bluuebunny/crossref_metadata_2025 huggingface dataset:
Circadian Fluctuations of Plasminogen Activator Inhibitor and Tissue Plasminogen Activator Levels in Plasma of Patients with Unstable Coronary Artery Disease and Acute Myocardial Infarction
Authors: Huber Kurt , Rosc Danuta , Resch Irene , Schuster Ernst , Glogar Dietmar H , Kaindl F , Binder Bernd R
Published: June 2018

A decrease in the fibrinolytic potential, mainly due to an elevation of plasminogen activator inhibitor (PAI), has been described in patients with stable coronary artery disease and a previous myocardial infarction. We investigated plasma levels of PAI and tissue plasminogen activator (t-PA) and their possible circadian variations in patients with unstable coronary artery disease (CAD). Sixty-three patients were studied for at least 2 consecutive days during their stay at the coronary care unit (CCU). Diurnal plasma fluctuations in PAI and t-PA and onset of further myocardial ischemic episodes were monitored. As controls we used 22 age-matched patients submitted to the clinic because of non cardiac chest pain or valvular disease who revealed no evidence of CAD. PAI levels were significantly elevated in patients with unstable CAD (p < 0.0001) but were not influenced by the extent of underlying CAD, history of previous myocardial infarction, known risk factors for CAD, or by extent of myocardial damage. The circadian variation of PAI levels with peak values between midnight and 6 A. M. found in controls was still present in patients but at a higher level. Preservation of circadian pattern in PAI plasma levels despite myocardial ischemic attacks indicates that elevation of PAI is rather not caused by a reactive phenomenon. On the other hand, elevated PAI levels and episodes of severe myocardial ischemia exhibiting a median time of onset at 10 A. M. seem to be closely related.
Synthesis of Factor VIII in Human Hepatocytes in Culture
Authors: Ingerslev J , Christiansen B Sloth , Heickendorff L , Petersen C Munck
Published: June 2018

Although several investigators have attempted to identify the site of synthesis of factor VIII (FVIII), the cellular species responsible for maintenance of plasma FVIII has not been clearly defined. Indications point at hepatocytes and certain endothelial cells. The present study investigated the FVIII coagulant antigen (VIII : Ag) of hepatocytes obtained by two-step collagenase digests of human liver pieces. Following Percoll gradient centrifugation, less than 1% of cells harvested were non-parenchymal. Lysates of freshly isolated and purified hepatocytes contained 165–250 mU of VIII: Ag/106 cells as defined by a two-site ELISA employing a haemophilic antibody against human FVIII. This material contained a single peak of VIII: Ag polypeptides as jugded from the VIII: Ag ELISA profile of Mono-Q fast protein liquid chromatography fractions. A haemophilic antibody specific for epitopes of the light chain of FVIII, employed in immunoisolation of VIII : Ag in lysate of human hepatocytes, extracted a polypeptide pattern that was studied in a reduced SDS-PAGE electrophoresis gel and compared to that of immunoisolate from normal plasma. After electroblotting onto nitrocellulose and reaction with a monoclonal antibody towards the light chain of FVIII, the appearance of a doublet at 78–79 kDa in both these materials indicated the presence of the light chain of FVIII in human hepatocyte lysate. During culture, human hepatocytes secreted 20–80 mU of VIII: Ag per 1 × 106 cells per 24 hours. Further, a significant secretion of VIII: Ag was found in media of cultured human hepatoma cells, Hep-G2, whereas human blood monocytes and human fibroblasts did not secrete detectable VIII: Ag. In all of these cell cultures, vWf : Ag was indetectable or present as trace. Our results suggest that the human hepatocyte is a production site of FVIII.
Insulin Stimulates the Synthesis of Plasminogen Activator Inhibitor 1 by the Human Hepatocellular Cell Line Hep G2
Authors: Alessi M C , Juhan-Vague I , Kooistra T , Declerck P J , Collen D
Published: June 2018

Secretion of plasminogen activator inhibitor 1 (PAI-1) by cultures of human umbilical vein endothelial cells and human hepatocellular cell line Hep G2 was evaluated after insulin stimulation. The secretion of PAI-1 antigen and activity was measured in the conditioned medium and the cellular extracts after incubation of confluent cultures with 1% serum medium for 24 hours.

Insulin induced a dose dependent increase of the PAI-1 secretion by Hep G2 cell line. At 10-8 M a two fold increase of PAI-1 antigen and activity were observed whereas a2 antiplasmin and fibrinogen were not significantly modified. No effect of insulin was observed on PAI-1 antigen and PAI activity production by human endothelial cells whereas endotoxin resulted in a two fold increase in PAI-1 secretion. In recent clinical studies we have demonstrated that the level of plasma insulin correlated with that of PAI-1. Thus we hypothesize that hepatocytes represent a physiological source of plasma PAI-1 which is modulated by plasma insulin level.
Platelet Agglutinating Protein p37 Causes Platelet Agglutination through its Binding to Membrane Glycoprotein IV
Authors: Lian Eric C-Y , Siddiqui Farooq A , Jamieson G A , Tandon Narendra N
Published: July 2018

A 37 kDa platelet agglutinating protein (PAP p37) has previously been shown to be present in a subset of patients with thrombotic thrombocytopenic purpura and has been purified from their plasma. Using solubilized platelet membrane proteins from normal donors, it was shown by Western blotting that r2sI-p37 bound to a membrane protein of 97 kDa (red/unred). Furthernore, the same protein was identified by reverse immunoblotting in which purified p37 was electrophoresed, transferred to the nitrocellulose sheet and incubated with solubilized normal platelet membrane proteins. The complex formed between p37 and the membrane protein was identified by autoradiography using polyclonal and monoclonal (OKM5) anti- GPIV antibodies, but was not detected by polyclonal antibody to GPIIIa. Similar studies with purified platelet GPIV under both reducing and non-reducing conditions demonstrated the binding of 125I-p37. Polyclonal and monoclonal antibodies to GPIV completely inhibited the platelet agglutination induced by TTP plasma containing p37, however, normal rabbit IgG, rabbit anti- GPIIIa IgG, and murine monoclonal anti-GPIIb/IIIa (10E5) antibodies had no effect. These data indicate that platelet GPIV is the receptor site for PAP p37.
A Monoclonal Antibody to the Human Platelet Glycoprotein II b/III a Complex Induces Platelet Activation
Authors: Modderman Piet W , Huisman Han G , Mourik Jan A van , Borne Albert E G Kr von dem
Published: June 2018

The platelet glycoprotein (GP) IIb/IIIa complex functions as the receptor for fibrinogen on activated platelets. The effects of two anti-GPIIb/IIIa monoclonal antibodies on platelet function were studied. These antibodies, 6C9 and C17, recognized different epitopes, which were exclusively present on the undissociated GPIIb/IIIa complex. Whereas C17 inhibited the binding of fibrinogen to platelets and platelet aggregation induced by adenosine diphosphate (ADP) or collagen, 6C9 caused irreversible aggregation of platelets, both in the presence and absence of extracellular fibrinogen. When incubated with unstirred (nonaggregating) platelets, 6C9 induced release of alpha and dense granule-constituents as well as binding of 125I-fibrinogen to platelets. The latter was evidently mediated in part by platelet-derived ADP, since it was inhibited to a large extent by apyrase, the ADP-hydrolyzing enzyme. F(ab’)2 fragments of 6C9 did not induce platelet-release reactions but caused (slow) aggregation of platelets in the presence of extracellular fibrinogen.

These results indicate that binding of an antibody to a specific site on the platelet GPIIb/IIIa complex may cause fibrinogen-mediated aggregation. The Fc part of the platelet-bound antibody appears to be involved in the induction of platelet release.
Platelet Function and the Bleeding Time in Progressive Renal Failure
Authors: Gordge M P , Faint R W , Rylance P B , Neild G H
Published: June 2018

Bleeding time and platelet function tests were performed on 31 patients with progressive chronic renal failure (CRF) due to non-immunological (urological) causes, and compared with 22 healthy controls. Patients were classified as mild (plasma creatinine <300 μmol/l), moderate (300-600 μmol/l) or severe renal failure (>600 μmol/l). Bleeding time was rarely prolonged in mild and moderate CRF and mean bleeding time significantly elevated only in severe CRF (p <0.005). Haematocrit was the only index which correlated with bleeding time (r = -0.40). Platelet counts, collagen stimulated thromboxane generation, and platelet aggregation responses to ADP, collagen and ristocetin were all either normal or increased in all three CRF groups, but thromboxane production in clotting blood was reduced. Plasma fibrinogen, C reactive protein and von Willebrand factor (vWF) were elevated in proportion to CRF. We found no evidence that defects in platelet aggregation or platelet interaction with vWF prolong the bleeding time in patients with progressive CRF.
Haemophilia A in a Female: Study of a Family Using Intragenic and Extragenic Restriction Site Polymorphisms
Authors: Piétu G , Thomas-Maison N , Sié P , Larrieu M J , Meyer D
Published: June 2018

Restriction fragment length polymorphisms(RFLPs) were studied in a large Algerian family which includes 6 haemophiliacs and a previously described case of female haemophilia A. The female propositus is 66 years old with a normal karyotype. Her parents are first cousins. Her 3 sons are haemophiliacs and her 3 daughters with affected children are obligate carriers. The proband has an excessive bleeding tendency and markedly reduced levels of F. VIII (VIII C 0.03 U/ml, VIII Ag 0.01 U/ml) with elevated vWF Ag (2.30 U/ml), similar to the levels observed in affected males from the family. Four RFLPs can be identified by Southern blotting after digesting genomic DNA with the restriction enzymes Bcl I, Bgl I, Kpn I/Xba I and Taq I and hybridization with a 647 bp Stu I/Sca I F. VIII genomic probe, a 1.8 Kb EcoRI F. VIII cDNA probe, a 1.0 Kb EcoRI/Sst I fragment of intron 22 and the extragenic probe ST 14, respectively. With these four RFLPS, the propositus was found to be homozygous for the alleles segregating in this family with the abnormal X-chromosome. The carrier status was proven in a granddaughter and excluded in another. In conclusion, this RFLP linkage analysis is another argument to suggest that the propositus, a rare case of female haemophilia, is homozygous for the abnormal gene.
Investigation by HPLC of the Catabolism of Recombinant Tissue Plasminogen Activator in the Rat
Authors: Harris Roy , Frade Louis Garcia , Creighton Lesley J , Gascoine Paul S , Alexandroni Maher M , Poole Stephen , Gaffney Patrick J
Published: June 2018

The catabolism of recombinant tissue plasminogen activator (rt-PA) was investigated after injection of radiolabelled material into rats. Both Iodogen and Chloramine T iodination procedures yielded similar biological activity loss in the resultant labelled rt-PA and had half lives in the rat circulation of 1 and 3 min respectively. Complex formation of rt-PA was investigated by HPLC gel exclusion (TSK G3000 SW) fractionation of rat plasma samples taken 1-2 min after 125I-rt-PA injection. A series of radiolabelled complexes of varying molecular weights were found. However, 60% of the counts were associated with a single large molecular weight complex (350–500 kDa) which was undetectable by immunologically based assays (ELISA and BIA) and showed only low activity with a functional promoter-type t-PA assay. Two major activity peaks in the HPLC fractions were associated with Tree t-PA and a complex having a molecular weight of ̴ 180 kDa. HPLC fractionation to produce these three peaks at various timed intervals after injection of 125I-rt-PA showed each to have a similar initial rate half life in the rat circulation of 4-5 min. The function of these complexes as yet is unclear but since a high proportion of rt-PA is associated with a high molecular weight complex with a short half life in the rat, we suggest that the formation of this complex may be a mechanism by which t-PA activity is initially regulated and finally cleared from the rat circulation.
Anti-thrombin III and Heparin Clotting Times in Thrombosis and Atherosclerosis
Authors: O’Brien J. R
Published: June 2018

A survey of the literature shows that when whole blood clots thrombin is formed and there is a decrease in anti-thrombin III (Anti-Th. Ill) and an increase in heparin neutralizing activity (HNA) which is probably identical to platelet factor 4 derived from platelets. Many studies of atherosclerosis and of arterial and venous thrombosis using various tests thought to measure Anti-Th. Ill and HNA report a decrease in Anti-Th. Ill or an increase in HNA or both. We have measured both in patients with atherosclerosis and survivors of myocardial infarction. The HNA was increased and the serum anti-thrombic activity was decreased relative to controls and there was an inverse correlation between the two measurements. All this evidence suggests that some kind of mild chronic intravascular coagulation may occur in atherosclerosis. These changes could be related to the cause or the result of atherosclerosis.
Alteration of the Kinetics of Thrombin-catalyzed Hydrolysis of Amino Acid Ester Substrates by Sodium Cholate and Other Steroids
Authors: Cole Edmond R.
Published: June 2018

Thrombin-catalyzed hydrolysis of TAME proceeds by an initial zero-order phase which later falls off into an apparent first order reaction as substrate becomes limiting. Optimum amounts of sodium cholate not only accelerated TAME hydrolysis but also altered its kinetics to apparent zero-order to complete substrate hydrolysis. As this implies, the rate of hydrolysis in the presence of cholate was found to be independent of substrate concentration, provided concentrations of TAME and cholate were low enough to prevent precipitation of some TAME-cholate as an insoluble complex. The formation of a soluble complex composed of polymeric molecules of TAME and cholate may explain both the acceleration and the change in reaction order. Although the pH and temperature optima for TAME hydrolysis by thrombin were not altered by the presence of cholate, the degree of acceleration increased with rising pH and temperature on the ascending portion of the curves. This is believed to be due to the greater solubility of the TAME-cholate complex. The effects of cholate on thrombin-catalyzed hydrolysis of other arginine esters as well as esters of lysine, histidine and phenylalanine were also studied.

Solutions of sodium desoxycholate and androsterone-3-sulfate accelerated TAME hydrolysis as did supensions of testosterone, etiocholanolone, androsterone, androsterone-3-hemisuccinate and pregnandiol-3-glucuronidate. However, isoandrosterone, progesterone, pregnandiol, estradiol, estrone, estriol, estrone-3-sulfate, cholesterol, corticosterone, hydrocortisone and hydrocortisone-3-phosphate had no significant effect on TAME hydrolysis by thrombin. The ability of the androgenic hormones to accelerate hydrolysis appeared to depend to some extent on the configuration of the substituent group at C3 and the hydrogen at C5. Androsterone-3-hemisuccinate was, like cholate, able to accelerate the hydrolysis of TAME at apparent zero-order kinetics to complete substrate hydrolysis.
Effect of Dextran on Experimental Venous Thrombosis in Rabbits
Authors: Ah-See A.-K , Arfors K.-E , Bergqvist D , Tangen O
Published: June 2018

An experimental model combining controlled stasis and endothelial destruction by sodium morrhuate has been used to investigate the effect of dextran on venous thrombus formation in rabbits. Dextran 70 (1 g/kg body weight in a 10% solution), given 3 hours before thrombosis induction, significantly reduced the incidence of venous thrombosis when compared to a saline control. Dextran 40 (1 g/kg body weight in a 10% solution) and dextran 70 (0.6 g/kg body weight in a 6% solution) had no significant effect under these conditions. When given 10 or 120 minutes before initiation of thrombosis none of the dosages of dextran had any preventive effect. The results indicate that the haemodynamic effects of dextran were not involved in its preventive effect in this study. The effect of dextran was probably due to a combination of an effect on platelet reactivity and a change of the structure of fibrin formed during growth of the thrombi.
Dose-response Curves in the Fibrin Plate Assay Fibrinolytic Activity of Proteases
Authors: Haverkate F , Traas D. W
Published: June 2018

In the fibrin plate assay different types of relationships between the dose of applied proteolytic enzyme and the response have been previously reported. This study was undertaken to determine whether a generally valid relationship might exist.

Trypsin, chymotrypsin, papain, the plasminogen activator urokinase and all of the microbial proteases investigated, including brinase gave a linear relationship between the logarithm of the enzyme concentration and the diameter of the circular lysed zone. A similar linearity of dose-response curves has frequently been found by investigators who used enzyme plate assays with substrates different from fibrin incorporated in an agar gel. Consequently, it seems that this linearity of dose-response curves is generally valid for the fibrin plate assay as well as for other enzyme plate bioassays.

Both human plasmin and porcine tissue activator of plasminogen showed deviations from linearity of semi-logarithmic dose-response curves in the fibrin plate assay.
Acute Deep Vein Thrombosis Treated with Porcine Plasmin
Authors: Storm O , Ollendorff P , Drewsen E , Tang P
Published: June 2018

The thrombolytic effect of pig plasmin was tested in a double blind trial on patients with deep venous thrombosis in the lower limb. Only patients with not more than three days old thrombi were selected for this study. The diagnosis of deep vein thrombosis was made clinically and confirmed by phlebography. Lysofibrin Novo (porcine plasmin) or placebo (porcine plasminogen) was administered intravenously to the patients. The enzyme and the placebo were delivered as lyophilized powder in labelled bottles - the contents of the bottles were unknown to the doctor in charge of the clinical administration of the trial. An initial dose of plasmin/plasminogen of 30 unit per kg body weight given slowly intravenously (1-1% hours infusion) was followed by a maintenance dosis of 15 per cent the initial dose per hour for the following 5-7 hours. In most cases a similar maintenance dosis was given the next day. In all patients heparin was administered after ending the plasmin/plasminogen infusion. The results of the treatment was evaluated clinically as well as by control phlebo- grams the following days.

A statistically significant improvement was found in the plasmin treated group compared with the placebo (plasminogen) treated group. Thrombolysis was obtained clinically and phlebographically in 65 per cent of the plasmin treated group, but only in 15 per cent of the control patients were improvements found.

This study has thus demonstrated that plasmin treatment according to a standard scheme was able to induce thrombolysis. There were only a few and insignificant side effects. Allergic reactions have not been seen and only very simple tests are required.
Efleet of Endotoxin on the Activity of Coagulation Factors VII, VIII and IX Produced during Organ Perfusion
Authors: Dodds W Jean , Evensen Stein A.
Published: June 2018

The effect of bacterial endotoxin on the production of factor VII, VIII and IX activities in isolated rabbit livers, spleens and kidneys was investigated. The organs were perfused for 4 hours with fluid devoid of coagulation activity and containing either endotoxin or glucose-saline diluent. A series of parallel experiments included an initial hour of perfusion to ensure removal of tissue-stored coagulation activity, and perfusion with platelet-rich plasma and protein synthesis inhibitors. Endotoxin increased factor VII production in all organs but especially in livers not subjected to the initial perfusion; this effect was abolished by actinomycin D and puromycin. Endotoxin had little effect on hepatic factor VIII and IX activities but it inhibited production of both factors in the kidney and of factor VIII in the spleen. Initial perfusion prevented this inhibition of splenic factor VIII activity. These data suggest that endotoxin increases factor VII synthesis and causes consumption of factors VIII and IX in perfused kidneys and, to a lesser extent, spleens. Also, initial perfusion appears to prevent the subsequent consumption of splenic factor VIII by endotoxin. This response may involve activation of some tissue-bound or stored factor(s) or may be due to removal of an inhibitor substance.
Severe Factor V Deficiency with Prolonged Bleeding Time
Authors: Bkeederveld Kees , van Royen Eric A. , ten Cate Jan W.
Published: June 2018

A patient with two congenital defects, i.e. a severe factor V deficiency and an aortic coarctation is described. Arguments for a dominant mode of inheritance of the factor V deficiency are presented. A coinciding haemostatic defect consisting of a prolonged bleeding time, which could be corrected by fresh plasma and cryoprecipitate, and a decreased platelet retention in the glass bead test disappeared after surgical correction of the coarctation.

Extensive platelet studies failed to show any further defect.
Lysosomal Enzyme Activities in Platelets of Normal Individuals and of Patients with Gaucher’s Disease
Authors: Yatziv S , White M , Eldor A
Published: June 2018

Five platelet lysosomal enzyme activities were estimated in 22 normal individuals and in 5 patients with Gaucher’s disease: ß-D-galactosidase, N-Acetyl-ß-D-glucosa-minidase, N-Acetyl-ß-D-galactosaminidase acid-phosphatase, and ß-D-glucuronidase.

In the 5 patients with Gaucher’s disease the specific activities for the first four of these enzymes were significantly higher than in normal individuals. The activity of acid-phosphatase, on the other hand, was lower in G. d. than in normals. The release of the 5 lysosomal enzymes from platelets upon incubation with thrombin was also examined in the same two groups. The only lysosomal enzyme that was not released from normal platelets was acid-phosphatase. In Gaucher’s patients, on the other hand, acid-phosphatase was invariably released from platelets when incubated with thrombin. The release of the other four enzymes from platelets of Gaucher patients was similar to that of normal controls.
Frog Thrombocytes Aggregation and the Release Reaction
Authors: Stiller Ronald A. , Belamarich Frank A. , Shepro David
Published: June 2018

Frog thrombocytes were shown to aggregate in response to thrombin, collagen, and adenosine triphosphate (ATP). Microscopic observations indicated that collagen and thrombin induced aggregates were cell-to-cell in nature and not due solely to passive adherence to collagen or fibrin strands.

The ability of ATP to promote aggregation suggested that the release of this nucleotide might be the mechanism by which frog thrombocytes aggregate ; however, there was no evidence to support this hypothesis. Enzymatic hydrolysis of ATP by hexokinase had no effect on thrombin-induced aggregation, and ATP could not be identified in the supernatants of aggregated thrombocytes. Nevertheless, the ability of these cells to aggregate in response to these aggregating agents indicates that significant parallels may exist between platelet aggregation and the aggregation of non-mammalian thrombocytes.
Structure-Activity Relationships for the Inhibition of Plasmin and Plasminogen Activation by Aromatic Diamidines and a Study of the Effect of Plasma Proteins on the Inhibition Process
Authors: Geratz J. D
Published: June 2018

1. Structure-activity relationships for the inhibition of human plasmin were established for a large series of aromatic diamidines. The compounds are reversible competitive inhibitors and block the amidase and fibrinolytic activities of the enzym. The results confirm pentamidine (4,4’-diamidino α ω-diphenoxy-pentane) as the leading inhibitor (Ki = 3.3 (μM) and show distinct differences in the inhibitory spectrum of diamidines against plasmin as compared with trypsin, pancreatic kallikrein and thrombin.

2. Diamidines are potent inhibitors of the SK-dependent activation of human plasminogen and of the activation of bovine plasminogen by the SK-human plasmin activator complex. Pentamidine is again the most powerful inhibitor of these systems.

3. In fibrinolytic assays of plasmin and in plasminogen activation tests the relative strength of diamidines as compared with E-ACA is greatly influenced by the test conditions. The decisive factor is the presence in the incubation mixtures of lesser or greater amounts of plasma or serum proteins which bring about a fall in the absolute strength of diamidines and an increase in the absolute strength of E-ACA. In the fibrinolytic assay of plasmin, this modifying effect of added serum is based on a time- dependent interaction with the enzyme, thereby presumably altering its susceptibility to inhibition.
Calibration of Five Different Thromboplastins, Using Fresh and Freeze-dried Plasma
Authors: Bangham D. R , Biggs R , Brozović M , Denson K. W. E
Published: June 2018

The results of this trial confirmed that the five different thromboplastin preparations could be calibrated in terms of thromboplastin 67/40 (2) in respect to their sensitivity to coumarin induced defect.

Using thromboplastin 2 (67/40) as reference and assigning to it, by definition, a TSR of 2.0, the other four thromboplastins can be assigned the following TSRs: thromboplastin 1 (69/223) 1.9: thromboplastin 3 (68/434) 2.0; thromboplastin 4 (70/115)2.0; and thromboplastin 5 (70/178) 1.5.

In view of the great amount of collaborative work which has been invested in the study of these preparations, they should be kept primarily for the purposes of calibrating other reference preparations rather than be consumed as working standards. Thromboplastin 67/40 should be preserved for calibrating future batches of an international reference material or of national reference preparations of a similar type. The other four preparations could be used to calibrate similar types of thromboplastin: human brain without added bovine factors (69/223) for calibration of similar human thromboplastins; the bovine thromboplastin (68/434) for bovine preparations, and the two different rabbit brain preparations (70/115) and (70/178) for calibration of the rabbit brain reagents. In this manner each of the preparations would be compared to a material of the same species and similar type, and the stocks of these materials conserved to last over a longer period of time.

Carefully prepared freeze-dried pooled plasma from patients on oral anticoagulants can be used instead of a large number of individual fresh plasmas to simplify the calibration procedure of selected thromboplastins.

In the present state of knowledge about the coumarin induced coagulation defect, artificially prepared abnormal plasmas appear to have little use in the calibration of thromboplastin preparations for control of anticoagulant therapy.
Neuraminidase Injections in Rabbits
Authors: Grøttum A. K , Jeremic M
Published: June 2018

Two types of neuraminidase from CL perfringens were injected intravenously in rabbits. Blood platelet electrophoretic mobility was markedly reduced 4 hours after the injection of the enzyme. A marked thrombocytopenia developed gradually with a mean lower level of 55 and 27% of the preinjection number 48 hours after the injec- - tion. White blood cell count and factors V and VIII were reduced.

The results indicate that rabbit platelets with a reduced surface charge5 due to cleavage of sialic acid from the platelet membrane, are not immediately aggregated, trapped or destroyed in vivo. We suggest that the enzyme-induced platelet membrane changes rendered the platelets more susceptible to destruction in the reticuloendothelial system with appearance of thrombocytopenia after some hours.
Intravascular Coagulation in Experimental Acute
Authors: Wardle E. N
Published: June 2018

The occurrence of intravascular coagulation and fibrin deposition in the kidneys in situations known to produce acute renal failure has been assessed quantitatively by radio-fibrinogen studies. Apart from the Shwartzman reaction a primary process of fibrin deposition in the kidneys due to intravascular coagulation has been found in glycerol haemoglobinuric nephrosis, BSA glomerulonephritis and some cases of placental abruption and tourniquet shock, and as a secondary process in mercuric chloride intoxication. Fibrin in glomeruli may explain in part the oliguria of acute renal failure and fibrin deposition in the peritubular circulation be the cause of tubular necrosis. Inhibition of fibrinolysis in the course of the defibrination of glycerol nephrosis and placental abruption actually caused renal cortical necrosis.
Molecular Variant of Factor VII
Authors: Briët Ernest , Loeliger Emil A. , van Tilburg Nico H. , Veltkamp Jan J.
Published: July 2018

Two patients with severe factor VII deficiency appeared to be different with respect to the presence of inactive factor VH-like material in their plasma.

The inactive material, demonstrated in one patient’s plasma and adsorbable onto BaSO4, inhibits the thrombotest-system and is capable of neutralizing a heterologous antibody against factor VII in a fluid phase system.
The Clotting of a Snake (Crotalus Viridis Helleri) Plasma and its Interaction with Various Snake Venoms
Authors: Denson K.W.E
Published: July 2018

The clotting of C. V. Helleri plasma is not accelerated by the factor X activator or throm-bin-like enzymes from its own venom. Clotting of the plasma is accelerated by the factor X activator from Russell’s viper venom, but not by the thrombin-like enzyme from Agkistrodon Rhodostoma venom (“Arvin”).

The prothrombin activator from the Taipan venom clots C. V. Helleri plasma equally well as human plasma, but the thrombin which is produced has a marked specificity for its own fibrinogen, and clots bovine fibrinogen more slowly.

C. V. Helleri plasma contains an inhibitor which progressively inactivates bovine factor Xa and thrombin, but the inhibitor is not potentiated by heparin. The slow, protracted clotting of the snake plasma either alone or when mixed with human plasma or bovine fibrinogen suggests that this inhibitor may interfere with the polymerisation of fibrin monomer.
Increased Protein Synthesis by Human Platelets during Phagocytosis of Latex Particles in Vitro
Authors: Bessler Hana , Agam Galila , Djaldetti Meir
Published: July 2018

A three-fold increase of protein synthesis by human platelets during in vitro phagocytosis of polystyrene latex particles was detected. During the first two hours of incubation, the percentage of phagocytizing platelets and the number of latex particles per platelet increased; by the end of the third hour, the first parameter remained stable, while the number of latex particles per cell had decreased.

Vincristine (20 μg/ml of cell suspension) inhibited platelet protein synthesis. This effect was both time- and dose-dependent. The drug also caused a decrease in the number of phagocytizing cells, as well as in their phagocytotic activity.
Treatment of Amyloidosis Associated Factor X Deficiency
Authors: Spero Joel A. , Lewis Jessica H. , Hasiba Ute , Ellis Lawrence D.
Published: July 2018

This is the tenth patient in thirteen years to be reported with the findings of an isolated factor X deficiency associated with primary amyloidosis. A favorable response to factor IX concentrate was manifested by temporary clinical and laboratory correction of her diathesis. This mode of treatment, therefore, provides an approach to therapy for bleeding complications in this group of patients who have previously failed to respond to fresh frozen plasma.
The Dissociation of Factor VIII by Reducing Agents, High Salt Concentration and Affinity Chromatography
Authors: Peake I. R , Bloom A. L
Published: July 2018

Incubation of a factor Vlll-rich fraction of plasma with a high concentration of salt confirmed the production of both high (HMW) and low (LMW) molecular weight factor VIII clotting activity (FVIIIC) as determined by agarose gel filtration but with considerable overlap. The electrophoretic mobility of factor VIII related protein (FVIIIRP) detected by precipitating rabbit antiserum was not affected by this treatment and LMW FVIIIC devoid of FVIIIRP was apparently produced. At low concentration the reducing agent dithiothreitol (DTT) altered the electrophoretic mobility of FVIIIRP. At higher concentrations it altered both its mobility and antigenicity and an LMW FVIIIRP was produced. Contrary to the findings of other workers no LMW FVIIIC devoid of FVIIIRP was produced. In further studies factor Vlll-rich plasma fraction was treated with sepharose beads to which had been coupled a non-coagulation inhibitory precipitating rabbit antibody to FVIIIRP. Both FVIIIRP and FVIIIC were taken up by the beads but after elution with 1.5 M NaCl, FVIIIC of LMW and devoid of FVIIIRP was selectively removed. Antisera raised to LMW FVIIIC produced with 1.5 M NaCl either by the gel filtration or affinity chromatography methods inhibited FVIIIC and precipitated with HMW factor Vlll-rich fractions. The results were consistent with the possibility that factor VIII clotting activity and FVIIIRP exist in plasma as a non-covalently bound complex.
Reactivity of Fibrinogen Crosslinking Sites in the Absence of Thrombin
Authors: Seelich Thomas , Furlan Miha , Beck Eugen A.
Published: July 2018

The reactivity of fibrinogen crosslinking sites with thrombin-free, preactivated factor XIII (F. Xllla) was investigated under different conditions such as increased ionic strength, presence of urea, protamine sulfate (PS) or of varying concentrations of monodansylcadaverine (MDC). Crosslinking and incorporation of MDC into fibrinogen or fibrin γ- and α-chains were evaluated by SDS-Polyacrylamide gel electrophoresis.

According to our results, rates of crosslinking of, and of MDC incorporation into, both γ-and α-chains of fibrinogen were low under physiological conditions; they were not significantly influenced by the presence of either 1.0 M NaCl or 1.0 M urea. In contrast, 0.01 % PS precipitated fibrinogen, and, simultaneously, significantly increased the rates of crosslinking and of MDC incorporation into both γ- and α-chains. MDC, at concentrations above approximately 6 mM, also precipitated fibrinogen, and, up to a concentration of about 9 mM, markedly enhanced the reactivity of acceptor crosslinking sites.

Our results suggest that solubility of fibrinogen and the conformational arrangement of its subunit chains are closely interdependent; the reactivity of crosslinking sites with F. Xllla seems to be a function of this conformational state.
Action of Coagulation Factors on Experimental Thrombogenesis and Their Changes after Thrombosis
Authors: Marbet G. A , Duckert F
Published: June 2018

The action of ellagic acid, serum and acetylsalicylic acid on the frequency of thrombosis has been studied in a modification of the thrombosis model of Blake et al. (1959). Thrombi were formed in the external jugular veins of the rats by irritation with a formalin-methanol solution. The rate of thrombosis is highest with ellagic acid and lowest with acetylsalicylic acid. None of the groups of treated rats differed significantly from the control group. A short activation of coagulation with either ellagic acid or serum does not greatly enhance the thrombosis rate. The action of acetylsalicylic acid in the prophylaxis of thrombosis is not very convincing. Species differences may play a role.

Exhaustive coagulation studies before and after operation showed in each group a specific behaviour of the coagulation factors. There were significant differences between thrombosis positive and thrombosis negative animals. With ellagic acid, however, there was no difference between animals with and without thrombosis.
A Simple Indirect Method to Evaluate Platelet
Authors: de Gaetano G , Vermylen J , Verstraete M
Published: June 2018

A simple indirect method to evaluate platelet aggregation is described: the Clot Retraction Inhibition (CRI)-test, based on the observation that clot retraction is inhibited by previous in vitro platelet aggregation ; a highly significant correlation exists between the inhibition of clot retraction and each of the three parameters of aggregation curves which have been evaluated by turbidometry.

The CRI-test is more sensitive in certain circumstances (thrombocytopenia, hyperlipaemia, contamination with red or white cells), than turbidometry.

The CRI-test can also be satisfactorily performed on citrated total blood.
Measurement of the Platelet Response to Laser- induced Microvascular Injury
Authors: McKenzie F. N , Arfors K.-E , Matheson N. A
Published: June 2018

A study has been made of the biochemical factors underlying the platelet response to laser-induced microvascular injury. A platelet aggregating substance is produced at sites of laser-induced injury which markedly stimulates platelet activity at a site of injury inflicted a short distance downstream. Distal sites of injury are not similarly influenced if the distance between the injuries is increased or if the proximal site no longer shows platelet-stimulating activity. The stimulating effect of an adjacent proximal injury on platelet activity at a distal site is inhibited by local intra-arterial infusion of adenosine. Measurements of arterial blood pressure and microvascular blood flow velocity during adenosine infusion showed that its inhibitory effect on platelet activity is largely independent of its vasodilator properties. The effect of infusion of different adenosine phosphates (AMP, ADP, ATP) was also studied. Very small amounts of ADP markedly stimulated platelet activity and the emboli formed were similar to those normally produced at sites of laser injury. At high concentration AMP inhibited while ATP stimulated platelet activity in vivo. The results emphasise the fundamental role of ADP as a mediator of the platelet response at sites of laser- induced microvascular injury.
Effects of Vitamin E Administration on Platelet Function and Serum Lipid Peroxides in DOCA-Salt Hypertensive Rats
Authors: Umegaki Keizo , Saegusa Hiromi , Kurokawa Masato , Ichikawa Tomio
Published: July 2018

Effects of vitamin E on platelet function and serum lipid peroxide levels were investigated in DOCA-salt hypertensive rats. In the hypertensive rats, ADP- and collagen-induced platelet aggregation in whole blood were markedly attenuated and accompanied by a reduction of serotonin content as compared with the normotensive controls. These facts indicated the appearance of exhausted platelets, which have already been activated in vivo, due to the hypertension. Platelet vitamin E levels were decreased by 50%, while serum lipid peroxide levels were increased 3.6-fold in the hypertensive rats. Vitamin E administration (10 times the dietary intake) during the experimental periods did not influence either the aggregability or the serotonin content of platelets from the hypertensive rats. However, vitamin E administration significantly prevented the elevation of serum tipid peroxides due to the hypertension. These results suggest that vitamin E administration has little effect on platelet activation in vivo due to DOCA-salt hypertension.
Recombinant Tissue Factor as Substitute for Conventional Thromboplastin in the Prothrombin Time Test
Authors: Tripodi Armando , Arbini Arnaldo , Chantarangkul Veena , Mannucci Pier Mannuccio
Published: July 2018

Relipidated recombinant tissue factor (r-TF) has been assessed in comparison with conventional rabbit brain thromboplastin (Manchester Reagent) for its suitability for measurement of prothrombin time (PT). The International Sensitivity Index (ISI) of r-TF calibrated against the International Reference Preparation BCT/253 (human plain) was found to be 0.96 and 1.12 with instrumental and manual techniques. Our study of plasmas from patients with congenital deficiencies of clotting factors covering a wide range of severity demonstrates that r-TF is able to detect even minor deficiencies of factors involved in the extrinsic and common coagulation pathways. Patients with liver diseases were correctly diagnosed with a prevalence of abnormal results comparable for both reagents. Between-assay reproducibility expressed as coefficient of variation was 2.3 % and 3.9 % at normal and abnormal PT levels.

In conclusion, our evaluation shows that relipidated r-TF possesses the necessary requisites of sensitivity, diagnostic accuracy and reproducibility which make it a suitable candidate for PT determination both for monitoring oral anticoagulant therapy and diagnosing congenital and acquired clotting factor deficiencies. Moreover, being a highly defined reagent it may constitute a step forward in the standardization of PT testing.
Haplotype Analysis of Identical Factor IX Mutants Using PCR
Authors: Green Peter M , Montandon A Jane , Ljung Rolf , Nilsson lnga Marie , Giannelli Francesco
Published: July 2018

We have detected the mutations in the factor IX genes from all of the haemophilia B patients registered at Malmo haemophilia centre (45) and are currently examining the entire UK haemophilia B population. From these studies we have found 13 base substitutions which have recurred in 1-6 other, presumably unrelated, patients. In order to determine the minimum number of independent repeats of each mutation we have used PCR to examine the five factor IX polymorphisms forming the most informative combinations and we have characterised the haplotype of each patient. Patients with different haplotypes are assumed to be unrelated and thus to carry independent mutations. All but one of the 13 mutations occur in at least 2 haplotypes thus pinpointing 12 mutational hotspots and mutations that can be clearly considered detrimental. Two of the 13 substitutions occur at non-CpG sites.
Reaction of Acetaldehyde with Human Platelets
Authors: Spertini Olivier , Hauert Jacques , Bachmann Fedor
Published: July 2018

Platelet function defects observed in chronic alcoholics are not wholly explained by the inhibitory action of ethanol on platelet aggregation; they are not completely reproduced either in vivo by short-term ethanol perfusion into volunteers or in vitro by the addition of ethanol to platelet-rich plasma. As acetaldehyde (AcH) binds to many proteins and impairs cellular activities, we investigated the effect of this early degradation product of ethanol on platelets. AcH formed adducts with human platelets at neutral pH at 37° C which were stable to extensive washing, trichloracetic acid hydrolysis and heating at 100° C, and were not reduced by sodium borohydride. The amount of platelet adducts formed was a function of the incubation time and of the concentration of AcH in the reaction medium. At low AcH concentrations (<0.2 mM), platelet bound AcH was directly proportional to the concentration of AcH in the reaction medium. At higher concentrations (≥0.2 mM), AcH uptake by platelets tended to reach a plateau. The amount of adducts was also proportional to the number of exposures of platelets to pulses of 20 pM AcH.

AcH adducts formation severely impaired platelet aggregation and shape change induced by ADP, collagen and thrombin. A positive correlation was established between platelet-bound AcH and inhibition of aggregation.

SDS-PAGE analysis of AcH adducts at neutral pH demonstrated the binding of [14C]acetaldehyde to many platelet proteins. AcH adduct formation with membrane glycoproteins, cytoskeleton and enzymes might interfere with several steps of platelet activation and impair platelet aggregation.

This in vitro study shows that AcH has a major inhibitory action on platelet aggregation and may account for the prolonged ex vivo inhibition of aggregation observed in chronic alcoholics even in the absence of alcoholemia.
Rapid Isolation of Human Endothelial Cells from Whole Blood Using S-Endo1 Monoclonal Antibody Coupled to Immuno-Magnetic Beads: Demonstration of Endothelial Injury after Angioplasty
Authors: George F , Brisson C , Poncelet P , Laurent J C , Massot O , Arnoux D , Ambrosi P , Klein-Soyer C , Cazenave J P , Sampol J
Published: July 2018

The presence in whole blood of circulating endothelial cells (EC) has been a subject of debate for many years. It could represent a good marker of vessel injury. We demonstrate here that human endothelial cells can be directly isolated and identified in circulating blood by means of an endothelial cell specific monoclonal antibody, S-Endol, coupled to micromagnetic beads. The specificity and efficacy of the assay were established using normal blood samples with cultured EC added. Specific rosettes formed between EC and beads could subsequently be isolated with a magnet. The rosetted cells were recovered with a yield >80%. Their endothelial origin was confirmed by the positive labelling of von Willebrand factor and thrombomodulin, as well as the presence of Weibel-Palade bodies. We applied this method to demonstrate significantly increased levels of EC in venous and arterial human blood samples in patients undergoing heart catheterization. This new whole blood immuno-separation method may be useful in determining endothelial cell injury in vascular disorders.
Processing and Characterization of Recombinant von Willebrand Factor Expressed in Different Cell Types Using a Vaccinia Virus Vector
Authors: Meulien P , Nishino M , Mazurier C , Dott K , Piétu G , Jorieux S , Pavirani A , Girma J P , Oufkir D , Courtney M , Meyer D
Published: July 2018

The cloning of the cDNA encoding von Willebrand factor (vWF) has revealed that it is synthesized as a large precursor (pre-pro-vWF) molecule and it is now clear that the prosequence or vWAgll is responsible for the intracellular multimerization of vWF. We have cloned the complete vWF cDNA and expressed it using a recombinant vaccinia virus as vector. We have characterized the structure and function of the recombinant vWF (rvWF) secreted from five different cell types: baby hamster kidney (BHK), Chinese hamster ovary (CHO), human fibroblasts (143B), mouse fibroblasts (L) and primary embryonic chicken cells. Forty-eight hours after infection, the quantity of vWF antigen found in the cell supernatant varied from 3 to 12 U/dl depending on the cell type. By SDS-agarose gel electrophoresis, the percentage of high molecular weight forms of vWF varied from 39 to 49% relative to normal plasma for BHK, CHO, 143B and chicken cells but was less than 10% for L cells. In all cell types, the two anodic subbands of each multimer were missing. The two cathodic subbands were easily detected only in BHK and L cells. By SDS-PAGE of reduced samples, pro-vWF was present in similar quantity to the fully processed vWF subunit in L cells, present in moderate amounts in BHK and CHO and in very low amounts in 143B and chicken cells. rvWF from all cells bound to collagen and to platelets in the presence of ristocetin, the latter showing a high correlation between binding efficiency and degree of multimerization. rvWF from all cells was also shown to bind to purified FVIII and in this case binding appeared to be independent of the degree of multimerization. We conclude that whereas vWF is naturally synthesized only by endothelial cells and megakaryocytes, it can be expressed in a biologically active form from various other cell types.