bluuebunny/crossref_metadata_2025_split huggingface dataset:
ACTIVATION OF HUMAN ENDOTHELIAL TYPE PLASMINOGEN ACTIVATOR INHIBITOR (PAI-1) BY NEGATIVELY CHARGED PHOSPHOLIPIDS
Authors: Lambers J W J , Cammenga M , Konig B , Pannekoek H , van Mourik J A 
Published: August 2018

The endothelial cell type plasminogen activator inhibitor (PAI-1) may exist in an active, latent form that can be converted into an active form upon exposure to denaturants such as sodium dodecyl sulphate (SDS), guanidine-HCl or urea. Here we show that latent PAI-1 can be activated with lipid vesicles, consisting of the negatively charged phospholipids phosphatidylserine (PS) or phosphatidylinositol (PI). The presence of a net negative charge on the phospholipid headgroup is essential for activation. Incubation with lipid vesicles, consisting of the zwitterionic phospholipids phosphatidylcholine (PC) and phosphatidylethanol-amine (PE), does not result in activation of the inhibitor. In the presence of PS vesicles, the capacity of PAI-1 to inhibit tissue type plasminogen activator (t-PA) is 50-fold higher than that of the untreated protein. For comparison, the activity of PAI-1 was enhanced 25-fold by treatment of the protein with SDS. PS induces activation of the inhibitor at much lower concentrations than SDS. For example, to achieve 50% inhibition of t-PA with a more than 100-fold excess of PAI-1, 0.25 nmoles of PS are required, whereas L.60 nmoles of SDS are necessary to reach half maximal inactivation of t-PA. Activation of PAI-1 by PS can be reversed by the addition of Ca2+-ions, suggesting that Ca2+-ions interfere with the interaction of PAI-1 with the negatively charged lipid surface, thus preventing its activation. The lipid-induced activation of PAI-1 points to a possibly important role of phospholipids in fibrinolysis; regulation of the fibrinolytic activity in blood plasma may ultimately be determined by the extent to which these phospholipids activate the inhibitor of t-PA.This study was supported by the Netherlands Thrombosis Foundation.
PLATELET AND ENDOTHELIAL CELL CYTOADHESINS ARE BIOSYNTHESIZED AND PROCESSED VIA SIMILAR PATHWAYS
Authors: Polack B , Duperray A , Berthier R 
Published: August 2018

The cytoadhesin family represents a group of heterodimeric adhesion receptors with common structural, fonctional and immunochemical properties. Platelet and endothelial cell (EC) GPIIbIII a related proteins exemplify two members of this family. In the present study the biosynthesis and processing of EC GPIIbIII a were examined and compared with that of platelet GPIIbIIIa to verify whether the diversity of these cytoadhesins was related to post translational events.Endothelial cells of human umbilical cord vein origin were metabolicaly labeled with 35S-methionine. The newly synthesized proteins were analyzed and immunoprecipitated with polyclonal antibodies against the purified platelet GPIIbllla complex and the isolated GPIIb and GPIIIa. Under non-reducing conditions three bands were detected at 135 kD, 125 kD and 90kD with the anti GPIIbIIIa. Samples obtained from pulse chase experiments and analysed under non reducing conditions indicated that the 135 kD band derived from the 125 kD band. Under reducing conditions the 135 kD generated two bands at 118 kD and 25 kD. The 125 KD band also gave a 118 kD band and the 90 kD band shifted to 100 kD. These results indicated that the mature form of 135 kD is composed of two polypeptidic chains which derive from a common single chain precursor similar to that observed in the megakaryocyte. The mature protein and the precursor molecule were not recognised by anti GPIIb antibodies. Also immunoprecipitated by polyclonal anti GPIIIa antibodies was a single chain protein of 100kD. In addition, endoglycosidase treatement showed that both EC GPIIIa and platelet GPIIIa were glycosylated protein of the high mannose type.These results indicate that although structural differences exists between platelet and endothelial cell GPIIbllla, the two membrane glycoproteins have a similar cellular transit.
MONTREAL PLATELET SYNDROME: DECREASED ACTIVITY OF PLATELET CALPAINS ASSOCIATED WITH AGGREGATION ABNORMALITIES
Authors: Okita J R , Frojmovic M M , Kristopeit S , Wong T , Kunicki T J 
Published: August 2018

The Montreal platelet syndrome (MPS) is an inherited disorder of platelet function characterized by a) severe thrombocytopenia, b) formation of "giant" platelets upon physical or biochemical stimulation, c) spontaneous aggregation (stir-induced microaggregate formation) and d) a lack of aggregation in response to thrombin. The Bernard-Soulier syndrome (BSS) is similar to MPS in that both syndromes are characterized by "giant" platelets and an abnormal aggregation response to thrombin. BSS patients have a deficiency of specific platelet glycoproteins (GPs). From our investigations we conclude MPS patients have apparently normal amounts of major platelet GPs. However, a defect in calpains (calcium-activated neutral proteinases) was detected in MPS platelets. The specific activity of calpains was decreased by 70% (p<0.001) in the cytosolic fraction of platelets from MPS patients as compared to that of platelets from normal control donors. The calpain activity of platelets from BSS patients was within the normal range.During the course of the biochemical studies, platelets from the MPS patients were shown to exhibit concurrent functional defects, i.e., stir-induced spontaneous aggregation and reduced to absent aggregation response to thrombin. It is concluded that MPS can be distinguished from BSS at the molecular level as follows: 1) MPS platelets contain normal amounts of GPs Ib, V and IX which are decreased or absent in BSS platelets; 2) The specific activity of calpains is reduced in MPS platelets but normal in BSS platelets. Supported by NIH (HL-33925, HL-32279), Amer. Heart Assoc. (85—GA—67, 83-186), Canadian Med. Res. Council (248-59) and Quebec Heart Fndn.
A COMPARISON OF THE BINDING OF ANTITHROMBIN III AND HEPARIN COFACTOR II TO HEPARINS, NATURALLY OCCURRING GLYCOSAMINOGLYCANS AND OTHER SULPHATED POLYMERS
Authors: Dawes J , Pepper D S 
Published: August 2018

Antithrombin III (ATIII) and heparin cofactor II (HCII) are currently thought to be the most important protein mediators of the anticoagulant and antithrombotic activities of glycosamino-glycans. A simple, quantitative method for assessing the affinity of a protein for a sulphated polymer in the liquid phase, based on competition with immobilised heparin, has been developed. Using this technique, the binding of ATIII and HCII to a wide range of glycosaminoglycans and other sulphated polymers have been compared, and the contributions to binding of size, degree of sulphation and backbone structure of the polymers analysed.In the presence of the high protein concentrations found in plasma, unfractionated heparin inhibited the binding of ATIII to immobilised heparin with a Ki of 1 x 10-6. Binding was destroyed by N-desulphation. 1 Results with a range of low molecular weight (LMW) heparins and heparan sulphates are consistent with the view that they all contain the ATIII-binding sequence, but at a lower molar ratio than heparin. Highly sulphated synthetic polymers such as dextran sulphate bound ATIII by a different mechanism, which was molecular weight-dependent.The affinity of HCII for heparins increased markedly with heparin chain length. Binding was largely, but not entirely, mediated by sulphate residues. HCII bound to heparan and dermatan sulphates with lower affinities than to heparin, and to synthetic sulphated polymers with similar or higher affinities. Pentosan polysulphate (SP54) bound HCII as effectively as did heparin. Binding of HCII to dextran sulphate was highly dependent on molecular weight. The affinity of HCII for a sulphated polymer appears to depend both on its chain length and density of sulphation.Thus the profiles of binding of ATIII and HCII to glycosaminoglycans and other sulphated polymers are quite different. This technique is useful both for investigating the interactions of existing therapeutic anticoagulants and assessing new products.
MOLECULAR CONFORMATION OF THE PENTASACCHARIDE CORRESPONDING TO THE BINDING SITE OF HEPARIN TO ANTITHROMBIN III
Authors: Choay J , Petitou M , Perly B , Casu B , Ragazzi M , Ferro D 
Published: August 2018

The knowledge of the tridimensional structure of the sequence required in heparin for binding to anti thrombin III (AT) is essential for the understanding of the molecular basis of the anticoagulant and antithrombotic activity of this glycosamino-glycan. This problem was approached by a combined experimental (chemical synthesis and Nuclear Magnetic Resonance, "NMR") and theoretical (force field calculations) study of the pentasaccharide I reproducing the minimal sequence for high affinity binding to AT.NMR data (interproton coupling constants and nuclear-Over-hauser-enhancement) were used to test a number of possible conformation of I proposed on the basis of-molecular energy calculation. In particular, these studies, confirming the results obtained by force field calculations, showed that the sulfated iduronic residue (Is) conformation is an equilibrium between the chair 4C4 and the skew boat2S0, and permitted to discard models with the Is residue in the alternate chair 4C1. It is of interest that the relative population of the 4C4 and 2S0 conformers of IS in sequence I (40:60) is reversed with respect to that found for the same residue in the regular sequences of heparin (60:40), such a reversal being largely associated with electrostatic interactions caused by the unique 3-0-sulfo group ot the preceding amino sugar residue. The most favoured models of I are characterized by a cluster of anionic groups including all the sulfate groups proposed as essential for full expression of the activity (full line square on the formula) but one (dotted line square on the formula), plus the carboxylate group of the glucuronic acid residue.
VON WILLEBRAND FACTOR (vWF) PRO-POLYPEPTIDE IS REQUIRED FOR vWF MULTIMER FORMATION
Authors: Verweij C L , Hart M , Pannekoek H 
Published: August 2018

The von Willebrand factor (vWF) is a multimeric plasma glycoprotein synthesized in vascular endothelial cells as a pre-pro-polypeptide with a highly repetitive domain structure, symbolized by the formula:(H)-D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-(0H).A heterologous expression system, consisting of a monkey kidney cell line (C0S-1), transfected with full-length vWF cDNA, is shown to mimic the constitutively, secretory pathway of vWF in endothelial cells. The assembly of pro-vWF into multimers and the proteolytic processing of these structures is found to oro-ceed along the following, consecutive steps. Pro-vWF subunits associate to form dimers, a process that does not involve the pro-polypeptide of pro-vWF. This observation is derived from transfection of C0S-1 cells with vWF cDNA, lacking the genetic information encoding the pro-polypeptide, composed of the domains D1 and D2. Pro-vWF dimers are linked intracellularly to form a regular series of multimeric structures that are secreted and cannot be distinguished from those released constitutively by endothelial cells. The presence of the pro-polypeptide, embedded in pro-vWF, is obligatory for multimerization since the deletion mutant lacking the D1 and D2 domains fails to assemble beyond the dimer stage. It is argued that the D domains are involved in interchain interactions.
INCREASED IN VIVO FORMATION OF THROMBOXANE AND PROSTACYCLIN IN HUMANS AFTER AORTIC REPLACEMENT WITH SYNTHETIC GRAFTS
Authors: Swedenborg J , Greén C , Lewin J , Vesterquist O 
Published: August 2018

Replacement of arteries with synthetic grafts causes activation of both plasma coagulation and platelets. In order to measure platelet activation the in vivo production of thromboxane A2 (TxA2) and prostacyclin (PGI2) were measured in patients following graft replacement of the abdominal aorta for aneurysmal disease.Specific methods based on gas chromatography-mass spectrometry using tetra-deuterated internal standards/carriers were used to measure the urinary excretion of 2,3-dinor-TxB2 and 2,3-dinor-6-keto PGF1α, the two major urinary metabolites of TxA2 and PGI2. The excretion of the metabolites increased ten-fold and fortyfold respectively on the first postoperative day and remained elevated up till 10 days postoperatively. In patients undergoing cholecystectomy only minor changes of shorter duration were seen. A marked decrease in platelet count occurred concomitanly with the increase in the urinary metabolites. Platelet counts returned to normal or supernormal values after 10 days when the excretion of 2,3-dinor TxB2had returned to normal values.It is concluded that synthetic grafts cause prolonged increase in the in vivo formation of TxA2 and PGI2 concomitantly with a decrease in platelet count. The reason for the increased TxA2 formation may be platelet interaction with the foreign surface but the increase of PGI2 is unexplained. The latter increase could be part of a vascular defense against the induced thrombotic activity.
ACUTE THROMBOSIS IN STENOTIC AREAS: IMPORTANCE OF THE VASCULAR MATRIX EXPOSED TO BLOOD
Authors: Badimon L , Badimon J J , Fuster V 
Published: August 2018

The platelet response to angioplasty or spontaneous plaque rupture leads to acute thrombotic occlusion under certain conditions. We analyzed the role of local shear rate (flow and vessel cross-section related), the nature of the exposed matrix and the effect of thrombin inhibition in platelet acute response to injury. Collagen type I (exposed in plaque rupture) and de-endothelialized pig aorta (mild injury) were exposed to pig blood in a tubular perfusion chamber with well characterized flow conditions, placed within an extracorporeal circuit in swine (N=20). Platelet deposition was measured by labeling autologous platelets with Indium and optical morphometry of epoxy embedded specimens. Selected specimens were analyzed by electron microscopy. Unanticoagulated blood and blood from animals treated with 300u/Kg of heparin were perfused over the substrate for 3 and 10 min at local shear rates typical of unobstructed arteries (212s™1 - 424s™1) and of stenotic vessel (824s™1 - 1690s™1). Platelet deposition (Platelets × 106/cm2 ± SE) for 3 min perfusions were:Platelet deposition is dependent on the reactivity of the vascular matrix exposed to blood and on the local shear rate. The greatest rate of thrombus growth is observed with collagen and high shear rate conditions which may precipitate acute thrombotic occlusion in stenotic regions, mainly when the coagulation pathway is not inhibited (255×l06 platelets/ cm2 in 3 minutes. The relative contribution of rheology and the isolated components of the atherosclerotic plaque matrix exposed to blood in the onset of acute coronary syndromes will be differentiated with this experimental model.
DETECTION AND QUANTITATION OF CLEAVED AND UNCLEAVED HIGH MOLECULAR WEIGHT KININOGEN IN PLASMA BY LIGAND BLOTTING WITH RADIOLABELED PLASMA PREKALLIKREIN OR FACTOR XI
Authors: Lämmle B , Zuraw B L , Heeb M J , Schwarz H P , Curd J G , Griffin J H 
Published: August 2018

A method for the quantitative assay of native single chain and kallikrein cleaved two-chain high molecular weight kininogen (HMWK) in plasma has been developed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of whole plasma is followed by electroblotting of the electropherogram to nitrocellulose membranes and detection of the inmobilized HMWK with its physiologic ligands, plasma prekallikrein or factor XI. Using 1251-prekallikrein or 125I-F.XI overlay nitrocellulose bound HMWK can be visualized by autoradiography.Using unreduced SDS-PAGE cleaved two-chain HMWK (Mr 107,000 and 95,000) is electrophoretically separated from uncleaved single chain HMWK (Mr 150,000). Counting the radioactivity of the nitrocellulose pieces corresponding to cleaved HMWK permits its quantitative measurement by comparison with standards consisting of decreasing amounts of fully dextran sulfate activated normal human plasma. Single chain HMWK is similarly assayed using reduced SDS-PAGE and unactivated normal human plasma standards.This technique is highly specific and sensitive to ˜ 50 ng of either cleaved or uncleaved HMWK. Varying concentrations of cleaved HMWK were found in plasmas from patients suffering from various systemic inflanmatory conditions. Higher levels of in vivo cleaved HMWK were observed during acute attacks of hereditary angioedema due to Cl-inhibitor deficiency.This technique may be useful for the assessment of the degree of in vitro or in vivo activation of the contact system of plasma.
TUMOR NECROSIS FACTOR INCREASES THE PRODUCTION OF PLASMINOGEN ACTIVATOR INHIBITOR IN HUMAN ENDOTHELIAL CELLS IN VITRO AND IN RATS IN VIVO
Authors: van Hinsbergh V W M , Kooistra T , Fiers W , Emeis J J 
Published: August 2018

The vascular endothelium plays an important role in fibrinolysis by producing tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI). The production of these factors by cultured endothelial cells (EC) is under separate control and influenced by various mediators, such as interleukin-1 (IL-1). Similar to IL-1, the monokine tumor necrosis factor (TNF) induces in EC various membrane bound components: tissue factor, HLA-A,B antigens and leukocyte adhesion molecules. We here report that TNF increased the production of PAI by human EC and increased PAI plasma levels in rats.In the presence of serum, TNF increased the production of PAI by cultured human EC from umbilical vein (2-fold) and from foreskin microvessels (2 to 10-fold). This was demonstrated by titration of t-PA to a fixed amount of EC conditioned medium, by reverse fibrin autography, and by immunoprecipitation with specific anti-PAI-1 IgG. No change in t-PA activity was found by fibrin autography. The stimulation of PAI activity by TNF was found at 4 U/ml and reached a maximum at 500 U/ml; it was not prevented by the addition of polymycin B. Stimulation of PAI production by TNF or IL-1 was observed after 2 h and sustained for at least 24 h. Separate addition of TNF or IL-1 gave similar maximal stimulation of PAI production by EC at 500 U/ml and 5 U/ml, respectively, while the addition of both mediators resulted in a 2-fold larger increase. This indicates an additive effect of TNF and IL-1. TNF did not change PAI production by human hepatocytes.To evaluate the effect of TNF in vivo, rats received a bolus injection of 250,000 U TNF/kg. Two h after injection, a 5-fold rise of circulating PAI levels was found (compared to control rats). Thereafter, the levels returned to basal values over a 10 h period. A decrease in circulating white blood cells was observed during the initial 3 h. The number of circulating platelets did not change.We conclude that stimulation of the vascular bed by TNF not only results in a change in surface characteristics of the endothelium, but also can result in systemic changes. The increase in PAI levels by TNF may decrease fibrinolysis.
EFFECT OF Ca2+ and Mg2+ ON PLATELET ACTIVATING FACTOR (PAF) INDUCED AGGREGATION AND SPECIFIC BINDING TO HUMAN PLATELETS
Authors: Chesney C M , Pifer D D 
Published: August 2018

Gel filtered human platelets (GFP) collected in Tyrode's buffer containing 0.5 mM Ca+2, ImM Mg+2, and 0.35% albumin exhibit high affinity binding of 3H-PAF with a Kd of 0.109 α 0.029 nM (mean α SD; n=13) and 267 α 70 sites per platelet. When fibrinogen (1.67 mg/ml final concentration) is added to these GFP preparations biphasic aggregation is observed with PAF (4 nM). Normal aggregation is also observed with other platelet agonists including ADP, epinephrine, collagen, arachidonic acid, A23187 and thrombin. If GFP is prepared without added Ca+2 or Mg+2 in the presence of 3mM EDTA, platelets do not aggregate in response to PAF. However the number of specific binding sites remains unchanged (387 per platelet) with some decrease in affinity of binding (Kd = 0.2l4nM). In the presence of ImM Mg+2 there is no significant difference in binding kinetics over a range of Ca+2 concentrations (0-2mM). On the other hand the calcium channel blocker verapamil (5-10uM) exhibits competitive inhibition of 3H-PAF as analyzed by Lineweaver-Burk plots. Specific binding of 3H-PAF to GFP in the presence of ImM Mg+2 and ImM EGTA shows Kd of 0.l66nM but with increase in specific binding sites to 665. Despite increase in number of sites and no change in binding affinity, GFP under these conditions does not exhibit platelet aggregation with PAF in doses up to 80 nM.From these data it appears that external Ca+2 is not necessary for specific binding of 3H-PAF to its high affinity receptor. However, calcium does appear to be necessary for second wave aggregation with PAF. While Mg+2 appears to enhance 3H-PAF binding to platelets Mg+2 cannot substitute for Ca+2 in PAF induced platelet aggregation. Although verapamil appears to competitively inhibit binding of PAF to GFP it is not clear whether the inhibition is due to competition at or near the actual PAF receptor or at a site involving the calcium channel.
HUMAN PLASMA PAF-ACETYLHYDROLASE, NORMALLY PRESENT IN LOW DENSITY LIPOPROTEINS, IS ASSOCIATED WITH HIGH DENSITY LIPOPROTEINS IN A PATIENT WITH LDL DEFICIENCY
Authors: Surya I E , Akkerman J W N 
Published: August 2018

Platelet Activating Factor (l-0-alkyl-2-acetyl-sn-glycerol-3-phosphocholine; PAF) plays an important role in allergic and inflammatory reactions and activates platelets in the nanomolar range. One of the main factors that controls PAF activity in blood is an enzyme that hydrolyzes the acetyl-chain thereby converting PAF to biologically inactive lyso-PAF. The enzyme is acid labile and normally associated with apo B-containing low density lipoproteins (LDL, density 1,006-1,063 g/ml).We investigated whether a deficiency in LDL would affect the enzyme activity. PAF-inactivating activity was measured in plasma from a patient with abetalipoproteinemia, a rare autosomal recessive disorder, characterized by the absence of apo B and apo B-containing lipoproteins (chylomicrons, VLDL and LDL). Plasma triglyceride was 0,2 mmol/1 (normal 1,40-2,20 mmol/1) and cholesterol 1,3 mmol/1 (normal 5,60-7,70 mmol/1). Separation of lipoproteins by density gradient centrifugation revealed a slightly decreased HDL content whereas VLDL and LDL were below the detection limit (0,20 mmol/1; based on cholesterol content).Despite the absence of LDL, PAF-inactivating activity in plasma of the patient (measured by (1) the decrease in aggregation inducing activity of PAF after incubation, (ii) the conversion of 3H-acyl-PAF to lysa PAF, separated on TLC, (iii) the liberation of 3H-label from 3H-acetyl PAF) was present and even slightly higher than in normal plasma (hydrolysis of 3
3H-PAF after 20 minutes incubation was 78 ± 4% and 65 ± 6% in patient and normals, respectively, n = 4). Subfractionation revealed that the enzyme activity was present in fractions with densities of 1,065-1,214 g/ml, which are typical for HDL.These results indicate that PAF-acetylhydrolase, although normally present in LDL, binds to HDL in a patient with extreme LDL-deficiency.Supported by the Dutch Heart Foundation (grant 85082)