bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphorylation Of Platelet Actin-Binding Protein During Platelet Activation
Authors: Carroll Roger C , Gerrard Jonathan M
Published: July 2018

We have followed the 32P-labelling of actin-binding protein as a function of platelet activation. Utilizing polyacrylamide sodium dodecyl sulfate gel electrophoresis to resolve total platelet protein samples we found 2 to 3 fold labelling increases in actin-binding protein 30 to 60 seconds after thrombin stimulation. Somewhat larger increases were observed for 40,000 and 20,000 apparent molecular weight peptides. The actin-binding protein was identified on the gels by coelectrophoresis of purified actin-binding protein as well as cytoskeletal cores prepared by detergent extraction of activated 32p-iabelled platelets. In addition, these cytoskeletal cores indicated that the 32P-labelled actin-binding protein was closely associated with the activated platelet's cytoskeleton. Following the 32P-labelling of actin-binding protein over an 8 minute time course revealed that in aggregating platelet samples rapid desphosphorylation to almost initial levels occurred between 3 and 5 minutes. A similar curve was obtained for the 20,000 apparent molecular weight peptide. This rapid dephosphorylation was shown to be dependent on platelet aggregation in the absence of external calcium or in thrombastenic platelets lacking the aggregation response to activation. These results suggest that phosphorylation of actin-binding protein initiates its association with the platelet cytoskeleton during activation.
In Vivo Platelet Activity In Young Patients With Cerebral Ischemia And Mitral-Valve Prolapse
Authors: Scharf R E , Hennerici M , Bluschke V , Schneider W
Published: July 2018

Recent studies have demonstrated a sixfold higher incidence of mitral-valve prolapse (MVP) in young patients with cerebral ischemia compared to an age-matched control population. To examine whether there is a pathogenetic relationship between MVP and cerebral ischemia, we studied 47 patients (12 males, 35 females) under 45 (mean age 35.4 ± 6.6) years with transient ischemic attacks (TIA) and stroke who had failed to show a cause from extracranial Doppler examination, cranial computerized tomography and cerebral angiography. In vivo platelet activity was evaluated by measuring the B-thrcmboglobulin (β-TG) concentration in the platelet poor EDTA-theophylline-PGE1-plasma using the Amersham RIA kit.

The β-TG levels of the patients (54.9 ± 31.4, x ± SD ng/ml) were significant higher than those of an age- and sex-matched control group (n=40, 19.7 ± 6.4 ng/ml, p<0.001) MVP was demonstrated in 28% of the patients (13 of 47) in contrast to 7.5% of the controls (3 of 40). The difference of the β-TG levels of patients with MVP (n=13, 52.9 ± 25.5 ng/ml) and of patients without MVP (n=34, 55.7 ± 33.7 ng/ml) was not significant (p < 0.40). There was no correlation bet ween duration or extent of the neurological deficit and the individual β-TG level.

Our results confirm that the incidence of MVP is higher in young patients with cerebral ischemia of unknown cause than in asymptomatic young people. The significantly elevated plasma β-TG concentrations in the patients' group nay indicate an increased platelet activity in vivo. There was io significant difference between β-TG levels of patients tfith and without MVP. Thus, MVP can not be the cause for the altered platelet activity.
Release Of Lyso-PAF-Acether From Platelets
Authors: Benveniste J , Le Couedic J P
Published: July 2018

The release of PAF-acether from platelets, stimulated by thrombin or the ionophore A 23187, is now well-documented. PAF- acether, 1-O-alky 1-2-ace tyl-sn-glyceryl-phosphorylcholine, may represent the molecular intermediate for the third pathway (i.e. non-ADP, non-thromboxane A2~dependent) of platelet aggregation. We now report the release from stimulated platelets of the non-ace ty la ted compound, l-0-alkyl-2-lyso-sn-glyceryl-3-phos- phorylcholine (lyso-PAF-acether). We have already published the structural identification of this molecule released from blood leukocytes.

Washed rabbit platelets (5 x 108/ml) were incubated in Tyrode’s for up to 7 min in the presence of 2.5 uM ionophore or 2.5 U/ml thrombin. PAF-acether was measured by aggregation of washed aspirin-pretreated rabbit platelets in the presence of ADP scavengers. Lyso-PAF-acether was measured by the same method after its chemical acetylation into PAF-acether, followed by treatment with lipase from Rhizoppus arrhizus. Results are given in g/ml, by comparison to known amounts of totally synthetic PAF-acether. PAF-acether and lyso-PAF-acether were released with similar kinetics, reaching a maximum at 5 min. Maximal releases of PAF- acether were 79.0 ± 16.9 and 12.4 ± 8.5 ng/ml (mean ± 1 SD) and that of lyso-PAF-acether were 2.6 ± 0.6 and 1.9 ± 0.8 ug/ml, i.e. about 4 uM, for the ionophore (3 exp.) and thrombin (7 exp.) respectively. PAF-acether directly released from platelets and that obtained through acetylation of lyso-PAF-acether maintained their aggregating activity after treatment with the lipase, thus behaving like the leukocyte-derived compound.

The release of lyso-PAF-acether implies the activation of a phospholipase A2 acting on a membrane alkyl-ether phospholipid. Biosynthetic incorporation of acetate into platelet PAF-acether has been reported. Therefore, lyso-PAF-acether may be an intermediate for PAF-acether synthesis ; also potent biological activities of lyso-PAF-acether can be anticipated.
Arachidonate Metabolism And Platelet Disaggregation (DA)-Reaggregation (RA)
Authors: Rao Gundu H R , White James G
Published: July 2018

Previous work has shown that platelets irreversibly aggregated by ADP or thrombin (T) can be dissociated by various agents and that the refractory state of disaggregated cells can be reversed immediately by treatment with epinephrine (E). In the present study we have evaluated the influence of drugs which affect different steps in the process of prostaglandin (PG) synthesis on platelet DA-RA. Aspirin and indomethacin did not cause DA of platelets in the process of aggregation nor did they prevent reversal of the refractory state by E and subsequent RA of previously dissociated platelets. Imidazole, which inhibits conversion of endoperoxide to thromboxane A2, also failed to influence DA or restoration of sensitvity and RA of disaggregated platelets. On the other hand, chemicals which interfere with release of AA from the membrane of activated platelets, such as mepacrine, chlorpromazine and trifluoperazine, caused rapid DA. Products of PG synthesis, such as PGE1, PGD2 and PGI2, which usually inhibit platelet aggregation, also caused rapid DA. The refractory state of platelets dissociated from aggregates by most of these agents could be reversed by E treatment. However, trifluoperazine disaggregated platelets could be reaggregated only by the combination of E and AA. Agents which block the a-adrenergic receptors did not cause dissociation of aggregating platelets, but prevented correction of the refractory state of dissociated platelets by E. Thus interference with AA release, even after aggregation, can cause DA of clumped platelets, but blockade of peroxidase, cyclo-oxygenase and thromboxane synthetase do not cause reversal once it is in progress. A membrane linked mechanism associated with AA availability, but not metabolism, regulates DA and restoration of membrane sensitivity for RA.
Stimulation Of Prostacyclin Release By A New Vasodilatory Coumarin Derivative (AD6)
Authors: Dejana Elisabetta , de Castellamau Conxita , Rotilio Domenico , de Gaetano Giovanni
Published: July 2018

AD6 (8-monochloro-β-6-diethylarnino-ethyl-4-methyl-7-ethoxy-caxbonyl-methoxy-coumarin) reportedly exerts specific coronary vasodilatory activity and reduces platelet aggregation in laboratory animals. We investigated whether AD6 stimulates prostacyclin (PGI2) synthesis by the vessel wall. Segments of thoracic aorta were obtained from rats 30 min after i.v. injection of AD6 (4 mg/kg) . PGI2 production measured by bioassay and confirmed by RIA of 6-Keto-PGF]a was significantly higher in the vessels of treated (3.27+0.3 ng/mg wet tissue), than of control animals (2.09+0.2 ng/mg wet tissue) . l^C-arachidonic acid (14C-AA) was incubated with vascular specimens for 4 hours, the free excess removed and the metabolites produced during the following 5 hours were studied by thin layer radiochromatography. Lhe vessels from AD6-treated animals converted significantly more 14C-AA to 6-Keto-PGF1α (3.7% total radioactivity) and PGE2 (2.1%total radioactivity) than controls (1.1% and 0.6%, respectively). In vitro incubation of the drug with the vessels had no demonstrable effect on PGI2 production at any concentrations
used (0.01-1 mM).
Decreased Prostacyclin Or PGI2 Production In The Infant Of The Diabetic Mother (IDM). Correlation With Maternal HbA1C
Authors: Stuart M J , Sunderji S G , Allen J B
Published: July 2018

Platelet and vascular prostaglandins are important regulators of normal hemostasis. Platelets produce endoperoxides and thromboxanes which are proaggregatory and prothrombotic, whereas vessels produce PGI2 which is antiaggregatory and antithrombotic. Since the IDM has an increased predisposition to thrombosis, we evaluated arachidonic acid (AA) metabolism and PGI2 production in the umbilical vessels of 12 control infants and 9 IDM of similar gestational age. Mean uptake of 14CAA into vascular tissue of controls and IDM were similar at 9157 ±1096 (1SE) and 12036 ± 1691 cpm per 30 mgm vascular tissue resp. Thrombin stimulated release of 14CAA was similar in the controls (5.0 ± 0.7) when compared to IDM (5.5 ± 1.3). However in the IDM, the production of vascular 6-Keto-PGF1αxa(the stable end product of PGI2) was decreased (p<0.02). Controls incorporated 5003 ± 533 cpm (5.2 ± 0.4%) into 6-Keto-PGF1αia when compared to 3033 ± 490 cpm (3.2 ± 0.6%) in the IDM. No correlation was observed between maternal blood glucose levels and fetal PGI2 production. However, a significant inverse correlation (r=0.87; p<0.02) was observed between maternal HbA1c levels and the conversion of AA to 6-Keto-PGF1α in the IDM. Rigorous control of maternal diabetes appears to be the best prophylaxis against the decrease in PGI2 in the IDM. We have previously shown that platelet endoperoxides are increased in the IDM. Thus, the normal balance between proaggregatory platelet) and antiaggregatory (vascular) prostaglandins is disrupted in the IDM, a factor which favors thrombosis.
Improved Assay For 6-Keto Prostaglandin F1α In Biological Fluids By Reversed-Phase High-Performance Liquid Chromatography In Conjunction With Radioimmunoassay
Authors: Jones P B B , Wark J D , Russell R G G
Published: July 2018

Prostacyclin production has been proposed as a fundamental mechanism by which intact vascular endothelium maintains a non-thrombogenic surface. Interest in a reliable assay for its stable breakdown product 6-keto prostaglandin F1α (6-KPGF1α) is, therefore, considerable. Previous studies have emphasised the need to reduce sample complexity prior to the quantification of PG’s by radioimmunoassay (RIA). This has been achieved by extraction of PG’s into organic solvents or onto resin columns followed by separation of PG metabolites by reversed-phase high-performance liquid chromatography (HPLC), but retention times are long if adequate resolution is to be obtained. By using an 8-min gradient elution programme with acetonitrile/water/acetic acid and a 6 ml/min flow rate through Waters reversed-phase (Radial Pak A) column under radial compression good separation of 6-KPGF1α from its closely-related metabolites 13,14-dihydro 6-KPGF1α 6, 15-diketo PGF1α as well as from other PG species was achieved as detected by ultra-violet absorbance spectrometry. In applying the method to the measurement of 6-KPGF1α in urine, PG’s were extracted onto a resin column, eluted in methanol and the extract subjected to HPLC as described. Good resolution of immunoassayable PG metabolites and known cross-reacting species was achieved. This method provides a significant improvement on existing PG assays by reducing sample-processing time dramatically, and is suitable for routine and repetitive clinical use.
In Vivo Clearance Of Prostacyclin In Rabbits
Authors: Skrinska V , Lucas F , Chisolm G , Thomas G
Published: July 2018

In vivo studies of prostacyclin (PGI2), a potent inhibitor of platelet aggregation, have been limited by its instability during isolation. A method has been developed in which PGI2 can be extracted from whole blood with negligible hydrolysis to 6-keto prostaglandin Fia (6-K-F1α) during collection and analysis. Bolus injections of 3H-PGI2 in ice cold saline were made through a cannula in the marginal ear vein of conscious male New Zealand white rabbits. Blood samples were drawn through the cannula at timed intervals into tubes containing Na2C03, 10 mg/ml. The PGI2 and 6-K-F1α were extracted from the plasma by passing an aliquot directly through an octadecylsilane reverse phase column. The prostaglandins were eluted with a boric acid-acetonitrile solvent system. The PGI2 and 6-K-F1α were then separated by high pressure liquid chromatography and samples of the injectate and blood were counted for radioactivity. Roughly half of the total radioactivity was collected from the bladder at termination, twenty minutes after the injection. Greater than 90% of the labeled PGI2 was cleared from the circulation in five minutes. The ratio of PGl2:6-K-F1α decreased faster in the blood in vivo than in an j[n vitro incubation of whole blood. This indicates a slower distribution or elimination of 6-K-F1α from the blood compartment and/or a more rapid distribution or metabolism of PGI2 in vivo.
The Effect Of Dietary Fish Oils On Hemostasis, Thrombosis And Some Underlying Processes
Authors: Haddeman E , Hornstra G , Don J A
Published: July 2018

Epidemiological evidence indicate fish oils to be beneficial in the prevention of ischemic heart disease. We therefore investigated the (anti-)thrombotic properties of two of these oils in rats. The animals were fed diets containing 5 energy % (en%) sunflowerseed oil(SO) + 45 en% cod liver oil (CLO) or another fish oil(FO). As compared with a diet containing50 en% SO, both fish oils prolong the bleeding time significantly (tail tip transection). CLO, but notFO, lowers arterial thrombosis tendency (thrombotic obstruction of aorta prosthesis). Both fish oils lower the arachidonicacid content of platelet- and vascular phospholipids, as well as the production of platelet TxA2 (measured as MDA-spectrofotometry- or HHT- GC/MS) and vascular PGI2 (measured by platelet aggregation bio-assay or as 6 keto-PGF1α-GC/MS). TxA3 and PGI3 formation from endogenous precursors does not occur in detectable amounts. Thrombin induced platelet aggregation in vitro is enhanced upon fish oil feeding, whereas collagen-induced aggregation is depressed. ADP-induced aggregation is not affected when measured in vitro. In circulating blood, however, it appea red enhanced (only CLO was tested). CLO- (but not FO-) feeding results in a lower vessel wall induced clotting response. As compared with SO-feeding, both fish oils indu ce a significant depression of intrinsic (APTT) and extrin sic(PT) plasma coagulability. Thus the effects of dietary fish oils on hemostatic parameters are manifold and greatly dependent on the origin (and composition?) of the oil. Their depressing effect on the generation of platelet prostanoids may provide a rationale for their dietary use to lower thrombotic risks. Further studies are indicated, however, to explain the discrepancies between the various fish oil sand to explore their long-term physiological and pathological effects.
Thromboxane Synthesis In Fatty Acid-Modified Human Platelets
Authors: Needleman S W , Spector A A , Hoak J C
Published: July 2018

Epidemiologic evidence suggests that ingestion of polyunsaturated fatty acids (PUFA) retards arterial thrombo-occlusive diseases. Consequently, we investigated whether PUFA might influence production of platelet thromboxane A2, a potent agonist of platelet aggregation. A culture system containing 2.7μM PGE1 was devised in order to maintain viability of washed human platelets for as long as 24 hr at 30°C during supplementation with linoleic (18:2) or oleic (18:1) acid. Platelets were then washed, aggregated with 0.12 U/ml thrombin and the media was assayed for thromboxane B2 (TX) by radioimmunoassay. 24 hr control platelets released comparable TX to freshly washed platelets (283 pmol/ml ± 44[SEM] vs 205 ± 40). At 200μM fatty acid concentrations, there was a 30-55% decrease in TX for 18:2 and a 20–30% decrease for 18:1 enriched platelets compared to control. Inhibition was not observed earlier than 4 hr after exposure to fatty acid. Inhibition was more pronounced when the fatty acids were incubated at 37°C than at lower temperatures. At lower concentrations of fatty acid, less inhibition was observed, and in some instances, enhancement of TX release was actually noted. Aggregation was recorded on a Payton aggregometer and analyzed for the slope of the initial linear phase (Mag). Mag for 24 hr control and freshly washed platelets was comparable. At 200μM fatty acid, for 18:2 compared to control, Mag was decreased 23-56%, where as for 18:1 Mag was decreased 22-34%. These findings are in agreement with our previous report of diminished prostacyclin synthesis in similarly modified endothelial cells, and suggest that changes in PUFA availability may influence TX synthesis in the intact human platelet.
Influence Of An Orally Active Thromboxane Synthesis Inhibitor On Platelet Function In Man
Authors: Walter E , Zimmermann R , Lehle R , Weber E
Published: July 2018

Several studies have shown that elevated levels of circulating thromboxane and a decreased capacity of the vessel wall to synthesize prostacyclin do occur in patients with cardiovascular diseases and in diabetes. In contrary to the effect of aspirin, selective inhibition of thromboxane synthesis of the platelets would preserve the production of prostacyclin. This selectivity is offered by UK-37, 248, a new imidazole derivative.

In 11 patients with cardiovascular diseases and en -hanced platelet aggregation a single dose of 100 mg UK-37,248 was tested. Before and 1, 2, 4, 6 and 24 h after intake, blood pressure, pulse, bleeding time were controlled, and blood was taken for in-vitro platelet aggregation and for measurements of plasma as well as serum TXB2 . The plasma TXB2 -level decreased from 130.9±14.2 pg/ml to 85.7±4.6 pg /ml (p< 0.005) after 6 h. Serum TXB (319.3±31.4 ng/ml) was lowest 1 h after drug intake (32.9±5.9 ng/ml, p< 0.001). 5 h later the level was still significantly decreased (182.1±24.3 ng/ml,p<0.005). The drug had no influence on blood pressure and pulse rate in the lying as well as standing position. Bleeding time was minimally prolonged in 7 patients, the mean prolongation was about 10 %. No effect could be observed on spontaneous, ADP-and co11agen-induced platelet aggregation.

The inhibition of thromboxane synthesis in man by UK-37,248 after a single dose of 100 mg could very well be demonstrated. There was no or only a minimal effect on platelet aggregation and bleedingtime. Evaluation of long-term administration is needed.

We thank Dr. Tyler, Sandwich, for TXB2-measurements.
A Thromboxane Synthetase Inhibitor Which Prolongs Bleeding Time In The Rat At Oral Doses Which Fail To Inhibit Platelet Aggregation In Vivo Or Ex Vivo
Authors: Butler K D , Maguire E D , Turnbull A A , Wallis R B , White A M
Published: July 2018

The role of thromboxane A2 (TXA2) in haemostasis was investigated with the use of a selective inhibitor of platelet thromboxane synthetase used in conjunction with radioimmunoassay of thromboxane B2 (TXB2). N-Carboxyheptylimidazole is such an inhibitor having no effect on platelet cyclooxygenase. An oral dose of this substance (10 mg/kg) to rats resulted in 85% (P < 0.001) suppression of platelet TXB2 production induced by collagen ex vivo while the ED50 and maximum rate of platelet aggregation were unchanged. It also caused a prolongation of tail bleeding time from 153±13 to 284±22 secs (P < 0.01). The thrombocytopenia resulting from the Arthus reaction in rats was unchanged, and the prothrombin and activated partial thromboplastin coagulation times were not affected by either 10 or 30 mg/kg p.o. It is concluded that the role of TXA2 in prevention of rat tail bleeding is not as an activator of platelet aggregation or blood coagulation. It is more likely that TXA2 prevents bleeding via its potent vaso-constricting properties. In addition the increased bleeding time may be due to change in the equilibrium of other vasoactive prostanoids.
Inhibition Of Human Tissue Plasminogen Activator By Human Plasma : No Evidence For A Specific Antiactivator
Authors: Korninger C , Collen D
Published: July 2018

Human tissue plasminogen activator (TPA) was highly purified (one-chain or two-chain form) from the culture fluid of a melanoma cell line and labeled with 125

Gel filtration of mixtures of human plasma with trace amounts of labeled and 10 to 1,000 units/ml of unlabeled TPA revealed formation of two radioactive complexes with apparent Mr of 150,000 and 800,000, which were devoid of lytic activity of fibrin plates. These radioactive complexes were precipitated (90 and 63 percent) by antisera against α2 antiplasmin (α2AP) and α2-macroglobulin (α2M) respectively, but not by antisera against other known plasma protease inhibitors. In plasma specifically depleted in α2AP formation of the complex with Mr ≃ 150,000 did not occur, whereas removal of α2M abolished formation of the complex with Mr ≃ 800,000. The initial rates of formation of these two complexes in plasma were very similar to those obtained with mixtures of TPA and 1 μM α2AP or 3.5 μM α2M respectively. Complex formation was completely abolished by blocking the active site serine of TPA.

In purified systems TPA was inhibited by α2AP with a rate constant of 140 M-1s-1 and by α2M with a rate constant of 30 M-1s-1. These rate constants correlate well with the rate of formation in plasma of the Mr ≃ 150,000 complex and the Mr ≃ 800,000 complex respectively.

All these data indicate that TPA is slowly inhibited in human plasma by α2AP (tl/2 ≃ 60') and by α2M (tl/2 ≃ 120'). We found no evidence for the existence of another significant inhibitor in plasma. Both the one-chain and two-chain forms of TPA behaved very similarly in all experiments.
Delayed Elimination Of Fibrin From The Lungs In Rats Treated With The Primary Fibrinolysis Inhibitor α2-Antiplasmin
Authors: Carlin G , Einarsson M , Saldeen T
Published: July 2018

Patients with the adult respiratory distress syndrome after trauma (the delayed microembolism syndrome) show fibrin deposition in the lungs and delayed elimination of this fibrin. This delayed elimination of fibrin has been supposed to play a key role in the pathogenesis of this syndrome.

In the present investigation we have studied the effect of treatment with purified α2-antiplasmin on the elimination of fibrin from the lungs in rats with thrombin-induced intravascular coagulation. The amount of α2-antiplasmin injected was chosen as to simulate the posttraumatic increase of this inhibitor in man.

Methods. Labelled fibrinogen was injected into a tail vein 24 hours before the experiment. Human α2-antiplasmin, 1.2 mg corresponding to the normal amount in about 12 ml human plasma, dissolved in 1 ml saline was injected into a tail vein 3 minutes before the thrombin injection. Thrombin, 350 NIH units/kg was injected during 5 minutes into a tail vein. The rats were killed 5 or 35 minutes after the end of the thrombin injection and the amount of fibrin in the lungs was determined.

Results. Rats not given α2-antiplasmin showed heavy fibrin deposits determined from radioactive incorporation in the lungs 5 minutes after the thrombin injection. Thirty-five minutes after the thrombin injection most of the fibrin had disappeared from the lungs. Rats given α2-antiplas-min showed the same amount of fibrin in the lungs 5 minutes after the thrombin injection whereas the fibrin amount in the lungs 35 minutes after the thrombin injection was not significantly reduced.

The results may argue for the increase of α2-antiplasmin being of importance for the delayed elimination of fibrin seen in patients in the posttraumatic phase.
Partial Primary Structure Of Human α2-Antiplasmin
Authors: Lijnen H R , Wiman B , Van Hoef B , Collen D
Published: July 2018

α2-Antiplasmin (α2AP), the main physiological inhibitor of plasmin in human plasma, is a single–chain glycoprotein with a molecular weight of 67,000 consisting of about 510 amino acids and containing 13 percent carbohydrate.

A tryptic digest on 400 mg of reduced, carboxymethylated and citraconylated purified α2AP was performed. Peptides were separated by combinations of ion exchange chromatography, gel filtration and high performance liquid chromatography, and sequenced using the manual Edman degradation. Some peptides were further digested in order to establish overlaps. At the time of submission of this abstract we have sequenced 7 out of the approximately 21 arginyl peptides completely (each between 3 and 21 residues) and are working on the others. At present we have about 200 residues of sequence. Here we only report the stretches of 10 amino acids or more, which may be useful to compare the structure of α2AP with that of other serine protease inhibitors.
α2–plasmin Inhibitor And α2-Macroglobulin-Plasmin Complexes In Plasma. Quantitation By An Enzyme-Linked Immunosorbent Assay
Authors: Harpel P
Published: July 2018

An enzyme-linked immunosorbent assay has been developed for the quantitation of α2-plasmin inhibitor-plasmin and α2-macroglobulin-plasmin complexes. In this method, the inhibitor-plasmin complex is bound to a surface by an inhibitor specific antibody, then the plasmin bound to the inhibitor is quantified by a second antibody labeled with alkaline phosphatase. Inhibitor-enzyme complexes were generated in plasma by the addition of plasmin or of urokinase (UK). The concentration of plasmin added was well below the plasma concentration of α2-plasmin inhibitor or of α2-macroglobulin so that neither inhibitor would be fully saturated with enzyme. Under these conditions, increasing amounts of plasmin generated an increase in both α2-plasmin inhibitor-plasmin and α2-macroglobulin- plasmin complexes. Varying amounts of plasmin were incubated with each of the purified inhibitors and the complexes that formed were quantitated by immunoassay. These studies made it possible to quantitate the distribution of plasmin between the two inhibitors in plasma. In plasmin-treated plasma, 10% or less of the plasmin bound to both inhibitors was in complex with α2-macroglobulin. In contrast, between 19-51% of the plasmin generated in UK-activated plasma was bound to α2-macroglobulin. Thus major changes in the distribution of plasmin were observed depending upon whether plasmin was added to plasma or whether plasminogen was activated endogenously. 23 normal individuals had low levels of α2-plasmin inhibitor-plasmin complexes (4.1±3.5 fmol/ml) whereas six patients with laboratory evidence for DIC demonstrated a 16 to 35-fold increase in the concentration of these complexes. These data indicate that a useful new probe for the study of the fibrinolytic enzyme system has been developed.
Preparation Of Antithrombin, High Activity Heparin And A Heparin-Antithrombin Complex By A Rapid Two-Step Affinity Method
Authors: Jordan R , Zuffi T , Fournel M , Schroeder D
Published: July 2018

The tight binding affinity of antithrombin for heparin makes possible a relatively selective purification scheme based on salt elution from heparin-Sepharose. We have found, however, that purity can often be greatly increased if the elution is carried out with soluble heparin instead. This heparin can be removed from the antithrombin, either in whole or part, by a second affinity step on Concanavalin A Sepharose. The antithrombin, which binds to the matrix through its glycosidic moieties, retains its ability to bind heparin at physiological ionic strengths. Thus, the complex of antithrombin and heparin is readily isolated free of unbound heparin species. The complex can be eluted intact with low ionic strength buffers containing sugars which compete for binding to the lectin. Alternatively, the high activity heparin (400–500 units/mg) can be obtained separately by a 1 M NaCl wash which is then followed by a carbohydrate wash to obtain the purified antithrombin.

We have made certain preliminary biochemical and anticoagulant characterizations of these materials. Not unexpectedly, both the high activity heparin and its complex with antithrombin show significantly greater in vitro potency in comparison to unfractionated heparin. In vivo anticoagulant efficacy, as evaluated in a rabbit infusion model, confirmed the in vitro findings and further suggests some potential therapeutic benefit may be derived from infusion of a preformed heparin-antithrombin complex.
Familial Antithrombin III Deficiency Associated With Recurrent Arterial Thromboembolism
Authors: Hale C S , Mattson J C , Zuhlke J A
Published: July 2018

A 28-year-old male with a strong family history of thromboembolic disease sustained three arterial thromboembolic occlusions during a 7 mo. period. The patient had antithrombin III (AT III) levels of 19.2 mg/dl (N=17-30 mg/dl) by RID and an immediate (heparin activated) antithrombin level of 77% (N=88-120%). Crossed IEP showed normal electrophoretic mobility of the patient's AT III. Of 30 family members tested, 10 demonstrated decreased AT III by both immunologic and functional assays. Two children of the propositus and three children of the sister of the propositus were tested and none were found to be abnormal. Because of the unusual presence of arterial thrombotic events in this family, platelet function studies were performed on the propositus and on two AT III deficient family members. A 19-year-old brother of the propositus, with no history of thrombotic events but with 13 mg/dl AT III and 83% activity had normal platelet aggregation studies and demonstrated no evidence of hyperaggregability with suboptimal concentrations of aggregating agents. He did, however, demonstrate a slight increase in platelet adhesion in a collagen adhesion assay. The second family member, a 65 year-old aunt of the propositus with 13.7 mg/dl AT III and 75% activity was on Coumadin at the time of evaluation of platelet function. Platelet aggregation and adhesion studies were normal in this individual. The propositus was also tested while on Coumadin and showed no abnormality of platelet aggregation, no hyperaggregability and no increase in platelet adhesion.

In view of our experience, we recommend screening for AT III deficiency in patients with unexplained recurrent arterial thromboembolism as well as those with venous thromboembolism, especially if the family history is suggestive.
Antithrombin III (ATIII) And Fibrin(Ogen) Fragment E (FgE) In Diabetic Nephropathy
Authors: Chan Vivian , Yeung C K , Chan T K
Published: July 2018

We measured plasma and urine ATIII and FgE levels by specific and sensitive radioimmunoassays (RIA) in 25 patients with diabetic nephropathy (DN) (proteinuria > lg/day) and in 17 patients with non-diabetic glomerulonephritis (GN), matched for degree of proteinuria. Plasma ATIII in DN (mean ± SD, 19.37 ± 2.40 mg/dl) were lower than in diabetics without renal involvement (21.84 ± 2.86 mg/dl). Total urine ATIII was directly related to proteinuria and inversely to creatinine clearance. In GN patients, plasma ATIII levels were even lower (16.84 ± 3.78 mg/dl), but the amount of urine ATIII fell when creatinine clearance decreased to below 37 ml/min. Serum FgE levels were elevated in both groups and this was associated with increased total urine FgE excretion. In DN, serum and urine FgE were directly related to proteinuria but inversely to creatinine clearance, indicating an increase in intraglomerular fibrin deposition as the disease progressed. These findings suggest that in DN, intravascular thrombosis might play an intermediary role as mediator of glomerular injury. Furthermore, the monitor of urine ATIII and FgE reflected the severity of DN and could be useful indices of the progression of the disease.
Generation Of Platelet Aggregating Activity By Adsorption Of Low-Molecular-Weight Factor VIII- Related Protein To Colloidal Gold
Authors: Furlan M , Horisberger M , Perret B A , Beck E A
Published: July 2018

Both the ristocetin cofactor (VIII:Rcof) and the platelet aggregating factor (PAF) are preferentially associated with the largest complexes of human and bovine factor VIII, respectively. It is not known whether the functional deficiency of smaller species of factor VIII is due to their size or quality. Binding of small oligomers of factor VIII-related protein, isolated by gel filtration from cryoprecipitates or prepared by disulfide reduction of multimeric factor VIII, onto gold granules (diameter 20-30 nm) generated platelet aggregating activity; on the other hand, adsorption of large factor VIII multimers onto gold particles impaired the initially high VIII: Rcof and PAF. Aggregation of human platelets by human factor VIII-coated gold particles required ristocetin and was not competitively inhibited by unbound low-molecular-weight factor VIII. We conclude that essential properties of factor VIII, necessary for its “von Willebrand activity”, are present potentially in the smallest species recovered from plasma or even after disulfide reduction. Factor VUI-coated gold granules can be used as an electron-dense label for surface receptors for factor VIII on human platelets.
On The Pharmacology Of Hirudin
Authors: Markwardt F
Published: July 2018

Hirudin, the anticoagulant substance obtained from medicinal leeches, was isolated and chemically characterized by us 20 years ago. In order to estimate the potential therapeutic effect, its pharmacodynamic and pharmacokinetic properties were studied in animal experiments. Pure hirudin is well tolerated and, except its anticoagulant effect, it proved to be a pharmacologically inert substance. According to its physico-
chemical properties as a polypeptide ( MW 7600) it is not absorbed enterally in active form and does not penetrate the skin. After parenteral administration hirudin distributes in extracellular fluid. It is eliminated with a half-life of 70 min and 80 % are excreted in unchanged form through the kidneys.

The specific inhibition of thrombin by hirudin in the blood prevents not only fibrinogen conversion but also the thrombin-catalyzed activation of other clotting factors and platelets. The antithrombotic effect was demonstrated in experimental animals (rat, rabbit, dog). The incidence of venous clotting thrombi induced by stasis and of arterial deposition thrombi induced by lesion of the vessel wall was prevented. Of special importance is the effect of hirudin to prevent
microthrombosis in DIC induced by infusion of thrombin or endotoxin. In this case hirudin is superior to heparin.

Pre-clinical testing showed that hirudin is a potential antithrombotic agent.
Kinetic Study Of Factor X (FX) : Potential Value For The Initiation Of Oral Anticoagulant Treatment
Authors: Fiessinger J N , Aiach M , Nussas C , Capron L
Published: July 2018

FX has been claimed to be of clinical relevance in monitoring long term treatment with anticoagulants. The aim of this investigation was to study the behaviour of FX at the beginning of the treatment.

Seventeen patients under heparin therapy received a daily dose of 3 mg of Acenocoumarol. Blood samples were obtained at 1, 2, 4, 6, 8 days (Dθ - 8), after starting the treatment. Heparin was stopped when prothrombin time (PT) was less than 40 per cent of normal value. FX was evaluated using an automated amidolytic method. Factor VII (FVII) and PT were measured in a clotting assay.

The disappearance of FX from plasma could be fitted to an exponential decay pattern during 6 days in 17 patients, 8 days in 11 patients. Concerning FVII, the exponential decay was limited to the first 2 days, then a stable level was reached. The decrease rates of FVII and FX during the first two days were correlated (r = 0,76). The regression line In (FX) = f (D) calculated from D0, D1, D2 could be used for predicting FX concentration at or D8. A highly significant linear correlation existed between the observed and predicted levels (D8 : r = 0,97) (D6 : r = 0,92).

These results suggest that FX amidolytic assay could be useful for monitoring the initiation of anticoagulant treatment. The decrease rate of FX could be a valuable guide for an early determination of the maintenance dose of anticoagulant.
Intra-Operative Antithrombin III, Plasminogen And α2 Antiplasmin Behaviour
Authors: Büller H R , Henny Ch P , Ritzen M , Kahlé L H , ten Cate J W
Published: July 2018

Previously we reported that major surgery induces a marked but transient decline (30%) of antithrombin III (AT III) and plasminogen (PLG) in the first post-operative days .α2AntiPlasm: in (α2AP) alone decreases on the day of operation. In tne study presented we focused on the intra-operative behaviour of AT III, PLG and α2AP in order to establish whether the decrease is gradual or that even lower values are reached during surgery, in different groups of patients. AT III was studied as a marker of activation of the coagulation system and PLG and α2AP as indicators of fiorinolytic activity. Blood samples were taken pre-operatively, every thirty minutes intra-operatively, and within one hour after surgery.

The observations were related to hematocrit, blood loss, transfusion and duration of surgery. It is concluded that major surgery is associated with a profound decline of AT III, PLG and α2AP, which may play an important role in the development of intra operative thrombosis.
The Effects Of Chymotrypsin And Of ADP On The Binding Site For Bovine Factor VIII On Human Platelets
Authors: Kirby Edward P , Mills David C B
Published: July 2018

The aggregation of human platelets by bovine Factor VIII (Platelet Agglutinating Factor-PAF) is inhibited by exposure of the cells to ADP or Chymotrypsin. We have investigated the mechanism of these effects using washed platelets. The washing procedure was modified from the method of Mustard et al. (Brit. J. Haematol. 22:193, 1972), omitting heparin and using a protein-free Tyrode’s solution for the final resuspension. The washed platelets were stable and responded to ADP (0.1-1 μM) with a shape change and, if fibrinogen was added, with aggregation. Bovine Factor VIII was purified to >90% homogeneity and was labeled with 125I (approx. 1 atom/subunit) by the IodoGen procedure, with no loss of activity. Aggregation was measured in the aggregometer in the presence of 7 mM EDTA. Binding was measured after incubation of labeled Factor VIII with washed platelets in the presence of 7 mM EDTA for 5 min at 37° without stirring.

Treatment of washed platelets with Chymotrypsin progressively destroyed their ability to bind Factor VIII and to be agglutinated by it. Responsiveness to Factor VIII disappeared before any alteration was detected in the ability of platelets to undergo ADP-induced shape change. Treatment of platelets with ADP, however, inhibited agglutination induced by Factor VIII without affecting the binding of Factor VIII to the platelets. Agglutination by wheat germ agglutinin or phytohemagglutinin was not inhibited by ADP treatment. We conclude that Chymotrypsin probably inhibits Factor VIII- induced agglutination by destroying the platelet binding site for Factor VIII, but that ADP must act at a point distal to Factor VIII binding. Agglutination of metabolically intact platelets by Factor VIII may not be a simple process, because ADP can specifically inhibit it without affecting Factor VIII binding or aggregation of the platelets by lectins.
Randomized Study Of Ticlopidine On Cerebral Vasospasm Following Ruptured Aneurysm
Authors: Mizukami M , Kikuchi H , Ono H , Taneda M
Published: July 2018

Based on our hypothesis for the mechanisms of chronic cerebral vasospasm following subarachnoid hemorrhage (SAH), we have made a randomized study of Ticlopidine in preventing cerebral vasospasm in 71 patients with ruptured aneurysm.

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.

Materials and Methods: Ticlopidine is a potent inhibitor of platelet aggregation and adhesiveness. The inhibitory nature of this drug is distinct from that of Aspirin which not only is an inhibitor of platelet aggregation butalso suppresses PGI2 generation in the vascular wall. Fourty one patients were given Ticlopidine (400 mg/day) and 30 patients served as a control group. All patients underwent direct intracranial operation and 46 of them were operated upon 4 days after SAH. High density which indicates subarachnoid clot was observed on pre- and postoperative CT scans in the majority of both groups.

Results: Mortality was 12% in Ticlopidine group and 17% in control group. As for morbidity, excellent results were obtained 57% in Ticlopidine group and 42% in control group in Grade 1-3. In order to know the effects of Ticlopidine on cerebral vasospasm and ischemic neurological deficit caused by vasospasm, 32 patients who did not show preoperative neurological deficit and underwent surgery within 4 days after SAH were analized in relation to the presence or absence of high density on postoperative CT. The development of postoperative vasospasm was almost same (71.4-76.5%) in two groups, but permanent ischemic neurological deficit was 28.6% in Ticlopidine group and 66.7% in control group when postoperative CT scan showed high density in subarachnoid space.

This preliminary randomized study indicates that Ticlopidine may be effective on prevention of postoperative ischemic neurological deficits due to vasospasm.
Ultrastructural Study Of Experimental Venous Thrombosis
Authors: Thomas D P , Merton R E , Hiller K F , Hockley D
Published: July 2018

Venous stasis is known to be a necessary but usually not a sufficient cause of venous thrombogenesis. The nature of the additional factor(s) required is uncertain, but the two most likely candidates are local generation of thrombin and vessel wall damage. Autopsy studies show that most venous thrombi develop in apparently normal vessels, although this evidence is based primarily on light microscopy. If thrombin generation is the additional factor, it is uncertain whether such thrombin acts solely on the blood or also damages the endothelium, leading to platelet deposition on exposed subendothelium. We studied the effect of locally injected thrombin on the jugular veins of rabbits, using transmission and scanning electronmicroscopy. One unit of thrombin was sufficient to clot blood contained in an isolated venous segment within a few minutes. The thrombus so formed was then embolised and autologous 11 indium-labelled platelets were injected. Blood flow was re-established for 30 minutes, following which the venous segment was fixed in situ and removed for study.

We found no evidence of significant vessel wall damage, as judged by ultrastructural studies or the deposition of labelled platelets. At least 10 u. of injected thrombin were required before the radioactivity in the thrombosed segment exceeded that found in a control vein. Even then, the endothelial lining appeared intact by electronmicroscopy. Following administration of aspirin (10 mg/kg) there was a marked increase in radioactivity present in the thrombosed venous segment, suggesting that inhibition of vessel wall cyclo-oxygenase had led to increased platelet deposition.
We conclude that a fresh stasis thrombus resulting from the direct action of thrombin on platelets and fibrinogen does not damage the endothelium, which appears to be relatively resistant in vivo to even high local concentrations of thrombin.
Chromogenic Substrates For Granulocyte Elastase, Chymo-Trypsin And The Subcomponents Of Cl-Esterase
Authors: Simonsson R , Arielly S , Friberger P , Aurell L
Published: July 2018

In order to improve and facilitate studies of various proteases and their possible interactions with the thromboembolic mechanisms, chromogenic substrates for granulocyte elastase, chymotrypsin, and Cls and Clr have been studied.

Substrates of the type Suc-Ala-Ala-Ala-pNA or Ac-Ala-Pro- Ala-pNA (S-2483) are useful for pancreatic elastase but not particularly sensitive towards granulocyte elastase. By placing valine in the PI position, the specificity requirements of granulocyte elastase are better satisfied. Thus pGlu-Pro-Val-pNA (S-2484) was found to be a suitable substrate for this enzyme. In tris buffer pH 8.2 I 0.40 Km was 0.4 mmol/1 with S-2484 and the substrate was approximately 30 times more sensitive than Suc-Ala-Ala-Ala-pNA.

The enzyme concentration of 4 nmol/1 gave a change in absorbance per minute of approximately 0.01.

In our search for water soluble chromogenic substrates for chymotrypsin, tripeptide derivatives of p-nitroanilin were synthezised where the aliphatic amino acid residues in the P3 position present in most chymotrypsin substrates earlier described were substituted with arginine. In the PI position both tyrosine, phenylalanin and tryptophan were used. The two substrates Sue- and Suc(OMe)-Arg-Pro- Tyr-pNA (S-2558 and S-2586 respectively) were found to be the most potent ones. In tris buffer pH 7.8 I 0.40 and with 3 mmol/1 of Ca2+ Km was 0.05-0.1 mmol/1 and kcat 50-60 per sec. This type of substrates would allow chymotrypsin assays of biological samples without the presence of organic solvents.

By studying purified preparations obtained from two different laboratories it was concluded that Cls and Clr have a similar specificity pattern towards pNA-derivatives of tripeptides. In all cases, the substrates split by these preparations have a C-terminal arginine residue. Some already known substrates seem to be sensitive enough to be utilized in biochemical studies of these enzymes.
FVIII:C Inhibitor Assays By Amidolytic Methods
Authors: Mackie I J , Seghatchian M J
Published: July 2018

Numerous problems are associated with the present assays of FVIII inhibitors, which give discrepant results between and within laboratories. We have attempted to remove some of the problems by using a FVIII assay based on FXa generation using a chromogenic substrate for end point detection instead of measuring a clotting time.

Nine plasmas from FVIII inhibitor patients were assayed several times by Bethesda and New Oxford methods, using both clotting and chromogenic FVIII assays. For the latter, the incubated samples were diluted in glyoxaline buffer and mixed with Diagen FVIII reagent and the synthetic substrate S2222. The reaction was left to proceed until suitable amounts of PNA were produced and then stopped with acetic acid. An azo dye technique was used to intensify the colour, which was measured at 540 nm, parallel line bioassays were then performed. The table summarises the inhibitor unitages (mean values).

A good correlation was obtained between clotting and amidolytic methods, except that the latter gave overall, slightly lower results. A marked discrepancy existed between Bethesda and New Oxford methods whichever type of FVIII assay was used. The chromogenic method therefore seems to measure a similar entity to clotting methods, and has the advantages that it may be modified in terms of incubation time and substrate concentration to suit the demand, and most importantly it requires minimal technician time. Most of the steps can be automated.