bluuebunny/crossref_metadata_2025 huggingface dataset:
ISOLATION AND CHARACTERIZATION OF cDNA CLONES FOR HUMAN FACTOR V
Authors: Dahlbäck B , LundWall A
Published: August 2018

Coagulation factor V is a single chain, 330 kDa glycoprotein functioning as a cofactor to factor Xa in the activation of prothrombin. Thrombin cleaves factor V into four major fragments, out of which the N-terminal (105kDa) and the C-terminal (71-74kDa) fragments together constitute the active factor V species. To isolate cDNA clones a λ-gt 11 liver library was screened with a polyclonal, monospecific antiserum against human factor V. Four positive clones (two "weak", Aland A2 and two "strong", A3 and A4) were identified and isolated. Al(0.7kb), A2 (1.25kb) and A4 (0.85kb) reacted strongly with an antiserum against the 105 kDa, N-terminal fragment (heavy chain of factor Va), whereas A3 (1.25kb) gave the best signal with an antiserum against the 71-74 kDa, C-terminal fragment (light chain of factor Va). A1 hybridized with A2 and A4, whereas A2 only hybridized with Al. A3, which did not hybridize to any of the other clones, was used to rescreen the library and 9 positive clones (Bl-9) were isolated. B9 (3kb) coded for the entire C-terminal factor V fragment and the 3' noncoding sequence. B8 (1.8kb) partially overlapped B9 but extented the 5' sequence with 0.8kb. In a third screening round Al was used in combination with B8 and a 1.1 kb clone (CIO) was identified which hybridized to both. C10 did not hybridize with A2. The following overlapping cDNA clones can be orderedfrom the 5´end: A2-A1-C10-B8-B9 and together they cover 6 kb of coding sequence
FEMORO-POPLITEAL ARTERY THROMBOLYSIS WITH INTRA-ARTERIAL INFUSION OF RECOMBINANT TISSUE-TYPE PLASMINOGEN ACTIVATOR (rt-PA)
Authors: Verhaeghe R , Wilms G , Mombaerts P , Vermylen J , Baert A , Verstraete M
Published: August 2018

The efficacy and tolerance of intra-arterial rt-PA infusion was tested in 27 patients with a thrombotic occlusion of the femoro-popliteal artery. The mean length of the occluding thrombus was 10 cm (range : 2-25 cm). The occlusion was recent (<1 week old) in 7 patients; in 2 it existed for more than 6 months. The rt-PA solution was infused through an angiographic catheter embedded into the thrombus at a rate of 10 mg/hr in the first 11 patients, 5 mg/hr in the next 11 and 3 mg/hr in the last 5. The maximal dose foreseen in the protocol was 50 mg; the mean dose infused was 42 mg. Heparin (400 IU/hr) was infused concomitantly. Thrombolysis occurred in all 27 patients. Angiographic restoration of patency was obtained in 25 (93%); it first appeared after a mean dose of 27 mg rt-PA (range : 10 to 50 mg). In 21 patients, a percutaneous transluminal angioplasty was needed to dilate a residual stenotic lesion or remaining mural thrombi. This secondary procedure initiated reocclusion in 2 patients by causing a distal embolus and a subintimal dissection, respectively. Early rethrombosis occurred spontaneously in 3 other patients. Thus, 20 (74%) patients had a clinical improvement at discharge from the hospital.

rt-PA infusion was complicated by bleeding in 10 (37%)
patients: a groin hematoma at the catheter entry site occurred in 9 patients, a hematoma from a previous venous in 2 and gingival oozing in 3. None required blood transfusion. Premature interruption of the infusion because of local hematoma formation was the cause of failure in one patient.

This pilot trial confirms the feasibility of thrombolysis with local infusion of rt-PA in peripheral arterial thrombosis. The early clinical results and the incidence of bleeding complications appear similar to those observed with local low-dose streptokinase, although initial patency seems easier to restore with rt-PA. A prospective trial comparing rt-PA to streptokinase in this condition is thus warranted
ROLE OF PLATELET MEMBRANE GLYCOPROTEIN IV IN PLATELET-COLLAGEN INTERACTION: A MICROTITER ASSAY TO STUDY PLATELET ADHERENCE
Authors: Tandon N N , Jamieson G A
Published: August 2018

The role of platelet glycoprotein IV (GPIV) in platelet function has not been elucidated. We have now isolated GPIV (Mr 88,000) from platelet membranes in homogeneous form by a series of steps involving (i) phase partitioning in Triton X-114, (ii) ion exchange chromatography on DEAE-cellulose, (iii) lectin affinity chromatography on WGA-Sepharose, and (iv) size exclusion chromatography on Ultrogel AcA44. Purified GPIV inhibited platelet shape change and aggregation induced by collagen (2 ug/ml; 7 nM as tropocollagen) in a dose-dependent fashion (ID50 ∼ 1 ug/ml; 10 nM) but did not affect aggregation induced by thrombin, ADP, epinephrine, arachidonate or ionophore A23187. To study the role of GPIV in platelet interaction with collagen we have developed a microtiter assay involving (i) coating acid soluble or fibrillar Type I collagen onto microtiter plates, (ii) incubation of coated collagen with 51Cr-labeled platelets and (iii) quantitation of platelet adherence by analysing the radioactivity of the SDS lysate of the adhered platelets. In this assay system, Fab fragments of anti-GPIV antibody inhibited platelet adherence by 75% to both acid soluble and fibrillar Type I collagen while nonimmune serum was without effect. Fab fragments also inhibited collagen-induced aggregation and secretion (ID∼ 10 ug/ml; 200 nM) and, slightly less effectively, aggregation by ADP and epinephrine (ID∼ 300 NM), but did not affect platelet activation by thrombin, arachidonate or ionophore. Fab fragments also inhibited platelet attachment to collagen-Sepharose columns by 80%. These results suggest a role for GPIV in the interaction of platelets with collagen, probably at the level of primary platelet adherence.
PADGEM PROTEIN EXPRESSION IN HUMAN ERYTHROLEUKEMIA (HEL) CELLS
Authors: Yeo E , Furie B C , Furie B
Published: August 2018

PADGEM (Platelet Activation-Dependent Granule ⇒ External Membrane) glycoprotein, a platelet alpha granule integral membrane protein with a molecular weight of 140,000, is translocated to the plasma membrane during granule secretion. PADGEM protein is expressed solely on activated platelets, but is not on the surface of resting platelets. Because HEL cells contain platelet alpha granule-like organelles and proteins (e.g. platelet factor 4, von Villebrands factor, β-thromboglobulin) and express certain platelet membrane proteins (e.g. GP IIb/IIIa, GPIb), we evaluated induced and uninduced HEL cells for the synthesis and expression of PADGEM protein. HEL cells were induced with 1.25% DMSO for 3-4 days, then grown in the absence of DMSO for 1-3 weeks. After eight cycles of DMSO exposure, the induced HEL cells were found to increase the expression of PADGEM, in contrast to the uninduced cells. Intact fixed and unfixed induced HEL cells were observed by immunofluorescence, utilizing KC4, a monoclonal anti-PADGEM antibody, to express PADGEM while non-induced HFT. cells expressed low levels of PADGEM. Both induced and uninduced HEL cells bound A2A9, an anti—GP Ilb/IIIa monoclonal antibody. Quantitative analysis by fluorescence activated cell sorting demonstrated a 2.5—fold increase in mean surface expression of PADGEM and 3.3—fold mean increase in GP IIb/IIIa surface expression compared to uninduced cells. By fluoresence microscopy, 70% of induced HEL cells expressed PADGEM protein versus 20% of the uninduced cells. GP-IIb/IIIa expression inoreased from 40% in noninduced cells to 90% in induced cells. The induced HEL cells contained PADGEM with a molecular weight identical to that of platelets, as demonstrated by Western blotting using the KC4 antibody. Direct binding experiments with 125I-KC4 antibody demonstrated that surface binding was specific, saturable, and time-dependent. Surface expression of PADGEM protein was not increased with platelet agonists (thrombin, epinephrine, ADP) nor cytokines (IL-1, IL-2, tissue necrosis factor). The surface density of PADGEM protein on induced HEL cells and activated platelets appears similar. HEL cells should provide a useful model to assist in the elucidation of the structure, function and biology of PADGEM protein.
IMMUNOLOGICAL AND CHEMICAL DETECTION OF VAC IN CULTURED ENDOTHELIAL CELLS
Authors: Reutelingsperger C P M , Buurman W , Horn-stra G , Horn-stra H C
Published: August 2018

Recently we reported the presence in human umbilical cord vessels of an anticoagulatory protein (VAC, Mr = 32,000) which inhibits phospholipid dependent procoagulant reactions through a high affinity binding, in the presence of calcium, to the phospholipid surface. The mechanism of anticoagulation differs fundamentally from those of the well-known physiological anticoagulants.

Polyclonal antibodies, raised in rabbits against purified VAC, bind in cultured endothelial cell lysates to an antigen with Mr = 32,000, as was revealed with immunoblotting techniques. It is demonstrated with chemical techniques, that this antigen is identical to VAC.

VAC appears to be an intracellular protein, attached to fine granular structures, which are located outside the nuclear area. Quiescent endothelial cells do neither secrete VAC in detectable amounts, nor contain detectable VAC on the extracellular side of their plasma membrane. In the presence of 1 mM calcium, endothelial VAC binds reversibly, as was indicated with EDTA, to the subcellular structures of the endothelial cell. Once VAC is bound to the subcellular structures, their apparent procoagulant activities are diminished, as was shown in a one-stage coagulation assay.

Based on these findings, we propose that the presence of VAC in endothelial cells supplies the endothelium with an additional anticoagulatory mechanism, which can be activated after cell injury, when intracellular structures become exposed to plasma constituents. VAC then controls the formation of procoagulant complexes, localized to the subcellular structures.
PRODUCTION OF A HIGH-PURITY FACTOR VIII CONCENTRATE BY MEANS OF FOROUS SILICA
Authors: In der Maur D W , Hoëk P J , Piet M P J , de Jonge E R , Jordan R E , Over J
Published: August 2018

Adsorption of proteins contaminating Factor VIII in cryoprecipitate to porous silica has been evaluated as a means to produce a high-purity Factor VIII concentrate for clinical use. At first, optimal conditions for the adsorption step have been assessed. A pore size of the silica of 1000 A, a pH of 6.6 and a contact time of 30 minutes gave the best results, while ionic strength and temperature were of no importance. The specific activity of Factor VIII in the effluent was directly proportional to the ratio of silica over cryoprecipitate solution, but the Factor VIII recovery was inversely proportional. At ratio's of about 2:1 the recovery was about 90% with a specific activity of 1.5 - 2.0 IU/mg protein.

Several techniques to concentrate Factor VIII from the effluent were evaluated. Of those tried (precipitation by ammonium sulphate, polyethyleneglycol or a combination of NaCl and glycin, freezedrying, and ultrafiltration using several types of membranes) ultrafiltration using hollow-fiber cartridges meant for kidney dialysis proved to be optimal: when stabilized by a mixture of amino acids the Factor VIII solution could be concentrated five-fold within 60 minutes at a 80 - 95% yield of Factor VIII with some additional purification. The stabilizers also allowed the final preparation to be heat-treated in lyophilized state for 72 hours at 60 °C or 30 hours at 70 °C.

The process scheme (cryoprecipitation, porous silica adsorption, ultrafiltration, sterile filtration, freezedrying and heat treatment) has been tried out 8 times at 200 - 300 liter plasma scale and showed highly consistent results. On average, the specific activity was 1.8 IU/mg, the Factor VIII recovery 56% relative to the Factor VIII content of the starting cryoprecipitate, and the Factor VIII concentration 28 IU/ml. The process is now being scaled up to 1,000 - 1,500 liter plasma, after which clinical evaluation will follow. It is anticipated that at full production scale the porous silica method combines good purifying capacity with a relatively high yield of Factor VIII
HEPARIN-INDUCED THROMBOCYTOPENIA: IN VITRO STUDIES WITH LOW MOLECULAR WEIGHT HEPARINOID, ORG 10172
Authors: Chong B H , Ismail F , Cade J , Gallus A S , Gordon S , Chesterman C N
Published: August 2018

Heparin-induced thrombocytopenia (HIT), an adverse effect of heparin therapy, may be associated with serious thrombosis resulting in severe disability or death. An IgG heparin-dependent antibody may be demonstrated in HIT by platelet aggregation studies with patient serum/plasma and standard (s) heparin. A recent study showed high cross-reactivity of the antibody with low molecular weight (LMW) heparins as most of the 22 patient sera tested gave a positive reaction with various LMW heparins including CY222, CY216, PK10169 and Kabi 2165 (Messmore HL et al, Blood 64(5), 1984 suppl). However, cross-reactivity rate with Org 10172, a LMW heparinoid which has strong anti-Xa but negligible antithrombin activity, is unknown. We tested the plasma of 17 patients with HIT for cross-reactivity with Org 10172. Although all 17 patient plasmas reacted positively with s.heparin (0.2
1.0 IU/ml), only 3 patient plasmas gave a positive but weaker reaction with Org 10172 (0.2-1.0 IU/ml).

Further studies were performed to investigate the effect of adding Org 10172 (0.2 to 2.0 anti-Xa U/ml) to a reaction mixture of normal platelet-rich plasma, patient plasma and s.heparin (0.2 IU/ml). With 7 patient plasmas which showed no cross-reactivity with Org 10172, the antibody-induced platelet aggregation was inhibited when the concentration of Org 10172 exceeded 0.5 to 1.0 anti-Xa U/ml. When the study was repeated with other s.heparin concentrations (0.05, 0.1, 0.4 IU/ml), this inhibitory effect was again present provided the ratio of Org 10172 concentration (anti-Xa U/ml) to heparin concentration (IU/ml) exceeds 2.5 to 5. However, this inhibitory effect was not observed with the 3 patient plasmas which showed cross-reactivity with the heparinoid whqp. the same concentrations of Org 10172 were added. This inhibitory effect appeared to be specific for platelet aggregation induced by the heparin-dependent antibody as Org 10172 (<10 anti-Xa U/ml) did not affect platelet aggregation induced by collagen (2 ug/ml) and ADP (2.5 uM). These findings together with our experience of 3 cases of HIT successfully treated with Org 10172 suggest that this LMW heparinoid may be a useful drug for the treatment of HIT.
ANALYSIS OF PLATELET REACTIVE ANTIBODIES USING MONOCLONAL ANTIBODIES
Authors: Kiefel V , Santoso S , Mueller-Eckhardt C
Published: August 2018

The characterization of platelet reactive alloantibodies and autoantibodies is mandatory for the diagnosis of posttransfusion purpura, neonatal alloimmune thrombocytopenia, autoimmune thrombocytopenia and for the selection of platelet donors prior to platelet transfusions in immunized polytransfused patients. The platelet immunofluorescence test is suitable for the detection of platelet reactive antibodies. In many cases, however, mixtures containing different platelet reactive antibodies have to be dissected.

In order to analyze these sera, we have developed a novel enzyme immunoassay based upon monoclonal antibody specific immobilization of platelet antigens (MAIPA). In brief, platelets are incubated simultaneously with the (human) serum to be investigated and a monoclonal (mouse) antibody directed against an epitope on the same platelet membrane glycoprotein (GP). Platelets are then washed and solubilized in TRIS buffered saline containing NP40. The lysed platelets are then pipetted into the wells of microtiter plates, coated with goat anti mouse IgG where mouse anti GP-complexes are immobilized. Human platelet reactive antibodies on the same GP are detected using enzyme labelled goat anti human IgG, IgM, or IgA, respectively. Using mab Gi5, mab FMC25, mab w6.32 directed against epitopes on the glycoprotein complex IIb/IIIa, glycoprotein Ib and HLA class I molecule, respectively, and a panel of typed platelet donors, even sera containing different platelet reactive antibodies are readily analyzed. Results of experiments with platelet specific alloantibodies (anti P1A1, anti P1A2 and anti Bak(a)), autoantibodies (against the GP Ilb/IIIa complex and GP Ib) and a drug dependent antibody show that this assay allows to discriminate all these different platelet reactive antibodies.
THE ACTIVATION OF PROTHROMBIN: A LINKED REACTIVE KINETIC MODEL
Authors: Gentry R , Nemerson Y
Published: August 2018

Prothrombin activation by factors Xa and Va is complex, involving five linked reactions occurring either in a bulk fluid phase or in a local phospholipid (PL) surface phase, each controlled by both the fundamental energetics of the species interactions and the relevant reactant concentrations. The significance of each reaction and in particular the kinetic efficiency of the PL surface reactions is yet to be resolved. Previous essentially Michaelis-Menten kinetic treatments of prothrombin activation have been derived without concern for the kinetic influence of the order of reactant assembly or possible nonlinear effects induced by complex trapping of reactants, as observed in factor VIIa reactions (Nemerson and Gentry, Biochem. (1976) 25, 4020). We propose a general model to analyze and explain this and similar PL associated coagulation reactions. The mathematical model consist of a system of nonlinear first order differential equations:

dX/dt - F(X(t),C(t),K); dC/dt - G(X(t),C(t),K), where the functionals F and G are derived from standard kinetic principles. To model prothrombin activation in this general setting the fluid and bound reactions are described by two parallel but linked systems, denoted S1(X,C,K) and S2(X*,C ,K*), respectively. In the linked relationship the primary constituents transfer between the systems, governed by a transfer component -T, resulting in a system of “linked-reactive” equations of the form:

dX/dt = -F(X, C, K) - T ; dX*/dt = F*(X*,C*,K*) + T* , etc. A key consideration is to ensure that the concentrations of reactive species are relative to the reactive volume in which the kinetic interactions are to occur. The detailed equations for prothrombin activation involve for the bulk phase three primary variables X1, X2, and X3 corresponding to factors Va, Xa, and prothrombin, four complexes, Ci, and a duplicate stared set for the PL phase. After condensation the resultant system of 11 differential equations involves 28 kinetic parameters; this has significant implications for the ability to uniquely identify the parameters but poses no difficulties for its use as a predictive model via its numerical simulation for specific parameter values.
DETERMINATION OF NUMBER OF γ-CARBOXYGLUTAMIC ACID (GLA) RESIDUES INVOLVED IN FORMING THE TWO HIGH AFFINITY METAL BINDING SITES IN PROTHROMBIN AND FACTOR X
Authors: Brodsky G L , Bajaj S P
Published: August 2018

Prothrombin and factor X possess two high affinity and several low affinity lanthanide ion binding sites. In both proteins, the association constant of the high affinity sites is at least 50-fold greater than that of the low affinity sites. Moreover, metal bound to these high affinity sites is extremely difficult to displace. It has been proposed that one of the two high affinity sites in factor X involves Gla residues while the other involves β-hydroxyaspartic acid and no Gla residues. It is also known that ^H can be specifically incorporated into Gla residues at an acidic pH. We have determined that under nondenaturing conditions when Gla (synthetic or in proteins) is complexed to metal at pH 5.5, this specific 3H incorporation is blocked. Furthermore, we have found that β-hydroxyaspartic acid does not incorporate in the presence or absence of metal. When we incubated prothrombin or factor X (41 μM) with 3H2O in the presence of Tb3+ or Gd3+ (82 μM), we blocked 5.6 Gla residues per prothrombin and 5.5 Gla residues per factor X from 3H incorporation. Under these conditions, we calculated that >95% of the high affinity sites are occupied by metal. Thus, in prothrombin, an average of 2.8 Gla residues are involved in forming each high affinity site. If the Gla residues in factor X participate in forming only one of the two high affinity sites, then all 5.5 Gla residues blocked from incorporation must be involved in forming that site. However, this seems highly unlikely. We conclude that, as in prothrombin, both high affinity sites in factor X involve Gla residues (average 2.75/site). However, our data does not exclude the possibility of existence of a heterologous site containing both β-hydroxyaspartic acid and Gla residues.
CONSTRUCTION AND EXPRESSION OF HYBRID PLASMINOGEN ACTIVATORS PREPARED FROM TISSUE-PLASMINOGEN ACTIVATOR (t-PA) AND UROKINASE (u-PA) GENES
Authors: Lee S G , Kalvan N K , Wilhelm J , Hum W-T , Rappaport R , Chenq S M , Dheer S , Urbano C , Levner M , Hung P P
Published: August 2018

A covalent hybrid plasminogen activator was prepared from the sulfhydryl forms of the NH2-terminal A chain of human plasmin (Pln^) containing the fibrin-binding domain, and the COOH-terminal B chain of tissue plasminogen activator (t-PAB) containing the catalytic domain. The PlnA (SH)2 and t-PAB(SH) chains were mixed in a 1:1 molar ratio, and hybridization was allowed to proceed at 4 °C for 6 days. The covalent PlnA-t-PAB hybrid activator was isolated from the mixture by a two-step affinity chromatography method, with L-lysine-substituted Sepharose and Zn-chelated agarose. The protein yield of purified hybrid was 10% with a major component (77%) of Mr ∼92,000. The covalent PlnA-t-PAB hybrid activator, contained 1 mol of each chain; after reduction, it gave the two parent chains, PlnA and t-PAA, also shown to be present by double immunodiffusion. The specific plasminogen activator activity, with soluble fibrin, and the specific amidolytic activity, of the purified covalent hybrid activator was determined to be 200,000 IU/mg of protein, about 40% of the specific activity of the parent t-PA. In a fibrin clot lysis assay, the covalent hybrid activator and t-PA have similar specific fibrinolytic activities, 500,000 IU/mg of protein; however, the clot lysis time curves were not parallel. The binding of the covalent PlnA-t-PAB hybrid activator and t-PA to forming fibrin were found to be similar; at physiological fibrinogen concentrations, binding of both activators to forming fibrin was about 90%.
PROPERTIES OF A HUMAN RECOMBINANT FUSION PROTEIN OF THE ‘FINGER’ DOMAIN OF TISSUE-TYPE PLASMINOGEN ACTIVATOR (t-PA) AND A TRUNCATED SINGLE CHAIN UROKINASE-TYPE PLASMINOGEN ACTIVATOR (scu-PA)
Authors: Gheysen D , Piérard L , Jacobs P , Lijnen H R , Bollen A , Collen D
Published: August 2018

A hybrid between human tissue-type plasminogen activator (t-PA) and human single chain urokinase-type plasminogen activator (scu-PA) was obtained by ligation of cDNA fragments encoding the NH2-terminal amino acids 1 to 67 of t-PA and the COOH-ter-minal amino acids 136 to 411, of scu-PA. Both this chimaeric cDNA and cDNA encoding scu-PA were expressed in a mammalian system (HAK-cells) using bovine papilloma virus (BPV) derived vectors. Two stable cell lines were obtained which secreted the recombinant hybrid and the scu-PA at 1 μg/ml and 2 μg/ml u-PA related antigen respectively into the culture medium. Following purification by Zinc chelate Sepharose, immunoadsorption chromatography, benzamidine-Sepharose and Ultrogel AcA44 gel filtration, highly purified proteins were obtained with a yield of about 200 μg/1. SDS gel electrophoresis under reducing conditions showed single bands with M 43,000 and M 50,000 respectively. Following conversion to urokinase with plasmin, both proteins had a specific amidolytic activity comparable to that of natural scu-PA. Both proteins activated plasminogen directly with km1.4 and 0.5 μM and k2 0.0034 s and 0.0027 s . Neither protein bound specifically to fibrin.

Thus the fusion of the finger-like domain of t-PA to the COOH-terminal part of scu-PA does not confer fibrin affinity of t-PA to this chimaeric protein. However, peptide material can be fused to the COOH-terminal part of scu-PA without perturbing its enzymatic properties.
5-LIPOOXYGENASE ACTIVITY IN THE HUMAN VESSEL WALL
Authors: De Caterina WALL R , Giannessi D , Lazzerini G , Mazzone A , Azzara A , Mosca F
Published: August 2018

5-lipooxygenase products have been identified from a variety of cells and may play a role in the progression of atherosclerosis and in its clinical manifestations (spasm, thrombosis). We investigated whether human vascular fragments, freshly obtained at surgery, are able to produce leukotriene (LT) B4, a definite end product of 5-lipooxygenase, provided with biological activity. Fragments obtained from human saphenous veins (n=21) or aorta (fibrous plaques, n=15, atheromas, n=16) were incubated in buffer at 37°C with mechanical agitation sequentially in the absence (15 min) and in the presence (15 min) of 10 jjM calcium ionophore A-23187. At the end of each incubation, the buffer was sampled to be assayed by a specific radioimmunassay (RIA) for LTB4 (sensitivity 4.3+0.9 pg). Validation of the assay was performed by comparison with a chemotactic bioassay in Boyden chambers, by interpolation of a standard curve evaluating the chemotactic response of neutrophils to a standard LTB4 preparation. RIA resulted the only practicable method to detect concentrations lower than 2.5 ng/ml, compared both to bioassay and to HPLC, all three performed in the incubation media from 8 vascular fragments. Incubations were also performed in a chamber with selective exposure of the endothelial surface in order to detect possible production of LTB4 on the luminal site of the vessel. Both unstimulated and ionophore-stimulated LTB4 were higher (P< 0.01) in atheromas (2.7±1.2 and 6.3±1.8) than in fibrous plaques (0.51±0.22 and 1.19±0.38) or saphenous veins (0.74±0.34 and 3.07±1.39) (ng/g wet weight, mean±SD). Detectable spontaneous and stimulated LTB4 productions were also found in the incubation media of the chamber with atheromas (40±14 and 324±85 pg/cm2 area, respectively). Histology of the fragments confirmed a higher cellularity (macrophages, atherocytes) in atheromas as compared to fibrous plaques and veins. The human vascular wall is a definite site of 5-lipooxygenase activity, possibly arising from white cell infiltration. LTB4 production, able to reach the inner vessel surface and the blood stream, is a possible factor in the progression of the lesion by increasing vascular permeability or recruiting white blood cells.
EFFECTS OF 13-H0DE AND HETEs ON TUMOR CELL/ENDOTHELIAL CELL INTERACTIONS
Authors: Bastida E , Almirall L
Published: August 2018

We and others reported that endothelial cells (ECs) convert linoleic acid into 13-hydroxyoctadecadienoic acid (13-H0DE) under basal conditions, and arachidonic acid into 15-hydroxyeicosatet-raenoic acid (15-HETE) following stimulation (1,2). We also reported that lipoxygenase metabolism influenced platelet (PLT) interactions with ECs, tumor cells (TCs) and extracellular matrix (BM) (1,3,4). Thus, we performed studies to determine i) if TCs also produce 13-H0DE and HETEs, and ii) the effect of TC and EC 13-H0DE and HETEs synthesis on TC/EC adhesion. We measured i) the ratios of 13-H0DE:HETE in 5TC lines, under basal and stimulated conditions, in metastatic and non-metastatic TCs of the same cell line, and TCs treated with salicylate (SAL) or dipyridamole (DIP), and ii) their relationships with TC adhesion to ECs and BM. 13-H0DE and HETEs were assayed by HPLC. TC adhesion was assayed as the # radiolabelled TCs adherent to ECs or BM. cAMP was assayed by RIA. Under basal conditions, TCs produced 13-H0DE and HETEs, the intracellular ratio of which markedly affected their adhesivity; e.g. the least adhesive TC (U87MG glioblastoma) produced 21Xs more 13-H0DE than HETE’s, while a more adhesive TC (A549, adenocarcinoma) produced 4Xs more HETEs than 13-H0DE. Non-metastatic TCs preferentially produced 13-H0DE while metastatic TCs of the same cell line, produced HETEs. Stimulation of TCs or ECs decreased 13-H0DE, and increased HETE synthesis and TC/EC adhesion. Inhibiting intracellular 13-H0DE synthesis in either TCs or EC (SAL RX) enhanced TC/EC and TC/BM adhesion. Enhancing 13-H0DE synthesis by elevating cAMP (DIP RX) inhibited TC/EC and TC/BM adhesion. We conclude that 1) in vitro TCs produce 13-H0DE and HETEs, 2) the ratio of 13-H0DE:HETEs in TCs and ECs affects their adhesivity; and 3) the ratio of intracellular 13-H0DE:HETEs depends upon cAMP. This suggests that 13-H0DE:HETE ratios in TCs and ECs influence the adhesion process in the pathogenesis of thrombosis and metastasis in vivo. (1) Buchanan et al, JBC 30:1985. (2) Hopkins et al, JBC 29:1984. (3) Bastida et al, Int. J. Cane. 1987. (4) Buchanan et al, Prost. Leuk. Med., 1986.
RELATIONSHIP BETWEEN VESSEL WALL 13-HODE PRODUCTION AND SUBENDOTHELIAL BASEMENT MEMBRANE THROMBORESISTANCE: INFLUENCE OF SALICYLATE
Authors: Weber E , Haas T A , Hirsh J , Buchanan M R
Published: August 2018

In previous studies, we have reported that i) the basement membrane (BM) underlying endothelial cells was initially throm-boresistant; ii) 13-hydroxyoctadecadienoic acid (13-HODE) synthesized by endothelial cells from linoleic acid via the lipoxygenase pathway, contributed to the thromboresistance of the endothelium; and iii) salicylate (SAL) increased injured vessel wall thrombogenecity. Therefore, we performed studies to determine the relationship between injured vessel wall thrombogenecity, vessel wall 13-HODE and cAMP levels, and salicylate treatment. Injured vessel wall thrombogenecity was measured as the number of H-adenine platelet (3H-PLT) adherent to the subendothelial BM exposed by air injury in carotid arteries of rabbits treated with 0 or 100 mg/kg of SAL bid, given orally2. Vessel wall 13-HODE was measured as the amount of 13-HODE/cm produced by the vessel wall following stimulation with 10μ/M linoleic acid, and measured by HPLC. Vessel wall cAMP levels were measured by RIA. Four hours after air injury, there was 25.4 ± 2 3
2H-PLT/cm2 of exposed BM. This was associated with 15.9 ng/cm2 of 13-HODE and 308 pM/cm2 of cAMP (Table 1). In contrast, in rabbits treated with SAL, there was a 2-fold increase in platelet adhesion onto the injured carotid arteries. The increase in platelet adhesion was associated with a 65% decrease in 13-HODE production by the vessel wall and a modest (20%) decrease in cAMP level.

We conclude that the lipoxygenase derived linoleic acid metabolite, 13-HODE contributes not only to the thromboresis-tance of the endothelium, but also to its underlying basement membrane.
THE EFFECT OF ARTERIAL DE-ENDOTHELIALIZATION ON IN VIVO AND EX VIVO PLATELET FUNCTION TESTS IN THE BABOON
Authors: Kotze H F , Badenhorst P N , du A , Heyns P , Heyns P , van Wyk V
Published: August 2018

The effect of endothelial cell injury on platelet function was evaluated in eight baboons with a normal vasculature. Endothelium was removed with a balloon catheter. On each baboon, the left carotid artery was de-endothelialized on day 0 and the right carotid artery and abdominal aorta on day 7. Mean platelet density (MPD; gm/ml), circulating platelet aggregate ratio (CPA), the number of electron-dense bodies per platelet (DBP) , and the plasma concentration (ng/ml) of platelet factor 4 x(PF4) and beta thromboglobulin (bTG) were determined on day -7 (baseline). These ex vivo measurements were repeated on days 1, 9 and 16. The mean lifespan (MPLS) and sites of sequestration of In-111-labelled platelets were determined from day 7 onwards. The results of the ex vivo platelet function tests were:

De-endothelialization of only the left carotid artery did not affect ex vivo platelet function. More extensive denudation decreased MPD, and increased CPA, PF4 and bTG. CPA were still abnormal 7 days after the insult. MPLS, 120±21 hours, was shorter (p<0.05) than normal, 145±15 hours. The sequestration pattern of senescent platelets was normal. We conclude that a decrease in the MPD, an increase in CPA, bTG and PF4, and a shortened MPLS are the most sensitive tests for in vivo platelet activation.
PATCH ANGIOPLASTY INCREASES PLATELET DEPOSITION FOLLOWING CAROTID ENDARTERECTOMY
Authors: Meek A C , Jarvis P , Harper R A , McCollum C N
Published: August 2018

Patch angioplasty with vein or Dacron may be required to prevent narrowing when the arteriotomy is closed following carotid endarterectomy. We studied the effect of such patches on intraluminal thrombus formation by measuring radiolabelled platelet uptake in patients following simple closure and patch angioplasty.

Following unilateral carotid endarterectomy in 33 patients, the arteriotomy was closed by direct suture in 23, Dacron patch in 6 and saphenous vein patch in 4. Autologous 111In-platelets were infused on the second postoperative day and platelet uptake over the carotid measured on daily gamma camera images for 3 days. Radioactivity over the operated and contralateral carotids were compared as the Carotid Uptake Ratio.

Mean (±sem) counts per gamma camera cell at 24 hours in the operated carotid of 43.6±3.0 were consistently higher than the reference artery of 35.4±2.4 (p<0.001). The overall carotid uptake ratio was 1.21±0.04 with that of 1.41±0.07 in patch angioplasty significantly higher than 1.14±0.04 found following standard arteriotomy closure (p<0.01). All 10 patch angioplasties were easily visible as "hot spots" on gamma camera compared to only 11 out of 23 with simple closure but there was no significant difference between platelet uptake with vein or Dacron patches with ratios of 1.47±0.35 and 1.37±0.15 respectively (p>0.5).

The greater local platelet accumulation with patch angioplasty may be due to turbulence from excessive widening of the vessel combined with the thrombogenic surface following endarterectomy. Until the significance of platelet accumulation is established platelet inhibitory therapy should be considered when patch closure is unavoidable.
ISOLATION AND CHARACTERIZATION OF cDNA CLONES FOR THE PLATELET MEMBRANE GLYCOPROTEINS IIb and IIIa
Authors: Butler-Zimrin A E , Bennett J S , Poncz M , Schwartz E , Surrey S , Eisman R , Heidenreich R A , Vilaire G
Published: August 2018

The platelet membrane GPIIb/GPIIIa complex on activated platelets contains receptors for fibrinogen, von Willebrand factor, and fibronectin. GPIIb and GPIlia also appear to be members of a family of membrane receptors involved in cell-cell and cell-matrix interactions. To study the structure of GPIIb and GPIIIa, we have constructed an expression library in the vector lambda gtll using mRNA from the HEL cell line and screened it with polyclonal antibody against each platelet protein. HEL cells constitutively express proteins similar to platelet GPIIb and GPIIIa. A 3.2kb GPIIb cDNA clone was identified that encodes for all 1008 amino acids of GPIIb including the known N-terminal amino acids of the α Cand βsubunits. This confirms that GPIIb is synthesized as a single chain polypeptide that is cleaved into two disulfide-linked subunits posttranslation. Analysis of the amino acid sequence revealed a major C-terminal transmembrane domain in the βsubunit, two potential transmembrane domains near the N-terminus of the αsubunit, and four possible N-linked glycosylation sites. Approximately 30% amino acid identity was found between GPIIb and the available amino acid sequences for the larger chains of the fibronectin and vitronectin receptors. Initial sequence analysis of a 3.8kb cDNA for GPIIIa included the known N-terminal amino acids of the platelet protein. Northern blot analysis was performed using HEL cell total RNA. The GPIIb cDNA hybridized to a 4.1kb mRNA while the GPIIIa cDNA hybridized to a 5.8kb mRNA. This indicates that the two cDNAs do not cross-hybridize and suggests that GPIIb and GPIIIa are encoded by separate genes. The availability of these cDNA for GPIIb and GPIIIa will facilitate study of the structure and function of the proteins and will aid in clarifying their relationship to other adhesive protein receptors.
EFFECT OF RABBIT THROMBOMODULIN ON THE INHIBITION OF THROMBIN BY ANTITHROMBIN IN PRESENCE OF EXOGENOUS HEPARIN: ROLE OF THE ACIDIC DOMAIN OF THROMBOMODULIN
Authors: Bourin M C , Bjôrk I , Boffa M C , Lindahl U
Published: August 2018

Thrombomodulin (TM) is an endothelial cell membrane protein chat acts as a cofactor for Protein C (PC) activation by thrombin (T). Rabbit TM also prevents fibrinogen clotting by T (direct anticoagulant activity) and accelerates T inhibition by antithrombin (AT-dependent anticoagulant activity). Rabbit TM was previously found to contain an acidic domain (presumably a heparin-like polysaccharide), separated from the PC cofactor site but required for the direct and AT-dependent anticoagulant activities. Although binding of TM to T modifies the specificity of T, it does not involve a modification of the catalytic site or a global conformational change of the enzyme.

A non-acidic form of rabbit TM was obtained by limited proteolytic degradation of the acidic form. The non-acidic form retained only the PC cofactor activity. The acidic and non-acidic forms of rabbit TM were compared with regard to their effects on the inhibition of T by AT in presence of exogenous heparin (hep). The AT-heparin complex was preformed by incubation for 5 min at 37°C of AT (0.1 μM) with high-affinity heparin (HA-hep, 250 ng/ml) and then added to a solution of T (90 nM) or T preincubated with either the non-acidic form (250 nM) or the acidic form (160 nM) of TM. Residual T activity was determined after various times for up to 5 min. Controls were performed by substituting buffer for TM. It was found that T bound to the non-acidic form of TM was inhibited at the same rate as free T (90% T inhibited within 30 sec), whereas T bound to the acidic form of TM was inhibited at a much slower rate (40 to 50% T inhibited after 5 min). Similar results were obtained even when HA-hep was used in excess (up to 750 ng/ml). The data suggest that the acidic domain of rabbit TM is primarily responsible for the retardation of T-AT complex formation in presence of exogenous hep. It is proposed that the polyanionic (endogenous heparin-like) component of rabbit TM blocks a site on T required for binding of T to hep in the ternary T-Hep-AT complex.
THROMBOMODULIN ON EXTRAVASCULAR MEMBRANES
Authors: Boffa M C , Burke B , Haudenschild C C
Published: August 2018

The distribution of thrombomodulin (TM) antigen (Ag) was studied in the rabbit using an affinity-purified antibody raised against rabbit TM in a goat. Tissues were obtained from 8 New Zealand rabbits. Paraffin-embedded sections were prepared from various organs. Staining was performed using the avidin-biotin peroxi dase method. TM was found to be best and most consistently preserved after perfusion of the vasculature with fixative (buffered formalin) i.e. perfusion of the systemic vasculature via the ascending aorta and individual perfusion of the pulmonary, portal, coronary vasculature via the appropriate artery or vein. A positive reaction was observed on the entire endothelial surface of the vascular system: arteries, veins ahd capillaries. In contrast, parenchyma, secretory epithelia, connective tissue, cartilage, bone and nerve tissue were not stained.

Interestingly, TM antigen was also found on the serosae: peritoneum, pericardium and epicardium and in the intralobular folds of the pleura, on the synovial membrane of the knee joint and on the entire surface of the spinal and cerebral arachnoid. The reaction was maximal on the intima of the large arteries, on the arachnoid and on the synovial membrane. The intensity of the reac tion did not depend on the organ examined but on the perfusion quality, except for arachnoidal and synovial membranes which were stained even without initial perfusion. The presence of TM on the membranes of body cavities was confirmed by the recovery of TM activity (as cofactor of protein C activation by thrombin in a chromogenic assay) in intraperitoneal lavage in vivo and in fluid used to rinse the brain arachnoidal surface ex vivo.

These findings suggest the presence of potent anticoagulant mechanisms in the systems of slow circulating fluids such as the cerebrospinal and synovial fluids and in the virtual spaces of the serosae. While no thrombin is present there in physiological conditions, anticoagulant activity in these locations may be very important in case of even mild permeability changes, such as in inflammation. As anticoagulant systems assure the fluidity of blood in the vasculature, the TM(-PC) system on body cavities linings may assure free mobility and absence of adhesion of the membranes.
HUMAN THROMBOMODULIN cDNA:SEQUENCE AND TRANSLATED STRUCTURE
Authors: Beeler D , Fritze L , Soff G , Jackman R , Rosenberg R
Published: August 2018

A 750 bp bovine Thrombomodulin (TM) cDNA fragment was used as an hybridization probe to screen an oligo-dT primed Lambda gtll. cDNA library prepared from human umbilical vein endothelial cell mRNA. A 2.4 kb positive human clone was isolated which showed an 80% nucleotide sequence homology with bovine TM cDNA. This clone and a 550 bp fragment from its 5' end were used to further screen the oligo-dT primed library as well as randomly primed library prepared from the same mRNA. The cDNA clones obtained allow us to describe the overall structure of human TM and reveal that it is extremely similar to the structure of bovine TM, especially as the bovine TM is organized like the receptor for low density lipoprotein (LDL R). Both TM and LDL R exhibit short cytoplasmic C-terminal tails which are either neutral or negatively charged. Other coated pit receptors such as the insulin receptor or the epidermal growth factor (EGF) receptor have very large cytoplasmic regions with a complex tyrosine kinase segment as well as multiple sites for phosphorylation. Both TM and LDL R possess a transmembrane region and an immediately adjacent extracellular serine/threonine rich region which in LDL R has been shown to bear 0-1inked sugars. Both TM and LDL R contain a more distal area of cysteine rich repeats, first noted in the EGF precursor and termed EGF type B. However, the TM EGF type B repeats appear to have been duplicated in TM resulting in their being 6 of them rather than the 3 found in LDL R. The N-terminal half of LDL R is thought to contain the ligand binding region of the receptor and is constructed from multiple cysteine rich repeats similar to those of Complement factor C9. The structure of this region of TM is quite different from that of LDL R, possessing few cysteines. We suspect that protein C and/or thrombin may bind to this unique domain of TM.
A LOW pH PREVENTS DENATURATION OF HEAT TREATED ANT I -HAEMOPHILIC CRYOPRECIPITATE
Authors: Skjonsberg O H , Gravem K , Kierulf P , Godal H C
Published: August 2018

The AIDS epidemic has necessitated heat treatment of FVIII:C products in order to inactivate HIV-virus. When antihaemophilic cryoprecipitate (CP) is heated (68°C, 24 hours), the solubility and the concentration of FVIIIrC, von Willebrand factor and fibrinogen are substantially reduced. Such heat denaturation is prevented by addition of synthetic amino acids (SyntaminR 17) prior to lyophilization and heat treatment (Ref.rMargolis and Eisen, Skjønsberg et al.). The exact mechanism of this protective effect is not known, but in our opinion, the large buffering capacity of Syntamin is of major importance. Thus, in the presence of Syntamin (4 mg/unit FVIIIrC), a pH of 7.4 was maintained during lyophilization and heat treatment, while in ordinary CP, pH rose from 7.8 to 8.6 during the same procedure. Moreover, heat denaturation of CP was prevented by replacing Syntamin with a phosphate buffer, keeping a stable pH below 7.4. It is of interest to notice that apart from the effect on pH, we were unable to observe any influence of Syntamin on heat denaturation of plasma proteins in solution, on degradation of FVIII:C and fibrinogen by thrombin or plssmin or on the solubility of fibrin in plasma and CP.

Heat treatment of CP at various pHs indicated that the :est product was obtained when pH .was kept between 6.5 and 6.3. By using an acidic buffer instead of Syntamin, the disadvantages of the latter, such as increased residual moisture leading tc discolouration, a probable stabilizing effect on virus and increased costs, may be avoided.
ANTI-HIV AFTER HEATED CLOTTING FACTOR CONCENTRATES IN HEMOPHILIACS
Authors: Mariani G , Ghirardini R , Verani P , Mandelli F , Rossi G B , Mannucci M P , Butto S
Published: August 2018

In Italy, heated concentrates became the only source of hemophilia therapy since July 1985, when a government act enforced their use instead of nonheated concentrates. Since then 63 anti-HIV seronegative hemophiliacs treated with heated concentrates were followed-up prospectively, focusing on the development of anti-HIV. Anti-HIV (documented by persistent positivity for ELISA and WB) occurred in 6 patients who had no other risk factor for HIV infection. For 3, anti-HIV was first found in Sept., Oct. or Nov. 1985 i.e. within 4 months of the last infusion of unheated concentrates (July 1985). For another patient, anti-HIV was found in Sept. 1986, but no other sample was available after the last negative test (Nov. 1985). For these 4 cases, therefore, we cannot exclude that seroconversions are due to nonheated concentrates used until July 1985. For 2 patients, however, anti-HIV occurred in July 1986, i.e. 11 months after change to heated concentrates. For both a hemophilia A patient (treated exclusively with a concentrate dry-heated for 72 hr at 68°C) and a hemophilia B patient (treated with both a steam-heated concentrate and a concentrate dry-heated for 72 hr at 68°C) the last seronegativities were found in March 1986, i.e. 7.0 and 7.5 months after commencing the use of heated concentrates or 3.5 and 4.0 months before the first seropositivity. The overwhelming majority of heated concentrates were prepared from non-donor-screened plasma. In conclusion, two anti-HIV occurred in previously seronegative patients treated exclusively with heated concentrates. Intensity and duration of concentrate exposure to heating were greater than those for the commercial dry-heated concentrate (60°C for 30 hours) that caused two reported seroconversions.
NO ANTI-HIV SEROCONVERSION AFTER REPLACEMENT THERAPY WITH PASTEURIZED F VIII CONCENTRATE. A STUDY OF 151 PATIENTS WITH HEMOPHILIA A OR VON WILLEBRAND'S DISEASE
Authors: Schimpf K l , Kraus B , Kreuz W , Brackmann H H , Haschke F , Schramm W
Published: August 2018

Transmission of hepatitis viruses and HIV has proven to be a risk of replacement therapy in hemophilia. As regards F VIII products a concentrate (Hemate HS or P) in which viruses are inactivated by heat-treatment over 10 hours at 60° C in aqueous solution is available since 1979. Our clinical studies have shown that this product does not transmit HBV and HNANBV. As the product was manufactured by 80% from US plasma it was necessary to prove that it also does not transmit HIV. As it is, for ethical reasons, not possible to treat a control group with non-virus-inactivated F VIII, non-transmission of HIV can only proven if anti-HIV seroconversion does not occur in larger groups of patients treated exclusively with this virus-inactivated product.We collected data from 151 patients treated with Hemate HS (P) who had never before received blood or blood products. Therapy was started between Feb. 1979 and Jan. 1986 (median July 7,1983). The median length of observation till the last anti-HIV testing was 24 (3 - 83) months. 112 patients were observed longer than 13 months. The median total dosage was 17,000 (500 -2,155,375) IU of F VIII, the median patient age was 6 (0,5 - 68) years. In none of these patients anti-HIV seroconversion (ELISA test) was observed. According to the rule of three, the upper 95% confidence limit for a random sample of 60 cases with zero events would be 3/60 or 5%. For greater numbers of n cases, as in our study, the range of confidence narrows increasingly. The period of observation of this study is hitherto the longest.