bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of a Mode of Specific Binding of Fibrin Monomer through its Amino-Terminal Domain by Macrophages and Macrophage Cell-Lines
Authors: Shainoff John R , Stearns Deborah J , DiBello Patricia M , Hishikawa-Itoh Youko
Published: July 2018

The studies reported here probe the existence of a receptor-mediated mode of fibrin-binding by macrophages that is associated with the chemical change underlying the fibrinogen-fibrin conversion (the release of fibrinopeptides from the amino-terminal domain) without depending on fibrin-aggregation. The question is pursued by 1) characterization of binding in relation to fibrinopeptide content of both the intact protein and the CNBr-fragment comprising the amino-terminal domain known as the NDSK of the protein, 2) tests of competition for binding sites, and 3) photo-affinity labeling of macrophage surface proteins. The binding of intact monomers of types lacking either fibrinopeptide A alone (α-fibrin) or both fibrinopeptides A and B (αβ-fibrin) by peritoneal macrophages is characterized as proceeding through both a fibrin-specific low density/high affinity (BMAX ≃ 200–800 molecules/cell, KD ≃ 10−12 M) interaction that is not duplicated with fibrinogen, and a non-specific high density/low affinity (BMAX ≥ 105 molecules/cell, KD ≥ 10−6 M) interaction equivalent to the weak binding of fibrinogen. Similar binding characteristics are displayed by monocyte/macrophage cell lines (J774A.1 and U937) as well as peritoneal macrophages towards the NDSK preparations of these proteins, except for a slightly weaker (KD ≃ 10−10 M) high-affinity binding. The high affinity binding of intact monomer is inhibitable by fibrin-NDSK, but not fibrinogen-NDSK. This binding appears principally dependent on release of fibrinopeptide-A, because a species of fibrin (β-fibrin) lacking fibrinopeptide-B alone undergoes only weak binding similar to that of fibrinogen. Synthetic Gly-Pro-Arg and Gly-His-Arg-Pro corresponding to the N-termini of to the α- and the β-chains of fibrin both inhibit the high affinity binding of the fibrin-NDSKs, and the cell-adhesion peptide Arg-Gly-Asp does not. Photoaffinity-labeling experiments indicate that polypeptides with elec-trophoretically estimated masses of 124 and 187 kDa are the principal membrane components associated with specifically bound fibrin-NDSK. The binding could not be up-regulated with either phorbol myristyl acetate, interferon gamma or ADP, but was abolished by EDTA and by lipopolysaccharide. Because of the low BMAX, it is suggested that the high-affinity mode of binding characterized here would be too limited to function by itself in scavenging much fibrin, but may act cooperatively with other, less limited modes of fibrin binding.
Extraction of Protein-Bound ATP and ADP from Human Platelets in Plasma
Authors: Beurling-Harbury Christina , Harbury Pehr B
Published: July 2018

Actin is the major ATP and ADP binding protein in platelets, 0.9–1.3 nmol/108 cells, 50–70% in the unpolymerized state. The goal of these experiments was to develop a method for extracting all protein-bound ATP and ADP from undisturbed platelets in plasma. Extraction of actin-bound ADP is routine while extraction of actin-bound ATP from platelets in buffer has been unsuccessful. Prior to extraction the platelets were exposed to 14-C adenine, to label the metabolic and actin pools of ATP and ADP. The specific activity was determined from the actin-bound ADP in the 43% ethanol precipitate. Sequential ethanol and perchlorate extractions of platelet rich plasma, and the derived supernatants and precipitates were performed. ATP concentrations were determined with the luciferase assay, and radioactive nucleotides separated by TLC. A total of 1.18 nmol/108 cells of protein-bound ATP and ADP was recovered, 52% ATP (0.61 nmol). The recovery of protein-bound ADP was increased from 0.3 to 0.57 nmol/108 cells. This approach for the first time successfully recovered protein bound ATP and ADP from platelets in a concentration expected for actin.
Factor IX Chongqing: a New Mutation in the Calcium-Binding Domain of Factor IX Resulting in Severe Hemophilia B
Authors: Wang N S , Zhang M , Thompson A R , Chen S-H
Published: July 2018

A Chinese patient with sporadic, severe hemophilia B was found to have a low level of total factor IX antigen (3.5 U/dl), but less apparent antigen in an assay using a calcium-dependent antibody fraction (1.1 U/dl). This suggested a defect in the factor IX Gla domain coded mainly by exon 2 of the factor IX gene. Exon 2 was therefore amplified and sequenced. An A to T substitution was found at nucleotide 6455 of the patient’s factor IX gene. This transversion changes the codon for Glu 27 in normal factor IX to a codon for Val. Since Glu 27 becomes an essential Gla residue, the defect should result in altered calcium-binding or calcium-dependent conformation of the patient’s factor IX. The introduction of a hydrophobic side chain also appears to affect the hemophilic protein’s stability.

In leukocyte DNA from the patient’s mother, the nucleotide sequence of exon 2 was entirely normal. Thus, barring somatic mosaicism within her germ cells, the new mutation occurred in oogenesis of her ovary.
Characterization of the Anticoagulant Activities from a Brazilian Arrow Poison
Authors: Jacobs John W , Petroski Christopher , Friedman Paul A , Simpson Ellen
Published: July 2018

A recent report indicated that an arrow poison used by the native Indians of Rondonia, Brazil, to kill small animals was associated with profuse bleeding (1). The arrow poison was prepared from the bark of a tree, known locally as Tike-Uba. We have obtained bark and sap specimens from this tree and have characterized a potent anticoagulant activity in both the crude bark and sap samples as well as in more highly purified preparations. An aqueous extract of the bark significantly prolongs both prothrombin times and activated partial thromboplastin times in plasma based assays. Further fractionation of the bark extract and sap by molecular weight indicated that all of the anticoagulant activity could be isolated in a molecular weight fraction of equal to or greater than 30,000 daltons. The anticoagulant activity was also further purified by C-18 reverse phase chromatography. When highly purified preparations of the anticoagulant activity from the Tike-Uba tree were examined in specific blood coagulation enzyme assays utilizing chromogenic substrates, the highest inhibitory potency was found versus thrombin, followed by factor Xa. These studies establish the presence of a compound(s) in a Brazilian arrow poison, which potently disrupts mammalian blood clotting, and which may account for some of the observed toxicities associated with the arrows.
Detection of both Type 1 and Type 2 Plasminogen Activator Inhibitors in Human Monocytes
Authors: Castellote Joan C , Grau Enric , Linde Maria A , Pujol-Moix Nuria , Rutllant Miquel LI
Published: July 2018

Increasing evidence suggests the involvement of leukocytes in the fibrinolytic system. Monocytes secrete pro-urokinase (Grau, Thromb Res 1989; 53: 145) and it has been shown that these cells have specific receptors for urokinase and plasminogen (Miles, Thromb Haemostas 1987; 58: 936). The aim of this study was to analyse the presence of plasminogen activator inhibitor(s) in platelet-free suspensions of human peripheral blood monocytes and polymorphonuclear leukocytes (PMN). SDS-PAGE and reverse fibrin autography showed an inhibitory band of 50 kDa in the monocyte extracts (Triton X-100) but not in the PMN extracts. Urokinase (u-PA) was mixed with increasing amounts of monocyte extract for 10 min and the mixtures were added to 125Ifibrin coated wells containing plasminogen. A dose-dependent decrease in the u-PA fibrinolytic activity was observed. The amount of inhibition increased when the monocyte releasates were preincubated with u-PA (40% inhibition after 5 min preincubation and 80% after 15 min), indicating a direct interaction between this activator and an inhibitor(s). After SDS-PAGE of monocyte extracts, immunoblotting and peroxidase staining identified both PAI1 and PAI2, with an apparent molecular weight of 47-50 kDa. Monocyte-associated PAI1 formed complexes with single chain t-PA with a molecular mass 50 kDa higher than the molecular mass of the free PAI1. However, a significant amount of PAI remained unbound to t-PA. This inactive PAI1 could have come from a rapid inactivation of the primary active PAI1. These PAI1 and PAI2 detected in human monocytes may be transcendent in the regulation of the fibrinolytic system.
A Plasma Clot Lysis Assay Based on the Release of Fibrin Degradation Products: Application to the Diagnosis of Hypofibrinolytic States
Authors: Gaussem Pascale , Gandrille Sophie , Molho-Sabatier Pascale , Capron Loïc , Fiessinger Jean-Noël , Aiach Martine
Published: July 2018

Using a monoclonal antibody-based assay, we measured the fibrin degradation product release in the supernatant of plasma clots obtained before and after venous occlusion (VO) in 30 patients with definite or suspected vascular thrombosis (19 definite and 2 suspected deep vein thrombosis, 6 recurrent superficial thrombophlebitis, 3 arterial occlusions of lower limbs). tPA and PAI-1 concentrations were determined using ELISA assays; the post-occlusion values were corrected for haemoconcentration. The increase in tPA during VO was correlated with haemoconcentration (r = 0.74), but 3 patients had ineffective VO (<2% increase in proteins). The fibrinolytic response to VO was evaluated using the shortening of the time necessary for the release of 200 μg of fibrin degradation products per mg of fibrinogen (Δ T 200). Two among the 27 patients with effective VO were bad responders with a Δ T 200 <3 h (whereas all the others had Δ T 200 >10 h). These patients had respectively a deficient tPA release (Δ tPA = 1 ng/ml) and an elevated PAI-1 level at rest (33 ng/ml). Several other patients were bad responders in terms of tPA release or of shortening of the euglobulin clot lysis time but they had a normal Δ T 200. This plasma clot test reflects the ability of free tPA to bind to fibrin (the amount of which depends on the level of tPA and PAI-1), and may be useful in the diagnosis of a hypofibrinolytic state.
Effect of Angiotensin II on Collagen-Induced Platelet Activation in Normotensive Subjects
Authors: Swartz Stephen L , Moore Thomas J
Published: July 2018

We examined the effect of angiotensin II (AII) on platelet activation in 15 normotensive volunteers. By measuring the initial phase of platelet aggregation on the optical density aggregation tracing and generation of thromboxane production, we demonstrated that AII enhances collagen-induced platelet activation. This enhancement in platelet activation is significantly greater when subjects are on a high salt than on a low salt diet. Furthermore, the increase in platelet activation parallels an increase in platelet AII receptor number. These parallel changes in platelet activation and platelet AII receptors provide a possible mechanism through which AII may affect platelet function.
A Resolution of Reported Discrepancies in the Characteristics of Platelet Glycoproteins IV (GPIV) and III b (GPIII b)
Authors: Yamamoto Naomasa , Romeuf Christophe de , Tandon Narendra N , Jamieson G A
Published: July 2018

The terms glycoprotein IV (GPIV) and glycoprotein IIIb (GPIIIb) have been used interchangeably and reports in the literature have indicated this glycoprotein as having a molecular weight variously described as either 88,000 or 97,000, a fast anodal mobility on crossed electrophoresis and either 13 or <1 methionine residues on amino acid analysis of the purified glycoprotein. To resolve these discrepancies, we have evaluated the characteristics of GPIV both in whole platelets and after isolation. These studies have shown that the term GPIV defines a protease-resistant platelet surface glycoprotein with Mr 88,330 ± 2,240 which is immunologically identical with the CD36 differentiation antigen, which migrates with a relatively slow anodal mobility on crossed immunoelectrophoresis and which contains ∼13 methionine residues per mole.
The Effect of Red Blood Cells on the ADP-induced Aggregation of Human Platelets in Flow Through Tubes
Authors: Bell David N , Spain Samira , Goldsmith Harry L
Published: July 2018

The effect of red blood cells, rbc, and shear rate on the ADPinduced aggregation of platelets in whole blood, WB, flowing through polyethylene tubing was studied using a previously described technique (1). Effluent WB was collected into 0.5% glutaraldehyde and the red blood cells removed by centrifugation through Percoll. At 23°C the rate of single platelet aggregtion was upt to 9× greater in WB than previously found in platelet-rich plasma (2) at mean tube shear rates Ḡ = 41.9,335, and 1,920 s−1, and at both 0.2 and 1.0 µM ADP. At 0.2 pM ADP, the rate of aggregation was greatest at Ḡ = 41.9 s−1 over the first 1.7 s mean transit time through the flow tube, t, but decreased steadily with time. At Ḡ ≥335 s−1 the rate of aggregation increased between t = 1.7 and 8.6 s; however, aggregate size decreased with increasing shear rate. At 1.0 µM ADP, the initial rate of single platelet aggregation was still highest at Ḡ = 41.9 s1 where large aggregates up to several millimeters in diameter containing rbc formed by t = 43 s. At this ADP concentration, aggregate size was still limited at Ḡ ≥335 s−1 but the rate of single platelet aggregation was markedly greater than at 0.2 pM ADP. By t = 43 s, no single platelets remained and rbc were not incorporated into aggregates. Although aggregate size increased slowly, large aggregates eventually formed. White blood cells were not significantly incorporated into aggregates at any shear rate or ADP concentration. Since the present technique did not induce platelet thromboxane A2 formation or cause cell lysis, these experiments provide evidence for a purely mechanical effect of rbc in augmenting platelet aggregation in WB.
Demonstration of Secondary Lysosomes in Bovine Megakaryocytes and Platelets Using Acid Phosphatase Cytochemistry with Cerium as a Trapping Agent
Authors: Ménard Michèle , Meyers Kenneth M , Prieur David J
Published: July 2018

The ultrastructure of lysosomes from bovine megakaryocytes (MK) and platelets was characterized using acid phosphatase cytochemistry with beta-glycerophosphate as substrate and cerium as a trapping agent. The technique was easily reproducible; cerium-phosphate precipitates were uniform, readily visualized, and there was a virtual absence of nonspecific reaction product. Acid phosphatase was localized in the trans aspect of the Golgi complex and/or granules of less than 50 nm to 650 nm diameters in MK at all stages of maturation. Forty percent of the MK lysosomes contained inclusions of variable shapes, sizes and electron-density and were classified as secondary lysosomes. Twenty-four percent of the platelet sections contained acid phosphatase-positive granules. Fifty-four percent of these were secondary lysosomes. This is the initial report demonstrating
secondary lysosomes in either resting MK or platelets using acid phosphatase cytochemistry. These findings suggest that MK and platelet lysosomes have an intracellular function in resting MK and platelets.
The Decreased Circulating Platelet Mass and Its Relation to Bleeding Time in Chronic Renal Failure
Authors: Michalak E , Walkowiak B , Paradowski M , Cierniewski C S
Published: July 2018

The mean platelet volume, measured optically and by the electrical impedance method, was analysed in the patients with chronic renal failure. All parameters describing the circulating platelet biomass except the platelet volume heterogeneity were decreased in these patients. There is a strong inverse relation between the Ivy bleeding time and the platelet mass in the patient group.

Our results indicate that the smaller platelets circulating in the patients with chronic renal failure may contribute to the uremic bleeding diathesis, and disturbed thrombocytopoiesis occurs in chronic renal failure.
Factor VII Hyperactivity in the Elderly
Authors: Kario Kazuomi , Matsuo Takefumi , Nakao Kazukiyo
Published: July 2018

To investigate the age-related increase in coagulation factor VII (FVII) and its significance in the elderly, we measured FVII coagulation activity (FVIIc), FVII antigen (FVIIag), and D-dimer levels in 150 normal subjects ranging in age from 60 to 98 years. We also measured blood lipid fractions and serum cholinesterase activity (ChE), os an indicator of hepatic protein synthesis. FVIIc (141 ± 36%), FVIIag(136 ± 28%), and D-dimer (0.150 ± 0.372 αd/ml) levels were significantly higher in the elderly than in younger controls (p <0.01). Both FVIIc and FVIIag levels were significantly higher in elderly women than in elderly men (p <0.01). FVIIc levels significantly correlated with FVIIag levels, but not with D-dimer levels. FVIIag was more closely correlated with ChE levels in both sexes (men: r = 0.425, womenr r = 0.365, p <0.001) than with the lipid fractions. When the elderly subjects were divided into atherosclerotic and nonatherosclerotic groups, both FVIIc (p <0.01) and FVIIag (p <0.05) levels were higher in the former group. Moreover, the FVIIc/FVIIag ratio and ChE levels were higher in both the elderly men and women with atherosclerosis. These results suggest that in elderly subjects, especially with atherosclerosis, hepatic FVII synthesis and the activation of FVII zymogen are both accelerated.
Subtle Differences in Active Site Structure between Bovine and Human Thrombins: ESR and Fluorescence Studies
Authors: Nienaber Vicki L , Berliner Lawrence J
Published: July 2018

The primary structures of bovine and human a-thrombins are highly homologous yet their x-ray structures are not yet complete enough to distinguish differences. In order to probe and compare their dynamic conformations in solution, we examined bovine and human α-thombins with a series of active site directed fluorosul-fonylphenyl spin labeled inhibitors and fluorophores which probe a region within 10-15 Å of the catalytic serine residue. Overall, the nitroxide moieties were more immobilized in the bovine vs human derivatives reflecting either more apolar binding regions or steric obstructions to the motion of the nitroxide in bovine thrombin. Most of the labels which distinguish indole (apolar ligand) binding in human thrombin were found to display similar interactions in bovine thrombin, although slight differences in the general topography of this region were suggested. The two active site directed fluorophores, dansyl fluoride and p-nitrophenyl anthranilate showed differences in both λem
max and λex
max of the complexes with bovine and human-a-thrombin, respectively. Several of the effects observed i.e., ligand binding (indole or benzamidine) and the subtle hydrophobic interactions between the nitroxide moiety and the protein active site would be difficult to assess from an x-ray structure determination alone.
Interaction of Plasma Kallikrein with Protein C Inhibitor in Purified Mixtures and in Plasma
Authors: España Francisco , Estelles Amparo , Griffin John H , Aznar Justo
Published: July 2018

The interaction between plasma kallikrein (KK) and protein C inhibitor (PCI) and the influence of KK on the complex formation between activated protein C (APC) and PCI was studied in purified systems as well as in plasma in order to assess the significance of these reactions in the plasma milieu. PCI complexed to KK (KK: PCI) or to APC (APC: PCI) was measured by sandwich ELISA’s using antibodies directed against each protein in the complexes. The formation of KK: PCI complexes assayed by this method paralleled the inhibition of KK amidolytic activity by PCI in purified system. Incubation of normal plasma (NHP) at 4 °C, which can induce prekallikrein activation due to cold activation, resulted in PCI inactivation and appearance of KK: PCI complexes. PCI activity fell to 35% of the NHp and 1.2 μ/ml of KK: PCI complex was formed. However, incubation of NHP at room temperature or of prekallikrein deficient plasma at 4 °C did not result in significant decrease of PCI activity. Thus the PCI inactivation was associated with prekallikrein activation and complexation to PCI following cold activation. Incubation of exogenous purified KK with NHP resulted in PCI inactivation and complexation with KK in a temperaturedependent manner. Addition of 2.8 μ/ml KK to plasma at 4 °C resulted in the inactivation of 55% of plasma PCI and the formation of 0.9 μ/ml KK: PCI which represents 21% of the KK added, whereas at 37 °C PCI was inactivated to 30% and only 0.30 μg/ml KK: PCI complexes were measured. These results indicate that PCI is a major KK inhibitor at 4 °C. At 37 °C, PCI accounted for aborfi 7% of the inhibition of the KK added. In separate experiments, following addition of 2.5 μg/ml APC to NHR more than 1 μg/ml of APC: PCI complex was formed in 3 h. When NHP was prior incubated with KK, PCI activity decreased to 10% of that of the normal plasma. Subsequent addition of APC to the plasma treated with KK resulted in formation of only 35 μg/ml of APC: PCI complex compared to 1,350 μg/ml when plasma was not previously incubated with KK. These results indicate that PCI could play a physiological role in the inhibition of plasma KK, and that, in turn, plasma KK can either complex to or inactivate plasma PCI. Thus, KK could modulate the PCI inhibition of APC in plasma.
Characterization of a Mouse Factor IX cDNA and Developmental Regulation of the Factor IX Gene Expression in Liver
Authors: Yao Shou-Nan , DeSilva Audrey H , Kurachi Sumiko , Samuelson Linda C , Kurachi Kotoku
Published: July 2018

A mouse factor IX cDNA was isolated and characterrzed. The cDNA was 1,837 bp in length and contained the coding region as well as short 5’ and 3’ untranslated sequences. Northern blot analysis of liver RNA showed two mRNA species of 3.2 kb (major) and 2.2 kb (minor) for the mouse factor IX. An antisgnse RNA probe prepared from the mouse cDNA was employed to determine the steady state level of factor IX mRNA in mouse liver at various developmental stages. The factor IX mRNA level was very low (2–5% of the adult level) during the gestational period until day –3 (gestational day 17) followed by a rapid increase at day –2 through birth. This phase of rapid increase was followed by a gradual increase before it reached the adult level at around 20 to 24 days. At birth, the factor IX mRNA level was found to be at about 43% of that of the adult. The rnRNA levels in mouse liver agreed well with the plasma factor IX activity levels. These results indicate that reduced factor IX activity in newborns is due to the low levels of factor IX mRNA available for translation.
The Influence of Infusions of 1-Desamino-8-D-Arginine vasopressin (DDAVP) In Vivo on the Anticoaguhht Effect of Recombinant Hirudin (CGP39393) In Vitro
Authors: Ibbotson S H , Grant P J , Kerry R , Findlay V S , Prentice C R M
Published: July 2018

Hirudin is a specific, potent inhibitor of thrombin that may be a valuable antithrombotic agent. The aim of this study was to investigate the hypothesis that the haemostatic effects of DDAVP counteract the coagulation defect induced by hirudin. The effect of DDAVP was studied in vivo on the anticoagulant action of recombinant hirudin (CGP39393) in vitro. Blood samples were taken at intervals from 10 normal volunteers infused with DDAVP. Factor VIII: C rose from (mean) 0.68 IU/ml before DDAVP to 2.L9 and 2.16 IU/ml after 30 and 60 min infusion, respectively. Samples taken during DDAVP infusion showed a dose related decrease in the hirudin (0.5 and 1.0 αM) induced prolongation of the APTT that occurred at FVIII: C concentrations of up to twice normal. At higher concentrations of hirudin no effect on the APTT occurred. These results demonstrate that DDAVP infusion elevates factor VIII: C levels with an associated significant reduction in the anticoagulant effect of hirudin in vitro.
Modulation of Endothelial Cells Fibrinolytic Activity by Platelets
Authors: Menashi S , He L , Soria C , Soria J , Thomaidis A , Legrand Y
Published: July 2018

Interaction between endothelial cells (EC) and platelets in culture was shown to regulate the fibrinolytic system of the aortic EC. Untreated porcine EC from aorta exhibited almost no net fibrinolytic activity and zymographic assay have shown a single fibrin lysis band of 105 kDa corresponding to a tPA-PAI complex. Incubation of aortic EC with intact platelets stimulated a cell-associated fibrinolytic activity of the urokinase type as evidenced by a plasminogen-dependent fibrin independent amidolytic activity, and the appearance of a new 48 kDa lysis band on zymography. However, in the culture medium of platelet-treated aortic EC, a new lysis band of. 92 kDa appeared with no associated amidolytic activity suggesting that the 48 kDa plasminogen activator secreted by the aortic EC after treatment with platelets is complexed to the inhibitor PAI1. This modulation of fibrinolytic activity depends on the EC origin sin0e it is not observed with pulmonary artery EC, and represents a new concept in fibrinolysis regulation by cell-cell interaction.
Physical Exercise Induces Enhancement of Urokinase-Type Plasminogen Activator (u-PA) Levels in Plasma
Authors: Dooijewaard G , de Boer A , Turion P N C , Cohen A F , Breimer D D , Kluft C
Published: July 2018

The enhancement of the blood fibrinolytic potential by physical exercise is generally attributed to the release of tissue-type plasminogen activator (t-PA) from the vessel wall. In this study we have investigated the possible contribution of urokinase-type plasminogen activator (u-PA).

Six healthy male volunteers (age 21–25 years) were screened for their ability to perform maximal exercise for their age-group for 12 min on a bicycle ergometer. Subsequently, on one occasion they were required to remain supine for 2 h (from 8.30 a. m. onwards) and on another they performed maximal exercise (from 9.00 a.m. onwards). During exercise an increase in u-PA antigen and plasmin-activatable pro-urokinase (proUK) activity, concurrent with t-PA antigen and euglobulin t-PA activity, was observed in all six volunteers, while at rest these parameters remained unaffected. Mean u-PA- and t-PA antigen increased, respectively, from 4.2 ± 1.0 ng/ml and 5.8 ± 2.1 ng/ml before exercise to 9.8 ± 3.0 ng/ml and 18.3 ± 3.8 ng/ml (peak). Mean plasminactivatable proUK activity and t-PA activity increased, respectively, from 2.1 ± 0.4 ng/ml and 0.3 ± 0.2 ng/ml before exercise to 4.3 ± 1.7 ng/ml and 7.2 ± 4.0 ng/ml (peak). The increases were statistically significant throughout (paired t-test, pre vs post, antigen P <0.005 and activity P <0.02). After cessation of exercise u-PA and t-PA declined concurrently to normal values with a 50"/" decay in about 5 min. In conclusion, we found that both u-PA antigen and plasmin-activatable proUK activity are, concurrently with t-PA, enhanced upon exercise and, therefore, we consider that u-PA also contributes to – and co-operates in – the enhancement of the blood fibrinolytic potential and activity under these conditions.
The Acute Effect of Insulin on Tissue Plasminogen Activator and Plasminogen Activator Inhibitor in Man
Authors: Landin Kerstin , Tengborn Lilian , Chmielewska Joanna , Schenck Henning von , Smith Ulf
Published: July 2018

The present study was performed to elucidate the acute effect of insulin on levels of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor of endothelial cell type (PAI-1). Nine middle-aged, non-obese and non-smoking men were studied during a hyperinsulinemic, euglycemic glucose clamp for 2 h. Plasma insulin level during the clamp averaged 84 ± 12 mU/l and euglycemia was maintained at 4.9 ± 0.6 mmol/l. The t-PA activity gradually increased (75% mean increase after 2h, p <0.001) and the PAI-1 activity decreased (49% mean decrease after 2 h, p <0.001) during the clamp. t-PA activity decreased and PAI-1 activity increased after the insulin infusion was ceased, but they were still 48% higher and 38% lower, respectively, after 60 min. PAI-1 and t-PA activities were not affected by saline infusion for 2h.

Thus, acute changes in the insulin levels lead to alterations rapid in the fibrinolytic system even when euglycemia is maintained. These effects may be induced by insulin itself or by the concomitant activation of the sympatho-adrenal system during the euglycemic clamp.
Determination of Human Prothrombin Activation Fragment 1+2 in Plasma with an Antibody against a Synthetic Peptide
Authors: Pelzer Hermann , Schwarz Angela , Stüber Werner
Published: July 2018

The present investigation describes a novel approach to prepare a specific antibody against prothrombin activation fragment 1+2 (F 1+2). The antibody discriminates between native prothrombin and F 1+2 in plasma. A synthetic peptide from the negatively charged region of F 1+2, which becomes the carboxyterminal sequence after cleavage of prothrombin by factor Xa, was used for immunization of rabbits. Obtained antiserum was immunopurified and an enzyme-linked immunosorbent assay (ELISA) was constructed for determination of F 1+2. The test system follows the sandwich principle and uses two different antibodies directed against F 1+2 and prothrombin, respectively. The ELISA was calibrated with purified F 1+2 added to F 1+2-poor plasma. The lower limit of sensitivity of the assay was 0.02 nmol/1. Coefficients of variation of 6.9 to 10.4% (intraassay) and 6.7 to 11% (interassay) were found for F 1+2 concentrations between 0.08 and 4.9 nmol/1. A reference range from 0.32 to 1.2 nmol/l was calculated from 95 healthy donors (mean value ± SD: 0.67 ± 0.19 nmol/l). In patients with deep vein thrombosis (n = 7) confirmed by phlebography and in patients with pulmonary embolism (n = 8) confirmed by lung scan, F 1+2levels were found up to 1.5 to 9.5 nmol/l. In plasma samples of patients under oral anticoagulant therapy in the stable state F 1+2 concentrations were found to be in the range of 0.08 to 0.5 nmol/l. The results indicate that the antibody is specific and highly sensitive for quantification of F 1+2 in plasma. It can be supposed that the ELISA we have developed is a valuable tool for detection of both hypercoagulable as well as hypocoagulable states.
Thrombolytic and Pharmacokinetic Properties of Human Tissue-Type Plasminogen Activator Variants, Obtained by Deletion and/or Duplication of Structural/Functional Domains, in a Hamster Pulmonary Embolism Model
Authors: Collen D , Lijnen H R , Vanlinthout I , Kieckens L , Nelles L , Stassen J M
Published: July 2018

A pulmonary embolism model in hamsters was used for the quantitative evaluation of the thrombolytic and pharmacokinetic properties of variants of tissue-type plasminogen activator (t-PA). A 25 μi 125I-fibrin labeled human plasma clot was made in vitro and injected into the jugular vein of heparinized hamsters. The extent of thrombolysis within 90 min was determined as the difference between the radioactivity injected in the jugular vein and that recovered in the heart and lungs.

Recombinant t-PA (home-made rt-PA or Activase®) infused intravenously over 60 min caused dose-dependent progressive thrombolysis. The results of thrombolytic potency (clot lysis in percent versus dose administered in mg/kg) and of specific thrombolytic activity (clot lysis in percent versus steady state plasma level in μg/ml) were fitted with an exponentially transformed sigmoidal function y = 100 c/(1+e−a(ex−eb)) and the maximal percent lysis (c), the dose or plasma level at which maximal rate of lysis is achieved (b) and the maximal rate of lysis (z = ¼ ac .eb) were determined. With rt-PA, these parameters were c = 72 ± 6% (mean ± SEM), b = 0.19 ± 0.08 mg/kg, z = 68 ± 25% lysis per mg/kg, with corresponding values of 87 ± 5%, 0.07 ± 0.03 mg/kg and 150 ± 38% lysis per mg/kg for Activase® (p = NS). Deletion of the finger and growth factor domains in rt-PA ( rt-PA-ΔFE) was not associated with marked alteration of the thrombolytic potency (c = 90 ± 30%, b = 0.34 ± 0.35 mg/kg, and z = 54 ± 14% per mg/kg), but was associated with a significant reduction of the specific thrombolytic activity. Substitution of the kringle 1 domain by a second copy of the kringle 2 domain in rt-PA and in rt-PA-ΔFE did not significantly alter the thrombolytic potency as characterized by a simultaneous decrease of the b value and an increase of the z value (b = 0.07 ± 0.03 mg/kg and z = 210 ± 70% per mg/kg for rt-PA-ΔK1∇K2 and b = 0.13 ± 0.02 mg/kg and z = 210 ± 29% per mg/kg for rt-PA-ΔFEK1∇K2). Deletion of either the finger or the growth factor domain in rt-PA-ΔK1∇K2 also did not significantly alter the thrombolytic potency (b = 0.08 ± 0.008 mg/kg and z = 320 ± 33% per mg/kg for rt-PA-ΔEK1∇K2 and b = 0.05 ± 0.01 mg/kg and z = 270 ± 48% per mg/kg for rt-PA-ΔFK1∇K2). Likewise, none of the variants had a higher specific thrombolytic activity than wild-type rt-PA. Plasma clearance rates were reduced 10- to 20-fold for the rt-PA mutants lacking the FE domains, and 3- to 5-fold for rt-PA-ΔK1∇K2.
PAI-1 Synthesis in the Human Hepatoma Cell Line Hep G2 Is Increased by Cytokines - Evidence that the Liver Contributes to Acute Phase Behaviour of PAI-1
Authors: de Boer J P , Abbink J J , Brouwer M C , Meijer C , Roem D , Voorn G P , Lambers J W J , van Mourik J A , Hack C E
Published: July 2018

The acute phase behaviour of the fast inhibitor of tissue-type plasminogen activator (PAI-1) in vivo has been attributed to increased synthesis by endothelial cells. However, most other acute phase proteins in vivo are synthesized in the liver, which process is regulated by cytokines and can be studied in the hepatoma derived cell line HepG2.

In this study, we investigated whether the synthesis of PAI-1 by HepG2 cells is regulated by the cytokines recombinant IL-1, rlL-6 and rTNF. Recombinant IL-1 and rTNF each increased PAI-1 synthesis by Hep G2 cells two to three fold, whereas rIL-6 hardly had an effect. Mixtures of rIL-1, rIL-6 and rTNF increased PAI-1 synthesis up to eleven fold. The effects observed were not due to non-specific effects on Hep G2 cell metabolism, since synthesis of alpha-2-antiplasmin was not effected by any of those cytokines, whereas fibrinogen synthesis was increased three to four fold by rIL-6, but was unaffected by rIL-1. Thus, our results demonstrate that synthesis of PAI-1 by Hep G2 cells is regulated by cytokines and implicate that the acute phase behaviour of PAI-1 in vivo at least in part may be due to an increased synthesis by the liver.
Evaluation of the Buccal Bleeding Time and Platelet Glass Bead Retention as Assays of Hemostasis in the Dog: The Effects of Acetylsalicylic Acid, Warfarin and von Willebrand Factor Deficiency
Authors: Brassard Jacqueline A , Meyers Kenneth M
Published: July 2018

The study evaluated two hemostatic assays in the dog, a modified version of the buccal mucosal bleeding time (BMBT) and the platelet glass bead retention (PR), to describe the aspects of hemostasis measured by these assays. Von Willebrand factor (vWf)-deficient Doberman pinscher dogs were used in evaluating the effects of altered platelet adhesion. Normal dogs were treated with either acetylsalicylic acid (ASA) or warfarin to evaluate the effects of altered platelet aggregation and coagulation. There was significant prolongation of the BMBT and reduction of the PR in vWf-deficient dogs as compared to normal dogs. In ASA treated dogs the BMBT was slightly prolonged; the PR was significantly reduced. The change in ASA-induced BMBT did not correlate with the sensitivity of the dog platelets to arachidonic acid. In warfarin treated dogs there was no change in the BMBT; however, the PR was significantly reduced. The BMBT is a test of hemostasis that is sensitive to platelet adhesion and aggregation deficits. The PR is useful in detecting general abnormalities in hemostasis including platelet adhesion defects due to reduced vWf.
Subcellular Distribution and Phosphorylation of Vinculin lsoforms in Human Blood Platelets
Authors: Bruin Taco , Asijee Guus M , Prins Arie , Wouter ten Cate Jan , Sturk Augueste
Published: July 2018

In this study we investigated human blood platelet vinculin microheterogeneity, the subcellular localization and phosphorylation of the different isoforrns before and after platelet stimulation. At least 5 vinculin isoforms could be detected, as well as metavinculin. These isofonns did not demonstrate a specific subcellular localization, i. e. their relative content was similar in cytoskeleton, membrane skeleton and cytosol. Upon platelet stimulation with thrombin a small increase in α’-vinculin was noted in all platelet subfractions. The cytoskeleton of non-stimulated platelets contained a minor quantity of vinculin. Upon thrombin stimulation of the platelets the cytoskeletal vinculin content increased significantly; previously we already reported a maximal n% incorporation of the total platelet vinculin content into the cytoskeleton upon stimulation. A phosphorylation of a minor vinculin-isoforrl, i. e. at the α’/α location was mainly detected in the cytoskeleton. This phosphorylation was observable in the non-stimulated platelet cytoskeletal vinculin. These findings argue against a regulatory role for vinculin phosphorylation in the uptake of the main isoforms of this protein in the platelet cytoskeleton upon thrombin stimulation. The function of the phosphorylated cytoskeletal vinculin remains to be established.
Heparin, DDAVP and the Bleeding Time
Authors: Schulman Sam , Johnsson Hans
Published: July 2018

The effect on the bleeding time of heparin, followed by an infusion with desmopressin (DDAVP) 0.3 Μg/kg or placebo in a randomized, double-blind set-up, was investigated in 20 patients treated for acute venous thromboembolism. The bleeding time was on the average prolonged by 90% (95% confidence interval 46% -134%) after at least 1 day of treatment with heparin. At that point DDAVP shortened the bleeding time by 23% with a confidence interval of −35% to −11% whereas the effect of placebo was −2% with a confidence interval of −23% to +20%. There was also a shortening of the APTT after DDAVP but not after placebo. It is concluded that DDAVP induces a partial reversal of the effect of heparin on the bleeding time, and that DDAVP should be tried in case of hemorrhagic problems.
Platelet Heparin-Neutralizing Factor (Platelet Factor 4)
Authors: Lüscher E. F , Käser-Glanzmann R
Published: July 2018

The heparin-neutralizing platelet factor (PF4) is released from platelets under the influence of inducers of aggregation and nucleotide-release in the form of a high-molecular weight complex. The heparin-neutralizing activity is carried by a basic protein of molecular weight 29,700, four of which combine with a proteoglycan carrier, which in turn consists of 4 chondroitin sulfate A residues and a peptide moiety.

The carrier-PF4-complex dissociates at higher ionic strength into proteoglycan and PF4 proper ; both forms (bound and free) are active in the inactivation of heparin, which displaces PF4 from its natural mucopolysaccharide carrier.

PF4 is localized in human platelets in storage organelles, most likely in the α-granules and not in the adenine nucleotide and serotonin containing dense bodies. It is released simultaneously with the contents of these latter organelles.

The physiological significance of PF4 is mainly seen in the interference with the inhibition of the clotting system by heparin within and in the vicinity of platelet aggregates. Released in circulation in the course of intravascular platelet damage, it may contribute to a facilitation of thrombin formation and action, by removing heparin.

Adequately purified PF4 does not interact with fibrinogen and its derivatives and therefore is not directly involved in the induction of intravascular coagulation or platelet aggregation.
A Case against Heparin Therapy of Intravascular Coagulation
Authors: Straub P. W
Published: July 2018

Administration of a potentially dangerous drug like heparin should not be based on theoretical considerations and innumerable case reports but 1) on a firm diagnosis and 2) on a critical evaluation of the clinical benefit.

The validity of criteria for the diagnosis of intravascular coagulation (IC) is discussed. It is emphasized in particular that loss of fibrinogen into extravascular spaces can only be excluded by assays of the level and disappearance rate of serum proteins not subject to the proteolytic action of thrombin or plasmin.

Even when the diagnosis can be reasonably established, heparin therapy in a particular condition should only be advocated if its clinical benefit has been demonstrated in controlled clinical trials.

Our conviction that heparin has as yet a small if any place in the management of patients with so-called intravascular coagulations is based mainly on two sets of arguments, one stemming from a critique of the diagnosis of IC, the other from a general attitude in the evaluation of therapeutic procedures.
A Scanning Electron Microscopic Study of Platelets of Certain Animal Species
Authors: Jain N. C
Published: July 2018

Comparative morphology of platelets of the dog, cat, cow, horse, sheep, and goat was studied with the scanning electron microscope and was found generally similar. Most of the platelets were slightly biconvex or flat with relatively smooth surface and even contour. Some of these platelets had few short marginal filaments, or small shallow depressions or tiny protuberances on the surface. A small number of platelets exhibited surface irregularity and few long pseudopods which were thought to result from platelet activation during the processing of blood. The number of such irregular platelets increased in stored blood and in blood fixed at 4° C.
Protective Effect of Pantethine on Experimental Thrombocytopenia in the Rat
Authors: Ashida Shin-ichiro , Abiko Yasushi
Published: July 2018

The effects of pantethine on circulating platelet counts and platelet functions were studied in normal and experimentally produced thrombocytopenic rats.

Administration of pantethine to normal animals did not cause any alterations in both platelet count and function except for a slight enhancement of intravascular platelet aggregation induced by collagen or neuraminidase.

Injection of anti-rat platelet rabbit serum into rats resulted in acute thrombocytopenia. Administration of pantethine prior to the antiserum promoted recovery from the thrombocytopenia in a dose dependent manner, but administration of the drug after development of the thrombocytopenia was not effective. A similar result was obtained with a transient thrombocytopenia induced by exchange transfusion with platelet poor blood. Regardless of whether animals were treated with pantethine or not, the platelets newly generated during the course of recovery from thrombocytopenia were essentially normal in the function tested in vitro.

A more chronic thrombocytopenia induced by repeated injections of the antiserum was prevented, to some significant degree, by daily administration of pantethine throughout the experimental period.

In contrast to these, such effect of pantethine was not observed with the thrombocytopenia models produced by nitrogen mustard N-oxide and neuraminidase.

These findings were discussed in relation to mechanism of the action of pantethine and to possible clinical application of the drug to thrombocytopenia.
Sodium Acetate Buffer
Authors: Chohan I. S , Vermylen J , Singh I , Balakrishkan K , Verstraete M
Published: July 2018

Sodium acetate buffer, 0.12 M, pH 7.4 as a diluent in the low temperature technique of dilute clot lysis time, is more effective in accelerating the velocity of lysis than phosphate buffer of similar pH and molarity. A uniform shape of the clot is maintained throughout the digestion in sodium acetate buffer and the end point of lysis is characteristically marked by an abrupt and sharply defined disintegration. Sodium acetate buffer can be employed advantageously in this technique not only to improve the observation but also to shorten the lysis times.
The Inhibition of Urokinase by Aromatic Diamidines
Authors: Geratz J. D , Cheng Michael C.-F
Published: July 2018

1. Structure-activity relationships have been established for the inhibition of urokinase by aromatic diamidines. In an assay system employing purified urokinase and human plasminogen the most potent inhibitor was found in 4′,4″-diamidino-2-hydroxy-1,4-diphenoxybutane which proved 5600 times more active on a molar basis than epsilon-aminocaproic acid (E-ACA).

2. 4′4″-diamidino-2-hydroxy-1,4-diphenoxybutane behaved as a competitive inhibitor of the urokinase catalyzed hydrolysis of Nα-acetyl-L-lysine methyl ester. At pH 7.85 and 37° C the Ki value was determined as 3.18 × 10–6 M which compares with a value of 6.79 × 10–5 M for p-aminobenzamidine and 3.57 × 10–2 M for E–ACA.

3. In two fibrinolytic tests including urokinase as activator the superiority of diamidines over E–ACA was less marked than in the pure plasminogen activation system. This was due to the presence of certain plasma proteins in the fibrinolysis assays which augmented the inhibitory strength of E-ACA. The order of effectiveness of diamidines in the lysis tests was also different from the one in the activation test. In a human fibrin clot lysis test the most active inhibitor was 3′3″-diamidino-2-hydroxy-1,4-diphenoxybutane which was 1700 times more effective on a molar basis than E-ACA. In a human plasma clot lysis test the strongest inhibitor, 2-hydroxy-stilbamidine, was 70 times more powerful than E–ACA.
Molar Concentrations of Fibrinolytic Components, Especially Free Fibrinolysin, in Vivo
Authors: Nanninga L. B
Published: July 2018

The levels of fibrinogen and of Profibrinolysin (plasminogen) in urokinase-treated plasma as a function of time of incubation were measured. The Profibrinolysin concentration was estimated through its complete conversion to fibrinolysin and the inhibition of the enzyme by crystalline soybean trypsin inhibitor. The dissociation constant of the FL-STX complex was determined to be 7 × 10–9 M. The average concentration of Profibrinolysin in normal human citrated plasma was found to be 8 × 10–7 M. From the decrease of fibrinogen with time in the urokinase-treated plasma, the free fibrinolysin was calculated. Free fibrinolysin in normal human blood in vivo was estimated from the half-life of fibrinogen and other data obtained in this study to be present at a concentration of 1.7 × 10–10 M. The plasmakinase activity in vivo, expressed as urokinase molarity, is also about 2 × 10–10 M.
Essential Athrombia
Authors: Inceman Şeref , Tangün Yücel
Published: July 2018

A constitutional platelet function disorder in a twelve-year-old girl characterized by a lifelong bleeding tendency, prolonged bleeding time, normal platelet count, normal clot retraction, normal platelet factor 3 activity and impaired platelet aggregation was reported.

Platelet aggregation, studied turbidimetrically, was absent in the presence of usual doses of ADP (1–4 μM), although a small wave of primary aggregation was obtained by very large ADP concentrations (25–50 μM). The platelets were also unresponsive to epinephrine, thrombin and diluted collagen suspensions. But an almost normal aggregation response occurred with strong collagen suspensions. The platelets responded to Ristocetin. Pelease of platelet ADP was found to be normal by collagen and thrombin, but impaired by kaolin. Platelet fibrinogen content was normal.

The present case, investigated with recent methods, confirms the existence of a type of primary functional platelet disorder characterized solely by an aggregation defect, described in 1955 and 1962 under the name of “essential athrombia.”