bluuebunny/crossref_metadata_2025 huggingface dataset:
FUNCTIONAL CHARACTERIZATION OF PROTEIN C INHIBITORS (PCI) FROM PLASMA AND URINE
Authors: Geiger M , Heeb M J , Binder B , Griffin J H
Published: August 2018

Immunoblotting studies showed that human plasma and urine contain components that form heparin-dependent complexes with activated protein C (APC) and urokinase (UPA) and that the presence of either active enzyme decreases complex formation by the other (Geiger et al. 1986, Circ. 74:11-234). To investigate whether these findings are due to competition of APC and UPA for one or more heparin-dependent inhibitors, a functional assay for PCI was developed to study the time course of inhibition of protein C (PC) in purified or crude systems in the presence or absence of stimulating or interfering substances. Microtiter plates coated with monoclonal anti-protein C antibodies were incubated with diluted plasma supplemented with 125I-pc. After activation of bound PC with Protac C, the wells were washed and then incubated with samples containing PCI in the presence or absence of heparin for varying times. The remaining amidolytic activity on the substrate S-2366 was determined as Δ A405/time/cpm bound. Using plasma as a source of PCI, APC inhibition followed pseudo first order kinetics and a linear relation between the pseudo first order rate constant, k, and plasma concentration was observed from 6% to 40% plasma with k = 0.012/min at 20% plasma. A dose dependent increase in k was observed in the presence of heparin, with half maximal stimulation at 2 to 4 U/ml and maximal stimulation (k = 0.05/min at 20% plasma at heparin concentration > 10 U/ml. Urinary PCI was partially purified vising heparin Sepharose chromatography from which it coeluted at 0.35 M NaCl with UPA inhibitory activity. Partially purified urinary PCI inhibited APC and UPA, and competitions similar to those obtained with plasma PCI were observed. The effect of UPA on the inhibition of APC by PCI in plasminogen-depleted plasma and by urinary PCI was studied. In both systems, UPA caused a significant decrease in the inhibition of APC in the absence or presence of heparin. These results and our previous immunobloting data demonstrate competition of APC and UPA for common heparin-dependent PCIs in plasma and urine.
IMMUNOLOGICAL SIMILARITIES BETWEEN PLASMA AND URINARY PROTEIN C INHIBITORS (PCIs) AND URINARY UROKINASE INHIBITOR (UKI)
Authors: Heeb M J , Espana F , Geiger M , Collen D , Stump D C , Griffin J H
Published: August 2018

Plasma PCIs have similar MW (∼ 57K), amino acid composition, and heparin dependence (Suzuki et al 1983, JBC 258:163) as urinary UKI (Stump et al 1986, JBC 261:12759). Urinary PCI of ∼ 50K MW has a similar heparin dependence and urokinase (UK) competes with activated protein C (APC) for this PCI (Geiger et al 1986, Circ. 74:11-234). For comparison, three forms of PCI, one from urine and two from plasma, were purified, and each exhibited heparin-dependent UK and APC inhibitory activity and formed heparin-dependent complexes with APC. The APC-PCI complexes were visible on immunoblots (nondenaturing gels) developed using: A) monoclonal anti-UKI + 125I-antimouse IgG; B) polyclonal anti-plasma PCI + 125I-plasma PCI; and C) monoclonal anti-protein C + 125I-protein C. The three forms of purified PCI were detected by methods A and B. Two new bands of APC-inhibitor complexes were seen upon incubation of plasma with APC in the presence of heparin, and the same pattern was visualized by methods A, B, and C. In the absence of heparin, only one APC-inhibitor band was visualized by methods A and B, but two bands were visualized by method C. Plasma immunodepleted of UKI by monoclonal anti-UKI-Sepharose showed no detectable antigen or complexes with APC as visualized by methods A and B. However, the UKI-depleted plasma contained components which formed a reduced amount of complexes with APC as visualized with protein C antibodies, i.e. method C. Heparin stimulates tenfold the PCI activity of normal plasma. Based on amidolytic assays of APC using S-2366, the UKI-depleted plasma was very deficient (< 15%) in heparin-dependent PCI activity, whereas the weak heparin-independent PCI activity was slightly reduced. This indicates that the majority of heparin-dependent PCI activity of plasma is immunologically.related to UKI. These studies suggest that the two slightly different forms of plasma PCI, the urinary UKI, and the urinary PCI are very similar if not identical proteins and that plasma may contain a minor heparin-independent PCI which is not immunologically related to these proteins.
A SECOND INHIBITOR OF ACTIVATED PROTEIN C (APC)
Authors: van der Meer F J M , van Tilburg N H , van der Linden I K , Briét E , Bertina R M
Published: August 2018

The inactivation of APC in plasma, as measured with the chromogenic substrate S2366, follows, in the absence of heparin, pseudo-first order kinetics. The ti of about 20 minutes is independent of the APC concentration (31-500 nM) and increases linearly with the dilution of the plasma. These observations suggest that the concentration of the APC inhibitor in plasma is much higher than 500 nM, which is much higher than the concentration of 88 nM reported by Suzuki for the protein C inhibitor (PCI).

In the presence of heparin (5 IU/ml) the inactivation of APC becomes biphasic. Fast inactivation with an apparent ti of 8 minutes is followed by slower inactivation with a ti of 20 minutes.

Removal of PCI from the plasma with α-PCI antibodies (kindly provided by Dr. Suzuki) has no effect on APC inactivation in the absence of heparin. However, in this plasma APC inactivation could not be stimulated by addition of heparin (absence of fast phase of APC-inactivation). These data suggest the presence of two APC inhibitors in plasma: the heparin dependent PCI (PCI-I), earlier reported by Suzuki, and a sofar unknown heparin-independent inhibitor (PCI-II).

Further experiments showed that this PC I-11 has a molecular weight of 60 kD and is different from the APC-binding protein reported by Kisiel. Using Heparin-Sepharose affinity chromatography we could separate PCI -II from both PCI-I and the APC binding protein. In this PC I-11 preparation APC inactivation was accompanied by the formation of an APC-PCI -11 complex of about 105 kD as demonstrated by immunoblotting with a-PC antibodies after SDS-PAGE. The identity of PCI-II is unknown; however, it is different from antithrombin III, heparin cofactor II, α1-antitrypsin and β2-antiplasmin.

The demonstration of the presence of two APC inhibitors in plasma will require a re-evaluation of the current functional assays for the APC inhibitor.
STRUCTURAL AND FUNCTIONAL COMPARISON OF THROMBOSPONDIN FROM PLATELETS, ENDOTHELIAL CELLS AND FIBROBLASTS
Authors: Clezardin P , McGregor J L
Published: August 2018

Thrombospondin (TBSP) is a 450 kDa glycoprotein secreted by a wide range of cells including platelets, endothelial cells and fibroblasts. Using non-denaturating conditions, we recently reported that platelet TBSP was structurally different from endothelial and fibroblast TBSP (P. Clezardin et al., Eur. J. Biochem., 1986, 159, 569-579). The aim of this study was to compare the structure of TBSP purified from platelets, endothelial cells and fibroblasts using denaturating conditions. Moreover, the interaction of fibrinogen with these three forms of TBSP was also investigated. TBSPs were first purified by heparin-Sepharose and immunoaffinity chromatography followed by Mono O anion-exchange chromatography on a FPLC system. Thermolysin digests of purified TBSPs were analysed by SDS-polyacrylamide gel electrophoresis under reducing conditions and the subsequent electrophoresed proteolytic fragments identified by Coomassie and silver staining. The interaction of undigested and digested TBSPs with solid-phase adsorbed fibrinogen was investigated by enzyme-linked immunosorbent assay using an anti-TBSP monoclonal antibody (P10). when using Coomassie staining, a 70 kDa proteolytic fragment of thermolysin-treated platelet TBSP was absent from the endothelial and fibroblast TBSP digests. Moreover, a 18 kDa fragment from thermolysin-treated endothelial and fibroblast TBSP was undetectable in the platelet TBSP digest when using silver staining on SDS-polyacrylamide gels. The binding of undigested TBSPs to solid-phase adsorbed fibrinogen was different from that obtained with digested TBSPs. These results indicate that the observed structural differences might induce functional differences between platelet and the two other forms of TBSP.
IMMUNOCHEMICAL IDENTIFICATION OF A THROMBOSPONDIN -LIKE ANTIGEN IN ARTERIAL THROMBOGENIC MICROFIBRILS
Authors: FAUVEL-LAFEVE F , LEGRAND Y J
Published: August 2018

The biochemical structure of arterial microfibrils (MFS) is unknown. Presently, the most probable hypothesis is that elastin associated MFS contain several antigenic determinants with MW varying between 31 and 200 KD.

From our previous studies we know that MFS extracted by 6 M GuCl contain a major glycoprotein with a 128 KD MV (GP128). GP 128 is essential for the reactivity of MFS towards blood platelets but due tc the high insolubility of the extracted material it was not possible to isolate and study this GP 128. We have used immunoblotting to determine if MFS contain determinants recognized by antibodies against connective tissue glycoproteins such as fibronectin, type VI collagen or anti-platelet thrombospondin (TSP). 'The results showed that MFS do not contain fibronectin or type VI collagen but that anti-TSP IgG reacted with GP 128. Furthermore, the Fab fragments from anti-TSP IgG inhibited platelet aggregation induced by MFS but not by collagen or ADP . In a second step,to raise antibodies against GP 128, we prepared blots from entire MFS, the nitrocellulose band corresponding to GP 128 was cut, dissolved in DMSO, and wrs injected to rabbits. Such obtained antibodies recognized only GP 128 in arterial MFS and also TSP in a platelet lysate confirming that GP 128 and TSP have a common antigenic structure. IgG from anti-GP 128 inhibit platelet aggregation induced by MFS but not by collagen or ADP. Previously reported observations showed that tissue TSP and endothelial cells derived GP 128 have a similar affinity for chromatography supports and have the same effect on platelet-MFS interactions. All these results led us to propose that TSP, GP 128, and MFS recognize a common determinant on platelet membrane. This assumption would be strenghened if GP 128 indeed is derived from tissue TSP.
PENETRATION OF FACTOR Xa INTO PHOSPHOLIPID LAYERS
Authors: LECOMPTE M F
Published: August 2018

Negatively charged phospholipids (PL) at the platelet surface play a crucial role in the conversion of prothrombin into thrombin in the prothrombinase complex. Interactions of prothrombin and factor Xa (FXa) with PL are generally thought to occur through ionic interactions via Ca++ bridges. However we have shown by various independent approaches that prothrom bin (as well as FV and FVa) actually penetrates into the PL layer. We show that this is also the case, in the presence of Ca++ , for the other vitamin K-dependent, Gla-containing protein of the complex, FXa.

Electrochemical surface measurements indicate a reversible increase in the capacitance of condensed PL monolayers (containing various concentrations of phosphatidylserine (PS)), upon interaction with bovine FXa (kindly provided by Dr. C.M. Jackson). Reduction of the FXa disulfide bridges at the contact of the electrode further demonstrates penetration Parallely, radioactive surface measurements allow to follow the adsorption of FXa which is quite reversible on PS gono-layers and to determine the association constant, 6.106 1/mol, very similar to the Ka of FX, obtained by various different techniques.

Finally, penetration of FXa with PS containing PL vesicles was tested using the radiolabelled apolar probe (INA) which binds selectively to the lipid embedded domains of protein. FXa was radioactively labelled and the label preferentially found on the active site containing FXa heavy chain Moreover, interaction of FXa with PS vesicles decreases its Km for S 2222.

Supported by grant INSERM n° 845016
ASSOCIATION OF HEPARIN AND FACTOR Xa: INFLUENCE ON THE RATE OF INHIBITION OF FACTOR Xa BY ANTITHROMBIN III-HEPARIN
Authors: Owen B A , Owen W G
Published: August 2018

Association of heparin non-covalently with bovine factor Xa was analyzed by Superose-12 gel chromatography. In 0.05 M NaCl, 0.02 M Tris, pH 7.5, DEGR-Xa (factor Xa inactivated by dans-Glu-Gly-Arg-CH2Cl) was eluted as a single, sharp peak at Ve/Vt=0-65 (elution volume/internal volume). Mixtures of heparin and DEGR-Xa were eluted as two partially resolved peaks of protein at Ve/Vt=0.59 and 0.65. The fraction of DEGFUXa in the leading peak was directly proportional to [heparin], and at 100 yM heparin the leading peak contained more than half the total protein. When 0.02 M HEPES was substituted for Tris a single, slightly broadened peak at Ve/Vt=0.64 was obtained on chromatography of 100 μM heparin and 10 μM DEGR-Xa. In a buffer system comprising 0.02 M Tris, 0.02 M HEPES, 0.03 M NaCl, pH 7.5, two peaks were eluted at Ve/Vt=0.59 and 0.65. Therefore, Tris increases the affinity of DEGR-Xa for heparin.

Solutions buffered with Tris or HEPES were compared for effects on the kinetics of inhibition of factor Xa by antithrombin III-heparin. Reaction mixtures containing 1 nM factor Xa, 30 nM heparin and 600 nM antithrombin III were assayed with S-2222 at intervals of 2-10 sec. Reagent concentrations were chosen (a) to assure pseudo-first-order kinetics, (b) to have [heparin]<< Kq for factor Xa-heparin, and (c) to bind virtually all available heparin to antithrombin III. The same second-order rate constant, Kobs=2.5×107 M−1s−1, was obtained in both buffer systems. We conclude that the association of factor Xa with heparin observed directly by gel chromatography does not contribute to the reaction rate of factor Xa with antithrombin III-heparin.
INHIBITION OF HUMAN BLOOD COAGULATION FACTOR Xa BY α2-MACROGLO-BULIN
Authors: Meijers Joost C M , Tijburg Pim N M , Bouma Bonno N
Published: August 2018

The inactivation of activated factor X (factor Xa) by α2 macroglobulin (α2M) was studied. Irreversible inhibition was observed with the initial formation of a reversible enzyme-inhibitor complex The secopd-order rate constant for the reaction was 8.4 × 104 M−1 min−1. The binding ratio was found to be 2 mol factor Xa/ mol α2M. Interaction of factor Xa with α2M resulted in the appearance of four thiolgroups/molecule α2. The apparent second-order rate constants for the appearance of thiolgroups were dependent on the factor Xa concentration. Sodium dodecyl sulphate gradient polyacrylamide gel electrophoresis was used to study complex formation between α2M and factor Xa. Under non-reducing conditions four factor Xa - α2M complexes were observed. Reduction of these complexes showed the formation of two new bands. One complex (Mr 225000) consisted of the heavy chain of the factor Xa molecule covalently bound to a subunit of α2M, while the second complex (Mr A00000) consisted of the heavy chain of factor Xa molecule and two subunits of α2M. Factor Xa was able to form a bridge between two subunits or α2M, either within one molecule of α2M, or by linking two molecules of The role of the light chain of factor Xa in this process remains to be elucidated. For this purpose, monoclonal antibodies specific for the light chain of factor Xa were prepared. Sodium dodecyl sulphate agarose electrophoresis studies showed that complexes involving more than two molecules of α2M were not formed.
IMPROVEMENT OF T-PA PROPERTIES BY MEANS OF SITE DIRECTED MUTAGENESIS
Authors: Haigwood N , Pâques E-P , Mullenbach G , Moore G , DesJardin L , Tabrizi A
Published: August 2018

The clinical relevance of tissue-plasminogen-activator (t-PA) as a potent thrombolytic agent has recently been established. It has however been recognized that t-PA does not fulfill all conditions required for an ideal thrombolytic pharmaceutical agent; for example, its physiological stability and its short half life in vivo necessitate the use of very large clinical doses. We have therefore attempted to develop novel mutant t-PA proteins with improved properties by creating mutants by site-directed mutagenesis in M13 bacteriophage. Seventeen mutants were designed, cloned, and expressed in CHO cells. Modifications were of three types: alterations to glycosylation sites, truncations of the N- or C-termini, and amino acids changes at the cleavage site utilized to generate the two chain form of t-PA. The mutant proteins were analyzed in vitro for specific activity, fibrin dependence of the plasminogen activation, fibrin affinity, and susceptibility to inhibition by PAI.

In brief, the results are: 1) some unglycosylated and partially glycosylated molecules obtained by mutagenesis are characterized by several-fold higher specific activity than wild type t-PA; 2) truncation at the C-terminus by three amino acids yields a molecule with increased fibrin specificity; 3) mutations at the cleavage site lead zo a decreased inhibition by PAI; and 4) recombinants of these genes have been constructed and the proteins were shown to possess multiple improved properties. The use of site directed mutagenesis has proved to be a powerful instrument to modulate the biological properties of t-PA.
CATALYTIC ACTIVITIES OF ONE-CHAIN tPA AS REVEALED BY AN ANALOGUE RESISTANT TO PLASMIN CLEAVAGE
Authors: Petersen L C , Johannessen M , Foster D , Kumar A , Mulvihill E
Published: August 2018

Substitution of Arg275 with Gly in the activation site of tPA provides a one-chain recombinant analogue, tPA-Gly275, which is very resistant to cleavage by plasmin. The amidolytic activity of tPA-Gly275 with simple synthetic substrates was investigated and compared to the kinetics obtained with authentic one- and two-chain tPA. Both one-chain (zymogen) forms possess enzymatic activity, however, in the absence of fibrin it is much lower than that of two-chain tPA. Fibrin enhances the activity of the one-chain tPA forms, but not of two-chain tPA.

A chromogenic assay was developed for measurement of plasminogen activation. Due to the presence of a high affinity plasmin substrate (D-Val-Phe-Lys-pNA) and aprotinin in the reaction mixture, the assay ensures a low steady-state concentration of free plasmin during the measurement. With this assay and with tPA-Gly275 it is possible to measure plasminogen activation kinetics of one-chain tPA without any significant two-chain tPA formation even in the presence of fibrin. The intactness of tPA-Gly275 was confirmed by direct measurement of the one-/two-chain tPA content by means of reduced SDS-PAGE combined with Western blotting after exposure to plasmin digestion in the presence and absence of fibrin. The results suggest that one-chain tPA possesses enzymatic activity also with plasminogen as the substrate, however, the activity is much lower than that of two-chain tPA. Addition of fibrin profoundly enhances the plasminogen activation rate of both tPA-Gly275, one-chain, and two-chain authentic tPA to approx. the same maximal level. Taken together these observations indicate that fibrin binding can induce an activated state of the intact tPA ‘zymogen’
STRUCTURAL DIVERSITY AMONG CELLULAR ADHESION RECEPTORS: FIBRINOGEN BINDING IS A NOVEL BIOLOGICAL PROPERTY OF THE MONOCYTE DIFFERENTIATION ANTIGEN OKM1
Authors: Altieri Dario C , Bader Rossella , Mannucci Pier M
Published: August 2018

A family of related glycoproteins (GP) mediate the interaction between the circulating adhesive proteins and a variety of cells (cytyoadhesins). In this study we have compared two cell-surface antigens which share the property to bind fibrinogen: the platelet GP IIb/IIIa, prototype of the cytoadhesins, and the receptor for fibrinogen costitutively synthesized by monocytes. Two anti-GP IIb/IIIa monoclonal antibodies (Mabs) (LJP9, LJP5), recognizing functionally distinct epitopes of the GP IIb/IIIa did not react with monocytes nor inhibited 125I-fibrinogen binding to monocytes. Similarly, an Arg-Gly-Asp containing peptide which completely abolished platelet-fibrinogen interaction, had no effect on monocytes. Structurally, the monocyte fibrinogen receptor was dimeric and composed of two subunits with molecular weight (Mr) of 155,000 and 95,000. This structural organization was different from that of the GP IIb/IIIa (Mr= 116,000), but in close analogy with the family of leukocyte differentiation antigens OKM1, LFA-1. Therefore, this possible relationship was investigated. A Mab to OKM1 antigen (10 μg/ml) completely suppressed fibrinogen binding to monocytes while it was ineffective on plateles. Iodinated monocyte lysate subjected to immunoprecipitation with OKM1 Mab (60 μg/ml) showed a dimeric antigen with the same molecular size of the monocyte fibrinogen receptor. Moreover, preclearing of the monocyte lysate with OKM1 Mab removed the immunoprecipitate corresponding to the monocyte fibrinogen receptor. These data indicate that the immunologic differentiation antigen OKM1, in addition to function as a complement receptor, displays also the novel biological adhesion property to mediate the binding of fibrinogen to monocytes.
BOW CAN WE INHIBIT 5HT-INDUCED PLATELET AGGREGATION AND WHY SHOULD WE BOTHER?
Authors: Bevan Jane , Heptinstall S
Published: August 2018

Platelets are induced to aggregate when 5-hydraxytryptamine (5HT) is added to citrated whole blood and the extent of aggregation can be measured using a Whole Blood Platelet Counter. We have used this method to study a) 5HT-induced platelet aggregation in normal human blood and the effects of 14 5HT receptor antagonists and 2 Ca++-channel blockers, and b) aggregation in blood from patients with peripheral vascular disease (PVD). Previous studies of platelet aggregation in platelet-rich plasma have indicated an increased platelet sensitivity to 5HT in PVD, and a multicentre study of ketanserin (a S2 antagonist) is in progress.

5HT induces a transient reversible aggregation in human whole blood which can be prevented by 5HT receptor antagonists. The inhibitory effects of 7 relatively potent antagonists (IC50 7 -41nM, e.g. ketanserin and pizotifen) could not be surmounted by increasing the concentration of 5HT, but the inhibitory effects of 7 less potent antagonists (IC50 0.28 - 53uM, e.g. mepyramine and amitriptyline) could be surmounted by 5-HT. One Ca++-channel blocker (verapamil) inhibited platelet aggregation but another (amlodipine) had very little effect. Verapamil inhibited 5HT-induced aggregation at much lower concentrations (IC50 1.6μM) than those required for aggregation induced by PAF, adrenaline or ADP (IC50 values 32, 33 and >100uM respectively) and the inhibition was insurmountable.

5HT-induced platelet aggregation in blood from patients with PVD does not differ qualitatively or quantitatively from aggregation in blood from healthy, age- and sex-matched controls: patients (n = 13), aggregation 30 seconds after adding 5uM 5HT = 55.1% ± 13.3(s.d.); controls (n = 13) aggregation = 48.3% ± 18.9(s.d.). Neither was the platelet aggregation induced by ADP, U46619 or PAF different in patients and controls.

We conclude that different 5HT receptor antagonists inhibit 5HT-induced platelet aggregation with different potencies and, apparantly, different mechanisms of action, and that verapamil has a selective effect on 5HT-induced aggregation at relatively low concentrations. Results obtained in PVD do not encourage the use of a 5HT antagonist in this condition.
EFFECTS OF SULFINPYRAZONE AND ITS METABOLITE G25671 ON PLATELET ACTIVATION AND DESENSITIZATION AND ON BRONCHOCONSTRICTION INDUCED BY THE PROSTAGLANDIN ENDOPEROXIDE ANALOGUE U46619
Authors: Hatmi M , Del Maschio A , Lefort J , De Gaetano G , Varqaftiq B B , Cerletti C
Published: August 2018

In previous studies we have found (Br. 3. Pharmac. 85, 849, 1985) that a) human platelets pre-exposed to arachidonic acid or to the endoperoxide analogue, U46619 and then washed and resuspended, fail to respond to a second challenge by both arachidonic acid and U46619; b) desensitization by arachidonic acid and U46619 occurs at a site sensitive to endoperoxides / thromboxane (Tx) receptor antagonists; c) the desensitizing effects of U46619 are direct, whereas those of arachidonic acid are mediated by a cyclooxygenase-dependent metabolite. Sulfinpyrazone (100 μM) and its thioether metabolite G25671 (50 μM) are known to suppress arachidonic acid-induced platelet aggregation and TxB2 formation (Eur. 3. Pharmac, 101, 209, 1984). We now demonstrate that the presence of sulfinpyrazone or G25671 during platelet exposure to arachidonic acid or U46619 prevents desensitization. Platelet activation by the endoperoxide analogue U46619 is also prevented by sulfinpyrazone or G25671 (0.3-1 mM). The threshold aggregating concentrations of arachidonic acid and U46619 in healthy subjects before and after oral treatment with sulfinpyrazone were elevated by 2-3 fold and a good correlation between ex vivo and in vitro findings was established. We finally examined the actions of sulfinpyrazone and G25671 on the bronchoconstriction in vivo and parenchymal lung strip contraction in vitro induced by U46619. Neither drug had any preventive effect.

Our results demonstrate that sulfinpyrazone and its metabolite G25671 are not only cyclooxygenase inhibitors but can also act as endoperoxide/Tx antagonists and indicate clearly that antagonism of U46619 by both drugs is selective for platelets.
PRESYSTEMIC DEACETYLATION OF LOW DOSES OF ENTERIC COATED ASPIRIN IN A PIG MODEL
Authors: Lloyd J V , Rodgers S E , Siebert D M , Bochner F , McIntosh G H , James M
Published: August 2018

The antithrombotic effect of aspirin might be enhanced if platelet cyclooxygenase could be inhibited in the portal ciculation while sparing cyclooxygenase in the systemic vascular endothelium. This might be achieved by modifying the dose and formulation to maximise presystemic aspirin clearance by the liver. To test this hypothesis low dose enteric coated aspirin (Astrix, 50mg single dose, lOOmg single dose and lOOmg daily for 1 week) was orally administered to pigs with permanent indwelling arterial and portal vein catheters. Plasma aspirin concentrations were measured by high performance liquid chromatography in blood obtained simultaneously from the artery and portal vein for 6 hours after dosage. Platelet aggregation and thromboxane generation were measured in 4 pigs before and after the lOOmg chronic dosage regimen. Aortic prostacyclin production was measured in aspirin treated (lOOmg daily for 1 week) and untreated pigs after sacrifice. After the 50mg single dose the arterial:portal areas under the plasma concentration versus time curve (AUC) ratio was 0.63±0.09 (n=6). In 3 pigs which received all 3 dosage regimens the arterial:portal AUC ratios were 0.48±0.05 after 50mg single dose, 0.52±0.02 after lOOmg single dose and 0.47+0.03 after lOOmg daily for 1 week. Platelet aggregation in response to sodium arachidonate (1.65mM) was completely abolished after chronic aspirin administration. Thromboxane production (pg/106 platelets) by this stimulus decreased from 536±117 before aspirin to 57±14 after aspirin (n=4; p=0.017). Aortic prostacyclin synthesis (ng/disc after 10 min incubation) was 1.66±0.28 (n=4) in untreated pigs and 0.97±0.25 (n=4) in treated pigs (p=0.06).

With this slow release aspirin formulation there was substantial but incomplete clearance of aspirin by the liver. This may not be sufficient to spare cyclooxygenase in the systemic vessels from the effect of aspirin.
IMMUNOCYTOCHEMICAL LOCALIZATION OF FIBRINOGEN DURING THROMBIN-INDUCED AGGREGATION OF HUMAN PLATELETS
Authors: Suzuki H , Kinlough-Rathbone R L , Packham M A , Tanoue K , Yamazaki H , Mustard J F
Published: August 2018

The association of fibrinogen (Fbg) with washed platelets was studied during thrombin-induced aggregation in Tyrode-albumin solution with 2 mM Ca2+ or no added Ca2+. Platelets were fixed, embedded in Lowicryl K4M, sectioned, incubated with goat antihuman Fbg, washed, reacted with gold-labelled rabbit anti-goat IgG and prepared for electron microscopy. To ensure that Fbg could be detected with this method, platelets were pretreated with chymotrypsin and aggregated with Fbg; gold particles were apparent on the surface and between adherent platelets and in the alpha granules. In a Ca2+-containing medium in the absence of external Fbg, washed platelets did not have Fbg on their surface although there was extensive gold labelling of the platelet alpha granules. Thrombin (0.05 U/ml) caused platelet aggregation, centralization and apparent fusion of alpha granules. By 60 sec large aggregates had formed, many platelets appeared degranulated but few gold particles were seen between adherent platelets. At 5 min, little Fbg remained in the aggregated platelets. In a few regions gold accumulated in fused granule material, and in occasional clusters between adherent platelets. In the presence of external Fbg (0.4 mg/ml) thrombin caused aggregation, centralization and fusion of granules, and discharge of granule contents. However, numerous gold particles were readily detectable between adherent platelets and on the platelet membrane. Fibrin formed and was abundantly labelled with immuno-gold. Similar findings were obtained in the medium without added Ca2+ (20 μM Ca2+). These results agree with observations obtained from measurements of 125I-Fbg binding and show that in the presence of external Fbg thrombin causes Fbg binding to platelets during aggregation. In the absence of added Fbg, thrombin aggregates platelets without extensive binding of released Fbg to the platelets.
THE SURFACE EXPRESSION OF ALPHA GRANULE PROTEINS FOLLOWING THROMBIN STIMULATION
Authors: Gralnick H R , Magurder L M , Hansmann K , Vail M , Marti G , McEver R , Williams S
Published: August 2018

We have studied the platelet glycoproteins (GP) GPIb and the GPIIb/IIIa and the expression of alpha granule proteins (AGP) on the platelet (P) surface following thrombin (T) stimulation. The platelets were separated from plasma proteins on a arabinogalactan gradient. The P were stimulated with purified alpha T 0.1u/108p. Either monospecific polyclonal or murine monoclonal antibodies were used to detect the P glycoprotein and AGP. The platelets were analyzed on an EPICS V Flow Cytometer. Resting P had small amounts of AGP (2-8%) present on their surface. Within 1-3 min. after T stimulation significantly increased amounts of PF4 (26%) vWf (8%) Ig (10%) and the 140 kD alpha granule membrane (70%) were present on the P surface. The peak expression of all the AGP occurred within 5 mins. The 140 kD activation protein remained stable over 3-60 mins, in contrast the PF4 and the vWf expression peaked at 5 mins. and then decreased to near baseline levels. The GPIb and GPIIb/IIIa showed different patterns after activation. The GPIb intensity and number of positive cells decreased over time, while the GPIIb/ IIIa increased in flourescent intensity and the number of positive cells. These studies indicate that T stimulation of AGP on the P surface. vWf and P4 have a transient appearance on the P surface while Ig and the 140 kD activation protein both appear to become stable components of the P plasma membrane. This technique of detecting platelet activation is a specific, sensitive, and rapid method.
MONOCLONAL ANTIBODY (ANTI-2B6D4) TO PLASMA FIBRONECTIN INHIBITS COLLAGEN AND THROMBIN INDUCED AGGREGATION OF WASHED PLATELETS
Authors: Kenneally D A , Thurlow P J , Cornellan J M
Published: August 2018

Fibronectins(Fns) constitute a family of large glycoproteins which are known to bind to a wide range of biological molecules eg.collagen, gelatin, fibrin, heparin and DNA,and to many cells including platelets via discrete structural domains. A murine monoclonal antibody (anti-2B6D4) produced by imnunizing BALB/c mice with plasma fn, was used to study the structure and function of fn and its platelet interaction Anti-2B6D4 reacted specifically with plasma fn was unreactive with FVIII/vWF,BTG,PF4 collagen and fibrinogen nor was it reactive with platelets (unstiraulated), human PBLs or a range of tumour cell lines. Iirmuno-blotting studies( 8% SDS-PAGE) using thermolysin-digested plasma fn with anti-2B6D4 indicated that the 2B6D4 epitope was present on only 2 of the 7 fragments detected by Indian ink. The 2 fragments had an Mr of 145 and 155 K dal tons and have been reported to each contain domains which bind cells, DNA and heparin. These fragments were studied further by examining the effect of anti-2B6D4 (Fabs) on the binding of 125I-fn to thrombin-stimulated platelets and demonstrated that anti-2B6D4 binding was inhibited by 50% thus implicating the 2B6D4 epitope as a platelet binding site within the two cell binding domains. Competitive binding analysis of 125I-fn to solid-phase macromolecules i.e.collagen, gelatin, fibrin, heparin, DNA and Con A demonstrated that anti-2B6D4 (Fabs) inhibited the binding of fn to DNA by 50%,but not to the other macromolecules. Therefore, either the DNA and platelet binding sites are shared or the inhibition is due to steric hindrance. However, as Fab fragments of anti-2B6D4 were used, it is more likely that the binding sites are shared. Functional studies were performed to investigate the role of 2B6D4 in platelet-platelet interaction. Anti-2B6D4 totally blocked the aggregation of washed platelets stimulated by low dose collagen (1.6ug/ml) and thrombin (0.05U/ml), partially inhibited arachidonic acid (250ugs/ml) induced platelet aggregation and had no effect on aggregation induced by A23187 (30uM). One other report had demonstrated that fn is a requirement for A23187 and low dose thrombin induced platelet aggregation. We conclude that fn plays an essential role in platelet aggregation induced by low dose collagen and thrombin.
THE MOLECULAR ORGANIZATION OF HUMAN ALPHA 2-MACROGLOBULIN. AN IMMUNO ELECTRON MICROSCOPIC STUDY WITH MONOCLONAL ANTIBODIES
Authors: Delain E , Barrav M , Tapon-Bretaudière J , Pochon F , Van Leuven F
Published: August 2018

Electron microscopy is a very convenient method to localize the epitopes of monoclonal antibodies (mAbs) at the surface of macromolecules for studying their tree-dimensional organization.

We applied this immuno-electron microscopic method to human ct2-macroglobulin (ct2M). 29 anti-α2M mAbs have been tested with the four different forms of a2M : native and chymotrypsin-transformed tetramers, and the corresponding dimers, obtained by dissociation with divalent cations. These mAbs can be classified in three types : those which are specific for 1) the H-like transformed molecules, 2) the native molecules, and 3) those which can react with both forms of α2M.

1) Among the H-like α2M specific mAbs, several react with the 20 kD-domain which is recognized by the cellular receptor of transformed a2M. This domain is located at the carboxyterminal end of each monomer. One IgG binds to the end of two adjacent tips of the H-like form.

The other mAbs of this type bind to the α2M tips at non-terminal positions. Intermolecular connections built polymers of alternating α2M and IgG molecules.

2) Among the native a2M-specific mAbs some are able to inhibit the protease-induced transformation of the native α2M. The binding sites of these mAbs are demonstrated on the native half-molecules. One of these mAbs was also able to react with transformed dimers, in a region corresponding very likely to an inaccessible epitope in the tetrameric transformed α2M molecule.

3) Among the mAbs of this type, only two were able to inhibit the protease-induced transformation of α2M. Obviously, their epitopes should be close to the bait region of α2M. The other mAbs reacting with both α2M forms did not inhibit the α2M transformation.

All these mAbs can be distinguished by the structure of the immune complexes formed with all forms of α2M. The epitopes are more easily located on the dimers and on the H-like transformed α2M than on the native molecules.

From these observations, we propose a new model of the tree-dimensional organization of the human α2M in its native and transformed configurations, and of its protease-induced transformation.
GENE DELETIONS IN THE FACTOR IX LOCUS
Authors: Matthews R J , Anson D S , Peake I R , Bloom A L
Published: August 2018

Previous studies have indicated that the majority of haemophilia B patients who produce anti-factor IX inhibitors (antibodies) have some kind of deletion of the factor IX gene. We have analysed DNA from nine haemophilia B inhibitor patients using the Southern blotting method and hybridisation with (i) factor IX cDNA and intragenomic probes (ii) probes originating from flanking sequences up to 60kb 5' and 170kb 3' to the factor IX gene that have been isolated by gene walking experiments (D.S.Anson and G.G.Brownlee, unpublished observations).

Two patients who are brothers (haemophilia B (Chicago I)) were shown to have a presumably identical complex rearrangement of the factor IX gene involving two separate deletions. The first deletion is approx. 5.0kb and removes exon e. The second deletion is between 9 and 29kb and removes exons g and h but leaves exon f intact. An abnormal TaqI fragment at one end of the deletions junctions acted as a marker for the inheritance of haemophilia B in the patients' family. Furthermore, an abnormal llkb Bglll fragment (detected with an intragenomic probe containing exon f) in DNA from both patients and their mother acted as a marker for the presence of both deletions. Since the patients' grandmother only showed the normal 12kb Bgl II fragment then both deletions appear to have arisen at the same time. We believe that haemophilia B (Chicago 1) is the first observation of a natural gene rearrangement involving two separate deletions within the same gene.

Patient haemophilia B (Jersey 1) was revealed to have a deletion of at least 170kb including the entire factor IX gene and > 60kb of 5' flanking sequence. The 3' breakpoint of this deletion was mapped to between 80 and 140kb 3' to the factor IX gene. One further patient, haemophilia B (Boston I) was shown to have a deletion of > 230kb including the factor IX gene, > 60kb of 5' flanking sequence and >140kb of 3' flanking sequence. Five other inhibitor patients had a structurally intact gene as detected by this method.

Although all nine haemophilia B inhibitor patients studied did not have a detectable plasma factor IX only in four of them is this absence due to a large deletion of the factor IX gene.
MOLECULAR BASIS OF HEMOPHILIA B: IDENTIFICATION OF THE DEFECT IN FACTOR IX VANCOUVER
Authors: Geddes V A , Louie G V , Brayer G D , MacGillivray R T A
Published: August 2018

Factor IX Vancouver (fIX-V) is the cause of a moderate form of hemophilia B. An individual presenting with this disorder had 2.6% of normal procoagulant activity in his plasma but had 62% of the normal factor IX antigen level. Specific antibodies showed that fIX-V contains epitopes for both the heavy and light chains of factor IXa. To identify the defect involved, DNA was isolated from the lymphocytes of the male hemophiliac. Southern blot analysis using a full-length factor IX cDNA as a hybridization probe showed no gross differences between the fIX-V gene and the normal factor IX gene. The DNA from the hemophiliac was then partially digested with Sau3A and the resulting fragments (10-20kbp in size) were ligated into the BamHI site of λEMBL3. The DNA was then packaged into phage particles in vitro, and the recombinant phage were screened with the factor IX cDNA as a probe. Eight phage were isolated that contained overlapping DNA covering the complete gene for fIX-V. DNA sequence analysis of the protein-encoding regions, the intron/exon junctions and 5'-and 3'-flanking sequences revealed a single nucleotide change from the normal factor IX gene. The codon for amino acid 397 was changed from ATA (lie) to ACA (Thr). This mutation is in the catalytic domain of factor IXa and is novel amongst those hemophilia B mutations reported to date. Based on the known three dimensional structures of the pancreatic serine proteases, trypsin, elastase and chymotrypsin, models have been constructed for the structures of the catalytic domains of both the normal and Thr-397 mutant of factor IXa. These results suggest that the Thr-397 mutation may alter the conformation of the substrate binding region in the active site of factor IXa Vancouver through the formation of a hydrogen bond between the hydroxyl group of the Thr-397 side chain and the main chain carbonyl group of Trp-385. The postulated conformational change would lead to reduced binding affinity for the factor IXa substrate resulting in a reduction in the catalytic activity of fIXa-Vancouver.

Supported in part by grants from the Medical Research Council of Canada (to GDB and RTAM).
PATIENTS WITH ARTIFICIAL BUT NOT BIOLOGICAL HEART VALVE PROSTHESIS PRESENT A HYPERCOAGULABILITY RELATED TO THE INTENSITY OF ANTICOAGULATION
Authors: Pengo V , Boschello M , Peruzzi P , Pagotto D , Schivazappa L , Dalla Volta S
Published: August 2018

Long term anticoagulant therapy is mandatory for patients with artificial heart valve prosthesis and is suggested for some patients with biological heart valve prosthesis. Oral anticoagulants reduce but not abolish thromboembolic complication in these patients. They act lowering the level of vitamin K-dependent coagulation factors and that in turn should result in a depression of "in vivo" thrombin formation. Fibrinopeptide A (FpA) is a good marker of thrombin formation and therefore we ascertained in several occasions the thrombin formation in 43 patients with artificial and 18 with biological heart valve prosthesis, all the patients being on oral anticoagulant treatment at least from 1 year. FpA was significantly higher in patients with artificial (determinations n = 138) with respect to biological (n=73) heart valve prosthesis (p 0.01). The FpA level in biological valves was close to that obtained in 22 not anticoagulated healthy subjects. When we divided FpA values in artificial heart valves according to the intensity of anticoagulation, we obtained a decreasing FpA mean levels with the increase of the degree of anticoagulation. In particular FpA values with an INR 4.5 were close to values obtained in healthy subjects. These data support the concept that patients with artificial heart valves are at higher risk of thromboembolism and therefore the intensity of anticoagulation should be different with respect to biological valves and probably a little higher than that recommended at the Leuven Consensus Conference.
A PROSPECTIVE RANDOMISED CONTROLLED STUDY OF MINI-DOSE WARFARIN PROPHYLAXIS OF DEEP VEIN THROMBOSIS IN MAJOR SURGERY
Authors: McKernan A , Thomson J M , Poller L
Published: August 2018

A prospective randomised study has been undertaken to assess the clinical effectiveness of oral anticoagulation using minidose warfarin (1 mg daily for 2-4 weeks before major gynaecological surgery) compared with conventional oral anticoagulant prophylaxis and an untreated randomised control group. The conventional oral anticoagulant prophylaxis was based on a therapeutic range of 1.5 - 2.0 INR at the time of operation and 2.0 - 3.0 INR post-operatively. Overall the mini-dose warfarin group showed no pre-operative prolongation of the prothrombin time with the Manchester Reagent although a minority of patients showed a 1-2 second prolongation of the prothrombin time before operation. Post operatively the mini-dose warfarin group showed an exaggerated prolongation of the prothrombin time which normally occurs after operation and was observed in the untreated controls. Factor VII assays paralleled these findings. Minidose warfarin, while not prolonging the prothrombin time before operation, resulted in delayed platelet aggregation with the Chandler's tube technique in almost all patients.

The incidence of deep vein thrombosis has been reduced in both mini-dose and conventional dose oral anticoagulant series compared with the untreated- group. It appears that the minimal changes in the prothrombin time, factor VII and platelet aggregation tests, observed in the mini-dose warfarin group, may offer sufficient protection against post-operative thrombosis in a moderate risk group undergoing abdominal or pelvic surgery.
USE OF INR FOR THE EXPRESSION OF PT IN PATIENTS UNDER ORAL ANTICOAGULANTS : COMPARISON OF RESULTS OBTAINED WITH FIVE THROMBOPLASTINS AND THREE DEVICES
Authors: Roussi J H , Francois D , Delemme J , Goguel A F
Published: August 2018

The aim of our study is to know if INR (International Normalized Ratio) is really the best way to express PT (Prothrombin Time) in patients under oral anticoagulants (AVK). In this way, PT of 85 patients treated by AVK and 30 normal controls were determined with 5 different reagents and three devices : manual technic, KC10 (AHS Dade), Coag A Pet 200 (Gal Diagnostics, Organon). The reagents were : Thromborel S (Behring) extracted from human placenta, Thrombotest (Nyegaard) from ox brain, and 3 rabbit thromboplastins : Neo-plastine (Diagnostica Stago), Thromboplastine calcique (Bio-Merieux), Simplastin + (Gal Diagnostics). The results are expressed in seconds, percentage according to the dilution curve and INR with ISI given by the manufacturers.

Analysis of results shows :

- Influence of the technic : Whatever the technic is, mean results obtained with the same reagent, expressed in % are very close ; expressed in INR mean results are dispersed with all the reagents, ex :

- Influence of the reagent : Results performed with the same technic whatever the reagent is expressed in INR are very close, whereas in % the dispersion is large.

Analysis of variance and Student test show highly significant differences between all reagents for expression in %, whe reas expressed in INR (KC10) the difference is not statistical ly significant.

INR leads an improvement in the dispersion of results but it seems necessary to have at least two ISI for each reagent, one determined by manual technic or device like KC10, the other one determined on photo-optical device.