bluuebunny/crossref_metadata_2025 huggingface dataset:
Purified Factor XII Has a Higher Specific Activity than the Parent Molecule in Plasma
Authors: Wuillemin Walter A , Furlan Miha , Huber Isabella , Lämmle Bernhard
Published: July 2018

The specific clot promoting activity of factor XII (F XII) in plasma samples from 50 healthy adults was between 30 and 48 U/ mg, whereas the specific activity of purified F XII ranged from 55 to 66 U/mg. This difference was neither due to partial proteolytic activation during purification of F XII nor to the influence of plasma protease inhibitors. Purified F XII showed normal size and charge, as demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing, respectively. The increase of the specific F XII activity during the purification process mainly occurred after anion exchange chromatography on DEAE-Sephadex and after the final gel filtration step. Upon dextran sulfate activation, proteolytic cleavage of F XII and generation of kallikrein-like amidolytic activity was faster in F XII deficient plasma containing purified F XII than in F XII deficient plasma containing a corresponding amount of pooled normal plasma (NHP). The binding to kaolin was similar for both, purified F XII and plasma F XII.

In conclusion, purification alters the properties of F XII in an unknown way, resulting in an increased specific clot promoting activity.
Blood Group A and B Determinants Are Expressed on Platelet Glycoproteins IIa, IIIa, and Ib
Authors: Santoso S , Kiefel V , Mueller-Eckhardt C
Published: July 2018

We report the localization of A, B blood group determinants on intrinsic glycoproteins using anti-A, B IgG antibodies. By radioimmunoprecipitation, anti-A and anti-B precipitated three bands with apparent molecular masses in sodium dodecylsulphate- polyacrylamide gel electrophoresis (SDS-PAGE) of 159, ~ 120 and 85 kDa under non-reduced conditions and 145, ~ 126 and 97 kDa under reduced conditions. In two-dimensional gel electrophoresis (isoelectric focussing/SDS-PAGE) these three bands could be resolved into six spots and fulfilled previously defined citeria for platelet membrane glycoprotein complexes Ia/IIa, Ic’IIIa, Ib/IX and IIb/IIIa. The results were supported by data obtained by an assay employing antibody-specific immobilizatisn of platelet antigens (MAIPA). By this technique, blood group A, B determinants were shown to be immobilized by monoclonal antibodies specific for GPIa, Ic’, IX, IIb/IIIa and strongly by mab specific for GPIIa, but not by mab specific for HLA class I molecules.

The more precise localization on platelet glycoproteins was achieved by immunoblotting technique by which blood group A determinants could be assigned to GPs IIa, IIIa and Ib.
The Effect of Human Platelets on Lipolytic Activity in Plasma
Authors: Vik-Mo Harald
Published: July 2018

A method for assay of lipolytic activity in platelet-poor plasma (PPP), platelet-rich plasma (PRP) and washed human platelets is presented. The lipolytic activity in PRP was about twice the activity in PPP. A significant correlation between lipolysis in platelets and platelet number was established. Molar sodium chloride strongly inhibited lipolytic activity in platelets and a washing procedure did not significantly change the lipolysis. The results indicate that a lipoprotein lipase is bound to human platelets and may play a role in metabolism of triglyceride in platelets.
Plasma Components which Interfere with Ristocetin-induced Platelet Aggregation
Authors: Baugh Robert F , Brown James E , Hougie Cecil
Published: July 2018

Normal human plasma contains a component or components which interfere with ristocetin-induced platelet aggregation. Preliminary examination suggests a protein (or proteins) which binds ristocetin and competes more effectively for ristocetin than do the proteins involved in ristocetin-induced platelet aggregation. The presence of this protein in normal human plasma also prevents ristocetin-induced precipitation of plasma proteins at levels of ristocetin necessary to produce platelet aggregation (0.5–2.0 mg/ml). Serum contains an apparent two-fold increase of this component when compared with plasma. Heating serum at 56° for one hour results in an additional 2 to 4 fold increase. The presence of a ristocetin-binding protein in normal human plasma requires that this protein be saturated with ristocetin before ristocetin-induced platelet aggregation will occur. Variations in the ristocetin-binding protein(s) will cause apparent discrepancies in ristocetin-induced platelet aggregation in normal human plasmas.
The Activation of Human Factor IX
Authors: Østerud B , Laake K , Prydz H
Published: July 2018

The activation of factor IX purified from human plasma has been studied. Factor XIa and kallikrein separately activated factor IX to factor IXa. In both cases factor IX a had an apparent molecular weight of about 42–45000 in sodium dodecyl sul-phate-polyacrylamide disc gel electrophoresis compared with a molecular weight of about 70000 for the native factor IX. The activation by XIa required Ca2+-ions whereas Ca2+-ions did not influence the activation by kallikrein. A mixture of tissue thromboplastin and factor VII or RusselPs-viper venom alone did not activate factor IX. Trypsin activated and plasmin inactivated factor IX.
Serologic Evidence for Hepatitis B Virus Infection in Patients with Hemophilia B
Authors: Hoofnagle Jay H , Aronson David , Roberts Harold
Published: July 2018

Among sera from 160 patients with hemophilia B from 9 centers in Europe and North and South America, 2.5% were positive for hepatitis B surface antigen (HBsAg), 60% for antibody to HBsAg, and 31% for antibody to the hepatitis B core antigen. Evidence of exposure to the hepatitis B virus appeared to be related to severity of disease and age rather than the source and method of manufacturer of factor IX concentrate.
Post-transfusion Hepatitis Following the Use of Prothrombin Complex Concentrates
Authors: Roberts Harold R , Blatt Philip M
Published: July 2018

The incidence of post-transfusion hepatitis in patients receiving concentrates containing the prothrombin complex factors is reviewed. A survey of the literature indicates a high incidence of hepatitis in such patients.
Heterogeneity of Rabbit Platelets
Authors: Freedman Michael L , Karpatkin Simon
Published: July 2018

Intravenous injection of Phenylhydrazine into intact rabbits resulted in a thrombocytosis of 1.85 fold and megathrombocytosis (large platelets) of 2.30 fold. Splenectomy markedly impaired this response. The intravenous injection of red blood cell hemo-lysates gave a response similar to that of Phenylhydrazine in both intact and splenec-tomized animals. Basal data obtained from intact and splenectomized animals; as well as kinetic data obtained following apparent splenic blockade secondary to Phenylhydrazine hemolysis revealed the presence of a splenic platelet pool with preferential splenic sequestration of megathrombocytes.

It is suggested that the thrombocytosis and absolute megathrombocytosis of hemolysis is due to splenic blockade by red blood cell hemolysate.
The Platelet-Endothelial Cell – VIII Axis
Authors: Nachman R. L , Jaffe E. A
Published: July 2018

Cultured human endothelial cells synthesize and secrete a protein(s) which has factor VIII antigen and von Willebrand factor activity. Subcellular membrane and granule fractions derived from human platelets also contain the factor VIII antigen and von Willebrand factor activity. Circulating platelets constitute a significant reservoir of VIII antigen containing approximately 15 % of the amount present in platelet-poor plasma. Thus normal platelets contain surface bound as well as intracellularly stored von Willebrand factor, a protein synthesized by endothelial cells which is required for normal platelet function.
The Young Platelet Population in Children with Cyanotic Congenital Heart Disease
Authors: Goldschmidt Béla , Sørland Svein Jan
Published: July 2018

Some morphological, biochemical and functional parameters of platelet population in children with cyanotic congenital heart disease (CCHD) were studied by making comparisons of the normal platelet population in both CCHD patients and controls. The mean volume of the platelets from cyanotic patients was greater than from normals. The platelet size distribution curves demonstrated a shift towards larger than normal size in the case of CCHD. The mean protein content, as well as the mean PF3 content of platelets was increased in CCHD. Following addition of kaolin, PF3 release was more rapid and of shorter duration with platelets from CCHD patients as compared to normal platelets. They also released more PF3 than did normal platelets. After addition of ADP, collagen, or adrenalin, platelets of CCHD patients were more responsive than similarly treated platelets from normals. Platelets from CCHD showed an increased initial rate of aggregation and greater maximum aggregation. These data suggested that the platelet population of CCHD patients consists of larger, younger and functionally more active platelets than does the platelet population of normals.
The Effect of Beta Adrenergic Blockade upon Exercise-Induced Changes in Blood Coagulation and Fibrinolysis
Authors: Britton B.J , Wood W.G , Smith Muriel , Hawkey Christine , Irving M.H
Published: July 2018

Exercise stress is known to stimulate blood coagulation and fibrinolysis possibly as a result of sympatho-adrenal stimulation of the beta adrenergic receptor. In order to test this hypothesis five men exercised on four separate occasions with and without prior beta adrenergic blockade with oxprenolol, propranolol and pindolol. The increase in plasma adrenaline and noradrenaline concentration was much greater during exercise under beta blockade but activation of fibrinolysis was enhanced as well. Factor VIII levels did not change significantly during any of the experiments. These results suggest that the activation of fibrinolysis by exercise is not mediated by the beta adrenergic receptor. The influence of beta adrenergic blockade on the change in factor VTII with exercise was inconclusive.
Role of Surface Glycoproteins in Human Platelet Function
Authors: Nurden A. T , Caen J. P
Published: July 2018

Glycoproteins present at the external surface of cells probably play specific roles in cellular function. Increasing evidence suggests that the glycoproteins span the plasma membrane with the bulk of the bound carbohydrate asymmetrically distributed on the outer surface. Micellar association of glycoproteins in membranes leads to pore formation and functional roles in transport through the membrane, while surface glycoproteins have been shown to be enzymes, to determine cell specificity and contribute to the cell surface charge. The platelet plasma membrane contains 3 major glycoproteins, glycoproteins I, II and III as characterized in order of their decreasing molecular weight. Glycoprotein I appears to have the highest sialic acid content and to give rise to a platelet specific acidic macroglycopeptide on trypsin digestion. Specific glycoprotein abnormalities in the platelets of patients with Glanzmann’s thrombasthenia suggest that the glycoproteins play a role in the mechanism of platelet aggregation. A much reduced content of glycoprotein I in the platelets of 2 patients with the Bernard Soulier syndrome may be associated with their defective adhesion to subendothelium and indicates a possible relationship on the platelet surface with the von Willebrand factor protein. Preliminary evidence suggests that in common with other plasma membranes the platelet membrane has a fluid structure and that the organization of the glycoproteins on the platelet surface is extremely sensitive to stimuli and susceptible to change.
Importance of Protease Inhibition in Studies on Purified Factor VIII (Antihaemophilic Factor)
Authors: Beck Eugen A. , Bachmann Peter , Barbier Peter , Furlan Miha
Published: July 2018

According to some authors factor VIII procoagulant activity may be dissociable from carrier protein (MW~ 2 × 106) by agarose gel filtration, e.g. at high ionic strength. We were able to reproduce this phenomenon. However, addition of protease inhibitor (Trasylol) prevented the appearance of low molecular weight peak of factor VIII procoagulant activity both at high ionic strength and elevated temperature (37°C). We conclude from our results that procoagulant activity and carrier protein (von Willebrand factor, factor VIII antigen) are closely associated functional sites of native factor VIII macro molecule. Consequently, proteolytic degradation should be avoided in functional and structural studies on factor VIII and especially in preparing factor VIII concentrate for therapeutic use.
The Effects of Hepes Buffer on Clotting Tests, Assay of Factors V and VIII and on the Hydrolysis of Esters by Thrombin and Thrombokinase
Authors: Roberts Phyllis S. , Hughes Haywood N. , Fleming Patricia B.
Published: July 2018

Shorter clotting times were found in the presence of 50 mM Hepes (N-2-hydroxyethylpiper-azine-N1-2-ethanesulfonic acid) buffer than of 50 mM Imidazole buffer in one-stage assays of factors V and VIII, in modified APTT and PT tests and in tests of the clotting of human plasma by purified human thrombin. All tests were performed at ionic strength 0.155 in the presence of either Hepes. NaOH or Imidazole. HC1 buffer, pH 7.4 at 37°. The faster clotting in the presence of Hepes buffer, therefore, is probably due, at least in part, to acceleration by Hepes of thrombin’s enzymatic action on fibrinogen and/or of the polymerization of the fibrin monomers.

Hepes may also have effects on other blood clotting reactions. Rates of hydrolysis of TAME or BAME (p-toluenesulfonyl-or benzoyl-L-arginine methyl ester) at pH 7.4, 37° by purified human or bovine thrombin were essentially the same in 200 mM Hepes as in 250 mM Tris. HQ buffer (rates in Hepes. NaOH or Hepes. KOH buffers were compared with those in Tris. HQ plus NaCl for KC1). However, with purified bovine thrombokinase, rates of TAME hydrolysis in Hepes buffer were accelerated and rates of BAME hydrolysis slightly inhibited. Hepes, therefore, reacts with thrombokinase but whether this accelerates (or inhibits) the rate of converting prothrombin to thrombin remains to be determined. In addition, Hepes has an inhibitory effect on clotting since increasing the concentration of Hepes from 50 mM to 200 mM inhibits clotting in the PT, APTT and bovine thrombin-human plasma tests.

Hepes buffer is being added to some plasmas and to some reagents used in clotting tests. It is, therefore, important to realize that its concentration must be monitored closely or erroneous results may be obtained in clotting tests and assays of clotting factors.

The clotting times were the same in the presence of 50 mM Tris. HC1 as in Imidazole. HC1 buffers in APTT tests at three ionic strengths but they differed slightly in plasma-thrombin tests. Depending upon the ionic strength, 17 mM Barbital Sodium. HC1 buffer inhibited APTT tests but accelerated plasma-thrombin tests. All the buffers tested, therefore, have individual effects on the clotting tests.
Study of a Proposed International Standard for Blood Coagulation Factor IX
Authors: Brozović Milica , Kirkwood T. B. L , Robertson Iris
Published: July 2018

An International Collaborative Study was organized to establish a standard for factor IX. Two freeze-dried concentrate preparations, C1 and C2, and one freeze-dried plasma P were compared with each other, with fresh normal plasmas and with local standards in 13 laboratories. One of the concentrate preparations (C1) contained heparin and this gave rise to non-parallel assays in laboratories testing concentrate C1 in dilutions containing more than 0.05 i.u. of heparin per ml.

Assays of factor IX showed good precision for both plasma and concentrate in all laboratories; no systematic effect of method, operator or day of assay was detected.

The plasma preparation P and the concentrate preparation C2 were compared with 59 individual fresh normal plasma samples, and a mean potency ratio of 0.78 (95% confidence Hmits 0.73-0.84) for plasma and 5.62 (95 % confidence limits 5.13-6.16) for the concentrate C2 obtained. Only 21 estimates of concentrate C1 in terms of fresh plasma were obtained giving a mean potency ratio of 3.85 (95% confidence limits 1.87—7.92).

The estimated loss of potency for freeze-dried plasma stored at — 20° C is approximately 0.4% per year. The concentrate C2 is apparently more stable and only very small losses occurred even at higher storage temperatures.

All participants agreed that the preparation C2 would be suitable to serve as an International Standard for factor IX; they also agreed that the figure assigned for the unitage should be based on the number of ml of ‘average fresh normal plasma’ estimated to contain the factor IX activity of one ampoule of the preparation. It is proposed to recommend to the World Health Organization that the preparation of factor IX concentrate C2, in ampoules coded 72/32, be considered for establishment as the International Standard for factor IX, and that the international unit for factor IX be assigned on the basis of 5.62 units per ampoule of this preparation.
Effect of Platelet Antiserum on the Precipitation of Soluble Fibrin by Endotoxin
Authors: Müller-Berghaus Gert , Kramer Wilfried
Published: July 2018

Rabbits were injected with a platelet antiserum to examine the role of thrombocytopenia on the precipitation of soluble fibrin by endotoxin. Platelet antiserum removed more than 98% of the circulating platelets, and the resulting thrombocytopenia with platelet counts below 10,000 per μl persisted during the entire 10 hr-period of the experiment. Leukocyte counts were not significantly influenced by the platelet antiserum. Since the rabbits were treated with high doses of heparin, activation of intravascular coagulation by the antiserum did not occur.

Precipitation of soluble fibrin was achieved by injection of endotoxin into rabbits with ancrod-induced circulating soluble fibrin. Thrombocytopenia did not prevent the occurrence of glomerular microdots after ancrod and endotoxin administration. On the contrary, if endotoxin was injected into antiserum- and heparin-treated rabbits with circulating soluble fibrin, glomerular microdot formation occurred even in a higher percentage than in control rabbits treated with absorbed platelet antiserum. This investigation indicates that platelet antiserum-induced thrombocytopenia does not protect against precipitation of soluble fibrin by endotoxin.
The Estimation of the Number of Binding Sites for Antibody on Antigen Molecules with Reference to Factor VIII and its Antibody
Authors: Pool Judith , Biggs Rosemary , Miller R. G
Published: July 2018

The theoretical basis for determining the number of antibody sites on antigen molecules is examined. The theoretical considerations are applied to factor VIII molecules. Examples based on data available at the Oxford Haemophilia Centre are calculated to illustrate the approach. It is concluded that there are few sites on each factor VIII molecule for human antibody. The three antibodies for which reasonable data were available suggest 1–3 sites for human antibody. The data for rabbit antibody suggest 5–6 sites per factor VIII molecule.
The Influence of the Thyroid Function on the Metabolic Rate of Prothrombin, Factor VII, and Factor X in the Rat
Authors: Oosterom Allan T. van , Mattie Herman , Hermens Wim Th , Veltkamp Jan J.
Published: July 2018

The influence of the thyroid function on the metabolic rate of prothrombin, factor VII, and X was studied in the rat. Disappearance rates of the three coagulation factors were measured after synthesis had been blocked with appropriate doses of warfarin, and reappearance rates were assessed upon induction of synthesis by high doses of vitamin K1 injected into rats displaying coumarin induced hypocoagulability.

No statistically significant difference in the disappearance and production rates of any of the factors could be found between normal euthyroid rats and thyroxin-treated hypothyroid rats proven to be euthyroid. The differences between the two euthyroid groups and the hypothyroid group were highly significant, however: hypothyroidism results in an approximately 50% decrease of the metabolic rates of the three coagulation factors under study.

The reappearance of the three factors, under euthyroid as well as hypothyroid conditions, showed a biphasic pattern: in the first two hours after vitamin K1 administration to warfarin treated rats, a rapid reappearance was observed, to the same extent for all three factors, in hypo- as well as euthyroid rats. This finding suggests that in vitamin K1 deficiency an intracellular accumulation of precursor proteins (PIVKAs) occurs, which after rapid conversion into biologically active coagulation factors by vitamin K1 are shed into circulation.

The subsequent phase of reappearance is much slower and reflects the synthesis rate of coagulation enzymes. It is characteristic for each factor and clearly slower in hypothyroid rats than in euthyroid rats. From this an influence of thyroid function on the synthesis rate of the protein moiety of coagulation factors can be inferred.

An apparent difference between disappearance and reappearance rate of the coagulation factors in the plasma, particularly pronounced for factors VII and X in euthyroid rats, could theoretically be explained as the consequence of the model used for derivation of these rates.
The Variability of Platelet-Collagen Interaction. Its Influence on Aspirin-Induced Inhibition of Platelet Aggregation
Authors: Sack E. S , Quiroga E
Published: July 2018

A study of platelet aggregation induced by progressive concentrations of collagen was performed in 40 normal persons, equally grouped by sex, immediately before, and 90 min after, the ingestion of 0.500 g aspirin. Simultaneously, serum ASA-esterase activity and plasmatic salicylic acid levels were investigated in the pre- and post-ASA samples respectively. Wide individual variations in platelet responsiveness to collagen were found when the minimal amount of the aggregating agent necessary to provoke significant aggregation was located. The percentage of aggregation and the slope were the most sensible indicators of both the individual variances and the ASA-induced inhibition. Correlation was found in both groups between the minimal amounts of collagen required for initiating aggregation pre- and post-ASA. Also a positive correlation was found between the minimal collagen concentration required to initiate aggregation pre-ASA and the aspirin-induced inhibition in the aggregation produced by a higher collagen concentration. Important individual and sexual differences were detected in the serum content of ASA-esterase activity. A negative significant correlation was demonstrated between the ASA-esterase activity and the differences in collagen concentrations necessary to start aggregation pre- and post-aspirin in the men group. Failure to consider individual variances in platelet reactivity may lead to false conclusions about the ASA affective-ness as an antithrombotic agent.
Spontaneous Platelet Aggregation in Arterial Insufficiency: Mechanisms and Implications
Authors: Wu Kenneth K. , Hoak John C.
Published: July 2018

To investigate the clinical implications and mechanisms of spontaneous platelet aggregation (SPA) in man, 150 normal subjects, 22 patient controls and 130 patients with vascular insufficiency were studied. SPA was negative in normal subjects and patient controls whereas it was positive in 36 of 66 (54%) patients with transient ischemic attacks, 6 of 32 (19%) patients with stable angina, 7 of 10 (70%) patients with acute myocardial infarction and 11 of 14 (80%) patients with acute peripheral arterial insufficiency. The SPA was inhibited with aspirin in vivo, and inhibited competitively in vitro by low concentrations of aspirin, 2-chloroadenosine, prostaglandin E1 or apyrase but only by high concentrations of heparin or hirudin. Addition of platelet-poor plasma from patients with positive SPA did not cause normal platelets to aggregate. Treatment of patients who had acute peripheral arterial insufficiency with aspirin and dipyridamole prevented SPA with notable clinical improvement of the ischemic changes.
Effects of Endotoxin in Cortisone-Treated Rabbits with a Hereditary Deficiency of the Sixth Component of Complement (C6 Deficiency)
Authors: Stafford Benjamin T. , Rapaport Samuel I. , Shen Stephen M.-C.
Published: July 2018

Endotoxin was infused into normal rabbits and C6 deficient rabbits prepared with cortisone for the generalized Shwartzman reaction. Endotoxin produced profound granulocytopenia and moderate thrombocytopenia in both normal and C6 deficient rabbits. In normal rabbits endotoxin consistently produced extensive intravascular clotting. In C6 deficient animals endotoxin resulted in intravascular clotting of variable extent. In one group of eight C6 deficient rabbits mean fibrinogen levels fell 0.67 g per 1 over 6 hrs after endotoxin and four of eight animals developed a generalized Shwartzman reaction. In a second group of seven C6 deficient rabbits mean fibrinogen level fell only 0.17 g per 1 over 6 hrs and one animal developed a generalized Shwartzman reaction. Values for mean fibrinogen consumption, calculated from plasma fibrinogen levels and rate of disappearance of 25I-fibrinogen, were as follows: normal animals infused with saline, 10 mg per kg; C6 deficient animals infused with endotoxin, 24 mg per kg; normal animals infused with endotoxin, 58 mg per kg. Fibrinogen consumption after endotoxin was found to be related to granulocyte levels prior to endotoxin, which determined the number of granulocytes disappearing from the blood after endotoxin. The data indicate that C6 deficiency in the rabbit does not prevent intravascular clotting and the generalized Shwartzman reaction.
Multi-Center Calibration of the Second Reference Material for Thromboplastin, Rabbit, Plain, Coded CRM 149R
Authors: Besselaar A M H P van den , Bertina R M
Published: July 2018

In a collaborative trial of eleven laboratories which was performed mainly within the framework of the European Community Bureau of Reference (BCR), a second reference material for thromboplastin, rabbit, plain, was calibrated against its predecessor RBT/79. This second reference material (coded CRM 149R) has a mean International Sensitivity Index (ISI) of 1.343 with a standard error of the mean of 0.035. The standard error of the ISI was determined by combination of the standard errors of the ISI of RBT/79 and the slope of the calibration line in this trial.

The BCR reference material for thromboplastin, human, plain (coded BCT/099) was also included in this trial for assessment of the long-term stability of the relationship with RBT/79. The results indicated that this relationship has not changed over a period of 8 years. The interlaboratory variation of the slope of the relationship between CRM 149R and RBT/79 was significantly lower than the variation of the slope of the relationship between BCT/099 and RBT/79. In addition to the manual technique, a semi-automatic coagulometer according to Schnitger & Gross was used to determine prothrombin times with CRM 149R. The mean ISI of CRM 149R was not affected by replacement of the manual technique by this particular coagulometer.

Two lyophilized plasmas were included in this trial. The mean slope of relationship between RBT/79 and CRM 149R based on the two lyophilized plasmas was the same as the corresponding slope based on fresh plasmas. Tlowever, the mean slope of relationship between RBT/79 and BCT/099 based on the two lyophilized plasmas was 4.9% higher than the mean slope based on fresh plasmas. Thus, the use of these lyophilized plasmas induced a small but significant bias in the slope of relationship between these thromboplastins of different species.
Abnormal Platelet Functions in a Patient
with Abetalipoproteinemia
Authors: Surya Ingrid I , Mommersteeg Marlene , Gorter Gertie , Willem Erkelens D , Akkerman Jan-Willem N
Published: July 2018

Studies with isolated lipoproteins and washed platelets suggest that lipoproteins may affect platelet functions. We investigated platelet-rich plasma (PRP) from a patient with abetalipoproteinemia (ABL), whose plasma lacks apo-B containing lipoproteins (VLDL, LDL and chylomicrons). ABL-PRP aggregated poorly with different agonists and failed to respond to arachidonate. Thromboxane. B2 (T×B2) formation was severely impaired. After gel-filtration most of the aggregation defects persisted in agreement with reduced metabolism of endogenous arachidonate. However, arachidonate-induced aggregation and T×B2 production partially normalized. Normal platelets suspended in ABL plasma showed similar defects in aggregation and T×B2 production but arachidonate-induced aggregation was much lower than expected on the basis of T×B2. We conclude that the abnormal platelet functions in ABL-PRP are caused by (i) an intrinsic platelet abnormality due to reduced arachidonate mobilization and (ii) a property in ABL plasma that inhibits aggregation partially by trapping the arachidonate and partially by an unidentified mechanism. The latter properties may be the result of the abnormal lipid composition of ABl-plasma.
Superoxide Dismutase Cooperates with Prostacyclin to Inhibit Platelet Aggregation: a Comparative Study in Washed Platelets and Platelet Rich Plasma
Authors: Salvemini Daniela , Nucci Gilberto de , Vane John R
Published: July 2018

The role of superoxide anions (O2
−) in human platelet aggregation in Krebs’ buffer or plasma was investigated. In indome thacin (10 μM)-treated washed platelets superoxide dismutase (SOD; 60 U/ml) or ferricytochrome c (FCC; 70 μM) inhibited platelet aggregation by thrombin but not that by collagen or ADP. In addition, in indomethacin (10 μM)-treated washed platelets, SOD significantly potentiated the anti-aggregatory activity of prostacyclin (PGI2) or iloprost when thrombin but not collagen was used as the aggregating agent. In platelet rich plasma, SOD (60 U/ml) did not inhibit platelet aggregation nor did it potentiate the anti-aggregatory activity of iloprost when ADP, collagen or thrombin were used as aggregating agents. Thus, O2
− participate in the aggregatory activity of thrombin but not collagen or ADP and PGI2 or iloprost, by reducing the sensitivity of platelets to thrombin, co-operate with SOD to inhibit thrombin-induced platelet aggregation

The interpretation of the use of SOD in experiments involving endothelium-derived relaxing factor (NO) is discussed
Effects of a Monoclonal Antibody to Glycoprotein IIb/IIIa (P256) and of Enzymically Derived Fragments of P256 on Human Platelets
Authors: Stuttle A W J , Powling M J , Ritter J M , Hardisty R M
Published: July 2018

The anti-platelet monoclonal antibody P256 is currently undergoing development for in vivo detection of thrombus. We have examined the actions of P256 and two fragments on human platelet function. P256, and its divalent fragment, caused aggregation at concentrations of 10−9−3 × 10−8 M. A monovalent fragment of P256 did not cause aggregation at concentrations up to 10−7 M. P256–induced platelet aggregation was dependent upon extracellular calcium ions as assessed by quin2 fluorescence. Indomethacin partially inhibited platelet aggregation and completely inhibited intracellular calcium mobilisation. Apyrase caused partial inhibition of aggregation. Aggregation induced by the divalent fragment was dependent upon fibrinogen and was inhibited by prostacyclin. Aggregation induced by the whole antibody was only partially dependent upon fibrinogen, but was also inhibited by prostacyclin. P256 whole antibody was shown, by flow cytometry, to induce fibrinogen binding to indomethacin treated platelets. Monovalent P256 was shown to be a specific antagonist for aggregation induced by the divalent forms. In–111–labelled monovalent fragment bound to gel-filtered platelets in a saturable and displaceable manner. Monovalent P256 represents a safer form for in vivo applications
Presence and Possible Origin of ɛ(γ-Glutamyl)Lysine Isodipeptide in Human Plasma
Authors: Harsfalvi J , Tarcsa E , Udvardy M , Zajka G , Szarvas T , Fesus L
Published: July 2018

ɛ(γ-glutamyl)lysine isodipeptide has been detected in normal human plasma by a sensitive HPLC technique in a concentration of 1.9-3.6 μmol/1. Incubation of in vitro clotted plasma at 37° C for 12 h resulted in an increased amount of isodipeptide, and there was no further significant change when streptokinase was also present. Increased in vivo isodipeptide concentrations were also observed in hypercoagulable states and during fibrinolytic therapy.
Characterization of the Original Christmas Disease Mutation (Cysteine 206 → Serine): From Clinical Recognition to Molecular Pathogenesis
Authors: Taylor Sherryl A M , Duffin Jacalyn , Cameron Cherie , Teitel Jerome , Garvey Bernadette , Lillicrap David P
Published: July 2018

Christmas disease was first reported as a distinct clinical entity in two manuscripts published in 1952 (1, 2). The eponym associated with this disorder, is the surname of the first patient examined in detail and reported by Biggs and colleagues in a paper describing the clinical and laboratory features of seven affected individuals (3). This patient has severe factor IX coagulant deficiency (less than 0.01 units/ml) and no detectable circulating factor IX antigen (less than 0.01 units/ml). Coding sequence and splice junctions of the factor IX gene from this patient have been amplified in vitro through the polymerase chain reaction (PCR). One nucleotide substitution was identified at nucleotide 30,070 where a guanine was replaced by a cytosine. This mutation alters the amino acid encoded at position 206 in the factor IX protein from cysteine to serine. The non conservative nature of this substitution, the absence of this change in more than 200 previously sequenced factor IX genes and the fact that the remainder of the coding region of this gene was normal, all provide strong circumstantial evidence in favour of this change being the causative mutation in this patient. The molecular characterization of this novel mutation in the index case of Christmas disease, contributes to the rapidly expanding body of knowledge pertaining to Christmas disease pathogenesis.
The Amount of Plasminogen, Tissue-Type Plasminogen Activator and Plasminogen Activator Inhibitor Type 1 in Human Thrombi and the Relation to IVVO-TNO Lysibility
Authors: Loon B J Potter van , Rijken D C , Brommer E J P , van der Maas A P C
Published: July 2018

Thrombolytic therapy successfully reopens obstructed blood vessels in the majority of cases. However, it is not known why a substantial amount of thrombi are resistant to lysis by a fibrinolytic agent. In vitro studies have demonstrated that tissue-type plasminogen activator (t-PA) and plasminogen incorporated in the clot (during formation) increase lysibility. To test whether lysibility of in vivo formed human thrombi is related to their composition, we studied 25 venous thrombi obtained at autopsy and 21 arterial thrombi obtained during embolectomy.

Plasminogen activator inhibitor-1 (PAI-1) antigen was measured in a phosphate-buffered saline (PBS) extract of each thrombus; t-PA antigen and plasminogen antigen were determined in a 6 M urea extract of the thrombus, representing bound proteins. Lysibility was measured as weight reduction during 8 h of incubation in PBS containing streptokinase (SK) 100 U/ml, corrected for spontaneous lysis, reflected by weight loss in PBS without SK. In addition, lysibility in SK was compared with lysibility in urokinase (UK) 100 U/ml and in t-PA 200 U/ml.

Spontaneous lysis amounted to 29 ± 5% (mean ± SEM) and 33 ± 5% in venous and arterial thrombi, respectively, and inversely correlated with the PAI-1 content of thrombi (r = —0.43, p <0.01). Lysibility amounted to 76 ± 6% in venous and 90 ± 4% in arterial thrombi (venous vs. arterial: p = 0.051). PAI-1-, plasminogen- and t-PA-content of venous thrombi were 902 ± 129 ng, 34.3 ± 4.8 pg and 26.7 ± 3.0 ng per gram of wet thrombus respectively; for arterial thrombi these values were 2,031 ± 401 ng/g (p = 0.011), 64.1 ± 11.4 pg/g (p = 0.088) and 62.2 ± 8.3 ng/g (p = 0.0001), respectively. A correlation was found between t-PA and plasminogen (r = 0.74, p <0.001). Lysibility by SK related to plasminogen content in both venous (r = 0.60, p <0.002) and arterial (r = 0.44, p <0.05) thrombi; PAI-1 and t-PA did not correlate with lysibility. Lysibility in the chosen concentrations of SK, UK and t-PA were similar.

We conclude that spontaneous lysis of thrombi in saline is dependent on PAI-1 content and that susceptibility of thrombibi to lysis by SK ex vivo is dependent on the plasminogen content
The d-Enantiomer Form of Indobufen Totally Accounts for the Anti-Cyclooxygenase and Antiplatelet Activity Ex Vivo and for the Increase in Bleeding Time by Indobufen in Man
Authors: Caterina Raffaele De , Sicari Rosa , Yan An , Bernini Walter , Giannessi Daniela , Lazzerini Guido , Efthymiopoulos Costantin , Strolin-Benedetti Margherita
Published: July 2018

Indobufen is an antiplatelet drug able to inhibit thromboxane production and cyclooxygenase-dependent platelet aggregation by a reversible inhibition of cyclooxygenase. Indobufen exists in two enantiomeric forms, of which only d-indobufen is active in vitro in inhibiting cyclooxygenase. In order to verify that also inhibition of platelet function is totally accounted for by d-indobufen, ten patients with proven coronary artery disease (8 male, 2 female, age, mean ± S.D., 58.7 ± 7.5 years) were given, in random sequence, both 100 mg d-indobufen and 200 mg dl-indobufen as single administrations in a double-blind crossover design study with a washout period between treatments of 72 h. In all patients thromboxane (TX) B2 generation after spontaneous clotting (at 0, 1, 2, 4, 6, 8, 12, 24 h), drug plasma levels (at the same times), platelet aggregation in response to ADP, adrenaline, arachidonic acid, collagen, PAF, and bleeding time (at 0, 2, 12 h) were evaluated after each treatment. Both treatments determined peak inhibition of TXB2 production at 2 h from administration, with no statistical difference between the two treatments (97 ±3% for both treatments). At 12 h inhibition was 87 ± 6% for d-indobufen and 88 ± 6% for dl-indobufen (p = NS). Inhibition of TXB2 production correlated significantly with plasma levels of the drugs. Maximum inhibitory effect on aggregation was seen in response to collagen 1.5 pg/ml (63 ± 44% for d-indobufen and 81 ± 22% for dl-indobufen) and arachidonic acid 0.5-2 mM (78 ± 34% for d-indobufen and 88 ± 24% for dl-indobufen) at 2 h after each administration. An effect of both treatments on platelet aggregation after 12 h was present only for adrenaline 2 μM (55 ± 41% for d-indobufen and 37 ± 54% for dl-indobufen), collagen 1.5 pg/ml (69 ± 30% for d-indobufen and 51 ± 61% for dl-indobufen), arachidonic acid 0.5-2 mM (56 ± 48% for d-indobufen and 35 ± 49% for dl-indobufen). The extent of inhibition of TX production and the extent of residual platelet aggregation were never significantly different between treatments. Bleeding time prolongation was similar in the two treatment groups without showing a pronounced and long lasting effect (from 7.0 ± 2.0 min to 10.0 ± 3.0 min at 2 h and 8.0 ± 2.0 min at 12 h for d-indobufen; from 6.0 ±1.0 min to 8.5 ± 2.0 min at 2 h and 8.0 ± 1.0 min at 12 h for dl-indobufen). These results demonstrate that the biological activity of dl-indobufen as an antiplatelet agent in vivo is totally accounted for by d-indobufen.
The Fibrinolytic System and Haemostasis
Authors: Chesterman C. N
Published: July 2018

The fibrinolytic enzyme system appears to be a parallel development with the plasma coagulation pathways involved in haemostasis. Both systems have common activation mechanisms, inhibitors and the enzymes involved have considerable homology. In addition, blood cells show capabilities for involvement in each of these systems.

There is disagreement over the possible contribution of the fibrinolytic mechanism to normal fibrinogen catabolism either by the lysis of continually forming fibrin, or by degradation of circulating fibrinogen.

The occurrence of systemic fibrinolysis is associated with a haemorrhagic tendency which may be due to a number of factors including degradation of coagulation factors, the anticoagulant effect of F.D.P. and the lysis of α-chain crosslinking sites. A clinical study of patients treated with streptokinase indicates that a fall in plasma fibrinogen levels and increase in serum F.D.P. levels are correlated with the haemostatic defect.
Gastric Fibrinolysis
Authors: Nilsson I. M , Bergentz S.-E , Hedner U , Kullenberg K
Published: July 2018

Gastric juice from 15 normals, 20 patients with gastric ulcer and 4 patients with erosive haemorrhagic gastroduodenitis was investigated in respect of its activity on unheated and heated fibrin plates and its content of FDP and plasminogen or plasmin with immunochemical methods. Gastric juice from normals showed no activity on unheated and heated fibrin plates, and no FDP or plasminogen could be demonstrated. In the patients with gastric ulcer the gastric juice showed little or no fibrinolytic activity on fibrin plates except in 2, who had regurgitation of duodenal juice and neutral pH of the juice. These patients had equally high activity on heated as on unheated plates and no plasmin could be demonstrated. It was shown that this activity was not due to fibrinolysis, but to non-specific proteolytic activity (probably trypsin). The patients with erosive haemorrhagic gastroduodenitis exhibited quite a different picture. The gastric juice from these patients showed extremely high activity on fibrin plates, the activity was higher on unheated than on heated plates. The activity was inhibited in vitro by addition of EACA and in vivo after administration of AMCA. The occurrence of plasmin could be demonstrated directly immunologically in the gastric juice. By comparison of plasmin and trypsin in various assays it could further be proved that the gastric juice in these cases contained plasminogen activator and plasmin. The patients with erosive haemorrhagic gastroduodenitis showed no increase in fibrinolysis in the blood, but low values for plasminogen and α2M, and the serum contained FDP. These findings in the blood and gastric juice were interpreted as signs of local fibrinolysis in the stomach and duodenum. There is reason to assume that this gastric fibrinolysis contributes substantially to the bleeding tendency. The effect of administration of AMCA on fibrinolytic activity and the haemorrhage lends support to the assumption of such a mechanism.
Studies in Experimental Animals on Disseminated Intravascular Coagulation (DIC)
Authors: Markwardt F , Nowak G , Meerbach W , Rüdiger K.-D
Published: July 2018

Changes in the clotting system, as well as morphological and functional alterations corresponding to that of the pathologic phenomenon of disseminated intravascular coagulation (DIC) or consumption coagulopathy, were produced by thrombin infusion (550 NIH U × kg−1 × h−1) in rats and simultaneous inhibition of fibrinolysis by PAMBA (100 mg/kg).

Changes in the fibrinogen level and platelet count as well as the appearance of fibrin monomers and the formation of microthrombi in several organs were evaluated. Simultaneously, the function of the respiratory system was investigated by continuous measurement of oxygen consumption as well as elasticity and water content of the lung. From the time course of the alterations in the several parameters, conclusions can be drawn for the pathogenesis and the possible therapeutic influence on DIC.
A Standard for Human Plasmin
Authors: Kirkwood T. B. L , Campbell P. J , Gaffney P. J
Published: July 2018

An international collaborative study involving ten laboratories was undertaken to assess the suitability of a preparation of human plasmin in 50% glycerol to serve as a single standard for the bioassay of plasmin activity. The study included examination of the stability of the proposed standard and comparisons between assay methods. The results suggest that the human plasmin preparation coded 72/379 is suitable to serve as a standard. It is sufficiently stable to define the unit of plasmin activity for several years at or below 4° C and for short periods at about 20° C. The preparation has been assigned a potency of 8 units per ml. The International Committee on Thrombosis and Haemostasis (Basle, 1974) has recommended the use of this material as a standard.
Colchicine Uptake and Binding by Human Platelets
Authors: Kenney Dianne M , Chao Francis C , Tullis James L , Conneely Gail S
Published: July 2018

The uptake and binding of antimitotic alkaloid colchicine has been demonstrated in washed preparations of human platelets. A silicone oil technique was adapted so that both uptake and binding of 14C-colchicine were examined in the same platelet preparations. The time dependence and amount of colchicine taken up and bound by different platelet preparations during a 90 to 120 min incubation period were highly reproducible. Both colchicine uptake and binding by intact platelets, and colchicine binding by preparations of lysed platelets were specific and temperature dependent. Colchicine uptake was slowly reversible. Magnesium and GTP enhanced colchicine binding by lysed platelet preparations but calcium decreased binding.

Exposure of platelets to either cold (4° C) or to thrombin, which disrupt platelet microtubules, produced significant increases in colchicine uptake and binding. The thrombin effect was maximal at 37° C and resulted in a greater increase in uptake and binding than that produced by either cold treatment alone or, by cold treatment followed by incubation with thrombin at 37° C. The amount of increase in uptake and binding produced by thrombin was independent of both thrombin (1–5 Units/109 platelets) and colchicine concentrations (1–50 × 10−6M).It is postulated that thrombin may initiate the formation, or make available, colchicine binding sites (microtubule subunits) within platelets.
Density functional theory calculations for magnetic properties of Co3W systems
Authors: Yuan H. K. , Cui H. , Liu B. , Tian C. L. , Kuang A. L. , Chen H.
Published: July 2018

Cheaper permanent magnetic nanostructures with magnetic properties equivalent to those of noble-metal or rare-earth nanomagnets have been experimentally developed for their potential applications in ultrahigh storage densities in magnetic memory. To date, their intrinsic magnetic properties are not well understood under the micro-level of local atomic arrangements and electronic structures. In this work, we performed theoretical investigations on the Co3W bulk, the clean surface, nanoclusters, and the Co|Co3W bilayers and superlattices for their geometrical structures, magnetic moments, and magnetic anisotropy energies (MAEs). We found that the Co3W nanostructures we constructed are stable and have the local minima in the energetic landscape, whose stabilities increase with increasing proportion of W and cluster size. The Co and W atoms in clusters are antiferromagnetically coupled, and their local magnetic moments decrease with increasing proportion of W. The breakdown of the Hund’s third rule in W atoms observed in experiment can be interpreted as the competition between the intra-atomic spin-orbit coupling in W atoms and interatomic Co—W hybridizations. The highest MAE of about a few tens of meV is obtained in small cluster sizes, whereas it is an order of magnitude reduction in large cluster sizes. The magnetic systems of Co3W clean surface, Co|Co3W bilayer and superlattice can present large MAEs, and their easy-axes of magnetization are perpendicular to the (001) surface. Our calculated MAEs are of the same order of magnitude as that of the experimental measurements, and the electronic origin is revealed through the second-order perturbation method.