bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction of Prothrombin with phospholipids monolayers In Presence of Ca++
Authors: Lecompte M , Miller I
Published: May 2019

The activation rate of prothrombin and its conversion into thrombin through a proteolytic degradation is enhanced by phospholipids in the presence of Ca++. In this work, we could elucidate stoechiometric and structural aspects of the interaction between prothrombin, phospholipids and Ca++. We studied the adsorption eguilibria of Ca++and of prothrombin on phospholipids monolayers by a new approach which combines two complementary methods of surface measurement.

Electrochemical investigations on lipid monolayer interacting with prothromoin showed penetration of prothrombin in presence or not of Ca++(by increasing the lipid layer capacitance), with a preferential orientation. The adsorption of Ca++and prothrombin to the lipid layer was measured by counting the surface radioactivity of 45Ca and 3H-prothrombin. Synergetic attachment of Ca++and prothrombin to the phospholipid water interface was observed and the number of Ca++per attached prothrombin was determined. The two methods give comparative results for the proposed ionic-hydrophobic interaction.
Investigation of the Catalytic Properties of the Dysthrombin, Thrombin Quick
Authors: Henriksen R , Owen W , Nesheim M , Mann K
Published: May 2019

Thrombin Quick (TQ) may be isolated following treatment of Prothrombin Quick [Owen, et al, Mayo Clinic Proceedings, 53: 29-33, (1978)] with Taipan venom, phospholipid and ca2+. The clotting activity of TQ with fibrinogen is 1/200 that of nornar thrombin (T). The activation of Factors V and VIII, and the aggregation of platelets by TQ occurs with an effectiveness of about 1/50 that of thrombin. when incubated with antithrombin III, both T ad TQ fom inhibitor complexes as determined by dodecylsulfate gel electropheresis. Titration of T and TQ with the fluorescent inhibitor dansylarginine-4-ethylpiperidine amide indicates an equivalent number of active sites based on protein absorption at 280 nm. However, the two enzymes may be distinquished by the decreased fluorescence enhancement observed with TQ relative to T, indicating an increased polarity in the inhibitor binding site of TQ. With the substrate benzoylarginine ethylester, TQ has a Km = 4.5 × 10-5M and kcat= 6.93 compared to Km = 4.0 × 10-5M and kcat= 17.7 for T. This indicates that the defect in TQ esterase activity is in the catalytic mechanism itself and not in substrate binding. The rate of inhibition of TQ by diisopropylphosphofluoridate is decreased. Decreased acylation and deacylation rates for TQ relative to T are observed for hydrolysis of the active site titrant 4-methykl-umbelliferyl-p-guanidinobenzoate
Acquired Factor X (Stuart Factor) Deficiency in a Patient with Mycoplasma Pneumonial Infection
Authors: Peuscher F , van Aken W , Swaak A , Sie L , Statius van Eps L
Published: May 2019

An isolated deficiency of factor X is known to occur in a hereditary form, the Stuart-Prower disease, and in an acquired form in some patients with para-proteinaemia and sporadically in systemic amyloidosis. Transient deficiency of factor X in the presence of normal levels of factors II, VII and V appears to be rare. In the literature, only three cases have been described. We have studied a patient with a severe haemorrhagic diathesis and concomitant mycoplasma pneumonial infection. The bleeding tendency proved to be due to isolated factor X deficiency. No circulating inhibitors of factor X were present. Systemic amyloidosis could not be demonstrated. Factor X-related antigen could not be detected (this test was kindly performed by Dr.Daryl S.Fair, Scripps Clinic and Research Foundation, La Jolla, U.S.A.). Treatment with vitamin K, prothrombin complex concentrate, fresh plasma and whole blood proved not to influence factor X activity in the patient’s plasma. However, 20 days after admission⋅to hospital both factor X activity and antigen spontaneously returned to normal. These results suggest that the synthesis of factor X was transiently defective. Since other conditions known to affect factor X activity could not be demonstrated, it is postulated that the acquired factor X deficiency in this patient was related to the infection with mycoplasma pneumoniae.
Sulfinpyrazone or Acetylsalicylic Acid to Prevent Postoperative Thrombus Formation in Arteriovenous Fistulas of Haemodialysis Patients
Authors: Albert F , Schmidt U
Published: May 2019

The effect of sulfinpyrazone (200 mg three times a day) and acetylsalicylic acid (500 mg three times a day) on the incidence of thrombosis of arteriovenous shunts was investigated in a controlled clinical trial. In 36 patients with chronic renal failure scheduled to begin haemodialysis the same operating team constructed a subcutaneous fistula in the distal forearm. During the first six weeks after the operation the antithrombotic efficacy proved to be good for both substances. No differences of thrombotic events between the two treatment groups were statistically significant. But in contrast to acetylsalicylic acid sulfinpyrazone made no significant inhibition of platelet - aggregation; sulfinpyrazone probably will prevent the clot formation by prolonging the shortened platelet survival in uraemic patients. In a high rate of patients given acetylsalicylic acid (10 out of 17) there were local bleeding and gastrointestinal side effects. In consequence we should prefer sulfinpyrazone, because in the sulfinpyrazone group side effects were minimal and in none patient withdrawal from the study was necessitated.
Relative reactivity of platelets towards nonendothelialized surfaces
Authors: Hanson S , Harker L , Ratner B , Hoffman A
Published: May 2019

Rates of platelet consumption (PC) by subendothelium, vascular grafts and artificial surfaces have been measured in nonhuman primates. Studies included 1) 18 monkeys (M. nemestrina)with balloon catheter deendothelialization of 10 cm or 20 cm segments of the aorta, 2) 24 baboons with Dacron velour (USCI Sauvage) aortic grafts, and 3) 113 baboons with femoral A-V cannulae. In all studies PC was directly proportional (r =0.935) to the area of nonendothelialized surface. Platelet-surface reactivities(plats/cm2/day × 10-8± 1 SD) were: subendothelium, 176 ± 35; aortic grafts, 42 ± 9; cannulae 1 ± 1 to 27 ± 3. Whereas serial measurements of PC by cannulae were unchanged, PC by subendothelium and aortic grafts decreased progressively over 3-5 weeks as a result of reendothelialization. PC by eight acrylic polymer cannulae ranged from 4.4 ± 1.5 to 27.2 ± 2.7 (54 studies). PC by five commercial polyurethanes ranged from 2.7 ± 1.0 to 24.3 ± 0.8 (25 studies). PC by Teflon, Tygon, Silastic and polyethylene was less than 2.5 (34 studies). Cannula PC was independent of variable blood flow rates, circulating platelet concentrations, or heparin anticoagulation but correlated directly with surface polarity (as determined from surface spectroscopy and water sorption measurements). 125I-fibrinogen kinetics were normal. Cannula PC was completely interrupted by dipyridamole (10 mg/kg/day), sulfinpyrazone (100 mg/kg/day) but not by ASA (100 mg/kg/day). We conclude that platelet kinetic measurements provide an objective in vivo measure of the reactivity of nonendothelialized surfaces and the efficacy of antithrombotic therapy.
Solution Studies Of Bovine Prothrombin Fragments Using High Resolution Nuclear Magnetic Resonance Spectroscopy
Authors: Esnouf M , Israel E , Pluck N , Williams R
Published: May 2019

High resolution nuclear magnetic resonance spectroscopy (NMR) is now a widely used technique for investigating the solution structures of proteins. In particular, proton NMR provides information on the entire molecular structure and is not confined to the study of a few amino acid side chains with specific properties. We have used this technique (at 270 MHz) to investigate the solution behaviour of bovine prothrombin fragment 1. Large variations in temperature and pH are seen to have little or no effect on its spectrum. Indeed, the spectra are all very similar to those expected for a “random coil” protein. The protein has little tertiary structure. Calcium binding has been monitored and small changes involving tryptophan intensity have been seen in agreement with the results of other physical methods. However, NMR shows there to be no significant change in tertiary structure in fragment 1 on binding calcium. Prothrombin fragment 2 contains the second ,Kringle region - a large amount of its sequence being homologous to that in fragment 1. Preliminary experiments have shown bovine fragment 2 to have some degree of folded structure but the temperature dependence and chemical modification studies suggest that this is not very extensive.
A New Radioimmunoassay-Technique For Fibrinopeptide-A In Human Plasma
Authors: Harenberg J , Zimmermann R , Haas F , Schmidt-Gayk H
Published: May 2019

Fibrinopeptide-A (FpA) is thought to be the most sensitive parameter to indicate hypercoagulability with increased fibrin formation in man. Previously described radioimmunoassays for FpA are very time consuming. We present therefore a modification of the assay, which is less time consuming, sensitive, reproducable and reliable. The following modifications were made on the original method: 1. plasma dialysis was omitted 2. performance on microtiter-plates 3. double antibody method.

The sensitivity was improved to 0.16 ng FpA / ml plasma. The mean concentration of FpA in dialysed and undialysed plasma did not differ significantly (p<0.001). Effective separation of fibrinogen was achieved by aethanol extraction alone. The mean concentration of FpA correlated highly, when the tracer was separated by charcoal or second antibody (r = 0.96). The recovery was improved to 85%.

In healthy persons 0.16-2.5 ng FpA/ml plasma were measured. In patients with venous thrombosis or myocardial infarction in the history FpA was elevated significantly to 2.0 ng/ml-19.6 ng/ml, indicating that the FpA is a usefull tool in diagnosis of hypercoagulability.
Pharmacological distinction between sulphinpyrazone and inhibitors of Prostaglandin Synthesis
Authors: Butler K , Smith J , White A
Published: May 2019

Injection (i.v.) of 0.5 ml anti-Forssman antiserum to guinea pigs resulted in an irreversible thrombocytopenia (95%) but a transient thrombocytopenia (50%) ensued after 0.2 ml which was complement dependent. During the onset of the thrombocytopenia there was marked platelet sequestration in the vasculature of the lung which was reversed when the circulating platelet count returned to normal. This sequestration was associated with a biphasic bronchospasm which was both platelet and complement dependent. Sulphinpyrazone (100 mg/kg i.v.) administered 30 or 60 min before the antiserum inhibited the thrombocytopenia by 59% and 100% respectively, as well as the platelet sequestration in the lung and the bronchospasm. Aspirin (100 mg/kg i.v.) and indomethacin (10 mg/kg i.v.) inhibited the bronchospasm but not the thrombocytopenia, while phenylbutazone was inactive in all cases. This model thus distinguishes between sulphinpyrazone and the other inhibitors of prostaglandin synthesis.
Studies on Nature of So-Called Antiplasmin Measured By Chromogenic Substrate S-2251 and Its Automatic Determination With ABA 100
Authors: Kato M , Fujimaki M , Fukutake K , Maki A , Hibino M
Published: May 2019

Using chromogenic substrate S-2251, authors studied on the nature of so-called antiplasmin and on the application of Coatest to automatic determination. As to several protease inhibitors, correlation performed vs single radial immunodiffusion yielded a regression of Y=19.5X-18.6 and r=0.91 for α2PI ;r=-0.15, 0.13, 0.46 0.28 for α2M, α2AT, AT-III and CIINA respectively. Purified protease inhibitors except α2PI did not exhibit immediate inhibition to human plasmin. The automation of chromogenic substrate method was studied with ABA Bichromatic Analyser Model 100 and auxiliary dispenser. 25 microliter of diluted plasma and 250 microliter of plasmin of known activity were added simultaneously to 100 microliter of substrate S-2251. Reaction rate was assayed with the use of two wavelengths, 450/415. Excellent correlation with manual protocol was observed, r=0.985 and Y=0.88X=8.7. Determination of within-day precision of automatic assay for three sample concentrations (Antiplasmin 25%, 100%, 150%) gave C.V. of 1.5%, 1.71% and 4.46% respectively. Thus, authors arrived at the conclusion that 1) α2PI is mainly determined as protesae inhibitor with the use of S-2251, 2) ABA 100 was successfully employed for automatic determination of antiplasmin with the use of simultaneous addition of plasma and plasmin to substrate solution.
A New Automatic Instrument to Investigate the Rheological Behaviour of Red Blood Cells
Authors: Teitel P , Mussler K
Published: May 2019

Based on the principles already described (Blood Cells, 1977, 3, 55) a completely automatic instrument has been constructed for the investigation of the flow behaviour of packed red blood cells at low shear stresses (below 1 Pa). Functioning as a simple polymicroviscometer the instrument features : (1) four cylindrical sample holders with filter mountings, (2) four opto-electronic level measuring devices, (3) a microprocessor controlling the functions of the whole instrument (including the successive scan of the samples in discrete steps), (4) a digital display of the actual sample level in each holder, (5) a memory unit to store the scanning results and (6) a logarithmic chip. At the end of the investigation the data stored can be retrieved either in analog or in digital form. The analog retrieval over a potentiometric recorder generates a "filtration curve" on which the type of flow behaviour (Newtonian or more or less non-Newtonian) can be directly visualized. The digital data retrieved over a printer can be fed into a computer to calculate the equation of the flow curve. The instrument can provide information either on the forces of aggregation between individual erythrocytes or on their rigidity (washed RBC packed in buffered isotonic saline containing 1 % human albumin). These information might help illuminate the contribution of RBC rheological factors in the pathogenesis of vascular occlusion.
Red Cell Filterability and Cardiovascular Risk Factors
Authors: Lagrue G , Marcel G , Faucher G , Grumel J , Beiler D , Branellec A
Published: May 2019

Red cell deformability, which allows cells of 7 µ diameter to flow through capillaries not larger than 3 µ, can be approached by the measure of blood filterability on Nuclepore 5 µ filters (Reid H.L. et al. J. Clin. Pathol. 1976 ; 29 : 855). Filterability seems reduced in certain states predisposing to thrombosis. We have, in 72 patients, correlated red cell filterability (using two batches of filters) expressed in µl/s, with the number of cardiovascular risk factors present (high blood pressure, overweight, diabetes, hyperuricemia, hyperlipemia, smoking). Statistical analysis has been performed using regression analysis for each batch of filters, and both significance levels have been combined using Fisher's method. The table illustrates the relationship between the number of risk factors and filterability. There is a statistical difference between groups as a whole (p<0.01). The difference is highly significant between 0 and 4 risk factors (p<0.0005). Filterability decrease is also directly correlated with the number of cigarettes smoked per day (p<0.05). This is significant in men (p<0.05), but not in women.
Affinity Chromatography of Factor VIII Subunits
Authors: Lane J. L. , Ekert H.
Published: May 2019

Gel filtration of factor VIII resulted in elution of a fraction which possessed procoagulant activity (VIII:C), ristocetin cofactor (VIIIrRCF) and antigenic activity (VIII:Rag). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of this preparation showed that no protein entered the gel. Eight bands ranging from 30,000 to 230,000 molecular weight (mw) were seen when 400μg reduced protein was loaded onto the SDS-PA geL Attempts to assay the functional properties were made in order to investigate whether the multiband structure was a property of factor VIII or due to contaminants. These proved unsuccessful, as reduction and SDS-PAGE of factor VIII destroyed its functional properties. Affinity chromatography with human and rabbit antibodies to factor VIII provided an alternative method of relating the subunit structure to functional properties. Insolubilized human antibody inactivated VIII:C and did not bind VIII:Rag. The protein bound to human antibody was dissociated with NH SCN and reduced. SDS-PAGE showed heavy protein staining, with 95% of the protein present in 35,000 and 62,000 mw bands. Similar treatment of a control human IgG column showed minimal protein in the same mw region. Using insolubilized rabbit antibody to human factor VIII, all 8 bands were observed after N^SCN dissociation, reduction and SDS-PAGE. These results suggest that contaminat^ely that the low bands observed on heavily loaded gels are due to protein contamination.
Generation of Factor VIII :C By Isolated Pig and Rat Livers
Authors: Owen C. A. , Walter Bowie E. J. , Fass D. N.
Published: May 2019

When isolated neonatal pig livers were perfused with blood from pigs with von Willebrand’s disease, no factor VIII coagulant activity (VIII:C) emerged in the perfusate. When purified porcine Willebrand factor, lacking VIII:C, was added to the perfusate it rapidly diminished, presumably being taken up by the liver. Concurrently VIII:C rose steadily in the blood during the 5 hours of perfusion. Similarly, isolated adult rat livers released no VIII:C when they were perfused with rat erythrocytes suspended in Tyrode’s solution containing 6% bovine serum albumin. VIII:C did emerge if cryoprecipitate from aged rat plasma were added. The neo-VIII:C was rapidly inactivated by plasma from a patient with classic hemophilia who had developed an antibody to VIII:C Addition of cycloheximide to the rat before extirpation of the liver, as well as to the perfusate, prevented the emergence of VIII:C. We suspect that VIII:C is derived from the liver but only when Willebrand factor is present.
Potential Sources of Emboli in Transient Cerebral and Retinal Ischaemia
Authors: de Bono D. , Warlow C. , Hyman N.
Published: May 2019

91 consecutive patients with transient ischaemic attacks (TIAs) were examined for potential source of emboli using echo cardiography and ambulatory ECG monitoring in addition to clinical examination and angiography. Patients were divided into those with amaurosis fugax and/or carotid TIAs (45),“hemiphenomena” TIAs (26) and vertebrobasilar TIAs (20).

“Structural” cardiac defects (of which mitral valve prolapse accounted for just under half) occurred in 34% of patients, significantly more than in age-matched controls. 40% of patients with amaurosis fugax and/or carotid TIAs had carotid artery lesions, as did 30% of those with “hemiphenomena” attacks. 6 patients had both cardiac and carotid lesions. Arrhythmias were detected in 15% of patients and a similar proportion of controls. Hypertension and ischaemic heart disease were more common in the patients.

Structural cardiac abnormalities, which may not be haemodynamically significant, might account for cerebral and retinal emboli in about a third of patients presenting with TIAs. The natural history and the most effective therapy of this subgroup require evaluation independently of patients with predominantly carotid atheromatous lesions.
An Assay for Activators of Platelet Adenylate Cyclase Present in Rabbit Blood:Evidence That (PGI2) Is Not A Circulating Hormone
Authors: Haslam R. J. , McClenaghan M. D.
Published: May 2019

Measurement of cyclic [3 H]AMP in washed rabbit platelets labelled by preincubation with [3H]adenine permitted the detection and assay of activators of platelet adenylate cyclase (e.g. PGI2). Cyclic [3H]AMP was extracted from the platelets with TCA and isolated by chromatography on neutral alumina and Dowex 50. in 30 s incubations at 37°C, increases in platelet cyclic [3H]AMP were detected with > 0.4 nM PGI2 in the absence of a cyclic AMP phosphodiesterase inhibitor and with > 0.04 nM PGI2 in the presence of 1 mM 3-isobutyl- 1-methylxanthine (MIX). Log/log plots of the increases in cyclic [3H]AMP in the presence of MIX were linear with 0.04-4.0 nM PGI2. Unlike bioassays for PGI2 based on inhibition of platelet aggregation, this assay could be applied to whole blood. Mixtures of 0.4 ml citrated rabbit blood, 0.04 ml labelled platelet suspension (108 platelets; 1 μCi H) and 0.06 ml of other additions including MIX were incubated for 30 s at 37°C. The same values for platelet cyclic [3H]AMP were obtained with arterial blood assayed 30 s or 30 min after its removal from rabbits, indicating the absence of detectable PGI2 which had a half-life of about 7 min in blood at 37°C. The effects of different concentrations of added PGI2 on the cyclic [3H]AMP in citrated rabbit arterial blood containing labelled platelets showed that fresh blood contained less than 0.1 pmol PGI2/ml. as > 0.4 nM PGI2 was required to inhibit aggregation of rabbit platelets in plasma-containing systems, the results suggest that the circulating concentration of PGI2 in rabbits is too low to affect platelet function.
Prostaglandins (PG) Inhibit Human Platelet Aggregation By PG Receptor Antagonism as well as Adenylate Cyclase Stimulation
Authors: Macfarlane D. E. , Salzman E. W.
Published: May 2019

11-Deoxy and/or 15 or 16 alkyl substitution confer platelet aggregating activity to bis-enoic PG’s: e.g., ll-deoxy-PGE2(threshold (T)-5μM); ll-deoxy-15(S)-15-methyl-PGE2(T= O.lμM); ll-deoxy-15(S)-16-methyl-PGE2(T=0.1μM). Responses induced by such PG’s mimic those induced by PGH2 and PGH2 analogues (e.g., U44069). N0164 competitively inhibits aggregation induced by PG’s and suppresses “secondary” responses induced by low concentrations of ADP or arachidonic acid, suggesting involvement of a specific PG stimulatory receptor in platelet aggregation. We compared non-aggregatory PG’s as inhibitors of primary aggregation induced by ADP or U44069. PG’s containing 11-deoxy and/or 16-alkyl groups selectively inhibited aggregation induced by U44069. Mean I50 values (n=4) against ADP and U44069 respectively were PGA2(>100μM; 0.3μM); PGB2(>100μM; 5μM). PGE1(60μM; 2μM); PGE1(10nM; 5nM); 11-deoxy PGEX (60μM; 2μM); ll-deoxy-15-(R)-16-methyl PGE1(lOOμM; 2μM); 13,14-dihydro-16-methyl-PGE2 methyl ester (750nM; 15nM). A low ADP: U4406 I50 ratio (e.g., PGD2) indicates that inhibition is mainly due to adenylate cyclase stim ulation, and a high ratio (e.g., PGA2) that inhibition is mainly due to PG receptor antagonism. We have demonstrated a PG stimulatory receptor on the platelet, and its involvement in “secondary”aggregation. PG’s inhibit aggregation by combining with this receptor and/or by stimulating adenylate cyclase.
Soluble Fibrin in Plasma At 37°C Exists as Des-A Fibrin in Monomeric Form
Authors: Müller-Berghaus G. , Mahn I. , Krell W. , Kröhnke G.
Published: May 2019

We intended to find out in which state fibrin exists in plasma at 37°C. in order to generate fibrin, small amounts of thrombin were added to human plasma containing 125I-fibrinogen. Thereafter, thrombin was quenched with hirudin and 131I-fibrinogen added to observe possible 125I-fibrin-131I-fibrinogen complex formation. Gel filtration of this mixture on sepharose columns equilibrated with plasma revealed 2 peaks. At 20°C, but not at 37°C, peak A eluted in front of the fibrinogen peak contained fibrin-fibrinogen complexes. If isolated fractions of peak A eluted at 20°C were rechromatographed at 37°C, the material was mainly eluted in peak B (= fibrinogen peak). If isolated fractions of peak B eluted at 37°C were rechromatographed at 20°C, part of the 125I-radioactivity representing fibrin was eluted in peak A. as purified des-AB fibrin is eluted in polymeric form at 37°C and as purified des-A fibrin is eluted only in monomeric form at 37°C, we conclude from our experiments that fibrin generating in plasma at 37°c exists in monomeric form. Furthermore, the experiments demonstrate that des-A fibrin does not form complexes with fibrinogen or other proteins at 37°C. (Supported by the DFG).
Isolation and Partial Characterization of A Novel Circulating Antithrombin
Authors: Messmore Harry , Parvez Zaheer , Fareed Jawed
Published: May 2019

We have previously reported on a novel circulating anticoagulant (Clin. Res. 22:396 A, 1974. Thromb. and Haem. 38:77, 1977 (abstr) in a 42 year old female who has had repeated episodes of life threatening hemorrhage since the age of 3. Our preliminary studies showed it to be a protein with some of the properties of a heparin activated antithrombin III. This report is on additional studies to further characterize the inhibitor.

The patient’s plasma was fractionated on Sephadex G-200, and the fraction showing immediate acting antithrombin activity was further purified on heparin-sepharose and conconavalin A-sepharose affinity columns. The patient’s inhibitor did not bind to heparin sepharose, and was thus separable from her antithrombin III. It did bind to conconavalin A, and was eluted from the conconavalin A with α - D (+) methylglucoside.

It has very broad serine protease inhibitor activity, blocking the hydrolysis of synthetic chromogenic substrates by thrombin, factor Xa, plasmin and trypsin. It did not inhibit Reptilase.

Immunochemical assays show it to be α1 antitrypsin. Isoelectric focusing shows it to be a variant of normal, with its isoelectric point being different from a normal control, and pure α1 antitrypsin (commercial, human).

The total α1 antitrypsin level in this patient is about 50% of normal, and it has potent immediate acting antithrombin activity. It appears to be similar to a phenotype previously reported as Antithrombin Pittsburgh (Blood 51:129, 1978).
F.VIII R:Ag in Hepatitis
Authors: Kotitschke R. , Scharrer J.
Published: May 2019

F.VIII R:Ag was determined by quantitative immunelectrophoresis (I.E.) with a prefabricated system. The prefabricated system consists of a monospecific f.VIII rabbit antiserum in agarose on a plastic plate for the one and two dimensional immunelectrophoresis. The lognormal distribution of the f.VIII R:Ag concentration in the normal population was confirmed (for n=70 the f.VIII R:Ag in % of normal is = 95.4 ± 31.9). Among the normal population there was no significant difference between blood donors (one blood donation in 8 weeks; for n=43 the f.VIII R:Ag in % of normal is = 95.9 ± 34.0) and non blood donors (n=27;f.VIII R:Ag = 94.6 ± 28.4 %). The f.VIII R:Ag concentration in acute hepatitis B ranged from normal to raised values (for n=10, a factor of 1.8 times of normal was found) and was normal again after health recovery (n=10, the factor was 1.0). in chronic hepatitis the f.VIII R:Ag concentration was raised in the majority of the cases (for n=10, the factor was 3.8). Out of 22 carrier sera 20 showed reduced, 2 elevated levels of the f.VIII R:Ag concentration. in 5 sera no f.VIII R:Ag could be demonstrated. The f.VIII R:Ag concentration was normal for n=10, reduced for n=20 and elevated for n=6 in non A-non B hepatitis (n=36). Contrary to results found in the literature no difference in the electrophoretic mobility of the f.VIII R:Ag was found between hepatitis patients sera and normal sera.
Antithrombin III Deficiency in A Japanese Family
Authors: Nakagawa M. , Kawamura T. , Tsuji H. , Okajima Y. , Urano S. , Kitani T. , Iiichi H.
Published: May 2019

Antithrombin IEC has been reported to be decreased in the cases of several thrombotic disorders and the decreased Antithrombin III is known to induce the hypercoagulable state. This study was started from the 28 year-old male patient who developed the superior sagital sinus thrombosis after appendectomy and it was followed by deep vein thrombosis of extremities. Antithrombin III level was 19 mg/dl and activity was 68% by the progressive antithrombin assay and other laboratory examinations were within normal range excepts for the elevated serum lipids. Antithrombin III was assayed for his family members in three consecutive generations by single radial immunodifusion method, coagulation assay, and chromogenic assay. Four out of eight members were comfirmed to have low Antithrombin III level and activity ranging from 59%-68% of normal values, although the two of these four members bad no history of thrombosis. Mother of this propositus is deceased, but it was suspected^of having the defect of Antithrombin III. History of peptic ulcers were found in all members of this family. The inheritance pattern of Antithrombin III deficiency was characteristic of an autosomal dominant disorder.
Storage Pool Disease: Evidence for Clinical and Biochemical Heterogeneity
Authors: Weiss H. J. , Lages B. A. , Witte L. D. , Kaplan K. L. , Goodman DeW S. , Nossel H. L. , Baumgartner H. R. , Hoffmann-LaRoche F.
Published: May 2019

Patients with platelet storage pool disease have decreased numbers and contents (ATP, ADP, serotonin, calcium) of the dense granules. Our studies on 14 patients with this disorder suggest considerable clinical and biochemical heterogeneity. The most pronounced dense granule defect (lowest levels of ATP and ADP, undetectable serotonin) was found in the 5 albinos with the Hermansky-Pudlak syndrome. One non-albino patient is unique in showing a decreased content of β thromboglobulin (βTG), platelet factor 4 (PF4), and the platelet growth factor (PtGF) that stimulated the proliferation of cultured fibroblasts and arterial smooth muscle cells. Her platelets also contained a decreased number of α granules in addition to decreased dense granules, suggesting that βTG, PF4, and PtGF are localized in specific α granules which are morphogenetically related to dense granules. Another patient was unique in that ADP, epinephrine, and arachidonic acid evoked completely normal aggregation responses, associated with normal production of platelet malondialdehyde. Since his platelets were markedly deficient in ADP, these findings provide further evidence that ADP release is not an absolute requirement for ‘second phase’ aggregation and that PGG2 or thromboxane A2 may directly aggregate platelets independent of ADP release. Variable defects in malondialdehyde production in other patients suggest further heterogeneity of the release defect in storage pool disease.
Defective Ristocetin Aggregation Not Attributable to a Von Willebrand or Bernard-Soulier Type Defect
Authors: Wautier J. L. , Nurden A. T. , Michel H. , Caen J. P.
Published: May 2019

In von Willebrand’s disease the absence of platelet aggregation by ristocetin has been correlated with abnormalities in the von Willebrand factor (VIIIVWF), while in the Bernard-Soulier syndrome (BSS) a platelet membrane defect involving surface glycoproteins has been reported. During a 3 year period an absence of platelet aggregation induced by ristocetin was observed in a 53 year old Caucasian man with enlarged spleen, normal platelet count and eosinophilic leukaemia. The platelets react normally with collagen and ADP but the aggregation induced by thrombin and bovine VIIIVWF were reduced. The patient’s platelets were well agglutinated by an antibody reacting with a component absent in the BSS. The bleeding time, VIII levels, platelet adhesion to subendothelium (rabbit aorta) were normal. No abnormalities were detected in the surface glycoproteins as studied by SDS Polyacrylamide gel electrophoresis however the platelet sialic acid content was slightly reduced. It is concluded that an abnormality additional to those previously described may be the course of the defective ristocetin induced platelet aggregation in this patient.
A Rapid and Simple Method for Separation and Analysis of Pure Antihemophilic Factor from Plasma
Authors: Shulman N. R. , Tack K. M.
Published: May 2019

Current methods of purifying antihemophilic factor (AHF) are complicated and lengthy, leading to marked loss of factor VIII (f VIII) activity despite low temperature processing. Uncertainties concerning denaturation or cryo-aggregation hinder clear interpretation of relationships between the f VIII and von Willebrand factor (vWf) activities of AHF, some suggesting the two activities exist on one molecule, others that two molecules are complexed.

Our method utilizes a controlled-pore glass column of pore-size approximately 1000Å. AHF separates as an isolated peak in the void volume while all other proteins including cold insoluble globulin and fibrinogen are retained. We obtain pure AHF, judged by SDS acrylamide gel and immunologic criteria, from large volumes of citrated plasma by a single filtration within one hour of obtaining blood. Processing entirely at 37° or 22° gives the same results. Yields are 40–60% of plasma f VIII activity.

Increasing ionic strength (μ) of plasma to >0.3 progressively dissociates vWf and f VIII up to μ = 0.75, vWf remaining at MW >106 while f VIII is retained by pores as small as 330Å, indicating a MW of approximately 1.5 x 105. Human anti-AHF reacts exclusively with separated f VIII, not with vWf; and rabbit anti-AHF reacts primarily with vWf. Findings on normal and hemophilic plasma support the concept that f VIII and vWf are distinct components circulating as a weakly associated complex, and that hemophiliacs lack f VIII by functional and immunologic criteria.

Preparation of pure AHF by this technique on a scale appropriate for clinical use is feasible.
On the Primary Structure of Human Fibrinogen
Authors: Henschen A. , Lottspeich F. , Töpfer-Petersen E. , Warbinek R.
Published: May 2019

The aim of the present investigation is to elucidate the primary structure of human fibrinogen. Through the work of several laboratories including our own large parts of the structure are now known. Here will be reported the complete amino acid sequence of the γ-chain (409 residues). Furthermore, the carbohydrate linkage site in the β-chain and plasmin cleavage sites in the β- and γ-chains have been identified.

The peptide chains were isolated by CM-cellulose chromatography following mercaptolysis and alkylation. The γ-chain was cleaved in a way to produce large fragments suitable for automatic sequencing, e. g. with cyanogen bromide or trypsin after citraconylation. The sequences of the isolated fragments allowed reconstruction of the complete sequence of the γ-chain.

The carbohydrate linkage site in the β-chain could be isolated by affinity chromatography on concanavalin A-agarose following cleavage of the chain by trypsin or cyanogen bromide. The sequence of 21 amino acid residues around the carbohydrate attachment site was determined.

The plasmin cleavage site giving rise to N-terminal glycine in the γ-chain D-fragment was identified. The amino acid sequence linking plasmic fragments E and D in the β-chain was determined.
Factor VIII-Like Activity of Basic Amphoteric Polyelectrolytes
Authors: Exner T. , Rickard K. A. , Kronenberg H.
Published: May 2019

The broad-range Ampholine (LKB) 3.5–10 containing a mixture of materials with isoelectric points between pH 3.5 and 10 was found to correct the prolonged PTTK of factor VIII-deficient plasma, and this effect was investigated. Ampholines which are used as carrier ampholytes or pH gradient stabilizers in isoelectric focussing are condensation products of propionic acid and polyethylene polyamines.

The most effective of the commercial Ampholine mixtures were found to be those with the most alkaline isoelectric points. Ampholine 9–11 at 0.2% concentration was found to have factor VIII and factor IX activity equal to that of normal plasma as well as somewhat less activity as factor XI. It had no significant correcting effect on plasmas deficient in factors V, VII, X, II or XII. In contrast to polyglutamic and polyaspartic acids which have also been reported to correct for factor VIII deficiency in the PTTK, Ampholine 9–11 was effective whether added before or after kaolin contact activation of the test plasma. However, Ampholine 9–11 inhibited contact activation of plasma in the absence of kaolin and showed less correcting activity in partial thromboplastin time tests utilizing activators other than kaolin.
Cross-Linking of Factor VIII by Dimethyl Suberimidate: Structural Implications
Authors: Cockburn C. G.
Published: May 2019

A dilute solution of highly purified human FVIII was cross-linked by dimethyl suberimidate (negligible intermolecular cross-linking), incubated in 0.2m mercaptoethanol for 35 min. At 37 c, and eluted through a sepharose 4b column. Unreduced SDS-polyacrylamide gel electrophoresis (3.75%:1%) of the material applied to the column showed 6 clear bands believed to represent cross-linked FVIII oligomers (on the grounds that electrophoretic mobility was directly proportional to log oligomer number). The staining intensity of the bands was roughly as follows:

(FVIII)1 ≏ (FVIII)2 > (FVIII)3 ≏ (FVIII)4 >(FVIII)5 ≏ (FVIII)6

This indicates that a single FVIII subunit is in intimate proximity with up to 5 other subunits and suggests large areas of close contact between subunits.

After cross-linking virtually all the FVIII RAg was lost, but a little FVIIIC (4% yield) eluted at the void volume (Vo) of the sepharose column. No detectable protein in the Vo fraction penetrated very large pore size sds-polyacrylamide gels, indicating a very high molecular weight cross-linked structure.
Ionophoretic Action of Phospholipids on Human Platelets
Authors: Ikeda Y. , Kikuchi M. , Watanabe K. , Ando Y. , Hasegawa M.
Published: May 2019

The effects of phospholipids on calcium uptake by platelets were examined by using 45CaCl2. Platelet suspensions(3 ̴ 5 × 108/ml) were prepared in PIPES buffer at pH 6.8 and were incubated with 0.2 mM 45CaCl2(10 µCi/ml) in the presence or absence of various kind of phospholipids. The mixtures were centrifuged through silicone oil for 1 min. at 7,000g to separate the pellet. The radioactivity in the pellet was measured with liquid scintillation spectrometer.

Among the phospholipids tested, cardiolipin and phosphatidic acid exerted a significant increase of calcium uptake. The amount of calcium translocated into platelets after 60 min. incubation with cardiolipin, phosphatidic acid, sphingomyelin and phosphatidylcholine (0.4 mM) was 2.58, 1.33, 0.30 and 0.32 n mol/109plts, respsctively, while that without phospholipids was 0.31 n mol/109plts. For the sake of comparison, the effect of A23187 or thrombin on calcium uptake was examined. The amount of calcium taken up in the presence of 1 μM A23187 and 1 u/ml of thrombin was 1.84 and 1.33 n mol/109plts, respectively. Calcium uptake was dependent on cardiolipin concentrations and incubation time. LaCl3(1 μM) completely inhibited cardiolipin or A23187 induced calcium influx. However, when platelets were initially incubated with 45CaCl2 for 60 min. in the presence of 0.4 mM cardiolipin followed by addition of 1 μM LaCl3, 50 % of total calcium translocated still remained in platelets indicating that cardiolipin exerted an increase of calcium uptake into both lanthanum-displaceable and non-displaceable pool. Both cardiolipin (0.4 mM) and A23187(1 μM) induced a similar enhancement of platelet aggregation by ADP(2 μM) or epinephrine(1 μg) . LaCl3(1 mM) also inhibited such enhanced aggregabilities. The results suggest that phospholipids may potentiate platelet aggregabilities by their ionophoretic activities.
The Mechanism of Activation of Human Plasminogen by Urokinase
Authors: Castellino Francis J. , Violand Bernard N.
Published: May 2019

When native human plasminogen (Glu-Pg) is activated to plasmin by urokinase (UK), at least two peptide bonds are cleaved in the process. Cleavage of one bond (I), located in the interior of the molecule, results in formation of the well known, two polypeptide chain plasmin; in which the chains are stabilized by a single disulfide bond. Cleavage of another bond (II), located at the original amino terminus of native Glu-Pg, results in covalent release of a peptide (P), of molecular weight ca. 8,000. The final plasmin obtained (Lys-Pm), is a molecule in which both bonds are cleaved. An intermediate plasminogen (Lys-Pg), in which only II is cleaved, and an intermediate plasmin (Glu-Pm), in which only I is cleaved, can be isolated under appropriate conditions. Our studies indicate that the activator, UK, is not capable of catalysis of bond II cleavage in either Glu-Pg, Glu-Pm, or the native plasmin heavy chain (Glu-H), isolated from Glu-Pm. On the other hand, plasmin readily accomplishes cleavage of II from all of the above molecules. Bond I cleavage is catalyzed only by UK, in this activation system. Physical and kinetic studies leads us to propose a mechanism for Glu-Pg activation by UK involving the following steps: (1), an initial UK catalyzed cleavage of I, forming Glu-Pm; (2), a Glu-Pm catalyzed cleavage of II, in an autocatalytic step, forming Lys-Pm; (3), a Glu-Pm or, more likely, Lys-Pm catalyzed cleavage of II from the remaining Glu-Pg, forming Lys-Pg; (4), a UK catalyzed cleavage of I in Lys-Pg, forming Lys-Pm. (Supported by grants from the N. I.H. and A. H.A.).
von Willebrand Activity and Electrophoretic Mobility of Platelet Factor VIII Related Antigen in Patients with von Willebrand’s Disease
Authors: Sultan Y. , Lamazière J. , Maisonneuve P.
Published: May 2019

We have previously shown that patients with severe Von Willebrand’s disease (VWd), mainly homozygotes had no factor VIII related antigen (F VIIIRA) in their platelets as well as no Von Willebrand activity (F VIIIVW) as measured by the ristocetin cofactor assay. In all other patients with less severe form of the disease platelet F VIIIRA was detectable. In normal controls electrophoretic mobility of platelet F VIIIRA was found to be identical to the mobility of plasma VIII-RA either on the total platelet lysate or after isolation on sepharose 4B columns.

In the present study F VIIIVW and the electrophoretic mobility of F VIIIRA were compared in platelet extracts and the plasma of a number of patients. Seven of the ten patients tested were genetic variants of vwd with very low ratio of F VIIIVW/F VIIIRA suggesting a non functional protein with an increased electrophoretic mobility : one group of patients showed abnormalities of plateletF VIIIRA identical to those found in the plasma. However another group showed a high ratio of F VIIIVW/F VIIIRA suggesting a more active protein than the one in patient’s plasma although the platelet F VIIIRA had an increased electrophoretic mobility. These results are the first example of a F VIIIRA with abnormal electrophoretic mobility associated to high F VIIIVW activity. The loss of F VIIIVW in the patient’s plasma would be a secondary process occuring after F VIIIRA is released in the circulation.
The Action of Immobilized Thrombin on Factor VIII
Authors: Brown J. E. , Carton C. , Hougie C.
Published: May 2019

The utility of immobilized enzymes in blood coagulation studies is readily apparent. An enzymatic transformation can be stopped by centrifugation and removal of the enzyme beads. This could leave an “activated” factor free of the contaminating “activator”. The further degradation which prolonged exposure to the enzyme often produces can thus easily be stopped. The action of immobilized thrombin on factor VIII warranted testing, since we have observed that hirudin added to a factor VIII solution that has been maximally activated with thrombin prevents the subsequent destruction of “activated” factor VIII. Thrombin was covalently bound to agarose and shown to be active in hydrolyzing a synthetic tripeptide (S-2160) with a pH optimum of 8.5. Tested on human or bovine plasma, immobilized thrombin had no effect on factor VIII activity over 24 hours. Incubated with purified bovine factor VIII (100 units/ml) and tested at various concentrations, no activation of factor VIII activity could be observed. Slow destruction of activity was noted, so that in 16 hours only 20% of factor VIII activity remained compared with glycine-agarose beads as control.

It was assumed that a complex of factor VIII and thrombin was necessary for the activated state. To test this, thrombin was labeled with 125I. This iodinated thrombin maintained full factor VIII activating activity. A time dependent binding of factor VIII and thrombin was observed following chromatography on Bio-Gel A 1.5M. This observation, plus the absence of activation of factor VIII by solid phase thrombin, suggests that a factor VIII-thrombin complex rather than limited proteolysis is the mechanism of factor VIII activity enhancement by thrombin.

Supported by the National Hemophilia Foundation.
A Comparative Study of the Effects of Heparin and a Low Molecular Weight Semi-Synthetic Heparin Analogue Upon Platelet Function
Authors: Michalski R. , Lane D. A. , Kakkar V. V.
Published: May 2019

We have already reported O) some in vitro and in vivo properties of a low molecular weight glycosaminoglycan polysulphate. It was found that while this semi-synthetic heparin analogue (SSHA) was virtually inactive in a number of in vitro clotting assays, following intravenous or subcutaneous injection it has a more specific anti-Xa potentiating effect than heparin. In the present communication a comparison has been made of some effects of SSHA and heparin upon platelet function. In several of the in vitro tests performed, such as their potentiating effect on ADP and adrenaline induced aggregation and their effects on the aggregation of washed platelets by Factor Xa, heparin proved to be far more potent than SSHA. It was found that after intravenous injection of both drugs, PRP samples containing comparable anti-Factor Xa activities responded differently to the addition of thrombin as SSHA barely inhibited thrombin induced aggregation. Similarly, SSHA had little effect on the dilute thrombin clotting time of plasma, following intravenous injection. Heparin and analogue were neutralised to approximately the same degree by a crude PF4 preparation, and similar transient thrombocytopenia effects were observed with both drugs.
The Heparin Thrombin Clotting Time - A Measure of Platelet Activation?
Authors: O’Brien J. R. , Etherington M. D. , Oamieson Sandra , Shuttleuorth R.
Published: May 2019

The heparin thrombin clotting time (HTCT) of platelet poor plasma (PPP) measures non-specifically the total heparin neutralizing activity (HNA) of PPP. This HNA must include any platelet factor 4 (PF4) liberated in vivo from “activated” platelets. It is suggested that released PF4 is the main determinant of clinical abnormalities of the HTCT for the folloiijing reasons. (l)There is an inverse relationship between the intra-platelet PF4 determined by a clotting method and the HTCT (Thrombos. Haemostas., 36, 649, 1976). (2)There is minimal activity in plasma from aplasia and I.T.P. (3)There is a mild excess of activity in groups of patients at risk of thrombosis and (4) most activity during thrombosis, operations, acute myocardial infarction and infections, in all of ujhich conditions platelets presumably are involved and “activated”. There is a significant inverse correlation between the HTCT and (5) the plasma “low activity PF4” of Nieuiarouski and (6) the β thromboglobulin (radioimmunoassay).

In most healthy people this test is remarkably constant, suggesting a steady state. But there are marked inter-person differences ujhich could indicate different degrees of platelet activation and different thrombotic risks. This test can be routinely carried out in 2 minutes in a hospital laboratory with consistent results over the years. It is already of great clinical use and importance. Since this test can now be normalized therapeutically the question of patient benefit can be raised.