bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhanced “In Vitro“ Release of Platelet α-Granules After Acute Myocardial Infarction (AMI)
Authors: Pengo V , Boschello M , Prandoni P , Schiavon R , Bellotto F , Schivazappa L , Dalla Volta S
Published: July 2018

In vivo platelet activity was studied in 58 patients with AMI on admittance to the Coronary Care Unit, in 48 of these patients after 1 week, in 30 after 1 month and in 24 patients after 6 months. Patients were carefully selected and excluded if they had associated diseases known to increase platelet activity. In vivo activation was studied by evaluating the plasma concentration of beta-thromboglobulin (β-TG) and platelet factor 4 (PF4) in the same blood sample. On admittance (x = 58.5) and on day 7 (x = 52.9) β-TG values were significantly higher than those obtained in the control group (x = 29). β-TG values were moderately elevated after 1 month (x = 37.7) and then returned to values similar to those of the control group after 6 months (x = 27.9). The simultaneous assessment of PF4 shows a β-TG/PF4 ratio indicative for in vitro release (≤2.5) in many patients on days 1 and 7. Moreover, the β-TG/PF4 ratio in patients with AMI tends towards 2 when β-TG values are high. These results may indicate a greater tendency to an in vitro platelet release in the acute phase of AMI.
Increased Tissue Factor Activity Generation In Vitro by Canine Blood Leukocytes Associated with Allogeneic Kidney Transplantation and Rejection
Authors: Rothberger Henry , Meredith Jesse , Mutton Tom , Brown Jeffery , McGee Maria P
Published: July 2018

Using a canine model, leukocyte populations enriched for monocytes and lymphocytes were isolated from blood during three week periods after kidney allotransplantation corresponding to episodes of acute rejection. Relative to controls, these cells incubated in vitro for five hours were found to generate increased amounts of PCA (procoagulant activity) characterized as tissue factor, the extrinsic clotting pathway activator. Controls included comparable blood leukocyte populations isolated from kidney autograft recipients and healthy animals. Differences in results for these two control groups were insignificant. These contrasts observed between allografted animals and controls demonstrate that leukocyte PCA generation is stimulated by the allogeneicity of histoincompatible kidneys rather than by direct effects of organ transplantation or non-specific postoperative effects. Results of in vitro transfer experiments provide evidence that cellular stimulation or induction in vivo accounted for the PCA increases observed. Stimulation of leukocyte tissue factor generation as a consequence of allogeneic kidney transplantation may in part acccount for coagulopathies and fibrin deposition during kidney rejection.
High Levels of Plasma Protein C in Nephrotic Syndrome
Authors: Pabinger-Fasching I , Lechner K , Niessner H , Schmidt P , Balzar E , Mannhalter Ch
Published: July 2018

In patients with severe nephrotic syndrome determinations of plasma protein C : Ag levels (8 patients: 5 adults, 3 children) and protein C activity (3 out of 8 patients) revealed significantly elevated plasma protein C concentrations. Furthermore we observed a significant inverse correlation of protein C : Ag to AT III : Ag levels. No protein C : Ag could be detected in the urine of two patients studied. We conclude from our data, that changes of plasma protein C do not contribute to the high thrombotic tendency in nephrotic syndrome.
Formation of Stable Complexes Between Urokinase and a Human Urinary Component
Authors: Cieplak Witold , Hart David A
Published: July 2018

Human urine was found to contain multiple species of urokinase (UK)-like plasminogen activator (PA) activity when subjected to concentration and/or dialysis and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and zymography. Untreated, freshly voided urine contained only Mr = 52,000 and 35,000 UK while dialyzed or undialyzed urine concentrates contained additional PA activity at Mr = 80,000 and 95.000. SDS-PAGE of incubation mixtures of radioiodinated UK (Mr = 52,000 or 35,000) and urine concentrates revealed the presence of radiolabeled complexes with MrS of 80,000 and 95.000. The bond(s) involved in complex formation was also relatively resistant to heat and reduction. Treatment of radioiodinated UK with the serine proteinase inhibitor, p-nitrophenyl guanidinobenzoate, prior to incubation with dialyzed, concentrated urine prevented formation of the complexes. In addition, the enzymatic activity of the Mr = 80,000 and 95,000 species was unaffected by diisopropyl fluorophosphate. These results indicate that UK forms SDS-stable complexes with a urinary component that has a Mr of approximately 40,000. The results further suggest that these complexes express PA activity when analyzed by SDS- PAGE and zymography.
Characterisation of Epitopes on Human Tissue Plasminogen Activator Recognised by a Group of Monoclonal Antibodies
Authors: MacGregor Ian R , Micklem Lisel R , James Keith , Pepper Duncan S
Published: July 2018

Seven mouse monoclonal antibodies have been produced against human melanoma tissue plasminogen activator (t-PA). They were specifically bound to 125I t-PA but not 125I urokinase (u-PA) and inhibited t-PA, but not u-PA, activity in plasminogen- rich 125I fibrin wells. Three of the antibodies directly inhibited the amidolytic activity of t-PA and the two most effective also bound near the active site histidine residue as determined by competition experiments using active site blocking agents. Several antibodies interfered with the fibrin binding properties of t-PA. One antibody neither interacted with the active site nor inhibited fibrin binding but still effectively quenched t-PA activity in fibrin wells suggesting that it masks another region of the molecule necessary for effective biological activity.
ADP Induced Depolarization of Human Platelet Membrane Potential
Authors: Wencel-Drake June D , Feinberg Harold
Published: July 2018

The transmembrane potential of human blood platelets suspended in plasma was investigated by studying the distribution of a radiolabeled permeant ion [14C] thiocyanate. The membrane potential of resting platelets was found to be -54.50 mV ± 9.23 S.D. with a range of -39 to -76 mV (n = 27). The possibility that platelet activation alters membrane potential or that changes in membrane potential serve as an activation trigger was investigated. Stimulation by ADP (10 ¼M) resulted in a significant (p :0.05) depolarization of the membrane potential. Preincubation with 6 mM EGTA failed to inhibit ADP-induced depolarization even though EGTA effectively prevented primary and secondary aggregation but not shape change. Preincubation with PGE1 inhibited shape change, aggregation, and the ADP- induced depolarization. No significant change in membrane potential was observed following stimulation by epinephrine (50 ¼M). These results suggest that the initial interaction of ADP and its receptor may involve an inward positive current which can be determined by thiocyanate distribution.
Measurement of Autoantibodies Against Fibrinogen and Fibrin Degradation Products by Enzyme-Linked Immunoassay
Authors: Whitaker A N , McFarlane J R , Rowe E A , Lee K , Masci P P
Published: July 2018

We have devised a simple enzyme immunoassay to detect and quantitate autoantibodies against derivatives of fibrinogen. This assay has been applied with a range of antigens including a fibrinogen lysate (containing X, Y, D and E), D dimer, D dimerE and a preparation of high molecular weight complexes derived from crosslinked fibrin. We have found that autoantibodies interacting with these antigens can be detected in varying concentrations in most sera from both normal subjects and patients with a variety of diseases and are evidently of mixed Ig class. These autoantibodies are directed against at least several cryptic antigens which appear during fibrinogen/fibrin degradation and some appear to be directed specifically against cross- linked fibrin derivatives. No clear disease correlates have yet emerged but a relationship between elevated levels and prior infective, thrombotic, inflammatory or traumatic disorders is likely. It is suggested that these autoantibodies may contribute to the catabolism of fibrinogen derivatives, provide a marker of thrombosis, and sometimes produce pathologic effects.
Maternal and Fetal Platelet Responses and Adrenoceptor Binding Characteristics
Authors: Jones C R , McCabe R , Hamilton C A , Reid J L
Published: July 2018

Paired blood samples were obtained from mothers (venous) and babies (cord venous blood) at the time of delivery by caesarean section under epidural anaesthetic. Fetal platelets failed to aggregate in response to adrenaline in vitro although adrenaline could potentiate the threshold response to adenosine diphosphate (1 μM). Fetal platelet responses to collagen and 8 Arg vasopressin did not differ significantly from maternal responses. Maternal and fetal platelets also showed similar inhibition of aggregation after activation of adenylate cyclase (PGE1 and parathormone), in contrast to the inhibition of adenylate cyclase by adrenaline.

Alpha2 adrenoceptors were investigated using [3H] yohimbine binding receptor number and were reduced modestly but significantly on fetal compared to maternal platelets. The failure of fetal platelet aggregation in response to adrenaline appears to be related to a failure of receptor coupling and may represent a delayed maturation of fetal platelet alpha receptors or a response- to increased circulating catecholamines during birth.
Measurement of Fibrinogen Concentrations in Suspensions of Washed Rabbit and Human Platelets by Radioimmunoassays
Authors: Harfenist Elizabeth J , Wrana Jeffrey L , Packham Marian A , Mustard J Fraser
Published: July 2018

Although fibrinogen is a cofactor in platelet aggregation, washed rabbit platelets aggregate when stimulated with ADP even when no fibrinogen is added to the platelet suspension. Washed human platelets usually do not aggregate to a significant extent when stimulated with ADP unless fibrinogen is added. To study this phenomenon, radioimmunoassays for rabbit and human fibrinogens have been developed and used to measure fibrinogen concentrations in suspensions of washed platelets. The fibrinogen concentration in the suspending medium of rabbit platelets was 2.5 ± 0.9 μg/109 platelets, and upon stimulation with 9 μM ADP it increased to 10.7 ± 2.9 μg/109 platelets. The loss of fibrinogen from the platelets was significantly greater than the loss of 14C-serotonin (11% vs 2%). The presence of prostaglandin E1 reduced the fibrinogen concentration to approximately 1 μg/109 platelets and prevented aggregation and loss of fibrinogen when the platelets were stimulated with ADP. With human platelets, the extracellular concentrations of fibrinogen and β-thromboglobulin, expressed as percentages of the amount in the platelets, were similar, and the increase in fibrinogen concentration upon ADP stimulation (approximately 2%) was much lower than with rabbit platelets. We conclude that rabbit platelets may release fibrinogen from their α-granules when stimulated with ADP, and that a portion of the released fibrinogen becomes available to support aggregation. Smaller amounts of fibrinogen would become available in the case of human platelets.
A Comparison of Heparin Potency Estimates Obtained by Activated Partial Thromboplastin Time and British Pharmacopoeial Assays
Authors: Merton R E , Curtis A D , Thomas D P
Published: July 2018

Heparin samples from five manufacturers were assayed by the revised British Pharmacopoeia (BP) heparin assay and the results compared with those obtained using the activated partial thromboplastin time (APTT) assay. The United States Pharmacopeia (USP) reference heparin preparation and the 4th International Standard (IS) for heparin were also assayed by the two methods relative to the 3rd IS. The results obtained by the revised BP assay were in close agreement with those obtained by the APTT assay for all the heparins that were tested. The assays revealed that there is at least a 10% discrepancy between the International Unit for heparin and the USP unit.
The Effect of Diabetes Regulation on Platelet Release, Fibrinolysis and Coagulation Tests, Before and After Stimulation with DDAVP
Authors: Knaap J H v d , Boer A C de , Pannebakker M A G , Heerde W van , Ottolander G J H den
Published: July 2018

In this longitudinal study we measured β-TG, PF4, fibrinolytic activity (extrinsic and euglobulin fraction), fibrinogen, FVIII RAg and FVIII Rcof before and after i.v. DDAVP (FPA was only measured before DDAVP) in 20 patients with diabetes mellitus. These parameters were measured on three occasions: phase I: during disregulation, phase II: after three weeks of strict control, phase III: after nine weeks of good control. Twenty-two healthy volunteers served as normal controls.

No significant differences related to metabolic control were found for β-TG, PF4, FPA and fibrinogen. There was no change after i.v. DDAVP administration. Fibrinolytic activity showed a significant increase after i.v. DDAVP. Baseline values and post-DDAVP increase were not significantly different from our normal controls.

FVIII RAg and FVIII Rcof were both significantly elevated in diabetes mellitus. Both increased significantly after DDAVP. The FVIII RAg release (ΔFVIII RAg) was significantly less in the diabetics. Fibrinolytic activity, FVIII RAg and FVIII Rcof are independent of the degree of metabolic control in patients with diabetes.
Purification of a Specific Placental Plasminogen Activator Inhibitor by Monoclonal Antibody and Its Complex Formation with Plasminogen Activator
Authors: Åstedt B , Lecander Ingegerd , Brodin T , Lundblad A , Löw Karin
Published: July 2018

A monoclonal antibody of IgG2a-type was obtained against a specific fast acting plasminogen activator inhibitor found in placenta. The placental inhibitor was purified by affinity chromatography using the monoclonal antibody and additionally in a FPLC-system. A strong complex formation was found between the inhibitor and urokinase and also with the two-chain form of plasminogen activator of the tissue-type. A weaker complex was found between the placental inhibitor and the one- chain form of the tissue-type activator.
Changes in Plasminogen Activator Activity and Plasmin Inhibition in the Pig During Experimental Hypovolaemia
Authors: Smokovitis A , Wagner M , Starlinger M , Opitz A , Binder B R
Published: July 2018

A markedly decreased plasminogen activator activity (PAA) was seen in lung, heart and kidney after experimental hypovolemia in the pig. In 2 of 12 cases, an increased PAA was found in liver and spleen. In skeletal muscle, skin, sciatic nerve and in 10 of 12 cases in liver and spleen PAA was unchanged. A slightly increased plasmin inhibition was noted only in the lung. These changes in tissue PAA were reflected by an increase in the PAA of euglobulin fractions concomitant with a decrease in antiplasmin activity in the blood samples obtained during the bleeding experiments.
The Application of a Monoclonal Antibody to Factor VIII Related Antigen (VIIIRAg) in Immunoradiometric Assays for Factor VIII
Authors: Thomas J E , Peake I R , Giddings J C , Welch A N , Bloom A L
Published: July 2018

A two site solid phase immunoradiometric assay (IRMA) for VIIIRAg has been developed using a monoclonal antibody as both the solid phase ligand and the radiolabelled antibody. A range of normal plasmas and von Willebrand’s disease (vWd) plasmas has been assayed for VIIIRAg by this method and compared with VIIIRAg values measured by an IRMA using polyclonal antibodies and by immunoelectrophoresis and with ristocetin cofactor activity (VIIIRiCoF). It was found that the monoclonal IRMA correlated well with the polyclonal IRMA and the immunoelectrophoretic assay, but the correlation with the VIIIRiCoF assay was relatively poor, in spite of the strong inhibitory effect of the antibody on VIIIRiCoF activity.
Comparison of the Interactions of Fibrinogen and Soluble Fibrin with Washed Rabbit Platelets Stimulated with ADP
Authors: Harfenist Elizabeth J , Packham Marian A , Mustard J Fraser
Published: July 2018

Because fibrin, formed at a site of vessel wall injury, is involved in the formation and stabilization of a platelet aggregate or thrombus, we have studied reactions of fibrin with rabbit platelets. Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization, was used to prepare soluble fibrin. Fibrin alone did not cause aggregation of washed platelets, but addition of ADP caused aggregation and deaggregation identical to those observed in the presence of fibrinogen. Specific binding of 125I-fibrin to ADP- stimulated platelets was similar to that of 125I-fibrinogen, but 125I-fibrin did not dissociate, even in the presence of high concentrations of apyrase. High non-specific binding of 125I-fibrin was observed that was not associated with aggregation. EDTA, prostaglandin E1 (PGE1) and creatine phosphate/creatine phos- phokinase prevented ADP-induced aggregation in the presence of fibrin and caused rapid deaggregation when added after ADP. They also inhibited 125I-fibrin binding when added before ADP, and EDTA or PGE1 caused partial dissociation of bound 125I-fibrin. In vivo, fibrin may bind to stimulated platelets, polymerize, form a gel, and interact with components of the plasma, the platelet aggregate, and the exposed subendothelium.
Effects of Oral Contraceptives and Obesity on Protein C Antigen
Authors: Meade T W , Stirling Y , Wikes H , Mannucci P M
Published: July 2018

In 24 women using oral contraceptives, protein C antigen was higher than in 24 women of the same age and of the same degree of obesity who were not using OC. In the pooled data for all 48 women, there was an increase in protein C of about 1% (of standard) for each 1.0 mm increase in skinfold thickness. Protein C tends to be high rather than low in circumstances predisposing to thrombosis.
Fibrinogen Fragments X, Y, D and E Increase Levels of Plasma Fibrinogen and Liver mRNAs Coding for Fibrinogen Polypeptides in Rats
Authors: Princen H M G , Moshage H J , Emeis J J , de Haard H J W , Nieuwenhuizen W , Yap S H
Published: July 2018

Previously, we demonstrated that in vivo regulation of liver fibrinogen synthesis occurs via the fibrinogen mRNA level. However, the molecular regulatory mechanism of fibrinogen synthesis is still not well understood. Fibrinogen or fibrin degradation products might play an important role in regulating fibrinogen synthesis. In our present study, we have injected rats intraperitoneally with purified homologous fragments and measured the liver content of mRNA specific coding for fibrinogen. Increased levels of fibrinogen mRNA and elevated plasma fibrinogen concentrations were observed in rats after administration of fibrinogen degradation products X, Y, DEGTA Dcate or E. Fragment E or E’ has a less stimulatory effect than X, Y or Dcate, whereas cross-linked fibrin degradation product D dimer does not increase fibrinogen synthesis. This article reports for the first time a stimulatory effect of the high molecular weight fibrinogen degradation products on fibrinogen synthesis.
Methods in Dose Response Platelet Aggregometry
Authors: Thompson S G , Vickers M V
Published: July 2018

A standardized method for performing dose response platelet aggregation has been developed, and has been used in about 1000 subjects in a large prospective study of ischaemic heart disease. A computer program has been used to fit a sigmoidal relationship between primary aggregation response rate and the log dose of aggregating reagent. Estimates of the parameters of the sigmoid curve are used to characterize an individual’s platelet aggregability. Methods for investigating the maintenance of long-term quality control have also been developed.
Human Platelet Aggregation and Release Reaction Induced by Platelet Activating Factor (PAF-Acether) – Effects of Acetylsalicylic Acid and External Ionized Calcium
Authors: Cattaneo Marco , Canciani Maria Teresa , Mannucci Pier Mannuccio
Published: July 2018

The effects of the cyclo-oxygenase inhibition on PAF-acether- induced human platelet aggregation and secretion are controversial. We studied the above parameters on citrated platelet-rich plasma of 12 normal subjects before and after the in vivo administration of acetylsalicylic acid (ASA). Individual sensitivities to PAF-acether were highly variable. ASA completely inhibited the platelet secretion induced by low concentrations of PAF-acether, but caused only partial inhibition when platelets were exposed to high concentrations of PAF-acether. The concentration of PAF-acether which overcame the cyclo-oxygenase inhibition varied substantially, depending on the individual sensitivity of the platelets to it. The addition of CaCl2 2 mM to the samples did not affect the extent of the platelet secretion, but increased irreversible aggregation in samples taken both before and after the ASA administration. These data suggest that low concentrations of PAF-acether stimulate the human platelet secretion by activating the cyclo-oxygenase pathway, whereas higher concentrations also trigger other mechanism(s) that suffice to induce human platelet secretion and full aggregation.
Cold Promoted Activation and Factor XII, Prekallikrein and C1-Inhibitor
Authors: Czendlik C , Lämmle B , Ducket F
Published: July 2018

During incubation of plasma in the cold an amidolytic activity due to the kallikrein-α2-macroglobulin complex appears in the plasma of about 40% of the women under hormonal contraception. The factor XII and prekallikrein activity are significantly increased 151.9% and 112.4% respectively in the cold promoted activation positive plasmas (CPA pos) whereas the activity of C1-inhibitor is decreased, 76%. The quotient of the product of the C1-inhibitor and α2-macroglobulin values divided by the product of the FXII and prekallikrein values is significantly lower in the CPA pos plasma 0.49 than in CPA neg plasma 0.96 (p <0.05). These results alone do not explain the cold promoted activation, since a patient with a C1-inhibitor as low as 9% showed no increase of the amidolytic activity after a 24 hr incubation at 4° C. However, the addition of purified C1-inhibitor to a CPA pos. plasma inhibits the cold activation. Heparin at a concentration of 0.5 IU/ml delays the appearance of the amidolytic activity.
Influence of Cyproheptadine on Endotoxin-Induced Disseminated Intravascular Coagulation (DIC) in Weaned Pigs
Authors: Nowak G , Markwardt F
Published: July 2018

Changes in the coagulation system typical of consumption reaction, microthrombosis in the lungs and other organs and haemodynamic disturbances caused by infusion of endotoxin in weaned pigs were prevented by treatment with the serotonin receptor antagonist cyproheptadine.
Nomenclature of Secreted Platelet Proteins – Report of the Working Party on Secreted Platelet Proteins of the Subcommittee on Platelets
Authors: Kaplan Karen L , Niewiarowski Stefan
Published: July 2018

Standard nomenclature for a number of secreted platelet proteins was agreed upon by The Working Party on Secreted Platelet Proteins of the Subcommittee on Platelets. Platelet factor 4 will continue to be used for the molecule with high heparin affinity, subunit molecular weight of 7780, and the described amino acid sequence. β-Thromboglobulin will be used to designate β-Thromboglobulin (81 amino acids/subunit, β-mobility on cellulose-acetate electrophoresis, pI 7), low-affinity platelet factor 4 (85 amino acids/subunit, y-mobility on cellulose-acetate electrophoresis, pI 8), and platelet basic protein (94 amino acids/ subunit, pI 10) when these are measured immunologically in plasma, but that thromboglobulin with a superscript designation of the pI should be used when assays are conducted on samples after isoelectric focusing, and a subscript amino-terminal amino acid can be added when a purified protein is described. Thrombospondin will continue to be the designation for the high molecular weight trimer that has previously been called thrombospondin or glycoprotein G. Platelet derived growth factor will be used for the group of closely related proteins of molecular weight about 30,000 and isoelectric point about 10.
Influence of Low Molecular Weight Heparin on the Hemostatic System After Abdominal Surgery
Authors: Schmitz-Huebner U , Rüschemeyer C , Schulte H , Loo J van de
Published: July 2018

The hemostatic effect of two low molecular weight heparin fractions and of one unfractionated heparin preparation administered subcutaneously b. i. d. was examined in 6 healthy subjects and in 53 patients after major abdominal surgery. Among other tests platelet count, prothrombin time, fibrinogen, β-thrombo- globulin, antithrombin, antiplasmin, FPA and F-CB 3 related antigen, as well as various heparin activities, were repeatedly determined pre- and postoperatively. Under all tested conditions the low molecular weight fractions induced higher heparin levels, both in terms of anti-Xa and of anti-thrombin activity. No further significant differences of the laboratory results between the treatment groups were documented. Total blood loss measured at the first postoperative day was higher in patients with malignancy and negatively correlated with antithrombin and antiplasmin levels, while no relation was observed with the heparin activities and the other tested parameters. Whereas evidence for a hemorrhagic property of the tested low molecular weight heparin fractions was found, a particular mechanism underlying this effect could not be identified.
The Effect of the Synthetic Steroid ORG OD14 on Fibrinolysis and Blood Lipids in Postmenopausal Women
Authors: Walker Isobel D , Davidson J F , Richards A , Yates R , McEwan H P
Published: July 2018

Two groups of postmenopausal women were compared. One group of 13 was given the synthetic steroid Org OD14 to suppress climacteric symptoms and one group of 14 was given placebo. After a 2 week baseline period the subjects received daily for 12 weeks, either 2.5 mg Org OD14 or placebo. There were no significant differences between the groups pre-treatment. Compared with the placebo group, during treatment, the Org OD14 group showed the following significant differences: higher haemoglobin, haematocrit, platelet count, plasminogen, fibrinolytic activity on fibrin plates and antithrombin III and lower fibrinogen. No significant differences between the groups were found in α2 antiplasmin levels, total cholesterol, total triglycerides, bilirubin or transaminase levels but the Org OD14 group had significantly lower levels of HDL cholesterol. With the exception of the haemoglobin and haematocrit levels all of the differences had disappeared by 2 weeks post treatment.
Identification of Platelet Inhibitor Present in the Melon (Cucurbitacea Cucumis Melo)
Authors: Altman Raul , Rouvier Jorge , Weisenberger Hans
Published: July 2018

An active fraction was isolated from an aqueous melon extract (Cucurbitacea cucumis melo) and was shown that it inhibits human platelet aggregation induced by epinephrine, ADP, collagen, thrombin, sodium arachidonate, prostaglandin endoperoxide analogue U-46619 and PAF-acether. Identification of the active substance as adenosine was indicated by TLC which gave identical Rf value compared to adenosine, by the UV spectrum, because the inhibitory effect on platelet aggregation disappeared after the addition of adenosine-deaminase and because the substance under study and adenosine produced the same spectra in the mass spectroscopy.
Persistence and Progression of Immunologic Abnormalities in Haemophilia
Authors: Gill Joan C , Wheeler Dana , Menitove Jay E , Aster Richard H , Casper James T , Kirchner Phyllis , Montgomery Robert R
Published: July 2018

Immunologic parameters in 51 patients with haemophilia treated with cryoprecipitate from volunteer donors or with commercial lyophilized concentrate were studied on 2–7 occasions. Concentrate users had significantly decreased mean T4 (helper)/T8 (suppressor) lymphocyte ratios when the initial (1.5 ± .43) and most recent (1.25 ± .60) values were compared (p <.001), whereas in cryoprecipitate users mean values remained normal. Of the 22 concentrate users with initially normal T4/T8 ratios, 10 became abnormal during the study, an incidence of 45%. Fifteen of 28 (54%) patients with abnormal T4/T8 ratios had elevated circulating immune complexes (CIC), whereas all 16 patients with normal T4/T8 ratios had normal levels of CIC. Six patients have persistent lymphadenopathy.
Studies on the Effect of Platelet Inhibitors on Platelet Adhesion to Collagen and Collagen-Induced Human Platelet Activation
Authors: Krishnamurthi S , Kakkar V V
Published: July 2018

The effect of pyridoxal 5’-phosphate (PALP) and trifluoperazine (TFPZ), the calmodulin antagonist, on in vitro platelet adhesion to collagen and collagen-induced platelet activation was studied using platelet-rich-plasma (PRP) or washed platelets (WPL). Platelet aggregation and [14C]-5HT release induced by “threshold” or low concentrations of collagen (0.6 μg/ ml) in PRP were completely abolished by PALP (24 mM), TFPZ (250 μM) as well as indomethacin (10 μM). At higher concentrations of collagen (10–15 μg/ml) in PRP and WPL, the use of stirred and unstirred platelets treated with collagen enabled a distinction to be made between aggregation and adhesion- mediated release reaction. Platelet aggregation and the aggregation-mediated release reaction induced by these concentrations of collagen in stirred platelets were completely abolished by PALP, TFPZ and indomethacin although neither adhesion to collagen nor the adhesion-mediated release reaction of unstirred platelets was significantly affected by these inhibitors. Interestingly, both adhesion and the adhesion-mediated release reaction were abolished by concentrations of PALP 10–40 fold higher than those required to abolish aggregation. Collagen-induced platelet aggregation, but not platelet adhesion, was inhibited in resuspended platelets pretreated with PALP and NaBH4 indicating a separation in the membrane sites involved in aggregation and adhesion. The results further emphasize the distinction between adhesion and aggregation-mediated events with regards to collagen with the latter being more susceptible to inhibition by antiplatelet agents such as PALP and TFPZ.
Percutaneous Treatment with a Mucopolysaccharide Polysulphate of Experimentally Induced Subcutaneous Haematomas in Man
Authors: Larsson B , Fianu S , Jonasson A , Forsskåhl B
Published: July 2018

In a double blind trial on ten healthy volunteers, the efficacy of a topically applied mucopolysaccharide polysulphate* in the treatment of subcutaneous haematomas was assessed. Standardized haematomas were induced by subcutaneous injection of a mixture of autogenous blood and 125I-fibrinogen. The absorption of the haematoma, expressed as the removal of the labelled fibrin(ogen), could thus be objectively measured by the decrease in radioactivity.

The results show an exponential absorption of these haematomas. The absorption time was reduced to about half compared with placebo, when treated topically with mucopolysaccharide polysulphate. The difference is statistically highly significant and is also considered clinically relevant.
Reactivity of Factor VIII Inhibitors in a Micro ELISA for Factor VIII : CAg and in Solid Phase Immunoisolation of VIII : CAg
Authors: Nordfang O , Ezban M , Dinesen B
Published: July 2018

Factor VIII coagulant antigen (VIII : CAg) was measured in a sandwich-ELISA. Microplates were used as solid phase and peroxidase conjugated F(ab’)2 fragments of IgG isolated from inhibitor plasma was used as label without affinity purification. The capacity of the assay was high and the sensitivity for VIII : CAg was 0.002 U/ml. Using this assay it was possible to measure coagulation inactive VIII : CAg in samples from purification studies. Below 0.05 VIII : CAg U/ml these samples responded in parallel with standard plasma.

Seven of 7 inhibitor antibodies tested were able to inhibit binding of peroxidase-conjugate in the VIII : CAg assay, and the inhibitory capacity correlated with coagulation inhibition as measured by the Bethesda method. Using the highest titered antibodies bound to a solid phase, VIII : CAg was isolated and identified in SDS-PAGE as a doublet with a molecular weight of 77-80 kD.
Studies on the Binding of Proteins to the Human Platelet Surface: Relation to Platelet Activation
Authors: Endresen Gerhard K M , Førre Øystein
Published: July 2018

Several antibody fractions and sera from patients with rheumatoid arthritis, systemic lupus erythematosus and chronic idiopathic thrombocytopenic purpura were examined for their ability to bind to normal platelets using immunofluorescent staining techniques. Platelet aggregometry was used to study the activating capacity of the samples.

Both C1q, C1s, C1 inactivator, fibrinogen, factor VIII-related antigen, alpha1-acid glycoprotein, alpha1-antitrypsin, beta2-micro- globulin and isoantigens A and B, as well as fibronectin and plasminogen were found on the platelet surface. Only antibodies to C1q, C1s and beta2-microglobulin were able to induce platelet aggregation. Sera containing immune complexes or platelet autoantibodies revealed positive surface staining for IgG, or for IgG and IgM. These sera also induced aggregation of platelets. Sera not containing immune complexes or autoantibodies gave negative staining and aggregation results. Thus, only some of the ligand receptor interactions were able to induce platelet aggregation.
Assembly of the Intrinsic Factor X Activating Complex – Interactions between Factor IXa, Factor VIIIa and Phospholipid
Authors: van Dieijen Gerbrand , van Rijn Jan L M L , Govers-Riemslag José W P , Hemker H Coenraad , Rosing Jan
Published: July 2018

The activation of blood coagulation factor X by factor IXa is strongly stimulated by the non-enzymatic cofactors phospholipid, Ca2+ and activated factor VIII. In this paper we present a method by which we were able to determine binding affinities of factor IXa for phospholipids (either in the absence or presence of factor VIIIa) from kinetic measurements of factor X activation. It is shown that rates of factor X activation in the presence of phospholipids can be saturated with an excess factor VIIIa at limiting amounts of factor IXa and vice versa. Our data indicate that the enzymatic unit in the intrinsic factor X activator is a 1:1 stoichiometrical complex of factor IXa and factor VIIIa bound to phospholipid. Titrations with factor IXa at fixed concentrations of phospholipid and factor X show that the apparent dissociation constant of factor IXa for phospholipid is lowered from 10-6 M to 10-8 M by the presence of factor VIIIa. We conclude, that in analogy with the role of factor Va in prothrombin activation, phospholipid-bound factor VIIIa functions as a high-affinity binding site (»receptor«) for factor IXa in the intrinsic factor X activating complex. Therefore, factor VIIIa increases the observed Vmax of factor X activation by 1) enhancing the kcat of the reaction and 2) increasing the amount of phospholipid-bound factor IXa that participates in factor X activation.
Kinetics of In-111-Platelets in the Baboon: I. Isolation and Labelling of a Viable and Representative Platelet Population
Authors: Kotzé H F , Lötter M G , Badenhorst P N , Heyns A du P
Published: July 2018

A fully representative and viable platelet population was isolated from the blood of 15 baboons by a multiwash procedure, and labelled with In-lll-oxine. The recovery of the total platelet population in the circulation was 85% ± 9. Mean platelet life span was 146 hr ± 13. Correcting for plasma radioactivity (always less than 3.5%) did not significantly affect the estimate of platelet life span (145 hr ± 16) or recovery (85% ± 12). Platelet survival estimates, repeated at different times, were reproducible. In 5 baboons, platelets were also harvested by a single step differential centrifugation. The mean life span of a representative platelet population was significantly longer than that of platelets harvested by a single step. Recovery values of the representative and non-representative population were similar. We conclude that it may be important to harvest and label a fully representative platelet population for kinetic studies. The proposed method is simple and reproducible, and may be applied in studies in humans.
Aspirin Kinetics and Inhibition of Platelet Thromboxane Generation-Relevance for a Solution of the “Aspirin Dilemma”
Authors: Cerletti Chiara , Latini Roberto , Maschio Aldo Del , Galletti Ferruccio , Dejana Elisabetta , de Gaetano Giovanni
Published: July 2018

Six healthy male volunteers received aspirin (ASA) in a compressed (320 mg) and an enteric-coated (800 mg) formulation as single oral doses ten days apart. Ten plasma samples were obtained from each volunteer between 5 and 120 min after compressed ASA, and seven between 10 and 240 min after enteric-coated ASA.

ASA was undetectable (less than 100 ng/ml) in plasma from three subjects receiving compressed ASA and two receiving the enteric-coated preparation. Plasma levels and kinetic parameters of salicylate were the same in subjects with undetectable and detectable ASA plasma levels.

More than 98% inhibition of pre-drug serum TXB2 was noted in all samples collected one and four hours after either ASA preparation. TXB2 generation recovered on average by 3.5% at 24 hr with both preparations.

Thus inhibition of platelet TXB2 generation occurred independently of the amount of ASA reaching the peripheral circulation.

If this is due to inhibition of platelet function in the enterohepatic circulation followed by extensive first-pass deacetylation of ASA, vascular PGI2 synthesis could be spared.

A better knowledge of the kinetic parameters of ASA for each of the formulations used in thrombosis prevention trials might help in solving the “aspirin dilemma”.
Studies of Suloctidil in Experimental Thrombosis in Baboons
Authors: Hanson Stephen R , Harker Laurence A
Published: July 2018

Suloctidil has been evaluated in the baboon for its antithrombotic efficacy using models of both acute and chronic arterial thrombogenesis. Acute thrombus formation was initiated by Dacron vascular grafts inserted as extension segments into chronic arteriovenous shunts. 111In-platelet deposition was measured by scintillation camera imaging for one hour. The results after oral administration of suloctidil (100 mg/kg/d in two divided doses) were not different from control studies. Moreover, concurrent heparin anticoagulation did not affect 111In-platelet deposition compared with control data. In contrast, ticlopidine (20 mg/ kg/d) significantly decreased platelet deposition that was reduced further by the addition of heparin.

Chronic arterial-thromboembolism was initiated by segments of polyurethane (Biomer) cannula introduced into chronic arteriovenous shunts. Thrombus formation by the polyurethane cannula was measured as 111In-platelet turnover (corrected for removal of senescent platelets). Cannula platelet consumption was unaffected by suloctidil (20 mg/kg/d given in two divided doses for two days preceding and throughout the period of platelet survival measurement). In contrast, dipyridamole (10 mg/ kg/d) and sulfinpyrazone (100 mg/kg/d) completely interrupted cannula platelet consumption.

We conclude that suloctidil probably has little or no effect on platelet-dependent thrombus formation.
Platelet Adherence and Detachment - In What Ways Is Surface-Bound Albumin Different from Surface-Bound Fibrinogen?
Authors: Feuerstein I A , Kush J
Published: July 2018

The adhesion and detachment of platelets on surface-bound albumin and surface-bound fibrinogen were studied. Fluorescent video-microscopy of platelets labelled with mepacrine was used to provide continuous information. Glass tubes (I.D. 1.3 mm) were precoated with either human albumin or human fibrinogen before exposure to a suspension of washed platelets and red cells. Observations were made 0.5 cm from the tube’s entrance over a 1370 μm2 portion of lumen. The rate at which cells leave the measurement area and the percent of initially attaching cells which leave are independent of protein coating but increase with flow rate. The percent of initially attaching platelets which permanently adhere is equal for both protein coatings but the pathways leading to this result can be different. For the lower shear rate studied, 80 s−1, the percent of cells which permanently adhere on first contact is less for albumin than for fibrinogen; the percent of initially attaching cells which adhere and then move before permanent adhesion is greater for albumin. The mechanism of detachment and reattachment leads to the equality of the overall adhesion efficiencies for the two protein coatings at 80 s−1. For the higher shear rate studied, 456 s−1 the adhesion pathways for both coatings were the same.
Platelet Function in Experimentally Induced Pancreatitis in the Dog
Authors: Jacobs R M , Murtaugh R J , Fertel R H
Published: July 2018

Evidence suggests that changes in prostaglandins and disseminated intravascular coagulation accompany pancreatitis. Both may induce changes in platelet function. We wished to determine if experimentally induced pancreatitis in the dog was associated with altered platelet number and function, and whether there were concomitant changes in prostaglandins. Evidence for disseminated intravascular coagulation in the dogs with pancreatitis were red blood cell fragmentation, increased platelet turnover indicated by macro-platelets and the transient presence of fibrin degradation products in urine. There were no significant changes in platelet count. The platelets from dogs with pancreatitis showed a functional defect characterized by significantly decreased aggregation in response to adenosine diphosphate, arachidonic acid, and collagen. Release of adenosine triphosphate from platelets was reduced in collagen-stimulated aggregation. There were no changes in the plasma concentrations of thromboxane B2, 6-Keto-PGF1a, and PGE2. This defect may have been due to the generation of fibrin degradation products and platelet “exhaustion”.
Studies on the Release of a Plasminogen Activator Inhibitor by Human Platelets
Authors: Kruithof E K O , Tran-Thang C , Bachmann F
Published: July 2018

The relative contribution of platelets to plasminogen activator inhibitor (PA-inhibitor) activity in blood was investigated. From the difference in PA-inhibitor levels in platelet-poor plasmas of 12 donors (3 ± 1 U/ml, mean ±95% confidence limits) and in the corresponding platelet-rich plasmas after induction of platelet aggregation by collagen, ADP or epinephrine (7 ± 1 U/ml), it may be concluded that a greater amount of PA-inhibitor in blood is associated with platelets than with plasma. In collagen-stimulated platelets maximal release of PA-inhibitor and of beta-thromboglobulin (β-TG) was attained within fifteen seconds, whereas in ADP-stimulated platelets the release of both factors was slower. In platelet-poor plasma no correlation was found between the level of PA-inhibitor and that of P-TG. Thus, the PA-inhibitor found in plasma is not derived from platelets that had been stimulated after blood collection. The rate of complex formation and the Mr of the principal complexes of radioiodinated tissue-type plasminogen activator (t-PA) or urokinase (UK), in platelet-poor plasma, in platelet-rich plasma after platelet aggregation or in an extract of washed platelets was the same. Moreover, complexes of UK or t-PA with plasmatic PA-inhibitor or with the PA-inhibitor(s) from platelets bound to immobilized antibodies against bovine endothelial cell-derived PA-inhibitor. These results show that the PA-inhibitors in plasma and in platelets are very similar or identical.
Antithrombin III Alger: A New Homozygous AT III Variant
Authors: Fischer A M , Cornu P , Sternberg C , Mériane F , Dautzenberg M D , Chafa O , Beguin S , Desnos M
Published: July 2018

A qualitative abnormality of antithrombin III (AT III) was found in the plasma of a 41-year old patient. The plasmatic AT III antigen concentration was 130% and the progressive anti-F IIa and anti-F Xa activities were normal (105% and 137%). The plasma heparin cofactor activity was less than 10%, when measured by F Ila or F Xa inhibition. Crossed immunoelectrophoresis of AT III in the presence of heparin revealed in the plasma an abnormal slow-moving peak. When tested by affinity chromatography on heparin Sepharose, this abnormal AT III did not bind to heparin. Among the investigated relatives, 5 subjects had normal AT III levels, whatever the test used, the nine others having reduced levels of antithrombin heparin cofactor activity (45-61%) but normal levels of immunoreactive AT III (97-122%). Consanguinity was found in the family history. We therefore considered our patient as homozygous for an AT III molecular abnormality affecting the binding site for heparin.
Peripheral Venous Plasma Aspirin Concentrations and Platelet Aggregation Inhibition Produced by Enteric-Coated Aspirin Formulations
Authors: Brandon Roslyn A , Emmett J A G , Eadie M J , Curran A C W , Bunce I H
Published: July 2018

In a random cross-over design, six healthy consenting adult volunteers were given on separate occasions single doses of 300-650 mg of 3 different formulations of enteric-coated aspirin. Over various intervals for 48-54 h following dosage, plasma aspirin and salicylate concentrations were measured together with percentage inhibition of platelet aggregation activated by threshold concentrations of sodium arachidonate alone and combined with ADP and collagen. In all subjects each formulation delivered measurable quantities of aspirin to the peripheral circulation, the unchanged drug being detected at various times up to and including 28 h after dosage. Moreover, low aspirin concentrations were found to co-exist with unimpaired platelet aggregation. All 3 formulations yielded statistically significant (P <0.01) inhibition of platelet aggregation activated both by arachidonate and by the combination of aggregants when tested 24-29 and 48-54 h after dosage; there were no significant differences (P >0.05) between the 3 formulations in this regard. Two different patterns of delivery of unchanged aspirin to the systemic circulation from these enteric-coated formulations were apparent. These patterns may be important when considering which aspirin formulation might be most appropriate in chronic use for an antiplatelet effect. None of the enteric-coated formulations used in this study may be optimal in this regard.