bluuebunny/crossref_metadata_2025 huggingface dataset:
Altered Properties Of Human Kallikrein Purified From Cohn Fraction IV-1
Authors: Alving B M , Tankersley D L , Mason B L , Finlayson J S
Published: July 2018

Cohn fractions are often used as source material for the purification of coagulation zymogens and enzymes. To determine if such a source provided an unaltered kallikrein, we purified this enzyme from Cohn fraction IV-1 paste and compared its properties to those of kallikrein prepared from fresh plasma. The paste was dissolved in distilled water (final concentration ∼ 33%, w/v) and stirred at 22∼C for 10 h. Lipoproteins were precipitated by the addition of polyethylene glycol. The kallikrein in the supernatant was purified by successive chromatography on SP-Sephadex C-50, benzamidine-Sepharose, and Sephadex G-150. When compared to kallikrein prepared from fresh plasma, kallikrein from Cohn fraction IV-1 had a lower (by ∼ 6%) molecular weight, a lower isoelectric pH, and lower proteolytic activity. The decrease in activity was 10-fold when measured by ability to correct the clotting defect in Fletcher plasma and 30-fold when assessed by ability to increase vascular permeability in guinea pigs. By contrast, when the synthetic substrates D-prolyl-L-phenylalanyl-L-arginyl-p-nitroani-lide (S-2302) or Nα-benzoyl-L-arginine ethyl ester (BAEE) were employed, the kcat and Km of this enzyme were similar co those of kallikrein prepared from plasma. When tested against an antiserum to plasma kallikrein, this enzyme gave a reaction of partial identity with plasma kallikrein and appeared to lack antigenic determinant(s) exhibited by the latter. Thus human kallikrein purified from Cohn fraction IV-1 may represent a degraded product, for it has chemical, enzymic, and immunologic properties that differ from those of kallikrein prepared from fresh plasma.
Coagulation Factors Are Activators Of Human Plasma “Prorenin”
Authors: Osmond D H , Tatemichi S R , Wilczynski E A , Purdon A D
Published: July 2018

We have demonstrated that human plasma “prorenin”, an inactive precursor of the blood pressure regulating enzyme renin, can be activated by cold, e.g. -4 to +4°C for 1-30 days (Can. J. Physiol. Pharmacol. 51:705, 1973). Several workers have reported cold activation of the coagulation system. Suspecting a link between these two cold- activated enzyme systems, we established that in factor XII deficient plasma, the rate of cold activation of prorenin is halved (Lancet i, 1313, 1978). Trypsinization of plasma can mimic within 1 minute the effect of prolonged cold (Circ. Res. Suppl . 1, 41:171, 1977), and can overcome specific coagulation factor deficiencies in varying degrees. FXII, VII, V, and especially FX deficient plasmas, all have subnormal basal active renin levels, implying an impaired state of prorenin conversion in vivo. FXII deficientplasma activates least by cold, suggesting special importance of FXII for operation of cold activation. All the plasmas activate better with 0.5 mg trypsin/ml plasma than with cold, except FX, suggesting that it especially mediates tryptic activation. Increasing the trypsin concentration corrects for factor deficiencies in varying degrees, implying some non-specificity and interchangeability of factor requirements for prorenin activation. Our data point to a hierarchy of factor importance, and to a “cascade7#x201D; of prorenin activation, by which plasma renin content can be rapidly increased. Thus, plasma renin activity is a function of renal release of renin, plus renin formation from renal (and possibly extrarenal) prorenin by an activation process involving the coagulation system.
Endotoxins And Procqagulant Activities Of Ascitic Fluid
Authors: Murr H , Schüssler P , Rindfleisch G E , Eisenburg J , Hiller E
Published: July 2018

Procoagulant activities in ascitic fluid of patients with cirrhosis of the liver have been reported repeatedly. It has been suggested that they cause DIC after reinfusicn of ascitic fluid, however, the nature of these factors is unknown. Since endotoxins have been found in ascitic fluid and it is known that they can greatly augment the procoagulant activities of monocytes and macrophages, we assayed for the presence of endotoxin and compared the procoagulant activities in each of 15 specimens of ascitic fluid caused either by cirrhosis of the liver or by metastasizing tumors.

Endotoxins were assayed by the limulus test. Procoagulant activities were determined by thrcmbelastography, thrombin generation test, the presence of soluble fibrin monomer complexes ( SFMC ) and the ability to activate prothrombin complex and factor X.

Endotoxins were detected in 12 of 15 specimens of ascitic fluid caused by cirrhosis of the liver. In 11 of the endotoxin positive and in 2 of the 3 endotoxin negative samples procoagulant activity was present ( i.e. shortened reaction time, increased thrombin generation, activation of prothrombin complex ). In contrast, in only one out of the 15 samples of tumerous ascitic fluid the limulus test was positive. Nevertheless, in 9 of these 15 specimens procoagulant activity was present. In all specimens increased amounts of SFMC were found.

The precise mechanism for the activation of the coagulation system following ascitic fluid reinfusion remains to be established. In addition to stimulation of macrophages by endotoxin other factors like tissue thromboplastin release from tumor cells or injured tissues may account for the procoagulant activity of ascitic fluid.
Prostanoids And Endotoxaemia In The Mini-Pig
Authors: Westwick J , Krishnamurthi S , Scully M , Newman Y , Webb P , Kakkar V V
Published: July 2018

Infusion (iv) of endotoxin (E coli 0111 B4) at a cone, of 1, 10, 100 or lOOOng/kg/min into mini-pigs with sampling every 20 mins produced a dose related fall in WBC, platelets, blood pressure (BP) and early large increases in plasma thromboxane (Tx) B2 with only minor changes in plasma 6-oxo-PGF1α conc. In 5 pigs endotoxin infusion at 100 ng/kg/min for 40 min produced elevated plasma levels of TxB2 from pre-infusion value of 40±5 to 750±50pg/ml, while WBC, platelets and BP expressed as % of pre-infusion value were 40±8, 81±6 and 105±5% respectively. At the end of the endotoxin infusion (100 min) plasma TxB2 had decreased to 120±10pg/ml; WBC, platelets and BP were 22±4, 62±4, 63±5% of pre-infusion value. In 5 other pigs receiving saline only, no significant changes in any of the above parameters were observed.

Thus there is an excess of thromboxane production compared to PGI2 in endotoxaemia. We attempted to determine the contribution of this prostanoid imbalance to endotoxaemia, by treating groups of 5 to 9 mini-pigs with either PGI2 (125 ng/kg/min, continuously), or a potent and selective Tx synthesis inhibitor, UK, 37, 248 (7.5mg/kg as a bolus 10 min before) or lignocaine (20mg/kg as bolus 10 mins before followed by an infusion of 2mg/min) prior to endotoxin infusion. Both PGI2 and lignocaine maintained the BP at pre-endotoxin values, but did not have a significant effect on the leucopenia and thrombocytopaenia or on the elevation of plasma TxB2 While the Tx synthesis inhibitor produced a significant (p<0.05) but not a maximum inhibition of the drop in BP, a marked inhibition of plasma TxB2 formation, but with no effect on the leucopenia or thrombocytopaenia induced by the endotoxin infusion.

Although Tx formation is a sensitive indicator of endotoxaemia and precedes the development of hypotension, it is not contributory to the thrombocytopaenia or the leucopenia. How ever both PGI2 and the Tx synthesis inhibitor by correcting the prostanoid balance appear to be therapeutically useful in endotoxaemia.
Prevention Of Shwartzman’S Phenomenon In Factor XIII Deficiency
Authors: Young Lee Soo , Keun Chang Sung , Chung Soo II
Published: July 2018

Cross-linked clots formed in vitro are reported to be more resistant to fibrinolysis but the relevance of these observations to the situation in vivo is uncertain. The possible role of Factor XIII in the formation of diffuse intravascular fibrin deposition was examined in experimentally induced Factor XIII deficient rabbits. Factor XIII deficiency was induced by intravenous infusion of IgG isolated from goat anti-rabbit platelet Factor XIII. Control received normal goat IgG. The Shwartzman reaction was produced by two injections of bacterial endotoxin given 24 hours apart.

The most striking histological differences were observed after 48 hours. A large number of glomerular loops were enlarged and engorged with red blood cells and platelet- fibrin thrombi; extensive bilateral cortical necrosis was observed in 8 out of 10 endotoxin injected control rabbits but none in the Factor XIII deficient group.

Fibrinogen levels in control rabbits were increased 3-4 fold (1.1g/100 ml), at 24 hours and slightly decreased at 48 hours after endotoxin injection, whereas in Factor XIII deficient animals, the rate of increase was slower but reached similar levels at 48 hours. Fibrinolytic activity in vivo, studied by the degradation of infused 125I-fibrinogen, was significantly increased in both endotoxin injected groups, irrespective of Factor XIII levels.

These results strongly suggest that cross-linked thrombi are more resistant to fibrinolysis in vivo as well as in vitro.
Diagnosis Of Venous Thrombosis: The Case For Phlebography
Authors: Salzman E W
Published: July 2018

Radiographic phlebography is the most accurate test for diagnosis of deep vein thrombosis (DVT) in the lower extremities. Its sensitivity is approached only by 125I- fibrinogen scanning in the calves, and it is far superior proximally. Its specificity is exceeded only by autopsy. Other attractive features of phlebography are its safety and objectivity, the fact that it requires no special equipment, the permanent record it provides, and its ability to distinguish fresh thrombi from old. On the other hand, phlebography is invasive and uncomfortable, requires physician’s skills and is expensive. Venous thrombosis may be induced by phlebography, most often in the superficial veins of the foot or ankle; the frequency of this complication is reduced by dilution of the contrast agent or the use of new non ionic media. Phlebography provides an immediate answer, unlike labelled fibrinogen scanning. It can be employed as an outpatient procedure in patients in whom DVT is suspected and can thus avoid unnecessary hospitalization.

The use of phlebography is essential to monitor thrombolytic therapy of DVT, in which complete dissolution of thrombi is necessary to maintain venous valvular function. It is also essential in patients with suspected recurrence or extension of DVT when noninvasive tests were previously positive. It is the preferred diagnostic procedure when anticoagulant therapy is hazardous or in other clinical settings in which the uncertainties associated with noninvasive methods might complicate management.
Treatment Of Venous Thromboembolism: Case For Heparin
Authors: Deykin D
Published: July 2018

Heparin is the only antithrombotic drug whose efficacy has been uniquivocally established *by controlled prospective studies. The premise of heparin therapy, whether the drug is given by intravenous or subcutaneous routes of administration is the same: that by accelerating the inactivation of activated clotting factors the deposition or growth of stasis thrombi is inhibited. The major complication of heparin therapy, bleeding, can be reduced but not prevented by close attention to details of administration and monitoring. Thrombocytopenia, another complication of heparin, may be more frequent than previously suspected. Osteoporosis, a consequence of long-term, high-dose heparin administration, occurs when doses in excess of 10,000 units/day are administered for more than a few months. Properly given and monitored, heparin remains the standard against which all other forms of antithrombotic therapy should be measured.
Determination Of Molecular Size Of VIII:C In Whole Plasma By Electron Irradiation
Authors: Harmon J , Jamieson G A , Rock G
Published: July 2018

Loss of activity during electron irradiation provides a means of determining the molecular size of specific molecules in complex biological mixtures. This technique has established a molecular weight of 200,000 for Factor VIII:C in concentrates prepared from citrated plasma in which calcium is sequestered by chelation (Aronson, et al. Thromb. Diath. Haemorrh. 8:270, 1962): this value is similar to that obtained by more conventional techniques. However recent data suggest that in heparinized plasma, where physiological levels of calcium are maintained, about half of the VIII:C activity has a molecular weight of 50,000 as determined by gel filtration and ultracentrifugation (Rock, et al. Thromb. Res. 13:85, 1978). When heparinized plasma was subjected to electron irradiation in the frozen state there was a perceptible loss of VIII:C activity at 1 megarad and 80% loss with 60 megarads of irradiation. Analysis of the course of inactivation showed a biphasic curve with 73% of the VIII:C activity having a target size of 40,000 daltons while 28% had a molecular weight in excess of one million. Similar results were obtained when blood was collected in citrate and rapidly processed (∼5 min) to platelet-poor plasma. Following electron irradiation, 62% of VIII:C activity showed a target size of 35,000 daltons while the remaining 38% gave a target size of 275,000. These results provide further evidence that circulating VIII:C activity in normal plasma has a molecular weight of about 40,000 and suggest that reports of higher molecular weights are an artifact of the chelation of calcium as evidenced by the biphasic decay of VIII:C activity in citrated plasma.
The Application Of Computed Tomography In The Diagnosis And Management Of Dissecting Aortic Aneurysms
Authors: Moncada Rogelio , Salinas Matias , Messmore Harry L , Fareed Jawed , Parvez Zaheer , Love Leon , Reynes Carlos , Demos T , Churchill Robert
Published: July 2018

Dissecting aortic aneurysm (DAA) is a well recognized clinical entity with a dramatic pathologic presentation and potentially lethal outcome. The diagnosis is generally suspected by a spectrum of classical symptoms and physical signs. Confirmation of this vascular disorder is usually accomplished through aortography. We have utilized computed tomography of the torso combined with simultaneous intravenous bolus injection of radiologic contrast media in sixteen patients suspected of having dissected aorta. All patients had subsequent correlative percutaneous aortography within twenty-four hours of the initial examination. Of the sixteen patients examined, four proved to be normal, one had an aneurysm of the thoracic aorta and eleven had aortic dissection; five Type I and six had Type III dissection. All eleven patients with aortic dissections were diagnosed by computed tomography and angiography; nine had spontaneous dissection and two had iatrogenic injuries to the aorta. Early experience with computed tomography-enhancement technique has reliably demonstrated normal as well as pathologic aortic wall morphology. We recommend that a CT enhancement study be the preliminary screening in all suspected cases or aortic dissection. Furthermore, this technique can be used for evaluation after medical or surgical treatment or in patients suspected of having rapid aortic expansion or rupture of the paracardium or the pleura.
Haemodialysis Without Heparin, A Possible Benefit From The Use Of Ticlopidine (TCP) In End Stage Renal Disease Haemodialysis (HD) Patients
Authors: Mion C , Chong G , Nguyen Q V
Published: July 2018

In order to test the efficacy of TCP as an anti- thrombogenic agent during HD, we attempted to suppress heparin in 10 stable end stage renal disease patients, treated for 4 months or more, and taking a 500 mg daily dose of TCP and no other anticoagulant therapy. The dialysis schedule was: 3 weekly 3-5 hours HD; single use Cordis HFAK IV dialyser, 1.3 m2 surface area; heparin dosage: 25-66 units per kg body weight per hour given 50% intravenously as a loading dose and 50% as a continuous infusion during dialysis. Two protocols were followed. In protocol I, the maintenance dose was progressively reduced (10% of the initial dose every 3 HD) down to complete suppression; thereafter, the loading dose was progressively reduced and eventually suppressed. Reduction in dialyser fiber bundle volume (ΔFBV) was measured on each dialyser. When FBV was >15%, heparin dosage was maintained at the lowest level obtained. In 3/5 patients, total suppression of heparin was obtained and 25 dialyses without heparin were realised (6, 10 and 9 dialyses in each patient); dialyser efficiency was maintained despite formation of heparin deposit on the outlet blood chamber of the dialyser. In 3 other patients, total heparin dose was reduced by 75%.

In protocol II, progressive reduction of heparin dosage was done simultaneously on loading and maintenance doses; AFBV was also measured in each dialysis and heparin was maintained at a level compatible with ΔFBV <10%. In 5 patients, a 70-80% decrease in heparin dosage was obtained without reduction in dialyser performance.

In conclusion, TCP made possible HD without heparin and allowed a striking reduction in heparin dosage without loss of dialyser performance.
The Interaction Of Platelet Active Drugs With Prostacyclin In Vitro And Against Biolaser Induced Intravascular Thrombosis
Authors: Fleming J S , Cornish B T , Buchanan J O , Buyniski J P
Published: July 2018

Prostacyclin and thromboxane A2, two of the physiologically most important end products of arachidonic acid metabolism, represent a basic control system which modulates platelet function. Decreased vascular prostacyclin is believed to play a role in the increased thrombotic tendency associated with various clinical diseases including diabetes and atherosclerosis. Compounds which either enhance the formation or release of prostacyclin or potentiate the activity of low levels of prostacyclin may be therapeutically useful in ameliorating this associated pathology. We have studied various inhibitors of platelet aggregation for their ability to potentiate the activity of low levels of prostacyclin both in vitro and in an in vivo model of experimental thrombosis. Anagrelide, aspirin, dipyridamole, sulfinpyrazone and ticlopidine all demonstrated interaction with prostacyclin in vitro against collagen-induced platelet aggregation. More limited interactions were observed against ADP-induced aggregation. Using isobolographic analysis most combinations demonstrated additive interaction. However, pronounced supra-additive interaction was observed vs. both aggregating agents in the case of prostacyclin (0.1-1 ng/ml) - anagrelide (8-90 ng/ml) combinations. Dramatic enhancement of the effects of prostacyclin on biolaser-induced thrombosis was also seen in anagrelide (0.5 mg/kg po) pretreated animals. Other inhibitors of platelet aggregation used in combination with prostacyclin produced less spectacular results. These findings suggest that aside from inherent antiaggregatory and antithrombotic activity, certain platelet active drugs may produce equally important effects by virtue of their ability to interact with prostacyclin in a clinically beneficial manner.
Platelet Activating Factor (PAF) Stimulates Protein Kinase (PK) Activity In Human Platelets
Authors: Chesney C M , Pifer D D , Cagen L M , Muirhead E E
Published: July 2018

Kishimoto et al.(J.B.C.255:2273,1980) have demonstrated a Ca2+ and phospholipid-dependent protein kinase (PK) from various mammalian tissues which is markedly stimulated by the addition of unsaturated diacylglycerol. PAF, an alkyl ether analogue of phosphatidyl choline induces platelet aggregation and secretion. We investigated the ability of the C18:0 analogue (l-alkyl-2acetyl-sn-glycero-3-phosphocholine) to stimulate Ca2+ dependent PK activity in homogenates of human platelets. Two hundred ml whole blood was collected in EDTA(5mM) and EGTA(5mM). PGl2(534nM) was included to prevent platelet activation during preparation. Platelets were washed twice in 0.05M Tris containing EDTA, EGTA,and PGl2, pH 7.5. On the 3rd wash PGI2 was omitted. The platelet pellet was then resuspended in the same buffer now containing leu- peptin(0.2mM) to inhibit proteolytic activation of PK. The suspension was sonicated and centrifuged at 100,000xg for 1 hr. PK activity was assayed in the supernatant and pellet suspension by measuring the incorporation of 32P into HI histone from [γ32P]ATP. The standard reaction mixture (0.3 ml) contained 250yl supernatant or pellet suspension, HI histone(60μg), [γ32P]ATP(3nmoles),magnesium acetate (13.3mM) diolein(500ng) Ca2+ and PAF for 3 min. at 30°.Basal PK activity was 14.6pmol/min/mg protein. PAF(0.8μg) which is just saturating dose for in vitro platelet aggregation, stimulate PK activity by 70% in the supernatant but was without effect on the pellet suspension. In the absence of Ca2+ and/or diolein there was no stimulation of PK by PAF. Phosphatidyl serine(PS)(5μg) also stimulated protein kinase by 100%.Stimulation of PK by both PAF and PS occurred at endogenous platelet Ca2+ concentrations (i.e. sufficient Ca2+ added to titrate EDTA and EGTA) and at higher Ca2+ concentration (by 0.2mM.)Supernatants from platelets prepared in the absence of PGI2 were not stimulated by PAF These data show that PAF activates a Ca2+ dependent protein kinase which may mediate its effects on human platelets.
Does Epinephrine Activate Platelets By Binding To A Receptor And Then Reducing Heme In A Membrane Enzyme To Transmit The Activating Signal
Authors: Peterson D A , Gerrard J M , Gerrard J M , Rao G H R , White J G
Published: July 2018

It is well established that epinephrine binds to an α- receptor on platelets. How the signal for cell simulation is transmitted is uncertain. Reduction of Fe3+-heme to Fe2+-heme was evaluated as described previously (Prost. Med. 4:73,1980). Compounds which are known to interact with the plateletoC-receptor reduced heme in the same rank order as their effectiveness as agonists epinephrine > norepinephrine> dopamine > phenylephrine. Phentolamine which binds to the platelet receptor but is an inhibitor not an agonist was ineffective at reducing heme. 1,10 phen- anthroline, 3-chloropyridine, 2,2’-dipyridyl and 4,4’-di- pyridyl which can bind to the Fe in heme all inhibited first wave epinephrine aggregation at lower levels than they inhibited first wave ADP aggregation (IC50s to epinephrine 0.31-0.95; IC50s to ADP 0.93-4.8). The results are consistent with the concept that epinephrine binds to its receptor primarily through interactions involving the methyl-amine and beta-hydroxy groups along with hydrophobic bonds involving the aromatic ring, while intrinsic activity is a function of the catechol moiety which reduces Fe3+ heme to Fe2+ heme in a membrane enzyme.
The Effect On Serum-Thromboxan B2 Production, Platelet Aggregation, Serotonin Release, PF-4 And 6-Keto-PGF1αOf A Novel Specific Thromboxan A2 Synthetase-Inhibitor In Patients With Hyperactive Platelets
Authors: Knudsen J B , Juhl A , Gormsen J
Published: July 2018

A novel, specific Thromboxan A2-synthetase inhibitor 4-1-2- (1 H-imidazol-l-yl)ethoxy-benzoic acid hydrochloride was given to nine patients with hyperactive platelets (defined by an aggregation threshold 0.05 ug/ml epinepherine) and nine controls. The effects on serum Thromboxan B2, platelet aggregation, serotonin release, PF-4, and 6-keto-PGF1 were evaluated in sequential blood samples from h to 24 h after single dose of loo mg. The serum-thromboxan production measured by RIA was reduced 96% ± 4.3 sd 1/2 h to 2 h after dosing. Platelet+aggregation was reduced 89 ± lo.2% with epinephrine, 92 ± 11.4% with collagen and 56 ± 14.3% with ADP. Serotonin release induced by ADP was reduced 65 ± 9.8%, while PF-4 showed no consistant changes. When crushed rat aorta or microsome preparations from human umbilical cord arteries were incubated with PRP from patients before and after dosing, and aggregation induced by 16 uM ADP, a 6 fold increase in 6-keto PGF1α production measured by RIA was observed. The Ivy-bleeding time was prolonged by 65±14sd %.

Conclusion: Specific inhibition of the platelet thromboxane synthetase in patients induces a highly effective inhibition of thromboxane production, and inhibition of platelet aggregability and serotonin release and an increase in Endoperoxide availability which by rat and human endothelial cell prostacycline synthetase can be utilized for increase prostacyclin production.
CIS-Unsaturated Fatty Acids Inhibit Platelet Function By Perturbation Of The Platelet Membrane
Authors: MacIntyre D E , Hoover R L , Karnovsky M J , Salzman E W
Published: July 2018

The uptake of free fatty acids into surface membranes has been shown to affect lymphocyte capping and fibroblast adhesion and growth. Using human gel filtered platelets (GFP), we examined the effects of saturated (S), cis-unsaturated (CU) and trans-unsaturated (TU) fatty acids (FA) on platelet morphology, shape change, aggregation, 5HT release, TxB2 production and diphenyl hexatriene (DPH) polarization, an index of membrane lipid organization. FA (1-30 μM) did not alter platelet morphology. CUFA (16:1, 18:1, 18:2, 18:3, 20:1, 20:2) but not TUFA (18:1, 18:2) or SFA (14:0, 16:0, 18:0, 20:0) inhibited thrombin (T)-induced platelet aggregation and 5HT release. Up to 1514 mninutes after addition of 14C-FA to GFP, >90% of platelet C was present as free FA. Under these conditions only CUFA inhibited T-induced platelet shape change and primary or secondary aggregation induced by ADP, U46619, collagen or arachidonate. Responses to A23187 were unaffected. T-induced TxB2 production was reduced by CUFA. The antiplatelet effects were evident despite the presence of inhibitors of adenylate cyclase (2',5'-dideoxy adenosine, 100μM) or cyclo-oxygenase (indomethacin, 30 μM), but were reversed by resuspension of FA-GFP in FA-free buffer. Analysis of fluorescence decay of DPH in platelet membranes revealed lifetime heterogeneity, indicative of distinct lipid domains. Only CUFA caused a decrease in DPH polarization of intact platelets and platelet membranes. These findings suggest that inhibition of receptor mediated platelet responses by CUFA is produced by perturbation of the platelet membranes in specific lipid domains.
Radioimmunoassay Of Bβ 15-42 Peptide From Human Fibrinogen
Authors: Kudryk B , Robinson D , Netré C , Hessel B , Blombäck M , Blombäck B
Published: July 2018

Early plasmin attack of fibrinogen or fibrin results in the removal of C00H- and NH2-terminal portions of the Aα and 3β chains, respectively. This paper describes a radioimmunoassay (RIA) for measurement, in clinical blood samples, of peptides containing the Bβ 15-42 sequence. Bβ 15-42 was prepared from the NH2-terminal CNBr fragment of human fibrinogen (N-DSK) by sequential proteolysis with Batroxobin and thrombin. Antiserum was obtained by immunizing rabbits with Bβ 15-42 peptide and BSA. The peptide was labeled with 125I by the chloramine-T method. A double antibody RIA was subsequently made.

Radiolabeled Bβ 15-42 (tracer) was bound by antiserum and binding was inhibited by excess cold peptide. Free tracer can be separated from bound by second antibody or by charcoal. Fibrinogen and N-DSK completely cross-react. However, the slope of the inhibition curve is different to that of free Bβ 15-42. Plasma, with Trasylol and heparin,must be treated with ethanol in order to precipitate fibrinogen prior to its use for RIA. The level of peptides with Bp 1542 sequence in treated plasma from normal men and women (20-43 year old) showed a mean value of 1.71±0.59 pmol/ml. Some patient plasma (e.g. uremics) showed 10-20 times higher values.

The immunoreactive material isolated by gel filtration from normal and patient plasma, as well as from streptokinase-plasmin digests of either plasma or purified fibrinogen, all,in gel chromatography, displayed the same molecular weight range.

This RIA is a rapid, specific and sensitive method to measure earliest stage of plasmin proteolysis and will be useful in clinical studies on all disease states associated with fibrino(geno)lysis.
Studies Of The Heterogeneity Of Purified Antithrombin III
Authors: Barrowcliffe T W , Eggleton C A , Mahmoud M
Published: July 2018

Deficiency of antithrombin III (At III), whether hereditary or acquired, is now recognised as a major predisposing factor for the development of venous thromboembolism. Purified At III concentrates are undergoing clinical trials in various conditions associated with At III deficiency; such concentrates may be given in addition to heparin and their potency is usually assessed by heparin co-factor assays. In an international collaborative study, a reference preparation of purified At III had a lower concentration by heparin co-factor than by immunological assays and this was shown to be due to the presence of non-heparin-binding antigens. In the present study we have examined purified At III from several manufacturers by heparin co-factor (amidolytic), progressive antithrombin (clotting) and immunological assays, and their heparin-binding abilities have been studied by crossed immunoelectrophoresis and heparin-agarose affinity chromatography.

There was good agreement between progressive antithrombin and immunological assays, but in some concentrates the heparin co-factor assays gave lower activity. The proportions of non-heparin-binding material varied considerably, from less than 5% to as much as 50% of the total At III antigen in some concentrates. The non-binding material isolated from a heparin column had little heparin co-factor activity, but was able to neutralise thrombin and Factor Xa. Gel filtration and polyacrylamide gel electrophoresis showed no major distinction between heparin-binding and non-binding antigens, indicating the absence of At III-protease complexes.

These studies show that some At III concentrates contain substantial amounts of partially denatured molecules, in which the heparin-binding ability of the At III has been impaired but its thrombin and Xa neutralising activity left relatively intact.
Mechanism Of The Inactivation Of Trypsin By Antithrombin III
Authors: Danielsson A , Björk I
Published: July 2018

The reaction between bovine antithrombin III (AT) and bovine trypsin was'studied and compared to the inactivation of thrombin by the inhibitor in order to elucidate general aspects of the mechanism of AT action. AT and trypsin formed inactive 1:1 complexes, as determined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/ PAGE) and trypsin activity measurements. In the absence of heparin, the reaction was about 100-fold faster than the AT-thrombin reaction. The reaction rate increased when AT was preincubated with heparin before trypsin was added. Purified AT-trypsin complex dissociated at pH 10 into free enzyme and two proteolytically modified forms of AT, while no intact AT appeared. In SDS/PAGE, the two modified inhibitors gave bands corresponding to apparent molecular weights of 52000 and 48000 under reducing conditions, while both forms co-migrated with intact AT (mol wt 56000) under non-reducing conditions. This indicates that each of the two modified forms of AT had been cleaved into two or more chains held together by disulfide bonds. Under reducing conditions, the larger of the modified AT chains co-migrated grated with the large chain of thrombin-modified AT, i.e. the form of AT which dissociates from the AT-thrombin complex and which is cleaved at the reactive Arg-Ser bond of the inhibitor. Control experiments showed that the smaller of the two chains was formed by tryptic cleavage of the larger chain. Antisera specific for thrombin-modified AT reacted with purified AT-trypsin complex, demonstrating that the inhibitor was present in the complex in a form immunologically identical to thrombin-modified AT. An analogous finding has been reported earlier for the anti thrombin- thrombin complex. Together, these results suggest the same general mechanism for inhibition of trypsin and thrombin by AT.
Kinetics, Redistribution And Fate Of Indium-111-Labelled-Platelets In Patients With Aortic Aneurysms
Authors: Heyns A duP , Lötter M G , Badenhorst P N , de Kock F , Pieters H , Herbst C , Nel C J C
Published: July 2018

Platelets of 7 patients with abdominal aortic aneurysms were labelled with In-lll-oxine prior to surgery. The platelets were reinjected with the patient positioned under a scintillation camera with a computer assisted imaging system. Images were acquisitioned daily, areas of interest selected with the computer, organ radioactivity- quantitated with a geometrical mean method and expressed as a percentage of whole body radioactivity. Platelet survival (PS) in the circulation was determined, and disappearance curves fitted to a gamma function “multiple hit” model.

Mean PS was shorthened to 143,2 ± 47h (normal 232<17); the dissappearance curves were exponential in all but the two patients who had PS within normal limits. The surgically removed aneurysms were dissected and radioactivity of different layers measured. In-111-activity was confined to the superficial layers of the aneurysm.

These techniques allow quantitative studies of the in vivo distribution of labelled platelets. Platelets are deposited in the aneurysms, this shortens PS, the disappearance curves become exponential, and the major sites of deposition of In-111-activity are in the liver and spleen. This indicates that although platelets are damaged and deposited in the aneurysm, the reticuloendothelial system remains a major site of platelet sequestration.
Platelet Survival In Patients With Aortic Dacron Prostheses Treated With Ticlopidine
Authors: Davies J A , Tindall H , Paton R C , Doig R C , McNicol G P
Published: July 2018

Ticlopidine inhibits platelet function ex-vivo and prolongs the bleeding time. We investigated its antithrombotic potential in patients with extensive peripheral vascular disease by measurement of survival of Na51Cr- labelled platelets before and following two months treatment with ticlopidine, 250mg twice daily. Platelet survival in six patients with aortic Dacron prostheses and one who had undergone aorto-iliac endarterectomy was 8.04 ± 0.14 days (mean ± SEM) calculated by linear regression and 7.08 ± 0.7l days by gamma function analysis. This is considerably shorter than the mean normal value of 9.98 ± 0.22 days for platelet life-span established in 40 normal subjects by the aspi rin-labelling technique, which correlates very closely with survival time of Na5lCr- labelled platelets in patients. During the eighth week of treatment with ticlopidine measurement of survival of Na5l Cr-labelled platelets was repeated. Mean platelet survival was not affected by ticlopidine treatment, with survival times of 8.12 ± 0.24 days by linear regression and 7.0 ± 0.54 days by gamma function analysis. Tablet counts indicated that patients complied with the treatment regime and single random blood sampling indicated that adequate plasma concentrations of ticlopidine were attained. It is concluded that ticlopidine 250mg twice daily does not prolong shortened platelet survival in patients with Dacron prostheses.
Interruption Of Malignancy-Associated Platelet Consumption With A Combination Of RA233 And Sulfinpyrazone
Authors: Slichter S J , Weiden P L , O’Donnell M R , Storb R
Published: July 2018

Fifty-five dogs with spontaneous tumors of varied histology were evaluated by platelet counts and 51Cr labeled autologous platelet survivals to determine if platelet hemostasis was altered. None of the dogs received cancer-directed therapy during the course of these studies. In 12 dogs with local tumors, platelet counts were 303,000/ul - 28,000 (normal 391,000/ul - 34,000, P > O.05), and platelet survival was 4.9 days - 0.2 (normal 5.6 days - 0.2, p < 0.05). In contrast, 23 dogs with metastatic tumor had platelet counts of 268,000/ul ± 26,000 (p < 0.005) and platelet survivals of 3.4 days ± 0.3 (p < 0.001). When the platelet survival was less than 4 days, the platelet count was also depressed with a correlation coefficient of 0.60.

After baseline studies, 17 additional dogs with metastatic disease were treated with drugs (RA233 and sulfinpyrazone) that interfere with two different enzyme systems. Although there was some improvement in platelet survived in 6 of 9 dogs treated with RA233 or sulfinpyrazone alone (2.4 days ± 0.3 baseline vs. 3.5 days ± 0.4 treated, p < 0.05), platelet counts were not elevated (241,000/ul ± 54,000 baseline vs. 201,000/ul ± 46,000 treated). However, when the two drugs were given in combination to 8 other dogs, all had increased platelet survivals (2.7 days ± 0.4 baseline vs. 4.4 days ± 0.6 treated, p < 0.02), and this was associated with improved platelet counts in 6 of 8 (259,000/ul ± 46,000 baseline vs. 367,000/ul ± 47,000 treated, p < 0.05). Five had completely normal platelet survivals

This study indicates that metastatic tumors, regardless of histology, are associated with significant platelet consumption resulting in depressed platelet counts. This consumption can be modified and platelet counts increased by treatment with a combination of two platelet active drugs that may act synergistically. We plan to determine whether similar treatment programs would maintain platelet counts and/or improve the survival of transfused platelets in human subjects with malignant disorders.
The Effect Of A Completely Synthesized Platelet-Activating Factor (S-PAF), 1-0-Octadecyl-2-Acetyl-Sn-Glyceryl-3-Phos-Phorylcholine, On Mammalian Platelets
Authors: Sheppard H , Tsien W-H
Published: July 2018

Studies Were Undertaken To Examine The Action Of S-Paf On Mammalian Platelets And Its Modification By A Variety Of Agents Known To Alter Platelet Aggregation. Racemic S-Paf Aggregated Washed Platelets Of Dogs, Humans, Rabbits And Guinea Pigs But Not Rats With Ec50 Values In The Nm And Sub-Nm Range. Albumin Was Necessary For The Stabilization Of Dilute Stock Solutions Of S-Paf But, Above 0.3% Final Concentration, Inhibition Of The Response Was Noted. In Prp, The Activity Of S-Paf Was Reduced Further To About 1/10 The Activity Seen In Buffered Solutions. Using Canine Platelets For Most Of The Remaining Studies It Was Noted That The Activity Of The S-Paf Resided In The Natural R Isomer. The Action Of S-Paf On Washed Platelets Could Be Inhibited By Camp Phosphodiesterase Inhibitors, Ibmx And Ro 20-1724, To The Extent Of 100% And 30%, Respectively, At 100 μM. The Activator Of Adenylate Cyclase, Pge1, Also Inhibited The S-Paf Effect Completely But With As Little As 1 μM. Colchicine, However, Was Inactive. Surprisingly, The Lipoxygenase (Lo) Inhibitors Etya (5,8,11,14-Eicoso- Tetraynoic Acid) And Ndga (Nordihydroguaiaretic Acid) At 100 μM Completely Blocked S-Paf Action While The Cyclooxygenase (Co) Inhibitor, Indomethacin, At The Same Concentration Reduced It By Only 50%. The Effect Of The Lo Inhibitors Was Reduced In 0.3% Albumin And Completely Prevented In Prp. The Action Of The Co Inhibitor, Indomethacin, Was Not Altered By The Presence Of Protein. Although The Action Of Indomethacin On S-Paf Disappeared At 10 μM, It Still Completely Blocked The Effects Of Arachidonic Acid (Aa) In Prp. Taken Collectively, These Data Suggest That S-Paf Can Be Blocked By Agents Which Are Able To Elevate Camp Or Inhibit The Lo Pathway Of Aa Metabolism.
Hypersensitivity To Exogenous Arachidonic Acid (Aa) And Redistribution Of Fatty Acids In Platelets From Women On The Pill
Authors: Minno G Di , Tacconi M T , Roncaglioni M C , Sadurska B , Pangrazzi J , Donati M B , Bizzi A , Silver M J
Published: July 2018

Oral contraceptive (OC) treatment is associated with an increased thrcmbotic risk, the mechanism of which remains still unclear. We have investigated the platelet response to exogenous AA and the plasma and platelet lipid composition in eight healthy women (aged 18-29 y) before and after six month OC treatment with a canbination of d-norgestrel (0.25 mg) and ethynilestradiol (0.05 mg). The threshold aggregating concentration of AA was significantly reduced in platelets of women after 6 months of OC treatment (0.30±0.04 mM versus 0.65 ± 0.08 mM, p < 0.001) whereas it was unchanged in a group of 10 control women, matched for age with the OC treated group and tested twice, with a 6-month interval (0.48 ± 0.04 versus 0.50 ± 0.04). In the same group of patients, seme clotting and fibrinolytic parameters, namely the Normotest, the biological and antigenic activity of Antithranbin III, the anti factor Xa biological activity, the diluted whole blood clot lysis time, were completely unmodified by OC treatment.

In platelets AA increased both in phospholipid and neutral lipid fractions. In plasma the pool of polyunsaturated fatty acids increased at the expense of the saturated pool. In particular, AA increased slightly but significantly in free-fatty acids and phospholipid fractions.

This data indicates that during OC treatment changes in in vitro platelet aggregatory responses may be associated with modifications of platelet and plasma fatty acid composition. Such changes may contribute to the thrcmbotic tendency associated with OC treatment.
Investigation Of The Relationship Between Preoperative Chromogenic Antithrombin III Assays And Hemorrhage Following Heart Surgery
Authors: Laveson J E , Winford J , Iden J , Marengo-Rowe A J , Race G J
Published: July 2018

The above study is part of an ongoing program to determine the mechanisms of hemorrhage in patients who have undergone extracorporeal circulation. Antithrombin III levels were measured in 850 patients (Quantichrom III) prior to undergoing cardiovascular surgery. Following surgery the maximum rate of blood loss was assessed by measuring chest tube drainage at hourly intervals. Patients whose blood loss exceeded 150 ml in any one hour were defined as “bleeders.” 257 patients had a maximum rate of blood loss exceeding 150 ml per hour, and this group is called the bleeder group. The bleeder group had a range of antithrombin III values of 16 to 148% and a mode 66% compared to a range of 24 to 170% and a mode 80% for the non-bleeder group. The means of two groups were 66% and 84% (p < 0.05) were statistically significantly different. 156 patients had an antithrombin III level of less than 60% of which 81 fell into bleeder group.

Possible mechanisms of this relationship are: (1) During massive surgical procedures disseminated proteolysis occurs and those patients with a lower level of proteolytic enzyme inhibitors such as antithrombin III have an impaired ability to control this process, which ultimately leads to hemorrhage. (2) During the massive surgery, an imbalance in protein metabolism occurs. Patients who are poor “protein producers” for pathological, genetic or nutritional reasons are less able to respond to this depletion and they respond less readily to the depletion of proteins such as the coagulation proteins with resultant bleeding. Antithrombin III may be an “indicator protein” for such patients. (3) Reduced levels of antithrombin III reduce the potency of heparin, and thus level of anticoagulation during surgery.
Ancrod In The Management Of Rapidly Progressing Glomerulonephritis
Authors: Glueck H I , Pollak V E , Weiss M A , Lebron-Berges A , Miller M A
Published: July 2018

Ancrod, (Malayan pit viper venom) which produces in vivo defibrination, has been shown to improve renal function and decrease fibrin deposition and crescents in experimental glomerulonephritis. Ancrod was given for 14 days to six patients with glomerulonephritis, moderate to severe renal functional impairment, crescents, and/or fibrin deposition in glomeruli. Ancrod treatment resulted in fibrinogen levels <50 mg/dl without bleeding, normalization of previously elevated factor VIII and von Willebrand factor levels, and normalization of in vitro platelet hyperaggregation. Renal function improved in two patients and was affected little in three; in the sixth deterioration of function was arrested. Serial renal biopsies showed a relatively rapid decrease of glomerular thrombi in three patients, and no increase in glomerular sclerosis. Ancrod administration appears to be safe, and may have a role in treatment of certain types of glomerulonephritis.
Suloctidil: Review Of Its Antiplatelet And Antithrombotic Properties In Man
Authors: Van Stalle F , Lambelin G
Published: July 2018

Suloctidil(S.) - 1-(4-isopropylthiophenyl)-2n octylamino- propanol - is a vaso-active compound endowed with antiplatelet and antithrombotic properties.

After administration to healthy volunteers it was found to inhibit thrombofax and collagen induced aggregation.

In a double blind cross-over set up-S. 200 mg T.I.D. vs. placebo - the induction of circulating platelet aggregates by venous occlusion was highly significantly inhibited.

Following positive results on P.S.T. in the baboon a.-v. shunt model, studies on P.S.T. were initiated in man: 23 patients with valvular prosthesis, all under coumarine therapy, were admitted to a double blind parallel study of 6 weeks duration cqnparing S. 200 mg T.I.D. (n=ll) to placebo (n=12). P.S.T. (51Cr-labeling) measured before and during the last week of the study, was unchanged in the placebo and significantly increased in the S. treated group. Similar results were obtained measuring platelet regeneration time according to a modified Stewart method in an open study (n=6) in valvular prosthesis and in a double blind parallel study (S. n=25; placebo n=27) in valvular prosthesis, coronary artery disease and myocardial infarction.

In patients with obstructive arteriopathy (Fontaine II & III) inhibition of thrombine induced platelet M.D.A. production and reduction of increased βTG levels have been observed under S. therapy at rest and after ischemia.

In 31 patients suffering from recurr. ven. thrombosis, admitted to a double blind cross-over study comparing S. 200 mg T.I.D. to placebo-2 periods of 3 months each - a striking difference in the clinical evolution (placebo 12 events, S. none) was paralleled by consistent changes in biological parameters such as platelet aggregation, PF4 and βTG.

The results obtained in pharmacological as well as in efficacy studies have justified the initiation of larger scale trials to evaluate the compounds antithrombotic potential.
Determination Of The Type Of Fibrinogen/Fibrin Degradation Products In The Preoperative Evaluation Of Pelvic Masses
Authors: Kaplan-Ikonicoff L , Engel A M , Roisman F R , Demattei A
Published: July 2018

It is known that no single method for the determination of fibrinogen/fibrin degradation products (FDP) indicates their nature which is important to establish the state of activation of the fibrinolytic system. Therefore we applied two simple and reproducible laboratory methods to study eleven preoperative patients with pelvic masses: 1) the latex fibrin polymerization inhibition test (Finkelstein test)and 2) the staphylococcal cumpling test. The normal values obtained were 0-3 polymerization inhibition units (PIU) and 2.5-4 μg fibrinogen equivalents per milliliter, respectively. Seven clinically healthy females of the same age range served as controls.

Out of seven cases with confirmed postoperative diagnosis of ovarian carcinoma, five (71%) had positive FDP by both methods with average values of 12.2 ± 5.9 PIU and 10.4 ± 4.6 μg/ml. The ratio between PlU/ml and μg/ml was determined and the range found was 0.53-1.75 which is indicative of the presence of FDP of the “late” type. The values are in agreement with previous reports of the incidence of late FDP in other malignancies. All the negative FDP, except two, corresponded to benign ovarian cyst, although there was a case of haemorrhagic cyst with positive FDP.

The presence of “late” FDP indicates an activated fibrinolytic process and could constitute a cautionary sign for the postoperative care of the patient. We consider that the application of two methods for FDP permits to detect an increased amount and also the type of the circulating FDP. The data presented here clearly warrant detailed investigation of the potential benefits of knowing the nature of FDP in monitoring the postoperative follow up of patients with pelvic masses.