bluuebunny/crossref_metadata_2025 huggingface dataset:
An Enhancing Effect Of Smoking On Fibrinolysis
Authors: Allen R A , Kluft C , Brommer E J P
Published: July 2018

The effect of smoking two cigarettes in 15 minutes on the fibrinolytic enzyme system in blood, was investigated. Samples were taken, using an indwelling catheter over a 1 hr period (starting at 9 a.m.), from 18 apparently healthy volunteers in fasting condition. The pre-smoking sample, considered to be a true base-line sample, was taken 30 minutes after the initial sample. In the subsequent 15 minutes two average (re: tar and nicotine) cigarettes were smoked; controls “smoked” unlit cigarettes. Samples were taken at the middle and at the end of the smoking period, as well as + 22.5 and + 30 minutes from the start of smoking. The heart rate was monitored throughout.

Haematocrit values, before and after smoking, did not change significantly, but carboxyhaemoglobin levels rose on average in inhaling smokers from 3.0 to 5.1% and the average heart rate increased from 64 to 76 beats/minute after smoking.

On screening euglobulin fractions of each sample on normal fibrin plates a small, but consistent, increase in fibrinolytic activity was seen, apparently due to smoking (an increase of up to 2.1 BAU). Activation of intrinsic systems, via factor XII, could not be demonstrated, but further analysis of the increase in fibrinolytic activity revealed quenching by anti-tissue activator antibodies and not by anti-urokinase antibodies.

Thus, in conclusion a small, but consistent increase in the circulating level of extrinsic plasminogen activator activity was detected after smoking 2 cigarettes.
Enhancement Of Plasma-Plasminogen Level By Stanozolol In Man
Authors: Kwaan H C , Robbins K C , Wohl R C
Published: July 2018

An anabolic steroid, Stanazolol, has been shown to increase AT-III concentration in subjects with both congenital and acquired AT-III deficiency (Thromb & Haem. 43:18, 1980; Thromb Diath Haem. 34:106, 1975). It is also claimed to have beneficial effects on idiopathic recurrent superficial thrombophlebitis and cutaneous vasculitis. In an attempt to elicit the pharmacologic action of this steroid on the fibrinolytic system, the plasma plasminogen of 15 subjects with no known thromboembolic disease were studied before and after administration of Stanozolol, 10 mg/ day for periods of 7-21 days. The plasma plasminogen level was found to increase from 15-63% (mean 35.4%) of baseline level within a period of 7 days, and up to 76%. by day 18. No significant changes were noted on the plasma fibrinogen level nor on the activities of alpha2 plasmin inhibitor, free protease, or plasminogen activator. In vivo plasmin generation by both streptokinase and urokinase was also studied in the plasma samples. The generation rate by either activators was found to be significantly increased along with the rise in plasminogen level. The plasmin generation index (plasminogen level x plasmin generation rate) representing potential amount of plasmin that can be generated in the plasma samples was found to be increased even more so, by Stanozolol administration. Since the successful outcome of thrombolytic therapy depends on the bioavailability of plasminogen, the present findings of an enhancement of plasminogen by Stanozolol has obvious clinical implications.
Anticoagulant Activity Of Heparan Sulfate From Cerebral Microvascular Tissue
Authors: Marcum J A , Fritze L , Rosenberg R D
Published: July 2018

Microvascular tissue (MT) was isolated by standard sieving techniques from the cerebral cortex of yearling calves. After proteolysis of MT, the cell debris was pelleted and the supernatant was charged to DEAE-Sepharose A-25 equilibrated with 0.1 M NaCl in 0.01 M Tris-HCl, pH 7.5. Uronic acid determination demonstrated that two peaks of mucopolysaccharides (MS) were eluted at an added salt concentration of 0.4 M NaCl and 0.8 M NaCl, respectively. Enzymatic and electrophoretic analyses of the first peak revealed the presence of a minimally sulfated MS with no ability to accelerate thrombin or factor Xa neutralization by antithrombin (AT). MS isolated from the second peak were degraded by chondroitin ABC lyase and Flavobacterium heparinase. This material migrated slightly behind NIH standard heparan sulfate (HS) and distinctly separately from NIH standard heparin (Hp) which had much greater electrophoretic mobility. Sequential degradation with chondroitinase and heparinase revealed that HS was responsible for the anticoagulant activity of the second peak. The antithrombin and anti-factor X activities of HS were 2.81 u/mg and 2.86 u/mg, respectively. Affinity fractionation of HS with AT resulted in an increase of antithrombin and anti-factor X activities to 36.6 u/mg (13 fold augmentation) and 41.3 u/mg (15 fold augmentation), respectively. Each of the biological activities required the presence of AT and disappeared upon exposure to heparinase. Electrophoretic analysis of the affinity fractionated material demonstrated that it migrated slightly behind NIH standard HS and 40% slower than NIH standard Hp. In conclusion, the ionic strength at which this affinity fractionated HS had been eluted from DEAE-Sepharose and its mobility during electrophoresis clearly distinguish it from classical Hp. These data demonstrate the existence of a specific species of HS from MT with significant anticoagulant activity. These components may in part, be responsible for the non- thrombogenic properties of vascular elements.
A Correlation Between Anticoagulant Activity, Molecular Weight And The Increase In Antithrombin-III Fluorescence Produced By Heparin Fractions
Authors: Mardiguian J , Aiach M , Trillou M , Michaud A
Published: July 2018

The aim of this study was to correlate the anticoagulant activity of heparin with its affinity for Antithrombin-III.

Eleven fractions of different molecular weight (3900 - 42000 daltons) were obtained by fractionation of a Commercial heparin on Ultrogel AcA 44. The anti-Xa and antithrombin activities of these fractions were measured by chromogenic assays and expressed on a molar basis. Their affinity for antithrombin-III was assessed by measuring their ability to increase bovine antithrombin-III tryptophan fluorescence.

A linear relationship was obtained between fluorescence enhancement (F), expressed on a molar basis, and molecular weight (M), (F = -26,50 + 0,01 M) as well as between anti-Xa activity, anti-thrombin activity and F.

This study shows that the anti-Xa and the anti-thrombin activities of heparin are increasing with its capacity to enhance the fluorescence of antithrombin-III and that this capacity is increasing with molecular weight.
Low Molecular Weight Heparin And Postoperative Deep Vein Thrombosis
Authors: Kakkar V V , Djazaeri B , Webb P , Scully M , Westwick J , MacGregor I
Published: July 2018

Recent studies have indicated that commercially available heparin is a hetrogenous compound consisting of the molecules of diverse chain length and different molecular weights. Low molecular weight (LMW) heparin has been shown in vitro to possess unique properties which should not only improve its efficacy for preventing post-op DVT but also reduce or eliminate the risk of bleeding complications. The aim of this paper is to report the results of in vivo studies performed to test this hypothesis.

Ten healthy volunteers received 5000 USP units of unfractionated heparin (mol. wt. 15,000 daltons) or equivalent amount of LMW heparin (mol. wt. 6000 daltons) subcutaneously. Blood samples were withdrawn 1/2 1, 3 and 5 hours later. These were analysed for anti-thrombotic effect using anti-factor Xa (anti Xa) assay and anticoagulant effect by estimating kaolin cephalin clotting time (KCCT) method. While both the preparations of heparin produced similar response in KCCT assay, LMW heparin was found to be 4 times more active in anti-Xa assay. 30 patients undergoing major abdominal surgery were randomly allocated to receive either 5000 units of unfractionated heparin or equivalent amount of LMW heparin every 12 hourly for 10 days. Blood samples were withdrawn before, during and immediately after surgery and every day during post-op period. These were analysed for anti-thrombin III and heparin concentrations, lipoprotein lipase, fibrinopeptide A, prekallikrein, TxB2 and 6-oxo-PGF1α to assess platelet effect. Significant differences were only observed in lipoprotein lipase and anti-thrombin III concentrations.
In ongoing trial 43 patients undergoing major abdominal surgery have received 1500 units of LMW heparin every 12 hourly for 7 post-operative days. Only 1 patient has developed DVT and another wound haematoma. If these results are confirmed in a larger number of patient, then LMW heparin may be ideal drug for prophylaxis against DVT.
In Vivo Studies On The Inhibition Of Coagulation By Fractionated Heparin
Authors: Schmitz-Huebner U , Balleisen L , Asbeck F , van de Loo J
Published: July 2018

Recent investigations suggest that low molecular weight heparin may have advantages over conventional heparin with regard to the prevention of venous thrombosis and haemorrhagic side effects.

High (HMW) and low (LMW) molecular weight heparin fractions with mean MWs of 16,000 and 8,800 respectively, obtained by gel filtration chromatography of sodium mucosal heparin (B. Braun Melsungen), were injected subcutaneously into six volunteers and compared with the unfractionated substance in a cross-over trial. Doses of 5,000 U were administered twice daily over a period of three days and heparin activity was controlled before injection and 2,4,8 hours afterwards by means of the APTT, the anti-Xa clotting test and a chromogenic substrate assay. In addition, the in vivo effect of fractionated heparin on platelet function was examined. The results show that the LMW fraction induced markedly higher anti-Xa activity than the other preparations. At the same time, APTT results did not significantly differ. Unfractionated heparin and the HMW fraction enhanced ADP-induced platelet aggregation and collagen-mediated MDA-production, while the LMW fraction hardly affected these assays, but potently inhibited thrombin-induced MDA production. All heparin preparations stimulated the release of PF IV, whereas the serotonin content of platelets determined at the same time increased.

It is concluded that s.c. injections of LMW heparin induce relatively high levels of anti-Xa activity without leading to sensitive platelet activation or to major effects on overall clotting tests.
Possible Mode Of Action Of A Semi-Synthetic Heparin Analogue . (SSHA)
Authors: Kakkar V V , Djazaeri B , Scully M F , Westwick J
Published: July 2018

It has been suggested that following parenteral injection, the anti-thrombotic effect of SSHA is possibly related to release of endogenous glycosaminoglycans probably from vascular endothelial cell surface. In this study, the mechanism of action of SSHA has been explored further.

Five healthy volunteers received 3 different doses of SSHA (75, 50, 35mgs) and 50mgs of heparin subcutaneously, four days apart, and venous blood samples were taken at 0,3,5 and 7 hours post injection. These were analysed for prothrombin time, KCCT, lipoprotein lipase, βTG, PF4 and prekallikrein levels. Heparin activity was measured by an anti-factor Xa assay. In addition, template bleeding times were done before and after the administration of 75mg of SSHA and 50mgs of heparin. Prothrombin time response, following addition of varying doses of SSHA and heparin to pooled blood was also studied.

Although SSHA prolonged the template bleeding time, the difference as compared with control value was not statistically significant. Heparin in a similar dose prolongs the template bleeding time to a lesser extent. No significant differences were observed in the levels of βTG, lipoprotein lipase, prekallikrein levels, KCCT and heparin activity following administration of SSHA and heparin. Unlike heparin, SSHA in a dose related manner significantly prolongs prothrombin time, the difference being significant at 1,3 and 5 hours following administration of each dose. These findings suggest that part of antithrombotic effect of SSHA may be mediated through inhibition of extrinsic pathway of activation of intravascular coagulation.
A Modified Stasis (Wessler) Rabbit Model To Evaluate The Antithrombotic Actions Of Heparin Fractions And Fragments With Low Anticoagulant – High Anti Xa Activities
Authors: Andersen A , Fareed J , Stulc J , Messmore H L , Choay J
Published: July 2018

Since its introduction the Wessler stasis rabbit model (Wessler, et al. J. Appl. Physiol. 14:(6), 943, 1959) has been widely used in the evaluation of the thrombogenic properties of prothrombin complex concentrates (activated and non-activated) and antithrombotic effects of various drugs. In order to study the antithrombotic actions of low molecular weight fractions and oligosaccharide fragments we used a modified stasis (rabbit) model with thrombogenic stimuli which produced a marked increase in circulating factor Xa levels. Male New Zealand rabbits ranging from 23 kg were injected (subcutaneous) with an analgesic muscle relaxant, xylazine (20 mg/kg) followed by ketamine (80 mg/ kg). Prothrombin complex concentrate (PCC) is injected within 2 minutes into a contralateral rabbit ear vein (25 units/kg) immediately followed by Russell’s viper venom (RVV). Within varying times after completion of the infusion, (1-15 minutes) previously exposed right and left jugular veins were gently isolated, kept in situ for 10 minutes, carefully excised and the clot formed was graded. Only minor changes were observed in the coagulation profile after the PCC/RW infusion, however measurable clots in both segments of the isolated vein were seen. Intravenous injection of heparin fractions (<500 μg/kg) and oligosaccharide fragments (<100 μg/kg)blocked the thrombotic effects of PCC/RW mixture in both the pretreatment and post-treatment regimens, whereas heparin at 500 μg/kg failed to show any antithrombotic effects. Bovine and human factor Xa concentrates, tissue thromboplastins, human serum, human and bovine a-thrombin, and arachidonic have also been employed as thrombogenic stimuli. Our studies show that the modified stasis rabbit model offers definite advantages over the existing model and provides a suitable in vivo standardized model to test antithrombotic effects of newly developed heparin fractions and fragments with high anti Xa and low anticoagulant action.
Is Warfarin Sodium Absolutely Contraindicated During Lactation?
Authors: McKenna R , Cole E R , Vasan U S
Published: July 2018

There is widespread concern regarding the secretion of a variety of drugs and toxins into human milk. A survey of the practices of obstetricians and neonatologists at 2 teaching medical centers regarding the appropriateness of allowing a mother on warfarin sodium to breast feed her infant revealed that almost all advised such mothers against breast feeding. This study reports the data on two mother- infant pairs who were evaluated with Prothrombin time activity (Protime), Factor II (FII) and Factor VII/X (FVII) levels simultaneously while the mothers were on warfarin sodium and the infants were exclusively breast fed. Both mothers had extensive serious venous thromboembolic disease necessitating the administration of anticoagulants; warfarin sodium was first started in both mothers in the post-partum period.

The prothrombin time activities on the first mother-infant pair, on blood samples drawn simultaneously are shown below.

Breast milk extracted from this mother on one occasion did not reveal any warfarin sodium by a spectrophotometric assay. Data on the second mother-infant pair on blood samples obtained simultaneously were as follows.

These data demonstrate conclusively that there is no inhibition of the coagulation mechanism in normal full term infants when they are breast fed by mothers who are therapeutically anticoagulated with warfarin sodium.
β-Propiolactone/Ultraviolet Irradiation: Quantitative Studies On Effectiveness For Inactivation Of Hepatitis B Virus
Authors: Prince A M , Stephan W , Brotman B
Published: July 2018

The efficacy of combined β-Propiolactone/Ultraviolet irradiation (βPL/UV) for inactivation of hepatitis B virus in labile blood derivitives has been reinvestigated. To permit quantitative estimation of process efficacy, a regression analysis of the relation between virus dose and incubation period was carried out. This has provided a means for estimating virus titer and for determining the accuracy of such estimates.

The data suggest that βPL/UV can reduce virus titer about 10,000,000 fold (10-7). The process efficacy for βPL/UV followed by the Aerosil adsorption procedure used in preparation of stabilized human serum (Biseko) containing most human serum proteins except for removal of factor VIII, the PPSB factors, fibrinogen and the lipoproteins was estimated to be about 10-8.

This degree of virus inactivation should be more than sufficient to sterilize the highest amounts of hepatitis B virus which could be expected in pooled human plasma which has been screened for HBsAg.
Prothrombin Fragment 1.2.3, A Major Product Of Prothrombin Activation In Human Plasma
Authors: Rabiet M J , Furie B , Furie B C
Published: July 2018

The conversion of human prothrombin to thrombin is associated with a number of cleavage intermediates and products whose appearance and concentration are dependent upon the prothrombin activation conditions used. In the current investigation, the fragments of prothrombin which appear in normal human plasma after activation of the blood coagulation cascade were studied. Radioiodinated human prothrombin was added to platelet-poor relipidated normal human plasma and clotting initiated with Ca(II) and kaolin. The radiolabeled prothrombin cleavage products which formed were analyzed by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate (SDS) and 2-mercaptoethanol (2-ME), A new product of prothrombin activation was observed. Its migration was more rapid than prethrombin 1 and slower than fragment 1.2. No previously identified products of prothrombin activation migrated to the same position in the gel.

The previously unrecognized fragment was identified as fragment 1.2.3 as follows. Prothrombin was activated by factor Xa in the presence of Ca(II) and phospholipid. The desired product was isolated by absorption to and elution from barium citrate and by DEAE cellulose chromatography. This purified material, migrating identically with the unknown plasma product was homogeneous upon SDS gel electrophoresis with 2-ME. The amino terminal sequence of the isolated material was identical to that of prothrombin. Digestion of this material with either factor Xa or thrombin yielded as major products fragment 1.2 and fragment 1. (Fragment 2 and fragment 3 eluted from the gels under the conditions employed). Amino terminal sequence analysis of the factor Xa digestion products of the isolated material indicated three amino acid residues at each cycle. The sequences of fragment 1, fragment 2, and fragment 3 are consistent with this sequence analysis. On this basis we suggest that fragment 1.2.3 is a prominent product of prothrombin conversion to thrombin in plasma.
Identification Of Two Dysthrombins Derived From Prothrombin Quick
Authors: Henriksen R A , Owen W G
Published: July 2018

A dysfunctional thrombin, Thrombin Quick, derived from the congenital dysprothrombin, Prothrombin Quick, had 3040 percent of normal thrombin activity on the low molecular weight substrates, benzoyl arginine ethylester, tosyl-gly-pro-arg-p-nitroanilide, and carbobenzoxylysine-p-nitro-phenylesier, but only 2-5 percent of normal activity in Factor V and Factor VIII activation and platelet aggregation. Titration with the high affinity competitive inhibitor of thrombin, dansyl-arginine-N-(3-ethyl-l, 5-pentanediyl)amide (DAPA) indicated an active site concentration of 0.4 per mole protein. Further studies indicated that the Thrombin Quick preparation could be eluted from sulfopropyl Sephadex as two components, one of which did not show fluorescence enhancement on treatment with DAPA nor did it possess tripeptidase activity. Further analytical chromatography on sulfopropyl Sephadex indicated that the two Thrombin Quick components are chromatographically distinct from thrombin and that their elution pattern is not altered by treatment with thrombin. Each of the Thrombin Quick components was rechromatographed in the presence of thrombin. There was no indication of interconversion of the two components. The results indicate that the presence of two components in Thrombin Quick is not related to the incomplete cleavage of a 13 residue peptide from the amino terminus of the thrombin A chain which occurs in normal thrombin. A synthetic substrate assay was used to determine relative activities of 0.38, 0.45 and 0.33 for Prothrombin Quick, Prethrombin 1 Quick and Thrombin Quick, respectively, compared to their normal counterparts. The simplest explanation for the occurrence of the two Thrombin Quick species is that they are derived from two genetic allomers of prothrombin.
An Acquired Inhibitor To Coagulation Factor II
Authors: Scully M F , Ellis V , Kakkar V V , Savidge G F , Williams Y F , Sterndale H
Published: July 2018

A 77 year old woman with symptoms of GI tract bleeding, haematuria, bruising and profuse bleeding after dental extraction was found to have a prolonged thrombin time (>90 secs) but normal reptilase time and fibrinogen level. Both prothrombin time and KCCT were prolonged to greater than twice normal values. She had no previous history of bleeding problems and no drug history apart from analgesics.

On investigation of citrated plasma samples an antithrombin III level as measured in a chromogenic heparin cofactor assay of 607% was found but measured immunologically of 120%. Upon chromatography on Ultragel AcA44 two peaks of antithrombin activity were observed one eluting in the position of normal antithrombin III immunologically. The activity of this fraction differed according to whether the antithrombin assay was a progressive type or heparin cofactor. The other much larger peak eluted in this second peak off AcA44 and the activity was not susceptible to heparin. The activity in this peak was pooled and chromatographed on DEAE Sephacel. It eluted in 0.0175 M Na (P) pH 6.8 as a single peak coincident with antithrombin activity. On SDS electrophoresis a single band was obtained. This fraction corresponds to IgG. Less than 10% of the activity was eluted with 0.08 M (P) pH 6.6 in the fraction containing IgG and IgA.

The purified IgG directly inhibited thrombin. When added to normal plasma and prothrombin measured using activated factor Xa, complete loss of prothrombin was observed at dilutions of the inhibitor corresponding to 25% of that found in the patient plasma. The inhibitor had no effect on the rate of generation of factor Xa in normal plasma (S2222) as initiated by thromboplastin nor on the activity of purified factor Xa. The evidence suggests that this is the first report of an acquired inhibitor specific for factor II.
Impaired Fibrinolytic Response To DDAVP And Venous Occlusion In A Subgroup Of Patients With Von Willebrand’s Disease
Authors: Niessner H , Korninger Ch , Lechner K
Published: July 2018

Fibrinolytic activity (Euglobulin lysis time, fibrin plate method) and factor VIII related properties (VIII :C , VIIIR : Ag , VIIIR : RCF) were determined in 8 healthy controls,in 21 patients with mild hemophilia A and in 15 patients with von Willebrand’s disease (vWD) before and after infusion of 0,4 μg/kg b.w. DDAVP(I-desamino-8-D-arginin vasopressin).Following infusion of DDAVP a marked increase of fibrinolytic activity occured in all controls and all patients with mild hemophilia A. However, in 7 out of 15 patients with vWD activation of fibrinolysis was absent or slight only. In these 7patients venous occlusion also did not induce activation of fibrinolysis. Activation of fibrinolysis was absent in all 5 patients with severe vWD (VIIIR:RCF < 3%) and DDAVP also did not raise the VIIIR:RCF level. In the mild cases of vWD fibrinolytic activation after DDAVP was impaired only in 2 out of 10 patients. In these mild cases an about 2 fold increase of VIIIR:RCF after DDAVP was equally observed in fibrinolytic “responders” and “non-responders”. The results indicate a combined endothelial cell defect in a subgroup of patients with vWD,resulting in impaired production/release of von Willebrand factor and impaired production/release of vascular fibrinolytic activator.
The Safety Of Ibuprofen In Factor VIII And IX Deficient Hemophiliacs
Authors: Inwood M J , Killackey B , Philp R B
Published: July 2018

Most anti-inflammatory analgesics, particularly ASA, are considered contraindicated in the treatment of hemophiliacs because of inhibition of in vitro hemostatic function. Nevertheless, it would appear reasonable to use such agents in the therapy of chronic or acute hemophiliac arthropathy, provided that the anti-inflammatory drug did not increase the incidence of hemorrhage.

Using moderately and severely affected factor VIII and IX deficient hemophiliacs, Ibuprofen was given, using a short (24 hrs) and long term (21 days) trial in order to assess the effect on in vitro hemostasis and the incidence of hemorrhagic symptoms compared to a comparable period prior to the ingestion of drug. An initial safety trial used 24 normal subjects and 12 moderately to severely affected factor VIII and factor IX deficient hemophiliacs. Within each group 600 mgs of Ibuprofen or lactose placebo was given in a random double-blind study. Over 24 hours no changes were seen in the bleeding time, peripheral blood counts or spontaneous bleeding patterns. Subsequently a 21 day trial was initiated with 15 moderately to severely affected hemophiliacs, using a dose of 2400 mg per os Ibuprofen per day. At 0,7,14 and 21 days, factor assays, bleeding times, platelet function and cell studies were performed. No clinically significant changes were found in any of the experimental variables, including pattern of spontaneous hemorrhages. Four of the 15 subjects experienced intermittent dyspepsia, controlled by antacids. 7 of 11 individuals with hemophilic arthropathy had a subjective decrease in symptoms from arthropathy.

It is concluded that Ibuprofen is not associated with clinically significant changes in vitro or in vivo hemostatic function in factor VIII or IX deficient hemophiliacs, and should be considered in the treatment of hemophilic arthropathy.
The Use Of Radiolabelled FVIII:CAg Antibodies To Characterise FVIII: C Abnormalities
Authors: Mackie I J , Seghatchian M J
Published: July 2018

The development of a simple method of characterising various forms of FVIII:C and identifying the functional abnormality in disease states is important for accurate diagnosis, treatment, and in the preparation of clinical concentrates. We report an electrophoretic method using radiolabelled antibodies to monitor the distribution of FVIII:CAg. Normal and patient plasmas were incubated with 125I-anti VIII:C (*IgG or *FAB), prior to electrophoresis in 1% agarose gels. The gels were then cut into fractions and the radioactivity was measured. Results were compared with crossed immunoelectrophoresis for FVIII:RAg. In normal plasma, bound *IgG/*FAB migrated in the same area as the slow moving side of the FVIII:RAg peak. In severe classical von Willebrands disease and haemophilia A (no detectable FVIII:CAg) plasmas, the radioactivity pattern was, as expected identical to running antibodies alone. Haemophilia A plasma with 100% FVIII:CAg activity (by fluid phase assay) gave a radioactivity pattern identical to normal plasma. Patients with variant von Willebrands disease (decreased levels of the larger FVIII multi- mers), also had antibody binding patterns similar to plasma. These preliminary findings show that the method is suitable for identifying FVIII:C abnormalities. The fact that FVIII: CAg was only seen in association with slow moving FVIII:RAg, could be caused by the method of preparing *IgG/*FAB; this may select antibodies with high affinity for more aggregated forms of FVIII. Better resolution between bound and free antibody was obtained when *FAB was used; increasing the gel strength and plate length may improve separation of smaller FVIII populations. The above method has been successfully applied to the identification of various FVIII forms in clinical concentrates, and has important implications in the management of patients with coagulation abnormalities. Since only small sample volumes are required, the method may be used on finger prick and foetal blood samples.
A Genetic Variant Gf Factor IX With An Abnormal High Molecular Weight
Authors: Bertina R M , van der Linden I K
Published: July 2018

In 1979 we reported a genetic variant of factor IX that behaved very similarly to acarboxy-factor IX (Br J Haematol 1979;42:623-35). With respect to normal FIX it showed an increased electrophoretic mobility in the presence of Ca2+ , a low specific coagulant activity, strongly reduced affinity for adsorption to AI(0H)3 and faint precipitation lines against anti-factor IX serum. These properties strongly suggested abnormal Ca2+ binding properties of this Fix-variant.

The FIX-variant was isolated from the patients plasma using affinity chromatography on anti-human FIX-Sepharose. heparin-Sepharose chromatography and DEAE-Sephadex chromatography. Purification of the FIX antigen was followed by counter imunoelectrophoresis or electroimmunoassay; 50-70% of the Fix-variant was recovered in the final preparation.

Analysis by SDS-PAGE revealed that the FIX-variant has an apparent MW of ca 85,000, which is much higher than that of either FIX or acarboxy-Flx (MW 63,003). After reduction two bands appear: one of 63,000 and one of 35,000. Apparently an additional polypeptide chain is associated with the FIX molecule in this variant. Incubation of the isolated FIX- variant with RVV-X, XIa or Xa does not result in cleavage of either α or β bond.

The abnormal MW of the FIX-variant was also demonstrated by gel-chromatography of the patients plasma or plasma of an obligatory carrier from the patients pedigree. Using a radioimmunoassay, which is specific for the FIX-zymogen, it was found that also in plasma the FIX-variant is not ‘activated’ by XIa.

It is hypothesized that the association of a FIX molecule with a 35,000 polypeptide interferes both with Ca2+ binding and with proteolytic degradation by its physiological activators.
Identification Of Variant Forms Of VIII:RAg In Von Willebrand’S Disease By Planimetric Analysis Of The 2 Die Pattern
Authors: Bone D J
Published: July 2018

Mathematical analysis of the antigen antibody precipitin arc produced by two dimensional immunoelectrophoresis (2 DIE) was used to identify variant forms of von Willebrand’s disease (vWd). 2 DIE was performed on glass plates using rabbit precipitating antibody to human factor VIII related antigen (VIII:RAg) and barbitone electrolyte pH 8.4. After the plates had been dried and stained the results of optically standardised planimetry of the area beneath the precipitin arcs were used to plot histograms. These quantified the distribution of VIII:RAg into slow, slow intermediate, intermediate, intermediate fast, fast, and very fast mobility bands. Application of statistical analysis to the mobility bands showed a significant difference in VIII:RAg distribution from normal for each patient tested so far and between each patient (N = 30). Reproducibility was good on multiple testing of the same sample. Samples taken on different dates from the same patient revealed some variability but overall a similar distribution across the full range of mobility bands was preserved. The technique has revealed much greater variation in the phenotype of vWd than previously suspected and may be useful in classification of variants.
Relationship Between Platelet Adhesion And Free Energies At Solid-Liquid Interfaces
Authors: McIver Donald J L , Schürch Samuel
Published: July 2018

Surface induced thrombosis remains an obstacle to extra-corpeal circulation and invasive investigation and therapy of the cardiovascular system. Although the molecular details of the blood-foreign surface interaction remain incompletely understood, from a thermodynamic viewpoint, thrombosis can only occur if thrombus formation lowers the free energy of the blood-surface system. We have recently reported a method of estimating surface free energies under physiological conditions from measurements of surface wetting by polar hydrophobic fluids. Here we report a correlation between interfacial energies and platelet retention on natural and artificial surfaces, and the effects on platelet retention of a synthetic coating developed in an attempt to mimic the thromboresistance of healthy endothelium. This coating has a lower surface energy than the artery and it appears to be equally thromboresistant.
Prostacyclin Release In Vascular Prostheses With Regard To The Non-Thrombogenicity Of The Neo-Intima
Authors: Fritsche H M , Holzmann Th , Wendt P , Erhardt W , Maurer P C , Blümel G
Published: July 2018

There is good evidence that internal surfacing of vascular prostheses by a non-thrombogenic biological layer is important for satisfactory long-term clinical results. However morphological and histological data obtained so far on the so called neo-intima provide little information about the anti-thrombogenic activity of the newly formed endothelium.

In order to characterize the functional activity of the neo-intima we have developed a device to measure prostacyclin (PGI2) release from the vascular wall into the lumen. Different types of vascular grafts (velour knitted dacron, P.T.F.E. length 10cm, diameter 4-6mm) were placed in the carotid arteries of sheep. After removal the grafts were cut in three transverse sections (proximal, medial and distal third) and were subsequently perfused with oxygenated buffer in order to restimulate the arachidonic acid metabolism in the newly formed vascular wall. Thereafter the sections were filled with a specific volume of buffer and incubated. In the incubated solution PGI2 release was measured using a collagen and ADP induced platelet aggregation model and a 6-Keto- PGF lalpha radioimmunoassay.

Within four months postoperatively PGI2 release increased especially in the proximal and distal sections of the prostheses up to the normal range, while in the medial sections of some grafts even 6 months postoperatively the PGI2 release remained weak. Simultaneous investigations obtained by scanning electron microscopy revealed that there is no clear correlation between the morphology of the newly formed endothelium lining different types of vascular prostheses and the functional activity of the neo-intima. This makes it desirable to characterize the functional activity of the neointima with regard to the non-thrombogenicity of the newly formed endothelium. Improvements in newly developed vascular prostheses can thus be quantitatively measured.
Comparison Of Human Smc From Aorta, Art. ILIACA, Vena Cava And Fetal Umbilical Cord Vein. A Tem-, Sem-, Light Microscopic And Functional Study
Authors: Thilo D G S , Heinrich D
Published: July 2018

Do smooth muscle cells (SMC) from various human origin show the same behavior in cell culture? Are there differences between arterial, venous and fetal SMCs? Explant cultures were established from human adult aorta, art. iliaca comm., vena cava. SMCs from the umbilical cord vein and art.iliaca were harvested by the collagenase method. All cells were grown in M 199, 20 mM Hepes, 20% fetal calf serum, pen.-strept, 37°C, without CO2. After sufficient outgrowth the cells were subcultivated. At various subpassages the cells were processed for TEM, SEM. For TEM the ruthenium-red-method were applied. At different subpassages growth curves were established. Growth pattern was monitored by phase contrast microscopy and staining with fluorescent dyes.--Results: V.cava SMC grow out of the explant faster, however, show slower proliferation rate than the art. SMCs. V.cava and fetal SMCs do not show the striking hill-valley growth pattern as the art. SMCs do. From fetal to adult SMCs the cell- and nucleus size increases. The varying nucleus sizes were monitored by impulscytophotometry. These results are reported in another abstract. From the art.iliaca comm, growing SMCs with up to 10 nuclei were harvested. Art. SMCs secret large amount of granular material which is not seen in venous SMCs. All cells were negative for F-VIII-antigen in the immunfluorescence.

Summary: Because of these marked differences between art. and venous SMCs from human origin evaluation of experiments with SMCs have to take this into consideration before drawing general conclusions on SMC-functions.
The Teflon Tubing Bleeding Time In Man
Authors: Didisheim P , Claypool D A , Stropp J Q , Baker R W
Published: July 2018

The skin bleeding time (BT)(Mielke, Ivy, Duke) is an important part of the diagnostic workup of bleeding disorders. Because of variable vascularity of the skin from one location to another, results may vary widely with the site and type of incision and the thickness of the skin. In some patients bleeding is difficult to stop. The discomfort of the test and the possibility of inducing scarring limit the frequency with which it can be repeated. A standardized BT that could be performed on the patient’s blood in the laboratory could avoid these problems. We have developed such a test, basing it on our previously described method in which a hole is made in a Teflon arteriovenous shunt in rats, and modified from the method of Blakely et al. A cylindrical hole of 75 μ diameter is made in a 50 cm length of 457 μ I.D. Teflon tubing with the aid of a needle, micromanipulator and dissecting microscope. Blood (2 U heparin/ml) from normal human subjects displaces buffer from the tubing at a rate (.6 ml/min) determined by a syringe pump. Pressure is controlled by raising the distal end of the tubing 5 cm above the hole. BT is recorded by an electronic drop rate counter (Payton BTR 400). Under these conditions, BT is 150 ± 47 sec, and bleeding rate from the hole, shear rate and shear stress on the wall of the hole are .11 ± .02 ml/ min, 40,700 ± 8,100 sec-1 and 1,830 ± 360 dynes/cm2 respectively. The BT can be varied from 0 to >15 min by lowering or raising pressure or flow in the system or by varying hole diameter. By phase microscopy the hole is seen to be occluded by a mass of aggregated platelets and occasional PMN. These observations show that hemostasis at small incisions is controlled by rheologic factors and platelets and does not require physical or chemical properties of a blood vessel. This new method may be useful in the diagnosis of bleeding disorders and in studying the effects of rheologic factors and drugs in hemostasis.
A Non-Invasive Method For Measuring Platelet Aggregation In Vivo
Authors: Morley J , Hoyle P M , Leyton J , Davies O J
Published: July 2018

In experimental animals, intravenous injection of ADP produces platelet accumulation in the lungs, together with thrombocytopenia. Since platelets can be isotopically labelled without gross functional disturbance, we have sought to develop a method for measurement of aggregation and disaggregation of labelled platelets in vivo.

Guinea-pig platelets are labelled with Indium-111 and injected into narcotised animals (Sagital, 37 mg/kg). Two collimated crystal scintillation probes are used to monitor the thoracic region (heart and lung) (Cl) and the vascular compartment (hind limbs) (C2). An indwelling cannula (25 SWG) is inserted into a foot vein and kept patent by small volumes of heparin (100 u/ml). Intravenous ADP (10 mg/kg) causes transient accumulation of platelets within the lung, as well as thrombocytopenia, within a two minute period. During a response, counts are monitored from both probes and retained in a dedicated microcomputer, so as to permit rapid display of results in tabular and graphical form. Usually, ninety consecutive 4 sec. counts are recorded, results being expressed both as a paired difference (C1-C2) and as a ratio (C1/C2). Repeated challenge can be made with ADP and a dose-related response is obtained over the range 10-30 mg/kg. Treatment with sulphinpyrazone or prostacyclin inhibits aggregation.
Platelet Factor 4 (PF4) Release In Vitro From Unstimulated Human Platelets
Authors: Cohen D S , Strohschein J M , Saunders R N , Cargill D I
Published: July 2018

Human venous blood was collected (9.5:0.5) in an anticoagulant (provided in the PF4 RIA kit, Abbott Labs.) consisting of 2.5% EDTA, 0.025% adenosine and 7.0% procaine HCl. Platelet rich plasma (PRP) was stirred (900 RPM) at 37°C in a Payton aggregometer for intervals of 3, 6, 9 and 12 minutes. Unincubated aliquots of PRP were taken as the 0 time controls.

A significant (p < 0.02) elevation in PF4 was observed with increasing intervals. Furthermore, from the data obtained, two populations of PF4 releasers were delineated: A high releasing (HR) group and a low releasing (LR) group. HR (n=10) displayed a baseline release of 10.3 ± 0.8 ng/ml and values of 36.1 ± 9.0 (p < 0.05 compared to baseline), 46.8 ± 8.2 (p < 0.01), 54.6 ± 7.9 (p < 0.01) and 87 ± 13.1 (p < 0.01) for the corresponding time periods. LR (n=7) exhibited significant but diminished release at each time period with a basal level of 10.1 ± 1.0 and 14.7 ± 1.4 (p < 0.05 compared to baseline), 15.3 ± 1.5 (p < 0.05), 19.7 ± 2.4 (p < 0.05) and 23.6 ± 2.0 (p < 0.01) respectively. Comparison of both populations revealed significant (p < 0.01) differences at the 6, 9 and 12 minute intervals. Total PF4 content from Triton lysed PRP displayed similar levels between groups: 360.0 ±22.6 (HR) and 380.2 ± 15.2 (LR).

We conclude that an endogenous release of PF4 occurs in vitro in unstimulated human PRP even in a milieu designed to deter release. This should be a concern when PF4 values are to be assessed.
Isolation Of Plasma Membrane Vesicles Of Human Platelet By Affinity Chromatography
Authors: Kobayashi T , Sakon M , Ohno H , Kambayshi J , Kösaki G
Published: July 2018

Platelets undergo a unique morphological changes leading to the formation of hemostatic plug. In recent years, its intermediaty metabolism has been extensively studied and the important function of plasma membrane in the platelet reaction has been recognized. The method of Barber and Jamieson has been employed in order to prepare plasma membrane vesicles of platelet of excellent quality but it is rather time consuming and the yield is relatively low. In this study, an attempt was made to isolate plasma membrane vesicles of human platelets by wheat germ agglutinin affinity chromatography.

Freshly collected human citrated blood was subjected to glycerol loading and hypotonic lysis to obtain lysed platelet suspension. Then, it was applied to the affinity chromatography and the fraction of plasma membrane vesicles was eluted by 0.2 M N-acetyl glucosamine. Electron micrograph of the fraction showed round membrane vesicles with some scattered intracellular organelles. Several marker enzymes were assayed in the fraction. No appreciable amount of β-glucuronidase or cytochrome c oxidase was detected in the fraction, indicating no contamination of mitochondria or α-granules. Relatively high activity of G-6-Pase was detected, suggesting possible contamination of endoplasmic reticulum. The yield was 11.6% in dry weight and 7.9% in protein.

By this method, the isolation was much faster than the centrifugal method and as low as 20 ml of human citrated whole blood may be used as starting material. Upon characterization of the plasma membrane fraction by electron microscopy and marker enzyme assays, the quality of the fraction was found comparable with the centrifugal method. The yield by this method was approximately two times higher than by the conventional method.
Histamine In Pig Platelet Dense Granules
Authors: Fukami M H , Ugurbil K , Holmsen H , Pakstis G L , Dangelmaier C A
Published: July 2018

In the course of high resolution nuclear magnetic resonance (n.m.r.) studies of dense granule storage complexes, an amine with two aromatic protons was observed in a proton n.m.r. spectrum of dense granules isolated from pig platelets. This amine was identified as histamine by the exact coincidence of the n.m.r. peaks of added histamine with the unknown peaks in the extract. The pH dependence of chemical shifts, paper chromatography and flurometric analysis after coupling with o-phthalaldehyde confirmed the identification. The concentration of histamine in isolated dense granules was about 700 nmol/mg of protein (n=3) or 1.6 times that of serotonin. In intact platelets, the histamine content was 11 nmol/mg compared to 7 nmol/mg of serotonin. The addition of 1 unit/ml of thrombin to suspensions of washed pig platelets resulted in the secretion of more than 90% of the histamine under conditions in which only 3.8% of thelactate dehydrogenase appeared extracellularly. These findings indicate that histamine is a major constituent of dense granules in pig platelets which can be released during hemostasis and thrombosis and may exert its pharmacological effects under these conditions.
The Infusion Study Of Neuraminidase: An Animal Model For The Study On Disialylation Of Platelet And Thrombosis
Authors: Meguro T , Yamada K , Miyaji Y , Inagaki M
Published: July 2018

In order to detect the influence of neuraminidase on thrombotic tendency, the infusion studies of neuraminidase were carried out in animal experiments. Recent studies in our laboratories has revealed higher activity of neuraminidase in plasma during acute stages of some inflammatory diseases in which thrombosis tended to be noticed with complications. New Zealand rabbit (N.Z.W.) were used for the animal experiments. Cl. Perfrigens type V or VI of neuraminidase (Sigma) was dissolved in saline and infused to the rabbits intravenously. In one group, one infusion of neuraminidase ranged from 0.5 U per kg to 1.0 U per kg per body weight was carried out. In other group, a continuous drip infusion with 2 U per kg of neuraminidase was given to the animal during 24 hours. In both groups, the blood specimens were obtained at every 24 hours and the examinations were sustained during more than 7 days after the infusions respectively. The neuraminidase activity of plasma was assayed by fluorescent method using synthetic substrate which was kindly made available by Dr. M. Flashner in Syracuse. The sialic acids of platelet was assayed by the method of M. A. Madoff, et al.

In the group of drip infusion, hyperfibrinogenemia and leukocytosis were observed in early stage, followed by thrombocytosis and acceleration of platelet aggregation, in accordance with decreased level of sialic acids of the platelet and increased level of free sialic acids in plasma. In the group of one infusion, most animals died with episodes of thrombocytopenia. Our results conclude that the drip infusion method devised by us would be useful and more applicable in the study of disialylation of platelet and hyperaggregablity and indicated the pathophysiology of some inflammatory diseases with thrombosis, more clearly compared with the one infusion technique.