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P Spangenberg

Publications and source records attributed to P Spangenberg.

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

ADP-induced platelet aggregation and actin polymerization. Involvement of GpIIb/IIIa and the effect of Mg2+.

We have investigated the effects of Mg2+ (added to platelet rich plasma [PRP] as 10mM MgCl2 or MgSO4) on the platelet aggregation and actin polymerization that occurs in response to adenosine diphosphate (ADP). The PRP was prepared from blood containing hirudin as anticoagulant. Mg2+ added before 1 microM ADP completely inhibited aggregation and markedly inhibited actin polymerization. Mg2+ (10mM) added before 10 microM ADP converted irreversible aggregation into a reversible response; similarly, apparently irreversible actin polymerization was converted to a reversible response in which polymerization was followed by some actin depolymerization. Mg2+ added after inducing platelet aggregation with 10 microM ADP produced parallel disaggregation of platelets and actin depolymerization. Actin polymerization occurs immediately on adding ADP to PRP (in association with shape change) and further polymerization occurs in association with platelet aggregation. When aggregation (and the actin polymerization associated with this) was prevented by adding M148, a monoclonal antibody directed at the GpIIb/IIIa complex, or simply by avoiding stirring the sample, Mg2+ had no effect on actin polymerization/depolymerization. Thus Mg2+ only affected the changes in actin that were associated with the aggregation response. This was in contrast to iloprost (which acts at the PGI2 receptor to stimulate adenylate cyclase) which induced rapid actin depolymerization when added after ADP stimulation of platelets under circumstances where platelet aggregation was avoided. These results show that Mg2+ affects actin polymerization as well as platelet aggregation, and that it affects the actin polymerization associated with aggregation but not that associated with shape change (in contrast to iloprost which inhibits both).(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1995

The effect of rapid thoracoabdominal compressions on intracranial pressure in newborn lambs.

Measurements of forced expiratory flow in infants can be obtained with the rapid thoracoabdominal compression (RTC) technique. To assess the transmission of the chest wall compression pressure into the cerebrum, we have performed intracranial pressure (ICP) measurements during RTCs in newborn lambs. Thirteen lambs were studied during the first 8 d of life. An epidural pressure transducer was implanted under general anesthesia. Respiratory measurements were performed under sedation with ketamine. RTCs were induced through an inflatable jacket wrapped around the lamb's trunk. RTCs were started with a pressure of 30 cm H2O. The compression pressure was increased in steps of 20 cm H2O up to 250 cm H2O. Flow and volume were determined with a pneumotachograph attached to a face mask. Transpulmonary pressure (Ptp) was measured with an esophageal balloon placed in the lower esophagus. RTCs resulted in proportional increases of Ptp and ICP. ICP was lower or equal to Ptp in all measurements. No changes in baseline ICP occurred throughout the study period. The maximal ICP observed during RTCs was within the range of pressures observed during crying in newborn infants. These data suggest that the RTC technique can be considered safe even at high compression pressures.

Animals↗
PubMed1995

Short-time rabbit model of endotoxin-induced hypercoagulability.

We describe a short-time endotoxin-induced rabbit model of hypercoagulability for the study of the coagulation cascade and the therapeutic effects of coagulation inhibitors. Cardiorespiratory function was maintained in rabbits under general anesthesia and standardized mechanical ventilation (tidal volume, 6 ml/kg; 60 breaths/min) via tracheostomy and low-dose inotropic support. Coagulation parameters such as prothrombin time, activated partial thromboplastin time, thrombin time, fibrinogen concentration, platelet count, fibrin monomers, D-dimers, antithrombin III and factor XIII activities, thrombelastography, and platelet aggregometry were measured during a 4-h period after sequential double endotoxin administration (80 and 40 micrograms/kg of body weight, intravenously). Mean arterial pressure and arterial and central venous blood gas tensions were monitored. Global clotting, activation parameters of coagulation, and leukocyte count deteriorated significantly in the endotoxin-treated animals but was mainly unaltered in controls (P < 0.05). Tissue specimens of the lungs, liver, brain, and kidneys were examined. Endotoxin-induced, disseminated fibrin deposition was found in the lungs and liver (P < 0.01). We conclude that this short-time model of hypercoagulability in rabbits reliably induced disseminated intravascular coagulation. Tracheostomy and mechanical ventilation provided a reproducible model in which the differences between the controls and the endotoxin-treated animals were exclusively due to administration of endotoxin and not to unforeseen complications of the respiratory system. This model allows the study of therapeutic effects of coagulation inhibitors on endotoxin-induced changes.

Animals↗
PubMed1994

Adhesion of activated platelets to polymorphonuclear leukocytes.

Polymorphonuclear leukocytes (PMNL) are components of the blood which as such interact extensively with other blood cells, with endothelial cells or with plasma. Here, we consider the interaction between PMNL and platelets which is efficient during adhesion of platelets to PMNL and which can be studied in vitro using the rosette formation assay. The adhesion of activated platelets to PMNL seems to be mediated mainly by a protein of platelets (CD62) and its counterreceptor on PMNL, but also other platelet receptors are involved. Here we demonstrate the participation of the glycoprotein IIb-IIIa complex (CD41a) in the adhesion of activated platelets to PMNL due to the following findings: a) inhibition of adhesion by monoclonal antibodies raised against CD41a, b) inhibition of adhesion by peptides such as RGDS and echistatin, c) inhibition of adhesion by dissociation of CD41a with EGTA and d) inhibition of adhesion using platelets from a thrombasthenic patient which have almost no CD41a in the surface membrane but a normal expression of CD62 upon activation. The adhesion of activated platelets to PMNL via CD41a seems to be mediated by fibrinogen due to the following findings: a) addition of fibrinogen to ADP-stimulated and fixed platelets increases significantly the rosette formation and b) the incubation of unstimulated platelets with fibrinogen and an antibody raised against glycoprotein IIIa which stimulates fibrinogen binding to the platelet surface results in an enlarged rosette formation.

Antigens, CD↗
PubMed1993

The platelet glycoprotein IIb/IIIa complex is involved in the adhesion of activated platelets to leukocytes.

The adhesion of activated platelets to leukocytes (rosette formation) seems to be mediated by CD62 on platelets and its counter-receptor (CD15 or a sialic acid-containing glycoprotein) on polymorphonuclear leukocytes (PMNL). However, neither treatment of platelets with an anti-CD62 antibody or fucoidan nor treatment of PMNL with anti-CD15 antibody or neuraminidase are able to inhibit completely the adhesion. Therefore, we have studied the platelet GPIIb/IIIa complex (CD41a) for its involvement in the adhesion of activated platelets to PMNL. The following evidences point to a participation of CD41a in the adhesion of activated platelets to leukocytes: a) inhibition of adhesion by monoclonal antibodies (mab) raised toward CD41a, b) inhibition of adhesion by peptides such as RGDS and echistatin, c) inhibition of adhesion by dissociation of the CD41a complex with EGTA, and d) inhibition of rosette formation using platelets from a thrombasthenic patient which have almost no CD41a in the surface membrane but a normal expression of CD62. It is likely that fibrinogen is involved in the adhesion of platelets to PMNL via CD41a, since fibrinogen increases the rosette formation of ADP-stimulated platelets. Furthermore, the incubation of unstimulated platelets with fibrinogen and an antibody raised against glycoprotein IIIa which stimulates fibrinogen binding to the platelet surface results in an enlarged rosette formation.

Amino Acid Sequence↗
PubMed1993

Functional studies on platelets of a patient with an acquired disorder of platelet function associated with autoantibodies against membrane glycoprotein IIB/IIIA complex.

Platelet functions have been studied of a 63 year old woman with a severe acquired thrombopathy. The platelets did not adhere to siliconized glass. Aggregation could not be induced by either ADP (1 microM) nor collagen (2 micrograms/ml), no release of serotonin was found under these conditions. Thrombin caused only a weak aggregation response. Quantitative analysis of platelet actin revealed a very low total actin content (473 micrograms/10(9) platelets) and an extremely low F-actin value (3% of total actin). Stimulation of platelets with 0.1 U/ml thrombin for 3 min resulted in an increase of only 5% F-actin, whereas ADP and collagen did not induce any actin polymerization. Ca2+ movement in the patient's platelets is severely impaired after ADP and collagen stimulation, whereas a normal Ca2+ movement was obtained by 0.1 U/ml thrombin. The inhibition of the functions of normal platelets (aggregation and actin polymerization) by addition of patient's serum (5-10% final concentration) points to receptor blockade by platelet autoantibodies in the patient's serum. The antibody was purified by adsorption on Protein-A-Sepharose. Addition of IgG-suspension (5% final concentration) to washed control platelets resulted in similar effects on aggregation and actin polymerization compared to the effects of patient's serum.

Actins↗
PubMed1992

Changes in G-actin after platelet activation in platelet rich plasma.

We have used the DNase I inhibition assay to study changes in G-actin after platelet activation in platelet-rich plasma (PRP) induced by ADP. Because of problems associated with depolymerization of F-actin after lysis of ADP-activated platelets in the presence of plasma, G-actin was measured using a lysis buffer that contained formaldehyde to prevent any depolymerization of F-actin. Different patterns of response were seen depending on the concentration of ADP used, and these were modified by avoiding aggregation by either not stirring the sample or by adding EDTA. The results show rapid conversion of G-actin to F-actin in association with shape change, and there is a further decrease in G-actin associated with irreversible platelet aggregation. Thus evidence is presented that actin polymerization occurs in two phases after ADP stimulation.

Actins↗
PubMed1991

Acquired disorder of platelet function associated with autoantibodies against membrane glycoprotein IIb-IIIa complex--1. Glycoprotein analysis.

A patient with idiopathic thrombocytopenic purpura developed after splenectomy a thrombasthenia-like severe haemorrhagic diathesis characterized by a normal or subnormal platelet count, prolonged bleeding time, strongly reduced platelet adhesion to glass and defective platelet aggregation in response to ADP and collagen. In contrast to hereditary thrombasthenia membrane glycoproteins (GP) IIb and IIIa were normally present in the patient's platelets. Immunoelectrophoretic analysis revealed an abnormal behaviour of the patient's GP IIb-IIIa complex. Autoantibodies against GP IIb-IIIa were detected in Triton-extracted washed platelets. Incubation of normal platelets with plasma from the patient resulted in a similar immunoelectrophoretic abnormality of the GP IIb-IIIa complex indicating that bound autoantibodies (IgG) are responsible for the abnormal immunoelectrophoretic behaviour of the patient's GP IIb-IIIa complex. Platelet fibrinogen was severely reduced similar to classical thrombasthenia suggesting that the GP IIb-IIIa complex is involved in platelet fibrinogen storage.

Autoantibodies↗
PubMed1991

The microfilamental system of human blood platelets.

The major microfilamental protein of human blood platelets is actin. It has been estimated to constitute 20-50% of total platelet proteins. The organization of actin filaments within the cell is regulated by their association with other proteins. This review will focus on the composition, the cellular organization and on the functions of the microfilamental system in unstimulated and stimulated platelets.

Actin Cytoskeleton↗
PubMed1991

Measurements of the G-/F-actin equilibrium in ADP-stimulated human platelets.

The amounts of the different forms of actin (G-actin, F-actin) can be measured biochemically following lysis of the cells by the DNase I inhibition assay. The existing methodology for studying G-actin in unstimulated platelets was found to be inappropriate for studies during ADP-induced platelet activation. However, this problem was overcome by a simple modification of the procedure in which formaldehyde was added to the buffer used to lyse the activated platelets. Using this modification the G-actin values obtained immediately after lysis did not change during storage of the lysates on ice for more than 30 minutes. The results show rapid conversion of G-actin to F-actin in association with the shape change during ADP-stimulated activation. Examination of unstimulated platelets using the modified procedure enabled identification of a pool of actin that is rapidly dissociated to G-actin in the absence of formaldehyde, the existence of which had not previously been recognised.

Actins↗
PubMed1991

Determination of platelet activation by assaying filamentous actin and detecting membrane alterations.

Thrombin stimulation of human gel-filtered platelets in an unstirred system (without aggregation) results in actin polymerisation. Concomitantly with actin polymerisation the activation markers (CD 63--glycoprotein 53, a 53 kD lysosomal protein and CD 62--GMP 140, a 140 kD alpha granule protein) increase on the platelet membrane as well as the specific binding of monoclonal antibodies to thrombospondin (P 10). Consequently, both assays run in parallel and indicate sensitively platelet activation. Our data also indicate that exposure of subcellular structures following granule fusion is a very early event when platelets are challenged by thrombin and involve a reorganisation of cytoskeletal structures. Cytochalasin E inhibits completely the thrombin-induced actin polymerisation and the platelet aggregation but only partially the thrombin-induced exposure of CD 63. The activation markers CD 62 and P 10 were not influenced.

Actins↗
PubMed1991

Evidence that the osmotically fragile yeast S. cerevisiae VY1160 is an actin mutant.

The phenotype of the osmotically dependent S. cerevisiae mutant VY1160 is caused by a single chromosomal mutation, termed srb, with pleiotropic effect. Compared with cells of the parental strain S288C, it was shown that the size and surface structure of the mutant cells are changed. The latter are sensitive to elevated cultivation temperatures as well as to hypotonic pressure and mechanical stress. In these cases, specific plasma membrane alteration were revealed by freeze-fracture electron microscopy. The total actin content is only 88% (21.4 micrograms actin/mg protein) of that of S288C cells. Remarkably, the mutant cells contain only 2.2 micrograms F-actin/mg protein, whereas the S288C cells have 10.3 micrograms F-actin/mg protein. Moreover, the level of reduced glutathione is found to be higher in the mutant cells (23.4 nmole/10(10) cells) than in the parental cells (15.2 nmole/10(10) cells). These results implicate that the srb mutation is localized in the actin gene.

Actins↗
PubMed1990

Platelet membrane defects in fawn hooded bleeder rats.

An inbred strain of fawn hooded rats with a congenital platelet defect shows a marked bleeding tendency with prolonged bleeding time. This haemorrhagic disorder has been exclusively related to a deficiency of nucleotides in platelet dense granules. When tested in cell electrophoresis platelets from fawn hooded bleeder rats showed a significantly lower electrophoretic mobility than normal rat platelets. Subsequent studies on the platelet membrane protein pattern by high resolution two-dimensional gel electrophoresis revealed the deficiency of a membrane glycoprotein (apparent molecular mass 90.000, isoelectric point 5.6), which is detectable in normal rat platelets after surface labeling by periodate-tritiated sodium borohydride. It seems likely, that this glycoprotein defect contributes at least partially to the disorder of platelet function in fawn hooded bleeder rats.

Animals↗
PubMed1990

Flow-cytometric detection of surface membrane alterations and concomitant changes in the cytoskeletal actin status of activated platelets.

Occlusive vascular diseases are promoted by a "prethrombotic state" with increased platelet activity. Polymerization of cytoskeletal proteins and exposure of subcellular structures or rebinding of secreted proteins have been characterized as early reactions after platelet activation preceding adhesion and aggregation. Here, we demonstrate the kinetic increase in specific binding of monoclonal antibodies to thrombospondin (P10) and to platelet membrane activation markers CD63 (GP53, a 53 kD lysosomal protein) and CD62 (GMP140, a 140 kD alpha granule protein) by using a flow-cytometric bio-assay and the related change in the actin status by using the DNase-I inhibition assay after stimulation of normal human platelets with 0.2 U/ml thrombin. F-actin was raised from 41% to 51% of total platelet actin content 30 s after stimulation and remained thereafter constant (50% at 60 s). Simultaneously, the percentage of P10, CD63, and CD62 positive platelets was elevated from 5.4%, 24.4%, and 9.1% to 67.4%, 80.2%, and 82.3% respectively. The mean number of P10, CD63, and CD62 antibody binding sites increased from 3,300, 1,715, and 2,146 to 6,400, 6,800, and 9,016 per platelet. Conclusively, changes in the organization of the cytoskeletal protein "actin" and exposure of subcellular structures indicating platelet secretion can be regarded as markers of early platelet activation. Thus, the parallel response in both analytical systems provides further support for the diagnostic concept of flow-cytometric detection of preactivated platelets in the peripheral blood by using fluochrome staining procedures detecting activation dependent structural alterations directly at the cellular level.

Actins↗
PubMed1990

[Effect of a phorbol ester (PMA) and forskolin of the G-/F-actin equilibrium in human platelets].

Phorbolester (PMA) and forskolin (FSK) cause a dramatic reorganization of microfilaments in cultured cells. We have incubated human blood platelets with PMA and FSK and we investigated the G-/F-actin equilibrium by the DNase I inhibition assay. PMA incubation (0.8 microM, 5 min, 37 degrees C) leads to an increase of filamentous actin (14.4 +/- 4.0%) compared to control platelets. The effect is rapid, dose-dependent and specific, since the biologically inactive derivative phorbol 12,13-didecanoate has no effect. FSK incubation (4 microM, 5 min, 37 degrees C) causes a decrease of F-actin (12.8 +/- 9.0%), the effect is rapid and dose-dependent too. Since 8-bromoadenosine 3':5' cyclic monophosphate also decreases F-actin in human blood platelets, the FSK effect seems to be mediated by cyclic AMP due to affecting the adenylate cyclase.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1990

[The cytoskeleton of platelets].

The platelet cytoskeleton is composed of microfilaments, microtubules and intermediate filaments (vimentin is only a minor component). Since actin is the major platelet protein, this review is restricted to aspects of the microfilamental system. The first part is devoted to the description of actin and of proteins thought to be associated with actin in platelets. The review will then focus on the organization of the microfilaments in unstimulated and stimulated platelets. Finally, the role of the microfilaments in maintaining unstimulated platelets in a functional form and in directing the responses of platelets to activation is discussed.

Actin Cytoskeleton↗
PubMed1989

Actin filament content in platelets--a sensitive index of cellular reactivity.

Blood platelets have the capacity to participate in a number of physiological as well as pathological processes within the circulation. In order to evaluate their cellular reactivity a number of platelet function tests have been developed. The main in vitro function tests are assessment of aggregation and adhesion, secretion, arachidonate metabolism, coagulant activities and the characterization of surface membrane glycoproteins (Day and Rao, 1986). Here we measure alterations of the G-/F-actin equilibrium of platelets. High F-actin values of unstimulated platelets indicate a hyperreactivity of the cell as examined in platelets from diabetics. Determination of the actin filament content in platelets can be considered as a new sensitive function test.

Actins↗