bluuebunny/crossref_metadata_2025_split huggingface dataset:
ENHANCED THROMBIN-INDUCED AGGREGATION AND INOSITOL TRISPHOSPHATE FORMATION OF PLATELETS FROM SPONTANEOUSLY HYPERCHOLESTEROLEMIC RATS
Authors: Winocour P P , Rand P D , Vickers J D , Kinlough-Rathbone R L , Mustard J F 
Published: August 2018

Platelets from rats with diet-induced hypercholesterolemia are hypersensitive to thrombin through a pathway independent of released ADP or thromboxane A2 (TXA2) formation. We examined if platelets from rats with spontaneous hypercholesterolemia (HC) are similarly hypersensitive. HC rats (plasma cholesterol: 130±4 mg/dl, n=15) were compared with their normocholesterolemic genetic controls (NC) (87±4 mg/dl, p<0.001, n=16). Total cholesterol/109 platelets was not different between the groups (HC: 0.314±0.032 μmole, n=7; NC: 0.357±0.046 ymole, n=7). Washed platelets were prelabelled with 14c-serotonin. In the presence of aspirin (to inhibit TXA2 formation) and creatine phosphate/creatine phosphokinase (CP/CPK) (to remove released ADP), HC platelets aggregated more (22±2%, n=ll) than NC platelets (10±4%, n=12, p<0.01) in response to thrombin (0.065 U/ml); 14C release was not different. Thrombin causes inositol mono-, bis-, and trisphosphate (IP, IP2, IP3) formation from phosphoinositides (PI, PIP, PIP2 respectively) in rabbit platelets; IP3 may be involved in Ca++ mobilization and the release of granule contents. We examined if enhanced inositol phosphate formation was associated with hypersensitivity of HC platelets. Platelets were prelabelled with 3H-inositol and stimulated with thrombin (0.057 U/ml) for 30 sec in the presence of aspirin and CP/CPK. Li+ (20 mM) was used to prevent degradation of inositol phosphates to inositol. 3H-IP, IP2 and IP3 were isolated by ion-exchange chromatography. The increase in radioactivity (dpm/109 platelets) in IP2 and IP3 following thrombin stimulation was greater in HC platelets (IP2: 2210±160, n=4; IP3: 1430±180, n=4) than in NC platelets (IP2: 660±150, n=4, p<0.001; IP3: 490±100, n=4, p<0.01); IP was not different.Thus platelets from spontaneously HC rats are hypersensitive to thrombin independently of released ADP or TXA2 formation. This hypersensitivity is associated with only moderate increases in plasma cholesterol and no detectable increase in total platelet cholesterol. Enhanced labelling of IP3 may indicate that enhanced activity of the pathways leading to IP3 formation is associated with this hypersensitivity.
IMMUNOLOGICAL ASSAYS FOR THE Ca(II)-DEPENDENT AND NONCa(II)-DEPENDENT CONFORMATIONS OFJtLUMAN PROTELN Z (PZ)
Authors: van der Meer F J M , van Tilburg N H , Poort S R , Briët E , Bertina R M 
Published: August 2018

Human PZ was purified using bariumcitrate absorption and elution, DEAE-Sephadex chromatography and gelfiltration on Sephadex G-200. The purified protein was used for the development of a specific rabbit antiserum, which could be used in a electroimmunoassay (EIA). Using immunoaffinity procedures, two populations of antibodies were isolated from the antiserum: one against the Ca(II)-dependent conformation of PZ(Ca(II)PZAg) and one against the nonCa(II)-dependent conformation (nonCa(II)PZAg). These antibody populations were used for the development of specific and highly sensitive immunoradiometric assays for Ca(II)PZAg and nonCa(II)PZAg. In healthy volunteers PZ levels varied from 0.28 to 1.96 U/ml (mean value 0.99 ± 0.34 U/ml). The observed variation was much more extensive than for other vitamin K-dependent proteins. Repeatedly testing of individuals with low PZAg levels gave comparable results, excluding acute phase reactivity of PZ. The mean value for the Ca(II)PZAg/nonCa(II)PZAg ratio was 1.04 ± 0.27 (range 0.51 - 1.58).Interestingly, PZ antigen levels were found to be severely reduced in 60 plasmas of patients stably treated with oral anticoagulants. At increasing intensity of treatment nonCa(II)PZAg decreased: 0.14 ± 0.04 (INR 2.6), 0.12 ± 0.04 (INR 3.0), 0.09 ± 0.03 (INR 3.4), 0.08 yp 0.03 (INR 3.8), 0.06 ± 0.01 (INR 4.2) and 0.06 ± 0.02 U/ml (INR >4.4).The decrease in nonCa(II)PzAg was much more extensive than observed for the nonCa(II)Ag of other vitamin K dependent factors (factors II, VII, IX, X, protein C and protein S).In general concentrations of Ca(11)PZAg were more reduced than those of nonCa(II)PZAg (ratio: ∼ 0.7). This ratio was virtually independent of the intensity of oral anticoagulant treatment.
MOLECULAR MARKERS OF HEMOSTATIC ACTIVATION: COMPARISON OF PLASMA AND URINARY LEVELS
Authors: Ashley H W , Fareed J 
Published: August 2018

We have compared plasma and urine levels for both normal control and aspirin treated groups (n=ll) using standard methods for fibrinopeptide, A (FPA), platelet factor 4 (PF-4), g-throm-boglobulin (β-TG), Bβ; 15-42 peptide (BβP), and tissue plasminogen activator (t-PA). Normal males and females of varying ages (22-50) made up the two groups. For aspirin treatment, 300 mgs/ day of aspirin was given PO for 3 days prior to the 24-hour urine and blood collection. The following data was generated:These results indicate that aspirin treatment produces a marked decrease of both plasma and urinary levels of molecular markers of hemostatic activation, suggesting that aspirin produces its effect at multiple sites within the hemostatic network. Profiling of urinary markers of hemostatic activation may also prove useful in the evaluation of prothrombotic states and evaluation of antithrombotic drugs.
MOLECULAR HETEROGENEITY IN FAMILIAL HEPARIN COFACTOR II DEFICIENCY
Authors: Andersson T R , Larsen M L , Abildgaard U 
Published: August 2018

In our normal material of 379 blood donors,3 indivi-uals had values below mean −2.5 SD (below 56%).Further studies revealed hereditary deficiency in two of these individuals. In the family study,5 out of 7 individuals had heparin cofactor II (HC II) values below 56%. In only one of these 5 a history of DVT was obtained. In deceased members of her family, however, frequent episodes of thromboembolic disease had occurred.Crossed immunoelectrophoresis (CIE) was performed in plasma from deficiency individuals from both families. Heparin in the first dimension gave a pattern similar to that observed with normal pooled plasma. The inactivation of thrombin by HC II is preferentially accelerated by dermatan sulfate (DS).HC II consumption in in vitro coagulation is increased by DS rather than by heparin. This prompted the addition ofTBS to the first dimension, which makes the antigen move faster, but produced no alteration in the antigenic pattern or size, neither in pooled plasma nor in a family member with normal activity. In family members with low HC II activity, DS in the first dimension, resulted in an abnormal CIE pattern with two distinct precipitation arcs with identity pattern. Molecular heterogeneity of HC II has previously not been reported.Screening 70 individuals who had sustained thrombosis before the age of 50,no values suggesting congenital deficiency were encountered.Conclusion. The CIE findings suggest that plasma of affected family members contain two types of HC II molecules of immunological identity. One type shows the normal accelerated mobility in the presence of DS and the other lacks this effect. The abnormal electrophoretic pattern becomes, however, first apparent when DS is added to the first dimension. When looking for molecular defects in congenital HC II deficiency, it is important that the first dimension is run with DS.
PROMOTOR MUTATIONS IN A PATIENT WITH HAEMOPHILIA B LEYDEN
Authors: Reitsma P H , Riemens A M , Bertina R M , Briít E 
Published: August 2018

Haemophilia B Leyden is characterized by low levels of factor IX antigen and activity before the age of 15, whereas after puberty factor IX levels rise at a rate of about 4-5% per year. To date such a genetic variant of factor IX synthesis has been reported in two (probably related) Dutch families, in a Greek and in an American family of Armenian descent. Laboratory and clinical investigations indicate that the factor IX protein is normal but that the regulation of factor IX synthesis has come under the control of the steroid hormone testosterone. We have started to investigate the factor IX gene in a patient from a Dutch family in an attempt to explain the aberrant regulation.Southern blotting revealed no gross deletions or insertions in the factor IX gene. Therefore the promotor region of the factor IX gene was cloned and subjected to a detailed restriction enzyme analysis. This also did not indicate that significant DNA deletions or insertions had occurred. Subsequently we established the nucleotide sequence of the DNA surrounding the first exon which encompassed about 600 basepairs of the promotor region. Two deviations from previously published sequence data were recorded. Firstly, an A T change was noted in the presumed "tata" box region 20 basepairs upstream from the start site of mRNA synthesis. Secondly, at position −423 a T C change was found which lies 13 basepairs upstream from a potential alternative "tata" box.The point mutation at position −20 might well explain the failure of gene expression during the prepuberal stage of the disease. Whether the same point mutation also leads to the testosterone effects or that a second sequence variation is a prerequisite for this phenomenon remains to be established from studies on the promotor region in representatives of the Greek and American families. Eventually the introduction of chaemeric genes, containing the various promotor regions, into testosterone responsive cells should delineate the promotor sequences responsible for the variations in factor IX gene expression.
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
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.
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.