bluuebunny/crossref_metadata_2025 huggingface dataset:
The Effects of Bedrest on Circadian Changes in Hemostasis
Authors: Rosenfeld Brian A , Faraday Nauder , Campbell David , Sakima Neal , Bell William
Published: July 2018

Venous stasis occurs when people are at bedrest, because of altered venous flow characteristics. This is commonly believed to be one etiology behind the development of deep venous thrombosis (DVT). The hemostatic effects of bedrest and their possible role in DVT development have not been fully examined. We hypothesized that bedrest would lead to increases in hemostatic function and that these increases could be important in the development of DVT.

Twelve non-smoking volunteers were studied during supine positioning for 36 hours. Platelet reactivity and plasma concentrations of fibrinogen, a2-antiplasmin, plasminogen, thromboxane (32, plasminogen activator inhibitor-1, tissue plasminogen activator and neuroendocrine hormones (cortisol, epinephrine and norepinephrine) were measured at 8:00 a.m., 10:00 a.m., 4:00 p.m. and 8:00 a.m.

Cortisol demonstrated an early morning increase while catecholamines were unchanged throughout. Fibrinogen, a2-antiplasmin, plasminogen and platelet reactivity were no different at any time point. Fibrinolytic proteins changed over time, manifested by decreased PAI-1 antigen and activity levels at 24 h.

Based upon the parameters measured, bedrest causes no increase in hemostatic function. In fact, bedrest causes the potential for enhanced fibrinolysis, that differs from that previously reported for normal activity over 24 h. This may represent a protective mechanism to counter the effects of stasis from bedrest.
In Vivo Release of Tissue-Type Plasminogen Activator across the Human Forearm during Mental Stress
Authors: Jern Christina , Selin Lena , Jern Sverker
Published: July 2018

We have previously shown that plasma levels of endothelium-derived tissue-type plasminogen activator (t-PA) increase during mental stress. The aim of the study was to investigate in vivo release in an intact human muscle vascular bed. Eleven healthy young males (22-36 yrs) were studied at rest and during 10 min of mental stress (forced arithmetic). Net release or uptake were assessed by arterio-venous (AV) concentration gradients across the forearm of t-PA antigen and t-PA activity, and plasminogen activator inhibitor antigen type 1 (PAI-1). Forearm blood flow was measured by venous occlusion plethysmography. At rest, there was a positive AV-difference of t-PA activity across the forearm indicating a net release of t-PA activity of approximately 3.7 fmol × min−1 × 100 ml−1 (Wilcoxon’s signed rank test vs 0, p = 0.01). However, t-PA antigen showed a variable release pattern. On the average, there was a net release of 0.17 ng × min−1 × 100 ml−1 after 60 min of rest (Wilcoxon vs 0, p = 0.07). PAI-1 antigen showed net release at rest. In response to stress, forearm blood flow increased from 1.9 to 2.9 ml × min−1 × 100 ml−1 (ANOVA, p = 0.007), and net release of t-PA activity increased to 9.8 fmol × min-1 × 100 ml −1 (ANOVA, p = 0.01 compared with rest). Arterial and venous plasma t-PA levels also increased significantly during stress (ANOVA, p < 0.01). t-PA antigen showed a similar but less pronounced release pattern during stress. PAI-1 antigen was unaffected by stress. During i. a. infusion of norepinephrine (6-1,200 ng/min), forearm blood flow decreased from 2.1 till 1.0 ml × min-1 × 100 ml−1 (ANOVA, p = 0.002), while net release of t-PA activity increased from 2.7 till 10.1 fmol × min-1 × 100 ml−1 (ANOVA, p = 0.002). The study demonstrates a net release of t-PA activity across forearm tissues at rest. Net release of t-PA activity increases in response to mental stress and i. a. infusion of norepinephrine, despite disparate effects on blood flow.
Platelet Activation Induced by lnterleukin-6: Evidence for a Mechanism Involving Arachidonic Acid Metabolism
Authors: Oleksowicz Leslie , Mrowlec Zbignelw , Zuckerman Dina , Isaacs Randi , Dutcher Janice , Puszkin Elena
Published: July 2018

The effect of IL-6 on in vitro platelet function was investigated. Platelet-rich plasma (PRP) incubated with IL-6 showed a dose dependent enhancement of agonist induced maximum aggregation (AIMA) and secretion of thromboxane B2 (TXB2) as measured by RIA, in short term incubations. Dazoxiben (0.2 to 160 (µM) pretreated PRP incubated with IL-6 and aggregated with ionophore A23187, showed a dose dependent inhibition of TXB2 secretion concomitant with a dose dependent abrogation of IL-6’s enhancement of AIMA. A similar abrogation of AIMA was observed when these experiments were repeated using indomethacin. Further, PRP incubated with IL-6 showed a dose dependent increase in TXB2 and BTG secretion as measured by RIA and an increased incorporation of actin binding protein, talin, and myosin into the cytoskeletal core (triton insoluble residue) as shown by SDS-PAGE. The integrin glycoprotein Ilia (GPIIIa) was also observed to be retained into the cytoskeleton by immunoblot. These results suggest that IL-6 activates platelets in vitro and enhances AIMA via a mechanism involving arachidonic acid metabolism.
The Antiaggregating Activity of Clopidogrel Is due to a Metabolic Activation by the Hepatic Cytochrome P450-1A
Authors: Savi P , Combalbert J , Gaich C , Rouchon M-C , Maffrand J-P , Berger Y , Herbert J-M
Published: July 2018

Clopidogrel and ticlopidine are two well known selective anti-ADP agents which are inactive in vitro and must be administered in vivo to fully exhibit their antiaggregating and antithrombotic effects. Since previous studies have clearly demonstrated that the activation steps take place in the liver, we examined the effect of specific induction or inhibition of cytochrome P450 subfamilies on the antiaggregating activity of clopidogrel. SKF 525-A, a global cytochrome P450 inhibitor, dramatically decreased the antiaggregating effect of clopidogrel, therefore indicating that cytochrome P450 enzymes are involved in the hepatic activation of clopidogrel. The efficacy of clopidogrel was increased in animals pretreated with 3-methylcholanthrene and (3-naphthoflavone, indicating that the cytochrome P450-1A subfamily pathway was mainly involved in the activating metabolism of clopidogrel. The use of specific antibodies directed against the various cytochrome P450 subfamilies ascertained this observation.
Gel Filtration Properties of Factors II, VII, IX, IXa, and X
Authors: Østerud Bjarne , Schiffman Sandra
Published: July 2018

The following gel filtration properties of the vitamin ढ dependent clotting factors have been established :

1. The elution patterns of factors II, VII, IX, and X in 0.5 M NaCl are the same in factor XII deficient native plasma, anticoagulated plasma, and BaSO4 eluates.

2. In 0.01 M Tris buffer pH 7.3 or 0.5 M NaCl, plasma factors II, IX, and X elute together; factor VII élûtes later as a smaller molecule. All 4 plasma activities elute as larger molecules in 0.01 M Tris buffer and smaller ones in 0.5 M NaCl.

3. In 0.01 M Tris - 0.005 M EDTA peak factor II activity elutes first, followed by factor IX, factor X, and then factor VII.

4. Activation decreases the apparent size of factor IX.
Studies on Activator Formation in Human Plasma with Streptokinase
Authors: Martin M ,   with the technical assistance of Frau R. Albrecht
Published: July 2018

The activator concentrations developing during a streptokinase infusion treatment were measured on a quantitative basis. The reference value (100 per cent activator) selected for this purpose was the activator effectiveness resulting from a mixture of 20,000 units of streptokinase and 1 millilitre of citrate plasma. The streptokinase treatment regimens used for our study consisted either of a continuous streptokinase infusion treatment extended over three days or of intermittent streptokinase infusions with a 16-hour streptokinase-free interval. The highest activator concentrations measured had developed between 1 and 24 hours after the streptokinase treatment had started, the values being in the order of 0.4 per cent. When treatment was continued, the activator concentrations dropped below that level. Under the intermittent streptokinase regimen, the activator concentrations measured during the second series of infusions were always lower than those measured during the first series. The activator concentration levels and the plasminogen values measured simultaneously acted in a reciprocal manner, i.e. a plasminogen decrease corresponded to an activator increase and vice versa. Seen under the aspect of the obtainable maximum of activator effectiveness, the intermittent streptokinase infusion regimen offers no advantage as against the continuous treatment form. It would certainly be desirable to achieve higher activator levels through modifications in the streptokinase dosage.
The Effect of Phospholipase C on Platelet Factor 3 in Human Blood Platelets
Authors: Otnaess A-B , Prydz H
Published: July 2018

Intact human platelets isolated by gel filtration have been treated with purified phospholipase C. The effect of the enzyme on available and total platelet factor 3 has been tested.

The available procoagulant platelet factor 3 was very low. A further small reduction was observed after incubation with phospholipase C when the enzyme was washed away before testing.

External attack on platelets by phospholipase C led to a marked inactivation of total platelet factor 3.
A Comparison of Coagulation Factor Replacement with and without Prednisolone in the Treatment of Haematuria in Haemophilia and Christmas Disease
Authors: Rizza C. R , Kernoff P. B. A , Matthews J. M , McLennan C. R , Rainsford S. G
Published: July 2018

A double-blind controlled study was carried out to investigate the effectiveness of treatment with factor VIII or factor IX concentrate and a reducing dose of prednisolone in controlling haematuria in patients with haemophilia and Christmas disease.

41 episodes of haematuria were studied in 30 different patients. No appreciable benefit was observed in the treated, as compared with the control group and this is at variance with the results of the few studies reported elsewhere.
Apparent Similarity of Mechanisms of Platelet Adhesion and Aggregation
Authors: Mason R. G. , Read M. S. , Saba S. R. , Shekmek R. W.
Published: July 2018

A correlation between effects of a series of compounds on platelet adhesion, aggregation, and retraction of the clot, and their effects on platelet electrophoretic mobility, ecto-ATPase activities, and AChE activity has been sought. Compounds which inhibited aggregation induced by ADP or epinephrine also inhibited platelet adhesion to glass. Six compounds which inhibited adhesion and aggregation did not inhibit clot retraction. The data suggest that the effect of thrombin on platelet electrophoretic mobility and ecto-ATPase activities may have less influence on platelet function than was formerly believed. However, the effects of test compounds on platelet AChE activity suggest that this enzyme may play a role in aggregation and adhesion. Platelet adhesion and aggregation may operate by similar mechanisms.
Fibrinogen Metabolism in Healthy Dogs and in Dogs with CCI4-Induced Liver Damage and with Portal Vein Occlusion
Authors: Tytgat G. N. , Collen D. , Oei L. S. , Van Damme B.
Published: July 2018

The metabolism of dog fibrinogen, labelled with radioactive iodine was studied in 16 healthy dogs on 28 occasions, in 12 dogs with CCl4-induced chronic liver damage and in 5 dogs with interruption of the hepatoportal blood flow by occlusion of the portal vein.

Results in healthy control dogs were: plasma volume 48±12 ml/kg ; plasma fibrinogen concentration 316±102 mg%; total plasma fibrinogen pool 168±42 mg/kg, representing 0.72±0.08 of the total body pool; fibrinogen half-life 2.54±0.23 day; fractional catabolic rate 0.39±0.05 of the plasma pool per day; absolute catabolic rate 58±25 mg/kg per day.

Results in dogs with CCl4-induced liver damage were: plasma volume 46 ± 12 ml/kg ; plasma fibrinogen concentration 334±97 mg%; total plasma fibrinogen pool 152±55 mg/kg, representing 0.70±0.04 of the total body pool; fibrinogen half-life 2.15±0.26 days; fractional catabolic rate 0.47±0.06 of the plasma pool per day; absolute catabolic rate 71±21 mg/kg per day.

Results in dogs with longstanding portal vein occlusion were: plasma volume 41 ±8 ml/kg; plasma fibrinogen concentration 330±23 mg%; total plasma fibrinogen pool 142±36 mg/kg, representing 0.70±0.06 of the total body pool; fibrinogen half-life 2.60 ±0.36 days; fractional catabolic rate 0.40 ±0.04 of the plasma pool per day; absolute catabolic rate 56±14 mg/kg per day.

A significant difference between control dogs and dogs with liver disease was observed for the fibrinogen half-life and the fractional catabolic rate. All fibrinogen turnover data in dogs with portal vein occlusion were comparable to control values.

Heparinization did not influence the fibrinogen half-life in most of the control dogs and in the dogs with portal vein occlusion ; however in 2 control dogs, a prolongation of their rather short fibrinogen survival occurred during anticoagulation. In contrast, a general trend towards prolongation and occasionally normalization of the fibrinogen life span during heparinization was observed in the dogs with liver damage, supporting the concept of accelerated fibrinogen consumption by a process of disseminated intravascular coagulation in liver disease.
Schwere serogene hämolytische Anämie verursacht durch ein Anti-D im Faktor-VIII-Konzentrat
Authors: Schkickeb K. Th , Schrenk K. H
Published: July 2018

Es wird über eine schwere serogene hämolytische Anämie berichtet, die unter der Therapie mit antihämophilem Globulin bzw. Cohnscher Fraktion aufgetreten ist und durch einen Rh-Antikörper (Anti-D) hervorgerufen wurde. Da eine Sensibilisierung durch Bluttransfusionen und eine echte autoimmunhämolytische Anämie als ursächliche Faktoren der Hämolyse ausgeschlossen werden konnten, muß das Anti-D im antihämophilen Globulin oder der Cohnschen Fraktion enthalten gewesen sein. Der Patient besaß das Antigen D, so daß der direkt mit dem Konzentrat zugeführte Rh- Antikörper (Anti-D) eine schwere hämolytische Reaktion auslöste.
Etude en Microscopie électronique de différentes Etapes de la Fibrinoformation
Authors: Pouit L , Marcille G , Suscillon M , Hollard D
Published: July 2018

Nous avons étudié en microscopie électronique par la technique de coloration négative : la molécule de fibrinogène, les étapes intermédiaires de la fibrinoformation et la fibre de fibrine. Nous avons constaté que la molécule de fibrinogène se présentait sous forme d’éléments globulaires, à pH 8,3 et pour une force ionique de 0,2, le diamètre moyen mesure 240 Â. L’observation des molécules de taille variable (entre 180 Å et 420 Å) et de filaments très minces nous a conduit à émettre l’hypothèse d’une molécule capable de se dérouler sous certaines conditions physiques. L’ensemble des clichés observés suggère qu’au cours de l’organisation périodique de la fibre, le matériel protéique change de structure. Ce phénomène se manifeste par une diminution des éléments globulaires qui constituent les bandes transversales (de 280 Å à 30 Å) et le développement à partir de ces éléments d’un réseau de filaments longitudinaux, très denses, porteurs de fins granules dont l’alignement forment des sous striations transversales. Il se produit aussi une diminution de la période qui passe de 300 Å à 230 Å.
Platelet Aggregation by Staphylococcal Toxins
Authors: Jeljaszewicz J , Niewiarowski S , Popławski A , Bławat L
Published: July 2018

Aggregation by some staphylococcal toxins resulted in the following observations. Staphylocoagulase in lower concentrations causes aggregation in a similar way as thrombin, whereas in higher concentrations, clotting occurs which is always preceded by quick platelet aggregation. Alpha-hemolysin aggregates human and rabbit platelets, whereas sheep platelets are not affected. Preincubation of susceptible platelets with this toxin, results in their lysis. Betahemolysin causes also aggregation. Leukocidin is inactive. All these phenomena are well visible visually and in the phase contrast microscope. Staphylocoagulase and alpha-hemolysin, when added to the platelet-rich plasma simultaneously with the adenosine diphosphate, interfere and the platelet aggregation is partly inhibited. Pathological aggregation of platelets by bacterial products and its possible significance is discussed.
Effects of Soy Bean Trypsin Inhibitor on Fibrin Clot Lysis
Authors: Egeblad K
Published: July 2018

The effect of soy bean trypsin inhibitor (SBTI) on fibrinolysis and blood coagulation was investigated. Clot lysis was recorded by means of thrombelastography. SBTI delays fibrinolysis induced by plasmin and by activators of plasminogen (SK-activator and urokinase). Activator-induced lysis is delayed by a combined effect on activator and plasmin. There appears to exist an equilibrium between highly dissociated compounds of inhibitor with the active agents. The inhibitory effect of SBTI is relatively decreased in clots containing human plasma probably caused by an equilibrium with inhibitory agents in the plasma. SBTI delays thrombin generation in recalcified plasma as well as the effect of thromboplastin, but the effect is weak and requires a concentration 70-100 fold the antifibrinolytic active. The effect on thrombosis is even weaker.
Orgaran (Org 10172) or Heparin for Preventing Venous Thrombosis after Elective Surgery for Malignant Disease? A Double-Blind, Randomised, Multicentre Comparison
Authors: Gallus A , Cade J , Ockelford P , Hepburn S , Maas M , Magnani H , Bucknall T , Stevens J , Porteous F ,   (ANZ-Organon Investigators’Group)
Published: July 2018

This double-blind, randomised, multicentre trial in 513 patients having elective surgery for intra-abdominal or intrathoracic malignancy compared the efficacy and safety of venous thrombosis (VT) prophylaxis using 750 anti-factor Xa units of Orgaran (a mixture of low molecular weight heparinoids) given subcutaneously (sc) twice-daily with that of twice-daily injections of 5,000 units Standard heparin. The main study endpoints were the development of postoperative VT detected by 125I-fibrinogen leg scanning, and the onset of clinically significant venous thromboembolism or bleeding. “Intent to treat” analysis showed a statistically non-significant trend towards less VT during Orgaran prophylaxis (10.4%) than after heparin (14.9%) and there was no difference in bleeding complications between the two study groups. Results remained similar if only patients who completed the intended course of therapy (“compliant patients”) were analysed. Other trials have shown that Orgaran prevents VT after hip surgery and stroke. We now show it is also safe and effective in patients having major surgery for cancer.
Capillary Fragility Induced By Psychotropic Serotonergic Drugs
Authors: Shepro D , Hechtman H B
Published: July 2018

In defining a mechanism for the putative platelet support of microvascular structural integrity, we have shown that serotonin (5-HT) injected into thrombocytopenic animals prevents petechial formation for as long as 6 h, and that psychomotor drugs inhibit this palliative action. Our data suggest that a platelet’s role is to deliver- release 5-HT into the cutaneous microcirculation in amounts physiologically above plasma levels, which acts as a signal to maintain “endothelial tone”. In this study psychotropic drugs known to inhibit 5-HT uptake, such as imipramine, amitriptyline, fluoxetine, were studied for their effect on endothelial structural integrity in normal adult hamsters. Adrenergic receptors, inhibitors and catecholamines were also used in conjunction with psychotropic drugs. Bleeding times and plasma levels of 5-HT were quantitated. Following a single injection or daily injection for 21 days of all substances tested, petechial sensitivity increased significantly in normal hamsters. Norepinephrine decreased and propanolol enhanced petechial formation. Microhemorrhages were observed within 5 min post-injection with fluoxetine (4.5 × 10-3 mg⋅100 g-1) and with amitriptyline (0.04 mg.100 g-1). Petechiae occurred for one week following a single injection of fluoxetine, which indicates that the stable metabolite of this antidepressant is biologically active. All other parameters measured were normal except bleeding times, which increased three-fold compared to controls. In published reports, 5-HT has been shown to alter microfilament orientation, especially at junctions. Based upon this observation and the present data, we theorize that the blockade of a 5-HT signal by the uptake antagonist effects endothelial cytoskeletal support of junctions.
The Coagulant Active Phospholipid(Pl) Content Of Prothrombin Complex Concentrates(Pcc) Is A Critical Determinant Of Their In Vivo Thrombogenicity
Authors: Giles A R , Nesheim M E , Tracy P B , Hoogendoorn H , Mann K G
Published: July 2018

It has generally been assumed that the thrombogenicity of PCC relates to their invariable contamination by activated products of their component clotting factors. In earlier studies we demonstrated a lack of correlation between the activated clotting factor content ᶓ in vivo thrombogenicity of some PCC. In contrast, in vitro measurement of coagulant active PL content appeared to be a more reliable determinant of subsequent in vivo activity. In recent studies we have confirmed the presence of PL of known coagulant activity in PCC ᶓ studied its activity both in vitro ᶓ in vivo after its extraction from the parent PCC.

PCCs were obtained from various agencies or prepared in our laboratory to a standard protocol either from fresh frozen plasma or plasma obtained from outdated platelet concentrates. Each was assayed for Xa (S-2222) ᶓ PL (thrombin generation assay with DAPA) ᶓ an index of thrombogenicity in vivo determined in a stasis model in rabbits. There was a significant correlation between the latter ᶓ PL (r=0.8727 p<0.01) but not Xa. Thin layer chromatography of an ethanol ether extract of the most thrombogenic PCC showed the presence of PL known to be coagulant active including Phospho- tidylserine ᶓ -choline. This extract retained full PL replacing activity in the DAPA assay in vitro but was non- thrombogenic in vivo. When, however, a highly purified preparation of Xa was added, in the same relative proportions as in the parent PCC, thrombogenicity in vivo was restored.

It is concluded that the content of coagulant active PL is a critical determinant of the potential thrombogenicity of PCC in vivo. It appears to act in concert with Xa ᶓ conceivably, other serine proteases perhaps by protecting them from inactivation by AT III. This may explain why relatively “non-activated” PCCs may still be associated with thromboembolic complications.
Clinical And Laboratory Variables In Prediction Of Venous Thrombosis After Elective Gastrointestinal Surgery
Authors: Lowe G D O , McArdle B M , Osborne D H , Smith A , Belch J J F , Carter D C , Forbes C D , Prentice C R M
Published: July 2018

Thirty-two clinical and laboratory variables were measured In 63 patients prior to elective major gastro-intestinal surgery, who were screened for deep vein thrombosis (DVT) using the 125I-fibrinogen uptake test. Twenty-one patients (33%) developed DVT in the six days following surgery. Five clinical variables were significantly associated with subsequent DVT - age, per cent overweight, varicose veins, female sex (all positive risk) and cigarette-smoking (negative risk). Using clinical variables a high-risk group (> 60% DVT) could be separated from a low- risk group (< 10% DVT). None of 20 laboratory variables was significantly associated with DVT, but several (fibrinogen, factor VIII, plasma viscosity, blood viscosity, and serum fibrin degradation products) were associated with malignancy, infection or jaundice, which limits their usefulness in prediction of DVT in this group of patients. An incidental finding, that postoperative fall in haemoglobin was associated with DVT, suggests that bleeding or haemodilution may promote thrombosis. The clinical index requires validation in a prospective study, but may have practical value in selection of high-risk groups for selective prophylaxis or research studies, particularly in view of its simplicity.
Declining Mortality From Pulmonary Embolism In Surgical Patients
Authors: Dismuke S E
Published: July 2018

The frequency and clinical characteristics of autopsied surgical patients dying with pulmonary embolism (PE) were studied at a University hospital from 1966-1976. During this period a standard autopsy protocol was in use. All patients in whom PE occluded the equivalent of at least one lobar artery were studied. Information collected in all adult deaths confirmed the year to year similarity of patient characteristics and likelihood of autopsy. In order to assess the role of PE in a patient’s death, cases were grouped separately by 2 criteria: 1) according to size of PE (3 groups: greater than 2/3 of pulmonary vasculature occluded, 1/2 - 2/3 occluded, less than 1/2 occluded); 2) weighing competing causes of death by having 3 physicians (randomly selected from a pool of 25) judge the likelihood of death after 1 month had PE been prevented (classifying them as primary PE death, contributory PE death, and death from competing causes). Sixty percent (900/1489) of surgical deaths were autopsied and 7.7% (69) had large emboli. The percentage of deaths from PE, using either criteria, has declined significantly (p<0.01) over time (1966-71 vs 1972-76): 10.5% to 4.5% for all cases studied and 5.5% to 2.1% in those where PE was the primary or a contributory cause of death. PE was the primary or a contributory cause of death in 3.9% (34 cases) of these autopsies. The clinical diagnosis of PE was considered in only 17% (6) of these cases but 63% (22) had no symptoms suggestive of PE prior to death. An infiltrate or effusion, however, was present in 60% (21) of cases and 80% (28) had 2 or more major risk factors for PE. In contrast to previous studies, this study demonstrates an impressive decline in surgical deaths from PE from 1966-1976 despite no routine use of prophylactic anticoagulation.
The Effects Of Estrogen On Von Willebrand Factor Production And Endothelial Cell Number In Culture
Authors: Harrison R L , McKee P A
Published: July 2018

Subconfluent and confluent endothelial cell monolayers derived from human umbilical veins were treated with pharmacologic levels of estrogen and compared functionally to control monolayers. The levels of von Willebrand ristocetin cofactor (vWF) present In media from cell cultures after three days of incubation were assayed by ristocetin-induced platelet aggregation (PA) as well as by a radioreceptor platelet assay (RR) for vWF. DNA content of each monolayer was determined fluorometrically as a measure of cell number. The incorporation of 3H-thymidine into 17β-estradiol (E2)-treated endothelial cell monolayers vs. control cultures was also quantitated. Whether measured by PA or RR, levels of vWF in media from E2-treated cells were reproducibly and significantly greater (1.2±0.33 μg vWF/ml/ μg DNA at 2 ng/ml E2) than in media from control endothelial cells (0.69±0.084 μg vWF/ml/μg DNA, p<0.01) and showed a dose-response effect with respect to the E2 added to the culture media. Compared to control cultures, E2-treated monolayers consistently contained greater DNA (2.0±0.10 μg DNA) than control cultures (1.7±0.12 μg DNA, p<0.001) and incorporated more 3H-thymidine into trichloroacetic acid insoluble material (30.2±6×103 CPM with E2, vs. 21.2±2×103 CPM control, p<0.05). However, differences in cell number between E2-treated and control monolayers did not account for the elevated vWF levels in the presence of exogenous E2. We conclude that estrogen has at least two effects on endothelial cell cultures: 1) estrogen directly stimulates the cells to increase their rate of production of vWF; and 2) estrogen causes an increased rate of endothelial cell replication.
Purification, Characterization And Radioimmunoassay Of A New Antiheparin Protein Secreted By Rabbit Platelets
Authors: Muggli R , Glatthaar B , Mittelholzer E , Tschopp T B
Published: July 2018

We isolated a platelet specific protein from rabbits for studying the usefulness of such proteins as marker for in vivo platelet activation in animals.

The protein was purified by chromatography on heparin-agarose and on Sephadex G75 from the soluble release products of thrombin aggregated washed rabbit platelets. On heparin- agarose the protein eluted in front of rabbit PF4 at a salt concentration of about 0.9 M NaCl, and had a MW of about 20,000 dalton as judged by gel filtration. The purified protein migrated in SDS PAGE in the presence or absence of mercaptoethanol as a single band with a MW of <12,000 dalton. It had a heparin-neutralizing activity of 0.16 mg Polybrene/ mg and a pI of >9.5.

A radioimmunoassay with second antibody precipitation was developed. Antisera were produced in goats. Plasma and serum levels were 26 ± 6 ng/ml (n = 16, x ± SD) and 2900 ± 960 ng/ ml (n = 8), respectively. Washed platelets contained 2.0 ng of the protein per 106 platelets. The protein was released in parallel to platelet aggregation induced by collagen. Plasma levels rose from 26 ± 8 ng/ml (n = 6) to 72 ± 29 ng/ml 30 s after i.v. injection of collagen into rabbits. After injection of the iodinated protein into rabbits (n = 4) the disappearance of radioactivity followed two-compartment kinetics with half-lives of 3.8 min and 137 min for the fast and slow component, respectively. No increase of plasma radioactivity was recorded after injection of 500 U heparin per rabbit after 3 hrs. At that time 51 ± 13 % of the injected radioactivity was recovered in the urine.

This new rabbit platelet secretory protein shows similar but not identical biochemical and in vivo behaviour as human βTG.
Increased Aggregation Of Human Platelets In A Vortex In Coronary Artery Disease
Authors: Karino T , Goldsmith H L
Published: July 2018

Previous studies of normal human platelets circulating in an annular vortex distal to a sudden expansion of a 150 μm into a 500 μm tube showed that collisions between cells led to spontaneous formation of aggregates without addition of any aggregating agent (Karino and Goldsmith, Microvasc. Res. 17: 217-237, 1979). Using the same technique, we have compared the reactivity of platelets from 27 patients with suspected coronary artery disease (CAD) at time of coronary angiography with that from 20 healthy volunteers. Platelet- rich plasma (PRP) was prepared from 40 ml of citrated blood and subjected to steady flow at a mean velocity of 7.5 cm/s in the 150 ym tube at which aggregation in the vortex was known to be maximal. The rate (time to form) and extent of aggregation (size of the largest aggregate) in the vortex were measured by observations through a microscope. Platelet aggregation was normal compared to volunteers in 5 of 6 patients found without CAD (largest aggregate < 20 μm formed in > 90s). By contrast, platelets in 12 of 21 patients with diagnosed CAD were markedly more reactive, forming aggregates > 50 μm in < 60s. The effect of propranolol therapy on aggregation was also investigated since we had found that both the active and inactive forms of the drug at 10-5 - 10-4M partially or totally inhibited aggregation of normal platelets in the vortex. Thus, it became apparent that both rate and extent of aggregation in PRP from CAD patients not taking the drug were appreciably higher (4 of 5 having hyper-reactive cells) than that of patients on the drug (8 of 16 with hyper-reactive cells). Finally, it was noted that platelets from patients with a previous record of myocardial infarction (MI) were more reactive than those of non-MI patients. The above technique may serve as a sensitive tool for measuring platelet hyperaggregability in certain disease states.
Studies On Glycocalicin And Glycoprotein Gp Ib
Authors: Solum N O , Sletbakk T , Hagen I , Gogstad G
Published: July 2018

Crossed immunoelectrophoresis (CIE) of extracts of human platelets in 1% Triton X-100 using antiserum to purified glycocalicin shows an immunopre- cipitate consisting of two peaks. Previous experiments have shown the small fast-moving peak to represent free glycocalicin whereas the slow-moving one corresponds to a larger amphiphilic protein, probably the integral membrane protein GP Ib. Glycocalicin is probably derived from the latter secondary to an activation of a calsium-dependent protease during platelet lysis. Further studies on these problems are presented. A gradual reduction of the concentration of Triton X-100 in the extraction buffer (tris-glycine, pH 8.7, 135 mOsM) gradually reduced the area of the slow-moving peak and increased that of the fast-moving one untill all was present as free glycocalicin. Reduction of the concentration of Triton in an extract already prepared with 1% Triton X-100 by adsorption to Bio-Beads SM-2 had no such effect. The presence of the protease inhibitor leupeptin during extraction at a low concentration of Triton (0.2%) reduced the peak corresponding to free glycocalicin. GP Ib was purified from Triton extracts by precipitation with con A, affinity chromatography of the supernatant on WGA-Sepharose and elution with NAGA, and gel filtration of the eluate on Ultrogel AcA 22. Triton X-100, EDTA and sodium azide were present at all steps. The characteristic change in mobility of GP Ib on SDS PAGE comparing unreduced to reduced sampLes deduced from studies on whole platelet proteins, was confirmed with the purified material, as was the correspondence between the GP Ib band on SDS and the slow-moving component of the CIE.
Selective Binding Of [3H]Yohimbine To The α2-Receptor Of Intact Platelets -- Comparison With [3H] Dihydroergocryptine Binding
Authors: Macfarlane Donald E , Stump David C
Published: July 2018

Analysis of the coupling of receptors and the adenylate cyclase is facilitated by radioligand analysis of receptor occupancy. The platelet α-receptor has been characterized by [3H]dihydroergocryptine binding to membranes, but poorly reproducible results are obtained with intact platelets. We incubated washed platelets with [3H]dihydroergocryptine and measured bound counts after 6-fold dilution with plasma and centrifugation. After 10 minutes, phentolamine (5μM) suppressible counts equivalent to 400 molecules per platelet were found with 1/2 saturation at 1.2nM. There was considerable intra-experimental variation, and non-specific binding “space” was greater than 10μl/108 platelets. Time course of displacement with cold dihydroergocryptine, phentolamine or yohimbine was biphasic with a rapid (2 min) component amounting to 20-30% followed by a further 20% over the next 60 minutes. These results, which confirm those of others, suggest that equilibrium is not reached in a reasonable period of time and that more than one binding mechanism may be involved. In contrast, [3H]yohimbine bound with simple first order kinetics (37°) giving k1 = 5.25 × 105M-1 and k-1 = 3.33 × 10-3sec-1. Scatchard analysis revealed kd = 4.8 nM, Bmax = 180 molecules per platelet. Non-specific binding “space” was less than 1μl/108 platelets. Binding was completely reversed or prevented by epinephrine, clonidine, ρ-aminoclonidine, phentolamine, dihydroergotamine and dihydroergocryptine in that order of potency. Prazosin, an α1-antagonist, was less potent than epinephrine. Yohimbine blocks the ability of epinephrine to potentiate and induce aggregation, and to inhibit the adenylate cyclase. It is concluded that this novel radioligand has clear advantages over dihydroergocryptine for analysis of receptor occupancy in intact platelets.
The Different Effects Of Aspirin On Experimental Arterial And Venous Thrombus Formation
Authors: Thiessen M , Zimmermann R , Weckesser G , Harenberg J
Published: July 2018

The influence of aspirin on platelet aggregation has given rise to frequent use as an antithrombotic agent. The knowledge of an unfavorable alteration of the thromboxane/prostacyclin ratio in the vessel wall at high dosage may favor therapy with low dosages of aspirin.

In order to obtain further information on the most effective dosage of aspirin the antithrombotic effect of repeated and different doses of aspirin was investigated in a standardized experimental model in 120 rabbits. In a 1. group aspirin 100 mg/kg were given 30 min before producing thrombosis. In two further groups aspirin was administered 12 hours and additionally 30 min before beginning the experiment. These rabbits received repeatedly either 10 mg (2.group) or 100 mg/kg (3. group) two times. In a 4.series aspirin was given at 50 mg/kg per day during three days.

A paradoxical thrombogenic effect with an increase of venous and arterial thrombus growth could be seen after a single high dose of 100 mg/kg aspirin but not after an additional one. If applicated two times a thrombus size minor than under repeated low doses of 10 mg/kg aspirin has been observed in the arterial and venous system (p< 0.05).Additionally after repeated treatment with 50 mg/kg an antithrombotic effect could be seen.

According to our data the paradoxical effect of only a single or first high dose of aspirin has to be taken into account but cannot be observed after repeated doses. At a low dosage aspirin a paradoxical effect was seen later,after a second dose. Animal thrombosis experiments does not provide sufficient evidence to support antithrombotic therapy with low doses of aspirin.
An Analog Of Thiazole Which Has Potential Antithrombotic Activity With Low Toxicity
Authors: Nishizawa E E , Rynbrandt R H , Mendoza A R , Balqoyen D P , Annis K A
Published: July 2018

Chemical modification of a compound found active in preventing death in mice following intravenous administration of collagen led to the development of an inhibitor of collagen-induced platelet aggregation which was active at 1 ng/ml in human PRP, namely 4,5-bis(p-methoxyphenyl)-2- (trifluoromethyl)-thiazole (TFT). It did not inhibit ADP, thrombin or PGH2-induced aggregation but inhibited the collagen-induced release of radioactivity from platelets labelled with radioactive serotonin. It was active in many animal species and in the rabbit it had a duration of activity of about 8 hours at 1 mg/kg (p.o.).

TFT has cyclooxygenase inhibitory activity, but unlike aspirin (A) or flurbiprofen (F), it showed no incidence of ulcers at 500 mg/kg p.o. in rats. However, again unlike A or F, it had very little anti-inflammatory activity as measured by the carrageenan-induced hind paw edema assay in rats. Its LD50 in mice (i.p.) was greater than 1 gm/kg.

Thus TFT appears to be a potential antithrombotic agent with high antiplatelet specificity and a wide therapeutic margin as compared with A or F. The anti platelet action of TFT may be due to its inhibition of cyclooxygenase.
Protection Against Epinephrine Exacerbated Platelet Thrombi Formation In Stenosed Dog Coronary Arteries With Chlorpromazine
Authors: Folts J
Published: July 2018

We have previously shown that platelet thrombi (PTH) form in stenosed canine coronary arteries in five to ten minutes producing reductions in coronary flow (RCF) and that these can be exacerbated by IV infusions of epinephrine (E) by an alpha adrenergic mechanism in spite of pretreatment with aspirin, indomethacin, ibuprofen, or sulfinpyrazone. We have also shown an apparent active involvement of lysed red blood cells (RBC) packed together proximal and in the area of stenosis releasing ADP and exacerbating the PTH formation. On high magnification using carstairs stain and fluoresceine conjugated antisera, fused platelets, lysed RBCs and some fibrin are noted. Eight dogs were prepared with an EMF probe in the circumflex coronary artery along with a fixed mechanical stenosis of 75% of the artery. RCF were noted and 5 ug of E/minute for ten minutes IV raised arterial blood pressure (ABP) 15 ± 6 mm Hg and exacerbated RCF in all dogs, but after administration of .6 mg/kg of chlorpromazine (C) IV the RCF were abolished and repeated infusions of E did not provoke RCA but did lower ABP 10 ± 6 mm Hg. In vitro platelet aggregation (ADP stimulus) decreased from 61 ± 11 LTU to 39 ± 13 LTU (p < .05). Red cell osmotic fragility tests were done on samples obtained before and after C and the curve was significantly shifted to the right after C suggesting more stable RBC membranes. We postulate that C may stabilize RBCs, preventing ADP release and inhibit platelet aggregation by an alpha adrenergic mechanism. C or an agent like it may prevent PTH from forming in patients with stenosed coronary or cerebral arteries who smoke or otherwise have elevated plasma E.
The Influence Of Physiological Parameters On The Plasma Concentration Of β-Thromboglobulin (β-TG) And Platelet Factor 4 (PF-4) In Normal Healthy Subjects
Authors: Duncan A , Didisheim P
Published: July 2018

The variability of normal ranges in different laboratories and the occurrence of occasional elevated values of β-TG and PF-4 in normal subjects are well established.
We examined the influence of factors such as age, sex, smoking, exercise, oral contraceptives and day-to-day variation in 148 healthy subjects. The β-TG and PF-4 assays were performed by commercial RIA’s modified by utilizing an EDTA- PGE1 -theophylline mixture as anticoagulant.

The statistical method used to compare our groups was a student t-test after log transformation of the data. Our baseline control group (N=63) were non smokers and not taking oral contraceptives (O/C) or any medications. There was no difference between sexes or a significant change with increasing age, although the mean β-TG and PF-4 does increase in cohosts of 20 years. [<30 years (21.65±8.35)(SD), 30-50 years (24.32±10.94) >50 years (27.88±14.3)-β-TG data].

Women on O/C were significantly different from those not on O/C for β-TG [35.06±22.45 (O/C) and 23.67±9.38 (N)] but not for PF-4 values [3.79±1.77 (O/C) and 3.91±1.63 (N)].

Strenuous exercise was the parameter that influenced both β-TG and PF-4 significantly, showing an elevated value after exercise for both β-TG [81.9±31.4 v 26.73±10.91] and PF-4 [7.08±1.71 v 3.73±1.91].

Day-to-day variations in β-TG and PF-4 were examined in 10 subjects for 7-10 days. The coefficient of variation (CV) amongst these subjects was 21-51%. In athletes day-today variation was the same unless a change in pattern of exercise occurred [CV-49-132%].

Our results confirm that parameters such as above do influence normal values and should be taken into consideration in establishing a normal range.

It also indicates the β-TG is a more sensitive parameter for delineating differences in physiological parameters in normal subjects than PF-4, and this may be applicable to clinical studies.
Platelet Factor 4 (PF4) And β-Thromboglobulin (BTG) In Atherosclerosis And Diabetes Mellitus
Authors: Kazmier F J , Bowie E J W , O’Fallon W M , Palumbo P J
Published: July 2018

Distinguishing between in vitro and in vivo release of platelet specific proteins has led to problems in interpretation of clinical studies with PF4, and with BTG.

As part of a prospective study of peripheral occlusive arterial disease in diabetes mellitus PF4 and BTG were measured together by specific radioimmunoassay in four groups of subjects age 50-70 years. Groups included (1) 106 normal subjects (NC); (2) 126 with clinical and objective evidence (transcutaneous doppler ultrasound and treadmill exercise) of peripheral arterial occlusive disease (ASO); (3) 244 with diabetes mellitus without ASO and (4) 101 with both diabetes mellitus and ASO.

PF4 did not distinguish among the four groups: NC vs ASO p=. 18, NC vs DM p=.52, NC vs DM+ASO p=.73, NC vs all diabetics p=.61, and NC vs all ASO p=.46. However, BTG did distinguish the groups with vascular disease from NC: NC vs ASO p=.02, NC vs DM+ASO p<.001, NC vs DM p=.32, DM vs DM+ASO p<.01, all ASO vs all nonASO p<.001.

Normal range for PF4 is 0-13 ng/ml with a median of 3.0 ng/ml, a mean of 3.0 ng/ml, and SD 1.97. Normal range for BTG is 14-46 ng/ml with a median of 26 ng/ml, a mean of 27.2 ng/ml, and SD 10.3. When PF4 and BTG are correlated (all groups included) r=.41 p<.001.

PF4 and BTG levels should be performed together. When done together low PF4 levels support the absence of in vitro platelet release due to sample collection and handling. BTG levels are significantly increased in subjects with peripheral arterial disease, supporting the active role of platelets in thrombotic occlusive arterial disease.
Fibrinopeptide A And Beta-Thromboglobulin Concentrations In Venous And Arterial Disorders
Authors: van Hulsteijn H , Bertina R M , Briët E
Published: July 2018

The purpose of this study was to assess the relative importance of thrombin generation and platelet activation in venous and arterial disorders. To this end Fibrinopeptide A (FPA) was used as an index of thrombin generation and Beta- thromboglobulin (BTG) as an index of platelet activation.
FPA and BTG were determined by specific radioimmunoassays.

In 80 controls (age 19-70, mean 41 yrs) mean FPA concentration was 0.72 ± 0.47 (ng/ml ± SD) and mean BTG concentration 28.2 ± 10.1 (ng/ml ± SD).

In 51 patients with the symptoms of deep vein thrombosis (DVT) or pulmonary embolism (PE), in which these disorders were confirmed by phlebography and/or Doppler ultrasound or perfusion lungscanning, mean FPA and BTG concentrations were clearly elevated compared to mean FPA and BTG concentrations in controls and in 25 patients with the symptoms of DVT or PE in which these disorders could be excluded by the above named techniques. Heparin and phenprocoumon lowered FPA concentrations into the normal range, while the BTG concentrations remained elevated.

In 20 patients with intermittent claudication, in which peripheral vascular disease (PVD) was confirmed by Doppler ultrasound mean FPA concentration did not differ from that in controls, while the mean BTG concentration was obviously elevated. This picture did not change after 2 months of phenprocoumon therapy.

In 33 patients with angina pectoris in which coronary artery disease (CAD) was demonstrated by arteriography mean FPA concentration did not differ from that in 7 patients with angina pectoris in which no coronary lesions were present and in controls contrary to the mean BTG concentration, which was clearly elevated. Results in patients on phenprocoumon or acenocoumarol therapy were not different from those without oral anticoagulant treatment.

So far the experimental results indicate that in venous disorders (DVT or PE) fibrin formation plays a more important role than in arterial disorders (PVD or CAD).
The Epidemiology Of Platelet Aggregation
Authors: Meade T W , Thompson S G , Vickers M V
Published: July 2018

Platelet aggregation has been studied in over 600 participants in the prospective Northwick Park Heart Study of ischaemic heart disease (IHD). Platelet-rich-plasma platelet counts are standardized to a value of 250 × 103/μl. The aggregating agents are ADP and adrenaline, in a full range of dilutions. Aggregation is detected by change in optical density. Dose-response curves and statistical parameters are calculated by computer. Although it will be some time before prospective data on platelet aggregation are available, some general epidemiological characteristics have been established. The most informative parameter is the ADP-ED50, i.e. the concentration of ADP at which aggregation occurs at half its maximum velocity. A low ADP-ED50 indicates easily aggregable platelets. An unexpected but highly significant finding is of a lower ADP-ED50 in women than men. Platelets become more aggregable (i.e. ADP-ED50 falls) with advancing age. There are no very striking associations between ADP-ED50 and smoking habit, alcohol consumption or obesity. There is a significant negative correlation between ADP-ED50 and plasma fibrinogen level - the higher the fibrinogen the more aggregable the platelets. There are no clear associations with factors II, VII, VIII or X, or with blood cholesterol or blood pressure. Some, though not all, of the epidemiological characteristics of ADP-ED50 are in line with current views about the role of platelets in IHD. The inverse relationship between fibrinogen and ADP-ED50 is a reminder that platelet function in vivo may be only partly determined by the intrinsic properties of the platelets themselves.