bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies on Blood Coagulation and Fibrinolysis in Patients Bitten by Bothrops jararaca (jararaca)
Authors: Maruyama M , Kamiguti Aura S , Cardoso J L C , Sano-Martins Ida S , Chudzinski Ana M , Santoro M L , Morena P , Tomy Sandra C , Antonia Luci C , Mihara H , Kelen Eva M A
Published: June 2018

The blood coagulation and the fibrinolytic systems of nine patients envenomed by Bothrops jararaca in São Paulo (Brazil) were studied. Five of the accidents were caused by young snakes (<50 cm). On admission, four patients had non-clotting and three partially-clotting blood. Fibrinogen levels were decreased due to the thrombin-like activity of the venom as expected. Consequent secondary activation of the fibrinolytic system was evident from the low levels of alpha-2-antiplasmin and the high titres of fibrin(ogen) degradation products. High titres of cross-linked fibrin fragment D (D-dimer) in seven patients together with decreased platelet counts and/or factor V, and/or factor VIII in some, suggests intrinsic thrombin formation as these factors are not consumed in the defibrinogenation induced by venom thrombin-like fractions such as Ancrod and Batroxobin. However, normal or increased levels of antithrombin III in all and normal levels of factor II in eight patients do not support this interpretation. The existence of variable concentrations of other proteins in the venom of B. jararaca such as botrocetin and thrombocytin isolated from B. jararaca and B. atrox or crotalocytin from Crotaliis horridus venom should be considered. Such proteins are known to activate factors V, VIII, XIII, and platelets without affecting prothrombin (factor II) and antithrombin III. Slower recovery of the haemostatic disturbances after antivenom administration to patients bitten by young snakes suggests a more severe coagulopathy in such accidents. This is supported by clinical observations.
Impaired Fibrinolytic Capacity in Patients with Inflammatory Bowel Disease
Authors: Gris J C , Schved J F , Raffanel C , Dubois A , Aguilar-Martinez P , Arnaud A , Sanchez N , Sarlat C , Balmès J L
Published: June 2018

The venous occlusion test was applied to 17 patients with inflammatory bowel disease (IBD; 7 cases of Crohn’s disease, 10 cases of ulcerative colitis). Results were compared to those obtained in 20 healthy matched control subjects. Patients with IBD had significantly decreased t-PA Ag release (p <0.001) and had no significant vWF Ag release. Residual PAI activity was evidenced after venous stasis in the IBD group but not in the control group. Hypofibrinolysis was more important in patients with an evolutive IBD than in patients with IBD in remission. Impaired systemic fibrinolytic capacity might contribute to an increased risk for thromboembolic complications and to the pathogenesis of inflammatory bowel disease.
Low Anticoagulant Activity of High Sulphated Heparan Sulphates
Authors: Kovensky J , Sassetti B , Fernández Cirelli A , Kordich L
Published: June 2018

Two high sulphated heparin-like polysaccharides (LI, MW 16,000 and L2, MW 11,700) were isolated from rat liver tissues, after DEAE-cellulose chromatography. Heparan sulphates from heart and lung tissues were isolated for comparison and fractionated according to their molecular weight. The anticoagulant activities in vitro were studied using clotting antifactor Xa, antifactor IIa, and APTT assay methods, falling in a narrow range (5-44 IU/mg) although the wide variability m molecular weight and sulphate content. The heparan sulphate nature of fractions L1 and L2 (sulphate/disaccharide ratio 2.05 and 2.48, respectively) has been verified by: a) low iduronic/glucuronic acid ratio; b) nitrous acid degradation followed by gel chromatography; c) heparinase treatment followed by gel chromatography; d) electrophoretic behaviour. Native proteoglycans have been isolated and the glycosidic chains compared with L1 and L2. Their anticoagulant activities in vitro and the fact that antiXa clotting activity was not neutralized by protamine sulphate are in accordance with the results of structural studies.
In Vitro and In Vivo Antiaggregant Effects of Magnesium Halogenates
Authors: Cantón R , Manzanares J , Alvarez E , Zaragozá F
Published: June 2018

The in vitro antiplatelet aggregating activity of magnesium and magnesium associated with soluble citroflavomJids (hesperidin and eriodictin, 1:1) is well-established.

The degree of inhibition of in vitro platelet aggregation activity produced by different concentrations of magnesium halogenates was determined. ADP (4 μM) was used to induce aggregation following Cardinal and Flowers’ (1) technique.

Antithtombotic activity was studied in vivo. The differencein duration. of ADP-induced respiratory dysfunction was compared between animals fed 25 mg/kg magnesium halogenates for 10 days before testing and controls.

An increase in circulating platelets was observed in rats treated with magnesium halogenates.
An Acquired Factor VIII Inhibitor Responsive to High-Dose Gamma Globulin
Authors: Green David , Kwaan Hau C
Published: June 2018

A 55-year-old previously well woman noted easy bruising and developed a swollen, painful leg after minimal trauma. A compartment syndrome was diagnosed, and medial and lateral fasciotomies were performed with evacuation of a massive hematoma. However, blood rapidly reaccumulated in the wound. The VILI : C level was 2%, and 4 Bethesda units of factor VIII inhibitor were detected. After initial treatment with clotting factor concentrates and corticosteroids failed to control bleeding or reduce inhibitor titers, gamma globulin, 25 g daily for 5 days, was administered. The inhibitor became undetectable, VIII :C levels rose, and bleeding stopped. However, 5 days later VIII :C levels were again low and bleeding recurred. A second course of gamma globulin, 50 g daily for 2 days, was accompanied by a prompt increase in VIII :C, and uneventful recovery. In conclusion, in this patient with an autoantibody to Vili : C, a response to gamma globulin was observed on two occasions, and the second response came when steroids were being tapered and the patient was on no other medication.
Binding of Aggregated Immunoglobulins to the Human Platelet Fc Receptors: A Mechanism of Platelet to Platelet Bridging
Authors: Parbtani Anwar , Clark William F , Caveney Anita , Reid Bruce
Published: June 2018

Aggregated immunoglobulins react with human platelets by occupying the Fc receptors present on their surface, inducing aggregation and the release reaction. We studied the effect of heat aggregated gammaglobulins (HAGG) on ADP-induced aggregation of platelets. We used the minimum concentration of ADP required to induce a reversible aggregation of platelets without any substantial amount of serotonin (14C 5HT) release. EDTA (5 mM) added at the peak of platelet aggregation resulted in rapid deaggrcgation ot these platelets. However, incubation of platelets with HAGG at a dose that did not by itself induce any aggregation or release reaction, followed by ADP addition resulted in an irreversible platelet aggregation of greater magnitude accompanied by a substantial release of 14C-5HT. The addition of EDTA at the peak of platelet aggregation failed to deaggregate these platelets. To determine whether the augmented aggregation response and the inhibition of deaggiega-tion was due to HAGG or a consequence of platelet release products, we used thrombin-degranulated platelets. The augmented aggregation response and the inhibition of deaggregation due to HAGG and ADP could be demonstrated using these platelets. To confirm that the binding of HAGG to the platelet Pc receptors was responsible for these observations, we incubated platelets with an excess of Fc fragments of IgG prior to the addition of HAGG and ADP. This abolished the aggregation response observed previously. From this study we conclude that interplatelet bridging by HAGG renders the platelets hyperag-gregable and appears to be a mechanism involved in maintaining platelet aggregates.
Calibration of Lyophilized Standards for Ristocetin Cofactor Activity of von Willebrand Factor (vWF) Requires vWF-Deficient Plasma as Diluent for Dose-Response Curves
Authors: Rodeghiero Francesco , Castaman Giancarlo
Published: June 2018

Calibration of local standards for ristocetin cofactor activity of von Willebrand’s factor (vWF : Rcof) against reference preparations is required to achieve a better standardization of this measurement. We have observed that in calibration assays (multiple line parallel bioassay) for vWF : Rcof, measured aggregometrically, the type of diluent used to obtain the dose-response curves exerted a critical influence on the final potency estimation when a fresh-frozen plasma against a lyophilized standard or two lyophilized preparations were to be compared. This pattern was observed with various commercial standards and also with the First International Reference Preparation for Factor VIII-Related Activités in Plasma. The choice of diluent was not relevant if two fresh-frozen plasma preparations were to be calibrated. Systematic experiments using as diluents Trissaline, Tris-buffered-albumin solution or vWF-deficient plasma (vW-plasma) showed that the ratio lyophilized/fresh-frozen preparations was invariably lower with albumin solution than with vW-plasma. Tris-saline partially corrected this discrepancy, but yielded less precise estimates. Preliminary results with cryosuper-natant-plasma seems to indicate that this material is a valid substitute for vW-plasma, but confirmatory experiments are required For the moment, plasma obtained from patients with severe type III vW-disease should be considered the “ideal” diluent for calibration assays involving lyophilized preparations.
Combined Use of DNA Probes in First-Trimester Prenatal Diagnosis of Hemophilia A
Authors: Sampietro M , Camerino G , Romano M , Cappellini M D , Fiorelli G , Brambati B , Guerneri S , Ferrari M , Travi M , Krachmalnicoff A , Mannucci P M
Published: June 2018

First-trimester prenatal diagnoses of hemophilia A were heretofore obtained by using either intragenic factor VIII markers or linked cxtragcnic polymorphic markers. Postulating that the combined use of all the available intragenic and extragenic markers can render such diagnoses more frequently feasible and more reliable, we carried out ten first-trimester prenatal diagnoses in male fetuses at risk for hemophilia A by DNA analysis of chorionic villus employing in combination the intragenic Bcl I polymorphism and the St 14 (DXS 52) or DX 13 (DXS 15) extragenic probes. A diagnosis of hemophilia was obtained in three fetuses, with a diagnosis of normal fetus obtained in the remaining seven. Seven diagnoses are confirmed by factor VIII assays carried out at the time of abortion, in the mid-Trimester or at birth. A factor VIII probe recognizing Bcl I polymorphism was useful in 4 of 6 diagnoses; St 14, in 5 of 6; and DX 13 in 3 of 5. In two cases, St 14 was the only useful probe for diagnosis. Even though no recombination between extragenic probes and factor VIII gene was detected in this study, when only extragenic markers were informative we advised diagnostic confirmation on fetal plasma obtained by fetoscopy. Hence, first-trimester prenatal diagnosis of hemophilia A is feasible for the great majority of fetuses at risk through combined use of all the available intragenic and extragenic probes, providing key family members are available.
Factor XII, Plasma Prekallikrein, α2-Macroglobulin and C1-Inhibitor Levels in Renal Allograft Recipients During Immunosuppression with Cyclosporin A – Sequential Measurements over Four Months in 17 Patients
Authors: Lohri A , Huser B , Lämmle B , Oberholzer M , Thiel G , Duckert F
Published: June 2018

Factor XII clotting activity (F XII), plasma prekallikrein amidolytic activity (PK), α2-Macroglobulin (α2-M) and C1-Inhibitor (C1-Inh) antigens have been measured in 17 patients immediately before and sequentially for up to four months after kidney transplantation. Before transplantation mean F XII and PK levels were normal (99 ± 27% and 102 ± 21%, respectively, mean ± S. D.) and α2-M and C1-Inh levels were slightly elevated (115 ± 55% and 129 ± 32%, respectively, mean ± S. D.). In the first two weeks after transplantation a significant decrease of F XII to 65 ± 27%, of PK to 67 ± 20% and of α2-M to 88 ± 42%, and a rise of C1-Inh to 201 ± 44% (mean ± S. D.) were observed (2 p <0.005). F XII levels four month after operation remained significantly (2 p <0.05) lower than preoperatively. PK and α2-M values, however, were significantly higher (2 p <0.05) at four months as compared to the pretransplant period. Mean F XII levels in the 17 patients at various time points after transplantation correlated positively with PK, α2-M and serum albumin and negatively with CyA level and dose and serum bilirubin. PK and α2-M correlated positively with each other and albumin and negatively with creatinine, bilirubin and CyA (2 p <0.01). Whether CyA has a direct influence on production or consumption of F XII, PK, α2-M and C1-Inh, or whether the changes merely reflect altered protein metabolism awaits further study.
Fibrinolytic Response to Venous Occlusion and Fibrin Fragment D-Dimer Levels in Normal and Complicated Pregnancy
Authors: Ballegeer V , Mombaerts P , Declerck P J , Spitz B , Van Assche F A , Collen D
Published: June 2018

The fibrinolytic response to venous occlusion was assessed in 29 women with normal or complicated pregnancy, by measurements of total t-PA and free t-PA with specific ELISAs. The release of t-PA from the vessel wall was 11±9 ng/ml in nonpregnant women (mean ± SD, n = 6) but was markedly reduced throughout pregnancy. Following venous occlusion, free t-PA increased by 12 ± 11 ng/ml in non-pregnant women but remained below the detection limit of 2 ng/ml towards the end of pregnancy. A markedly reduced t-PA release with absence of free t-PA was also observed during late pregnancy in patients with insulin-dependent diabetes mellitus, intra-uterine growth ietaida-tion and pre-eclampsia.

Plasma levels of fragment D-dimer of cross-linked fibrin were measured with a specific FITS A in 79 pregnant women. D-dimer levels were 129 ± 36 ng/ml (mean ± SD, n = 8) in non-pregnant women and increased to 400 ± 170 ng/ml (n = 25) and 440 ± 220 ng/ml (n – 22) during the second and third trimester of pregnancy respectively. Significantly higher levels than observed in uncomplicated third trimester pregnancies were found in 3 out of 6 diabetic and in 2 out of 7 pre-eclamptic women.

It is concluded that the t-PA release after venous occlusion is significantly reduced during pregnancy. In addition, released t-PA is rapidly inhibited. The levels of fragment D-dimer increase during pregnancy, suggesting that, notwithstanding the marked impairment of the fibrinolytic response to venous occlusion, the fibrinolytic system remains functionally active.
Intranasal and Intravenous Administration of Desmopressin: Effect on F VIII/vWF, Pharmacokinetics and Reproducibility
Authors: Lethagen S , Harris A S , Sjörin E , Nilsson I M
Published: June 2018

A comparison was made of intranasal administration of 300 μg desmopressin (DDAVP) by spray, with intravenous administration of 0.2, 0.3 and 0.4 μg DDAVP/kg in 10 healthy volunteers. The effect of DDAVP was measured on F VIII/vWF complex and on plasminogen activator release. In addition, plasma levels of DDAVP were determined using a specific and sensitive radioimmunoassay. Moreover, the reproducibility of the spray effect in 10 healthy volunteers was tested after administration of 300 μg DDAVP intranasally by spray on 5 different occasions.

Plasma levels of DDAVP showed a clear dose-response with the maximum levels at 0.4 μg/kg i.v. The effect of the spray approximated the 0.2 μg/kg response. However, the maximum response in both F VIII/vWF complex and plasminogen activator release was obtained after 0.3 μg/kg i. v. The response to 0.4 μg/kg i. v. was not significantly different from the response to 0.3 μg/kg indicating that maximum stimulation was reached with 0.3 μg/kg. There was no correlation between plasma levels of F VIII/vWF and DDAVP indicating that the biological response to DDAVP is subjected to saturation kinetics.

The reproducibility of the effect of the spray dose on VIII : C was 21% (c.v.) and 27% for the intra-individual and interindividual variation, respectively, and compared favorably with intravenous administration.

Intranasal DDAVP (300 μg) is as effective as 0.2 μg/kg intravenously and provides an accurate, reproducible and convenient alternative to parenteral administration.
Plasma Prekallikrein Activity in Patients with Total Hip Arthroplasty
Authors: Hoffmann J J M L , Kortmann J H J P M
Published: June 2018

The behaviour of the contact system was studied in 40 patients with total hip arthroplasty, by measuring plasma prekallikrein, spontaneous kallikrein activity and factor XII. In the literature it had been shown that patients with complications from this operation had decreased prekallikrein and increased kallikrein activity (M. Nakahara. Acta orthop scand 1982; 53: 591-6). In the present study, comprising patients with and without pain and proven loosening of the hip prosthesis, these findings could only partially be confirmed. Patients with a loosened prosthesis had significantly lower prekallikrein (mean 0.78 ± 0.28 U/ml; p <0.01) than patients without problems, but no detectable kallikrein activity in plasma. Patients with pain but no loosening had normal prekallikrein (1.04 ±0 0.26 U/ml) and also no demonstrable kallikrein activity. Factor XII was normal in all patient groups. It is concluded that decreased prekallikrein is limited to patients with a loosened hip prosthesis, with or without pain.
F VIII Subunits: Purification and Antigenic Properties
Authors: Nordfang Ole , Ezban Mirella , Hansen Jan J
Published: June 2018

Factor VIII-Light Chain (FVIII-LC) and FVIII-Heavy Chain (FVIII-HC) were purified from human plasma by the use of immunosorbents containing monoclonal antibodies or human inhibitor antibodies. FVIII-LC was subsequently isolated in essentially pure state by cation exchange chromatography. The preparations obtained contained 50 ng of protein for each unit of FVIII-LC antigen (FVIII-LC: Ag).

Affinity purified FVIII-LC and FVIII-HC preparations containing less than 0.3% of the opposite subunit were added in FVIILC inhibition assay of hemophilia A inhibitor antibodies. FVIII-LC was able to fully block the inhibitor activity in 6 out of 7 hemophilia A plasmas and partially block the inhibitor activity of one plasma. FVIII-HC only blocked FVIILC inhibiting antibodies form the plasma that was not fully blocked by FVIII-LC. It is suggested that FVIII-LC can be used for immunotherapy of the patients whose FVIILC inhibiting antibodies are directed towards FVIII-LC.

When FVIII-LC was coupled to Sepharose at a concentration of 4800 units of FVIII-LC: Ag per ml Sepharose, 0.2 ml of the immunosorbent was able to bind 900 Bethesda units from 100 ml hemophilia A inhibitor plasma. This opens the possibility to remove FVIII inhibitor antibodies from circulation by extracorporeal immunotherapy with FVIII-LC coupled to Sepharose.
Continuous High-Dose Factor VIII for the Induction of Immune Tolerance in Haemophilia A Patients with High Responder State: A Description of Eleven Patients Treated
Authors: Scheibel E , lngerslev J , Dalsgaard-Nielsen J , Stenbjerg S , Knudsen J B ,   The Danish Study Group
Published: June 2018

Eleven severely affected haemophilia A patients (aged 6-42 y) with F VIII : C inhibitor (high responders) were treated with highdose F VIII in order to eliminate the inhibitors. The patients comprise Danish high responder patients treated during the period 1977-1985.

In all patients the inhibitors decreased significantly. In six, the inhibitor apparently disappeared (detection limit 0.4 Bethesda Units per ml) (BU/ml), in four patients a low level inhibitor of 0.4-1.4 BU/ml persisted. One patient is still on high-dose ;schedule. The duration of high-dose treatments ranged from less than one month up to 18 months. In all patients the tendency to spontaneous bleedings vanished when a measurable VIII : C level appeared in the post-infusion sample. The inhibitor suppression has allowed for extensive physical training and rehabilitation orthopaedic surgery. The patients are now able to conduct a normal haemophilic life on self-administered prophylactic doses of F VIII.
Requirement for Thrombin Receptor Occupancy During Platelet Secretion Under Aggregating and Non-Aggregating Conditions
Authors: Koike Kazuo , Holmsen Holm
Published: June 2018

The requirement for receptor occupancy in thrombin-induced secretion in human platelets has been studied. When increasing concentrations of thrombin were added to gel-filtered platelets containing a constant, high concentration of hirudin, dense granule secretion was initiated at lower thrombin concentrations than those required for α-granule secretion and aggregation; acid hydrolase secretion required higher concentrations. A 62-fold excess of hirudin produced abrupt stop of dense granule secretion and a-granule secretion when added to non-aggregating (no stirring) platelets shortly after thrombin; it had no affect after these secretory process had reached about 30% of their maximal values. Acid hydrolase secretion was, however, abruptly stopped by hirudin at any stage. When the platelets were allowed to aggregate, the three secretory processes increased their rates and were abruptly stopped by hirudin at any stage. Aggregation (optical) occurred slower than dense granule and α-granule secretion, and was reversed by hirudin when added before it had reached 30% of its maximum.

It is concluded that a-granule secretion, like dense granule secretion, only requires a short receptor occupancy to be completed, in contrast to the requirement for sustained occupancy for acid hydrolase secretion. α-Granule secretion might, however, require longer occupancy than dense granule secretion. Aggregation is believed to potentiate secretion through close cell contact and the secretion processes were inhibited by hirudin through hirudin’s effect on aggregation.
Shortening of Bleeding Time by 1-Deamino-8-Arginine Vasopressin (DDAVP) in the Absence of Platelet von Willebrand Factor in Gray Platelet Syndrome
Authors: Pfueller Sharron L , Howard Margaret A , White James G , Menon Chandrasekhara , Berry Elizabeth W
Published: June 2018

The Gray platelet syndrome is a rare disorder characterised by the absence of platelet a-granules and their contents. We describe a new patient and the effects of infusions of l-deainino-8-aiginine vasopressin (DDAVP). The patient had a prolonged skin bleeding time and his platelets had reduced numbers of a-granules, increased vacuolation and reduced retention on glass beads. Flatelet von Willebrand factor antigen (vWf:Ag) was undetectable and levels of platelet fibrinogen, p-thioniboglobulin, platelet factor 4 and thrombospondin were reduced. All tests of plasma coagulation factors were normal, including Factor VIII (F. VIII: C), vWf: Ag, ristocetin cofactor (R: CoF) and botrocetin cofactor. Platelet ATP, ADP, platelet albumin, surface membrane glycoproteins and 14C-serotonin uptake were also normal. Infusions of DDAVP increased plasma F.VIII:C, vWf:Ag and R.CoF and sliuitened the bleeding time un two occasions. This suggests that DDAVP shortens the bleeding time by releasing vWf: Ag and/or other proteins from cellular storage sites other than the platelet.
A Multi-Centre Study to Evaluate Method Dependency of the International Sensitivity Index of Bovine Thromboplastin
Authors: Peters R H M , van den Besselaar A M H P , Olthuis F M F G
Published: June 2018

In The Netherlands, a particular coagulometer method for prothrombin time (PT) determination with reduced sample and reagent volumes is used by 62% of the laboratories controlling oral anticoagulant therapy. This “micro-method” has been calibrated against the manual tilt-tube technique for PT determination by six Dutch laboratories. Each laboratory tested 20 fresh normal blood samples and 60 fresh patient blood samples using both methods with the same batch of bovine thromboplastin reagent, according to a detailed protocol. Both methods were comparable as to their precision, but PTs measured by the micromethod were significantly prolonged (p <0.001, Student’s t-test) as compared to the manual method. This effect is stronger for samples of normal subjects than for patients’ samples. It was assumed that the International Sensitivity Index (ISI) of the bovine thromboplastin for the manual method was 1.00 in each laboratory. The ISI-values of the bovine thromboplastin for the micro-method determined by the six laboratories ranged from 1.00 to 1.07 (mean 1.03, SD 0.03). Our results indicate that any other laboratory, using this thromboplastin and the micromethod, should obtain accurate assessment of the International Normalized Ratio from their own mean normal PT and an ISI which is 3% higher than the ISI supplied by the thromboplastin manufacturer for the manual tilt-tube method.
Impaired Fibrinolysis as an Essential Contribution to Thrombosis in Patients with Lupus Anticoagulant
Authors: Tsakiris D A , Marbet G A , Makris P E , Settas L , Duckert F
Published: June 2018

Lupus anticoagulants (LA) are IgG or IgM antibodies against phospholipids which in vivo represent an important thrombophilic factor despite their in vitro anticoagulant activity. We investigated the fibrinolytic system of 20 patients with connective tissue disease and positive LA, compared to a control group of 24 age- and disease-matched patients without LA. There was no statistically significant difference of alpha2-antiplasmin, plasminogen, fibrinogen, t-PA activity, D-dimers and heparin cofactor II, between the two groups. Although t-PA was uniformly low in both groups, plasminogen activator inhibitor activity (PAI) was significantly higher in LA cases (p <0.001). Increased PAI levels represent an inhibitory factor of the fibrinolytic defense mechanism, which together with other functional deviations may contribute to the thrombophilic tendency of LA patients.
Induction of Endothelial Cell/Macrophage Procoagulant Activity: Synergistic Stimulation by Gamma Interferon and Granulocyte-Macrophage Colony Stimulating Factor
Authors: Zuckerman Steven H , Surprenant Yvonne M
Published: June 2018

Inflammatory mediators such as endotoxin can stimulate the expression of procoagulant activity on both endothelial cells and macrophages while the monokines Interleukin 1, IL-1, and Tumor Necrosis Factor, TNF induce procoagulant activity on endothelial cells. Incubation of murine peritoneal macrophages with suboptimal concentrations of endotoxin results in a two fold increase in procoagulant activity. Macrophages incubated with gamma interferon, IFN γ, or Granulocyte-Macrophage Colony Stimulating Factor, GM-CSF, for 16 hours prior to endotoxin stimulation demonstrated a synergistic increase in procoagulant activity. A synergistic increase in procoagulant activity was also observed with primary cultures of human umbilical cord endothelial cells incubated with recombinant human IFN γ for 16 hours prior to endotoxin, TNF, or IL-1 stimulation. Human GM-CSF had no stimulatory effect on endotoxin or monokine induced endothelial cell procoagulant activity. The augmentation of macrophage and endothelial cell procoagulant activity by IFN γ and GM-CSF may provide a novel explanation for the role of these cytokines in acute and chronic inflammation.
Demonstration of Ac-Arg-Gly-Asp-Ser-NH2 as an Antiaggregatory Agent in the Dog by Intracoronary Administration
Authors: Shebuski R J , Berry D E , Bennett D B , Romoff T , Storer B L , Ali F , Samanen J
Published: June 2018

This study compared the anti-platelet effect of Ac-RGDS-NH2 which is a peptide fragment from fibrinogen to Ac-RGES-NH2 in which the aspartic acid (D) of Ac-RGDS-NH2 has been replaced by glutamic acid (E). When Ac-RGDS-NH2 was infused intracoronary at concentrations of 100–400 mM, acute platelet dependent thrombus formation in the dog coronary artery was inhibited. However, infusion of Ac-RGES-NH2 intracoronary at similar concentrations to Ac-RGDS-NH2 failed to inhibit platelet dependent thrombus formation in the dog. Ac-RGDS-NH2 and Ac-RGES-NH2 were also tested for their ability to inhibit collagen-induced platelet aggregation in vitro. Ac-RGDS-NH2 elicited concentration-dependent inhibition of collagen-induced aggregation with no effect of Ac-RGES-NHz otr collagen-induced platelet aggregation. Thus, Ac-RGDS-NH2 is an effective antiplatelet agent after intracoronary administration in the dog and also inhibits collagen-induced platelet aggregation in vitro. Ac-RGDS-NH2 is a specific inhibitor of platelet aggregation as replacement of the aspartic acid in Ac-RGDS-NH2 with glutamic acid results in complete loss of biological activity.
Risks to the Fetus of Radiologic Procedures Used in the Diagnosis of Maternal Venous Thromboembolic Disease
Authors: Ginsberg Jeffrey S , Hirsh Jack , Rainbow Andrew J , Coates Geoffrey
Published: June 2018

The diagnosis of suspected venous thromboembolic disease during pregnancy is problematic because of the fear of fetal irradiation with routine diagnostic procedures. In order to develop rational guidelines, we have calculated levels of radiation exposure to the fetus for the common diagnostic procedures and conducted a literature review to examine the fetal risks associated with these levels of radiation. A small increase in the relative risk of childhood cancer is suggested by a literature review of outcomes following low dose (less than 5 rads), in utero radiation exposure. With careful use of the available procedures, a diagnosis of venous thrombosis is possible with fetal radiation exposure of less than 0.50 rads and a diagnosis of pulmonary embolism is possible with fetal radiation exposure of less than 0.05 rads. The risk of such exposure is small, both in relative and absolute terms.
Measurement of Aspirin Concentrations in Portal and Systemic Blood in Pigs: Effect on Platelet Aggregation, Thromboxane and Prostacyclin Production
Authors: Bochner F , Siebert D M , Rodgers S E , McIntosh G H , James M J , Lloyd J V
Published: June 2018

Low doses of enteric-coated aspirin were administered orally to pigs. Plasma aspirin concentrations measured in blood obtained simultaneously from permanent catheters in a systemic artery and portal vein for 6 hours after dosage showed a large variation in the plasma aspirin concentration : time profile between pigs. After 50 mg single dose the ratio of the arterial : portal area under the plasma concentration versus time curve (AUC) was 0.63 ± 0.08 (mean ± SE, n = 6). In three pigs which received all three dosage regimens, the arterial : portal AUC ratios were 0.48 ± 0.05 after 50 mg single dose, 0.52 ± 0.02 after 100 mg single dose and 0.47 ± 0.02 after 100 mg daily for 1 week. Platelet aggregation in response to sodium arachidonate (1.65 mM) was completely abolished after chronic aspirin administration of 100 mg daily. Thromboxane production (pg/ 106 platelets) induced by this stimulus decreased from 536 ± 117 before aspirin to 57 ± 14 after aspirin (mean ± SE, n = 4; p = 0.03). Aortic prostacyclin synthesis, measured as 6-keto PGF1α (ng/disc after 10 min incubation), was 1.66 ± 0.28 (mean ± SE, n = 4) in untreated pigs and 0.95 ± 0.25 (n = 5) in treated pigs (p = 0.07). Results from this study support the idea that a difference between aspirin concentrations in the portal and systemic circulations can be achieved. Whether this can be translated into a clinically useful differential effect on the vessel wall compared to the platelet remains to be determined.
Epitope Localization of Anti-Factor VIII Monoclonal Antibodies Determined by Recombinant peptides
Authors: Ware Jerry , Toomey John R , Stafford Darrel W
Published: June 2018

In order to define the epitopes recognized by anti-coagulation factor VIII (FVIII) monoclonal antibodies, we have constructed a recombinant DNA epitope library from random fragments of the FVIII cDNA. The characterization of 33 different clones producing recombinant antigens in the expression vector, λgt11, has identified the epitopes for six different anti-FVIII monoclonal antibodies. The antigenic determinant for each antibody was defined by the overlapping or shared DNA sequence of multiple immunoreactive clones. One weak inhibitor of FVIII coagulant activity binds within the gly701 ser750 sequence of the FVIII sequence. An antibody which recognizes the amino-terminus of FVIII heavy-chain (within trp14 - tyr46) does not inhibit FVIII activity. Two non-neutralizing antibodies both map within residues asp807 - ser817 while two other non-neutralizing antibodies bind within lys1673 - pro1688. The general usefulness of this strategy for mapping FVIII antigenic determinants is discussed.
Methylene Blue Enhances Lateral Association of Fibrin Resulting in Rapid Gelation and Thick Fiber Formation
Authors: Carr Marcus E , Powers Patrick L
Published: June 2018

Methylene blue (MB) has been suggested as a therapeutic alternative for heparin reversal in patients sensitive to protamine. We investigated the impact of MB on the assembly and structure of thrombin induced fibrin and plasma gels. MB (1,600 μg/ml) reduced the thrombin clotting time (TCT) of plasma by 30% and of purified fibrinogen by 46%. Above 1,600 μg/ml, TCTs were prolonged due to MB mediated fibrinogen precipitation. The presence of 5 mM CaCl2 masked the effect of MB in both purified and plasma systems and lowered the threshold for MB-induced purified fibrinogen precipitation to 800 μg/ml. MB shortened the lag phase prior to thrombin-induced turbidity increase, and enhanced final gel turbidity. The fibrin fiber mass/length ratio increased from 5.2 to 13.1 × 1013 dalton/cm in purified fibrin gels and from 3.2 to 10.4 × 1013 dalton/cm in plasma gels as the MB concentration increased from 0 to 200 μglml. Due to the photooxidant effect of MB on fibrinogen, rapid time-dependent loss of fibrinogen clottability was obvious at low MB concentrations (50 to 400 μg/ml). At high MB concentrations, intense MB light absorption partially protected fibrinogen within the sample. Accurate measurements could only be made, however, when MB was added just prior to thrombin and the assays were performed in the dark. While erythrocytes may reduce the impact of MB photooxidation in whole blood, plasma samples must be shielded from light if reproducible results are to be obtained.
Defibrinogenation by Arvin Reduces Air-Drying-lnduced Arteriosclerosis in Rat Carotid Artery
Authors: van Pelt-Verkuil E , van de Ree P , Emeis J J
Published: June 2018

The effects of lowering plasma fibrinogen levels by Arvin injections have been investigated in a rat model of arteriosclerosis. In this model air-drying-induced endothelial denudation of the left carotid artery results in myointimal lesion development. Arvin (2 × 50 Units/kg) was given at 4 and 2 hours prior to surgery, reducing plasma fibrinogen levels at surgery to about 20%. Fibrinogen levels then normalized within 24 hours. Myointimal lesion development was evaluated morphometrically at 14 days after surgery. Statistical analysis of the morphometric data showed that defibrinogenation had resulted in significantly smaller myointimal lesions. Low plasma fibrinogen levels might thus be considered a negative risk factor for intimal smooth muscle cell proliferation in this model.
Influence of Whole Blood on Standard Curve for Heparin Measurement - Possible Heparin Binding by Red Cells
Authors: Melissari E , Thye C , Scully M F , Kakkar V V
Published: June 2018

Measurement of heparin ex vivo is usually with reference to standard curve prepared with a “spiked” normal human plasma pool (NHP). When the calibration curve was prepared by addition of heparin to whole blood before plasma separation, although the linear relationship was maintained the slope was increased in comparison to the classical standard calibration curve. It is concluded that the preparation of the calibration curve by addition of heparin to NHP may give erroneously high heparin levels in treated patients’ plasma, leading perhaps to inappropriate dosage. It was also observed that when heparin was added to blood of different haematocrit (prepared by addition of washed RBC to plasma) and plasma prepared, the subsequent APTT was decreased with the fall in haematocrit; suggesting that the laboratory monitoring of heparin treatment should take into account the patient’s haematocrit.
DN 9693: A Phosphodiesterase Inhibitor with a Platelet Membrane Effect
Authors: Jackson C , Ball J , Peel J , Lawry J , Greaves M , Preston F E
Published: June 2018

We have examined the in vitro effects of DN 9693 (piperidinylimidazo-quinazolinone) on various aspects of platelet reactivity. Our results are consistent with its known function as a phosphodiesterase inhibitor in that it increased platelet cyclic AMP, particularly in conjunction with an adenylate cyclase stimulator, and exerted a profound inhibitory effect on platelet aggregation responses to a variety of agonists. DN 9693 also inhibited ristocetin-induced platelet agglutination (RIPA). We therefore examined its effect on ristocetin co-factor assays and on the binding of a monoclonal antibody (McAb) to platelet membrane glycoprotein lb (GPIb). The drug inhibited the binding of the monoclonal antibody in a dose-dependent manner. This suggests an effect of the drug on the platelet surface membrane with reduced expression of GPIb. Our results indicate that in addition to its anticipated inhibitory effect on platelet aggregation, DN 9693 may also inhibit platelet adhesion.
The Inhibitory Effect of Aspirin on Fibrinolysis Is Reversed by Iloprost, a Prostacyclin Analogue
Authors: Bertelé V , Mussoni L , Pintucci G , del Rosso G , Romano G , de Gaetano G , Libretti A
Published: June 2018

The reduced fibrinolytic response after aspirin intake may be due to prevention of prostacyclin production. The effect of iloprost (a stable prostacyclin analogue) was tested on the fibrinolytic activity (euglobulin lysis area on fibrin plate [E.L.A.], t-PA antigen, PAI activity and PAI-1 antigen) of plasma drawn after venous stasis test from six healthy male volunteers, who each received all the following treatments according to a single-blind randomized cross-over design: placebo, iloprost, aspirin + placebo, aspirin + iloprost. The mean E. L. A. value after venous occlusion was significantly higher than the basal level after every treatment, but aspirin. Within each treatment group the t-PA antigen levels in response to venous stasis were significantly higher than the basal ones. PAI-1 antigen levels did not change significantly before and after venous stasis either within or among the treatment groups. These data are consistent with the hypothesis that the mechanism related to aspirin’s effect on fibrinolysis is mediated by suppression of vessel wall prostacyclin production. Aspirin’s inhibitory effect on fibrinolysis was in fact prevented by replacing endogenous prostacyclin with iloprost. Iloprost enhances fibrinolytic activity reduced by aspirin, but not by promoting t-PA release or by inhibiting release of the specific inhibitor, PAI-1.
Evaluation of Fibrinolytic Capacity in Plasma during Thrombolytic Therapy with Single (scu-PA) or Two-chain Urokinase Type Plasminogen Activator (tcu-PA) by a Combined Assay System for Urokinase Type Plasminogen Activator Antigen and Function
Authors: Wojta Johann , Binder Bernd R , Huber Kurt , Hoover Richard L
Published: June 2018

A combined assay for urokinase type plasminogen activator (u-PA) activity and antigen determination in plasma samples is described. This assay is based on binding of u-PA to an antibody immobilized on a microtiter plate followed by determination of the enzymatic activity of the bound u-PA. Thereafter bound u-PA antigen can be quantified by means of a specific peroxidase labelled monoclonal antibody against u-PA. By use of this assay system u-PA activity and antigen can be determined with lower detection limits of 0.08 IU/ml and 1.0 ng/ml, respectively, and intraassay as well as interassay coefficients of variation of 10% and 12% for activity and 5% and 7% for antigen determinations, respectively. Normal plasma levels of u-PA antigen could be determined to be 1.88 nglml ± 0.61. Furthennore, this assay system allows specific quantification of u-PA antigen and activity during thrombolytic therapy.
Effect of Physiological Concentrations of Vasopressin on Components of the Fibrinolytic System
Authors: Hariman H , Grant P J , Hughes J R , Booth N A , Davies J A , Prentice C R M
Published: June 2018

High physiological concentrations of plasma vasopressin (aVP) when achieved by infusion cause an increase in plasma factor VIII coagulant activity and shortening of the euglobulin clot lysis time (ECLT). To investigate the effects of aVP on components of the fibrinolytic pathway and on thrombin generation, t healthy volunteers were infused with saline for 30 min followed by aVP for L hour and blood samples taken every 30 min for measurement of aVR ECtf tissue-type plasminogen activator (t-PA), t-PA inhibition (tPA-I), plasminogen activator inhibitor L (PAI-1 Ag), activated partial thromboplastin time (APTT), fibrinopeptide A (FPA), fibrinopeptide B 15-42 (FPBß 15-42) and crosslinked fibrin breakdown products (XL-FDP). Plasma aVP rose to a median of 75 pg/ml after 90 min and fell to 13.8 pdml 30 min later. The APTT fell from 43.5 to 35 sec (p <0.01) but there was no change in plasma FPA or in XL-FDP. Plasminogen activator activity (106/ECLT2) increased from 25 to 736 units (p <0.01) and t-PA from 200 to 1012 mlU/ml (p <0.01). tPA-I fell from 8.0 to 2.7 IU/ml at 90 min (p <0.05) but PAI-1 Ag remained unchanged. Plasma FPBß 15-42 was 2.4 and 1.2 pmol/ml before infusion with aVP and showed a small rise to 3.5 pmol/ml after 60 min (p <0.05). The results show the effects of aVP on fibrinolysis are mediated by an increase in t-PA. In the absence of thrombin generation the rise in t-PA was not accompanied by changes in XL-FDP.
In Vivo Measurements of Fibrin Formation and Fibrinolysis in Operable Breast Cancer
Authors: McCulloch P , Douglas J , Lowe G D O , Murray G , George W D
Published: June 2018

Fibrin formation and fibrinolysis were estimated in 89 breast cancer patients by measurement in plasma of Fibrin Fragment Bβ 15-42 and Fibrinopeptide A (FPA), serum Fibrin(ogen) Degradation Products (FDPs) and plasminogen activator by Fibrin Plate Lysis Assay. Results were compared with (a) 26 patients with benign breast diseases; and (b) 45 healthy factory workers. FPA, FDP and Bβ 15-42 Levels were elevated in both breast cancer patients and benign disease patients, but there were no significant differences between these two groups. Cancer stage, patient age and smoking habits did not affect these results, but Oestrogen Receptor (ER) positive patients had higher Bβ 15-42 values than ER negative patients (p = 0.017). These results show that fibrin formation is enhanced preoperatively in patients with either benign or malignant breast disease. The fibrinolytic response to activated coagulation may be relatively deficient in breast cancer. The roles of malignancy, stress and other factors in the causation of these abnormalities require further assessment.
Plasminogen Activators and Plasminogen Activator Inhibitors in Liver Deficiencies Caused by Chronic Alcoholism or Infectious Hepatitis
Authors: Tran-Thang Chiên , Fasel-Felley Josette , Pralong Gerard , Hofstetter Jean-René , Bachmann Fedor , Kruithof Egbert K O
Published: June 2018

Plasma concentrations of tissue-type plasminogen activator (t-PA), urokinase (u-PA), plasminogen activator inhibitor 1 (PAI-1) and PAI-2 were studied in 53 patients with liver deficiency caused by chronic alcoholism (n = 40), viral hepatitis (n = 10) or malignant disease of the liver (n = 3) and compared to that of a control group (n = 20) of healthy subjects. u-PA and PAI-1 levels were significantly increased in all patients with chronic alcoholism, whereas high t-PA was only observed in combination with disturbed liver function tests or with liver cirrhosis (two and sixfold above control values, respectively). A good correlation was observed between t-PA and gamma glutamyl transferase (r = 0.615; p <0.001). In patients with infectious hepatitis or with malignant disease of the liver t-PA was normal whereas u-PA and PAI-1 were increased.

PAI-2 levels were close to or below the detection limit (15 ng/ml) in the control group and in most patients. However, in two patients with alcohol induced cirrhosis PAI-2 levels were approximately 45 ng/ml and in one patient with hepatocarcinoma even 66 ng/ml. Thus, in liver disease, marked elevations of t-PA, u-PA and PAI-1 levels may occur, with increased PAI-1 as an early marker of liver defects and t-PA a marker of severe liver defects.
Synthesis of Vitamin K-Dependent Proteins by Cultured Human Tumor Cells
Authors: Al-Mondhiry Hamid , Wallin Reidar
Published: June 2018

The observation that warfarin inhibits the growth and metastasis of certain types of clinical and experimental tumors suggests a role for vitamin K in tumor biology. We have investigated synthesis of vitamin K-dependent proteins in four malignant (lung epidermoid carcinoma, melanoma, colon adenocarcinoma, and breast adenocarcinoma) and three normal (colon epithelium, breast epithelium, and fibroblasts) cell lines of human origin grown in tissue cultures. Our results show the following: 1) Vitamin K-dependent carboxylase activity is present in all of the malignant and normal cell lines studied. 2) The malignant as well as normal cell lines synthesize a family of vitamin K-dependent proteins. Microsomal precursors of these proteins with apparent molecular mass of 74, 62, and 34 kDa are common to all malignant and normal cell lines whereas precursors of higher and lower molecular mass seem to be synthesized by some but not all tumor cell lines. 3) The 74 kDa precursor synthesized by colon carcinoma and breast carcinoma was positively identified as a precursor of protein S.
Binding and Degradation of Tissue-Type Plasminogen Activator by the Human Hepatoma Cell Line Hep G2
Authors: Otter M , Van Berkel Th J C , Rijken D C
Published: June 2018

In this study, binding and degradation of tissue-type plasminogen activator (t-PA) by the human hepatoma cell line Hep G2 was investigated. Binding at 4° C was time-dependent and reached a maximum after ca. 2 hours. Scatchard analysis of saturation experiments showed about 170,000 high affinity binding sites for t-PA per cell with an apparent Kd of 90 nM. These binding sites were calcium-dependent. Part of the binding to the hepatoma cells was non-saturable, owing to a large amount of low affinity binding sites which are at least partially located on the extracellular matrix of the cells. Competition with mannose- and galactose-terminated glycoproteins had no effect on total binding of 125I-t-PA. Degradation products of 125I-t-PA were found in the supernatant after a short lag phase and then increased linearly for at least 5 hours at 37° C. Degradation could be inhibited by chloroquine, NH4Cl and NaN3. We conclude that the human hepatoma cell line Hep G2 has a specific binding mechanism for t-PA which is not mediated by known carbohydrate receptor systems. Binding is followed by cellular uptake and degradation in the lysosomes.