bluuebunny/crossref_metadata_2025 huggingface dataset:
Measurement Of Platelet Factor 3 Availability By Using Chromogenic Substrate
Authors: Hársfalvi J , Chmielewska J , Latallo Z S , Muszbek L
Published: July 2018

The measurement of platelet factor 3 (Pf 3) availability (the prothrombin converting procoagulant activity of platelet membran phospholipids) induced by collagen, ristocetin, arachidonic acid or other activating agents is a potentially valuable test in the diagnosis of platelet disorders. Its diagnostic potential, however is greatly impaired by the fact that RW clotting time method usually used for Pf 3 determination is influenced by many factors, its reproducibility is rather poor, and results are difficult to express in quantitative well defined biochemical terms.

In the present paper a new method measuring Pf 3 activity by using a chromogenic substrate was developed. Platelet rich plasma(PRP) was incubated by various activating agents, then prothrombin thrombin conversion was started by the addition of purified factor Xa. After a certain interval the reaction was stopped by soybean trypsin inhibitor and thrombin formed during the reaction was quantified by the chromogenic substrate S-2238 (Kabi). By varying incubation time, prothrombin, factor V concentrations, the optimal conditions, where platelet phospholipid was the rate limiting factor, were determined. In these conditions Pf 3 activity was a linear function of the number of activated platelets. The results are expressed as the ratio of thrombin generated in activated and unactivated PRP. The method is a rather simple quantitative photometric technique, has an improved reproducibility, and results showing the increase in thrombin generation have well defined biochemical meaning.
Interaction Of Plasma Proteins With Artificial Surfaces With Reference To Platelet Adhesion
Authors: Malhotra O P , Helmus M N , Gibbons D F
Published: July 2018

On occasion, fewer platelets from platelet rich plasma (PRP), adhered to hydrophilic (glass) surfaces exposed to platelet poor plasma (PPP) for 3 min than areas exposed for 3 s. The decrease was dramatic and consistent when platelet suspension (gel-PLS, obtained from PRP by 2B Sepharose gel filtration) was used instead of PRP. To further explore the factors which influence platelet adhesion, we used the following: for surfaces, a) sparkleen-cleaned glass (hydrophilic), b) acid-washed (somewhat hydrophobic), and c) siliconized (hydrophobic); for proteins, a) PPP, b) fibrinogen (96% clottable), c) defi-brinogenated (defib.) plasma, and d) defib. plasma plus fibrinogen; for platelet suspension, a) PRP, b) gel-PLS, and c) platelets in defib. plasma (defib. PLS).

From gel-PLS, non-siliconized surfaces exposed to fibrinogen for 3 s attached more platelets (F<0.05) than those exposed to PPP or defib. plasma plus fibrinogen. The latter two attracted more platelets (P<0.01) than defib. plasma. Hydrophilic sparkleen-cleaned glass previously exposed to PPP (or defib. plasma plus fibrinogen) attached a minimum of 10-fold as many platelets from gel-PLS than from PRP. Under similar conditions acid-cleaned surfaces attached 2-fold from gel-PLS, while hydrophobic glass did not show any change. By exposing the surfaces to PPP followed by gel-PLS, the sparkleen-cleaned glass showed the greatest decrease (P<0.001) in the number of platelets attached to areas exposed to PPP for 3 min as compared to 3 s, while siliconized showed no such decrease. If, however, the surfaces were exposed to defib. plasma, they all showed decreases in platelet attraction at 3 min.
Antiplatelet Drugs In Haemodialysis
Authors: Crow M J , Donaldson D R , Roberts T G , Davison A M , Rajah S M
Published: July 2018

Platelet and fibrin desposition on dialysis membranes remains a significant problem despite systemic heparinisa- tion. In randomised double blind cross-over studies the effect of three antiplatelet drug regimens have been investigated: 1) Placebo, 2) Low Dose Aspirin (ASA) and Dipyridamole (DPM) (110 mg + 75 mg) tds, 3) Standard Dose ASA + DPM (330 mg + 75 mg) tds, 4) Ticlopidine (250 mg) bd.

No antiplatelet drugs were given for 21 days prior to each study and the heparin schedule remained unchanged throughout all the studies. Seventeen patients received the ASA + DPM regimens for 7 days and the Ticlopidine regimen for 14 days. Investigations were performed at the last dialysis of each study period and comprised of platelet count pre and post-dialysis, post-dialysis heparin and drug concentrations, scanning electron microscopy (SEM) of the dialysis membrane and in addition platelet aggregation predialysis in the Ticlopidine group.

Standard Dose ASA + DPM significantly inhibited the reduction in platelet count during dialysis (p<0.01) and increased the post-dialysis heparin concentration compared to the other regimens (p<0.05). SEM demonstrated that only Standard Dose ASA + DPM produced consistent reduction in platelet and fibrin deposition. Ticlopidine significantly reduced platelet aggregation with ADP (p<0.05). Patient compliance was monitored by serum drug concentrations.

This study indicates that Standard Dose ASA + DPM significantly reduces the thrombogenic potential of dialysis membranes, hence improving overall dialysis efficiency. Low Dose ASA + DPM and Ticlopidine show marginal reduction compaired to Placebo. Standard Dose ASA + DPM exerted a deparin-sparing action.
Possible Pathways Of Arachidonic Acid Liberation As Studied With Purified Phospholipases
Authors: Chap H , Perret B , Mauco G , Plantavid M , Laffont F , Simon M F , Douste-Blazy L
Published: July 2018

Two kinds of informations about arachidonic acid (AA) metabolism in platelet phospholipids (PL) have been obtained from the use of purified phospholipases: 1) Beside the determination of PL sidedness in the plasma membrane, non-lytic degradation by phospholipase A2 + sphingomyelinase C showed that only 6 % of the total platelet AA is localized in the outer surface of the plasma membrane. This heterogeneous distribution is actually a consequence of PL asymmetry, since sphingomyelin and phosphatidylcholine, which predominate in membrane outer leaflet, contain only traces or relatively lower amounts, respectively, of AA than the internal lipids. It is further shown that incubating platelets with free AA specifically labels the large internal pool of AA, whereas the small external pool is renewed by a direct exchange of phosphatidylcholine with plasma lipoproteins. This offers a doublelabelling method allowing to explore the exact role of each AA pool.

2) Platelet aggregation by Clostridium welchii phospholipase C produces the same metabolic changes (accumulation of phosphatidic and lysophosphatidic acids) as those induced by thrombin. These observations have led to describe the existence of a cytosolic phosphatidylinositol-specific phospholipase C and a membrane-bound diglyceride lipase. Both enzymes, coupled to diglyceride− (and monoglyceride−) kinase(s), could achieve AA release and (lyso) phosphatidic acid accumulation. Some properties of these enzymes (subcellular localization, calcium and pH dependence, positional specificity) will be presented.
Clinical Application Of Blood Surface Interactions
Authors: Edmunds L H
Published: July 2018

Blood contact with non-endothelial cell surfaces activates Factor XII and platelets. Clinical applications of temporary or permanent devices placed within or in series with the circulation are limited by this fact. Depending upon whether or not coagulation is inhibited, thrombosis or blood injury occur.

In recent years improvements have been made in vascular prostheses, heart valves and assist devices. PTFE (Gortex) prostheses as small as 4 mm in diameter have been successfully used in selected high-flow applications such as aorto-pulmonary shunts. Improved mechanical heart valves have reduced the tendency of .valve thrombosis and possibly late emboli, but long-term anticoagulation is still required for patients with aortic bioprostheses and some patients with mitral xenografts. Development of durable relatively thromboresistant materials has led to wide and successful application of the intra-aortic balloon with few thromboembolic complications. Improved designs and development of durable, relatively thromboresistant materials have advanced experimental left ventricular assist devices to the stage of limited clinical application.

When coagulation is inhibited by heparin, extracorporeal perfusion systems injure formed and unformed blood elements. Platelets particularly are activated with the result that platelet function is depressed and postoperative bleeding times are prolonged after perfusion with either membrane or bubble oxygenator systems. Currently clinical trials using prostacyclin to protect platelets during the period of bypass are underway. Bleeding consequences of platelet dysfunction and heparin continue to limit clinical application of long-term extracorporeal perfusion applications for cardiac and respiratory assistance.
Clinical Information From Screening Programs For Coagulation And Fibrinolytic Factors And Inhibitor Deficiencies Using Automated Chromogenic Methods
Authors: ten Cate J W , Kahlé L H , Büller H R , Peters M , Weenink G H
Published: July 2018

The introduction of chromogenic substrates allowed the development of automated spectophotometric assays. After having developed such methods employing an automated kinetic enzyme and substrate analyser for coagulation factors II and X, antithrombin III (AT III), plasminogen (PG) and α2- antiplasmin (α2-AP) , we decided to apply these methods in well defined clinical projects in order to evaluate their future clinical potency. Results obtained thusfar revealed that 1. AT III and PG are predictors of gram negative septicaemia in patients with intraabdominal infection following major abdominal and vascular surgery, 2. The predictive value of AT III for gram negative septicaemia in prospective studies in premature neonates (all assays require only 0.4 ml of venous blood), 3. AT III remains unchanged in normal pregnancy. Decreasing values are observed in toxaemia and isolated deficiencies are observed as an early sign of intravascular coagulation in pre- ecclampsia, 4. Routine screening of AT III in female subjects on oral anticonceptive drugs is of no use, 5. Factor X assays may possibly be used for the control of oral anticoagulant therapy, 6. Screening of patients with postoperative bleeding disorders may disclose congenital factor X deficiency not reflected by routine prothrombin time measurements.
Definition And Evaluation Of A Therapuetic Range For The Control Of Oral Anticoagulant Treatment By Chromogenic Assays
Authors: Bertina R M , Loeliger E A
Published: July 2018

The evaluation of the usefullness of chromogenic assays in the control of oral anticoagulant treatment, often is hampered by the rather poor correlation between the new parameter and the reference one (Prothrombin Time determination) within the therapeutical range. This means that before large scale double blind prospective studies can be started, it should be checked that the use of the experimental method will not result in unacceptable deviations in prescribed dosage.

From 150 patients (each within therapeutic zone for more than 3 months) blood/plasma was analysed by at least 2 different chromogenic assays (FII, FX) and by the reference method (PT determination); the selection of patients was such to get an equal distribution over the total therapeutic range (2.0-5.0 in terms of proposed International Calibrated Ratio - ICR); on basis of these results a therapeutic range was defined for each of the experimental methods.

Next it was checekd to what extent the use of the experimental method would result in differences in prescribed dosages (when compared with the reference method) a. in 100 of the abovementioned patients at subsequent control periods, b. in previously stable patients with a PT < lower limit or > upper limit of the therapeutic zone, and c. in patients starting oral anticoagulant treatment (outclinic patients and hospitalized patients).

During the study the precision of the different chromogenic assays was checked using two lyophilized pooled patient plasmas.
A Rapid Screening Method To Increase Efficiency In Assaying Plasma Levels Of Factor VIII Inhibitors
Authors: White K S , Dombrose F A , Blatt P M
Published: July 2018

The plasma level of naturally acquired inhibitors to clotting Factor VIII activity (F.VIII: C) was assessed by the Bethesda Inhibitor Assay (BIA). At inhibitor concentrations outside the BIA working range (25% to 75% residual F.VIII: C), patient plasmas had to be diluted. However, when a rough estimate of the inhibitor concentration was unavailable, the choice of dilution was arbitrary and testing became tedious and inefficient. In an effort to improve the BIA, a screening test was devised to predict the appropriate plasma dilution. It is based on the partial thromboplastin time (PTT) of the incubated sample and is performed just prior to preparing dilutions for the F.VIII: C assay. Test plasmas were diluted serially in multiples of two, from 1:2 to 1:1024, and then incubated according to the BIA. After 90 min, samples were removed for PTT determinations to screen for the appropriate dilution to use in the F.VIII: C assay. The first two test dilutions to produce PTT values 21 sec (± 4 sec, 95% confidence interval) greater than the control resulted in a residual F.VIII: C for the BIA within the 25% to 75% acceptable range. When the log ratio of patient-to-control PTT determinations (specific PTT) for a given dilution was plotted as a function of log residual F.VIII: C at that test dilution, in 81 Bethesda assays a linear relationship was observed (p> .25; with r2 = .81). The screening test was 89% effective in predicting an appropriate dilution (range 0 to 1020 Bethesda units/ml). The accuracy can be enhanced further if, based on the first of three dilution screen tests in which the specific PTT >21 sec, the F.VIII: C assay is performed using the middle dilution value first. Depending on whether that dilution yields an acceptable residual F.VIII: C, either the higher or lower dilution is chosen in the second F.VIII: C assay.
Kinetics Of Factor Viii Inhibitors In Three Non-Hemophiliac Patients
Authors: McClellan S L , Hussey C V , Fobian J E , Pisciotta A V
Published: July 2018

Factor VIII (VIII) inhibitors were isolated from the gamma globulin fraction of the plasmas of three non-hemophilic patients. One of these patients is thought to have acquired the inhibitor secondary to Hodgkin’s disease while the other two were apparently drug-related. These patients presented without significant prior replacement therapy and showed elevated levels of von Willebrand factor (VIIIVW-R) as measured by Ristocetin aggregation of fixed washed platelets and elevated levels of antigenic VIII (VIIIAGN) using a CELIA technique. The procoagulant VIII (VIIIAHF) activities, however, ranged from 12-20% of normal activity (one-stage method) and the inhibitors were present in levels of 6-50 Bethesda Units/ml of plasma.

The kinetics of the interaction between these inhibitors and VIII were studied to further characterize the inhibitors. In vitro inactivation of VIII by the inhibitors demonstrated a two-step kinetic model similar to that proposed by Biggs. Iii vivo neutralization of transfused VIII appeared to follow the same model. The high levels of VIIIAGN and VIIIVW-R in the presence of low levels of VIIIAHF suggest binding of the inhibitor to the procoagulant moiety. Further, two-step inactivation of the procoagulant activity suggests binding of more than one molecule of inhibitor near rather than at the active procoagulant site which results in increasing stearic inhibition of the active site. This is in contrast to simple direct binding of a single molecule of inhibitor as described with bimolecular second order kinetics.

Finally, the presence of high levels of VIIIAGN with markedly attenuated VIIIAHF activities implies relatively slow clearance of the inhibitor-VIII complex.
Deleterious Effect Of Aspirin In The Asymptomatic Patient With Spontaneous Factor VIII Inhibitor
Authors: Klein M E , Akman S A , Wiernik P H
Published: July 2018

A patient with spontaneous acquired circulating inhibitor to factor VIII and factor IX is reported. This 80 year old previously well black female developed spontaneous bleeding in the right forearm after ingestion of 650 mg of aspirin the day of the bleed. Acquired anticoagulants were identified by factor assay. Factor VIII and factor IX activities were 1.7% and 60% of normal. Dilution analysis revealed a factor VIII antibody titer of 1: 100. Diagnostic evaluations for collagen vascular disease, rheumatoid arthritis, inflammatory bowel disease, and paraproteinemias were negative. Treatment was instituted on day 1 with prednisone (80 mg per day) and azothiaprine (150 mg per day). Hemorrhage ceased within two days of therapy. After one week of therapy, factor VIII level had risen to 8%. On day 16 azothiaprine therapy was discontinued and prednisone was tapered. There was no further spontaneous bleeding. Aspirin therapy may result in clinically significant hemorrhage in otherwise asymptomatic patients with spontaneous factor VIII inhibitors.
Ristocetin Induced Platelet Factor 3 Availability, A Possible New Tool In The Diagnosis Of Von Willebrand’S Disease
Authors: Muszbek L , Losonczy H , Hársfalvi B , Nagy I
Published: July 2018

Ristocetin induced platelet aggregation (RIPA) is a highly valuable technique in the laboratory diagnosis of von Willebrand’s disease (vWD) and together with the estimation of bleeding time, F VIII procoagulant activity and F VIII related antigen in most cases it makes also division of vWD into phenotypic subgroups possible. We have shown that ristocetin makes platelet factor 3 available and ristocetin induced platelet factor 3 availability (RIPF3) similarly to RIPA also depends on a plasma factor. In the present paper it was examined if RIPF3 can be applied as a laboratory test in the diagnosis of vWD. PRP from normal controls, patients with various release defects and von Willebrand patients was incubated and continuously stirred with ristocetin or phys. saline and after 20 min the RW clotting times were determined. RIPF3 activity was expressed as the ratio of the clotting times obtained following incubation with phys. saline and ristocetin.

In healthy controls and patients with release defect (n=21) this ratio was higher than 1.5, and in most cases between 1.8-2.5. In patients with vWD (n=10) the ratio was well below 1.5 (with one exception it ranged between 0.95-1.2). In 9 of the lO patients the absence or strong diminution of RIPA and RIPF3 showed close paralellism. In one patient only the slope of first vawe of ristocetin aggregation was decreased while at the same time an almost total inhibition of RIPF3 could be observed. The results indicate that RIPF3 is a valuable quantitative diagnostic test in von Willebrand disease. It can be used if expensive aggregometer is not available and it may also have an importance as additional test to RIPA in the division of vWD into subgroups. An attempt was made to develop this test further by using chromogenic substrate for the measurement of RIPF3.
The Binding Of 125I-Von Willebrand Factor To Human Platelets In The Presence Of Ristocetin
Authors: Silber P , Finlay T H
Published: July 2018

The effect of ristocetin on the binding of 125I-porcine von Willebrand factor to human platelets was studied. Previously, we had shown that 125I-porcine von Willebrand factor binds to human platelets in the absence of ristocetin. The present work demonstrates that binding is stimulated by ristocetin and this stimulation is maximal at a ristocetin concentration of 2 mg/ml. At a ristocetin concentration of 0.5 mg/ml, Scatchard analysis indicates a binding constant of 5.18 × 10-9M and the presence of 105,000 binding sites. This compares with our previous finding, in the absence of ristocetin, of a binding constant of 2.92 × 10-7M and 4760 binding sites. These binding data assume the porcine von Willebrand factor to be a tetramer with a molecular weight of 9 × 105. This study indicates that ristocetin causes tighter binding and increases the number of binding sites on human platelets for porcine von Willebrand factor. Unlabelled porcine von Willebrand factor competitively inhibits the specific binding of the labelled protein and gives a binding constant of 0.17 × 10-9M. Similar results were obtained using human von Willebrand factor.
Study On The Effect Of Ddavp On Factor VIII- Related Properties In Hemophilia And VWD
Authors: Ciavarella N , Solinas S , Pilolli D , Ranieri P , Corrao D , Antoncecchi S , Mariani G
Published: July 2018

Twenty-one patients affected by mild and moderate Hemophilia A as well as 9 patients with the classic form of vonWillebrand’s disease (vWD) were given a total of 58 infusions of DDAVP. Concerning Hemophilia a three fold mean raise ( x = 3.0, sem O. 19; range of ratios post/preinfusion 1.35 - 5.55) of factor VIII : C levels was observed after the infusion of 0.3 μg/Kg b.w. A mean raise of 3.44 ( sem 0.48, range 2.20 - 6.7) after the infusion of 0.4/ug/Kg was found. The difference between the two regimens is not statistically significant (p < 0.5). As to the vWD 18 infusions were given. In 6 patients the changes of factor VI1I: C, VIIIR: Ag and VIII: vWF were roughly consensual ( ratios post/preinfusion ranging from 2.2 to 4.0 for VIII: C; from 1.8 to 3.5 for VIHR:Ag and from 3.1 to 6.2 for VIII: vWF). In the remaining 3 patients a very strong response of VIII : C ( ratios post/preinfusion 12.0, 15.1 and 6.5) was observed. Also the other properties related to factor VIII underwent to relevant increase. In one of these patients a modified electrophoretic mobility of factor VIII was found; the other two (father and doughter) had a normal factor VIII mobility after stimulation with DDAVP.
The Factor VIII Response To High Purity Porcine Factor VIII Concentrate In Acquired Von Willebrand’s Disease
Authors: Preston F E , Malia R G
Published: July 2018

Two patients with acquired von Willebrand’s disease were given an infusion of 2000 units of high purity porcine factor VIII (Hyate) which we previously have shown contains only negligible amounts of FVIIIR: Ag. The disappearance of half life of FVIII:C was 2 hours and 3½ hours. In one patient a marked secondary rise in FVIIIC occurred 1 hour after completion of the infusion. In both patients ristocetin-induced platelet aggregation rose from zero to 10 per cent and 70 per cent respectively between the first and second hour with accompanying rise in human FVIIIR:Ag measured by specific non-crossreacting immuno-radiometric assays. These observations suggest that porcine FVIII:C induced de novo synthesis or release of human FVIIIR:Ag. However an alternative ejqplanation would be dissociation of a FVIIIR:Ag-antibody complex since we have demonstrated antibody directed against the Factor VIII complex in these and other patients with acquired von Willebrand’s disease.
A Possible Role Of Factor VIII Coagulation And Cyclic Nucleotides In Promoting Endothelial Renal Injury
Authors: Bertaglia E , Vertolli U , Pengo V , Baggio B , Tessarin C , Belmonte P , Zanella R
Published: July 2018

To evaluate a possible interaction between coagulation factors, inflammation, and immunological disorders in the pathogenesis of glomerulonephritis (G.N.) factor VIII clotting activity (VIII:C), related-antigen (VIIIR:AG), urinary fibrinogen degradation products (F.D.P.) excretion and cyclic nucleotides (cAMP) in 21 patients affected by chronic G.N. with normal renal function, was investigated. These patients were followed-up for two years. For the assay of factor VIII:C, a factor VIII deficient plasma was used. Factor VIIIR:AG were assayed by rocket electroimmunodiffusion in agarose gel, urinary F.D.P. by Thrombo-Wellcotest and cAMP by radioimmunoassay. The same tests were performed in 25 healthy controls. Patients' clotting activity, measured at first admission, showed a significant decrease in comparison with the values obtained at the end of the follow-up period (t=-2.61 p<0.02).The clotting activity of the renal patients was lower than in controls (t=2.09 p<0.05). However, factor VIIIR:AG was significantly increased in the same patients. A significant direct correlation between the urinary excretion of cAMP and plasma concentration of factor VIIIR:AG (r=0.82 p<0.001) as well as between urinary F.D.P. and cAMP excretion (r=0.49 p<0.05) was found, when the patients were classified according to the morphological findings of the kidney biopsy. Most probably a circulating thrombin excess leads to consumption of factor VIII:C and promotes the extrusion of cyclic nucleotides from intracellular sites. The relationship found between factor VIIIR:AG, cAMP and F.D.P. besides showing a linkage between inflammation and coagulation, seems also to suggest a role for both factors in promoting a renal injury.
Crossed Concanavalin A Affinoimmunoelectrophoresis Of Factor VIII - Related Antigen In Whole Plasma
Authors: Krauss J S , Sheard M
Published: July 2018

Normal human factor VIII is precipitated by the lectin concanavalin A (con A). In order to study this interaction pooled normal plasma has been subjected to crossed affinoimmunoelectrophoresis with con A in the first dimension and commercial antiserum to factor VIII in the second dimension. Both decreased anodal migration and decreased precipitin height of the factor VIII - related antigen (FVIIIR:ag) confirmed the precipation of FVIIIR:ag by con A.

This technique, performed in conjunction with crossed immunoelectrophoresis (CIEP) of FVIIIR:ag, should prove useful in the analysis of von Willebrand disease (vWd) variants whose FVIIIR:ag has decreased carbonhydrate and, therefore, is poorly precipitated by con A.
Relationships Between Factor XII-Dependent Systems And The “Prorenin”-Renin System In Human Plasma
Authors: Wilczynski E A , Purdon A D , Osmond D H
Published: July 2018

Treatment of plasma with cold (-4°C,72 hr), and with trypsin (0.5 mg trypsin/ml plasma), are well established in-vitro techniques used to activate plasma prorenin. Various clotting factor deficiencies have been found to impair the conversion of prorenin to renin in plasma. Studies with factor XII deficient plasma, in which marked reduction in both cold and tryptic activation was seen, led to further studies on the role of clotting factors and other factor XI I-dependent systems in prorenin activation. Removal of factors II, VII, IX, and X by adsorption onto BaSO4, and subsequent exposure of the residual plasma to cold (-4°C, 48 hr) and trypsin (1 mg/ml), resulted in a decreased capacity for prorenin activation when compared to control plasma, more so in cold than in trypsin-treated plasma. Plasminogen-free plasma responded similarly and, while increased concentrations of trypsin could enhance its prorenin activation to near-normal levels, prolonged cold incubation could not. This suggests that trypsin, added in an appropriate concentration to deficient plasma, may be able to substitute for the missing factor(s), while cold activation is limited by availability of one or more crucial factors. Unmanipulated Fletcher plasma (prekallikrein deficient) has a low level of active renin, and elevated prorenin, symptomatic of a block of prorenin conversion in-vivo. However, cold and tryptic activation were, if anything, relatively greater than normal, especially for trypsin, suggesting that enzymes other than kallikrein are important activators, in-vitro, and can substitute for the missing kallikrein. Thus, neither kallikrein, nor any other single factor studied here, including factor XII, is solely responsible for the activation of plasma prorenin.
Presence Of Complement Factor C4 And HMW Kininogen In Prothrombin Complex Concentrates
Authors: Steinbuch M , Pejoudier L , Boffa M C
Published: July 2018

The protein contaminants of prothrombin complex preparations obtained by adsorption onto ion exchangers and namely DEAE-Sephadex were studied.

Among them a high amount of C4 accompanied by some C4- binding protein was noticed. Certain preparations contain as much as 20 times the normal plasma concentration of C4. This complement component is able to form an anaphylatoxin. Its molecular integrity has been checked by immunoelectrophoresis and rocked electrophoresis. Its degradation by several enzymes is shown.

The clotting component concentrates contain also high amounts of kininogen. The enrichment of high molecular weight (H.M.W.) kininogen is comparable to the one observed for C4. Its presence was assessed with an antiserum specific for this protein and the ability of the kininogen to supply kinin was checked on guinea pig ileon.

The potential thrombogenicity of prothrombin complex preparations has been attributed to contact factors. It is shown in this study that factor XII and its derivatives as well as PK can be easily removed during the processing of the fraction. However, kininogen displays the same affinity for the ion exchanger than the clotting components.
Incomplete Fibrin Formation And Highly Elevated Factor XIII Levels In Multiple Myeloma
Authors: Klingemann H G , Egbring R , Heimburger N , Havemann K
Published: July 2018

In some patients suffering from multiple mye - loma a defect in fibrin formation has been suggested as one cause for bleeding tendency. However nothing is known about the commoness of this disorder or about the correlation to factor XIII activity.

As pointed out in this investigation the defect in fibrin formation, as proven in SDS-PAA electrophoresis, is due to a lack of α- chain polymerization in 5/11 patients with IgG - myeloma and 2/5 with IgM paraproteinemia. No disturbed fibrin formation could be observed in IgA myeloma (n=6). Factor XIII concentration of subunit A and to a smaller extent of subunit S (Laurell technique) were highly elevated in all cases with regular fibrin formation. Comparable values were obtained measuring the transamidase activity as incorporation of C 14 labelled putrescin into casein. Levels up to 600 % of normals could be evaluated. In contrast all patients with a lack of α- chain polymerization had a factor XIII activity within the normal range. Addition of factor XIII concentrate to plasma with defective fibrin formation led in 5 out of 7 cases to a partial crosslinking of α - monomers. A complete α- chain polymerization could not be achieved, even when high concentrations of factor XIII were used. We conclude that in some cases paraproteins can inhibit the factor XIII activity and prevent its action on fibrin. Patients, who are able to produce factor XIII, especially the active part subunit A in excess, can achieve a regular fibrin formation. Treatment with factor XIII in case of bleeding should be taken into consideration.
In Vitro Comparison Of Activated & Nonactivated Prothrombin Complex Concentrates (PCC)
Authors: Lazerson J , Abildgaard C F , Harrison J
Published: July 2018

In order to determine whether any in vitro test is related to clinical efficacy of PCC we compared nonact ivated PCC (Konyne-Cutter, Proplex-Hyland; Prothromplex- Immuno) with activated PCC (Autoplex-Hyland, FEIBA-Immuno, Antiinhibitor Correctional Concentrate (AlCC)-Cutter). The non-activated partial thromboplastin time (NAPTT), factor VIII correctional activity (FVIIICA) by the Hyland method, and factor VIII inhibitor bypassing activity (FEIBA) by the Immuno method were performed on all PCC according to the manufacturer’s specifications, utilizing their reagents. The results are reported as averages as interlot differences were minimal.

No differences in results were obtained in FVIIICA assay of Autoplex whether inhibitor plasma or noninhibitor (factor VIII deficient) plasma was used as substrate, in contrast to the FEIBA assay where substitution of noninhibitor plasma resulted in a threefold decrease in final units of FEIBA (i.e. 5 units vs 16 units).

It is concluded that the non-activated PCC contain 1/3 to 1/2 the factor VIII correctional activity of the activated PCC and that the NAPTT depending on the dilution selected is only a poor correlate of either. The relationship of the assays to clinical efficacy remains unknown.
Identification Of Thrombin In Human Serum By Iso- Tachophoresis
Authors: Principe D Del , Biancini G , D’Arcangelo C , Mancuso G , Persiani M , Menichelli A
Published: July 2018

We investigated the nossibility of detection of thrombin in blood by analytical isotachophoresis. This technique is a relatively new electrophoretic method for separation of different ions. At the equilibrium the ions move with the same speed and are spaced into consecutive zones between the leading and terminating electrolytes according to their mobilities. The instrument used for analytical isotachophoresis was equipped with an UV and thermal detectors. The leading was MES, Ammediol, hydroxypropylmethyl cellulose 0.25%, pH 9.2; the terminating was EACA, Ammediol, adjusted with Ba(OH2) to pH 10.8. Carrier ampholytes,pH 3.5-8 range were employed as spacers.

Free thrombin was not detected by analytical isotachophoresis in 30 normal sera, while it was demonstrated in 2 children with laboratory and clinical evidence of DIC.

Our data show that the detection of free thrombin by isotachophoresis is due to an in vivo decrease of the serum neutralizing capacity following either an enzyme excess or an inhibitor loss.

The isotachophoresis allows the assay of microquantities of sample(2 μl)in short time(15-20 min) and may reveal itself useful in clinical diagnosis.
Permeability Enhancing And Chemotactic Activities Of Lower Molecular Weight Degradation Products Of Human Fibrinogen
Authors: Tanaka K , Sueishi K
Published: July 2018

Fibrinogen degradation products were investigated for leukocyte chemotactic activity and enhancement of vascular permeability. Fragments X, Y, D and E, and non-dialysable lower molecular weight degradation products were fractionated from plasmin digests of human fibrinogen by gel filtration, zone electrophoresis and ion exchange chromatography. They were assayed for chemotactic activity for human granulocytes by the modified Boyden chamber and for vascular permeability increasing activity in rabbit skin injected intravenously with pontamine blue.

Both activities increased progressively with plasmin digestion of fibrinogen. Molecular weights of the permeability increasing and chemotactic activity fractions were 25,000-15,000 and 25,000 respectively. Both fractions had much higher activities than the fragment X, Y, D and E. The low molecular weight break down product with chemotactic activity showed the highest activity in rabbit skin in vivo as well. Granulocytic infiltration was most prominent 6-12 hr after intradermal injection and apparent even at an intradermal dose of 0.5/μg. Permeability enhancement of the active fraction reached to its muximum level at 5-10 min and was dose-dependent. Electron microscopic observations of the small blood vessels in rabbit skin correlated an increased permeability with the formation of characteristic gaps between adjacent endothelial cells and their contraction. Carbon particles used as a tracer passed into the vascular walls through these intercellular gaps.

These observations add more support for the hypothesis that lower molecular weight degradation products of fibrinogen may be influential in contributing to granulocytic infiltration and enhanced permeability in lesions associated with deposits of fibrin and/or fibrinogen.
A New Automated Fibrinogen Assay By Using A Standard Prothrombin Time Test Procedure In An Automated Photoelectric Clot Timer
Authors: Musumeci V , Zappacosta B , Elamthuruthil J , Carloni L , Marra R
Published: July 2018

A new electronic system was developed for measuring fibrinogen in undiluted plasma during a clotting reaction. The new electronic circuit can pick up the signal coming from the photoelectric sensor of an automated coagulizer (in our case a Dual Channel of Warner Lambert Company) without interfering with its normal function. The signal produced by the clotting reaction is electronically elaborated and then sent to a four digit display which shows directly after adequate scale adjustment the concentration of fibrinogen in mg%. Electronic details of this system are patent pending. Fibrinogen electronic assay can be performed by using as reagent a tissue thromboplastin (Simplastin Automated) in the standard test procedure for prothrombin time. This system permits to obtain simultaneously in the same cuvette the determination of prothrombin time and fibrinogen. The time required for fibrinogen determination by fibrinogen electronic assay with tissue thromboplastin (FEATT) depends on prothrombin time and is about 50 sec in samples showing a prothrombin time of 30 sec. Fibrinogen can be correctly measured even in samples showing a prothrombin time as long as 100 sec. Reproducibility of FEATT showed a C.V. of 0.8 and 4% respectively at fibrinogen levels of 500 and 100 mg%. Fibrinogen concentrations as low as 10 mg% could be measured by FEATT. Heparin up to a concentration of 4 u/ml of plasma did not influence fibrinogen assay by FEATT. A good correlation was found between FEATT and methods currently used for fibrinogen assay performed on normal and abnormal samples (Parfentiev method, r=0.957; Clauss method, r=0.869; biuret assay of clottable protein, r=0.943).
Fibrinogen In Liver Cirrhosis. Sialic Acid Content Of Fibrinogen In Moderate And Severe Cirrhosis And Relation To Thrombin Time
Authors: Palareti G , Poggi M , Coccheri S
Published: July 2018

An increased content in sialic acid in the carbohydrate moiety of fibrinogen (fibrinogen sialic acid, FSA) as described in a number of different liver patients has been accounted for the polymerization defect often found in conditions of liver damage.

In 22 cases of liver cirrhosis divided into “moderate” (n = 11) and “severe” (n = 11) according to standardized criteria, FSA was measured with the method of Warren after purification of fibrinogen with β-alanine and glycine, and expressed in mg per 100 mg of fibrinogen.

In the whole group of cirrhotics FSA was 0.79 mg ± 0.17 (controls n = 10, FSA 0.70 mg ± 0.13, p > 0.05). However, FSA was markedly increased in “severe” cases (0.90 mg ± 0.16) both vs controls (p < 0.001) and vs “moderate” cases (0.69 mg ± 0.10, p < 0.005). Thrombin time (TT) although prolonged in cirrhotics (p < 0.001) was not significantly different between the subgroups. FSA correlated inversely with fibrinogen level (p < 0.01) but did not correlate with TT.

These results show that the increase in FSA is a feature of severe liver cirrhosis especially in presence of a low fibrinogen level. Although probably involved in the polymerization defect, this increase alone cannot properly explain the prolongation of TT which is known to occur also in absence of serum FDP.
Lyophilized, Reconstituted Human Plasma-, A Better Substrate Than Purified Fibrinogen For Thrombin Activity Assay
Authors: Brosstad F , Godal H C
Published: July 2018

The clotting time of purified fibriiwgen/thrcinbin mixtures is regularly used for assay and standardization of thrcmbin activity. However, considerable batch-tobatch variations as well as difficulties in obtaining precise determination of the visual gel point are nharasteristics of purified fibrinogen preparations, making them suboptimal for these purposes. In contrast, thrombin (1 NIH U) clotting time of pooled, human citrated plasma varies only insignificantly from batch to batch,and the readability of the gel point and storage stability at -20°C are excellent . For a more general use of pooled plasma as substrate for thrcnibin assay a lyophilized specimen would obviously be more practicable. To study this, pooled citrated hunan plasma was either lyophilized directly, or subsequent to dialysis against 0.2M citrated buffer pH 7.4 and reconstituted to original volume with water after storage at either +20°C or -20°C for varying periods of time.

Conclusion: Only plasma dialysed prior to freeze-drying shewed a pH and a thrombin clotting time identical to that of unprocessed plasma. Such lyophilized plasma can be kept at rocm temperature for at least 1 year (our present limit of observation) wihtout any sign of deterioration, and represent a premising alternative to presently used thrombin substrates.
Comparison Of Thrombelastogrpahy With Common Coagulation Tests
Authors: Caprini J A , Zuckerman L , Cohen E , Vagher J P , Woodward E , Mitchell J , Llama R Wonder
Published: July 2018

Thrombelastographic (TEG) results were compared with hematocrit (HCT), platelet count (PLT), prothrombin time (PT), partial thromboplastin time (PTT), fibrinogen concentration (FIB), and fibrin split products (FSP) concerning the ability of the test or tests to differentiate between 141 normal adult volunteers and 121 patients with histologic evidence of malignancy. The cancer patients were studied because they represent a contrasting group with respect to coagulation abnormalities. Using canonical correlation, the normal subjects’ TEG and coagulation tests captured all significant information in a single dimension, indicating that a single variation in the coagulation tests would result in a single change in the TEG. In the cancer patients, 3 pairs of functions were required to capture the correlations between the TEG and the other tests.

1. The first, which accounts for 40.8% of the correlation, links the abnormal rates and magnitudes of fibrin polymerization in the TEG to static constituents (HCT, PLT, and FIB)

2. The second, accounting for 22.6% of the correlation, relates the kinetics and magnitude of clot formation in the TEG to both dynamic and static measurements (PLT and PTT)

3. The third equation relates the kinetics of coagulation to kinetic assays (PTT, PT, and FSP)

This implies that 3 independent processes may be occuring in the cancer patients and this information is captured by the TEG. In addition, discriminant function analysis demonstrated that the TEG alone was capable of a 96.7% discrimination between the two groups, while all the other tests combined could only predict 72.3%. Thus, due to its ability to be simultaneously sensitive to different processes, the TEG may add valuable information to the coagulation testing regimen.
Plasma Hypercoagulability After Immuntherapy With Corynebacterium Parvum
Authors: Baumgärtner A , Harenberg J , Fritze D , Zimmermann R
Published: July 2018

Toxic side effects after i.v. application of co- rynebacterium (c.) parvum in the experimental immuntherapy of malignant disease are known. Since hypercoagulability is proven in cancer patients a controlled trial was carried out in 18 patients with metastatic breast cancer to investigate the influence of c. parvum i.v. on platelet count, blood coagulation and fibrinolysis.

Patients were allocated randomly to one of the three treatments:1)0.4mg c.parvum i.v. and cytostatic therapy (vincristine, adriblastin and en- doxan), 2) 0.4mg c.parvum i.v. and 0.9% saline infusion at day 15 of the cytostatic cycles and 3) only cytostatic therapy. Parameters were controlled before and 1, 2, 4, and 24 hrs after beginning of therapy. Platelet count decreased in a rate of 15% within 4 hrs.in all groups. Factor VIII, fibrinogen, prothrombin time, aPTT, thrombin clotting time, reptilase time, ethanol gelation test and AT III remained uneffected in all groups. Fibrinopeptide A (FPA) determined radioimmunological ly increased in group 1 from 1.1 to 18.6 ng/ml (median)two hrs after the application of c.parvum and in group 2 from 4.5 to 57.5 ng/ml (p 0.05). Within 24 hrs FPA declined to the initial values. In group 3 no changes were measured. The euglobulinlysis time, plasminogen, α-1-antitrypsin and α-2-macroglobulin were not influenced in all treatment groups.

The data indicate, that c.parvum i.v. enhances or initates even in small doses plasma hypercoagulability in patients with malignancy. This may contribute to development of thrombemblic complications in patients with increased risk factors.
Specificity And Sensitivity Of Some Coagulation Tests In Various Thrombotic Diseases
Authors: Rodzynek J J , Schoenfeld P L , Léautaud P , Martin T , Wettendorff P , Delcourt A
Published: July 2018

Fibrinogen degradation products (FDP), Ethanol gel test (EGT), citrated whole blood thrombo elastogram (TEG) and its several parameters (r, k, am, IFT), Raby’s transfer test (RTT) and Wu’s circulating platelet’s aggregates (CPA) were studied in 52 healthy volunteers acting as normal controls, in 205 consecutive patients admitted in a general hospital for various non-thrombotic pathological situations (group 1), and in 14 consecutive patients admitted for deep vein thrombosis, pulmonary embolism or acute coronary insufficiency (group 2).

In conclusion : 1. TEG and CPA are of doubtful help in separating thrombo-embolic situations from other pathological conditions.

2. EGT disclosed a better specificity (6% false positive) but its sensitivity is poor (64% false negative).

3. Good specificity altogether with sensitivity in diagnosing thrombo-embolic situations is better achieved by FDP (11% false positive and 43% false negative) and even better by RTT (11% false positive and 29% false negative).
Antiticiombin III Deficiency In A Large Family with 41 Affected Members
Authors: Stibbe J , Adhin S , Ong G L , Panday R S , Peters S H , Smith S J , Snel L A , Went L N
Published: July 2018

Hereditary Antithrombin III (AT III) deficiency was found in a large Hindustani family, living partly in the Netherlands, partly in Suriname. Of 201 members investigated 35 were found to affected: AT III activity (chromogenic substrate) and AT III antigen (immuno-electrophoresis according to Laurell) were about 45 %. Analysis of this fanily clearly demonstrated the autosomal dominant inheritance of the condition. Six non-investigated members (1 living, 5 non-living) were diagnosed as being affected on the basis of affected offspring.

Seventeen affected members had no signs of thrombo-embolic(TE) processes (age group 0-10 years old, n=2; 11-20, n=5; 21-30, n=4; 31-40, n=4; 41-50, n=2). Thirteen showed clinical or proven signs of TE processes (first time in age group 0-10 years old, n=0; 11-20, n=l; 21-30, n=G; 31-40, n=4; 41-50,n=l; 51-60, n=0; 61-70, n=l). No clinical information is yet available on the remaining affected members. Deep venous thrombosis (DVT) occurred in 9 patients (age group 21-30, n=5; 31-40, n=3; 61-70, n=l). Triggering factors were none 4, surgery 1, oral contraceptives and preg- nancy4. Pulmonary embolism occurred in 6 patients (2 clinical, 4 proven) and was fatal in 4; ages were 19, 21, 26, 37, 48 and 68 years old. Pregnancy was uncomplicated in 3 women (total of 4 pregnancies), one of these women was treated prophylactically with anticoagulants during pregnancy (1 pregnancy). Two women (9 pregnancies) had a thrombotic episode (1st and 3rd pregnancy respectively) and 1 woman died suddenly 7 days after her 7th childbirth. DVT occurred in 2 of 4 women who used oral contraceptive pills.

In some symptomless patients (age 22, 26, 32, 33, 40 years old) impedance plethysmography (n=5), 125I-fibrinogen leg scanning (n=3), 125I-fibrinogen T½(n=3) and 51C-platelet survival (n=l) were normal
Antithrombin III In Patients Treated With Subcutaneous Or Intravenous Heparin
Authors: Conard J , Lecompte T , Horellou M H , Cazenave B , Samama M
Published: July 2018

Plasma Anti thrombin III (AT III) has been measured by Mancini method and serum anti thrombin (AT) activity by Von Kaulla's method in 49 patients treated with intravenous (10 cases) or subcutaneous (39 cases) heparin at therapeutical doses. Among the 39 subcutaneous treated patients, 19 were re-examined after cessation of heparin.

A statistically significant decrease in plasma AT III and in serum AT activity has been observed in both groups of heparin-treated patients. In addition, in the 19 subcutaneous treated patients, the plasma AT III and serum AT activity which were decreased under treatment, increased within normal limits after cessation of the treatment : consequently, a congenital AT III deficiency could be ruled out in these patients

In conclusion, we did find a statistically significant decrease in plasma AT III and in serum AT activity in heparin-treated patients, whatever the route of administration.