bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional Properties of Factor VIIa/Tissue Factor Formed with Purified Tissue Factor and with Tissue Factor Expressed on Cultured Endothelial Cells
Authors: Almus Fanny E , Mohan Rao L Vijaya , Rapaport Samuel I
Published: July 2018

Factor VIIa (F. VIIa)/tissue factor (TF) function was examined using purified human TF reconstituted into mixed phospholipid vesicles and TF expressed on cultured human umbilical vein endothelial cells (HUVEC) treated with thrombin. In reaction mixtures containing either type of TF, F. VIIa, 10 nM, either 3H-factor X or 3H-factor IX, 88 nM, and Ca2+, 5 mM, F. VIIa/TF activated factor X (F. X) several fold faster than it activated factor IX (F. IX). Adding heparin, 1 U/ml, increased rates of activation of both substrates and F. X remained the preferred substrate. Adding plasma at concentrations of 5%;or above inhibited factor VIIa/TF catalytic activity. Inhibition was shown to require F. Xa as a cofactor, was prevented by antibodies to extrinsic pathway inhibitor (EPI), and was reversible by decalcification. Thus, with factor VIIa/TF formed with both types of TF, EPI appeared responsible for inhibition induced by plasma. Our data indicate that functional properties of factor VIIa/TF as delineated in reaction mixtures made with purified TF reconstituted into mixed phospholipid vesicles also hold for factor VIIa/TF activity on the surface of cultured HUVEC.
Alpha-2-Macroglobulin Is an Important Progressive Inhibitor of Thrombin in Neonatal and Infant Plasma
Authors: Schmidt Barbara , Mitchell Lesley , Ofosu Frederick A , Andrew Maureen
Published: July 2018

Antithrombin III (ATIII) is the main inhibitor of thrombin in adult plasma; α2-macroglobulin (α2M) and heparin cofactor II (HCII) are of lesser importance. The relative contributions of these inhibitors to the inactivation of thrombin may differ during the neonatal period and infancy, when plasma concentrations of α2M are about twice as high as those of ATIII. We therefore compared the relative importance of these anti-proteases for the inhibition of 125I-thrombin in defibrinated pooled adult and neonatal plasma. Observations were also made in pooled plasma of 6 months old infants. 125I-thrombin-inhibitor complexes were quantitated after SDS-PAGE and autoradiography by scanning densitometry. Thrombin (2.5 NIH U/ml) was inhibited more slowly in neonatal than in adult plasma. However, both plasmas inhibited 88% of the added thrombin by 5 minutes. α2M inhibited consistently a larger fraction of thrombin in neonatal than in adult plasma. Consequently, the ratio of thrombin bound to ATIII over thrombin bound to α2M was significantly lower in neonatal (<2.5) than in adult plasma (>4.5; p <0.0001). In infant plasma, this ratio was <2.0. Upon addition of therapeutic amounts of heparin (0.4 U/ml), differences between the contributions of ATIII and α2M to the inhibition of thrombin were no longer apparent, as over 90% of complexed thrombin was bound to ATIII in heparinized plasmas of all age groups. We conclude that α2M is an important progressive inhibitor of thrombin in young infants. This finding may explain why healthy newborns rarely suffer from thrombosis, despite their low plasma ATIII levels.
Plasminogen Interactions with Immobilized Fibrinogen
Authors: Adelman Burt , Ouynn Patricia
Published: July 2018

This report describes the binding of plasminogen to fibrinogen adsorbed onto polystyrene wells. Binding was determined by enzyme linked immunosorbent assay. Both glu- and lys-plasminogen bound to immobilized fibrinogen in a dose-dependent fashion. However, more lys- than glu-plasminogen bound when equal concentrations of either were added to immobilized fibrinogen. Plasminogen binding was inhibited by epsilon aminocaproic acid indicating that binding was mediated via lysine-binding regions of plasminogen. Soluble fibrinogen added in excess of immobilized fibrinogen did not compete for plasminogen binding but fibrinogen fragments produced by plasmin digestion of fibrinogen did. Treatment of immobilized fibrinogen with thrombin caused a small but significant (p <0.01) increase in plasminogen binding. These studies demonstrate that immobilized fibrinogen binds both glu- and lys-plasminogen and that binding is mediated via lysine-binding regions. These interactions may facilitate plasminogen binding to fibrinogen adsorbed on to surfaces and to cells such as platelets which bind fibrinogen.
Elimination of Native and Carbohydrate-Modified Tissue Plasminogen Activator in Rabbits
Authors: Einarsson Monica , Häggroth Lena , Mattsson Christe
Published: July 2018

The elimination of native and carbohydrate-modified tissue type plasminogen activator (t-PA) after an i. v. bolus injection was studied in rabbits. t-PA with a low content of mannose (mant-PA) was obtained by treatment with α-mannosidase, which cleaves terminal mannose from the carbohydrate side chains of the molecule. About 50% of the total mannose was removed from the native t-PA.

Clearance of t-PA in rabbits was followed by measurements of both the fibrinolytic activity of the activators and the radioactivity of the iodine-labelled compounds. A biphasic mode of elimination of the fibrinolytic activity as well as of the radioactivity was found with both the native and the carbohydrate modified t-PA. The initial half-life (t½α) was the same, about 1.5 minutes, for both compounds, irrespective of method of analysis. The late half-life (t½β) was also the same, about 15 minutes, for both compounds. However, the β-elimination of native t-PA could only be determined accurately from radioactivity data in the dosages tested, i.e. up to 2 mg of t-PA. No dose dependency in the elimination pattern was observed.

In gel filtration experiments, with plasma from the rabbits, it was demonstrated that in addition to fibrinolytically active t-PA and man-t-PA, both fibrinolytically inactive high molecular weight complexes and low molecular weight degradation products of the activators were present in plasma after injection of the compounds.

The second phase of elimination .(β) was much more pronounced after mannosidase treatment of the t-PA. The proportion of molecules eliminated with a longer half-life was increased 6-7 times, from about 4% for native t-PA to about 25% for carbohydrate-modified t-PA.

Thus, a simple reduction of terminal mannose in the carbohydrate side chains of t-PA does not result in altered half-lives. However, a significantly greater proportion of molecules were eliminated in the late β-phase after the carbohydrate modification. The data also support our recent finding that the mannose receptor on the liver cells is at least in part responsible for the clearance of t-PA.
Eicosapentaenoic Acid Interferes with U46619-Stimulated Formation of Inositol Phosphates in Washed Rabbit Platelets
Authors: Chetty N , Vickers J D , Kinlough-Rathbone R L , Packham M A , Mustard J F
Published: July 2018

Eicosapentaenoic acid (EPA) inhibits platelet responsiveness to aggregating agents. To investigate the reactions that are affected by EPA, we examined the effect of preincubating aspirintreated rabbit platelets with EPA on stimulation of inositol phosphate formation in response to the TXA2 analogue U46619. Stimulation of platelets with U46619 (0.5 μM) caused aggregation and slight release of dense granule contents; aggregation and release were inhibited by preincubation of the platelets with EPA (50 μM) for 1 h followed by washing to remove unincorporated EPA. Incubation with EPA (50 μM) for 1 h did not cause a detectable increase in the amount of EPA in the platelet phospholipids. When platelets were prelabelled with [3H]inositol stimulation with U46619 of control platelets that had not been incubated with EPA significantly increased the labelling of mos1tol phosphates. The increases in inositol phosphate labelling due to U46619 at 10 and 60 s were partially inhibited by premcubat10n of the platelets with 50 μM EPA. Since the activity of cyclo-oxygenase was blocked with aspirin, inhibition of inositol phosphate labelling in response to U46619 indicates either that there may be inhibition of signal transduction without a detectable change in the amount of EPA in platelet phospholipids, that changes in signal transduction require only minute changes in the fatty acid composition of membrane phospholipids, or that after a 1 h incubation with EPA, activation of phospholipase C is affected by a mechanism that is not directly related to incorporation of EPA.
Purified Human Plasma Kallikrein Does Not Stimulate but Primes Neutrophils for Superoxide Production
Authors: Zimmerli Werner , Huber Isabelle , Bouma Bonno N , Lämmle Bernhard
Published: July 2018

In patients with septicemia and septic shock the contact phase of blood coagulation is activated. It has been suggested that polymorphonuclear leukocytes (PMN) are directly activated by purified plasma kallikrein. This has been recently questioned because granulocytic elastase release induced by recalcification of normal and prekallikrein-deficient plasma was similar. We studied the interaction of different preparations of purified human plasma kallikrein with PMN. Cytosolic calcium shifts were measured with the quin2 method, PMN aggregation was assayed in an aggregometer, and superoxide production was quantitated as superoxide dismutase inhibitable cytochrome c reduction in a continuous assay. No increase of cytosolic free calcium was found during at least 5 min after adding 10 μg/ml plasma kallikrein to PMN. Similarly, highly purified plasma kallikrein from two different sources did not induce PMN aggregation at all, nor did it stimulate superoxide production. However, sequential exposure of PMN to plasma kallikrein and formylpeptide increased the superoxide production compared to stimulation with formylpeptide alone. This phenomenon which is called priming was observed at plasma kallikrein concentrations ≥7 μg/ml. The active site of the molecule was required for the priming, because plasma prekallikrein, active site-inactivated plasma kallikrein, and soybean trypsin inhibitor treated kallikrein did not prime PMN. This indicates that the contact activation system may play a role in host defence against bacterial infection.
A Method for Blood Platelet Homogenization Using the Aminco-French Pressure Cell
Authors: French Paul C. , Holme Randi
Published: July 2018

A rapid effective method for platelet homogenization using a pressure cell is described. With this method, almost complete homogenization of human platelets occurred without excessive solubilization of enzymes as compared to a pestle homogenization procedure where there was incomplete homogenization. Electron microscopy of pressure homogenates and fractions revealed well preserved organelles.
Patterns of Incorporation of 75Se-Methionine into Fibrinogen and Other Plasma Proteins in Rabbits Stimulated by Different Test Conditions
Authors: Seligsohn Uri , Rapaport Samuel I. , Zivelin Ariella
Published: July 2018

Rabbits were injected with 75Se-Methionine (75SeM) 4-8 hr after being subjected to a variety of experimental conditions: injection of ACTH, growth hormone, glucagon, adrenalin, endotoxin, turpentine, hydrocortisone and laparotomy. All of these experimental conditions except injection of glucagon were associated with increased incorporation of 75SeM into fibrinogen. Three patterns of incorporation of 75SeM into plasma proteins were recognized: 1. the pituitary pattern, which was observed in animals injected with ACTH, growth hormone or endotoxin, and which was characterized by increased incorporation of 75SeM only into fibrinogen and by a delayed incorporation of 75SeM into α2 and β1 globulins; 2. the tissue injury pattern, which was characterized by a markedly increased incorporation of 75SeM into fibrinogen and no alteration in incorporation of 75SeM into α2 or β1 globulins; and 3. the pharmacologic corticosteroid pattern, which was characterized by a moderately increased incorporation of 75SeM into fibrinogen and a strikingly increased incorporation of 75SeM into α2 and β1 globulins.
Experimental Study of Rabbit Fibrinogen Degradation Products
Authors: Soria J , Soria C , Vairel E , Prost R. J , Warneson G
Published: July 2018

An experimental study of human and rabbit fibrinogen degradation products, carried out with a parallel assay of plasma plasminogen has allowed us to show that in rabbits the absence, or only slight increase, of serum fibrinogen degradation products during experimental intravascular coagulations was only the consequence of the lower plasminogen level in rabbits than in human beings.
Effects “in Vitro” of Some Cardiovascular Drugs and Other Agents on Human Platelet Aggregation
Authors: Sacchetti G , Bellani D , Montanari C , Gibelli A
Published: July 2018

The in vitro effect of the following substances on human platelet aggregation is studied: K 4423 (a ß-blocking drug), isoproterenol, phentolamine, papaverine, methamidoline (a myolytic agent).

None of these substances causes aggregation. All inhibit aggregation induced by ADP or noradrenaline, except for phentolamine, which is active only on noradrenaline- induced aggregation.

Hypothesis on the mechanism of action of the compounds tested are proposed.
Insulin and Blood Fibrinolytic Activity
Authors: Hedlin Anne M
Published: July 2018

The effect of insulin on fibrinolysis was studied in hospital patients who were subjected to an insulin tolerance or glucose tolerance test. Blood samples were obtained at frequent intervals over a 2-3 hour period and fibrinolytic activity determined by the dilute whole blood lysis test. Normal control subjects were tested in both fasting and in non-fasting states. A marked increase in fibrinolytic activity was produced during the insulin tolerance test. However, as this increase did not appear until the blood glucose had been reduced to 30-40 mg % it was considered to be due, not to the insulin which had been administered, but rather to the action of those hormones which would be released in response to the hypoglycemia. On the other hand, the results of the glucose tolerance test demonstrated a gradual increase in fibrinolysis during the test period. Again this increased activity could not be attributed to the action of insulin because a similar effect was apparent in the samples obtained from the control subjects. Therefore, it was concluded that the enhancement of fibrinolysis observed in both test and control subjects was not due to the action of insulin.
Enhancement by ADP of Thrombin-induced Aggregation in ADP-refractory Platelets
Authors: Simard-Duquesne Nicole
Published: July 2018

In ADP-refractory rat platelets, thrombin-induced aggregation is enhanced in the presence of ADP and inhibited in the absence of ADP. Two mechanisms are proposed for thrombin-induced platelet aggregation: one through ADP release which may become refractory, and a second mechanism in which thrombin acts synergistieally with ADP.
The Determination of Optimum Glycine Concentration for the Preparation of Human Fibrinogen at Ambient Temperatures
Authors: Silberstein Edward B. , Ingraham Samuel C. , Kereiakes James G.
Published: July 2018

A glycine-precipitation method is presented allowing the preparation of human fibrinogen of high clottability and stability at ambient temperature (25 ± 3° C) with a minimum of specialized equipment. From barium sulfate adsorbed plasma, the optimum yield of clottable fibrinogen is found to occur with a 65% relative glycine saturation of plasma.
Plasminogen Depletion during Administration of Epsilon-aminocaproic Acid
Authors: Nibbelink D. W , Jacobsen C. D
Published: July 2018

Sixteen patients with acute onset subarachnoid hemorrhage were treated with intravenous administration of epsilon-aminocaproic acid. This drug inhibits activation of plasminogen and, to some extent, the effect of plasmin as well. In this study a marked decrease in immunological quantities of plasminogen was observed. This depletion of plasminogen occurred without concommitant quantitative changes in platelets or fibrinogen. Hence, the antiftbrinolytic effect of epsilon-aminocaproic acid is not only through inhibition of plasminogen activation and of plasmin itself, but also through a marked reduction in plasminogen.
The Possible Role of Platelet Coagulant Activities in the Pathogenesis of Venous Thrombosis
Authors: Walsh Peter N
Published: July 2018

Recent studies of the role of platelets in blood coagulation have shown that platelets can trigger intrinsic coagulation by two alternative pathways, protect platelet-associated active clotting factors from inactivation by plasma inhibitors and catalyze intrinsic coagulation reactions on the platelet surface to form fibrin. These platelet coagulant activities (i.e., contact product forming activity, collagen-induced coagulant activity, intrinsic factor-Xa forming activity and platelet factor 3 activity) were found to be increased in patients with deep vein thrombosis developing after hip surgery, in patients with established retinal vein thrombosis and in patients with established deep vein thrombosis. It is suggested that increases in platelet coagulant activities concerned with triggering and catalyzing intrinsic coagulation reactions may play a role in the pathogenesis of venous thrombosis.
The Effect of Dipyridamole and RA 233 on Human Platelet Function in Vitro
Authors: Rifkin Paul L. , Zucker Marjorie B.
Published: July 2018

Dipyridamole (Persantin) is reported to prolong platelet survival and inhibit embolism in patients with prosthetic heart valves, but its mechanism of action is unknown. Fifty jxM dipyridamole failed to reduce the high percentage of platelets retained when heparinized human blood was passed through a glass bead column, but prolonged the inhibition of retention caused by disturbing blood in vitro. Possibly the prostheses act like disturbance. Although RA 233 was as effective as dipyridamole in inhibiting the return of retention, it was less effective in preventing the uptake of adenosine into erythrocytes, and more active in inhibiting ADP-induced aggregation and release. Thus there is no simple relation between these drug effects.
Three Distinct Point Mutations in the Factor IX Gene of Three Japanese CRM+ Hemophilia B Patients (Factor IX BMNagoya 2, Factor IX Nagoya 3 and 4)
Authors: Hamaguchi Motohiro , Matsushita Tadashi , Tanimoto Mitsune , Tekahashi Isao , Yamamoto Kohji , Sugiura Isamu , Takamatsu Junki , Ogata Kanji , Kamiya Tadashi , Saito Hidehiko
Published: July 2018

Enzymatic DNA amplification and complete sequence analysis were used to investigate human factor IX coding sequences in three CRM+ hemophilia B patients. In a patient with severe hemophilia B and a markedly prolonged ox-brain prothrombin time, a C to T transition in exon VI changed the codon for Argl80 to Trp (factor IX BMNagoya 2). This mutation would impair the cleavage by factor XIa required for activation of the zymogen. In a patient with mild hemophilia B, a G to A transition in exon VI changed the codon for Argl45 to His (factor IX Nagoya 3). This substitution also would be predicted to preclude the cleavage of factor IX by factor XIa at this peptide bond (Argl45-Alal46). Furthermore, this point mutation creates a new NlaIII restriction site which provides a quick and reliable method for carrier detection in the affected family members. A patient with severe hemophilia B (factor IX Nagoya 4) had a G to A transition in exon II changing the codon for Glu21 to Lys. This novel point mutation is assumed to impair the function of factor IX by disrupting the calcium binding of factor IX.
Dependence of Blood Clot Lysis on the Mode of Transport of Urokinase into the Clot - A Magnetic Resonance Imaging Study in Vitro
Authors: Blinc A , Planinšič G , Keber D , Jarh O , Lahajnar G , Zidansěk A , Demsar F
Published: July 2018

Magnetic resonance imaging was employed to study the dependence of clot lysing patterns on two different modes of transport of urokinase into whole blood clots. In one group of clots (nonperfused clots, n1 = 10), access of urokinase to the fibrin network was possible by diffusion only, whereas in the other group (perfused clots, n2 = 10) bulk flow of plasma containing urokinase was instituted through occlusive clots by a pressure difference of 3 .7 kPa (37 cm H2O) across 3 cm long clots with a diameter of 4 mm. It was determined separately that this pressure difference resulted in a volume flow rate of 5.05 ± 2.4 × 10−2 ml/min through occlusive clots. Perfused clots diminished in size significantly in comparison to nonperfused ones already after 20 min (p <0.005). Linear regression analysis of two-dimensional clot sizes measured by MRI showed that the rate of lysis was more than 50-times faster in the perfused group in comparison to the nonperfused group. It was concluded that penetration of the thrombolytic agent into clots by perfusion is much more effective than by diffusion. Our results might have some implications for understanding the differences in lysis of arterial and venous thrombi.
Thrombolysis with an Escherichia coli-Produced Recombinant Plasminogen Activator (BM 06.022) in the Rabbit Model of Jugular Vein Thrombosis
Authors: Martin Ulrich , Fischer Stephan , Kohnert Ulrich , Opitz Ulrich , Rudolph Rainer , Sponer Gisbert , Stern Anne , Strein Klaus
Published: July 2018

The recombinant plasminogen activator BM 06.022 consists of the kringle 2 and the protease domains of human t-PA and is unglycosylated because of the expression in Escherichia coli The thrombolytic and pharmacokinetic properties as well as the hemostasis effects of BM 06.022 were investigated in the rabbit model of jugular vein thrombosis. The thrombi were 125I-fibrin labeled. Intravenous bolus injection of 50, 100, 200, and 400 kU/ kg BM 06.022 or 400, 800, and 1600 kU/kg alteplase over 15 s to six rabbits/dose produced a dose-dependent increase of thrombolysis determined 2 h post injection. The dose-response curve of BM 06.022 was located left compared with that of alteplase. The effective dose of 50% thrombolysis (ED50) obtained by half-logarithmic regression analysis was 163 kU/kg (=0.28 mg/kg) for BM 06.022 and 871 kU/kg (= 1.09 mg/kg) for alteplase. At equipotent doses (50% thrombolysis), the residual concentration of fibrinogen was 74.2% and 76.5%, that of plasminogen 66.7% and 69.4%, and that of α2-antiplasmin 47.3% and 46% for BM 06.022 and alteplase, respectively. Pharmacokinetic analysis for plasma activity at a dose of 400 kU/kg revealed a half-life of 18.9 ±1.5 min for BM 06.022, whereas alteplase was distributed with a half-life of 2.1 ± 0.1 min, accounting for 86.7 ± 1.9% of the total AUC, followed by a β-phase with a half-life of 13.8 ± 0.9 min. Plasma clearance of BM 06.022 was 4.7 ± 0.7 ml min-1 kg-1 compared with 20 ±1.2 ml min-1 kg-1 for alteplase. Due to its lower clearance rate, we conclude that BM 06.022 requires a 5.3-fold lower activity dose than alteplase to achieve an effect of 50% thrombolysis in rabbits. At equipotent doses, BM 06.022 is fibrin selective to the same degree as alteplase.
Functional Characterization of a Variant Factor XII (F XII Locarno) in a Cross Reacting Material Positive F XII Deficient Plasma
Authors: Wuillemin Walter A , Furlan Miha , Stricker Hans , Lämmle Bernhard
Published: July 2018

The plasma of a healthy woman was found to contain half normal factor XII (FXII) antigen level (0.46 U/ml) without any FXII clotting activity (<0.01 U/ml). The variant FXII in this plasma, denoted as FXII Locarno, was partially characterized by immunological and functional studies on the proposita’s plasma. FXII Locarno is a single chain molecule with the same size (M
r = 80 kDa) as normal FXII. Isoelectric focusing suggested an excess of negative charge in the variant FXII as compared to normal FXII. In contrast to FXII in normal plasma, FXII Locarno was not proteolytically cleaved upon prolonged incubation of proposita’s plasma with dextran sulfate. Adsorption to kaolin was similar for both, abnormal and normal FXII. Incubation of the proposita’s plasma with dextran sulfate and exogenous plasma kallikrein showed normal cleavage of FXII Locarno outside of the tentative disulfide loop Cys340-Cys467, but only partial cleavage within this disulfide loop. Furthermore, plasma kallikrein-cleaved abnormal FXII showed neither amidolytic activity nor proteolytic activity against factor XI and plasma prekallikrein.

These results suggest a structural alteration of FXII Locarno, affecting the plasma kallikrein cleavage site Arg353-Val354 and thus formation of activated FXII (a-FXIIa).
Changes in Haematological Indices, Blood Viscosity and Inhibitors of Coagulation during Treatment of Endometriosis with Danazol
Authors: Ford I , Li C , Cooke I D , Preston F E
Published: July 2018

The effects of treatment with danazol (600 mg daily) on haemostatic and haematological function were investigated in 18 pre-menopausal women with endometriosis. Blood samples were taken at 2 pre-treatment visits, at 6, 12 and 24 weeks on treatment, and at 6 weeks after discontinuation of the drug. Haemoglobin, red cell count, haematocrit and platelet count all rose significantly during treatment with danazol (p <0.01 vs. baseline). Plasma fibrinogen levels fell significantly (p <0.01), while whole blood viscosity increased during treatment and remained significantly elevated at follow-up. The prothrombin time shortened, but remained within normal limits, and there were no significant changes in factor VII:C, Vila, or fibrinopeptide A. No significant changes were found in platelet function. Plasma B-beta 15-42 increased significantly. Functional levels of protein C, protein S, and antithrombin III, all rose significantly, above the normal range, while C4b-binding protein levels fell. We conclude that the observed changes in coagulation inhibitors and fibrinolytic activity may be considered beneficial in the context of venous thromboembolism. The rheological effects, however, indicate a degree of caution in the use of the drug in individuals considered to be at risk from arterial cardiovascular disease.
Activation of Blood Coagulation after Abrupt or Stepwise Withdrawal of Oral Anticoagulants - A Prospective Study
Authors: Palareti G , Legnani C , Guazzaloca G , Frascaro M , Grauso F , De Rosa F , Fortunato G , Coccheri S
Published: July 2018

The occurrence of a “rebound hypercoagulable state” in patients after discontinuation of oral anticoagulants is still a matter of debate and no definite recommendation can be made on the best procedure for anticoagulant withdrawal. The present study investigated the changes in the levels of markers of activated blood coagulation in 32 patients (pts) in whom warfarin treatment (for venous thromboembolic disease) was randomly withdrawn abruptly (n = 17, group A) or gradually (n = 15, group B: ⅔ of initial dose the 1st week, ⅓ the 2nd weeks and nothing from the 3rd week on). Blood was sampled at baseline, once a week for the first three weeks and after 2 months. At the 1st week group A had significantly higher F1+2 and TAT values (p <0.001); at the 2nd week F1 + 2 levels remained higher (p <0.05) though INR values were not different from those of group B. After baseline, higher than normal F1+2 levels were recorded in 32/66 (48%) controls in group A vs 15/60 (25%) in group B (p <0.01); at the 2nd week, 10/17 (59%) patients in group A vs 1/15 (7%) in group B still had higher than normal F1+2 levels (p <0.01 ). The values of areas under curve (AUC) and maximum concentrations of all variables were not statistically different in the two groups; however, very high levels were observed in a few cases of group A. Thrombotic events (one DVT recurrence and one thrombophlebitis in a varicose vein) occurred in 2 pts of group A, both with high F1+2 and TAT AUC values. In conclusion, the present study shows that withdrawal of oral anticoagulants elicits low grade transient clotting activation, which is more intense and longer lasting after abrupt discontinuation. In single cases, however, such activation is particularly intense. It is possible that these cases are at greater risk of thrombotic complications.
Multicenter Evaluation of a Kit for Activated Protein C Resistance on Various Coagulation Instruments Using Plasmas from Healthy Individuals
Authors: Rosén S , Johansson K , Lindberg K , Dahlbäck B ,   (for the APC resistance study group)
Published: July 2018

Recently a new hemostatic disorder has been described which appears to be an important risk factor for familial thromboembolism. The disorder is characterized by a poor anticoagulant response to activated Protein C (APC) and has been shown to be due to lack of an APC cofactor activity which is a property of factor V.

A kit for determining the response of plasma samples towards addition of APC in an APTT-based assay - COATEST APC Resistance -has been evaluated on 35 coagulation instruments in a multicenter study involving 32 laboratories. A lyophilized normal plasma and identical plasma aliquots from 20 individuals, one of whom had a borderline resistance to APC, were analysed in each laboratory and the sensitivity of each plasma to APC was determined as the ratio between the clotting times obtained in the presence and absence of APC (APC ratio).

The plasma from the individual with a borderline resistance to APC activity was correctly classified as the lowest responder in each laboratory, with an APC ratio in the range 1.6-2.4. In comparison, plasmas from individuals with a pronounced response to APC activity resulted in APC ratios above 3.4 in most cases. Interestingly, although the actual APT time for a plasma from a given individual showed a more than 10 s difference due to the type of instrumentation used, the variation in the APC ratio was limited. A similar discrimination was also obtained from evaluation of the actual prolongation of the clotting time in the presence of APC.

The intra-laboratory coefficient of variation for the clotting times were on average 2.0% and 3.9% in the absence and presence of APC, respectively, indicating that the precision for the prolonged clotting times obtained in the presence of APC is sufficient to allow a safe assignment of the APC response. The APC ratio for the lyophilized normal plasma was 2.7 ± 0.2 (2 S.D.) illustrating a narrow distribution between instruments which shows the feasibility of including such plasma for assay validation. Altogether, the results indicate that all the coagulation instruments included in the study can be used for detection of individuals with resistance to APC activity through determination of the APC ratio or the prolongation time.
Results of a Multicenter Study Assessing the Status of Standardization of a Recombinant Thromboplastin for the Control of Oral Anticoagulant Therapy
Authors: Tripodi A , Chantarangkul V , Braga M , poller L , ten Cate J W , van den Besselaar A M H P , Mannucci P M
Published: July 2018

An international collaborative study involving 20 laboratories was undertaken to evaluate the status of standardization of a commerical recombinant thromboplastin. The results show that the reagent fulfills the WHO requirements for thromboplastin calibration against two International Reference Preparations (IRP) from different species, because there was a linear relationship of log-transformed prothrombin times as measured by the recombinant reagent and the two IRPs in normal individuals and patients stabilized on oral anticoagulants. The regression lines drawn through the patient data points passed through the normal data points in the majority of instances. The average ISI values for three different batches were close to unity. On the average, the ISI value calculated against BCT/253 (human, plain) was slightly smaller than that against RBT/79 (rabbit, plain). The between laboratory precision of calibration (CV = 5-6%) did not change whether BCT/253 or RBT/79 was used, whereas it was substantially improved when the calibrations of batches 2 and 3 were performed against batch 1, arbitrarily taken as standard (CV ≤ 1.7%). Statistical analysis to test for differences between slopes revealed no significant between-batch differences in 16 of 20 laboratories. However slight differences for the third batch (2.8% less than the average ISI value of batches 1 and 2) leave room for further improvement in the standardization of the reagent.
Potentiating Effect of Adenosine on Other Inhibitors of Platelet Aggregation
Authors: Murakami M. , Odake K. , Takase M. , Yoshino K.
Published: July 2018

Adenosine was rapidly incorporated into human platelets, and the inhibitory effect of adenosine on platelet aggregation was correlated with the incorporation process. Adenosine potentiated the inhibitory action of other inhibitors, such as dipyridamole, prostaglandin E1 and Y-3642. The inhibition of aggregation was associated with the preservation of platelet adenine nucleotides and the prevention of ADP release. On the other hand, the radioactive adenine nucleotide pattern of platelets was not substantially affected by inhibitors. The relation of inhibition of aggregation with ADP release was discussed.
Activation of Hageman Factor by Cardiac Arrest
Authors: Latour J. G. , McKay D. G. , Parrish Mary H.
Published: July 2018

The induction of cardiac arrest in rabbits for a period of 3min, followed by reestablishment for the circulation, resulted in a 27 % decrease in circulating Hageman factor. The maximum depletion occurred within 30 to 60 min and normal levels were restored by 4 h. Anti-contact product activity remained unchanged. Circulating platelets dropped by 17 % and small arterial thrombi were found predominantly in the lungs. It is concluded that cardiac arrest followed by re-circulation of the blood causes activation of Hageman factor. The proximate mechanism of this activation in experimental generalized anoxemia remains to be determined. Administration of a potent glucocorticoid prior to cardiac arrest markedly inhibited activation of the clotting mechanism.
Measurement of Fibrin Proteolysis In Vivo by a Radioactive Fibrin-Millipore Tube Technique
Authors: Cihak R. W , Shaw S. T
Published: July 2018

Millipore tubes containing radioactive fibrin can be used to measure fibrin proteolysis. Experiments described herein indicate that loss of soluble radioactivity from fibrin- containing tubes during in vivo incubation can be used to measure relative rates of lytic activity at specific locations within an organism under various conditions.

Preparation of urea-insoluble 125I-fibrin and its apparent purity and uniformity of label have been described. This preparation has been shown to be stable over a period of several months, when properly stored, in its response to protease activity and lack of spontaneous degradation. Contained within Millipore tubes, the fibrin is protected from direct cellular attack while it is susceptible to enzymes from the environment which can enter the porous chambers. Enzymatic degradation of fibrin releases diffusible radioiodinated peptides from the tubes. Thus, determination of fibrin-tube radioactivity before and following in vivo incubation may be used as a measurement of proteolytic rate within the organism.
Clot Lysis in a Perfusion System
Authors: Bouvier C. A , Gruendlinger J , Berthoud S
Published: July 2018

Most information on clot lysis is derived from in vitro methods whereby various components of the clotting and fibrinolytic systems are mixed before an actual clot is formed. This situation bears little relationship to thrombolysis in vivo. Therefore several techniques have been recently proposed, in which pre-formed clots are exposed to the effects of active agents by contact and diffusion rather than by intimate mixing prior to clotting. We describe an apparatus whereby a perfusion is delivered at controlled rates to clots of standard size and volume formed in calibrated tubes. The composition of the clots can be varied as well as the rate of perfusion and the content of perfusate. The surface of contact between the fluid and the fibrin gel is kept constant throughout and the clot-perfusate relationship is as close as possible to the in vivo situation during thrombolytic therapy. Under these conditions clot lysis by Streptokinase appears as a linear function of time, and the rate of lysis is directly related to kinase concentration. Since the clot intrinsic plasminogen-proactivator content is sufficient to ensure lysis, the lysis time finally depends upon the rate of diffusion of the kinase into the gel. Inhibition obtained with various amounts of E-aminocaproic acid incorporated to the clots or added to the perfusion fluid also suggests that diffusion problems are of major importance in physiological and therapeutic thrombolysis.
Human Antihemophilic Factor (AHF) Purification A Comparison of Two Procedures
Authors: Hurt J. P , Wagner R. H , Brinkhous K. M
Published: July 2018

A method is described for the precipitation from plasma of a human AHF fraction (Fraction AA) with beta alanine. The Fraction AA is further purified by reprecipitation with beta alanine or by adsorption with Florigel or bentonite. Reprecipitation with beta alanine is a convenient step as no additional equipment or reagents are required for the second precipitation. All reagents can be rendered sterile and free of pyrogens. When the precipitates are quantitatively dissolved, the yields are greater than 70% and purification ranges from 300-600 times. The adsorption procedure also has advantages. Adsorption adds only 45 minutes to a 6 hour fractionation. AHF recovery is as high as 70% with a purification of 3,000 to 5,000 times.
Some Effects of Fibrinogen Degradation Products (FDP) on Blood Platelets
Authors: Jerushalmy Z , Zucker M. B
Published: July 2018

“Early” fibrinogen degradation products are more potent inhibitors of thrombin-induced clotting than “late” products and also interfere with the ability of thrombin to release serotonin from platelets. “Early” and “intermediate” FDP cause moderate inhibition of platelet aggregation induced by adenosine diphosphate or connective tissue particles. Serotonin release by connective tissue particles is probably not inhibited by FDP.
Human and Bovine Plasminogen-Free Thrombin, Purified by Means of Gel Filtration and Ion Exchange Chromatography
Authors: Berg W , Korsan-Bengtsen K , Ygge J
Published: July 2018

A simple method for preparation of plasminogen-free human and bovine thrombin is described.

Crude thrombin was prepared in the usual manner from oxalated plasma by means of adsorption on BaSO4, elution with trisodium citrate and activating the eluate from BaSO4 with tissue thromboplastin.

This crude thrombin was purified by means of gel-filtration and chromatography on CM-Sephadex A-50.

The gel-filtration was performed on three types of Sephadex, G-75, G-50, and G--25. By means of Sephadex G-75 the thrombin was well separated from the main part of inert protein and this type of Sephadex was used for the purification in large-scale. Separation of thrombin from protein of higher molecular weight was also obtained with Sephadex G-50 but not with Sephadex G-25 indicating a molecular weight of thrombin between 4000 and 10,000.

The importance of using an elution buffer of sufficient high ionic strength for gel-filtration is shown. A great deal of the thrombin was adsorbed to the Sephadex if the gel-filtration was performed at a too low ionic strength.

The final preparation contained 30,000 NIH units of thrombin per mg tyrosin and no detectable plasminogen.

The commercial preparation “Topostasine” was also purified in the same manner, but the plasminogen content in “Topostasine” was high and could not be completely separated from thrombin.
Preparation of Human Plasminogen Suitable as Substrate for Quantitative Determination of Plasminogen Activators
Authors: Berg W , Korsan-Bengtsen K , Ygge J
Published: July 2018

A simple method for preparation of plasminogen with low spontaneous activity and soluble at a neutral pH and at physiological ionic strength is described. Euglobulin made from fresh, oxalated, BaSO4-adsorbed, human plasma was first purified by means of gel filtration on Sephadex G-200. After gel filtration, further purification and concentration was done on DEAE-sephadex A-50. The activity was 100-130 casein units per mg tyrosine.
Über den Nachweis der Plasminogenverarmung in retrahierten Vollblutgerinnseln und über deren Bedeutung für die Lysierbarkeit mit Streptokinase
Authors: Gottlob R , Blümel G
Published: July 2018

1. Blood clots are lysed by SK before the beginning of retraction. Retracted clots are only very little lysed.

2. Recalcified citrated blood is easily lysed since the excess of added calcium ions inhibits the retraction.

3. The determination of the water content of the clots gives information on their retraction state.

4. The determination of water content made it possible to prove that thrombus retraction takes place in vivo. Already one day after thrombosis the water content is clearly lower than in fresh clots. Later the water content increases again slightly.

5. Clots formed in vitro and in vivo are lysed by SK after retraction if they come into contact with fibrin or fibrinogen containing plasminogen.

6. Heated fibrin plates or heated fibrin tubes both with SK, casein degradation and an immunological method allowed to prove that whole blood clots loose their activable plasminogen during retraction. One assumes that plasminogen is expressed during retraction.

7. The possibility of a therapeutical fibrinolysis with SK is due to the fact that SK can penetrate into the clot and the SK containing thrombus continuously comes in contact with circulating blood rich in plasminogen. The importance of these results for thrombolysis with SK is discussed.
Platelet Aggregation and Fibrinogen Binding in Human, Rhesus Monkey, Guinea-Pig, Hamster and Rat Blood: Activation by ADP and a Thrombin Receptor Peptide and Inhibition by Glycoprotein IIb/IIIa Antagonists
Authors: Cook Nigel S , Zerwes Hans-Günter , Tapparelli Carlo , Powling Max , Singh Jagjit , Metternich Rainer , Hagenbach Alex
Published: July 2018

Platelet aggregation and fibrinogen binding in whole blood, induced either by ADP or by a 14 amino acid peptide mimicking an N-terminal region of the platelet thrombin receptor (TRP, thrombin receptor activating peptide), have been studied with blood from different species. Aggregation was assessed by counting the number of single platelets in blood before und after addition of the agonist with an automated cell counter. Both ADP (0.02-0.5 μM) and TRP (1-10 μM) were found to be potent agonists of platelet aggregation in human, rhesus monkey and guinea-pig blood, causing a near-maximal aggregatory response within 2 min of agonist addition. In contrast, hamster and rat platelets were much less responsive to both ADP and TRP in terms of the whole blood aggregation.

Echistatin, RGDW and a synthetic glycoprotein (GP) IIb/IIIa antagonist, Ro 43-8857, inhibited fibrinogen binding to purified immobilized human GP-IIb/IIIa with IC50’s of 1.6, 88 and 11.4 nM, respectively. In whole human blood, the respective IC50’s (as determined by flow cytometric analysis of platelet fibrinogen binding) were 4.4, 1700 and 29.5 nM. The affinities of these three compounds for inhibiting fibrinogen binding in whole blood from rhesus monkeys and guinea-pigs were similar to the affinities for human platelets, but with rat blood echistatin, RGDW and Ro 43-8857 were all around 100-fold less potent. Ro 43-8857 was a potent inhibitor of ADP- or TRP-induced platelet aggregation in human, rhesus monkey and guinea-pig whole blood (IC50 of 69-320 nM) but was much less active in hamster blood.

These results highlight important species differences in the response of platelets to activation by two different agonists and also in their inhibition by GP-IIb/IIIa antagonists. In particular, platelets from the rat and hamster were insensitive to agonists and antagonists, whereas guinea-pig and rhesus monkey platelets responded with an affinity similar to human platelets. Since these studies were performed in whole blood, the results should be representative of those expected in animal experiments. These recently developed methods for studying platelet responses in small aliquots of whole blood are simple to perform and provide important information concerning the optimal choice of species for subsequent in vivo studies with these compounds.
A Mutation in the Protein S Pseudogene Is Linked to Protein S Deficiency in a Thrombophilic Family
Authors: Ploos van Amstel Hans K , Reitsma Pieter H , Hamulyák Karly , Die-Smulders Christine E M de , Mannucci Pier M , Bertina Rogier M
Published: July 2018

Probands from 15 unrelated families with hereditary protein S deficiency type I, that is having a plasma total protein S concentration fifty percent of normal, were screened for abnormalities in their protein S genes by Southern analysis. Two probands were found to have a deviating DNA pattern with the restriction enzyme Mspl. In the two patients the alteration concerned the disappearance of a Mspl restriction site, CCGG, giving rise to an additional hybridizing Mspl fragment.

Analysis of relatives of both probands showed that in one family the mutation does not co-segregate with the phenotype of reduced plasma protein S. In the family of the other proband, however, complete linkage between the mutated gene pattern and the reduced total protein S concentration was found: 12 heterozygous relatives showed the additional Mspl fragment but none of the investigated 26 normal members of the family. The mutation is shown to reside in the PSβ gene, the inactive protein S gene. The cause of type I protein S deficiency, a defect PSα gene has escaped detection by Southern analysis. No recombination has occurred between the PSα gene and the PSβ gene in 23 informative meioses. This suggests that the two protein S genes, located near the centromere of chromosome 3, are within 4 centiMorgan of each other.
α2-Adrenoreceptor Status of Human Platelet Subpopulations Separated by Continuous Flow Electrophoresis
Authors: Crook M , Crawford N
Published: July 2018

Human platelets were separated into subpopulations using continuous flow electrophoresis, which uses electrophoretic mobility as the separative parameter. The platelets with the greatest electrophoretic mobility showed higher amounts of total sialic acid than the less electrophoretically mobile subpopulation (74.3 ± 18.2 nmol/109 platelets vs. 49.2 ± 20.1 nmol/109 platelets, p <0.05 paired Student’s t-test). Furthermore, neuraminidaselabile sialic acid was also elevated in the more electrophoretically mobile platelet subpopulation (29.1 ± 6.0 nmol/109 platelets vs. 21.8 ± 10.4 nmol/109 platelets, p <0.05 paired Student’s t-test). We also found that the sialic acid enriched platelet subpopulation had more α2-adrenoreceptors than the less electrophoretically mobile platelets (457 ± 104 vs. 302 ± 164 receptors per cell, p <0.05 paired Student’s t-test).
Prolongation Of The Bleeding Time And Increased Intraoperative Bleeding In Neurosurgical Patients Treated With High Dose ∈-Aminocaproic Acid
Authors: Green D , Glick R , Ts’ao C
Published: July 2018

∈-Aminocaproic acid (EACA) is an inhibitor of fibrinolysis which crosses the blood brain barrier. Several studies have shown that in doses of 24 G/day, EACA reduces the incidence of rebleeding in patients with intracranial aneurysms. Recently, however, intravenous doses of up to 48 G/day have been recommended. At this dose level, we observed rebleeding and increased intraoperative blood loss (> 1000 ml) in 5 of 8 patients, and in two of 5 patients on 36 G/day, whereas no such bleeding occurred in 4 patients on lesser doses. Laboratory investigation revealed that the template bleeding time was prolonged in all patients receiving EACA in daily intravenous doses of 36 or 48 G. Values of greater than 20 min were recorded in 4/8 patients on the 48 G dose and 3/5 on the 36 G dose, whereas no prolongation was observed with 24 G/day. Bleeding times returned to normal in all but one patient when the drug was stopped. Platelet aggregation studies showed impaired responses to ADP, collagen, and epinephrine during drug administration which returned to normal when the drug was discontinued. We conclude that EACA exerts a dose- dependent inhibitory effect on platelet function, and that patients receiving doses in excess of 24 G/day are at risk for serious bleeding. Patients receiving EACA should be monitored with serial bleeding time tests.