bluuebunny/crossref_metadata_2025 huggingface dataset:
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.
COMPARISON OF BLOOD, RED CELL AND WHITE CELL RHEOLOGY IN UNSTABLE ANGINA AND ACUTE MYOCARDIAL INFARCTION
Authors: Lowe G D O , Thomson G , Lennie S E , Anderson J , Cobbe S M , Forbes C D
Published: August 2018

In acute coronary artery thrombosis, the flow properties of blood might conceivably influence progression to (a) complete thrombotic occlusion and transmural myocardial infarction, or (b) resolution of ischaemia without infarction (unstable angina). To test this hypothesis, several rheological variables were measured in the following groups of patients, matched for age, sex and smoking habit: (1) acute transmural myocardial infarction (n=15); (2) unstable angina (ischaemic pain and ECG but no significant enzyme rise, (n=16); (3) non-cardiac acute chest pain (n=9); and (4) healthy controls (n=20). Patients with infarction had significantly elevated levels of blood viscosity at high and low shear rates (94 and 0.94 s™1, Contraves LS30) compared to all other groups, associated with significantly higher levels of haematocrit, fibrinogen, plasma viscosity and fibrinogen: white cell count was also significantly higher. These abnormalities could therefore predispose to complete thrombotic occlusion and infarction. Patients with unstable angina also had significant increases in fibrinogen, plasma viscosity and white cell count, intermediate between infarct patients and controls: however blood viscosity increase was prevented by a lower haematocrit, which may predispose to resolution of thrombosis and ischaemia. Red cell deformability (Contraves visconeter and St. George's Filtrometer with 5 μm Nuclepore filters) was normal in infarction and m unstable angina, but significantly decreased in non-cardiac chest pain. Polymorph leucocyte filtration in a positive-pressure system (Nuclepore 5 μm filters) was normal in infarction and unstable angina, and mononuclear leucocyte filtration was only slightly reduced in both groups. Hence the major rheological changes in myocardial infarction and unstable angina are increases m fibrinogen and cell counts, rather than altered cell deformability.
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.
BINDING OF HEPARIN TO H-KININOGEN
Authors: Björk I , Olson S T , Shore J D
Published: August 2018

The binding of heparin to kininogen was analyzed by competition of kininogen with anti thrombin for high-affinity heparin. Residual heparin binding to anti thrombin was quantified by the accelerating effect on the anti thrombin-thrombin reaction. The rate of the latter reaction was monitored by displacement of the fluorescent probe, p-aminobenzamidine, from the enzyme. A linear dependence of the observed pseudo-first-order rate constant (kobs) for the heparin-accelerated anti thrombin-thrombin reaction on heparin concentration was achieved by use of catalytic amounts (≤30 nM) of heparin, a 20-fold ratio of anti thrombin to thrombin and thrombin concentrations (0.25 μM) much below the apparent of heparin for thrombin at the high (1 mM) p-aminobenzamidine concentration used. The two-chain form of H-kininogen minimally affected the heparin-accelerated rate of the anti-thrombin-thrombin reaction at pH 7.4 in the absence of metal ions. However, at saturating concentrations of Zn2+ (10 μM), kobs was reduced to 50% at ˜15 nM kininogen and to that of the uncatalyzed reaction at ≥˜0.25 μM. Conversely, at saturating kininogen, a 50% decrease of kobs was observed at ˜0.6 μM Zn2+, i.e. in the plasma concentration range. Other metal ions were effective in the order: Zn2+˜Ni2+>Cu2+>Co2+˜Cd2+. Single-chain H-kininogen and H-kininogen light chain reduced the heparin enhancement in the presence of Zn2+ to the same extent as the two-chain form, whereas L-kininogen had no effect. In the absence of metal ions, the binding of heparin to two-chain H-kini-nogen increased with decreasing pH below 7.4 in a manner consistent with involvement of protonated histidine residues. Thus, heparin presumably binds to the histidine-rich region of the light chain portion of H-kininogen. The elution of two-chain H-kininogen from immobilized dextran sulfate at pH 7.4 was shifted to higher salt concentrations in the presence of 10 μM Zn2+, indicating that metal ions may also enhance H-kininogen binding to surfaces relevant to contact activation reactions. The sensitivity of H-kininogen-surface interactions to divalent metal ions and pH suggest regulation of the interactions by these factors. Like histidine-rich glycoprotein, H-kininogen may also compete with anti thrombin for heparin during heparin therapy.
TRANSIENT INDUCTION OF PLASMINOGEN ACTIVATOR INHIBITOR 2 (PAI-2) GENE TRANSCRIPTION BY THE TUMOUR PROMOTING PHORBOL ESTER PMA IN THE HUMAN MACROPHAGE-LIKE CELT, LINE U-937
Authors: Msdcalf R L , Kruithof E K O , Schleuning W-D
Published: August 2018

The human hematopoietic cell line U-937 differentiates to a macrophage/monocyte phenotype after exposure to the tumour promoter PMA. We have recently shown that PMA concomitantly induces PAI-2 biosynthesis in these cells. Now, we have employed an 1880 base pair cDNA to study PAI-2 biosynthesis on the level of transcription and mRNA stability. By in vitro elongation of initiated PAI-2 transcripts in isolated nuclei in the presence of 32p-labelled UTP followed by hybridization to cloned DNA (“run-on” transcription assay), we have demonstrated that PAI-2 gene transcription rates are induced 50-fold after exposure of the cells to PMA for 4 h in the presence of 5% fetal bovine serum. Transcription rates subsequently declined to base-line levels within 48 h. This transient increase of PAI-2 transcription rate was reflected by a transient induction of PAI-2 mRNA with maximal levels (60-fold) occuring after 16 h and subsiding to near base-levels after 48 h. Similar experiments performed in the absence of fetal bovine serum revealed that maximal levels of PAI-2 mRNA occured after only 4 hours exposure to PMA and were virtually absent after 16 h. Our results indicate that PMA induction of PAI-2 biosynthesis occurs at the level of gene transcription and is influenced by the presence of serum. Since PAI-2 is a major protein of U-937 cells, these date also suggest a role for hormonally modulated PAI-2 biosynthesis in macrophage physiology.
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.
INTERLEUKIN 1 (IL-1) AND TUMOR NECROSIS FACTOR (TNF) ACTIVATION OF VASCULAR ENDOTHELIUM: EFFECTS ON PLASMINOGEN ACTIVATOR INHIBITOR (PAI-1) AND TISSUE-TYPE PLASMINOGEN ACTIVATOR (tPA)
Authors: Schleef R R , Bevilacqua M P , Sawdey M , Gimbrone M A , Loskutoff D J
Published: August 2018

The regulation of the fibrinolytic system of cultured human umbilical vein endothelial cells (ECs) by two distinct monokines (IL-1 and TNF) was investigated. Conditioned media (CM) was collected from ECs cultured for 24 h in the presence of the monokines and analyzed in quantitative immunological assays for PAI-1 activity and tPA antigen. Both monokines induced a dose-dependent increase in extracellular PAI-1 activity, with a concomitant decrease in tPA antigen. Maximal effects were achieved with either 10 U/ml IL-1 or 200 U/ml of TNF, and resulted in a 4 fold increase in PAI-1 and a 50% decrease in tPA. The kinetics of the effects of both monokines on EC PAI-1 and tPA were similar, with maximal response detected at 24 h. Cell-associated PAI-1 also increased in response to these monokines. For example, a 24 h exposure of EC to TNF (250 U/ml) or IL-1 (5 U/ml) caused a 5-fold increase in cell-associated PAI-1. Northern blot analysis using a PAI-1 cDNA probe indicated that the monokines increased the levels of two RNA species, corresponding to PAI mRNAs of approximately 3.0 and 2.2 kb in length. To determine if the increase in cel 1-associated PAI-1 reflects a storage pool of rapidly releasable PAI-1, ECs were pretreated with IL-1 for 24 h, washed and the PAI-1 activity in CM measured after 5, 15 and 60 min treatment with known secretagogues (i.e., phorbol myristate acetate, calcium ionophore A23187). Although IL-1 treated ECs released PAI-1 at a rate which was 5-fold higher than controls, this rate was not increased further by treatment with phorbol myristate acetate or ionophore. The fact that both monokines act in a similar manner strengthens the hypothesis that the local development of immune and inflammatory processes could reduce endothelial fibrinolytic activity, thus leading to the pathologic formation of intravascular thrombi.
CHARACTERIZATION OF A 40 kDa FRAGMENT OF VON WILLEBRAND FACTOR THAT CONTAINS THE GLYCOPROTEIN IIb/IIIa-BINDING DOMAIN
Authors: Niiya K , Kostel P , Zimmerman T S , Ruggeri Z M
Published: August 2018

We have isolated a 40 kDa fragment of von Willebrand factor (vWF) that contains the glycoprotein (GP) Ilb/IIIa-binding domain. The Staphylococcus aureus V8 protease-generated fragment II was digested with trypsin (1:50 enzyme:substrate ratio on a weight-to-weight basis). After addition of a 100fold molar excess of (p-amidinophenyl)methanesulfonyl fluoride in order to inhibit any residual trypsin activity, the whole digest was subjected to ion-exchange and size-exclusion high pressure liquid chromatography. Two major fragments were separated. Analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) demonstrated that one of the two purified polypeptides had an apparent molecular weight of 40 kDa under both reducing and nonreducing conditions, suggesting that it was a single chain polypeptide. The other fragment had an apparent molecular weight of 22 kDa after reduction and 44 kDa unreduced, suggesting that it was a homodimer. Amino terminal sequence analysis of both fragments was performed by classical Edman degradation following electroelution from reduced SDS-polyacrylamide gels. The amino terminus of the 40 kDa fragment corresponded to residue Glu (1366) (as did the fragment II from which it was derived), while the amino terminus of the 22 kDa fragment corresponded to residue Val (1927) of the constituent 2050 residue subunit. The effect of both fragments on vWF binding to the platelet membrane GP IIb/IIIa complex was evaluated by measuring the residual binding of 125I-labeled vWF to thrombin-stimulated platelets in the presence of varying amounts of the unreduced fragments. The 40 kDa polypeptide inhibited 64 percent of vWF binding when tested at a concentration of 20 μK, whereas the 22kDa dimer was without effect. This study establishes that the GP IIb/IIIa-binding domain of vWF resides in a discrete, single-chain 40 kDa fragment derived from the 220 kDa, homodimeric fragment II generated by V8 protease. Moreover, we found evidence for the existence of inter-chain disulfide bonds within 22 kDa from the carboxyl terminus of the constituent subunit.
LOCALIZATION OF A FACTOR VIII BINDING DOMAIN ON THE N-TERMINAL PORTION (FRAGMENT SpIII) OF VON WILLEBRAND FACTOR
Authors: Takahashi Y , Girma J P , Kalafatis M , Sewerin K , Andersson L O , Meyer D
Published: August 2018

A new domain has been identified on the von Willebrand Factor (vWF) subunit. vWF binds to platelet glycoproteins GPIb and GPIIb/IIIa as well as to collagen and corresponding domains have been isolated. vWF also binds to Factor VIII (F.VIII). We show here that the corresponding domain is located on the N-terminal portion of the vWF subunit (residues 1 to 1,365). For this purpose, F.VIII was tested for its ability to bind to purified vWF degradation fragments obtained by digestion with S.aureus V-8 protease, ie a dimeric N-terminal fragment of 320 kd (SpIII) and a dimeric C-terminal fragment of 220 kd (SpII). Human F.VIII was purified from cryoprecipitate by immunoadsorption of F.VIII/vWF onto a monoclonal antibody (MAb) to vWF coupled to Sepharose, followed by elution using 0.25 M CaCl2. The F.VIII preparation contained 100 U/ml VIII:C and less than 0.001 XJ/ml vWFAg. The binding assay was performed using polystyrene tubes coated with 2 ug/ml of purified vWF, SpIII or SpII. Coated albumin, fibrinogen or fibronectin were used as controls. Purified F.VIII (0.1 to 2 U/ml VIII:C) was incubated in the coated tubes for 1 h at 37°C. Following washing, bound F.VIII was estimated in situ by one-stage clotting and chromogenic assays. Immunoradiometric assay with 125 I-MAbs to SpII or SpIII demonstrated that the amount of coated protein remained constant throughout the experiments. F.VIII bound in a dose-dependent manner to coated vWF and SpIII but not to SpII. Binding was specific for F.VIII as demonstrated by inhibition experiments. Bound F.VIII could be removed with 0.25 M CaCl2 and its coagulant activity inhibited by a MAb or an oligoclonal (homologous) antibody neutralising VIII:C. Binding of F.VIII to coated vWF or SpIII was also inhibited in a dose-dependent way by vWF or SpIII. In contrast, addition of SpII had no effect upon the binding. Binding of F.VIII to SpIII was confirmed using MAbs to vWF. Among 28 MAbs which bound SpIII and had no anti VIII:C activity, 12 inhibited binding of F.VIII whereas the others had no effect. Among the latter, 3 MAbs blocked binding of vWF or SpIII to GPIb and 6 MAbs inhibited binding of vWF or SpIII to collagen. Ten MAbs to SpII had no inhibitory effect upon binding of F.VIII. These results indicate that a F.VIII binding domain of vWF is located in the N-terminal portion of vWF (residues 1 to 1,365) and that it is distinct from the GPIb and collagen binding domains. The 12 MAbs to SpIII which block binding of F.VIII to vWF or SpIII should allow the precise localization of this new domain.
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.
PLATELETS PLAY A ROLE IN THE IMMEDIATE ALLERGEN-INDUCED BRONCHOCONSTRICTIVE REACTION IN ASTHMATICS
Authors: Akkerman J W N , Kloprogge E , Bruynzeel P L B
Published: August 2018

Platelet Activating Factor (PAF) is generally considered to play an important role in acute allergic reactions. A special example of such a reaction is allergic asthma and indirect evidence suggests that here too PAF may be involved. Direct proof for a role of PAF, however, is difficult to obtain since in blood PAF has an extremely short half-life due to its inactivation by an acetyl-hydrolase in plasma, its uptake and degradation by many cell types and its lipophilic properties making it easily stick to membranes. Despite these handicaps we report here that during an acute bronchoconstrictive reaction in asthmatics, PAF is liberated into the circulation and binds to platelets. Eight patients with allergic asthma were challenged by inhalation provocation. Four patients responded with an immediate bronchoconstrictive reaction, which was accompanied by a 40% decrease in freely accessible PAF-receptors on their platelets, collected 1 hour after bronchoconstriction (specific bindings of 3H-PAF decreased from 264 ± 44 to 164 ± 45 mol/platelet, p < 0.05). The patients who did qpt respond to allergen provocation failed to show a change in 3H-PAF binding. In both groups platelet counts and PAF-induced aggregation did not change significantly. In view of the high specificity of PAF-receptors on platelets (a 200-fold excess of histamine did not interfere) and the fact that in vitro3H-PAF-binding gradually becomes irreversible, we conclude that during an immediate bronchoconstrictive reaction circulating platelets make contact with PAF. Together with evidence that urtder similar conditions secreted products from platelets appear in the circulation, these data indicate that platelet activation by PAF forms an important step in the pathophysiology of allergic asthma.
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)
SEVERELY IMPAIRED SYNTHESIS OF PLATELET ACTIVATING FACTOR IN CHONDRO DYSPLASIA PUNCTATA RHIZOMELIA PATIENTS
Authors: Sturk A , Schaap M C L , Prins Heymans A , ten Cate J W , Wanders R J A , Heymans H S A , Schutgens R B H , van den Bosch H
Published: August 2018

The first steps of the de novo synthesis of alkoxyether lipids, like plasmalogens and platelet activating factor (PAF) are localized in the peroxisome. We have previously reported the severely impaired PAF synthesis in Zellweger patients. These patients lack cytochemically detectable peroxisomes, and have a severely impaired alkoxyether lipid synthesis. However, chondro dysplasia punctata (CDP) patients have also been shown to have an impaired alkoxyether lipid synthesis. We therefore investigated PAF synthesis in CDP patients.

Platelets and leucocytes were isolated from 3 CDP patients. Leucocytes from normal controls produced 4678 ± 2033 pMoles PAF/10 cells (n=6, range 1698-7058) when optimally stimulated with Ca2+-ionophore A23187. Normal control platelets produced 0.6 ± 0.3 pMoles PAF/10 cells (n=6, range 0.3-1.0) when optimally stimulated with thrombin. PAF synthesis by the leucocytes of the patients was severely reduced, but detectable. Leucocytes from patient 1, 2 and 3 synthesized 9, 660 and 325 pMoles PAF/10 cells respectively. Platelets from the patients 1, 2 and 3 synthesized 0.1, 0.2 and 0.2 pMoles PAF/10 cells respectively.

Platelet aggregation, induced by ADP, PAF, or thrombin (also in the presence of inhibitors of the first and second pathway of platelet activation) was normal.

We conclude that PAF synthesis is severely impaired in leucocytes and reduced in platelets from CDP patients. The residual platelet PAF synthesis may suffice to warrant normal platelet functioning.
SEPARATION OF FIBRINOGEN FRAGMENTS ON GLYPRQARGPROLYS-FRACTOGEL
Authors: Kuyas C , Haeberli A , Straub P W
Published: August 2018

The tetrapeptide GlyProArgPro, which corresponds to the newly exposed N-terminal sequence of fibrin a-polypeptide chain after the action of thraribin, has a binding site in the C-terminal part of the ;-chain as suggested by several authors. Using Gly-ProArgProLys-Fractogel (GPRPK) chrcmatography we tried to isolate a fibrinogen fragment, obtained with different enzymes and conditions, which includes the binding site for GPRP. Human fibrinogen was digested by plasmin in presence and absence of Ca-ions, and the resulting lysates were applied in 0.05 M triethanolamine (TEA), 0.1 M NaCl pH 7.4 to the GPRPK-Fractogel. The gel was washed extensively with TEA-buffer and the adsorbed protein was eluted with 6M urea in TEA-buffer. The protein containing fractions were analyzed iiununologically with anti-fragment E- and with anti-fragment D antibodies. Human fibrinogen was also digested with endopeptidase Arg-C in O.IM NaHco3 pH 8.0 at 37C over night. The enzyme cleaves fibrinogen to fragments, one of which comprising the y-chain sequence 275-375 which is said to contain the fibrin polymerization site. The Arg C-lysate was chromatographed on GPRPK-Fractogel. All fragments were analyzed by SDS-electrophoresis and by reversed-phase HPLC.

Fragment D1 was the only fibrinogen fragment which was adsorbed on GPRPK-Fractogel. All other assayed fibrinogen fragments obtained by enzymatic cleavage, showed no affinity to GPRPK-Fractogel. These results demonstrate that for the binding of GPRP to fibrinogen a conformationally intact γ-chain remnant of the fragment D is required.

One step chrcmatography using GPRPK-Fractogel can thus also be used to isolate fragment D1 in high purity fran plasmin lysates of fibrinogen.
DISCREPANCIES BETWEEN VENOGRAPHY AND REAL TIME B MODE ULTRASOUND IMAGING IN THE DIAGNOSIS OF DEEP VEIN THROMBOSIS
Authors: Elias A , Le Corff G , Bouvier J L , Villain Ph , Serradimigni A
Published: August 2018

Methods : in this prospective study, real time B Mode ultrasound imaging (USI) was compared to bilateral ascending contrast venography, double blindly, in 430 patients suspected of deep vein thrombosis (DVT) or pulmonary embolism.

A complete scan of the venous system from the inferior vena cava to the calf veins, was performed with a high resolution duplex system (DIASONICS DRF 400) and coupled systematically with a C.W. Doppler examination. The results obtained by USI were thus compared to the venograms performed on a total of 854 legs.

Results : there are corresponding results in 95% of the legs (808/854). If we consider venography as the standard of reference, the sensitivity of USI is 98% (325/333) and the specificity 94% (483/514). Isolated calf vein thrombosis are detected in 91% (84/92) of the legs and proximal DVT in 100% (241/241) in this series whatever the topography and the extension of the thrombosis and whatever the degree of the obstruction of the vein.

Discrepancies found in 46 legs are related to :

- 8 DVT located in the calf (6 in the presumed healthy leg) diagnosed only by venography.

- 27 DVT (18 distal, 9 femoral or iliac) detected only by USI

- 9 doubtful examinations with USI not confirmed by venography

- 2 doubtful venograms with negative USI test.

Comments : Calf vein thrombosis especially located in the soleal sinuses and the gastrocnemius with in most cases the direct image of the thrombus are more often detected by USI provided that the technique and the equipment are appropriate.

The absence of visualisation of venous segments with venography is not specific of venous thrombosis. These veins non affected by the thrombosis are not filled by the contrast medium when located above in occluded ilio-femoral or ilio-caval junction or when they are the site of extrinsic compression. The direct image of the vein and the surrounding structures obtained with USI enhances the diagnostic sensitivity and specificity and provides precision of the exact extension of the thrombosis.

Due to these differences, can venography still be considered as the standard of reference in the diagnosis of DVT and their precise localisation ?
COMPUTERIZED IMPEDANCE PLETHYSMOGRAPHY, A NEW PLETHYSMOGRAPHIC METHOD TO DETECT DEEP VEIN THROMBOSIS
Authors: Prandoni P , Vigo M , Huisman M V , Jonker J , Büller H R , ten Cate J W
Published: August 2018

Since the clinical diagnosis of deep vein thrombosis (DVT) is unreliable, several invasive and non-invasive methods have been developed recently. Of these, impedance plethysmography (IPG) is a widely employed technique based on measurement of changes in blood volume produced by temporary obstruction. IPG has been shovn in large prospective studies in symptomatic patients to be a safe and effective alternative to contrast venography, if used either in combination with 1251-fibrinogen legscanning or serially as a single test. Currently available impedance plethysmographs are limited by several technical and operational problems. Therefore, a new computerized impedance plethysmograph (CIP) was developed, having the following characteristics: portability, battery operated and fully automated. A prospective two-center study in 299 consecutive outpatients was done to compare the efficacy of CIP vs. venography in patients with symptomatic DVT. Using a blind design i.e. care was taken to insure that CIP and venography were performed and interpreted independently. The results in patients without venography proven thrombosis and those with proximal vein thrombosis were subjected to a discriminant analysis producing a line of best discrimination between normal and proximal vein thrombosis. In 14 patients it was not possible to obtain an adequate CIP tracing. 12 patients were not entered because of refusal to undergo venography and 15 patients were excluded from analysis because of poor opacification of the proximal veins. On the basis of discriminant analysis 138 of the CIP results were classified as normal and 120 as abnormal. 175 patients were normal on venography and 83 had proximal thrombosis. The sensitivity of CIP for proximal vein thrombosis was thus 95% while the speci-+ ficity was 77%. It is concluded that computerized impedance plethysmography is a potentially sensitive method to detect proximal vein thrombosis in patients with clinically suspected deep vein thrombosis.
RATE OF NORMALIZATION OF ABNORMAL IMPEDANCE PLETHYSMOGRAPHY IN PATIENTS WITH PROVEN DEEP VEIN THROMBOSIS: SIGNIFICANCE IN THE MANAGEMENT OF RECURRENT SYMPTOMS
Authors: Huisman M V , Buller H R , ten Cate J W
Published: August 2018

The diagnosis of deep vein thrombosis (DVT) by clinical signs and symptoms is unreliable, but contrast venography is expensive and invasive. Therefore, the use of non-invasive methods to detect DVT have become en vogue, of which impedance plethysmography (IPG), either in combination with 125x fibrinogen leg scanning or performed serially as a single test, have been demonstrated to be a safe and effective alternative. Since the principle of IPG is based on the measurement of venous outflow obstruction due to intravascular thrombus and since the aim of anticoagulant treatment is to facilitate recanalisation one might expect a gradual normalisation of IPG in the majority of patients. This information is important for patients presenting with recurrent signs and symptoms. If the IPG has normalized prior to presentation it is possible to separate complaints due to recurrent DVT from post phlebitic syndrome. In this prospective trial we studied 161 consecutive outpatients with abnormal IPG and venography proven DVT three monthly during one year to determine the rate of normalization and to estimate the utility of IPG testing in patients with recurrent symptomatic DVT. The IPG test had normalized in 101 of 151 patients (67%) by three months, in 126 of 148 (85%) after six months, in 133 of 145 (92%) while after one year 139 of 146 (95%) had their IPG normalized. During the one year follow-up 35 of the 161 study patients (23%) returned with recurrent symptoms. Of these 31 had normal IPG tests prior to the visit. In 18 patients IPG remained normal at repeat testing. In 13 patients IPG became again abnormal (venography showed acute on chronic DVT in 10 patients while 3 patients showed no acute DVT). It is concluded that a 95% normalization of IPG occurs and that IPG is useful in the management of recurrent symptoms.
SEMI-QUANTITATIVE RADIONUCLIDE PHLEBOGRAPHIC (RNP) ASSESSMENT OF DEEP VEIN THROMBOSIS (DVT) AND CHRONIC VENOUS INSUFFICIENCY (CVI)
Authors: Zahavi J , Zaltzman S , Firsteter E , Avrahami E
Published: August 2018

A semi-quantitative RNP using 99Technetium macroaggregated albumin for the evaluation and follow-up of DVT and CVI has been developed. Values were assigned to the deep veins of the calf, knee, tigh and pelvis based upon the localization and the characteristics of the images obtained: stasis, hot spots and collateral circulation. A maximum score of 18 reflected complete thrombosis of all 4 segments. 208 patients (mean age 53.7 years, range 18-92), 161 of whom had a proven risk factor for DVT were studied. 99Technetium was injected into the dorsal foot vein of 407 limbs with appropriate tourniquets and early and late imaging of the limbs, pelvis and lungs was performed. In 48 patients, 83 limbs, X-ray contrast phlebography (CP) was also done. The mean RNP score was 4.1 units (range 0.4-18) and higher in the left than the right lower limb. It was mostly high in patients with proximal recurrent DVT or in DVT superimposed on CVI. The score was easy to follow and helpful in the assessment of the extent of DVT. It was particularly helpful in 3 instances. 1) Assessment of venous patency following anticoagulant therapy. 2) Estimation of recurrent DVT. 3) Differentiation of recent DVT from venous insufficiency. Overall RNP method had a sensitivity of 87.6%, a specificity of 54% and an accuracy of 64.8%. The sensitivity was similar in above & below-knee thrombi. Yet the specificity was higher in above-knee thrombi. The highest accuracy (87.3%) was observed in pelvic and groin thrombi. The distribution of thrombi on CP was 19% below the knee, 31% above it and 50% both above and below the knee. Pulmonary embolism (PE) was initially observed in 54 patients (26%) with no clinical evidence of DVT and therefore untreated. This high level is most probably related to the high incidence of proximal DVT in the patients. 181 patients were treated with heparin & coumadin and the RNP score was decreased to 3.6 units (range 0.4-8.8). PE occurred during treatment in 11 (6.1%) and recurrent DVT in 16 (8.8%) patients. CVI was observed in 23 patients before treatment and in another 24 patients (13.2%) after treatment. These results indicate that the RNP method is a simple, semi-quantitative and useful technique for the evaluation and follow-up of DVT and CVI. It is most helpful in the assessment of the extent of DVT. It is also a rapid, noninvasive and cost effective techniaue.
MONOCLONAL ANTIBODY 13G11 RECOGNIZES PREKALLIKREIN, KALLIKREIN AND COMPLEXES OF KALLIKREIN WITH,C1-INHIBITOR AND. α2-MACR0GL0BULIN
Authors: Veloso D , Shapira M , Kueppers F , Colman R W
Published: August 2018

Abnormal prekallikrein (PK) levels in plasma can be due to decreased biosynthesis or increased activation either by surface-activated factor XIIa (e.g., in septicemia) or by other proteases (e.g., in pancreatites). To study the products of activation of PK in plasma, intact normal plasma and plasma exposed to either activating surfaces or factor XII fragment, was immunoblotted from SDS-gels. MAb 13G11 which recognizes purified PK, kallikrein (KAL) and the complexes of KAL with Cl-inhibitor (Cl-Inh) and α2macroglobulin (α2M) formed from purified proteins detected a doubLet (88- and 85-kDa) which comigrated with PK and KAL but was not visible in PK-deficient plasma. Transfer of either PK in normal plasma (25-125 ng) or KAL (50-300 ng) added to PK-deficient plasma was proportional to the amount of protein applied to the SDS-gels. Activation of plasma decreased the intensity of the PK bands with the formation of new bands with molecular weights similar to those of KAL-Cl-Inh and KAL-α2M. Identity was confirmed by MAb 4C3 (reacts with KAL-Cl-In, not with KAL) and a polyclonal antibody to α2M. Increase of incubation temperature from 24 to 37 increased KAL-Cl-Inh and decreased KAL-α2M. Addition of an excess of a2M before surface activation caused an increase of KAL-α2M complex and a decrease of KAL-Cl-Inh. Addition of an excess of Cl-Inh increased KAL-Cl-Inh and decreased KAL-α2M. In addition, activation of Cl-Inh-deficient plasma showed lower KAL-Cl-Inh and higher KAL-a2M than those when normal plasma was activated. When the deficient plasma was treated with CH3NH2 to inactivate α2M, an increase at KAL position was observed since no inhibitors were active. These studies indicate that 13G11 will be useful to detect changes in the distribution of PK, KAL, KAL-Cl-Inh and KAL-α2M associated with abnormal activation of PK and/or abnormal availability of inhibitors in disease.
C1 -INHIBITOR: STRUCTURE-ACTIVITY RELATIONSHIPS
Authors: de Agostini A , Barja F , Carrel S , Harpel P C , Schapira M
Published: August 2018

Cl-inhibitor [C1 -In] and other protease inhibitors of the serpin superfamily inactivate serine proteases by forming bimolecular enzyme-inhibitor complexes, a reaction that is associated with changes in the inhibitor conformation. To determine the significance of these changes, we have examined the influence of various treatments on the binding to C1-In of monoclonal antibody 4C3. This antibody was previously shown to bind to an epitope created during the reaction of C1-In with the Arg-specific protease plasma kallikrein [K]: the site for 4C3 was expressed on the K-C1-In complex, on C1-In cleaved at position Pi and released from K-C1-In [C1-In*], but not on unreacted C1-In. The binding of 4C3 to the various forms of C1-In was now measured by radioimmunoassay and Western blot. Following inactivation by C1-In of the Arg-specific enzymes factor XII active fragment [Xllf] or C1s, the binding site for 4C3 was detectable on XIIf-CI-In, C1s-C1-In and C1-In*. However, when K or Xllf were incubated with heat-inactivated C1-In, bo+h enzymes remained active, no complex was formed, and the site for 4C3 was not created. When C1-In was cleaved by neutrophil elastase [E] (a Met-orVal-specific protease that is not inhibited by C1 -In), the 1st cleavage product C1-In’ retained inhibitory activity (as shown by its ability to form a complex with Xllf) but did not bind 4C3. However, subsequent cleavage of C1-In’ by E at position P3 yielded C1-In’, a product which was inactive but bound 4C3. Thus, identical conformational changes of C1-In (as assessed by the emergence of the site for 4C3) are seen when Cl-In inactivates its target enzymes while being cleaved at Pi or when the inhibitor is catalytically inactivated by cleavage at P3. Therefore, these changes are necessary but not sufficient for observing enzyme inactivation.
FIBRIN-BINDING STUDIES OF PRO-UROKINASE (PRO-UK) USING SOLID PHASE FIBRIN PLATES
Authors: Angles-Cano E , Pannell R , Gurewich V
Published: August 2018

Pro-UK is a single chain urokinase-type plasminogen activator (scu-PA) which has fibrin selective thrombolytic properties. However, quantitative data on pro-UK binding to fibrin and on the mechanism of its fibrin enhanced activation of plasminogen have been difficult to obtain. In the present study, a well defined fibrin network constructed on glutaraldehyde-activated PVC plates (Anal. Biochem. 153 : 201-210, 1986) and highly purified pro-UK (99 % scu-PA) were used. Binding was investigated as follows : varying dilutions of pro-UK in the presence of a trace amount of I-labeled pro-UK in buffer without or with glu-plasminogen, plasmin or oC -thrombin and in urine, plasma or serum, were incubated overnight at 4°C and then 2 h at 37°C in the fibrin plates. After washing, the wells were cut out and counted in a gamma-counter. The labeled pro-UK and the effect of enzymes on scu-PA were investigated by SDS-PAGE and autoradiography. In parallel experiments, the activity of the fibrin bound and unbound products was investigated spectrophotometrically by adding glu-plasminogen and a synthetic substrate selective for plasmin. The binding of pro-UK to fibrin was 1.7 ± 0.1 % in buffer and 0.2 ± 0.08 % in plasma, as determined from isotopic and spectro-photometric measurements. This binding is similar to that of (0.13 ± 0.05%) two-chain urokinase (plasmin-transformed scu-PA), but is extremely low compared to the specific binding of tPA (68 - 4%). By contrast, in urine, 11.2 ± 4.47 % binding of pro-UK to fibrin was observed. Thrombin did not modify the binding but transformed scu-PA into a two-chain molecule which had lost activity. These data indicate that pro-UK has little affinity for fibrin under these conditions but that some binding may be induced by a co-factor which is present in urine. Confirmation that thrombin degrades scu-PA was obtained and it is suggested that this effect may help to regulate fibrinolysis.
FIBRIN ACTIVATABLE UROKINASE (FA-UK): A LATENT FORM OF UK IN URINE RELATED TO A COMPLEX WITH AN INHIBITOR/FIBRIN-BINDING CO-FACTOR OF UK AND POSSIBLY OF PRO-UK
Authors: Dwivedi C , Pannell R , Gurewich V
Published: August 2018

The plasminogen activator activity in urine, expressed in IU, was found to be consistently 2-3 fold higher by fibrin plate assay than against amidolytic substrate (S-2444). Moreover, when the S-2444 assay was preceded by incubation of urine with soluble fibrin, a similar 2-3 fold increase in activity was found. The fibrin effect was dose-dependent and specific for various forms of soluble fibrin but not fibrinogen. The fibrin activatable activity was inhibited by antibodies to UK and was termed FA-UK. About 1/3 of the total UK activity in freshly voided urine was composed of FA-UK. The relative FA-UK content of urine was found to be enhanced by concentration. The FA-UK bound to fibrin/Celite. By gel filtration (S-200) of urine or by zymography, the MW of FA-UK was - 100K. Pretreatment of the samples with soluble fibrin prior to SDS-PAGE enhanced the amount but not the position of the FA-UK activity on the zymogram, indicating that the complex was not dissociated by fibrin. Pretreatment with hydroxylamine (1M) eradicated the FA-UK activity in urine. Addition of UK or pro-UK to urine followed by concentration (X10), increased the 100K band on the zymogram. Under conditions of this experiment, it was shown that little conversion of pro-UK to UK occurred suggesting that complexation occurs with pro-UK as well as with UK. Moreover, a - 100K FA-UK band on zymography was demonstrated afer addition of pro-UK to urine treated with DFP (5mM) or GGAck (20juM) to inactivate UK.

It was concluded that a - 50K inhibitor in urine, with properties similar to an inhibitor described by Stump et al (JBC 261:12759, 86), acts as a co-factor for fibrin binding of UK and possibly also of pro-UK. It is speculated that this co-factor may contribute to the fibrin-specificity of pro-UK by localizing both it and its activated derivative, UK, to the fibrin surface.