bluuebunny/crossref_metadata_2025 huggingface dataset:
COMPARATIVE PHARMACODYNAMICS OF SUBCUTANEOUSLY ADMINISTERED HEPARIN (HEP) AND A LOW MOLECULAR WEIGHT HEPARIN (LMWH) AS STUDIED BY AN EX VIVO FIBRINOPEPTIDE A (FPA) GENERATION TEST
Authors: Spyropulos B , Fareed J , Emanuel R M , Hoppensteadt D
Published: June 2019

Subcutaneously administered heparins do not produce any sizeable effects on the global tests and their activities are currently measured using amidolytic anti Xa and anti Ila assays. These methods are sensitive only to the individual enzyme and do not reflect other biologic actions. We have used a thromboplastin C activated FPA generation test to measure the pharmacodynamic effects of subcutaneously administered HEP and a LMWH in primates. 400μl of citrated plasma was equilibrated at 37°C for 2 minutes and was activated with 100 μl thromboplastin diluted in .020M CaCl2. FPA was generated for 2 min, inhibited with 50 μl of an inhibitor cocktail and measured using an RIA kit (Mallinckrodt, St, Louis, M0). Marked dose dependent suppression of the FPA generation was noted for varying periods of time by both HEP and LMWH. Stronger effects were observed with LMWH, suggesting better subcutaneous bioavailability and functional effects. Significant FPA generation inhibition was observed even when there was no circulating antiprotease activity present in the plasma. These results suggest that FPA generation test is a sensitive test to measure the pharmacologic effect of heparins. Furthermore the FPA generation tests can be modified to activate the coagulation system at certain sites to study the effect of heparins.
SUBCUTANEOUS PHARMACOKINETICS/PHARMACODYNAMICS OF A LOW MOLECULAR WEIGHT DERIVATIVE (RD 11885) AND UNFRACTIONATED HEPARIN (PM 16885) IN PRIMATES
Authors: Norm R , Fareed J , Silber I , Belo A , Fenchel R , McGuire R
Published: June 2019

Subcutaneous pharmacokinetics/pharmacodynamics of a depolymerized low molecular weight heparin (RD 11885) and an unfractionated porcine mucosal heparin (PM 16885) were studied in primates (Macaca mulatta) at 0.5, 1.0 and 2.5 mg/kg/24 hr for 10 days after repeated administration. Ex vivo actions were determined using partial thromboplastin time (APTT), thrombin time (TT), IieptestR time (HT) , anti-factor Xa and anti-factor Ila assays at various time periods. Platelet counts and bleeding times were also measured. The cumulative bioavailability of RD 11885 calculated ex vivo was found to be 2-3 fold higher than PM 16885. The RD 11885 treated group exhibited a clear dissociation of the anti-factor Xa and anti-factor Ila activities. The biological half-life of RD 11885 was significantly greater than PM 16885 in all assays. No staircasing phenomenon was observed with either agent. A desensitization of the PM 16885 effects was observed. Neither agent produced any effect on the bleeding time or platelet count at any time. The pharmacokinetics/pharmacodynamics of RD 11885 were uniform and allowed the calculation of various pharmacologic parameters, whereas inconsistent results were obtained with PM 16885. These results demonstrate that this low molecular weight heparin exhibits better and more predictable bioavailability, in contrast to unfractionated heparin.
HEPARIN POTENTIATES PLATELET ADHESION AND AGGREGATION ON COLLAGEN SUBSTRATES. ABSENCE OF INHIBITION BY ASPIRIN
Authors: Mazurov A V , Si-nitsin V E , Repin V S
Published: June 2019

We studied the effects of two commercial heparin (H) preparations, from bovine lungs (HL) and pig intestine (HI), on the interaction of human plateletswith collagen substrates, and tested a possibility of modulating these effects by aspirin. PRP was incubated in the presence or absence of H with human collagens type IV and III (CIV and Cl I I) adsorbed ona plastic surface. Platelet adhesion and spreading on CIV and aggregation on Cl I I was quantitated by scanning electron microscopy. HL and HI were added to platelets in the concentrations approximating to those achieved in vivo at heparin therapy (0.5̲5.0 U/m 1) . HL and H|=I raised the number of adherent platelets, but do not alter the percentage of spread platelets on CIV. Both preparations had approximately equal effect, increasing platelet adhesion by 1.5 to2-fold at 2.5 U/ml . HL and HI potentiate the formation of. surface-bound aggregates on CIII, enlargingthe total substrate surface coated with aggregates. This effect is achieved due to increased size and number of aggregates. At 2.5 U/ml both preparations potentiate aggregation 2- to 3"fold.

Aspirin has no effect on platelet adhesion and spreading both in the absence and presence of HL/HI. In the absence of H, aspirin decreased platelet aggregation on CIII, reducing the aggregate-coated area by 40-50%. The inhibition was related mainly to reducednumber, not the size of aggregates. Aspirin failed to prevent potentiation of the Clll-induced aggregation by HL/HI.
IN VITRO DEMONSTRATION OF "HEPARIN REBOUND"
Authors: Taylor C , Baugh R F
Published: June 2019

"Heparin rebound", the in vivo appearance of measurable heparin anticoagulant activity following theapparent neutralization of heparin by protamine, hasbeen a problem sporadically associated with the use of heparin in cardiovascular surgery. A number of mechanisms have been proposed to explain rebound, and to some extent each may contribute to the phenomena. As yet no reliable, predictable method has been demonstrated for measuring, reproducing or quantifying "heparin rebound".

We have demonstrated and measured the appearance of heparin anticoagulant activity following neutralization with protamine in citrated whole blood. The reappearance of heparin anticoagulant activity was associated with a rapid loss of protamine. The loss of protamine followed 1st order enzyme kinetics, and was indicative of the action of an enzyme. The anticoagulant activity which eappeared could be titrated againwith protamine. The loss of protamine neutralizing activity, in whole blood, could be followed by titration with heparin using a recalcified activated clotting time. The rate of loss varied with both individual blood donors and with the type and source of protamine. The rate of loss of protamine was great enough to influence in. vivo heparin/protamine neutralization ratios, i.e. at 4 units of heparin/ml, 1 unit/ml anticoagulant activity was routinely recovered within 30 minutes following initial neutralization. The indications for cardiovascular surgery are:1)the in vivo neutralization ratio should be adjusted to account for loss of protamine activity, 2) the higherthe blood levels of heparin used during surgery, themore significant the potential for heparin rebound, and 3) protamines may be evaluated in an in vitro test which measures the stability of protamine neutralizing activity in whole blood.
VENOGRAPHY (VG) OF THE LOWER LIMBS IN THE MANAGEMENT OF PATIENTS WITH SUSPECTED PULMONARY EMBOLISM (PE)
Authors: Kruit W H J , Sing A K , den Ottolander G J H , de Beor A C , Jonker J J C
Published: June 2019

In a prospective cohort study, we evaluated X-ray VG in the management of non-surgical patients with clinically suspected PE. Thusfar follow up is available on 131 consecutive patients with suspected PE. In all patients a perfusion lungscan (PS) was carried out within 24 hours. In case of a normal PS (group A, n=32), no anticoagulant (AC) therapy was given. In case of an abnormal PS, AC therapy was started (heparin) and a bilateral ascending VG was carried out within 72 hours. In 46 patients (group C) venous thrombosis (DVT) was demonstrated by VG, and these patients were treated with AC for 6 months. In 53 patients with suspected PE and an abnormal lungscan, bilateral VG did not show DVT (group B). AC therapy was discontinued in these patients These patients were then screened for 14 days with fibrinogen legscanning and impedance plethysmography (IPG), followed by IPG alone every 2 months for at least 1 year. In group B, 6 patients died in the follow up period. None of the patients had signs of PE at autopsy. One additional patient in group B developed DVT documented by repeat VG, 6 months after entry into the trial. According to these preliminary data, it seems safe to base the decision whether or not to treat a patient with suspected PE with AC, on the presence or absence of DVT in the lower limbs as demonstrated by VG
SAFETY AND EFFICACY OF SERIAL IMPEDANCE PLETHYSMOGRAPHY IN THE DIAGNOSIS OF DEEP VEIN THROMBOSIS IN AN URBAN HOSPITAL: AN EXTERNAL VALIDATION STUDY
Authors: Heyermans H H S , Huisman M V , Büller H R , Laan J V D , ten Cate J W
Published: June 2019

The clinical diagnosis of deep vein thrombosis (DVT) is unreliable. Impedance plethysmography (IPG) has become accepted as a highly reliable non-invasive method for DVT detection in symptomatic patients.

It has a high sensitivity (95%) and specificity (96%) for proximal vein thrombosis. Studies with impedance plethysmography have however always been carried out in academic hospitals. To evaluate the safety and efficacy of serial impedance plethysmography alone in an urban hospital setting, a prospective study was done in an urban hospital, involving 234 consecutive outpatients with clinically suspected venous thrombosis. IPG was performed on days 1, 2 and 7. If all tesis remained normal the patient was not treated with oral anticoagulants. All patients were followed for 3 months. In 131 of the 234 patients (56%) IPG was repeatedly normal. Of these 131 patients, no patient died from venous thromboembolism during 3 months follow-up, completed in all patients, and no patient returned with signs of pulmonary embolism. One patient (0.8%) returned with objectively documented DVT after two months. In 103 of the 234 patients (44%) the IPG was abnormal. Venography confirmed the diagnosis of deep venous thrombosis in 92%.

It is concluded that serial IPG is a safe and effective method to detect DVT in clinically suspected outpatients, referred to an urban hospital.
IMPROVEMENT OF SICKLE CELL DEFORMABILITY BY PIRACETAM IN VITRO AND IN VIVO
Authors: Sonnet J , Gini E K
Published: June 2019

Piracetam (P) (2-oxo-pyrrolidine acetamide) has Theological properties and has been used at various dosages over the past decade for the management of psychosenescent syndromes. On maintenance therapy, at the oral dosage of 160 mg/kg/day, in four divided doses, P reduces the number of vaso-occlusive crises in sickle cell homozygous patients, to about a fifth of what could be expected without drug. After oral intake at the latter dosage P's bioavailability in the blood ranges from 0.5to 1 m mol/1. Microsieving on polycarbonate filters, 5 μ m por size, of diluted suspensions (haematocrit 1%) of oxygenated HbSS cells previously incubated with P 0.5 to 1 m mol/1, shows that the drug strongly improves their deformabi1ity. Similarly, microsieving of oxygenated HbSS cells obtained from patients on maintenance therapy with P, shows that the drug enhances the deformabi 1 ity of these cells as actively as it does in in vitro experiments in the same range of concentration. On the other hand the drug only poorly restores the loss of deformabi 1 ity of physiologically deoxygenated HbSS cells. From these experiments, it seems that P works rather on the outer viscoelastic properties of the HbSS red cell membrane than on the inner HbSS content of these cells.
COMPUTERGRAPHIC ANALYSIS OF OBSTETRIC HEMORRHAGE
Authors: Suzuki S , Ichinoe K
Published: June 2019

(Purpose) From the viewpoint of maternal and perinatal mortality, it is very important to manage the postpartum hemorrhage (PPH). In order to observe the riskfactors, especially the relationship between the obstetric factors, abnormal bleeding of PPH,. and blood -coagulation-system, we have tried to prognosticate the abnormal bleeding during labor.

Next, the relationship of the Hepaplastintest-value of umbilical venous blood and obstetric factors were examined by computer graphic analysis.

(Methods) 200 cases of non-DIC and 17 cases of DIC cases, Fibrinogen, FDP, Platelet, Factor XI and Factor XII et were tested in the 1st stage of delivery.

(Results)
(1) In the non-DIC cases no relation was proved between bloodcoag-ulation factors excluding Factor XI.

(2) The correlation-Index in the DIC cases, we have proved the following order.

Platelet (75 %) > Fibrinogen (65 %) > FDP (55 %)

(Conclusion)
(1) Even in normal delivery, the Kinin-Kallikrein system plays an important role in the contraction of the uterus, and the duration-of delivery.

(2) In the DIC-cases, the platelet count was reversely related and showed a linear relation with Postpartum-Hemorrhage. We also noted that Fibrinogen, FDP and Prothrombin Time are important factors from the viewpoint of DIC.
EFFECT OF ORAL CONTRACEPTIVES ON HEMOSTASIS
Authors: Farag A M , Bottoms S F , Mammen E F , Hosni M , Ali A
Published: June 2019

Retrospective statistical epidemiological studies have suggested a possible association between the ingestion of oral contraceptives (OC) and thromboembolic disease. Past analyses of the coagulation system have yielded controversial information

We studied a cross section of 131 women taking different kinds of OC and 36 controls for changes in hemostasis. No significant differences were noted in the levels of fibrino- peptide A (RIA), platelet factor 4, 8 thromboglobulin (RIA), fibrinogen (Multistat III (MCA), clottable), antithrombin III (MCA, S-2238), α2 antiplasmin (MCA, S-2251), pre-kallikrein (MCA, S-2302) and fibronectin (MCA, immune turbi-dometric). However, plasminogen (MCA, S-2251) and protein C antigen (Laurell) levels were significantly elevated (p < 0.001 and p < 0.01), respectively)

Canonical correlation analysis was used to examine correlations between hemostasis parameters measured and clinical risk factors, such as age, parity, weight, smoking, family history for thromboembolic diseases and estrogen-progesterone dose. There was a significantly negative correlation between family history for thromboembolisms and antithrombin III levels (p < 0.01). A positive correlation existed between obesity and fibrinogen and fibronectin levels (p < 0.001 for both). The hemostasis data seem to suggest that OC use does not introduce an imbalance in the hemostasis system which fosters "hypercoagulability", and that, if at all, possibly other risk factors determine the incidence of thromboembolisms in OC users. It is suggested that caution be exercised in the use of OCs in patients with a history of thromboembolic diseases and with obesity
EDTA-DEPENDENT MONOCLONAL ANTIBODIES TO HUMAN PROTEIN S
Authors: Marcovina S , Coppola R , Protti M P , Gelfi C , Mannucci P M
Published: June 2019

Splenocytes from a Balb/c mouse immunized with purified human protein S (PS) were fused with murine hybridoma cell line SP2/0-Agl4 and cultured in Iscove's medium without addition of fetal bovine serum. Hybrid supernatants were screened for the presence of specific antibodies by solid-phase ELISA and cloned by the limiting dilution technique. Pour clones, named S2, S3, S8, and S10, were selected, recloned several times, and injected intraperitoneally into Balb/c mice for the production of antibody-rich ascitic fluid. The monoclonal antibodies (Mabs), all of IgGl subclass with k light chain, were purified from ascitic fluid by Protein-A chromatography. The specificity of Mabs was controlled by the immunoblotting technique: the Mabs appeared to react only with two plasma proteins, one with a MW of about 70.000 dal tons comigrating with purified PS, and the other with a MW >300.000 da that is likely to be the C4b-binding protein-PS complex. No interaction has been observed with PS-depleted plasma. As tested by a fluid phase radio immunoassay, the binding of Mabs to PS was significantly higher in the presence of EDTA while was totally inhibited in the presence of Ca2+. Scatchard plot analysis of the binding between 125I-PS and Mabs showed that the binding affinity of the antibodies ranged from 108 to 109 1/mol. Each EDTA-dependent Mab was then immobilized on Sepharose 4B-CNBr and used to purify PS from barium precipitation of citrated plasma. The fraction eluted with 2 mmol of CaCl2 from the immunoadsorbent appeared to contain only two proteins when stained with Cocmassie Blue. By immuno blotting, all Mabs reacted with both proteins, one comigrating with purified PS and the other with a MW >300.000. Essentially homogeneous PS, free from the high MW component, was obtained when the barium citrate adsorbate was applied to a DEAE-Sephadex column and the protein peack containing the balk of PS was sussequently applied to the immunoadsorbent and eluted with 2 mmol CaCl2.

In summary, we have described an unusual set of EDTA-dependent monoclonal antibodies to PS and their use for the purification of homogeneous protein S from human plasma.
SUDAY ON A SUBSTANCE FUNCTIONALLY LIKE PROTEIN C IN PLASMA
Authors: TSUNEIZUMI I , MEGURO T , YAMADA K
Published: June 2019

The present report first deals with the isolation and characterization of a substance found to demonstrate protein C like activity(PCLA). When it was activated by thrombin, the PCLA substance prolonged the activated partial thromboplastin time(aPTT). The activated PCLA substance also hydrolyzed synthetic substrates such as S�2238, S�2366 and S�2266,while the PCLA substance was not cross reacted with anti-protein C serum(Behring Manheim). The PCLA substance was adsorbed by both aluminium hydroxide gel and barium sulfate. The level of Km, optimum pH and optimum I.S. in the amido-lytic reaction with synthetic substrate is shown in the table.

In the chromatography of Sepharose CL-6B gel, the PCLA substance was eluted in the fraction of higher molecular weight, while protein C was eluted with a lower fraction.This prompts an estimate that the molecular weight of the PCLA substance is approximately 200,000. Through DEAE-Sepharose CL-6B gel ion exchange chromatography, the PCLA substance was eluted by a lower ionic strength than was protein C.

The levels of a PCLA substance were low in the patients with a vitamin K deficiency and in newborn infants. It is expected that our findings regarding the PCLA substance will be as clinically significant as that which we know about protein C at present.
ASSOCIATION OF HEREDITARY DYSFIBRINOGENEMIA WITH PROTEIN C DEFICIENCY IN TWO PATIENTS WITH THROMBOTIC TENDENCY
Authors: Gandrille S , Priollet P , Capron L , Roncato M , Fiessinger J N , Aiach M
Published: June 2019

An abnormal fibrinogen was found to be associated with protein C deficiency in two unrelated patients. Both were symptomatic, one having severe complicated atherosclerosis, the other recurrent venous thrombosis. In both cases, the two abnormalities coexisted in several members of the family. The two abnormal fibrinogens (Poitiers and Argenteuil) were purified from the patients' plasma and compared to fibrinogens purified from several normal subjects. Polymerisation was abnormal in both cases in presence of reptilase and thrombin.

The kinetic of proteolysis by plasmin was studied during 24 hours using SDS-polyacrylamide gel electrophoresis. In the first case, the appearance of fibrinogen degradation products (FDPs) was slightly delayed. The most sliking abnormalities were observed with fibrinogen Argenteuil which still contains "early" FDPs (X and Y) at 24 h while the normal fibrinogens were all completely degraded.

The stimulating effect of fibrin on plasminogen activation by tPA was studied after insolubilization of the purified fibrinogen according to the SOFIA technique described by Angles-Cano (Anal. Biochem. 1985, 153, 201). Only fibrinogen Argenteuil was found to have a decreased catalytic effect on plasmin formation.

Thrombin binding by fibrin clots, studied according to Ha-verkate (Thromb. Haemostas. 1986, 55, 131) was found decreased in fibrinogen Poitiers and normal in fibrinogen Argenteuil.

Conclusion: In these two cases of hereditary dysfibrinoge-nemia least one of the natural antithrombotic function elicited by fibrinogen-fibrin transformation was found to be strongly abnormal . Both fibrinogens presented abnormal lysis by plasmin, particularly fibrinogen Argenteuil in which Tibnn degradation was also reduced. This functional abnormality have already been described in patients with thrombotic tendency. It is however difficult to conclude, as in both families the dysfibrinogenemia was associated with protein C deficiency.
THROMBO-HEMORRHAGIC SKIN NECROSIS DUE TO RAPID DEVELOPMENT OF SEVERE VITAMIN K DEFICIENCY ASSOCIATED WITH CHOLESTASIS
Authors: Michiels J J , Bertina R M
Published: June 2019

A female patient is presented, who developed thrombotic and hemorrhagic skin necrosis of the feet and toes during an acute episode of severe vitamin K deficiency due to cholestasis in the absence of coumarin treatment. Painful blue toes and feet progressed to erythematous swelling and bluish discoloration with blister formations and immanent gangrene. The histo-pathology of skin excisions from the erythematous skin lesions showed extravasation of erythrocytes and extensive fibrin thrombus formations in capillaries and venules as has been described in patients with coumarin skin necrosis. At the time of painful acrocyanosis the results of coagulation investigations (platelets 109 × 109/1, APTT 56/35 sec., PT 61/15 sec., ThrombotestR (TT) less than 3%, NormotestR (NT) less than 10%, fibrinogen 1,6 g/l, absence of fibrin monomers and degradation products, factor V 1.00 u/ml, antithrombin lil 1.03 u/ml and alfa2 antiplasmin 0.97 u/ml were consistent with severe vitamin K deficiency. Measurements of vitamin K dependent factors revealed very low levels for procoagulant factor VII (16%) and protein C (22%) antigen concentration and normal levels for procoagulant factor II (97%) and procoagulant factor X (87%) antigen concentration. After substitution of 5 mg vitamin K1 both the TT and NT normalized.These data confirm the hypothesis that an imbalance within procoagulant and anticoagulant vitamin K dependent factors (severe protein C and factor VII deficiency as compared to the procoagulant factors IX, X and II due to rapid development of vitamin K deficiency) contributes to the pathogenesis of thrombo-hemorrhagic skin necrosis and that the so-called coumarin necrosis is not due to drug toxicity.
Effects of Glyoxylate on Adenine Nucletodies and Aggregation of Human Platelets
Authors: Holmsen H. , Setkowsky C. A. , Day H. J.
Published: June 2019

Glyoxylate (G) reduces the ATP/ADP ratio and the adenine nucleotide pool in Ascites tumor cells, probably through inhibition of Krebs cycle and accumulation of citrate (JBC 246, 1607 1971). Incubation of human platelets in plasma or after gelfiltration with G reduced the level of metabolic ATP with a corresponding accumulation of hypoxanthine within the first 10 min after which no changes took place. The decrease in ATP levels is noticeable above 15 mM and is maximal (60–75% decrease) at 45 mM G. Oligomycin, antimycin A (AA), pyruvate, succinate, aspartate or glutamate did not alter the effect of G; 2-deoxyglucose (2-DG) and absence of extracellular glucose enhanced the G-induced reduction in ATP levels. Lactate production was not altered by 45 mM G. The same degree of ATP reduction was observed in the absence and presence of extracellular Na indicating that G did not cause depletion of the adenine nucleotide pool by activating the Na-dependent platelet AMP deaminase through increased Na uptake. The adenylate energy charge (AEC) remained constant (0.89–0.93) despite the large fall in the ATP levels. Primary and secondary platelet aggregation also remained unaffected by G-induced reduction of the ATP levels. Incubation with 2-DG plus AA reduced both ATP levels and AEC, with complete abolishment of secondary and primary aggregation at AEC lower than 0.85 and 0.80, respectively. Although the mechanism whereby G reduces the levels of metabolic ATP remains obscure, these experiments demonstrate clearly that the platelets’ ability to undergo primary and secondary aggregation does not correlate with the level of metabolic ATP, but may correlate with AEC.

(Supported by NIH Grant HL 14217–05.)
ADP-Induced Refractory State of Platelets in Vitro
Authors: Holme S. , Holmsen H. , Sixma J. J.
Published: June 2019

ADP-induced aggregation was determined at various times of pre-incubation with ADP in unstirred platelet rich plasma to which adenosine deaminase was added. In the early stages of incubation the shape change response was absent, the aggregation response was poor and not reversible. As incubation proceeded, these three parameters returned towards normal while there was still ADP in the system. Log dose response (rate of aggregation) curves for the platelets incubated with 1 μM of ADP had shifted to higher concentrations of ADP and there was a small decrease in the maximal height of the curves. Gelfiltered platelets in calcium-free Tyrode solution were incubated with 1 μM of ADP at 37° C. Log dose response curves obtained at different times of incubation showed a greater shift to higher ADP concentrations than those obtained with platelets in plasma. There was also a pronounced decrease in the maximal height of the curves. These two observations became more apparent as incubation proceeded. Addition of apyrase (0.1 mg/ml) at various times of incubation prevented the progessive impairment of the aggregation response and this even slowly increased towards that of the control. The time of incubation with ADP before addition of apyrase did not have any influence on the ability of the platelets to recover their aggregability towards ADP.
The Contribution of Individual Protease Inhibitors to Plasma Antiplasmin Activity
Authors: Crawford G. P. M. , Ogston D. , Douglas A. S.
Published: June 2019

The plasma proteins alpha-1-antitrypsin, alpha-2-macroglobulin, antithrombin III, Cl inhibitor and inter-alpha trypsin inhibitor have been shown to be capable of inhibiting plasmin activity. Whole plasma possesses both ‘rapid’ and ‘progressive’ plasminneutralising activity: the study was intended to assess the contribution of individual protease inhibitors to this plasminneutralising activity.

The antiplasmin properties, assessed as ‘rapid’ and ‘progressive’ by the neutralisation of plasmin in a caseinolytic assay, and the levels of the individual protease inhibitors, assayed by single radial immunodiffusion, were measured in the plasma of 35 subjects. Statistical analysis using zero order correlation coefficients and zero order partial correlation coefficients indicated that ‘rapid’ and ‘progressive’ antiplasmin activity correlated with the plasma concentration of alpha-2-macroglobulin and alpha-1-antitrypsin respectively, but not with antithrombin III, Cl inhibitor and interalpha trypsin inhibitor.

It is concluded that alpha-2-macroglobulin and alpha-1-antitrypsin are the major plasma inhibitors of plasmin and that immunological determination of these proteins provides a valid assessment of plasma antiplasmin activity.
A new Fibrinolytic Enzyme: the Prolixase. Physicochemical and Biological Properties
Authors: Hamouda Ben M. , Soria J. , Soria G.
Published: June 2019

A new fibrinolytic enzyme was discovered in hemolymph of rhodnius prolixus. Its isolation was performed and the physicochemical proterties were determined.

The activity of this enzyme was measured either by fibrinolysis of fibrin plates and by decrease of fibrinogen level induced by incubation of fibrinogen with the enzyme, or by its esterase activity on synthetic substrates.

The nature of fibrinogen degradation products (FDP) were analysed both by Immunoelectrophoresis and by SDS polyacrylamide gel electrophoresis and compared with those obtained by degradation of fibrinogen with plasmin.

Furthermore, considering the analysis of FDP after addition of mercapto-ethanol and urea, it was found that the enzyme induces in a first time a lysis of the α (A) chain of the fibrinogen molecule, in a second time a degradation of the β (B) chain and later a degradation of the γ chain. The activity of this enzyme was not inhibited by addition of Kunitz inhibitor. We propose to call this new enzyme: the prolixase.
Sterol Synthesis in Human Endothelial Cells
Authors: Evensen S. A. , Henriksen T.
Published: June 2019

Endothelial cells were isolated by collagenase digestion of the intima of the vein of umbilical cords and grown in medium 199 or RPMI 1640 supplemented with 20% FCS. After 3–5 days in primary culture fresh medium was added containing either sodium acetate-2-14C or mevalonic acid lactone-2-14C. Following further 24, 48 or 96 h incubation the harvested cells and media were extracted according to Folch and separated on TLC. About 35% of the total cellular radioactivity accumulated in the sterol fraction after 48 h incubation with labelled acetate. Delipidation of the medium resulted in markedly increased incorporation. Incorporation of mevalonic acid into cellular sterols was maximal at 48 h, and larger in RPMI 1640 than in medium 199. In the medium a progressive accumulation of labelled sterols with time was observed. Approximately 20% of the cellular radioactivity was found in the FFA fraction and was saponifiable. Whether this finding represents fatty acid synthesis from mevalonic acid or an intermediary product in sterol synthesis has not yet been clarified.
Reactions of Human Endothelial Cell Cultures Exposed to Adrenalin Concentrations
Authors: Wright Payling H. , Evans M.
Published: June 2019

Cultures of vascular endothelium obtained from fresh human umbilical veins and grown in vitro in fortified 199 medium for several days have been subjected to differing concentrations of adrenalin for various times. Their reactions to the drug, as seen microscopically, were recorded photographically. The viability of endothelial cells under these cultural conditions gives a measure of the maximal exposure to adrenalin which they are able not only to survive, but also to multiply. Their capacity to mitose was studied autor adiographically.

The significance of the findings will be discussed with reference to atherogenesis and particularly the possible link with infection and in “stress”.
Effect of Structural Changes of Collagen on Collagen-platelet Reaction
Authors: Yamanaka M. , Nagai Y. , Kodama M.
Published: June 2019

Removal of telopeptides from the acid soluble calf skin collagen with proctase did not affect the platelet aggregation. Limited digestion with tadpole collagenase at 20° C maintained the ability of collagen to aggregate platelets, when it was measured at 20°C., although its ability was completely abolished at 37° C. Neither α1 and α2-components of collagen did show the ability to aggregate platelets. Denaturation of the soluble collagen by heating at 45° C for 10 min destroyed its ability to aggregate platelets, although an initial shape change of platelets was observed.

These results suggest that there may be different mechanisms in relation to the structure of collagen in the adhesion of platelets and the aggregation of platelets as a sequential reaction and that the tertiary structure of the soluble collagen composed of three polypeptides chains with a some critical length may be necessary for the platelet aggregation.
A Method for the Quantitative Determination of the Antithrombin III (AT III) a Citivity in Plasma and Serum Using the Complex Formation with Heparin
Authors: Karges H. E. , Heimburger N. , Loechelt C.
Published: June 2019

A prerequisite of most AT III assays is the defibrination of the samples. Defibrination of plasma, however, is generally associated with a loss of AT III. This especially true of heat defibrination. Furthermore, according to our experience, most of the functional determinations don’t correlate well with the immunological ones, and don’t provide reproducible results. Therefore, it was the aim of our investigations to establish a method which omits the defibrination and yields reproducible results.

The method reported is based on the identity of AT III and heparin cofactor antithrombin II (AT II) respectively. AT III is converted by heparin from a progressive into an immediate inhibitor, allowing a plasma dilution of 1:50. Due to this high dilution, the defibrination step can be omitted and AT III determined in native plasma. To transform AT III completely into its heparin complex, 5 USP units heparin are used. α2-macro-globulin up to 400% of normal plasma concentration does not influence the assay. When determinations are performed at a certain pH value, good reproducibility is obtained. The mean error of determinations of the same sample does not exeed 4%. Maximal deviations were in the range of 5 to 10%. The results of functional determinations are compared with those obtained by two immunological methods. Deviations scarcely exeed the limits of methodical errors.
Kininogen Deficiency and Defective Surface Activation of Blood Coagulation and Fibrinolysis in a Kindred with Fitzgerald Trait
Authors: Donaldson V. H. , Glueck H. I. , Miller M. A.
Published: June 2019

A 28 year old black man who had never had a bleeding syndrome was found to have a long plasma activated partial thromboplastin time which could not be attributed to deficiencies of Hageman factor, Fletcher factor (a prekallikrein), PTA, Factor VIII, or factor IX. The prothrombin time was normal. In addition to delayed clotting, the release of kinin and generation of fibrinolytic activity upon exposure of the plasma to an activating surface was markedly impaired. Defective blood coagulation and kinin release were corrected by plasma from persons with Hageman trait, Fletcher factor deficiency, and PTA deficiency, but not by plasma from each of 2 patients with Fitzgerald trait. The addition of preparations of activated Hageman factor and of kallikrein to this defective plasma did not correct the coagulation defect. The concentration of kininogen in the plasma of the propositus was less than 1% of that in normal plasma, as measured by the release of kinin from the heated plasma during treatment with trypsin. The incidence of the defect in this kindred indicates that it is inherited as an autosomal recessive trait, for 5 of 13 relatives in 3 generations were partially deficient in Fitzgerald factor, determined with a coagulation assay, and in kininogen, quantified as kinin released by trypsin. Thus, Fitzgerald trait appears to be associated with a functional deficiency of a kininogen which may be important for the effective function of activated Hageman factor in generating clot promoting and fibrinolytic activity in plasma.
Postoperative Deep Vein Thrombosis in Patients on Oral Contraceptives
Authors: Sagar S. , Kakkar V. V. , Thomas Duncan P.
Published: June 2019

A causal relationship has been established between the use of oral contraceptives and venous thromboembolism occurring in previously healthy women. There is also evidence that the risk of thromboembolism is further increased if patients undergo a surgical operation while on oral contraceptives (M. P. Vessey et al., Brit. med. J., 3, 123, 1970). In the present study, activated Factor X inhibitor (Xal) levels were measured in blood samples withdrawn from 48 women before, during and after a minor dental operation. In 26 patients not on oral contraceptives, the mean preoperative Factor Xal level was 92.0% (S. E. M.±4.1), and no significant changes occurred in response to surgery. In 22 patients taking oral contraceptives, the mean preoperative Factor Xal level was 83.3% (S. E. M.±3.7) and during the operation there was a significant fall to a mean level of 70.6% (S. E. M.±4.8) (P < 0.05). Factor Xal levels returned to preoperative values within a week of operation. These findings indicate that even a minor operation causes a significant fall in Factor Xal levels in women on oral contraceptives.
ICSH-ISTH International Anticoagulant Control Study III. Implications of the Findings
Authors: Ingram G. I. C.
Published: June 2019

For general standardization of oral anticoagulant control, the results of tests performed with the present diversity of reagents must be referable to a common scale. To date, most comparative work has been related to the relative sensitivities of different thromboplastins to the anticoagulant defect. A “master” reference thromboplastin, against which working reference materials — thromboplastins and standardized plasmas — could be expertly calibrated, may well provide the backbone of an international scheme. For working-laboratories, anticoagulant treatment could then be controlled by using either a thromboplastin of known sensitivity or standard plasmas with calibrated responses, to refer patients’ clotting times to a common scale.

The relative merits of these two schemes will be discussed in the light of the findings of the international study described in the previous two abstracts.
Differences in Crosslinking Reactivity of Fibrin Molecules Formed During Differential Release of Fibrinopeptides A and B
Authors: Furlan M. , Seelich T. , Beck E. A.
Published: June 2019

Mixtures of human fibrinogen and preactivated factor XIII were incubated at pH 7.3 with either thrombin, reptilase or a procoagulant fraction of the southern copperhead snake venom. These three enzymes release either both fibrinopeptides, fibrinopeptide A, or preferentially fibrinopeptide B, respectively. Crosslinking of γ-chains and the size of initial γ-γ polymers were studied in the early stage of fibrinopeptide cleavage, using polyacrylamide electrophoresis in the presence of sodium dodecyl sulfate. Formation of α-chain polymers was also followed during a later stage. Similar experiments were performed with thrombin acting on fibrinogen at pH 6.0 and 9.0. Our results indicate differential effects of the release of fibrinopeptides A or B on fibrin crosslinking.
Clinical Thrombosis. A Subunit Study of the Fibrin
Authors: Strachau G. J. L. , Gaffney P. J.
Published: June 2019

The fibrins in venous and arterial thrombi, obtained by surgical intervention, were examined with respect to their subunit compositions. All the fibrin samples examined were totally crosslinked by the action of Factor XIII, e.g. the α chains were crosslinked as α chain polymers (αP) and the γ chains were crosslinked as γ chain dimers (γ-γ), while the β chains were uncrosslinked. The only difference between the subunits of the in vivo formed fibrins and those of in vitro plasma clots, was the consistent presence of some partly digested fibrin chains (of molecular weight 33,000) which were fragments of the β or γ chains, suggesting that the latter polypeptide chains may be more subject to lysis in vivo than the crosslinked α chains. The α chain polymers may be structural features stabilising the thrombus fibrin in vivo.

Brinase (a purified extract of Aspergillus Oryzae) was shown in vitro to rapidly lyse the in vivo formed thrombi, having the unique property of degrading the plasmin resistant crosslinked α chain regions of fibrin. The in vitro lysis of the pulmonary emboli by plasmin resulted in the production of soluble fragments called D dimer and E which are also associated with the lysis of plasma clots made in vitro. These similarities in the formation and lysis of in vitro and in vivo fibrins encourages the use of in vitro systems to help understand the initiation and progression of clinical thrombosis.
Comparative Effects of Platelet Suppressant Drugs on Platelet Survival Time
Authors: Genton E. , Ellis J. , Steele P.
Published: June 2019

The important role of platelets in thrombosis makes inhibitors of their reactivity potentially useful therapeutically. A number of laboratory tests have been identified which measure platelet reactivity, but it is not clear which test and which drug effect will correlate with thrombosis and thrombosis prevention. Platelet survival (SURV) correlates with thromboembolism in patients with valvular heart disease and is shortened in several other diseases. Therefore, it is of interest to identify drugs which prolong shortened SURV. Patients with arterial and venous thromboembolism and shortened SURV (51Chromium) were treated with platelet suppressants and restudied after 12 weeks. Sulfinpyrazone prolonged SURV(2.4±.04 to 3.1 ±.06 days; p < 0.001; n = 94; average ± SEM; normal, 3.7±.04 days) and 68 (72%) had some prolongation and 39 (42%) had normalization (> 3.3 days). Dipyridamole (100 mg qd) combined with aspirin (1200 mg qd) prolonged SURV (2.6±.11 to 3.2±0.12 days; p < 0.001; n = 13) and 9 of 13 (69%) had prolongation and 6 (46%) had normalization. Clofibrate altered SURV (2.6±.09 to 3.4±.14days;p < 0.001; n = 12) and 10 of 12 (83% ) had prolongation and normalization occurred in 6 (50%). Aspirin (1200 mg qd), cyproheptadine (32 mg qd) and propranolol (160 mg qd) failed to alter SURV.

Thus, of drugs which alter in vitro tests of platelet reactivity, only sulfinpyrazone, dipyridamole and clofibrate improve shortened SURV.
Tissue Antithrombogenic Inducing Factor: Experimental evidence and practical applications in the construction of cardiac valves and the artificial heart
Authors: Carpentier A. , Relland J. , Carpentier S. , Lessana A. , Fabiani J. N. , Deloche A. , Chauvaud S. , Schahmaneche L. , Gory G.
Published: June 2019

Thrombo-embolic complications remain the major problem in human organ support and replacement devices: artificial kidney, artificial heart, cardiac valves. Based on our 7 year experience with heterograft valves which did not present thrombo-embolic problems, we postulated the existence of an anticoagulant factor inherent in tissue valves and in the biopolymers extracted from these valves.

Methods:

- Biopolymers were extracted from cardiac valves according to the method of L. Robert and the various fractions were used for impregnation of a Rhodergon fabric trileaflet valve prosthesis.

- Three types of prosthetic valve were implanted in the mitral position in 31 sheep: glutaraldehyde preserved heterograft, valve (4), biopolymer impregnated prosthesis (22), non impregnated trileaflet prosthesis (5).

- Anticoagulation was not used in the post-operative period.

Results:

- None of the heterograft valves presented thrombo-embolic complications.

- Collagen impregnated prostheses thrombosed 4 to 12 days following implantation.

- Proteoglycans and glycoproteins impregnated prostheses did not present any thromboembolic complications.

- Non biopolymer impregnated prostheses thrombosed in the first few days.

Electron microscopic studies in the heterograft and proteoglycans valve group showed a transitory adhesion of protein and platelets followed after 6 hours by formation of a protein layer preventing further platelet adhesion. These data suggest that a Tissue Antithrombogenic Inducing Factor (T. A. I. F.) may exist in the proteoglycan fraction of heterograft valve tissue. The precise nature of this factor remains to be elucidated.
Prothrombin Binding” to Phospholipid Vesicle Surfaces and Phospholipid Acceleration of Prothrombin Activation
Authors: Jackson Craig M. , Dombrose Frederick A. , Ittyerah T. Roy , Gitel Sanford N. , Esmon Charles T. , Suttie John W.
Published: June 2019

Prothrombin binds to phospholipid vesicle surfaces via the Fragment 1 region of the prothrombin molecule. The physiological importance of the direct prothrombin binding to phospholipid has been demonstrated recently in comparative studies of the activation of bovine normal and abnormal (dicoumarol “induced”) prothrombins (Esmon, C. T., Suttie, J. W. and Jackson, C. M., In Press). The binding of prothrombin and isolated Fragment 1 to vesicles composed of a variety of different phospholipids has been quantitatively investigated. From these studies it has been shown that prothrombin binding involves 20 ± 2 calcium ions and by comparison with the binding of calcium to prothrombin and Fragment 1 in free solution, this binding is consistent with association via calcium bridges between protein and lipid. Comparison of the ability of phospholipid to accelerate prothrombin activation with the affinity of prothrombin for particular phospholipid surfaces underlines the quantitative importance of the lipid-protein-calcium interaction in the activation process.

The in vitro acceleration of prothrombin activation by phospholipid surfaces which can be readily quantitated and related to protein binding to the surface provides a new set of criteria for quantitatively assessing the thrombogenicity of surfaces in general. Although providing a limited definition of and scale for thrombogenicity, the specificity of such assay provides a means by which the multiplicity of factors contributing to surface thrombogenicity may be better understood.

(Supported by a grant to Washington University, HL-14147 for a Specialized Center on Research on Thrombosis).
Serum F.D.P. Measurement as a Screening Test for Thrombo-Embolic Disease
Authors: Vasilescu C. , Jones D. R. B. , Webber Anne , Webber R. G. , Allan N. C. , Ruckley C. V.
Published: June 2019

Serum F.D.P. values are elevated in venous thrombo-embolic disease. In order to define the place of daily serum F.D.P. estimation as a screening test 300 consecutive patients admitted to medical wards and 70 undergoing operation have been studied. All patients were screened for venous thrombosis (D.V.T.) with the 125I-fibrinogen test and if positive they were further investigated by lung scanning and phlebography. All had daily blood sampling for F.D.P. and fibrinogen. F.D.P. were estimated by the Staphylococcal Clumping test to provide rapid results, backed up by the Tanned Red Cell Haemag-glutination Inhibition Immunoassay (Wellcome). The mean F.D.P. in medical patients with no detectable thrombo-embolic disease was 10.5 ug/ml. In patients with positive leg scans the mean was 43.3 ug/ml. In surgical patients with negative leg scans the mean F.D.P. was 15.4 ug/ml and with positive leg scans 38.9 ug/ml. In those with positive lung scans it was 275 ug/ml.

We conclude that a finding of elevated serum F.D.P. in hospital patients strongly favours the diagnosis of venous thrombo-embolic disease. Low F.D.P. values do not exclude the condition.
Structural Studies of Fibrinogen by Ultrasound Irradiation
Authors: Albert A. , Ayuso M. R. , Usobiaga P.
Published: June 2019

Low frequency ultrasound irradiation of purified fibrinogen solutions produces a precipitation of protein and a loose on clottability due to molecular fragmentation, both depending in different ways on the irradiation dose. Precipitation seems to be closely related to free radical formation, the fragmentation being due mainly to the shearing forces appearing during cavitation.

The high molecular weight fragments obtained by ultrasound irradiation of fibrinogen have been isolated by gel filtration and studied by disc gel electrophoresis and analytical ultracentrifugation. They show molecular properties similar to those of the fragments obtained by endopeptidase incubation and the protein degradation proceeds in a similar way. All that can be interpreted according to a trinodular model for the fibrinogen molecule, with two elongated fibrous zones more easily cleaved by mechanical forces and less compact parts of the molecule that are mostly constituted by the α-chains.
Sugar-Amine Interactions in the Blood Clotting System and their Effects on Haemostasis
Authors: Mester L. , Kraska B. , Crisba J. , Mester M.
Published: June 2019

N-Glycosides and l-Desoxy-l-amino-2-keto-sugar derivatives (Amadori compounds) are formed from reducing sugars and amino groups of amino acids, proteins or simple amines in the first step of the Maillard reaction leading to the non enzymic browning of foodstuffs. Very little if any attention has been devoted to this reaction in the blood clotting system. Two examples are given to illustrate the possible role of this reaction in haemostasis.

Poly-L-lysine, often considered as a model for collagen, reacts easily with D-glucose or D-galactose to form Amadori compounds with the ε-amino group of poly-L-lysine (mol. w. from 2,000 to 30,000). As a consequence, poly-L-lysine looses its ability to aggregate platelets. Serotonin reacts with D-glucose to form a stable and strongly reducing Amadori type compound. No change in the platelet aggregating ability of this compound has been observed, but other biological properties of the amine are highly affected.