bluuebunny/crossref_metadata_2025 huggingface dataset:
Localization and Biosynthesis of Functional Thrombomodulin in Human Megakaryocytes and a Human Megakaryoblastic Cell Line (MEG-01)
Authors: Ogura Michinori , Ito Takahiko , Maruyama Ikuro , Takamatsu Junki , Yamamoto Shuji , Ogawa Kohei , Nagura Hiroshi , Saito Hidehiko
Published: July 2018

The localization and biosynthesis of functiosnal thrombomodulin (TM) on the cell surfaces of human platelets, megakaryocytes and a human megakaryoblastic cell line (MEG-01) were investigated. TM was demonstrated on the cell surfaces and in cytoplasms of human platelets, megakaryocytes and MEG-01 by an indirect immunofluorescent technique using monospecific rabbit anti-human TM serum. Immunoelectronmicroscopic analysis revealed that TM was localized in plasma membranes of MEG-01 cells as well as human megakaryocytes. 125I-monoclonal antithrombomodulin IgG binding assay showed that one MEG-01 cell possessed approximately 78,000 TM molecules on its cell surface. Thrombin-dependent protein C activating-cofactor activity was demonstrated on MEG-01 cells. Northern hybridization technique using cDNA probe of TM revealed that poly(A)+-RNA from MEG-01 cells showed a single band of 3.8 kb similar to that from human endothelial cells. These data suggest that human megakaryocytes synthesize functional TM, and thereby platelets possess TM on their surfaces. TM on platelets may participate in the activation of protein C at the site of a hemostatic plug.
Point Mutations in Four Hemophilia B Patients from China
Authors: Wang N S , Chen S H , Thompson A R
Published: July 2018

Point mutations in factor IX genes of four unrelated Chinese patients with hemophilia B have been identified by direct sequencing of amplified genomic DNA fragments. These four mutations occur in exon 8 of the factor IX gene. A C to T transition at nucleotide 30,863 changes codon 248 from Arg (CGA) to a new Stop codon (TGA), described in a previous family as factor IXMalmo3 (Green P M et al., EMBO J 1989; 8: 1067). A G to A transition, at nucleotide 31,051 changes codon 310 from Trp (TGG) to a nonsense or Stop codon (TGA; factor IXchongqing2)- A G to A transition at nucleotide 31,119 changes codon 333 which is for Arg (CGA) in normal factor IX, to one for Gin (CAA) in the variant previously described as factor IXLondon2 (Tsang T C et al., EMBO J 1988; 7: 3009) in a patient with moderately severe hemophilia B. The fourth patient has a novel C to A transversion at nucleotide 31,290, which corresponds to replacement of codon 390 which is for Ala (GCA) in normal factor IX, to one for Glu (GAA) in a patient with moderately severe hemophilia B (factor IXChongqing3)- DNA sequences of amplified fragments from mothers of three showed both their son’s variant and a normal nucleotide at the appropriate position, indicating that they are carriers. The fourth patient’s (factor IXMalmo3) mother, whose DNA was not evaluable, was most probably a carrier because of her low plasma factor IX levels.
Selenium Dependent Glutathione Peroxidase: A Physiological Regulatory System for Platelet Function
Authors: Perona G , Schiavon R , Guidi G C , Veneri D , Minuz P
Published: July 2018

In human platelets the selenoenzyme glutathione peroxidase (GSH-Px) acts as a scavenger of the peroxides generated during the burst of arachidonic acid (AA) metabolism. Such a mechanism inhibits the biosynthesis of both thromboxane A2 (TXA2) and lipoxygenase products. The same mechanism is not effective on the prostacyclin (PGI2) biosynthesis from cultured endothelial cells. In order to evaluate this effect in vivo, besides in vitro, we activated the enzyme in eight normal volunteers by increasing their daily Se intake for 8 weeks, monitoring: platelet GSH-Px activity, platelet aggregation induced by A A and U 44069, and concurrent malondialdehyde (MDA) and thromboxane B2 (TXB2) production, urinary excretion of renal and systemic TXA2 and PGI2 metabolites, platelet enzyme activities of the hexose monophosphate pathway and glutathione content, platelet adenine nucleotides, bleeding time, plasma Se concentration. We found: a) progressive platelet GSH-Px activation by Se paralleling an enhancement of platelet aggregation threshold values for AA, but not for U 44069; b) concurrent inhibition of platelet biosynthesis of TXA2 both in vitro and in vivo while the biosynthesis of systemic prostacyclin was unaffected; c) a progressive increase in the bleeding time, unmodified by aspirin. In conclusion, we believe that Se-dependent GSH-Px represents a physiological mechanism regulating the biosynthesis of prostanoids with implications in platelet function and that a Se dietary supplement might be considered in the prevention of arterial thrombosis.
Subcutaneous Desmopressin (DDAVP) Shortens the Prolonged Bleeding Time in Patients with Liver Cirrhosis
Authors: Cattaneo Marco , Tenconi Paola M , Alberca Ignazio , Garcia Vicente Vicente , Mannucci Pier Mannuccio
Published: July 2018

The intravenous infusion of 1-deamino-8-D-arginine vasopressin (DDAVP) shortens the prolonged bleeding time in patients with congenital or acquired bleeding disorders, including patients with uremia or liver cirrhosis. We carried out a double-blind, placebo-controlled crossover study in ten patients with liver cirrhosis to evaluate whether or not their prolonged bleeding times could be shortened by subcutaneous injections of DDAVP (0.3 μg/kg), a more practical route of administration than intravenous infusions. One hour after DDAVP injection the bleeding time was significantly shortened (p <0.05). After 4 h, however, the bleeding time shortening was no longer statistically significant. There was no bleeding time change after placebo. Plasma levels of von Willebrand factor antigen (vWF: Ag) did not significantly increase after DDAVP or placebo. The study shows that subcutaneous DDAVP is an alternative method for shortterm shortening of the bleeding time in liver cirrhosis.
A Specific Thromboxane Receptor Blocking Drug, AH23848, Reduces Platelet Deposition on Vascular Grafts in Man
Authors: Lane I F , Lumley P , Michael M F , Peters A M , McCollum C N
Published: July 2018

The antithrombotic effect of a specific thromboxane A2 receptor blocking drug, AH23848, on radio-labelled platelet deposition in mature Dacron aorto-bifemoral grafts has been evaluated in patients. Thirty patients were randomly allocated to AH23848 70 mg, aspirin 300 mg plus dipyridamole 75 mg or placebo 8-hourly for 9 days. AH23848 inhibited platelet aggregarion induced by the thromboxane ,A2 mimetic U-46619; no such effect was observed with aspirin plus dipyridamole. 111In-platelet uptake was measured as the thrombogenicity index (TI) which is a measure of the daily rate of accumulation of platelets by the graft. The mean (s.e. mean) value of 0.193 (0.029) on placebo was significantly reduced to 0.115 (0.022) by AH23848 (p <0.05) but only to 0.175 (0.028) by aspirin plus dipyridamole. There was no difference in mean platelet life span between the three treatment groups. The pronounced antithrombotic effect of AH23848 implicates thromboxane ,A2 in the process of platelet deposition in arterial prostheses and demonstrates the considerable promise of thromboxane receptor blocking drugs as antithrombotic therapy.
A Collaborative Study to Establish a Standard for High Molecular Weight Urinary-Type Plasminogen Activator (HMW/u-PA)
Authors: Gaffney P J , Heath A B
Published: July 2018

An international collaborative study involving eleven laboratories located in eight countries was undertaken to establish an International Standard for high molecular weight urinary-type
plasminogen activator (HMW/u-PA). The current International Reference Preparation (IRP code numbered 66146) for urinarytype plasminogen activator (u-PA) ot urokinase (see Nomenclature footnote) is a 66/34 molar ratio mixture of low molecular weight (LMW) - and high molecular weight (HMW) - u-PA’s and is considered unsuitable as a standard for homogeneous preparations of HMW/u-PA. The putative standard for HMWu-PA (code number, 87/594) was compared for potency in a clot lysis
assay with the current IRP for u-PA (code numbet, 66146) and a lyophilised preparation of single chain urinary-type plasminogen activator (SCuPA), the latter being used in the assay without
prior activation by plasmin to its active two chain form (TCuPA).

Both the proposed standard for HMWu-PA (871594) and the SCuPA compared in a statistically satisfactory manner in parallel line bioassays with the current IRP for u-PA (66146), thus allowing potency estimates to be obtained for these two materials in relation to defined international units. Data from the eleven laboratories indicated that each ampoule of the proposed standard for HMWu-PA contained 4,300 i. u. of activity and was stable for over 1 year at 4° C. Most participants indicated that SCuPA expressed only a small amount of its activity without a prior activator step and this suggests that SCuPA assays need to be preceded by a plasmin activation step

The Expert Committee on Biological Standardization of the World Health Organisation (Geneva, Oct 1989) established 871 594 as the International Standard for high molecular weight two chain urinary type plasminogen activator (HMW/TCuPA, of HMWu-PA), with an assigned unitage of 4,300 international units per ampoule.
Correlation between Progressive Adsorption of Plasminogen to Blood Clots and Their Sensitivity to Lysis
Authors: Sabovic M , Lijnen H R , Keber D , Collen D
Published: July 2018

The binding of plasminogen to preformed human plasma clots immersed in citrated human plasma was measured and correlated with the sensitivity of these clots to lysis with recombinant tissuetype plasminogen activator (rt-PA), recombinant single-chain urokinase-type plasminogen activator (rscu-PA) or two chain urokinase-type plasminogen activator (tcu-PA, urokinase)

When 0.15 ml plasma clots were compressed mechanically to about 1% of their original weight, and immersed in 0.15 ml plasma, 131I-labeled native plasminogen (Glu-plasminogen) adsorbed progressively from the plasma milieu onto the clot; binding was 3 ± l% (, - 10) after 1 h, 7 ± L% after 12h and
12 ± l% after 48 h. This was associated with an increased sensitivity of the clot to lysis; 50% clot lysis in 4 h was obtained with 65 ± 5 ng/ml (n= 3) rt-PA before and 30 ± 5 ng/ml (n = 3) after 48 h preincubation in plasma (p
 1,600 ng/ml and 220 ± 2A ng/ml (n = 3) for urokinase.

These results show that preincubation of compressed plasma clots or retracted whole blood clots in plasma causes a progressive increase in both the binding of plasminogen and the sensitivity to lysis with plasminogen activators. Increased plasminogen binding may be the result of partial fibrindigestion.
Continuous Flow and the Prothrombinase-Catalyzed Activation of Prothrombin
Authors: Schoen Pieter , Lindhout Theo , Willems George , Coenraad Hemker H
Published: July 2018

The activation of prothrombin by prothrombinase was investigated in a continuous flow system at 25 °C. A glass capillary, containing a continuous phospholipid bilayer attached to the interior surface, was first perfused with factor Va. The factor Va bound to the phospholipid surface functioned as sites for the formation of prothrombinase, when subsequently a factor Xa and prothrombin containing solution was perfused. Under the conditions used, steady-state rates of prothrombin activation were attained after 4 to 15 min. The rates of prothrombinase formation increased with increasing factor Xa concentrations and flow rates, which is compatible with the assembly of prothrombinase being dependent on the flux of factor Xa to the phospholipid-bound factor Va. As long as factor Xa and prothrombin were present in the fluid phase the assembly of prothrombinase was apparently irreversible; during at least 20 min no loss of activity occurred. The steady-state rate of prothrombin activation was dependent on the surface concentration of prothrombinase, at 1.0 ΜM prothrombin and a shear rate of 82 s−1 the average rate was 870 mol thrombin/min per mol prothrombinase. In contrast to test tube experiments it was observed that in this flow system, the formation of Α-thrombin is favoured above the formation of meizothrombin (des fragment 1).
The pH Dependence of the Binding of Pro-Urokinase to Fibrin/Celite
Authors: Pannell Ralph , Angles-Cano Eduardo , Gurewich Victor
Published: July 2018

A re-examination of the affinity of pro-urokinase (pro-UK), HMW and LMW-urokinase (UK) to fibrin/Celite was undertaken in order to explain how the chance purification of pro-UK from freshly voided urine by fibrin/Celite affinity chromatography may be reconciled with the subsequent observations that pro-UK failed to bind significantly to fibrin clots in plasma. A significant pH dependence of pro-UK binding to fibrin/Celite was found. Substantial binding of pro-UK (native or recombinant from E. coli), but not of the two-chains forms, was seen at about pH 6.5, which is in the normal pH range of pooled, freshly voided urine. By contrast, at pH 7.4 fibrin binding of pro-UK was much reduced, though it was still significantly greater than that of HMW or LMW-UK. This finding helps to explain the fibrin-binding of pro-UK in freshly voided urine but not in blood. In order to determine if this' pH dependence was the sole explanation for why pro-UK could not be isolated by this method from stored urine, the stability of pro-UK in urine was evaluated by incubating 125I-labeled pro-UK in urine: Incubation for up to 4 days (37° C) was not accompanied by any degradation of the single-chain pro-UK as evidenced by autoradiography under reducing conditions. It was concluded that the alkaline shift in pH which occurs in urine left standing, rather than the degradation of pro-UK, explained why freshly voided urine was found to be essential. Clot lysis studies at pH 6.5 and 7.4 showed no promotion of fibrinolysis at the pH which favored fibrin/Celite binding. Therefore, while the present study defines the conditions under which pro-UK may be purified from urine by fibrin/Celite chromatography, it provides no evidence that this binding phenomenon plays any role in fibrinolysis.
Synthetic RGDS-Containing Peptides of von Willebrand Factor Inhibit Platelet Adhesion to Collagen
Authors: Fressinaud Edith , Girma Jean Pierre , Sadler J Evan , Baumgartner Hans R , Meyer Dominique
Published: July 2018

We compared the effect of a synthetic dodecapeptide of residues 400-411 of the Γ chain of fibrinogen (Γ Fg 400-411) and of three synthetic peptides (15 to 18 aminoacids), of human von Willebrand Factor (vWF), containing the 1744-1747 Arg-Gly-Asp-Ser (RGDS) sequence, upon platelet adhesion to collagen in flowing blood. Both types of peptides are known to inhibit the binding of adhesive proteins to platelet membrane glycoprotein Ilb/IIIa (GPIIb/IIIa). Collagen was coated onto plastic cover slips and exposed in parallel-plate perfusion chambers to reconstituted human blood at various shear rates for 5 min at 37 °C. At a shear rate of 2,600 s−1, RGDS peptides inhibited platelet adhesion to collagen in a dose-dependent manner and appeared to be more potent inhibitors than the Γ Fg 400-411 on a molar basis. No synergetic effect between RGDS and Γ Fg 400-411 peptides was observed. These results suggest that the RGDS peptides affect adhesion by inhibiting the GPIIb/IIIa-vWF interaction and confirm the involvement of this platelet receptor in vWF-mediated platelet adhesion to collagen at high shear rate.
High Versus Ultra-high Purity Factor VIII Concentrate Therapy: Prospective Evaluation of Immunological and Clinical Parameters in HIV Seronegative and Seropositive Hemophiliacs
Authors: Varon D , Schulman S , Dardik R , Barzilai A , Bashari D , Martinowitz U
Published: July 2018

This study aimed at evaluation of the immunological status and the clinical course of both HIV seronegative and seropositive hemophiliacs treated with either an ultra-pure factor VIII product (UP-F VIII), or a high-purity F VIII (HP-F VIII) concentrate. Eighteen HIV seronegative patients were divided into two groups of therapy and their immune status was followed for 2 years. During the second year of the study 8 patients of the HP-F VIII and 6 from the UP-F VIII therapy groups were switched to the alternative F VIII concentrates. Eighteen asymptomatic HIV seropositive patients were also divided into therapy groups and their immune status and any development of HIV-related symptoms were followed for 4 years. Evaluation of the HIV seronegative patients during the first year did not reveal any differences between the groups in the CD4 or CD8 cell counts, in natural killer cell (NK) activity, or in the mitogenic responses of T lymphocytes to Phytohemagglutinin (PHA), and of B lymphocytes to Pokeweed mitogen (PWM). The switch of 8 patients from the HP-F VIII and 6 from the UP-F VIII groups to the alternative concentrate did not yield any changes in their immune profile during the second year of the study. The HIV seropositive groups differed in the initial CD4 count, with a lower CD4 count (193 ± 126 vs 437 ± 142) and a higher F VIII consumption (63,000 ± 17,000 vs 26,000 ± 10,000) in the UP-F VIII group. During four years of follow-up the annual decline of CD4 counts in the UP-F VIII group was 8 (5%) compared with 74 (17%) in the HP-F VIII group, with only the latter being significant. The decrease in delayed type hypersensitivity, was also significant in the HP-F VIII group alone. Cumulative HIV-related symptoms with an advancement from CDC category II to category IV were observed in 2 and 5 of the UP-F VIII and HP-F VIII treatment groups, respectively. Our data, taken separately or together with previously published studies, demonstrate the impact of purity of the F VIII concentrate on CD4 decline and possibly also on the clinical course in HIV seropositive hemophiliacs. In the seronegative group, we found no support for a beneficial effect on the immune system by switching to an ultra-pure F VIII concentrate.
Isolation and Characterization of Single-Chain Protein S
Authors: Meijer-Huizinga Fenny , Mertens Koen , van Mourik Jan A
Published: July 2018

Protein S is a vitamin K-dependent cofactor of activated protein C in the proteolytic cleavage and concomitant inactivation of the coagulation factors Va and Villa. Protein S is sensitive to proteolysis by thrombin which reduces its functional activity. Uncontrolled proteolytic breakdown, leading to the generation of a two-chain molecule, is commonly encountered during the purification of both human and bovine protein S. In this study we demonstrate that human, single-chain, intact protein S can be isolated from plasma in a single step by affinity chromatography using a monoclonal antibody, CLB PS 52, directed to an epitope located within the thrombin-sensitive region of protein S. The product of purification was readily cleaved by thrombin after Arg49, resulting in a two-chain molecule which demonstrated a lower reactivity towards CLB-PS 52 than the parent single-chain protein. This study for the first time shows that intact protein S can be isolated directly from plasma using a monoclonal antibody selected for its ability to recognize uncleaved protein S.
Inhibition of Microarterial Thrombosis by Activated Protein C in a Rabbit Model
Authors: Arnljots Björn , Bergqvist David , Dahlbäck Björn
Published: July 2018

Protein C is the key component in a natural anticoagulant pathway. After its activation by the thrombin-thrombomodulin complex, it degrades the activated forms of coagulation cofactors VIII and V, which leads to downregulation of the coagulation process. Due to its specific anticoagulant activity, activated protein C (APC) is potentially interesting as an antithrombotic agent. The effect of bovine activated protein C on thrombus formation and haemostasis was investigated in a rabbit model of microarterial thrombosis. Segments of both central ear arteries were prepared and blood-flow interrupted with double vascular clamps.

Longitudinal arteriotomies (7 mm) and deep vessel wall trauma (5 mm) were performed, whereafter the arteriotomies were closed with running sutures. Five minutes prior to opening of the clamps (reperfusion), boluses of APC (0.8 mg/kg body weight) or vehicle alone were administered to two groups, each of 10 rabbits, in a blind random fashion. Vessel patency-rates were drastically improved by the administration of APC compared to vehicle. Correspondingly, thrombus weights were significantly lower in the APC group than in the control group. The activated partial thromboplastin time was prolonged to approximately twice the baseline throughout the 2 h observation interval in the APC group. Levels of circulating platelets were unaffected by the APC infusion, but the arteriotomy bleeding times were significantly longer in the APC group. In summary, activated protein C exerted powerful and long-acting antithrombotic effects in a microarterial model of thrombosis in rabbits.
High Affinity Binding Sites for Activated Protein C and Protein C on Cultured Human Umbilical Vein Endothelial Cells
Authors: Bangalore Neelesh , Drohan William N , Orthner Carolyn L
Published: July 2018

Activated protein C (APC) is an antithrombotic serine proteinase having anticoagulant, profibrinolytic and anti-inflammatory activities. Despite its potential clinical utility, relatively little is known about its clearance mechanisms. In the present study we have characterized the interaction of APC and its active site blocked forms with human umbilical vein endothelial cells (HUVEC). At 4° C 125I-APC bound to HUVEC in a specific, time dependent, saturable and reversible manner. Scatchard analysis of the binding isotherm demonstrated a Kd value of 6.8 nM and total number of binding sites per cell of 359,000. Similar binding isotherms were obtained using radiolabeled protein C (PC) zymogen as well as D-phe-pro-arg-chloromethylketone (PPACK) inhibited APC indicating that a functional active site was not required. Competition studies showed that the binding of APC, PPACK-APC and PC were mutually exclusive suggesting that they bound to the same site(s). Proteolytic removal of the N-terminal γ-carboxyglutamic acid (gla) domain of PC abolished its ability to compete indicating that the gla-domain was essential for cell binding. Surprisingly, APC binding to these cells appeared to be independent of protein S, a cofactor of APC generally thought to be required for its high affinity binding to cell surfaces. The identity of the cell binding site(s), for the most part, appeared to be distinct from other known APC ligands which are associated with cell membranes or extracellular matrix including phospholipid, thrombomodulin, factor V, plasminogen activator inhibitor type 1 (PAI-1) and heparin. Pretreatment of HUVEC with antifactor VIII antibody caused partial inhibition of 125I-APC binding indicating that factor VIII or a homolog accounted for ∼30% of APC binding. Studies of the properties of surface bound 125I-APC or 125I-PC and their fate at 4°C compared to 37 °C were consistent with association of ∼25% of the initially bound radioligand with an endocytic receptor. However, most of the radioligand appeared not to be bound to an endocytic receptor and dissociated rapidly at 37° C in an intact and functional state. These data indicate the presence of specific, high affinity binding sites for APC and PC on the surface of HUVEC. While a minor proportion of binding sites may be involved in endocytosis, the identity and function of the major proportion is presently unknown. It is speculated that this putative receptor may be a further mechanisms of localizing the PC antithrombotic system to the vascular endothelium.
Differences in the Binding to Fibrin of Urokinase and Tissue Plasminogen Activator
Authors: Thorsen Sixtus , Glas-Greenwalt Pia , Astrup Tage
Published: July 2018

When present during clot formation, tissue plasminogen activator (TA, porcine) is strongly bound to fibrin, in marked contrast to urokinase (UK, human). The amount of bound activator is approximately proportional to the total concentration of activator present, suggesting the formation of a dissociable complex between activator and binding sites on the fibrin. Frozen sections of plasminogen-free fibrin clots prepared in the presence of TA or UK, studied with the histochemical fibrin slide technique, showed sites of fibrinolytic activity related to fibrin strands in the clot. The activity caused by TA was more uniformly distributed along the fibrin strands than that caused by UK.
Inhibition of Thromboplastin Activity in Plasma by Aluminosilicate Treatment
Authors: Chenkin Theodore , Moses Doris , Perel James M. , Weiner Murray
Published: July 2018

Permutit, a synthetic aluminosilicate is an ion exchange agent which, on mixing with plasma produces a prolongation of the clotting time, change in pH, and removal of some gamma globulin and alpha and beta lipoprotein. Heat-inactivation of the ion-exchange property of Permutit also eliminates these effects on plasma. Some of these changes also occur on mixing Permutit with extrinsic thromboplastins.

The evidence presented suggests that the prolonged clotting time is not due to the removal of one or more clotting factors from plasma treated with Permutit.

It is concluded that the prolongation of clotting time produced by Permutit treatment of plasma is due to the formation or release of a potent thromboplastin inhibitor.
Lack of Evidence for a Thrombolytic Effect of Sodium Gluconate in Arteries
Authors: Rasche H , Hiemeyer V , Schniep K
Published: July 2018

After Kopper (1966) reported that sodium gluconate induces thrombolysis, we tested the effect of this substance on 8 hrs old clots which had been artificially initiated in the femoral artery of cats. These clots can be lysed completely as previous experiments with streptokinase and urokinase have shown. During a 24 hrs continuous administration of sodium gluconate, in no animal a thrombolytic effect was observed. Kopper’s positive results, which were obtained primarily on venous clots, lead one to believe that possibly sodium gluconate liberates plasminogen activators from the endothelium of veins, where these are in high concentrations compared to the intima of arteries.
Potato Inhibitors of Intrinsic Prothrombin Activators
Authors: Worowski K
Published: July 2018

The potato protease inhibitors inhibit intrinsic prothrombin activators and trypsin activation of prothrombin. The inhibitors 5a and 5b are responsible for the intrinsic prothrombin activator inhibition. This inhibition is progressive. No inhibition by these inhibitors of tissue thromboplastin activation of prothrombin or thrombin activity was observed.
Analysis of the Effect of Musical Stimulation on Cortical Auditory Evoked Potentials
Authors: Lima Daiane , Regaçone Simone , Oliveira Anna , Alcântara Yara , Chagas Eduardo , Frizzo Ana
Published: July 2018

Introduction Cortical auditory evoked potentials (CAEPs) are bioelectric responses that occur from acoustic stimulations, and they assess the functionality of the central auditory system.

Objective The objective of the present study was to analyze the effect of musical stimulation on CAEPs.

Methods The sample consisted of 42 healthy female subjects, aged between 18 and 24 years, divided into two groups – G1: without musical stimulation prior to the CAEP examination; and G2: with stimulation prior to the examination. In both groups, as a pre-collection procedure, the complete basic audiological evaluation was performed. For the musical stimulation performed in G2, we used an MP4 player programmed to play Pachelbel's “Canon in D Major” for five minutes prior to the CAEP examination. To analyze the effect on the groups, the ear side and the ide–group interaction , a mixed analysis of variance (ANOVA) of repeated measures was performed. Box M test and Mauchly sphericity test were also performed.

Results Test differences were considered statistically significant when the p-value was < 0.05 (5%). Thus, it was possible to observe that there was a statistically significant difference of the P2 component characterized by the decrease in the amplitude of response in the left ear in G2 when comparing the responses of CAEP with and without prior musical stimulation.

Conclusion The result of the present study enabled us to conclude that there was a change in the response of CAEPs with musical stimulation.
The Control Of Antithrombotic Prophylaxis And Therapy: A Pro-Coagulant Effect Of Heparin
Authors: Szwarcer E , Giuliani R , Aquino E Martinez
Published: July 2018

For studying heparin effect on blood coagulation and on inhibitors, the drug was added at increasing amounts to a normal platelet poor plasma (PPP), and to plasmas of patients with variable amounts of clotting factors (cirrhotic, pregnant, etc) -IN VITRO STUDIES-, and infused to the same individuals -IN VIVO STUDIES-. Modifications on two clotting assays (KCCT-TT) were compared to heparin potentiating effect on AntiXa (Denson & Bonnar tech).

When studied IN VITRO, the sensibility of KCCT, TT, and AntiXa techniques for heparin measurement was similar. IN VIVO, an apparently greater sensibility using AntiXa technique was observed.

For determining if this phenomena was related to a specific enhanced potentiating effect of the inhibitor against Xa, exerted by heparin IN VIVO, experiences were repeated IN VITRO and IN VIVO, measuring heparin effect on KCCT, TT, and on the inhibitor, studied against Xa and thrombin. A personal technique was used for the measurement of Antithrombin III heparin potentiating effect, using diluted platelet poor test plasma, heated (56°C 15’) and incubated with thrombin during a fixed time, and reading residual thrombin on citrated human PPP. IN VITRO, all techniques were similar in their ability to show heparin presence.

IN VIVO, the potentiating effect of heparin on the inhibitor, measured against Xa or thrombin, was greater than the changes obtained on KCCT or TT.

So, AntiXa-Antithrombin III techniques seem to be more sensitive for heparin measurement IN VIVO.

This “dissociation” of results in between the potentiating effect on the inhibitor, that is not simultaneously exerted on global coagulation, is interpreted as a heparin pro-coagulant effect, exerted by the drug IN VIVO.
Effect Of Heparin In Hereditary Antithrombin III Deficiency
Authors: Losonczy H , Nagy I
Published: July 2018

Hereditary antithrombin III (AT III) deficiency was divided into three types: In type I,both quantity and function of AT III were diminished, in type II, AT III was normal in quantity but abnormal in function,and in type III, quantity and function were normal but activation of AT III by heparin was diminished. In the present study,the response to heparin of the different types of AT III deficiency was examined. Tests were carried out on 10 healthy volunteers and on 14 patients with known AT III deficiencies who had suffered from recurring thrombotic episodes. In addition, 7 relatives of these patients without a history of thromboembolism were examined. Three patients belonged to type 1,7 to type II, and 4 to type III. All patients and controls received an intravenous infusion of 10,000 I.U. heparin within 1 hour. On a second occasion, 20,000 I.U. heparin was given in the same way. Activities of AT III and plasma heparin levels were assayed by amidolytic methods (Coatest AT III and Coa- test Heparin, KABI).

AT III activity of the controls was between 80% and 130% of the normal average. This activity was not influenced by the two doses of heparin. In type I,averaqe AT III activity was 57.5% (minimum 25%, maximum 80%). After heparin,a further 20% decrease of AT III activity was observed. In type 11,the heparin-induced decrease of AT III activity averaged 15.4% whilst in type III AT III activity was not influenced by heparin. Though the difference of plasma heparin levels after the two different doses of heparin was comparatively small,there was a significant difference of the AT III decreasing effect.
Action Of SSHA And SP 54 On Blood Coagulation In Comparison With Commercial Heparin Preparations
Authors: Stemberger A , Merkl C , Haas S , Fritsche H M , Blūmel G
Published: July 2018

A semisynthetic heparin analogue (SSHA) and natrium-pento- usan-sulfate (SP 54) are discussed as potential drugs in preventing thrombosis. Action on coagulation was studied in comparison with commercially available heparin preparations and with fractionated heparins based on the assumption that 150 USPU heparin = 1 mg heparin.

The drugs were compared on weight using per ml plasma 2,5, 10 μg of the drugs in routine tests like thrombin-time,aPTT and prothrombin-time. With chromogenic substrates the inhibitory effects of the drugs were elucidated on the Xa-and thrombin level after exogen or endogen activation of the coagulation system. Inhibition of Xa and thrombin was studied using partly purified enzymes and AT III preparations.

These studies have shown that SSHA and SP 54 increase the Xa-AT III and dosedependent the IIa -ATIII reaction. The chromogenic substrate assays have further shown that commercial heparins differed slightly in their inhibitory capacity. An increase of the inhibitory effect could be observed using fractionated heparin preparations compared on USP-units.
Intermittent Infusions Of Arvin And Low-Dose Urokinase In The Treatment Of Deep Vein Thrombosis
Authors: Dubiel M K , Scully M F , Ham R , Djazaeri B , Kakkar V V
Published: July 2018

A preliminary study has been made on the thrombolytic therapy effectivity in seven patients with deep vein thrombosis, undergoing intermittent arvin/urokinase therapy. On the first day, patients received an infusion of 70 units of arvin over a period of 6 hours, followed by a bolus infusion of 70 units of arvin and half an hour later, a bolus infusion of 250,000 units of urokinase. After 6 hours 35 units of arvin were administered over a six hourly period followed by a bolus infusion of 250,000 units of urokinase. Subsequent four days consited of a six hourly infusion of 35 units of arvin followed by a bolus infusion of 250,000 units of urokinase. Urokinase dose was repeated twice at 8 hourly intervals. Blood samples were collected each day, pre arvin, post arvin and post first dose of urokinase.

No haemorrhagic complications were observed. Two out of four patients with incomplete occlusive thrombi showed 100% lysis, in the other two no lysis occurred. The three patients with complete occlusive thrombi showed no lysis.

After the initial infusion of arvin, circulating clottable fibrinogen fell to ≃35% of the preinfusion level. Urokinase had a small effect on the fibrinogen level reducing it to 12% of the preinfusion level. Plasma concentration of fibrinogen degradation products initially rose to levels >3 mg/ml but dropped to lower levels remaining there throughout the treatment. On the first day of treatment plasminogen levels dropped by ≃60% and antiplasmin levels by ≃80% of the pretreatraent level, after the administration of arvin and urokinase. Levels of urokinase rose to > 0.3u/ml post treatment but fell to≃0.01u/ml in the pretreatment samples.

These results suggest that this type of treatment involving a defibrinogenating agent arvin, and a plasminogen activator urokinase, may constitute a safe and effective way of lysing non-occlusive deep vein thrombin without any associated haemorrhagic complications.
The Effect Of Alpha-Tocopherol On Urokinase
Authors: Ogston D
Published: July 2018

Alpha-tocopherol (vitamin E) in the form of its succinate markedly inhibited the activity of urokinase (5 CTA u/ml) on fibrin plates when tested at a concentration of 10-5m . The amidolytic (S-2444) and esterase (N-α-acetylglycyl-L-lysine methyl ester) activities of urokinase were also inhibited by α-tocopherol. Inhibition was concentration and time-dependent. Sodium succinate (10-4M) did not influence urokinase activity.

The inhibition of urokinase by α-tocopherol was abolished in the presence of dilutions of whole plasma. All fractions of plasma separated by gel filtration on Sephadex G-200 interfered with the inhibition of urokinase by α-tocopherol indicating that the effect of plasma does not reside in a single protein. The inhibition of urokinase by α-tocopherol could be reversed by serum albumin.

It is concluded that α-tocopherol is a potent inhibitor of urokinase in purified systems, but the effect of protein on the reaction suggests that this inhibition has little physiological relevance.
Local Versus Systemic Fibrinolytic Therapy
Authors: Stowell V A , Mitchell J E , Caprini J A , Vagher J P , Zuckerman L
Published: July 2018

The purpose of this study was to examine fibrinolytic activation produced by streptokinase-plasmin complex in a dog with an artificially-induced thrombus. A labelled thrombus was produced by clotting a Wessler stasis segment in the external jugular vein with thrombin (100 u) and 1-125 Fibrinogen (100 μC) and occlusion maintained for 40 minutes. Streptokinase-plasmin complex (SKp) or saline was injected as a systemic bolus (femoral vein) or locally (at the jugular site) 60 minutes after the thrombus was formed. Clot dissolution was monitored by appearance of 1-125 in the circulation. Platelet count, hematocrit, partial thromboplastin time, fibrinogen, fibrin split products, thrombelastography, and Lee-White clotting times were performed before the surgical procedure and at 30 minute intervals. A systemic fibrinolytic state was achieved in both local and systemic experiments. However, 1-125 appearance and gross examination after the experiments showed that no appreciable clot dissolution had occurred when SKp was injected systemically in contrast to virtually complete lysis with local SKp injection. This total dissolution was apparent 40 minutes post injection. It was concluded that 1-125 appearance is a sensitive monitoring method for detecting the degree of lysis, and local administration of the fibrinolytic agent seems to be much more effective than systemic SKp injection.
The Fibrinolytic Response To Stanozolol In Normal Subjects
Authors: Greaves M , Preston F E
Published: July 2018

It has been recognized for a number of years that fibrinolytic activity in patients with various forms of vascular disease may be enhanced by stanozolol (17-β-hydroxy-17α-methyl adrostano {3.2,- c } pyrazole ). However, little is known of its effect in normal healthy individuals. Also it is not known how quickly the drug exerts its effects. Two groups of eight normal healthy adults were studied. Seven subjects were included in both groups. Group A received stanozolol 5mgs b.d. Group B received 5mgs daily. Before receiving the drug, two base line studies were performed on blood samples from each individual. Thereafter, blood samples were taken at 2,7, 10, 17, 24, 31 and 38 days after commencement of treatment. The following investigations were performed - haemoglobin, white cell count, platelet count, euglobulin clot lysis time (ECLT), fibrinogen (Fg), plasminogen (Pg), α2 macroglobulin (α2M), fibrin degradation products, serum albumin, liver enzymes. In Group A statistically significant increases in plasminogen activator activity, measured by the ECLT, and Pg were observed 2 days (P<0.05) and 10 days (P<0.05) respectively after administration of stanozolol; these remained elevated throughout the study. Also, significant reductions of plasma Fg (P<0.01) and α2M (P<0.05) occurred within 7 days of commencement of the drug and these also persisted throughout the six week period of the trial. Similar statistically-significant changes were also observed in Group B subjects in ECLT, Pg, α2M, although the induced changes were less predictable than those observed in Group A. These results indicate that stanozolol exerts significant and persistent enhancement of fibrinolysis in normal individuals.
Acquired α2 Antiplasmin Deficiency In Patients With Glomerular Proteinuria
Authors: Taberner D A
Published: July 2018

α2 antiplasmin has been measured in the plasma of 20 patients with a glomerularproteinuria. Significantly lower levels were found as compared to eight controls. The unconcentrated urine was also examined and α2 antiplasmin antigen was demonstrated in seven patients but in none of the controls. Deficiency of antithrombin III due to glomerular leak has been suggested as a major cause for tendency for venous thrombosis in these patients.

Acquired α2 antiplasmin deficiency also due to renal loss may tend to counterbalance this effect. Furthermore, the presence of this potent plasmin inhibitor in the urine in patients with nephrotic syndrome may be involved in other pathological mechanisms involving fibrinolysis in the renal tract.
The Estimation Of α2-Antiplasmin By Means Of Synthetic Substrates; Kinetic Analysis And Optimization Of The Method
Authors: Dooijewaard G , Kluft C
Published: July 2018

Generally the concentration of an enzyme inhibitor in plasma is estimated as follows: plasma, in decreasing dilutions, is incubated with a fixed amount of enzyme. When the inhibitor has been irreversibly converted to its enzyme- inhibitor complex, the excess of enzyme is estimated as the initial rate (v) of its activity with a synthetic substrate. The plot of v against plasma dilution results in a straight line which intercepts the abscissa at the point of equivalency.

Contrary to the above method, for the estimation of α2-antiplasmin (A) in plasma, the excess of plasmin (Pi) is measured immediately after the attainment of a fast equilibrium between Pi, A and the reversible complex PlA (equilibrium constant Ki). Since this is attained almost two orders of magnitude faster than the irreversible reactions of PI with inhibitors in plasma other than A, the assay,thus, becomes more specific for A.

The results of the kinetic analysis are; 1. The involvement of the reversible equilibrium causes the plot of v against plasma dilution to be a hyperbola instead of a straight line. The hyperbolic shape of the plot becomes more pronounced at higher ratios of K, over Pi concentration. Moreover, the synthetic substrati (S) shifts the equilibrium
to dissociation, thereby apparently increasing the Ki by a factor of (1 + S/Km). 2. Pi inhibitors in plasma other than A, interfere Relatively more at low plasma dilutions. Because, coincidently, this interference makes the hyperbola resemble a straight line, the contribution of the other inhibitors is largely overlooked.

It is concluded that the usual assay may have its merit as a routine bioassay, provided the conditions are chosen carefully. As an alternative approach it is recommended to titrate a fixed amount of plasma with increasing concentrations of Pi and to evaluate the initial velocities in a Scatchard plot. In this way a more reliable point of equivalency and in addition the Ki value can be obtained
Hemostatic Activation During Two-Syringe Technique
Authors: Caprini J A , Vagher J P , Yeager S , Harvey G , Mitchell J
Published: July 2018

These studies were done to compare APTT test results on the first and second aliquots of blood drawn by a 2 syringe technique using a 19 Ga. thinwall needle. Three ml of blood were drawn into a plastic syringe following a clean venapuncture and another 5 ml of blood were drawn into a second plastic syringe. The two specimens were immediately dispensed into polypropylene test tubes for TEG analysis. Polypropylene tibes containing buffered citrate (Exp 1) or pediatric blue-top Vacutainer tubes (Exp 2) were used for APTT analysis. The native whole blood specimens were placed in the TEG cups within 4 min. of venapuncture. The citrated specimens were centrifuged within 5 minutes and plasma APTT assays were done in duplicate. Test results were expressed as the absolute difference between the two samples (ΔAPTT and ΔTEG). Blood from the first syringe showed a TEG pattern visually and statistically different (ΔTEG) than the sample from the second syringe 100% of the time. ΔTEG mean (SD) in Exp 1 was 3.7 (2.9). The APTT values obtained from the first syringe showed considerable variation when compared to its second syringe counterpart. The variability in APTT (ΔAPTT) between the first and second aliquots was markedly increased when Vacutainers were used. In Exp 1 with 30 samples ΔAPTT was 7.6 sec mean (9.5sec SD) which was statistically different from Exp 2 with 15 cases which was 20.6 sec (37.7 sec). In addition, the variability between the two samples increased significantly as the APTT lengthened and this was further augmented by using Vacutainers. These results emphasize the importance of a 2 syringe technique in obtaining consistent APTT results. The variation in the first syringe may be due to contamination by tissue thromboplastin in the needle and apparantly disappears if at least 5 ml of blood are allowed to pass through before samples are taken. Inconsistencies seen with Vacutainers may be due to surface activation of the blood by glass or shear effects from the high velocity of the blood through the needle. These data indicate that TEG can be used to detect samples contaminated by poor drawing.
Altered Susceptibility In The Chediak-Higashi Syndrome (Aleutian) Mink To The Generalized Shwartzman Reaction
Authors: Bell T G , Carter W E , Aulerich R J , Mattson J C , Padgett G A
Published: July 2018

The Aleutian mink is known to have many characteristics similar to other species with Chediak-Higashi Syndrome (CHS) and is considered to have a form of CHS. The CHS mink is partially characterized by defective platelets that lack nucleotide-serotonin dense body storage and by impaired leukocyte function where chemotaxis, degranulation and bacterial killing are decreased. A normalization of CHS leukocyte microbicidal activity in vitro has been demonstrated following the addition of normal platelets. The influence of these platelet-leukocyte defects on the endotoxin induced generalized Shwartzman reaction (GSR) has never previously been examined.

A study was undertaken to determine differences in the endotoxin dose required for GSR induction and in clinical, gross and histopathologic findings in normal and CHS mink. Twenty mink, half normal and half CHS were administered 55:B5 Escherichia coli endotoxin or saline intraperitone- ally followed by a second identical injection 21 hrs. later. Forty-eight hrs. later or at the time of death all animals were necropsied and sampled for histopathologic examination. The results revealed that CHS mink were more susceptible to lethal effects of endotoxin and had different signs and lesions. The dual dosage ranged from 200 μg to 25 mg/kg. CHS mink were symptomatic at 400 μg/kg and were killed at 12.8 mg/kg or greater. Normal mink were symptomatic at 800 μg/kg and died at 25.6 mg/kg. Spleno- hepatomegally with extravasation was present at a dose of 1.6 mg/kg in CHS while it was not a lesion of normal mink or control CHS mink. Renal tubular necrosis was present in CHS mink. Thus, CHS mink were more susceptible to GSR induction which may be the result of CHS platelet/leuko-cyte dysfunction manifest after endotoxin induced vascular injury.
The Clinical Diagnosis Of Deep Venous Thrombosis
Authors: Briët E , Boekhout-Mussert M J , van Hulsteijn L H , Koch C W , Loose H W C , Koch C Th
Published: July 2018

Fifty-three patients were examined because of suspected deep venous thrombosis, by means of clinical examination, Doppler ultrasound and venography. Eighty-two legs were examined with all three methods. Venography was positive in 40 and normal in 42. The clinical examination was false positive in 4 legs and false negative in 6. The Doppler ultrasound studies gave false positive results in 3 legs and false negative results in 6. These results are better than those reported in the literature probably because the thrombosis extended to the popliteal vein or the more proximal veins in 38 of the 40 legs with deep vein thrombosis. This high percentage of upper leg vein thrombosis can be explained by the fact that 47 of the 53 patients were ambulant when they developed the signs and symptoms of thrombosis. It is concluded, that the clinical examination and Doppler ultrasonography can be used to diagnose deep vein thrombosis in ambulant patients in our clinic. We presume that the findings reported in the literature cannot be used indiscriminately as a basis for diagnostic strategies in other hospitals because of widely varying categories of patients, referral patterns and diagnostic criteria that are virtually impossible to standardize.