bluuebunny/crossref_metadata_2025_split huggingface dataset:
Reconstitution Of Liposomes Bearing Platelet Membrane Glycoproteins. A Functional Study With Von Willebrand Factor
Authors: Sié P , Gillois M , Chap H J , Boneu B , Biermé R , Douste-Blazy L 
Published: July 2018

Functional studies on isolated membrane glycoproteins (GP) are impaired by their poor solubility in the absence of detergent. The major glycoprotein of the platelet plasma membrane (GP lb) does not escape to the rule since its soluble derivative (glycocalicin or GP Is) does not exactly share the properties of the integral GP.This study aimed to develop a membrane model where the GP Ib was solubilized from fresh human platelet membranes by desoxycholate, isolated using wheat-germ-agglutinin (WGA)- affinity chromatography and finally reincorporated into phospholipid vesicles by removal of the detergent. As shown by electron micrography, the method yielded individual, large - sized, single bilayered vesicles : WGA reversibly agglutinated theses vesicles arguing for the incorporation of the GP in the wall of the vesicles and the accessibility of the oligosacharidic moiety. Additionally, a rapid and convenient method for assessing agglutination was developped using thin -layer chromatography on agarose gel.Since the GP Ib is the putative receptor for von Willebrand factor (vWF) we studied the effect of vWF on these vesicles. vWF was purified from cryoprecipitate by gel exclusion chromatography followed by an ion-exchange chromatography on DEAE-Sephacel. vWF, vWF plus ristocetin but not ristocetin alone induced a strong specific agglutination of the vesicles. This ristocetin-independent effect markedly differs from the natural behaviour of intact platelets and we speculate that the present model might help to analyse the factors which, at the platelet surface, refrain the plasmatic vWF from directly interacting with the exposed GP Ib.
Effect Of The Multimeric Structure Of The Factor VIII/Von Willebrand Factor Protein On Binding To Human Platelets
Authors: Gralnick H , Williams S , Morisato D J 
Published: July 2018

We have studied the characteristics of binding of intact factor VIII/von Willebrand factor (f. VIII/vWf) protein and 2-mercaptoethanol (2ME) treated f. VIII/vWf protein to human platelets. The purified f. VIII/vWf was radiolabelled with tritiated 3H potassium borohydride; 4.5 × 103 molecules of the intact radiolabelled material bound per platelet. Of these some molecules bound with a high affinity/low capacity (Kd 0.21 nM and 1.5 × 103 molecules) and another with a low affinity/high capacity (Kd 2.5 nM and 3 × 103 molecules). When the material was reduced with 2ME at 0.01%, it bound with an intermediate affinity of 1.6 nM with a capacity of 4.0 × 103 and a low affinity binding of 12.5 nM and a capacity of 4.0 × 103 . The 0.1% 2ME-treated material revealed only low affinity binding with a Kd of 15 nM and the number of molecules bound 13 × 103. Studies of competitive inhibition of the intact f. VIII/vWf binding to human platelets by the reduced materials revealed that the smallest f. VIII/vWf protein (i.e., 0.1% 2ME) was the least effective while the 0.01% 2ME material was intermediate between the 0.1% and the intact material. The differences noted in the ability to displace the intact material as well as in binding to the human platelet were paralleled by decreases in the vWf activity of these proteins.These studies aid in our understanding of the binding of the f. VIII/vWf to platelets in that the binding sites on platelets may be homogeneous while the ligand is heterogenous. These studies reinforce the structure/function relationships of f. VIII/vWf proteins which have been defined using ristocetin-induced platelet aggregation (i.e., the minimum molecular size of the f. VIII/vWf protein and the penultimate galactose residues on the carbohydrate side chain). We conclude that these defects of the f. VIII/vWf protein also interfere with the protein binding to its platelet receptor and that f. VIII/vWf binding to platelets is an important primary step in hemostasis.
Fibrinogen Bern I: A Hereditary Fibrinogen Variant With Defective Conformational Stabilization By Calcium Ions
Authors: Rupp C , Kuyas C , Häberli A , Furlan M , Beck E A 
Published: July 2018

Inherited hypodysfibrinogenemia (fibrinogen Bern I) was found in four members (two generations) of a family with no haemorrhagic or thrombotic history. Fibrin aggregation curves (350 nm, 37°C) with patient plasma or purified fibrinogen Bern I, after addition of thrombin, were normal at high calcium concentrations (5mM) but delayed at lower calcium concentrations (≤0.lmM). The release of fibrinopeptide A was normal. Whereas the polypeptide chains of fibrinogen Bern I were indistinguishable from normal fibrinogen by SDS-gel-electrophoresis, an abnormal γ-chain with a decreased negative charge was found by isoelectric focussing.Plasmic degradation o| normal fibrinogen, in the presence of calcium (≥ImM), results in only one terminal D fragment which is stabilized by calcium against further degradation of γ-chains. In contrast, degradation of fibrinogen Bern I, under the same conditions, yielded at least two additional smaller D fragments. In conclusion, fibrinogen Bern I is characterized by defective calcium binding in the D domain of the γ-chain.
The Role Of Glycoprotein V (GPV) In Thrombin Activation Of Human Platelets
Authors: Fujimura K , Maehama S , Kuramoto A 
Published: July 2018

The analysis of platelet membrane glycoproteins and platelet functions was conducted to disclose the role of GPI and V in the thrombin activation of platelet. Our previous study proved that native and HNB thrombin hydrolyzed GPV(M. W.8-9 × 104) selectively and released new glycoprotein fragment (M.W. 6.2-6.8 × 104 ) of GPV, resulting in the development of 14C-5HT release reaction and platelet MDA production. But DIP thrombin could not induce these phenomena.Membrane surface proteins of intact platelets were labeled with Na[3H]BH4 by neuraminidase and galactose oxidase method and analyzed by fluorography after SDS-PAGE.The high molecular weight glycoproteins, GPI, GPIII and GPV were diminished by trypsin treatment in correlation with the concentration and incubation time. In correspond to the diminution of these membrane glycoproteins, platelet release reaction was increased .Chymotrypsin treatment in various concentrations, release reaction and MDA production were not induced in spite of long incubation times. But the ristocetin aggregation was decreased in Chymotrypsin treated platelets whose membrane glycoproteins did not change significantly. The Chymotrypsin treated platelets whose GPI was modified functionally, showed normal release reaction and MDA production by thrombin stimulation. On the other hand, the thrombin treated platelets in low concentration previously whose GPV was hydrolyzed partially, demonstrated little release reaction and MDA production by thrombin or trypsin stimulation. From these results, the GPV was hydrolyzed specifically by thrombin and nonspecifically by trypsin but was not hydrolyzed by Chymotrypsin. It was concluded that the thrombin binds to the GPI and hydrolyzed GPV specifically, and hydrolysis of GPV might act as a signal to induce the platelet release reaction and prostaglandin metabolism.
Fibrinolysis And Coumarin Therapy: A Possible Relationship
Authors: Bauer J , Bachmann F 
Published: July 2018

It has been reported that a good proportion of patients suffering from idiopathic deep vein thrombosis (IDVT) exhibit a defective release of plasminogen activator after venous stasis (Isacscn & Nilsson, Acta Chir Scand 138:313-319, 1972).We have studied the 3 known activation pathways of physiological fibrinolysis in 36 patients with IDVT and 21 controls. The activity of the Hageman factor and contact phase Independant Intrinsic Fibrinolytic pathway (HIIF) was measured in the euglobulin fraction of a plasma obtained in fasting subjects after a 30 min rest period at 9 a.m. Activity of the Hageman factor Dependant Intrinsic Fibrinolytic pathway (HDIF) was measured after activation of the plasma by dextran sulfate. HDIF activities were expressed as A (after-before) dextran sulfate activation. Venous stasis induces the release of vascular activator. The latter triggers off the Extrinsic Fibrinolytic pathway (EF). Its activity was expressed as the Δ (euglobulin activity after venous stasis - HIIF activity).26/36 patients (72%) with IDVT showed impaired fibrinolysis of at least one pathway. 24 (67%) exhibited a defective HIIF: in 14 (38%), this was an isolated defect, in 10 (28%), impaired HIIF was associated with defective EF. An isolated defect of EF occurred in only 2 patients. HDIF was normal in all patients. 20 of 24 antiooagulated patients, but only 4 of 12 nonanticoagulated patients had impaired HIIF, either alone or in combination (X2=9; p < 0,005).These results strongly suggested an association of impaired HIIF and oral anticoagulation. When HIIF, HDIF and EF were measured before and 10 to 14 days after acenocouma- rol treatment in normal volunteers, there was marked decrease of HIIF activity, suggesting that an as yet non- identified vitamin K dependant factor participates in the HIIF pathway.
Plasminogen Activation In Normal Individuals And Patients With Thrombotic Tendency
Authors: Schulman S , Holmgren A 
Published: July 2018

Following the principles described by Wohl, Summaria and Robbins we have studied the kinetics of human plasminogen activation by streptokinase. Plasma plasminogen activation kinetic parameters were measured in the following way: Citrated plasma was mixed with a Tris/NaCl buffer, pH 7.4, and increasing amounts of streptokinase were added. After incubation at 37°C for 5 min, synthetic substrate, H-D-val- -L-leu-L-lys-p-nitroanilide was added and initial plasmin rates were recorded. We also determined the Michaelis-Man- ten parameters for the plasmin of the plasma samples following maximal activation of the plasminogen with streptokinase. This was done by incubating citrated plasma (diluted 1:200) with streptokinase (5000 U/ml) for 10 min at 37°C.In the normal individuals the zymogen.streptokinase complex gave a mean catalytic efficiency, kcat/KM, of 0.15 μM-1S-1. For the streptokinase activator species the average catalytic efficiency, kplg/Kplg, was 26 μM-1min-1, Iμ(95%) = {8.4; 44.5} μM-lmin-1.So far we have studied eight patients with a history of thrombosis. With the exception of one patient, all showed values within the range mentioned above. The deviating patient had a significantly lower concentration of plasminogen, 6.7 mg/dl, as measured by immunodiffusion, and a significantly higher kplg/Kplg-value, 54 μM-1min-1, but a similar kcat/KM-value, 0.2 μM-1S-1. The lower plasminogen concentration is thus matched by an apparent increase of streptokinase activation. We do not know if the observed effect is caused by a mutation in the enzyme. An explanation for the low concentration of plasminogen could be chronic fibrinolysis of thrombi.We will investigate this further and also screen more patients with a history of thrombosis for possible variants with lowered catalytic efficiency of zymogen activation.
Inhibitory Activity Of Heparin And Of A Heparin Side Fraction (Org.10172) On The Activation Of Thrombin In Human Plasma
Authors: Jonker R , van Houdenhoven F , van Dedem G 
Published: July 2018

The dynamics of thrombin generation and -inactivation in human plasma was studied in the absence and presence of heparin and of Org 10172, with the purpose of defining (a) mechanism(s) whereby anticoagulant action is accomplished.Plasma was activated with a standard stimulus of either thromboplastin and Ca++ or by kaolin-cephalin and Ca++ ; subsamples were taken at regular time intervals and assayed for amidolytic-(S 2238) and antithrombin-3 (AT3) activities. Similar experiments were done in the presence of heparin and of Org 10172.With heparin, 2 domains could be distinguished: a) a domain (at heparin concentrations below 1 U/ml) where heparin accelerates the inactivation of thrombin and b) a domain (at heparin concentrations above 1 U/ml) where no measurable free thrombin is generated and no measurable drop in AT3 levels could be detected.With Org 10172 the most pronounced effect was a dose- dependent delay in the onset of free thrombin generation and a weakly dose-dependent reduction in the free thrombin peak levels.In AT3-free plasma, thrombin generation after a standard stimulus was very rapid and the final constant level of am- idolytic activity was higher than in normal plasma. Heparin concentrations of 2 - 4 U/ml changed this pattern to a gradual increase of amidolytic activity to its final level.Org 10172 showed the same pattern as heparin and also delayed the onset of free thrombin generation in a dose- dependent manner.It is concluded that a) the anticoagulant effect of heparin in plasma, activated by a standard stimulus is due to at least 2 mechanisms, one of which is probably independent of AT3, and b) Org 10172 exerts its effect independently of AT3, possibly by inhibiting one of the positive feedback loops in the coagulation sequence.
Isolation And Characterization Of The Vitamin K Dependent Domain Of Human Prothrombin
Authors: Dodé C , Thiesce A , Labie D , Elion J 
Published: July 2018

Chymotryptic cleavage of human prothrombin (hPt) produces two fragments of 68000 and 5000 MW respectively. These two species were separated by Ba citrate adsorption and Sephadex G100 chromatography. The 68000 MW species corresponds to hPt (des 1-44) and has lost all the vitamin K dependent properties of hPt : adsorption on Ba citrate, Ca+2 and phospholipid dependent stimulation of the activation by FXa, presence of strong Ca+2 binding sites (as shown by dialysis equilibrium) and Ca+2 induced fluorescence quenshing. The 5000 MW species corresponds to the N-terminal portion of hPt (residues 1-41). It contains the 10 Gla residues and the 17-22 disulfide bond. This peptide is quantitatively adsorbed on Ba citrate. Activation of hPt in a mixture containing FXa, Ca+2 and phospholipid is drastically inhibited by the addition of peptide 1-41 (≃ 50 % inhibition for a 1/1 peptide/Pt ratio, ≃ 7 % for a 40/1 ratio).Ca+2 produces a quenshing of the intrinsic fluorescence of peptide 1-41 (λ emission = 355 nm). This quenshing plateausat 40 % of the initial fluorescence at 3 mM Ca+2. The plot of the fluorescence quenshing versus Ca+2 concentration however is not cooperative. Mn+2 and Mg+2 also induced fluorescence quenshing but to a lesser extent. Hence peptide 1-44 represents a functionnal domain in itself interacting with Ca+2 and phospholipid. It contains only one Trp (residue 41), showing directly the involvment of this residue in the Ca+2 induced fluorescence quenshing observed for Pt fragment (FI) and Pt. This isolated vitamin K dependent domain therefore retains many of the vitamin K dependent properties of FI or Pt, but shows differences in the Ca+2 induced conformational change in that it is no longer a cooperative process.
Preferential Binding Of Prothrombin Fragments On Monolayers Containing Phosphatidylserine
Authors: Lecompte M F , Miller I R 
Published: July 2018

Phospholipids from platelet membranes play a crucial role in the last step of the blood coagulation cascade, as a catalytic surface for the conversion of prothrombin into thrombin by the prothrombinase complex. It provides a favora ble configuration of the required factors ( factor Xa, prothrombin, factor Va ), concentrated on it’s surface. The maximal biological activity is observed with mixtures of phospholipids containing phosphatidylserine, in presence of Ca++.In order to understand at the molecular level, which part of prothrombin interact with phospholipids, we studied the correlation between the number of prothrombin molecules or fragments of it adsorbed ( by counting the surface radioactivity of 3H-protein ) and their ability to penetrate into the phospholipid monolayer ( measured by the increase of the lipid layer capacitance ), by two recent complementary methods of direct surface measurement ( radioactivity and electrochemistry ), in function of Ca++ concentration and phospholipid composition.We could detect parallely the number of cystine residues ( by electrochemical reduction ) of the penetrating parts of the adsorbed protein.lt occurs that adsorbed protein don’t penetrate. The number of S-S groups reduced per adsorbed protein molecule can be deduced and its variation indicate different orientation of the molecules on the phospholipid surface. Indeed, it is the case at different Ca++ concentration,for prothrombin and fragment 1, which contain γ-carboxyglutamic groups.We could also observe that penetration is specific of phospholipid composition for fragment 2 but not for thrombin and is not specific for both in function of the Ca++ concentration.Results indicate that the ratio of penetration to the total number of adsorbed prothrombin molecules and fragment 1 is important for an optimal catalysis.
Calcification At The Blood-Material Interface. A Study Employing X-Ray Diffraction And Other Methods In Various Pumps
Authors: Murray J D , Harasaki H , Gibbons D F , Kiraly R J , Nose Y 
Published: July 2018

Blood pumps assisting the natural heart have been tested successfully in calves for up to 9 months. The aim of current research is for an assist device to last 2 years or longer for clinical application. To achieve this, the problem of calcification on the blood contacting surface of the pump must be solved, or deterioration of the pump and a source of emboli could result.Scanning electron microscopy(SEM),energy dispersion x-ray analysis(EDAX).thickness mapping,and histology have been used to characterize the pseudoneointima(PNI),or cellular layer.depositied on the diphargm of the assist devices. Calcification in the cellular components in the flexing area of the diaphragm has been seen in 22/47 left ventricular assist devices implanted 12-227 days,and in 19/26 par- athoracic ventricular assist devices implanted 5-160 days. Hydroxyapatite,the final crystalline form of calcium phosphate in calcification,was detected using x-ray diffraction, which is the best means of determining crystal structure.In the rough, dacron fibril flocked surface of the PVAD, hydroxyapatite was first observed after 13 days,and in the smooth.biolized gelatin surface of the LVAD,hydroxyapatite was detected after 43 days. Relating to the pathogenesis of calcification, RBC and platelets were embedded separately in gelatin and reacted with calf serum in an in vitro experiment to determine the nidus for calcification. TEM,histology and x-ray diffraction were performed on the embedded material after 3,7,14,21,and 28 days. Hydroxyapatite was seen in 1 week in the RBC and after 3 weeks in platelets.The in vitro data support the in vivo evidence that calcification is dystrophic in nature.occurring in the degraded RBC and platelets contained in the PNI. It is also indicated that increased stress at the flexing region,thicker PNI,and high serum phosphorus level exacerbate calcification.
Assessment Of Thromboresistant Materials By 125I-Fibrinogen Scanning In Animals And Man
Authors: Lewis J L , Sweeney J , Houlihan M J , Baldini L , Salzman E W 
Published: July 2018

Further development of artificial organs requires improved methods to evaluate potentially thromboresistant materials, for which there is at present no simple method in man. We now report that thrombi associated with intravenous cannulae can be detected using 125I-fibrinogen and a portable gamma counter. FEP/Teflon cannulae were inserted percutaneously in 9 dogs receiving EACA and 1 sheep, and four sites over each cannula and over a control vein were counted before and at 30 to 45 minute intervals for up to 6 hours after cannula insertion. Animals were heparinized before sacrifice. Results were confirmed by autopsy. Nine of ten thrombi related to 12 cannulae were detected by a significant rise in counts (p<0.05, 3 way ANOVA). There were no false positives; the single false negative was related to a filmy thrombus with low gamma activity when counted directly. The smallest thrombus detected weighed 1 mg, representing fibrin arising from 500 μl of blood.Thirty-nine patients were studied. Radioactivity.over an IV catheter introduced after administration of 125I- fibrinogen was compared with independent blinded clinical appraisal by a trained nurse. Significant progressive rise in radioactivity over the catheter tip or within 2 cm downstream developed in 13 of 14 forearms with 16 or 18 gage cannulae, all of whom subsequently developed rubor, tenderness, palpable thrombus, or plugging of the catheter. The rise in 125I counts preceded the clinical signs by 12 to 36 hours. 22 gage cannulae produced clinical signs of phlebitis in only 42% of cases but increased 1251 counts in 92%. Phlebograms were confirmatory.
125I-fibrinogen external counting is a simple, safe, and sensitive technique to detect thrombosis induced by intravenous cannulae in man and should be suitable for the comparison of potentially thromboresistant materials.
Effects Of Short Time High Shear Exposure Upon Platelet Function And The Coagulation System Under Heparin Anticoagulation
Authors: Wurzinger L J , Opitz R , Blasberg P , Bialonski K , Schmid-Schönbein H 
Published: July 2018

The fact that high shear activates and damages platelets has been suspected to be a major cause of thromboembolism in artificial internal organs (AIO) or in arterial stenosis. In AIO wall shear stresses well above 50 Nm-2 have been computed to which blood cells are exposed for times in the order of milliseconds (ms). Unfortunately, the studies on this subject employing defined flow conditions operate with exposure times higher than 10 seconds. The pupose of the present study was to elucidate the effects of high shear exposure for ms upon platelet function (ADP induced platelet aggregation (PA), platelet procoagulant activity {PF- 3)) under heparin anticoagulation, which is also used in AIO.To apply shear rates ranging from 50 - 220 Nm-2 to heparinized PRP for defined exposure times between 7 - 700 ms a flow through Couette-viscometer was employed. Platelet factor 3 (PF-3) availability was estimated by using a modified Stypven time technique. Lactic dehydrogenase (LDH) liberation was taken as a measure for platelet destruction. All steps of the experimental procedure were carried out at 37°C.From our data we conclude that, in the presence of physiological calcium levels (heparin anticoagulation) shear stresses and exposure times that certainly occur in AIO are able to activate platelets and procoagulant potential of blood.