bluuebunny/crossref_metadata_2025 huggingface dataset:
DIETARY LIPIDS, INFECTION AND MACROPHAGE PROCOAGULANT ACTIVITY
Authors: van Dam-Mieras M C E , Muller A D , Hornstra G
Published: August 2018

It is generally accepted that the type of dietary fat influences arterial thrombosis and atherosclerosis. Although it is still largely unknown how the dietary lipid composition influences the process of atherogenesis, it is evident that several cell types are involved. Morphological evidence for the involvement of monocyte/macropages has been given.

We described before that the dietary lipid composition has striking effects on the procoagulant activity of macrophages. When macrophages were isolated from the spleens of healthy rats the procoagulant activity slightly decreased during the first few hours after isolation, and reached a plateau value after 4 hours. When, however, macrophages were obtained from animals infected with a pneumona virus (PVM) different results were obtained:

Experiments carried out with peripheral blood monocytes showed close resemblance to those described in the table.

These results show that:

- moncytes/macrophages isolated from PVM-infected animals increase their procoagulant activity during in vitro culture

- the differences in macrophage procoagulant activity found in cells obtained from healthy animals fed diets containing different lipids no longer were found in PVM-infected animals

This would implicate that the infection process has a more profound influence on macrophage procoagulant activity that the composition of the diet
MODULATION OF THROMBIN-MEDIATED REACTIONS IN HAEMOSTASIS- BY TREQUINSIN AND ITS ANALOGUES.
Authors: Blasko Gy , Blasko G , Sas G , Szantay Cs
Published: August 2018

A new pyrimido/6,1-a/-isoquinoline derivative ,/Ref.1 / developed by Hoechst and named Trequinsine has been reported to be a powerful inhibitor of cAMP phosphodiesterase and platelet aggregation having a potency nearly similar to prostacyclin. We synthesized Trequins-in (1), its indole derivative (4) , as well as their precursors (2 3) and subjected them to a study in order to evaluate their effects on haemostasis. Surprisingly they exhibited a modulatory effect only on reactions mediated mainly by thrombin. They inhibited platelet aggregation (ICc:q for Trequinsin: 13 + 2.5 ng/ml, for indole derivatives: 1-3 /ig/ml); 1, 2, 4 enhanced the amidolytic activity of CC-thrombin towards S-2238 substrate within a narrow range of submicromolar concentrations but they did not exert any effect on the activity or the inactivation of Factor Xa and plasmin.

Compound 2 proved to be an inhibitor of prostacyclin production of rat aortic tissue (Trequinsin has much less effect) but in contrary to this 3. and 4 exerted an enhancing effect on the total prostacyclin production.
ANTI-THROMBOTIC EFFECT IN CANINE CORONARY ARTERIES OF A COMBINED TXA2 synthetase/TXA2-prostaglandin ENDOPEROXIDE RECEPTOR INHIBITOR (R 68070)
Authors: Van de Water A , Xhonneux R , De Clerck F
Published: August 2018

The effects of R 68070 an oxime-alkane carboxylic acid derivative combining specific thromboxane A2 (TXA2) synthetase inhibition with TXA2/prostaglandin endoperoxide receptor blockade in one molecule, on thrombus formation in a coronary artery following electrically-induced endothelial injury and on its myocardial repercussions were examined in dogs. In an open-chest model in anaesthetized dogs, a stainless steel electrode was inserted into the left anterior descending coronary artery (LAD) distally (+ 1 cm) from an electromagnetic flow probe. ECG and heart rate were derived from limb leads. Serum TXB2 levels were measured by RIA on venous spontaneously coagulated blood (1 h, 37°C). Endothelial cell injury in the LAD coronary artery was induced by the application of an anodal current of 300 μA during 30 min; after an additional 60 min observation period, the thrombus wet weight was determined.

In comparison with solvent treatment (n = 8), R 68070 (1.25 mg/kg I.V. 10 min before electrical stimulation, n = 7), significantly reduced the thrombus mass (solvent : 43 mg; R 68070 : 18 mg median value, p < 0.05), the incidence of ECG changes indicative for myocardial ischemia (fibrillation : solvent 1/8; R 68070 0/7; arrhythmias : solvent 3/8; R 68070 2/7; ST changes : solvent 7/8; R 68070 1/7, p < 0.05) and the decrease in coronary blood flow after electrical stimulation (solvent : from 13 to 6.5 ml/min; R 68070 : from 13 to 11 ml/min median values, p < 0.05). Serum TXB2 levels were reduced by 92 % at 100 min after the injection of the active compound (median value, n = 7).

Heart rate and coronary blood flow measured before the induction of the endothelial injury were not modified by R 68070.

The present study thus demostrates that R 68070 exerts a potent anti-thrombotic effect in canine coronary arteries. The relative contributions to this effect of TXA2 synthetase inhibition and of TXA2/prostaglandin endoperoxide receptor blockade exerted by the compound are being investigated.
S2-SEROTONERGIC RECEPTOR INHIBITION (KETANSERIN), COMBINED WITH THROMBOXANE A2/PROSTAGLANDIN ENDOPEROXIDE RECEPTOR BLOCKADE (BM 13177) : ENHANCED ANTI-PLATELET EFFECT
Authors: De Clerck F , Xhonneux B , Van Gorp L , Beetens J , Janssen P A J
Published: August 2018

Subsequent to an initial activation, several products are released from the platelets, and -potentially- contribute to the recruitment of new cells to build up the eventual platelet haemostatic plug or thrombus. Among the various accussates, serotonin as well as the arachidonic acid derivatives thromboxane A2 (TXA2) and prostaglandin endoperoxides (PGEND) seem to be involved as causative mediators, in particular when acting in concert on the same target cells. Indeed, in vitro at concentrations achievable after oral administration to man, either ketanserin (K), an S2-serotonergic receptor antagonist or BM 13177 (BM), an antagonist at TXA2/PGEND receptors, pre-incubated separately for 5 rain at 37°C with human citrated platelet-rich plasma, reduces to some extent the rate of the second-wave, irreversible platelet aggregation elicited by critical concentrations (1 to 2 × 10−6 M) of ADP. However, the combindation of both agonists results in a significantly more pronounced inhibition than achieved with the single products (K 1 × 10−7 M : 26.1 ± S.E.M. 9.9 %, n = 8. p < 0.05; BM 1 × 10−77 M : 25.1 ± S.E.M. 8.7%, n = 8, p < 0.05; K ± BM at 1 × 10−7 M each ; 65.5 ± S.E.M. 10.3 %, n = 8; p < 0.05 versus K or BM separately). In vivo in rats, a modest prolongation of the bleeding time occurs with K (1.25 mg/kg orally -2 h : 410 sec, median values n = 9, p < 0.05) but not with BM (40 mg/kg orally, -2 h : 250 sec, n = 9) in comparison with controls (190 sec, n = 9); the amount of initial blood loss as a parameter for anti-vasoconstriction is significantly increased by K (140.4 μl/30 sec) but not by BM (77.2 μl/30 sec) in comparison with controls (35 μl/30 sec). Again, the combined treatment with both agonists results in a marked prolongation of the bleeding times (> 2065 sec, n = 9, p < 0.05) without further significant increase of the initial blood loss, indicating the interplay between serotonin and TXA2/PGEND to be of primary importance for the aggregation of platelets in damaged vessels.
EFFECTS OF SOME ORGANIC SOLVENTS ON GP lb AND ACTIN-BINDING PROTEIN IN BLOOD PLATELETS
Authors: Aakhus A M , Solum N O , Hagen I
Published: August 2018

Effects on filamentous proteins appear to be a central phenomenon in the neuronal toxic effects of organic solvents. We have therefore compared the effects of some organic solvents (particularly isopropylalcohol, IPA) to the previously observed effects of dibucaine (DBC) on platelet cytoskeletal proteins. Incubation of platelets with 6% IPA at 37° C, like DBC, initiates a degradation of actin-binding protein (ABP) as substrate for a calcium activated protease (CAP), shown by SDS-PAGE. IPA leads to an increase followed by a decrease in bovine von Willebrand factor-induced agglutination. The decrease is accompanied by a release of glycocali-cin from the GP lb α-chain. The process was also studied using CIE of Triton X-100 extracts of platelets against antiserum to glycocalicin. Incubation of platelets with IPA before extraction in the presence of 4.2 mM leupeptin leads to a time-dependent transformation of GP Ib-related immunoprecipitates from that of the slow-migrating peak III complex (probably between ABP and GP lb) to the faster migrating GP Ib-precipitate. Our working hypothesis is that IPA induces an activation of the CAP by mobilizing calcium. This leads to degradation of ABP and liberation of GP lb from the cytoskeleton accompanied by an increased tendency for agglutination. The following decrease is explained by degradation of the glycocalicin part of the GP lb enchain which contains the binding-site for von Willebrand factor. We conclude that IPA has a similar effect on GP lb and ABP as DBC. Preliminary studies with 1% DMSO and 0,005% toluene at 37° C revealed that these organic solvents have some similar effects on platelets as described for IPA. Possibly the described effects are characteristic of certain cells at an early stage in a process ultimately leading to cell lysis.
THE ROLE OF GPIIb-IIIa IN MODULATION OF ADHESION REACTIONS
Authors: Mattson J C , Estry D W , Peterson D , LaFevre R , Chirco J
Published: August 2018

We have previously reported that patients with Glanzmann’s Thrombasthenia (GT) fail to adhere to a carbon-formvar surface and undergo contact-induced shape change in a non-flow system. The ability of ADP to reverse this adhesion defect suggested that it may be secondary to defective dense granule release rather that a direct requirement for GPIIb-IIIa. To further assess the role of GPIIb-IIIa in adhesion, we examined the effect of two mouse monoclonal antibodies to the GPIIb-IIIa complex, AP2 (IgG, kappa) from T. Kunicki, Milwaukee Blood Center and MAb36 (IgM, lambda) from D. Peterson, Rice University. AP2 (1:50 dil) and MAb36 (1:200 dil) both completely abolished aggregation by ADP, collagen and epinephrine and prevented clot retraction. In a transmission EM (TEM) whole mount assay of adhesion and contact-induced shape change, both antibodies inhibited platelet attachment to the substrate and impaired spreading in those few platelets that did attach. This antibody-induced adhesion defect was reversed by the addition of 2×10−6 m ADP just prior to exposure of platelets to the activating surface. In parallel studies, antibody treated platelets demonstrated a dose-related defect in ATP release as measured in a Lumiaggregometer with total absence of release at antibody dilutions that abolished aggregation. Using a colloidal gold-fibrinogen probe, virtual absence of binding of exogenous fibrinogen was demonstrated in antibody treated platelets induced to. spread by ADP stimulation. These studies suggest that while GPIIb-IIIa may play a role in adhesion in non-flow systems, as suggested by the altered adhesion seen in GT platelets, adhesion and adhesion-induced shape change can be supported by ADP stimulation in the absence of fibrinogen binding to GPIIb-IIIa.
PRIMARY BINDING SITE OF VON WILLEBRAND FACTOR IN THE SUBENDOTHELIUM WHICH MEDIATES PLATELET ADHESION IS NOT COLLAGEN
Authors: de Groot Philip G , van Mourik Jan A , Sixma Jan J
Published: August 2018

We have studies the binding of von Willebrand factor (vWF) to extracellular matrices of endothelial cells and smooth muscle cells and to the vessel wall of human umbilical arteries in relation to its function in supporting platelet adhesion at high shear rates. CLB-RAg 38, a monoclonal antibody directed against vWF inhibits the binding of 125I-vWF extracellular matrices completely. The binding of 125I-vWF to subendothelium is not inhibited, because there are many different binding sites. CLB-RAg 38 inhibits platelet adhesion to extracellular matrices and subendothelium, in sofar as it is dependent on plasma vWF. CLB-RAg 38 has no effect on adhesion depending on vWF already bound to the matrix or subendothelium. CLB-RAg 38 does not inhibit binding of vWF to collagen type I and type III. Another monoclonal antibody against vWF, CLB-RAg 201, completely inhibits binding of vWF to collagen type I and type III. CLB-RAg 201 does not inhibit binding of 125I-vWF ot the extracellular matrices. CLB-RAg 201 partly inhibits platelet adhesion but this inhibition is also present when the adhesion depends on vWF already present in matrix or subendothelium, indicating that CLB-RAg 201 also inhibits the adhesion of platelets directly, this in contrast to CLB-RAg 38. The epitopes for CLB-RAg 201 and 38 were found on different tryptic fragments of vWF. These data indicate that vWF binds to subendothelium and to matrices of cultured cells by mechanism that is different from binding to collagen.
DETERMINATION OF THE DOMAINS OF FACTOR VIII ESSENTIAL FOR PROCOAGULANT ACTIVITY
Authors: Verbeet M Ph , Evers R F , Leyte A , Lamain H L , Van Ooyen A J J , Van Mourik J A , Pannekoek H
Published: August 2018

Factor VIII (FVIII) consists of an obvious domain structure that can be represented as A1-A2-B-A3-C1-C2 (Vehar et al., 1984, Nature 312, 337). In order to determine the domains involved in the procoagulant activity of FVIII, we constructed mutant FVIII cDNAs containing deletions in the coding sequence of the full-length molecule. In one of the mutants a large part of the B domain is deleted. In another one we made a deletion in the B domain that extends beyond the thrombine cleavage site. We used pSV-2 derived expression vectors and COS-1 cells in a transient expression system for the full-length and mutant recombinant proteins. Conditioned media (CM) were harvested.

In accordance with the described mutants of recombinant FVIII (Toole et al., 1986, PNAS 83, 5939), we demonstrated an increase in activity in the CM for these mutants as compared to the full-length activity. We also found that the specific activity of the mutants is similar to that of plasma FVIII. So, shorter chains lead to an increased amount of procoagulant protein.
HEPARIN AND NON-ANTICOAGULANT HEPARINS INHIBIT HEPARANASE ACTIVITY IN NORMAL AND MALIGNANT CELLS:POSSIBLE THERAPEUTIC USE IN PREVENTION OF EXTRAVASATION AND DISSEMINATION OF BLOOD BORNE CELLS
Authors: Eldor A , Bar-Ner M , Wasserman L , matzner Y , Fuks Z , Viodavsky I
Published: August 2018

Degradation of vascular subendothelium occurs in_vivo during the process of inflammation and tumor invasion. Various observations suggest that the capacity of some blood-borne cells to extravasate may depend in part on their ability to express hepara-nase activity. Incubation of human platelets, human nc-utrophils, or highly metastatic mouse lymphoma cells with sulfate-labeled extracellular matrix (ECM) results in heparanase mediated release of labeled heparan sulfate cleavage fragments (0.5<Kav<0.85 on Sepharose 5B) (J. Clin.Invest. 74: 1842 and 76: 1306; Cancer Res. 43: 2704). The present study was undertaken to test the heparanase inhibitory effect of heparin and non-anticoagulant species of heparin that might havea potential therapeutic use in preventing heparanase mediated extravasation ofblood-borne cells. We prepared totallyor N-desulfated heparins which were either left with their N-position exposed or were subsequently N-acetylated or N-resulfated. These heparins exhibited less than 5% of the anticoagulant activityof native heparin. It was found that total desulfation of heparin abolished its heparanase inhibitory activity whether desulfation was followed by N-acetylation or not. Inhibitory effect was restored by resulfation of the N-position. When only the N-sulfate group was desulfated, inhibitory activity was lost but could be restored by acetylation of the N-position. These results indicate that N-sulfate groups of heparin are necessary for its heparanase inhibitory activity but can be substituted by an acetyl group provided that the 0-sulfate groups are retained. Low Mr heparins (main Mr species of 2500 and 4500 daltons) and heparin fragments as small as the tetrasaccharide inhibited degradation of heparan sulfate in the ECM, albeit to a lower extent than native heparin. Similar effects of the different heparins were observed with heparanase activities from platelets, neutrophils and lymphoma cells. Preliminary in vivo experiments suggest that non-anticoagulant heparins interfere with tumor metastasis and experimental autoimmune diseases (some heparins were kindly provided by Inst. Choay, Paris and Kabi Vitrum, Stockholm).
SELECTIVE EXPRESSION OF PLATELET-DERIVED GROWTH FACTOR B-CHAIN mRNA BY HUMAN ENDOTHELIAL CELLS AND BY HUMAN PERIPHERAL BLOOD MONOCYTES, BUT NOT BY HUMAN SMOOTH MUSCLE CELLS
Authors: Sakaruassen K S , Powell J S , Raines E W , Ross R
Published: August 2018

Vascular injury may occur by a variety of mechanisms. Episodes of local hypoxia or conditions leading to local generation of thrombin may influence local cells to release growthregulatory molecules such as platelet-derivedgrowth factor (PDGF) in the surrounding connective tissue. The roles of the cells and of PDGF in these processes are not entirely understood, and this prompted us to investigate effects of hypoxia (5% O2) on cultured human saphenous vein endothelial cells andhuman thoracic aorta smooth muscle cells. Freshly isolated human peripheral blood monocytes were exposed to 3.0 U/ml a-thrombin. PDGF-A and PDGF-B mRNAs were analyzed by Northern blots, and their levels were assessed by dot blots utilizing 32P nick-translated cDNA probes. Selective expressionof PDGF-B mRNA occurred in endothelial cells during hypoxia and in monocytes exposed to thrombin. Genes coding for PDGF-A and PDGF-B are expressed cons tit utively, in endothelium, and after 48 hr of hypoxia a nine-fold increase of PDGF-B mRNA is detected (9 pg mRNA/ug total RNA). No detectable levels of mRNA encoding PDGF-A and PDGF-B were observed in freshly isolated monocytes; however, a 4-hr exposure to a-thrombin resulted in a selective and transitory increase in PDGF-B mRNA, amountingto 1 pg mRNA/ug toted RNA. No PDGF-B mRNA wasdetected after 20 hr. Hypoxic conditions did not trigger any selective expression of PDGF-B mRNA in smooth muscle, including arterialsmooth muscle derived from 1-day- and 3-month-old individuals, or from adult venous smoot muscle. However, constitutive expression of PDGF-A mRNA was observed in each of these, amounting to 0.4 pg mRNA/ug total RNA in the 1-day- and 3-month-old cells, and 0.2 pg mRNA/ugtotal RNA in the venous smooth muscle. Our datashow that endothelium and monocytes selectively express PDGF-B mRNA in vitro in response to conditions mimicking those encountered during vascular injury in some in-vivo situation.The data imply that both endothelial cells and monocyte/macrophages may be sources for mitogens that induce intimal hyperplasia and eventually plaque formation.
EFFECT OF ANTITHROMBIN III AND HEPARIN ON FACTOR X ACTIVATION BY FACTOR IXa
Authors: Pieters J , willems G , Hemker H C , Lindout T
Published: August 2018

The heparin-catalyzed inactivation of activated coagulation factors by antithrombin III (AT III) has mostly been studied for isolated serine proteases. However, we decided to study the action of heparin and AT III under more physiological conditions, i.e. during the activation of factor X by factor IXa in the presence of phospholipid and calcium. Thereby we made use of a mathematical model which describes the generation of factor Xa by factor IXa, phospholipid and calcium in the presence of AT III and heparin. Fitting the experimental factor Xa generation curve to a set of equations gave the pseudo-first-order rate constants of factor Xa and factor IXa. In a first approach we examined the effect of AT III alone on factor X activation. We found that the second order rate constant of inhibition of formed factor Xa was 2 x 10 5M-1min-1 , whereas that of factor Xa in free solution was 5 x 10 5M-1min-1 , indicating that phospholipid-bound factor X competes with AT III for factor Xa. The second order rate constant of inhibiton of factor IXa, either in the presence or absence of accessory components, was 8 x 103 M-1min-1. Unfractionated heparin (UFH; 168 USP units/mg) was found to stimulate the inhibition of generated factor Xa by AT III (200 nM) with 0.1 min-1 per nM of UFH, and a synthetic pentasaccharide (PS; 4000 anti-Xa units/mg) stimulated this inhibition with only 0.03 min-1per nM. Due to the presence of phospholipid-bound factor X this stimulation was 4-fold less when compared with factor Xa in free solution. At UFH concentrations higher than 3 nM, and PS concentrations exceeding 10 nM hardly any active factor Xa generation could be measured because of the rapid inactivation of factor Xa whereas factor IXa was not inhibited. Using a factor IXa assay we found that PS, even at relatively high concentrations, had no effect on factor IXa inactivation by AT III (200 nM), both in the presence and absence of accessory components. The inactivation of factor IXa by AT III (200 nM) during factor X activation was stimulated by UFH with 1.6 x 10 -2min-1 per nM of UFH. Surprisingly, this was 4-fold more when compared with factor IXa in the absence of accessory components. We established that calcium stimulates the heparin-dependent inhibition of factor IXa.
RELAXATIONS INDUCED BY LIPOXYGENASE METABOLITES OF ARACHI-DONIC ACID IN SPLENIC ARTERIES OF THE DOG
Authors: Van Diest M J , Verbeuren T J , Herman A G
Published: August 2018

The lipoxygenase metabolites of arachidonic acid, 15 hydroperoxyeicosatetraenoic acid (15HPETE) and its hydroxy derivative (15HETE) evoke contractions in a variety of isolated blood vessels. We recently were able to show that thromboxane A2 (TXA2)-receptor antagonists BM13177 and BM13505 suppress the contractions induced by lipoxygenase metabolites in canine splenic arteries, and that these compounds also inhibit the contractile effects of PGF2α and of the TXA2-mimetic U46619. We also reported that 15HETE and 15HPETE cause relaxations of isolated dog arteries when the tissues are contracted with prostaglandin F2α (PGF2α) or with U46619 and suggested that lipoxygenase metabolites may act as endogenous antagonists towards prostaglandin receptors. The present study was designed to investigate the effects of lipoxygenase metabolites of arachidonic acid in isolated splenic arteries in which the tone is raised by various agonists.

Segments of canine splenic arteries with or without endothelium were placed in organ chambers filled with Krebs-Ringer solution at 37°C for isometric tension recording. Responses to 15HPETE were obtained in segments which were contracted with serotonin, K+, PGF2α or noradrenaline (the latter with or without BM13505); concentrations causing comparable levels of contraction were selected. HPETE evoked relaxations in segments with and without endothelium, during the contractions evoked by K+, PGF2α and noradrenaline (with or without BM13505). At higher concentrations, 15HPETE caused relaxations only during contractions induced by PGF2α and noradrenaline with BM13505. Our results suggest that 15HPETE can cause relaxations by two different mechanisms :

(1) 15HPETE may act as an antagonist to PGF2α, which causes its contraction via the TXA2-receptor and,

(2) in presence of BM13505, it suppresses the contractions to noradrenaline via an endothelium independent mechanism.
CHARACTERIZATION OF THE FUNCTIONAL ADHESION PROPERTY OF THE MONOCYTE FIBRINOGEN RECEPTOR WITH A MONOCLONAL ANTIBODY (Mab) DIRECTED TO THE ACTIVATED STATE OF THE PLATELET GLYCOPROTEIN (GP) IIb/IIIa
Authors: Altieri Dario C , Bader Rossella , Mannucci Pier M
Published: August 2018

We recently showed that human blood monocytes bind fibrinogen through a genuine surface antigen only in part similar to the platelet GP Ilb/IIIa. Moreover, some anti-GP IIb/IIIa Mabs cross-react with monocytes. In this study we used the 7E3 Mab which preferentially binds to the activated conformation of the platelet GP IIb/IIIa to characterize the dynamic mechanism of "exposure" of the monocyte fibrinogen receptor. 7E3 Mab (25 μg/ml) completely suppressed the binding of i25I-fibrinogen to ADP (10μM)-stimulated monocytes. However, differently from the platelet GP IIb/IIIa, 125I-7E3 binding to unstimulated monocytes was a non-specific and non-saturable reaction. In contrast, after stimulation with ADP (10 μM), suspensions of human monocytes bound 125I-7E3 with saturation of 25-30 μg/ml of added Mab. Scatchard plot analysis was a single-affinity straight line revealing 97,400 binding sites/monocyte with a dissociation constant of 5.2×10−8 M. The monocyte surface antigen uniquely expressed after ADP-activation recognized by 7E3 was visualized by immunoprecipitation studies. Surface iodinated platelet lysate subjected to immunoprecipitation with 7E3 revealed a single band with molecular weight (Mr) of 116,000 corresponding to the platelet GP IIb/IIIa. In contrast, monocytes showed a dimeric surface antigen precipitated by 7E3 in two subunits with Mr=l55,000 and 95,000 respectively. These data indicate that the adhesion properties of the monocyte fibrinogen receptor defined by an anti-platelet GP IIb/IIIa cross-reacting Mab are structurally and functionally distinct from those of the platelet receptor.
SUSTAINED EXPRESSION OF FULL LENGTH AND VARIANT RECOMBINANT FACTOR VIII IN GENETICALLY ENGINEERED CELLS
Authors: Sarver Nava , Ricca George A
Published: August 2018

A major effort is presently underway to provide factor VIII (FVIII) in a form free of viral pathogens via a recombinant DNA approach. We have constructed two chimeric FVIII cDNA vectors based on the bovine papillomavirus mammalian expression system. The first vector (FVIII) contained a full length FVIII cDNA; the second vector (AFVIII) contained a cDNA insert with an extensive deletion, corresponding to amino acid residues 747 to 1560 in the region encoding the "B" domain. This internal region is removed during activation of the parental FVIII molecule and is believed not to be required for coagulant activity. We have found that recombinant FVIII produced by stable cell lines harboring either the full length or the variant FVIII was capable of restoring coagulant activity to FVIII deficient plasma in. vitro. This expressed activity was neutralized by anti-FVIII antibodies. Similar to observations with FVIII derived from human plasma, the two recombinant FVIII forms were (i) inactivated by the chelating agent EDTA, (ii) demonstrated a biphasic response of an initial activation followed by a decay in activity when treated with thrombin, and (iii) presented the expected peptide banding pattern by western blot analyses. A higher percentage of ΔFVIII transformants were isolated expressing coagulant activity compared to transformants harboring the complete FVIII cDNA. Among the positive transformants isolated, those harboring ΔFVIII produced higher levels of coagulant activity than their full length counterparts. Comparable steady state levels of FVIII specific transcripts were detected in FVIII and ΔFVIII transformants indicating that the difference in expression levels is due to a post transcriptional event(s). Our study demonstrates the efficacy of a full length and an abridged recombinant FVIII produced by stably transformed cells in correcting coagulation deficiency in. vitro. It further suggests the potential usefulness of other molecular variants for efficient expression in genetically engineered cells.
HOMOZYGOUS AND HETEROZYGOUS COMPLETE DELETIONS OF THE VON WILLEBRAND FACTOR GENE CODING REGION IN SEVERE VON WILLEBRAND DISEASE AND CARRIERS
Authors: Ngo K Y , Lynch D , Gitscher J , Ciavarella N , Ruggeri Z , Zimmerman T
Published: August 2018

Severe von Willebrand disease (vWD) is characterized by undetectable levels of von Willebrand factor (vWF), or trace amounts, in plasma and tissue stores. We have studied the genomic DNA of ten affected individuals from five families with this disorder using two cDNA probes. One probe extended from 175 base pairs of the 5’ untranslated region to the nucleotides encoding amino acid 618 of pro-vWF; the second extended from the nucleotides encoding amino acid 2225 of pro-vWF to 100 bp into the 3’ untranslated region. Three variants of the disorder were identified. Southern blots of restriction endonuclease digests and slot blots of undigested genomic DNA showed complete homozygous deletion of the vWF gene coding region in four affected siblings, three of whom had developed allo-antibodies. Gene dosage analysis performed with slot blots and laser densitometry were consistent with complete heterozygous deletions in both parents. The second variant was characterized by a complete heterozygous deletion of the vWF gene coding region in the propositus and one asymptomatic parent, suggesting that a different type of genetic abnormality was inherited from the other parent and that the patient was doubly heterozygous for distinct genetic abnormalities affecting vWF. In a third variant, no abnormalities could be detected. These techniques should prove useful in identifying carriers of severe vWD and also defining patients at risk of developing allo-antibodies to vWF.
CHANGES IN PLATELET SURVIVAL AND SIALIC ACID CONCENTRATION IN PLASMODIUM BERGEI INFECTED RATS
Authors: Essien E M , Inyang A L
Published: August 2018

Reduced circulating platelet count sometimes to thrombocytopenic levels in man and normally severe thrombocytopenia in animals are well known features of acute Plasmodium falciparum or experimental P. bergei infections in these respective organisms. Suggested mechanism(s), disseminated intravascular coagulation or immune mediated mechanism, thought to be involved in these observations are disputed. Shortened platelet survival has been reported in man.

We now present data on platelet survival and total platelet sialic acid concentration in P. bergei-infected Wistar rats. A total of 52 rats were used. For the platelet survival studies each of the 8 suckling test animals was infected by intraperi-toneal route with mouse-passaged P. bergei 4-5 days before inaction of Cr-labelled homologous rat platelets (50 μCi Na51 CrC4/rat) the platelets being obtained from adult Wistarrats. Blood samples were then collected 2 hr after the injection (zero hr sample) and subsequently at 17.0, 42.5 and 66 hr s.Platelet recovery and survival curves were determined on these samples. It was found dat fewer platelets (as % recovery) were obtained from each infected rat sample compared with control, the difference was significant in the 42.5 and 66 hr samples: 7.9 ± 8.1 (test) vs 41.4 ± 15.2% (C) for 42.5 hr and 2.8 ±4.1 (t) vs 26.8 ± 6.2% (C) for the 66 hr samples (p < 0.005 for each). For sialic acid determinations, 40 suckling Wistar rats (30 test, 10 control) were treated as for survival studies.At identical periods, blood was collected, washed platelets obtained, lysed and protein and total sialic acid determined by Lowry (1951) and Aminoff (1961) methods respectively. Total sialic acid of 7.02 ± 4.21 nM/mg protein at 42.5 hrs and 4.8 ± 2.14 at 66 hrs were significantly less than control value of 11.43 nm/mg protein and also showed a negative correlation (r = -0.95) with % parasitaemia.

It is concluded that P. bergei infection causes a reduction in total platelet sialic acid with resultant significant shortening of the platelet life span.
Enhancement by Activated Protein C of tPA-induced Lysis in a Cell-free System
Authors: Fredenburgh J C , Collen D , Nesheim M E
Published: August 2018

The profibrinolytic activity of human activated protein C (APC) was studied in a cell-free system using human plasma. Normal and Ba+* citrate adsorbed human plasmas were dialyzed against 150mM NaCl, 20mM Hepes, pH 7.4 and diluted to an A280 of 16. Reactions were initiated by the addition of aliquots of plasma to cuvettes containing human melanoma tPA and human thrombin at final concentrations of 1 and 30nM, respectively. The effects of Ca+* and varying concentrations of APC on clotlysis times were examined by monitoring turbidity at 600nM while maintaining the temperature at 37°C. The lysis time, defined as the midpoint of turbidity change, was 128 min for normal plasma containing 10 mM Ca+* and showed progressive and saturable shortening to about 90 min at > 50nM APC. In the absence of Ca+*, lysis time was 55 min for normal plasma and did not shorten in response to APC. With Ba+* citrate adsorbed plasma, the lysis time was 82 min in the presence of 10mM Ca+*, and shortened to 42 min without Ca+*. APC had no effect on lysis time in Ba+* adsorbed plasma either with or without Ca+*. Both bovine and human APC were equally potent. Electrophoresis in DodSO4 and autoradiography of plasma samples containing 125I-labelled plasminogen indicated enhanced rates of plasminogen activation in the presence of APC. These data indicate that APC decreases lysis time in vitro at the level of plasminogen activation. This effect is dependent on Ca+* and may involve additional vitamin K-dependent protein ( s).
INCREASED SECRETION OF A FIBRINOLYTIC INHIBITOR BY HUMAN MONONUCLEAR LEUKOCYTES PARALLELS THE PR0COAGULANT RESPONSE TO SPECIFIC ANTIGEN
Authors: Schwartz B S , Monroe M C
Published: August 2018

The presence of fibrin is a characteristic finding of Immune mediated tissue lesions. It is known that peripheral blood mononuclear cells (PBM) express tissue factor in response to recognition of a specific protein antigen. We have found PBM secrete a plasminogen activator (PA) Inhibitor (I) in parallel to expression of tissue factor upon exposure to a sensitizing antigen. Increased PA-I can be detected by Inhibition of urokinase (UK) in an 125I-fibrin plate assay, Inhibition of 125I-plasminogen cleavage, and formation of complexes between 125I-urokinase and UK-I. PA-I secretion is dose dependent, and antigen specific, i.e. a nonsensitizing antigen does not Induce a PA-I response.

The PA-I is secreted by monocytes, however the recognition of antigen is a T-cell function. Inhibition of PBM protein or RNA synthesis abrogates the PA-I response. The PA-I appears to be the type 2 Inhibitor, in that 1) it is a much more efficient Inhibitor of UK than of tissue type PA, 2) it is labile in acid and detergent, and 3) it is neutralized by IgG to human placental PA-I, but not by antiserum to PA-I of endothelial cells. It is concluded that PBM respond to a foreign stimulus by elaborating molecules that lead to both the deposition and persistence of ftbrln.
RELATION OP FIBRIN STIMULATION OF tPA MEDIATED PLASMINOGEN ACTIVATION AND FIBRIN BINDING TOWARDS FIBRONEKTIN AS REVEALED BY A MONOCLONAL ANTIBODY (MAB) AGAINST FCB-2 FIBRINOGEN FRAGMENTS
Authors: Meusburger S , Beckmann R , Wojta J , Binder B R
Published: August 2018

Fibrin binds to the finger domain of fibronektin via the C-terminal end of the chain and it was reported previously by us that in a fluid phase assay fibronektin inhibits fibrin enhancement of plasminogen activation by tPA. However, other have shown that tPA binds to fibronectin thereby possibly mediating enhanced matrix bound plasmin formation. In the present study we tried to further characterize the interaction between fibronectin and fibrin in regard to fibrin dependent enhancement of plasminogen activation by tPA. For fibrin binding to fibronectin we have developed an ELISA system using fibronectin coated plates and antibodies against fibrin(ogen) to quantify bound fibrin. For determination of plasminogen activation we used a coupled spectrophotometric fluid phase assay with Glu-plasminogen as substrate and H-D-Val-Leu-Lys-pNA to quantify the formed plasmin. Fibrin binding to coated fibronectin was linear between 500ng and 1 mg/ml for fibrin monomers (reptilase), FCB-2 fragments and thrombin (3.3 U/ml) treated fibrinogen, respectively. A monoclonal antibody directed against the FCB-2 fibrinogen fragment which also could be shown to recognize fibirn but not fibrinogen did not recognize fibronectin bound fibrin and inhibited also the fibrin stimulatory effect on plasminogen activation indicating that the epitope against which the antibody is directed is closely related to both the fibronectin binding site and the site involved in t-PA stimulation.
PLASMINOGEN ACTIVATOR INHIBITOR ACTIVITY AND ANTIGEN (PAI 1), RELATIONSHIP TO FIBRINOLYTIC RESPONSE AFTER VENOUS OCCLUSION IN 48 PATIENTS WITH DEEP VEIN THROMBOSIS
Authors: Nguyen G , Horellou M H , Conard J , Van Dreden P , Kruithof E K O , Samana M
Published: August 2018

Fibrinolytic parameters were studied before and after a 10 minute venous occlusion (VO) in 48 patients with confirmed deep vein thrombosis (DVT), at least 3 months after the last thrombotic episode. Congenital antithrombin III or protein C deficiencies were ruled out in these patients. The following tests were performed : euglobulin clot lysis time (ECLT), diluted whole blood lysis times (DWBLT), tissue plasminogen activator (t-PA) antigen (Biopool kit) and activity (fibrin plates), PAI activity (Verheijen et al.) and PAI i antigen (RIA, Kruithof et al.).

Patients were divided in 2 groups : group 1 (27 patients) with normal fibrinolytic response (ECLT and DWBLT less than 90 min. after VO), group 2 (21 patients) with defective fibrinolytic response at least twice with a few weeks time interval. In group 1, the mean t-PA antigen release after VO was 32.0 ± 27.2 ng/ml (post-occlusion value minus preocclusion value) and was associated with low or normal levels of basal PAI activity (7.1 ±3.1 IU/ml), and PAI 1 antigen (15.1 ±4.1 ng/ml, n = 7). These results could explain the good response to VO, and a PAI activity suppression after stasis (PAI activity undetectable in 20 out of 27 patients).

In contrast, in group 2, a low t-PA Ag increase after VO:10.5 ± 8.2 ng/ml as compared to group 1 (p <0.001) was associated with high levels of basal PAI activity : 22.4 ± 15.4 IU/ml (p <0.001), and PAI 1 Ag : 43.6 ± 24.9 ng/ml (n = 11, p <0.001). This association may account for the impaired fibrinolytic response aftep V0, and for persistant high PAI activity levels after stasis (17.6 ± 22.5 IU/ml). Before V0, PAI 1 Ag levels were in good correlation with PAI activity (r = 0.66, p <0.01), and , were significantly higher in group 2 as compared to group 1 (only 3 patients belonging to group 2 had normal PAI levels).

Since elevated PAI 1 antigen and PAI activity levels were associated with an abnormal fibrinolytic response to venous stasis, VO test could be restricted to patients with normal PAI levels, in order to detect hypofibrinolysis related to insufficient t-PA release.
ROLE OF MEMBRANE DEPOLARIZATION IN PLATELET ACTIVATION INDUCED BY THROMBIN
Authors: Yamaguchi A , Azuma H , Sekizaki S , Tanoue K , Yamazaki H
Published: August 2018

Though membrane depolarization is an imoportant step in signal transduction in living cells, it is immpossible to use microelectrode for analyses of the phenomenon in platelets due to the size limitation. Cetiedil, oc-cyclohexyl�3�thiophenacetic acid 2-(hexahydro-1H-azep in-1-yl) ester, is the drug which inhibits erythrocyte sickling by promoting net salt and water gain. It increases flux of both Na and K, and net Na gain exceeds net K loss. We used cetiedil as a tool to modify intracellular Na and K contents which are closely related to membrane potential.

Normal human platelets contained 23.5±3.7 nEq of Na and 82.7±5.6 nEq of K per 108 platelets measured by atomic absorption spectrophotometer (n=6, meantSE). When 0.5 µ/ml of thrombin was added, Na content increased to 200 % and K content decreased to 80% of the resting value after 15 sec. 22 Na spaces of platelets (0.3L±0.07 µl/l0° platelets in the resting stage, n=4) increased to 0.53±0.12 at 2 min after thrombin. After 5 min incubation with 50 µM cetiedil, Na contents increased and K contents decreased slightly. Under the cetiedil treatment, the increase in Na and decrease in K contents induced by thrombin were inhibited significantly. The increase in 22 Na spaces induced by thrombin was also inhibited. Thrombin-induced membrane depolarization measured by membrane potential probe dye, diS, also was inhibited by cetiedil. In the presence of 50 yM cetiedil, magnitude of depolarization was 27.6±1.0 mV (n=30). The depolarization was not affected by Na transport inhibitors, such as procaine and tetrodotoxine, but inhibited by low Na content in the suspension medium. Cetiedil also inhibited platelet aggregation and TXA2 synthesis by thrombin. However, the treatment did not induce any changes in platelet volume, their morphology under elecrton microscope, Ca content and LDH leakage. There is a concept that a small degree of depolarization can inhibit the stimulus-response coupling of various kinds of cells. A similar mechanism might be present in platelet membrane. A role of membrane depolarization in platelet activation is suggested
ROLE OF GUANINE NUCLEOTIDES IN Ca2+ - DEPENDENT LYSOSOMAL SECRETION FROM ELECTROPERMEABILISED PLATELETS
Authors: Athayde C M , Scrutton M C
Published: August 2018

Previous studies have shown that the maximal extent of Ca2+ dependent secretion of β-N-acetylglucosaminidase (B-N-AcGlc) from electropermeabilised human platelets can be enhanced by addition of thrombin or of 1-oleyl-2-acetylglycerol or 12-0-tetradecanoyl phorbol-13-acetate without a significant alteration in the EC^q for Ca2+. (Knight et al. Europ. J. Biochem.,143337 (1984)). We have found a similar Ca2+ dependent increase in the maximal extent of β-N-AcGlc and β-galactosidase secretion on addition of metabolically stable analogues of GTP (GTPγS and GppNHp) in the absence of thrombin or of GTP added in the presence of a nonsaturating concentration of thrombin. The EC50 values for GTP and GppNHp do not differ significantly for β-N-AcGlc and 3H-5HT secretion, but GTPγS is significantly more effective for 3H-5HT secretion.

The time course of β-N-AcGlc secretion induced by GTPγS shows a significant delay as compared with that induced by thrombin + Ca2+. No significant differences could be detected between the properties of β-N-AcGlc or β-galactosidase secretion in this system. The results are consistent with involvement of a GTP binding protein (Np) in receptor-phospholipase C coupling mediating lysosomal secretion, but provide no indication that an additional protein of this type (Ne) is involved as has been proposed for lysosomal secretion from neutrophils. We have thus far failed to find evidence for heterogeneity in lysosomal secretion in this system (supported by SERC and Ciba-Geigy).
AGGREGATES OF RED BLOOD CELLS, AND AGGREGATES OF PLATELETS UNDER ZERO GRAVITY: EXPERIMENT ON NASA SPACE SHUTTLE "DISCOVERY" STS 51-C, JANUARY l985
Authors: Dintenfass L
Published: August 2018

The aim of experiment "ARC" on the space shuttle "Discovery STS 51-C, was to define effect of zero gravity on kinetics and morphology of aggregation of red cells in blood obtained from patients suffering from ischaemic heart disease, colon cancer, insulin-dependent diabetes, hyperlipidaemia, IgG and IgM papa-proteins. Space-rated automated slit-capillary photo-viscometer contained a motorized infusion pump capable of handling eight different blood samples. Two cameras and a microscope allowed micro and macrophotography, and total of 500 photographs was obtained in space; and equivalent number on the ground, in the Kennedy Space Center, where a duplicate ground photo-viscometer was present. Identical blood samples have been used in the ground experiments. The slit had a gap of 12.5 microns (micrometers). Blood was anticoagulated with EDTA and adjusted to haematocrit of 0.30 using native plasma. Samples were kept at -5°C prior to the experiment, and at 25°C during experiment; duration of experiment was 91/2 hours. The same computer program was used in both instruments. Photography was carried out at set intervals up to six minutes from the moment of stasis. There was a drastic difference between aggregation on the ground and at zero gravity. Blood from patients was greatly sludged on the ground, but normal rouleaux were formed under zero gravity. Also, aggregates uikder zero g were much smaller. However, red cell shape was not changed. Blood samples from normal donors, which showed normal rouleaux on the ground, exhibited random swarm pattern under zero gravity. Platelets, which tended to aggregate on the ground, and tended to accummulate at the slit entrance, remained monodisperse under zero gravity and no pseudopodia have been noted; under zero g platelet moved through the slit. Subject to future confirmation, it is suggested that zero gravity affects cell-to-cell interaction, and probably causes a modification of the cell membrane. If this is true, a new vista opens in the studies of immunology and oncology under zero gravity.
REFRACTORINESS OF PLATELETS AFTER REVERSIBLE STIMULATION BY ADP, PAF AND AA
Authors: Voss R , Ohanes H D , Ditter H , Matthias F R
Published: August 2018

It has been observed that platelets which have been, stimulated by thrombin, ADP or platelet-activating factor (PAF) show an inhibited response to a subsequent stimulation by the same agonist. We performed a crossover stimulation of human platelets (PRP) with the four agonists ADP, PAF, arachidonic acid(AA) and collagen (COL); after a first stimulation with a dose giving a reversible aggregation (0,1-0,4×10-6M ADP, 0,1-0,5×10-8 M PAF) or a shape change (0,05-0, 1mM AA., 0,4 ug/ml COL) the platelets were again stimulated after 5 min with the same or a higher dose, usuallyresulting in an irK8
reversible aggregation (1,0-3,0×10-6 M ADP, 1,0-5,0×10-8 M PAF, 0,3-0,5mM AA, 1,0 ug/ml COL).

ADP-, PAF- and AA-stimulation were inhibited by a prestimulation with the same agonist (ADP/ADP 60% inhibition, PAF/PAF 100% inhibition, AA/AA 10% inhibition) ; ADP and PAF did not or only slightly inhibit each other. Prestimulation with AA changed a reversible ADP- or PAF-induced aggregation into an irreversible one, and a reversible AA-stimulation was increased to irreversible by ADP-prestimulation. COL-aggregation was not influenced by any prestimulation nor did a COL-presti-mulation influence the second stimulation by any of the four agonists.

The refractoriness of platelets for ADP and PAF after prestimulation with the same agonist may be explained by receptor internalization; in the case of ADP the effect lasted for at least one hour. The enhancement of ADP- and PAF-aggregation by an AA-prestimulation can be explained by other investigations which have shown that the arachidonic acid pathway acts as a positive feedback mechanism in platelets.