bluuebunny/crossref_metadata_2025 huggingface dataset:
PREOPERATIVE IDENTIFICATION OF PATIENTS AT HIGH RISK OF DEEP VENOUS THROMBOSIS DESPITE PROPHYLAXIS IN TOTAL HIP REPLACEMENT
Authors: Rocha E , Práamo J A , Alfaro M J , Cuesta B , Fernádez J , Herández M
Published: August 2018

Preoperative prediction of postoperativevenous thrombosis was investigated in 111 patients undergoing total hip replacement prophylactically treated with aspirin (1 g/d) or a combination of heparin (5000 IU) plus dihydroergotamine (0.5 mg) twice a day during 7 days. The followingpreoperative parameters were determined:age, sex, overweight percentage, previous thromboembolism, varicose veins,heartdisease, malignancy, plateletcount, platelet-crit, mean platelet volume, circulating platelet aggregates, platelet factor 4, β-thromboglobulin, fibrinogen, Factor Xa, VIII:C, AT III, fibrin monomers, FDP,euglobulin lysis time, α2-antiplasmin, tissue-type plasminogen activator (t-PA) and its inhibitor (PAI). Postoperatively deep vein thrombosis (DVT) developed in 16patients was detected by ascending venography. Stepwise logistic discriminant analysis was used to identifyfactors which predicted DVT. Three such factors, FDP, PAI and t-PA, were significantly associated with DVT and used to construct a predictive index. The predictiveindex, I = 2.09 + 0.46 (FDP) + 1.39 (PAI) - 0.24 (t-PA), was 100% sensitive and 95% specific in the prediction of DVT. This index could allow for identification of those patients in whom routine prophylaxis would be sufficient and forselecting those in whom more effective prophylactic regimens would be necessary.
VENODILATION AND DEVELOPMENT OF DEEP VEIN THROMBOSIS IN TOTAL HIP AND KNEE REPLACEMENT PATIENTS
Authors: Stewart G J , Lachman J W , Alburger P D , Ziskin M C , Philips C M , Jensen K
Published: August 2018

Postoperative deep vein thromboisis(DVT) is a frequent complication following total hip (THR) or knee (TKR)replacement but no test has been devised to identify specific patientswho will develop DVT. Because conventional prophylaxis does not significantly reduce the incidence of DVT, monitoring is widely used to detect evolving thrombosis. More intense anticoagulation (adjusted dose heparin,two step warfarin) may be effective but requires laboratory tests and carries increased risk of bleeding. Itwould be an economic and medical advantage to restrict prophylaxis and monitoring to patients who will develop DVT. Based on observations in a canine model of THR, we developed andtested a method that shows promise of being able toidentify, intraoperatively, patients who will develop DVT.

In the canine model we found characteristic venous lesions (gaping tears through endothelium and basementmembrane, localized to confluences,selectively infiltrated with platelets and leukocytes). Incidence of lesions correlated with intraoperative venodilation, measured by a modified ultrasound scanner. Lesionsmight serve as sites for initiationand anchorage of thrombi. Diagnostic ultrasound was used to monitor cephalic vein diameter in 25 THR patients and 12 TKR patients. In THR patients, 1 of 9 patients with venodilation of 6-16% developed DVT. At 21-57% venodilation 12 of 12 THR patients developed thrombi. In the intermediate range of venodilation (19%,20%), 2 of 4 patients developedDVT. In 12 TKR patients, 10 had venodilation of 0-16% and none developedDVT in the non-operated leg. In patients with 22% and 55% venodilation,one did and one did not develop DVT in the non-operated leg (expectedfrcxn equal distribution between legs in THR patients).DVT in the operated leg did not correlate with venodilation. We suggest that in THR patients substances released at the operative site circulate briefly, causing venous dilation. In TKR patients the tourniquet prevented substances from being circulated, reducing venodilation and DVT in the non-operated leg. Proximitv of surgicalwound to calf veins and tourniquet pressure mav have contributed to DVT in the operated leg.
ACTIVATION DEPENDENT ALTERATIONS IN THE CHEMICAL CROSSLINKING OF ARGINYL-GLYCYL-ASPARTIC ACID (RGD) PEPTIDES WITH PLATELET GLYCOPROTEIN (GP) GPIIb-IIIa
Authors: D’Souza S E , Ginaberg M H , Lam S , Plow E A
Published: August 2018

The platelet adhesive proteins, fibrinogen, fibronectin and von WillebrandFactor, contain RGD amino acid sequences; RGD-containing peptides inhibit the binding of these adhesive proteins to platelets; and a membrane receptor for these adhesive proteins binds to Arg-Gly-Asp and contains GPIIb-IIIa. The present study was undertaken to characterize the interaction of RGDpeptides with GPIIb-IIIa using a chemical crosslinking approach. A radioiodinated RGD-containing heptapeptide was bound to washed human platelets under conditions at which ≥ 85% of theinteraction was inhibited by excess nonlabeled peptide. After binding of the peptide to platelets for 45 min at22°, a homobifunctional crosslinking reagent was added, and the platelets were extracted and analyzed on polyacrylamide gels. With resting platelets,autoradiography of the gels revealedthat the peptide crosslinked tobothGPIIb and GPIIIa. This interaction wasinhibited by excess nonlabeled peptide but not by certain conservatively substituted RGD peptides. Stimulation of the platelets caused a dramatic increase in crosslinking of the peptide to only one of the two subunitsof GPIIb-IIIa. The stimulus dependentincrease in the crosslinking reactionwas specific and saturable as it was inhibited by RGD peptides in a dose dependent manner. In addition, peptides corresponding in structure to the carboxy terminus of the γ chain of fibrinogen also produced concentration dependent inhibition of the interaction. The increase in crosslinking induced by platelet stimulation was divalent ion dependent. Similar results werealso obtained with a second, larger RGD-containing peptide and with asecond chemical crosslinking reagent.Theseresults indicate that platelet stimulation in the presence of divalent ions causes a change which permitsmoreefficient crosslinking of RGD-containing peptides to only one of the two subunits of GPIIb-IIIa. The results are also compatible with a proximalrelationship of both subunits tothe RGD binding sites on the plateletmembrane.
PRODUCTION OF HUMAN MONOCLONAL ANTIBODIESSPECIFIC FOR PLATELET MEMBRANE GLYCOPROTEIN IIIa
Authors: Furihata Kenichi , Nugent Diane J , Bissonette Amy L , Vokac Elizabeth , Kunicki Thomas J
Published: August 2018

Human monoclonal antibodies specific for platelet membrane glycoproteins (GPs) arepotentially important reagentsfor studies of the immunogenicity of membrane glycoproteins. A human monoclonalautoantibody, 5E5, reactive with plateletGPIIIa has been developed (Nugent, et al.,Blood, 1987, in press). In this report, we describe the production of additional human monoclonal antibodies specific for GPIIIa. Peripheral blood lymphocytes fromone patient with post-transfusion purpur(PTP) and one woman who had delivered an infant with neonatal alloimmune thrombocytopenic purpura (NATP) were used as a source of antigen-specific lymphocytes. A B-lympho-cyte-enriched population was transformed with Epstein Barr virus, strain B95-8, and cultured in microtiter plates. After two weeks, culture supernatants were screened by an antigen-capture ELISA wherein murine monoclonal antibody specificfor the GPIIb-IIIa complex was used to holdcorresponding antigen from a lysate ofnormal platelets. B-lymphoblastoid cell lines producing IgG and/or IgM antibodies were expanded and either cloned by limiting dilution technqiue or hybridized with a HAT-sensitive, ouabain-resistant heterohybrid fusion line, F6, using polyethylene glycol. Hybridomas were selected in medium containing HAT andouabain. After twoweeks, hybridomas producing anti-GPIIb and/or anti-GPIIIa antibody were cloned by limiting dilution. Culture supernatants from cloned B-lymphoblastoid cell lines and cloned hybridomas were rescreened by ELISA wherein affinity-purified GPIIIa or other platelet GP weredirectly conjugatedto microtiter plates. One IgM antibody produced by acloned B-lymphoblastoid cellline (CH16) andtwo IgG antibodies produced bycloned hybridomas(Del5.19 and Del5.23) were shownto react with GPIIIa but not other GP. Further characterization of these human monoclonal antibodies, produced continuouslyin culture now for four months, is in progress.
UNSTABLE ANGINA : DEFINITION
Authors: Julian D G
Published: August 2018

A satisfactory definition of unstable angina continues to be elusive.Like stable angina, it is a clinicalsyndrome, and mustbe defined in those terms. In both cases, it is understood that myocardial ischaemiabut not infarction is responsible for the symptoms. For angina to earn the “unstable” label, theremust have been the recent development or deterioration of symptoms. Traditionally, “recent” has meant within the last month, but it has become increasingly clear thatthe time frame is of critical importance - the patient with a sudden irruption of severe chest pain in the last two days is likely to have a different pathology and prognosis from the individual who first developed exercise-induced pain two weeks ago, which has not worsened during this period. Likewise, the patient who hadhis last attack of pain one week agois very different from one who had his last attack one hour ago; indeed in the HINT study, there was a high incidence of myocardial infarction undetected on admission in the lattergroup (Br Heart J 1986;56:400-13).

A problem that has hardly been addressed is “When does unstable angina stop being unstable?”. The answer must be when it either becomes stable (i.e. stops getting worse),or proceeds to myocardial infarctionor death.

Most studies have shown that the vast majority of patients stablise quickly, often losing their symptoms completely. There is, however, a group of patients who fail to respond promptly to medical treatment, and it isthese patients who are most likely to go on to angiography, angioscopy, angioplasty, surgery, myocardial infarction or death. Thus, the enormously valuable information we have obtained from, for example, angiography and necropsy studies applies essentially only to this subset of unstableangina patients, albeit they are themost severe. Unfortunately, such studies tell us relatively little aboutthe more dynamic aspects of the disorder, such as the role of coronary vasomotion.It is only relatively recently that clinicians have appreciated that they have quite simple means of suspecting the mechanisms involved. Thus, the history of progressive exercise-induced angina on the one hand or angina only at rest on theother must tell us something of the underlying physiopathology. Likewise,the fact that the symptoms respond to rest, or beta-blockers, or calcium antagonists also provides evidenceas to causation.

Thus, while all would agree that angina is “unstable” whenit has recently developed or worsened, it must bediscussed in subsetsdefined by their history and response to treatment.In this way, we may be able to assign a patient to a particular clinicalgroup, which will indicate that it is likely that he has a particular physiopathology, which inturn will suggest the most appropriate management.
EDRF AND RESISTANCE VESSELS
Authors: Griffith T M , Edwards D H , Davies R L , Harrison T J , Evans K L
Published: August 2018

The influence of endothelium on vasomotionin resistance vessels was studied in an isolated, buffer-perfused rabbit ear preparation using novel microangiographic techniques and haemoglobin as a specific inhibitor of EDRF. In constricted preparations acetylcholine and Substance P, whose action is EDRF-dependent in large vessels, induceddilatation in vessels down to 25um in diameter which was inhibited by haemoglobin. Log (IC50>q) values calculated from diameter changes were the same in the central artery, GO, and its first three generations of branch vessels Gl, G2 and G3 (ie down to 7Oum) being -7.7 and -9.8 respectively. Consistent with this, almost identical valueswere derived from the pressure responses of the intact network. Such spatial homogeneity has not been found when the same vessels are studied in isolation. In contrast,constrictor responses to 5HT or histamine exhibited spatial heterogeneity (in the rank order G0>G1>G2>G3) which was exaggerated by inhibition of basal EDRF activity. Inerms of normalised diameter changes EDRF and its analogue GTN were equipotent in reversing these constrictor responses in GO to G3. Interms of hydraulic resistance however, dilator responses paralleled relative changesn resistance induced by the constrictor agents. EDRF and GTN thus appear more potentin vessels exhibiting high degrees of tone.

In control preparations (i.e. in the absence of pharmacological constriction) basal EDRF activity was found to exert maximal influence in vessels in which calculatedshear stress and hydraulic resistance werehighest, and continuously inhibited myogenic tope in Gl and to a lesser extent G2 and G3. In the absence of haemoglobin, diameter and flow were related by the expression QÒaD4+b in GO, Gl and G2 over an 8-fold range of flow rates. In the high-flow limit this implies constancyof pressure gradient and pressure drop inthese vessels so that the energy expended in delivering a given volume of perfusate to the terminal elements of the bed would be effectively independent of flow rate, rather than directly proportional to it as in a rigid tube.Basal EDRF activity also conferred identical flow-related distensibility in G0 through G3 in control and partially constricted preparations over the samerange of flow rates. This implies independence of flow distribution from flowrate. Flow dependent release of EDRF may provide a mechanism which links network topography with vessel function, thus optimising perfusion characteristics.
RECOGNITION SPECIFICITY OFADHESION RECEPTORS
Authors: Plow E F , Marguerie G A , Ginsberg M H
Published: August 2018

The cytoadhesins are a broadly distributedfamily of structurally, immunologicallyand functionally related cell surface molecules which participate in cellular adhesivereactions. The members of this family are heterodimeric in structure and consist of similar, if not identical, beta subunitsand related, but not identical, alpha subunits. A common functional property of the cytoadhesins is that they possess an arginyl-glycyl-aspartic acid (RGD) recognition specificity which mediates their capacity to act as receptors for adhesive proteins.The above assigned characteristics of thecytoadhesin family are based upon immunochemical analyses, primary structural determinations, deduced amino acid sequences fromcDNA clones and from dissectionof the recognition specificity of specificfamily members for their adhesive proteins.Platelet GPIIb-IIIa, the prototype of thecytoadhesin family, exhibits a very relaxed recognition specificity which permits itto interact with at least four distinct RGD-containing proteins: fibrinogen (Fg), fibronectin, von Willebrand Factor and vitronectin. RGD-containing peptidesas well as peptides corresponding in structure to the extreme carboxyl terminus of the gamma chain of Fg inhibit the binding of this adhesive protein set to GPIIb-IIIa.Both peptide sets interact with GPIIb-IIIaat either the same set of sites or intercommunicating sites. Nevertheless, using crosslinking reagents, certain RGD and gamma chain peptides can be crosslinked to different subunits of GPIIb-IIIa. This raises the possibility that a binding pocket for the adhesive proteins on GPIIb-IIIa may be directly comprised or situated in close proximity to both subunits of the cytoadhesin. Endothelial cells also express a cytoadhesin which is capable of binding Fg. Although both RGD and gamma chain peptides inhibit this interaction, the finerecognition specificity of the platelet and endothelial cell cytoadhesin for Fg are distinguishable. Thus, by combining very similar if not the same beta subunits with different alpha subunits, the RGD specificity of the cytoadhesins may bemodulated to achieve selectivity in the recognition of adhesive proteins.
ROLE OF BIOLOGICAL RESPONSE MODIFIERSIN THE REGULATION OF THROMBOPLASTIN SYNTHESIS IN MONOCYTES AND ENDOTHELIAL CELLS
Authors: Carlsen E , Prydz H
Published: August 2018

A rabbit polyclonal antibody which was monospecific for thromboplastin (TP) apoprotein from human brain according to a number of criteria was used to screen two human placenta cDNA libraries in the expression vector Agtll, one randomly primed and one oligo-dT primed, by the method of Young and Davis. 23 positive clones expressing TP related antigen were isolated and plaque purified. DNA from the different clones was isolated and the TPcDNA inserts released by EcoRl digestion. The inserts could be classified into11 size classes ranging from approx. 300-1100 base pairs.The largest insert (1100 bp) was subcloned intothe plasmid vector pGEM-1. When the nick-translated plasmid (pTP4-l) was used as a probe to screen the phage clones by slot blot hybridization all the 23clones hybridized to the 1100 bp insert. A XgtllTP4 lysogen expressed B-galactosidase- TP4 fusion peptide upon IPTG induction as shown by immunobinging studied using two different antibodies to TP apoprotein: the rabbit antibody originally used to screen the libraries and an antibody raised in goat against human brain TP purified by affinity chromatography on a Factor Vll-antiVII-agarose column.Cytokines mediate many of the cellular interactions in the inflammatory and immune response systems and have a variety of actions. We have investigated the effect of rIL-1a/$,rIL-2, rlFNa/y and rTNFa on thromboplastin synthesis (TPL) in monocytes (M) and human umbilical vein endothelial cells (HUVEC). Recombinant IL- 1a and IL-16 both induced a dose dependent increase in TPL activity of monocyte (8-fold) and HUVEC cultures (15-fold) at 6h. The increase levelled off at interleukinconcentrations of 50-100u/ml. Recombinant IL-2 at 50u/ml induced a 5-fold rise in monocytes TPL. The effect of rIL-2 on HUVEC TPL synthesis at 6 h was smaller than on monocytes but still clearly significant at dose dependent. Recombinant IFN-Y (10 -10 u/ml) increased.TPL activity in HUVEC at 6h and 16 h in adose dependent manner, whereas no effect of rIFN-Y and IFN-a (1-10 u/ml) on M TPL was seen. When LPS (5pg/ml) was used to induce TPL synthesis, additional stimulation with rIFN-Y further enhanced HUVEC TPL activity, but decreased M TPL activity. Recombinant IFNa also decreased LPS induced TPL synthesis in M andhad no effect on HUVEC TPL. Recombinant TNFa (0.3x104u/ml) increased HUVEC TPL 7-fold at 6h. There wasno effect on M TPL synthesis. No endotoxin was detected in any of these preparations.
CONCLUSIONS: Some biological response modifiers induced thromboplastin synthesis in monocytes (IL-ia, IL-13, IL-2) and in human umbilical vein endothelial cells (IL-ia, IL-18, IL-2, TNFa and IFNY). Some had no direct effect on TPL synthesis but inhibited the response to monocytes to other thromboplastin-inducing agents like LPS (IFNa and IFNY).
GLA-CONTAINING PROTEINS FROM CALCIFIED HUMAN ATHEROSCLEROTIC PLAQUES
Authors: Van Haarlem L J M , Hemker H C , Soute B A M , Vermeer C
Published: August 2018

Vitamin K-dependent carboxylase activity has been detected in human andbovine vessel wall. Studies comparingthe carboxylases from liver and vessel wall revealed that the enzyme systems may be regarded as isoenzymes withwidely different substrate specificities. The carboxylated product of vessel wall carboxylase has not yet been identified, but it seems plausible that it will be found amongst the Gla-containing proteins which are abundantly present in calcified atherosclerotic plaques (Gla= gammacarboxyglutamicacid, the abnormal amino acid formed by vitamin K-dependent carboxylase). Therefore we have started to characterize the protein constituents of hardened atherosclerotic plaques.

The calcified areas from human aortae were solubilized in EDTA and the proteins extracted were partly purified by batch-wise adsorption onto QAE and elution with high salt. The crudeplaque-extract did not contain prothrombin, factor X or protein C. This excludes the possibility that Gla-containing coagulation factors are bound non-specifically from blood. Osteocalcin accounted for 20% of the total amount of protein-bound Gla-residues.

Another Gla-containing protein waspurified from the crude plaque-extract by employing high performance liquid chromatography (HPLC). Gel filtration yielded a Gla-rich protein with anapparent Mr of 25 kD. In vitro boththe crude plaque-extract and the purified Gla-containing protein strongly inhibited the precipitation of calcium phosphate and calcium carbonate. A similar effect was not found with humanserum albumin nor with a thermallydecarboxylated plaque-extract. If also in vivo the Gla-containing proteinsproduced by vessel wall carboxylase prevent the precipitation of calcium salts remains to be investigated.
HIGH AND VERY HIGH DENSITY LIPOPROTEINS ADMINISTRATION INHIBITS PROGRESSION OF EXPERIMENTAL ATHEROSCLEROSIS IN THE RABBIT
Authors: Badimon J , Bodimon L , Fuster V
Published: August 2018

Epidemiologic studies have shown an inverse relationship between HDL and coronary artery disease. We have previously demonstrated that in vivo administration of HDL-VHDL inhibits thedevelopment of atherosclerosis in cholesterol (cho)-fed rabbits. In the present study we have analyzed whether high levels of the physiological cholesterol acceptor, homologous HDL-VHDL,could inhibit the progression of established atherosclerotic lesions. Atherosclerosis was induced by feeding rabbits a 0.5% cho-rich diet for 2 months (140g/day). At that moment, a subgroup of animal (N=4) was sacrificed and their aortas showed 30 ± 8% of aortic atherosclerotic involvement. The remaining animals, kept on the same atherogenic diet, were randomly divided in two identical groups (N=7): a control and a treated group administered with 50 mg of HDL-VHDL a week for 4 weeks. HDL-VHDL fraction wasisolated from normal rabbit plasma byultracentrifugation at a density range of 1.063-1.25g/ml. The amount of HDL-VHDL administered was determined byits protein content according to Lowry"s technique. The 50mg of HDL-VHDL, measured as protein, contained1.4mg of total cholesterol, 1.43mg oftriglycerides and 0.6mg of phospholipids. At sacrifice, the treated group showed a marked decrease on the extent of aortic by fatty streaks (20 ± 6%X ± 1SE) as compared to(36% + 6) inthe control group (p < 0.05). Similar results were obtained in aortic wall lipid accumulation (See table, results expressed as X±1SEM; rag/gr dry aorta.)

In conclusion, administration of HDL-VHDL induced a marked inhibition on the progression of atherosclerosis in cholesterol-fed rabbits.
MONOCYTE-MACROPHAGE PROCOAGULANT ACTIVITY AND ENDOTHELIAL THROMBOMODULIN IN RABBITS FED AN ATHEROGENIC DIET
Authors: Montemuro P , Pasquetto N , Curci E , Colucci M , Semeraro A
Published: August 2018

Mononuclear phagocytes (M) and vascular cellsmay participate in the events that lead tothe development of atherosclerotic lesions. We have studied the procoagulant activity (PCA) of M and thrombomodulin (TM)-like activityofendothelial cells in 15 rabbits fed an atherogenic diet for 4-5 weeks and in 15 rabbits fed a standard diet. Peripheral blood and spleen M were tested for PCA immediately after isolation (basal PCA) and following in vitro stimulation by bacterial endotoxin, using a one-stage clotting assay. TM-like activity was measured by the rate of (bovine) protein C activation induced by catalytic concentrations of thrombin in the presence of aortic rings (1cm long) and CaCl2 Blood M expressed negligible basal PCA (< 1 U/105 M) both in hyperlipaemic and controlrabbits. Endotoxin-induced PCA was not significantly different in the two groups. In contrast, dietary treatment resulted in a significant increase in the basal PCA of spleen M (67.6 ± 13.5 vs 26.5 ± 5.4 U/105 M, pcO.Ol). Moreover, spleen M fromtreated animals produced significantly more PCA than controls (p<0.01) in response to endotoxin. When rabbits were given a single injection of endotoxin, spleen M harvested 60 min after the injection from hyperlipaemicanimals expressed 3 times more PCA (p<0.05, n=6) than did cells from controls. In all instances PCA was identified as tissue factor. TM activity associated with the endothelium was not different in the two groups of animals notwithstanding the presence of fatty streaks on the aortic endothelium of treated rabbits. It is suggested that dietary fats maycause early functional changes in M that leadto increased PCA production both in vivo and in vitro.These data may be relevant to an understanding of the role of M in the pathogenesis of atherosclerosis.
HEPARIN STIMULATES FIBROBLAST GROWTH INDUCED BY PDGF
Authors: Dupuy E , Rohrlich P S , Tobelem G
Published: August 2018

Heparin binds to smooth muscle cells and endothelial cells. It inhibits the proliferation of the smooth muscle cells and modulates the growth of endothelial cells. Fibroblasts which represent an other cell type belonging to the vascular wall could also have their growth modified by heparin. We have at first, demonstrated that 125I unfractionated heparin bigds to cultured human skin fibroblasts with a Kd of 1.16 10 M.A low molecular weight heparin fraction (PK 10169) competed (50 %)with I unfractionated heparin, but at aless extent than cold unfractionated heparin(90%).As it has been reported with endothelial and smooth muscle cells, about 30% of the bound unfractionated heparin was internalized bythe fibroblasts. Heparin alone at the concentration ranges from 0 to 10-5M has no effect on fibroblast proliferation measured by the H thymidine uptake. When the cellproliferation was induced by pure PDGF, heparin potentiated markedly the fibroblast growth.The effgct started at 10-8 M heparinand reached a plateau from 10-6 M to 10-5 M. Similar stimulationwas observed when the growth was induced by FGF or EGF. Low molecular weight heparin enhanced the fibroblast proliferation induced by PDGF but at a less extent than unfractionated heparin, chondroltin sulfate has no effect. When added during the cell culture growth withhuman serum (5%), unfractionated heparin increased by 48 the cell proliferation as measured bycell counting at the 6th day of the culture. PDGF did not modify the heparin binding on fibroblast cultures either at 4°C or 37°C and did not alter the process of heparin internalization. JDGF binding to the cultured fibroblast (Kd 10.1 ± 3.4 10-10 M)was not modified by the presence of heparin when studied at 4°C.

In conclusion : i) cultured human fibroblasts bind and internalize heparin, ii) heparinand heparin fraction stimulate the fibroblastgrowth induced by PDGF, iii) since the binding of PDGF is not modified by bound heparin, the mechanism of stimulation remains unknown.
PLATELET-DERIVED GROWTH FACTORA-CHAIN GENE ACTIVATION AND GROWTH FACTOR PRODUCTION BY HUMAN AORTIC SMOOTH MUSCLE CELLS
Authors: Kanthou C , Parker C , Huber D E , Stroobant P , Kakkar V V , Pringle N , Richardson W
Published: August 2018

The many contributory factors leading to the development of cardiovascular disease are currently thought to induce a common pathological change involving smooth muscle cells, which migrate from the vessel wall, proliferate,accumulate at the sites of endothelial cell damage, and then secrete connective tissue proteins and lipids which contribute to the plaque which results in the occlusion of the vessel. According to the recently modified hypothesis of Ross (1), a key event in the development of atheroma may be the abnormal release of a number of growth modulatory polypeptides,including platelet-derived growth factor (PDGF), which can potentially originate from platelets, endothelial cells, monocytes or macrophages, and smooth muscle cells themselves.

We have isolated smooth muscle cell lines from 25 samples of human aorta, using digestion with collagenase and elastase. With DNA synthesis and Northern blot techniques, we examined them for both the production of PDGF-like proteins, and for the possible activation of the PDGF A-chain and B-chain genes. Severallines secreted a growth factor and were stillviable after culture for 57 days in serum-free medium. Parallel experiments using Northernblot analysis revealed the activation of the PDGF A-chain gene in all lines examined with no detectable B-chain gene transcripts.

These data raise the possibility that vascular damage may activate the gene encoding the A-chain of PDGF in adjacent smooth muscle cells. Such cells might then become capable ofautonomous growth, in an analogous manner tocells transformed by Simian Sarcoma Virus, whose sis oncogene encodes the B-chain of PDGF.
A SPECIFIC THROMBOXANE A2 ANTAGONIST, GR32191, REDUCES PLATELET DEPOSITION IN PTFE GRAFTS
Authors: McCollum C N , Harper R A , Lane I F , Meek A C
Published: August 2018

Platelet inhibitory therapy improves patency in arterial grafts but when aspirin is given over 20% of patients discontinue therapy. We evaluated a specific Thromboxane A2 antagonist (GR32191 - Glaxo Group Research) on graft platelet uptake and pseudo-intimal hyperplasia in a canine model.

Thirty greyhounds were randomised to orally administered placebo, GR32191 25mg, or aspirin150mg (ASA) plus dipyridamole 50mg (DPM) 12 hourly, commencing 48 hours prior to implantation of a 6cm length of 6mm-PTFE in the femoral artery. Autologous 111In-platelete were infused on the fifth postoperative day and platelet uptake measured by probe and ratemeter with the daily rise in graft radioactivity over reference expressed as Thrombogenicity Index (TI). Drugs were continued to 8 weeks when 111In-platelets were again infused and graft uptake measured on the excised graft and expressed as a ratio to blood. Percentage luminal stenosis was measured by grid microscopy.

GR32191 significantly reduced luminal stenosis and graft platelet uptake compared to placebo and although TI appeared lower with both platelet inhibitory regimens this did not achieve statistical significance (p<0.1>0.05). Thromboxane A2 antagonists reducethrombus formation on artificial surfaces andbeing specific may have less undesirable effects.
IRREVERSIBLE BLOCKADE OF THE THROMBOXANE A2/PROSTAGLANDIN H2 RECEPTOR OF HUMAN PLATELETS BY AZIDO-BSP
Authors: Zehender H , Witte E C , Stegemeier K , Patscheke A
Published: August 2018

Azido-BSP (4-[2-(4-azido-benzenesulphonylamino)-ethyl]phen-oxyacetic acid) is a photolabile derivative of the competitive thromboxane A2 /prostaglandin H2 (TXA2/PGH2) receptor antagonist sulotroban (=BM 13.177). If protected from short wave light, azido-BSP reversibly inhibited the platelet shape change induced by the PGH2 analogue U 46619 but notthe shape change induced by ADP or PAF. Schild analysis revealed an apparent KD=0.2 μM with washed platelets. The irreversible inhibition requiredirradiation of the platelet suspensionwith UVlight (254 nm) for 5 minutes in the presenceof azido-BSP. After this treatment,the platelets were washed twice and used forplatelet function tests. Treatment with 0.5 μM of azido-BSP suppressed the U 46619(10 μM)-induced (3H)serotonin release and 1 μM of azido-BSP was necessary to block the U 46619(2 μM)-inducedaggregation.The platelet shape change induced by U 46619 (0.01μM) was only partially inhibited, even at very high concentrations (50μM) of the antagonist.This suggests that a small portion of the TXA2/PGH2 receptors could not be blocked bythe photoaffinity treatment with azido-BSP. After treatment with 1 μM azido-BSP, the shape change stimulated by ADP or PAF was not reduced. This indicates a high specificity of thephotoaffinity ligand for the TXA2/PGH2 receptor. It is concluded that UV irradiation of azido-BSP generates anitrene intermediate that covalently links to the majority of the TXA2/PGH2 receptors. Azido-BSP provides a specific tool for tagging and subsequent purification of the TXA2/PGH2 receptor of platelets.(Supported by the Deutsche Forschungsgemeinschaft, Grant Pa263).
THE DIHYDROPYRIDINE CALCIUM CHANNEL AGONIST, BAY K 8644, AND THE ANTAGONIST, NIFEDIPINE, INHIBIT U46619-INDUCED HUMAN PLATELET ACTIVATION BY COMPETITIVE BINDING TO THE THROMBOXANE A22/PGH2 RECEPTOR
Authors: Johnson G J , Dunlop P C , Rabiet M J , Leis L A , From AH L
Published: August 2018

The dihydropyridine (DP) Ca2+ channel antagonist, nifedipine (NF), inhibits platelet aggregation .in vitro and ex vivo by an undefined mechanism. Inhibition of Ca2+ influx via Ca2+ channels is a postulated mechanism, but voltage-dependent Ca2+ channels have not been demonstrated in platelets. We previously observed that NF blocked thromboxane A2 (TXA2)-induced platelet aggregation and secretion. In order to further evaluate the mechanism of DP inhibition of platelet activation, we studied the effects of NF and BAY K 8644, (BAY), a DP with opposite (agonist) effects on muscle cells, on human platelet aggregation and secretion induced by the TXA2 mimic, U46619. We also observed the effects of DP on biochemical consequences of platelet activation: cytoplasmic ionized Ca2+ ([Ca2+]i) by fura-2 fluorescence; phosphorylation of 40,000 Dalton protein (40KP) substrate of protein kinase C by SDS-PAGE and [32p] counting; TXA2 formation by RIA of TXB2. 1μM BAY and 10μM NF inhibited the 2nd wave of platelet aggregation and secretion induced by ADP or epinephrine and blocked aggregation and secretion induced by U46619. A Schild plot gave a slope of -1 indicating competitive inhibition of U46619 by BAY (K1[=0.7μM).BAY and NF also blocked U46619-induced phosphorylation of 40KP, rise in [Ca2+]i and TXB2 formation. The (+)-(R) enantiomer of BAY (BAY+) was responsible for BAY inhibition. BAY, BAY(+), and the R enantiomer of another DP, 202-791, all functioned as competitive antagonists of [3H]-U4661 9 binding (K1[ for BAY=2.8 μM-comparable to known receptor antagonists, 13-azaprostanoic acid and BM 13.177; K1 for BAY(+)=0.69μM). Neither BAY nor NF inhibited[3H]-yohimbine binding to α adrenergic receptors.NF, BAY, BAY(+) and BAY(-) in nM concentrations slightly stimulated platelet aggregation,secretion and biochemical events induced by U46619 similar to their effects on muscle. Therefore, DP's do not inhibit platelet activation by blocking voltage-dependent Ca2+ channels. The mechanism of DP inhibition of TXA2-induced platelet activation is stereoselective, competitive binding to the TXA2/PGH2 receptor. DP's may exert similar effects on TXA2-induced vascular smooth muscle contraction.
PLATELET ACTIVATION AND AGGREGATION BY GAS BOBBLES IN VITRO
Authors: Thorsen Terje , Holmsen Holm
Published: August 2018

After diving, in decompression sickness and after extra-corporeal circulation in membrane oxygenators, there is often a reductionin the number of circulating platelets. Gas bubbles seem to be the common denominator for these quite different conditions, and it is assumed that gas bubbles activate the platelets although by unknown mechanisms. We have used gas bubbles as a platelet agonist in an aggregometer-like apparatus. The bubbles produced platelet aggregation similar to that caused by "classical" agonists,e.g.,ADP, epinephrine etc. The gas-liquidinterface was essential for this aggregation,and the bubble diameter, rather than thetype of gas or the total number of bubbles, determined the potency of this agonist. Electron microscopical studies revealed that singleplatelets and platelet aggregates with numerous, short pseudopods adhered to the bubble wall. The platelet aggregation caused bygas bubbles was abolished by metabolic blockers (2-deoxyglucose and antimycin A), EGTA,phosphodiesterase inhibitors (theophylline, caffeine, papaverine, dipyridamole), adenylate cyclase activators (PGE1 adenosine, AMP), or a combination of low concentrationsof phosphodiesterase inhibitors and adenylate activators. Theophylline which is regardedas a weak inhibitor of platelet activation in general, was an especially potent inhibitor of gas bubble-induced platelet aggregationwhereas cyclooxygenase inhibitors (acetyl salicylic acid, indomethacin), adrenoreceptor blocker (yohimbine), guanylate cyclase activators (azide, jiitroprusside) and trifluoperazine had little or no effect. Bubble-induced,aggregation is thus similar to theprimary,and not the secondary, aggregation caused byclassical agonists. However, the ineffectiveness of guanylate cyclase activators distinguishes bubble-induced aggegation from "classical" primary aggregation.
LYSINE-125 IS ESSENTIAL FOR HEPARIN BINDING TO ANTITHROMBIN
Authors: Peterson C B , Noyes C M , Pecon J M , Church F C , Blackburn M N
Published: August 2018

Identification of lysyl residue(s) in human plasma antithrombin required for binding of heparin was approached using chemical modification with the amino-group reagent, pyridoxal-5'-phosphate. Modification of antithrombin (AT) with limiting amounts of reagent yields an average incorporation of the phos-phopyridoxyl label into one lysine per protein molecule. Fractionation of the labeled AT by affinity chromatography on heparin-Sepharose separated a phosphopyridoxylated AT species devoid of heparin binding (Pool I) from modified protein which retained affinity for heparin (Pool II). Pool I contained an average of 1.6 mol phosphopyridoxyl label per mol AT, whereas Pool II was labeled to a lesser extent, with a ratio of 1.0 mol phosphopyridoxyl per mol protein. To generate peptide maps of the two AT species, the proteins were reductively denatured, S-carboxymethylated, and digested with trypsin. Fractionation of the tryptic digests by reverse-phase HPLC indicated one peak in the chromatogram of the non-heparin-binding species to be clearly different when compared to the chromatogram of the heparin-binding species. Upon sequencing of the unique peptide by automated Edman degradation, the modified residue was identified as Lys-125 in the primary sequence of AT. Additionally, AT was reacted with pyridoxal-5 ′-phosphate in the presence of added heparin for comparison with protein modified in the absence of heparin. Overall incorporation of the phosphopyridoxyl label was 3-4 mol reagent per mol protein, without inclusion of heparin during the modification reaction, and only 2-3 mol pyridoxyl per mol protein with heparin added. Tryptic peptide maps of these two modified ATs indicated that eight lysine residues are "protected" from modification by addition of heparin. The "protected" lysines identified, in addition to the essential Lys-125, are residues 11, 39, 133, 136, 257, 275, and 287. Identification of Lys-125 as the critical lysine for heparin binding strongly supports previous data which indicates that the heparin-binding domain of AT is located at the N-termi-nus within one of the disulfide cross-linked loops of the protein.
LOCALIZATION OF THE DOMAINS OF FIBRIN INVOLVED IN BINDING TO PLATELETS
Authors: Hantgan R R
Published: August 2018

The molecular basis of platelet-fibrin interactions has been investigated by using synthetic peptides as potential inhibitors of binding fibrin protofibrils and fibrinogen to ADP-stimulated platelets, adhesion of fibrin fibers to the platelet surface, and platelet-mediated clot retraction. Synthetic peptides RGDS and HHLGGAKQAGDV, corresponding to regions of the fibrinogen α and γ chains previously identified as platelet recognition sites, inhibited the binding of radiolabelled soluble fibrin oligomers to ADP-stimulated platelets with IC50 values of 12 and 40 μM, respectively. The IC50 values obtained with fibrinogen as the ligand were 3-fold higher. Synthetic GPRP and GHRP, corresponding to the N-terminal sequences of the fibrin α and β chains, were minimally effective in blocking soluble fibrin oligomer binding to ADP-stimulated platelets. The extent of fibrin:platelet adhesion was determined with a microfluorimetric technique which measures the quantity of fluorescein-labelled fibrin attached to the surface of platelets. The signal obtained from the brightly fluorescent platelet:fibrin adducts was time- and concentration-dependent, and was fully inhibited by a monoclonal antibody directed against the glycoprotein II:IIIa complex (HP1-1D, kindly provided by Dr. W. Nichols). Inhibition of fibrin:platelet adhesion by RGDS, HHGGAKQAGDV, and GHRP all exhibited a similar, linear dependence on the peptide concentration, reaching 1/2 maximum at about 200 μM, suggesting nonspecific effects. GPRP inhibited fibrin assembly but did not appear to have specific effects on fibrin:platelet adhesion. The time course of clot retraction was followed by right angle light scattering intensity measurements. Only RGDS affected clot retraction, causing a 4-fold decrease in rate at 230 μM. These results indicate that fibrinogen and fibrin protofibrils, which are obligatory intermediates in the fibrin assembly pathway, share a set of common platelet recognition sites located at specific regions of the α and γ chains of the multinodular fibrin(ogen) molecules. The RGDS site is also involved in mediating interactions between the three dimensional fibrin network and ADP-stimulated platelets.
LOCALIZATION CF FIBRIN POLYMERIZATION SITES BY PHOTQAFFINITY LABELING
Authors: Kuyas C , Sigrist H , Straub P W
Published: August 2018

Fibrin polymerization is competitively inhibited by the te-trapeptide GlyProArgPro. This peptide corresponds to the N-terminal sequence of the fibrin α-polypeptide chain, which is exposed upon release of fibrinopeptide A by thrombin. A binding site for GlyProArgPro was suggested to be located in the C-terminal end of the 411 amino acids containing γ-chain (Varadi and Scheraga, Biochemistry, 25, 519, 1986). In order to characterize the polymerization domain, GlyProArgProLys-azidoazobenzene, a photoactivable derivative of GlyProArgPro was synthesized. Photoaffinity label was bound to fibrinogen in the dark and photolysis was carried out at 0°C. After reduction and S-carboxymethy-lation of the photoaffinity labeled fibrinogen, the polypeptide chains (Aα, Bβ,γ ) were separated by ion-exchange chromatography. Photolabel binding was monitored imnunologically with anti-azo-benzene antibodies (ELISA, Western blot). Selective labeling of the γ-chain was observed. Labeled γ-chains were further digested with CNBr, and the resulting fragments were separated by reversed phase HPIC, immunologically characterized and identified by Edman degradation. GlyProArgProLys-azidoazobenzene was incorporated in the 18 kD CNBr-fragment (γ95-264). The CNBr-fragments arising from C-terminal end of the γ-chain were not labeled.

Our results indicate that the binding site of GlyProArgPro is localized exclusively on γ-chain, within the sequence γ95-264.
FACTOR VII-PHOSPHOLIPID COMPLEX IN MALE SURVIVIRS OF ACUTE MYCORDIAL INFARCTION
Authors: Arnesen H , Dalaker K , Smith P , Prydz H
Published: August 2018

The finding of coagulation factor VII-phospholipid complex in plasma of some men at high risk for cardiovascular disease has previously been reported (1). Factor VII present in such complexes is markedly activated, and this activitation is reversible upon treatment with phospholipase C.

We report here on the presence of this activated form of factor VII in 100 male survivors of acute myocardial infarction The subjects were recruited from 475 patients in the placebo group of the prospective double-blind Warfarin Re-Infarction Study (WARIS).

Two age groups were defined:55 years or younger, and older than 55. In each age group the 25 patients with the highest or lowest conventional risk factor scores (total cholestrol, systolic blood pressure, and smoking habits) were selected. The amount of factor Vll-phospholipid complex was estimated as factor VII activity before and after treatment with phospholipase C.

The factor VII-phospholipid complex was found to be responsible for 29% (mean value) of the total factor VII activity in the whole material. The level of this activated form of factor VII did not correlate with age or level of conventional risk factor score, but showed a highly significant positive correlation with serum triglycerides in all the subgroups, as well as in the total material (r=0,88, p<0.0001).

Presence of the factor VII-phospholipid complex contributes to the procoagulant state in hypertriglyceridemia, and its measurement may constitute an additional screening method for men at risk for cardiovascular disease.
Ref.: (1) Dalaker K., Hjermann I, and Prydz H.
Br J Haematol 1985,61,315
ALTERATION OF PROPTIDE SEQUENCE IMPAIRS BIOLOGICAL ACTIVITY OF HUMAN FACTOR VII
Authors: Busby S , Berkner K , Halfpap L , Gambee J , Kumar A
Published: August 2018

We have investigated the effect of altering the leader sequence of human factor VII on its biological activity. Factor VII is a vitamin K-dependent blood coagulation protein whose activity depends on the presence of gamma-carboxyglutamic acid (gla) residues in its amino terminal region. Since factor VII and other vitamin K-dependent proteins exhibit structural homology in the propeptide, it has been suggested that the propeptide is involved in gamma-carboxylation. Recently, two factor IX patients were identified with point mutations which prevented the processing of the propeptide and generated a factor IX with greatly reduced biological activity (Diuguid et al., PNAS 83; 5803; Bentley et al., Cell 45: 343). To examine this question using recombinant DNA technology, we altered the sequence of the factor VII propeptide by in vitro mutagenesis of the factor VII cDNA and then expressed the altered genes in baby hamster kidney (BHK) cells. For the 60 and 38 aa leader forms of factor VII, the arg (R) at -1 was changed to ser (S), yielding the sequence HRRRS before the +1 ala. In addition, for the 60 aa leader form, a ser was inserted after the arg at -1, resulting in the sequence HRRRRS before the +1 ala. As determined by ELISA, the mutant proteins were synthesized and secreted by BHK cells at levels comparable to the wild-type forms of factor VII. Analysis by radioimmune precipitation and SDS-PAGE indicated that substitution of arg by ser at -1 prohibits processing of the factor VII propeptide, whereas, insertion of a ser after the four arg's does not. However, all three proteins have reduced biological activity by approximately 5-fold when compared to the wild-type forms with the one-stage clotting assay. All three proteins are also quantitatively precipitated by Ba citrate, indicating they are at least partially gamma-carboxylated. These results suggest that the correct sequence of the propeptide, not just cleavage of the propeptide, is necessary for generating a biologically active molecule. The effect of these sequence alterations on gamma-carboxylation will be evaluated further by analysis of the amino acid sequence and composition of the mutant proteins.