for biotinylation and for visualization in the crossed immunoelectrophoresis gels are described. Further, surface-biotinylation of platelets was used to observe shedding of microparticles as a consequence of formation of the complement membrane attack complex. For this purpose the formation and composition of the biotinylated microparticles were observed by flow cytometry and crossed immunoelectrophoresis.

Antibodies, Monoclonal↗

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↗