bluuebunny/crossref_metadata_2025 huggingface dataset:
Antithrombin III “Northwick Park”: A Variant Antithrombin with Normal Affinity for Heparin but Reduced Heparin Cofactor Activity
Authors: Howarth D J , Samson Diana , Stirling Yvonne , Seghatchian M J
Published: July 2018

Further studies have been carried out in a previously reported family with congenital antithrombin III (AT III) deficiency due to an abnormal variant of AT III (AT III Northwick Park). The variant has been identified in five members of the family, three of whom had a history of venous thrombosis. Inheritance followed an autosomal dominant pattern. The affected family members have reduced levels of antithrombin heparin cofactor (41–67%) and progressive antithrombin activity (44–62%) but normal levels of immunoreactive AT III (91–162%). Two dimensional immunoelectrophoresis (2 DIE) of AT III in the absence of heparin revealed an abnormal fast-moving peak in addition to the normal peak but 2 DIE in the presence of heparin appeared normal. Further studies confirmed that the abnormal AT III binds completely to heparin but has no heparin cofactor or progressive antithrombin activity. These results would be consistent with a mutation affecting the binding site for thrombin.
The Effect of Ticlopidine on Platelet Functions in Acute Myocardial Infarction
Authors: Knudsen J Bjerre , Kjøller E , Skagen K , Gormsen J
Published: July 2018

A group of 43 consecutive patients with AMI were randomized to treatment with a novel platelet inhibitor, ticlopidine, or placebo in a double blind study. Treatment was started within 12 hr after onset of precordial pain. Patients who had taken drugs with known platelet inhibitory effect prior to the onset of therapy were excluded.

Platelet survival time (PS) was measured 24–36 hr after onset of precordial pain and after 3 months of treatment in both groups. In the early phase of AMI CK-MB and ASAT were taken twice daily for estimation of infarction size. Platelet function, coagulation factors and fibrinolysis parameters were followed sequentially for 21 days and repeated after 3 months.

In the placebo group a significant reduction in PS (5.62 ± 1.63 S. D. days) was measured in the acute phase of AMI compared to PS 3 months after infarction (8.03 ± 1.20 S.D. days). In the ticlopidine group PS was normal during the acute phase (8.35 ± 1.82 S.D. days). After 3 months of treatment PS was normal in both groups. During the first two weeks after AMI significant changes in coagulation parameters and fibrinolysis indicated an increased risk of thrombosis in both groups. These parameters were unaffected by the platelet inhibitory therapy. Estimated by peak CK-MB and ASAT, infarction size was significantly reduced in the ticlopidine group.
Smooth Muscle Proliferation in Chronically Injured Canine Pulmonary Arteries Is Reduced by a Potent Platelet Aggregation Inhibitor U-53,059
Authors: Schaub Robert G , Keith James C , Simmons Carol A , Rawlings Clarence A
Published: July 2018

Dirofilaria immitis (DI) infection chronically injures canine pulmonary arteries. This injury produces endothelial cell loss, platelet/leukocyte adhesion, and smooth muscle proliferation. In the present study we assessed the effect of the cyclooxygenase inhibitor, U-53,059, on platelet function, platelet kinetics, coagulation, and smooth muscle proliferation in DI infected dogs.

Platelet aggregation to the combination of arachidonic acid/ ADP was significantly inhibited by U-53,059. Coagulation and hematologic parameters were not effected by either DI infection or U-53,059 treatment. Platelet survival and the number of platelet dense granules were reduced in DI infection. Quantification of the lesions demonstrated that U-53,059 reduced both severity and density compared to non-treated dogs. U-53,059 is a potent and effective inhibitor of platelet aggregation which modifies smooth muscle proliferation produced by chronic vascular injury.
Epinephrine-Induced Aggregation of Rabbit Platelets Refractory to ADP
Authors: Keraly C Lalau , Kinlough-Rathbone R L , Mustard J F
Published: July 2018

The mechanisms involved in platelet aggregation induced by epinephrine are unclear. Although epinephrine does not aggregate washed rabbit platelets, platelets made refractory to ADP will aggregate in response to epinephrine in the presence of ADP. We have examined whether the mechanism(s) by which epinephrine induces aggregation of refractory platelets involves fibrinogen binding and Ca2+ association. With normal platelets, ADP causes aggregation, fibrinogen binding and Ca2+ association in a medium containing 0.2 mM 45Ca2+. After 3 min of incubation with ADP, fibrinogen dissociates from platelets, but 45Ca2+ does not. Epinephrine alone does not cause aggregation, fibrinogen binding or 45Ca2+ association. Platelets that are refractory to ADP do not aggregate and bind fibrinogen upon addition of ADP, but aggregate and bind fibrinogen in response to epinephrine, provided ADP is still present. These effects of epinephrine are mediated by the α-adrenergic receptor since they are blocked by phentolamine or verapamil and potentiated by propranolol. However, epinephrine-induced aggregation of platelets refractory to ADP does not involve further detectable increase in the amount of 45Ca2+ associated with the platelets.
Effect of Stanozolol on Factors VIII and IX and Serum Aminotransferases in Haemophilia
Authors: Greer I A , Greaves M , Madhok R , McLoughlin K , Porter N , Lowe G D O , Preston F E , Forbes C D
Published: July 2018

The treatment of haemophilia has been dramatically improved since the introduction of factor VIII and IX concentrates, however these concentrates have brought new problems such as hepatitis and A.I.D.S. An oral agent which could raise endogenous levels of factor VIII and IX would be of great benefit. Danazol, an anabolic steroid, has recently been shown to increase levels of factors VIII and IX in haemophilia. We therefore studied the effect of stanozolol, a closely related anabolic steroid, in 15 patients with haemophilia A or Christmas disease over a 2-4 week period. There was no consistent change in factor VIIIc or factor IX, and fibrinolysis was significantly enhanced. No effect was apparent on the incidence of spontaneous bleeds. However serum aminotransferases which were abnormal in 11 of the 15 patients at the start of the study fell significantly with stanozolol therapy. This raises the interesting possibility that anabolic steroids may be beneficial in patients with chronic liver diseases.
Kinetics of In-111-Platelets in the Baboon: II. In Vivo Distribution and Sites of Sequestration
Authors: Kotzé H F , Lötter M G , Badenhorst P N , Heyns A du P
Published: July 2018

The kinetics and sites of sequestration of a fully representative population of In-111-platelets were determined in 11 baboons. The in vivo method of quantification with computer assisted scintillation camera image analysis was validated by sacrificing 5 baboons and measuring and comparing the distribution of organ radioactivity. Recovery of platelets in the circulation was 87% ±7, and their mean survival time was 147 hr± 15. The mean splenic platelet pool was 16.0% ± 1.9. At equilibrium 15.8% ± 2.9 of the In-111-platelets were in the hepatic blood pool. Senescent platelets were destroyed in the reticulo-endothelial system. The major sites of sequestration were: liver (37.6% ±6.0), and the spleen (23.3% ±4.6). The bone marrow sequestrated 14.4% ± 1.7 of the labelled platelets, and 15.5% ±4.0 were present in various other tissues. We conclude that the in vivo method of In-111- quantification is accurate. Senescent platelets are mainly sequestrated in the reticuloendothelial tissue, with the liver, spleen and the bone marrow important sites of sequestration.
Metabolism of Sodium Pentosan Polysulphate in Man Measured by a New Competitive Binding Assay for Sulphated Polysaccharides – Comparison with Effects Upon Anticoagulant Activity, Lipolysis and Platelet α-Granule Proteins
Authors: MacGregor I R , Dawes J , Pepper D S , Prowse C V , Stocks J
Published: July 2018

Three human volunteers were injected with a range of doses of pentosan polysulphate, SP54, i.v. or s.c. A competitive binding assay (CBA) for sulphated polysaccharides was used to detect circulating SP54 after doses as low as 1 mg i.v. and a linear relationship was observed between the peak plasma concentration of SP54 measured by CBA and the administered dose. A comparison was made between the clearance of SP54 measured by CBA and its anticoagulant and lipolytic activities. SP54 was detectable by CBA after doses which caused no alteration in activated partial thromboplastin time (APTT) or anti-factor Xa activity but after which a small increase of lipase activity was measurable. After SP54 at 10 mg i.v. or 100 mg s.c. anti-factor Xa activity was 4-6 times greater than would be expected from the in vitro activity of the concentrations of SP54 measured by CBA. Like heparin and other heparin analogues, SP54 caused an increase in plasma concentrations of platelet factor 4 (PF4) without a concomitant rise in p-thromboglobulin (β-TG).

It is concluded that the newly developed CBA will provide a more sensitive means than conventional bioassays for the determination of plasma concentrations of SP54.
Blood Platelet Behaviour in Mothers and Neonates
Authors: Andrews N P , Pipkin F Broughton , Heptinstall S
Published: July 2018

Blood platelet behaviour was compared in mothers at birth and their babies, and in non-pregant, female controls. Platelet responses to arachidonic acid (AA) and to adrenaline were measured in platelet-rich plasma and the inhibitory effects of prostacyclin (PGI2) were determined. Platelets from the mothers differed from those from the neonates and controls in that lower concentrations of AA were needed to induce platelet aggregation and a release reaction. In addition, more PGI2 was needed to inhibit AA-induced platelet aggregation. Platelets from the neonates differed from the mothers and controls in that they were almost completely insensitive to adrenaline. They did not differ from the controls in their sensitivity to AA or PGI2 but the extent of the release reaction induced by AA was significantly reduced.
Localization of a Thrombin-Binding Site on Human Platelet Membrane Glycoprotein Ib Determined by a Monoclonal Antibody
Authors: Yamamoto K , Yamamoto N , Kitagawa H , Tanoue K , Kosaki G , Yamazki H
Published: July 2018

To determine a thrombin-binding site on GPIbα on platelet membrane, we have examined the binding activities of tryptic or chymotryptic fragments of purified GPIbα to a monoclonal antibody against GPIb (TM60) and thrombin using (immuno) affinity chromatography. When purified GPIba was digested with trypsin, two fragments (94-kDa, and 43-kDa) were obtained. The 43-kDa fragment was shown to bind to both affinity columns of TM60- and thrombin-Affi-Gel, while the 94-kDa fragment did not bind to either Affi-Gel columns. When trypsin fragments were incubated with TM60 and then applied to the column of thrombin-Affi-Gel, neither fragments were bound to the column. When the same experiment was performed using chymotrypsin, three fragments (94-kDa, 45-kDa and 39-kDa) were observed. On TM60- and thrombin-Affi-Gel columns, the smaller fragments (45-kDa and 39-kDa) were bound to the column. After incubation of these fragments with TM60, neither bound to the thrombin column. These results indicate (i) that the epitope for TM60 is located near, or on the thrombin-binding site of GPIba, and (ii) that the thrombin-binding site is located on the tail portion of GPIbα, especially on a chymotrypsin cleavage site.
Direct Evidence for the Inhibition of Platelet Aggregation and Release by Intracellular Cyclic AMP Produced with a New Photoactivatable Derivative
Authors: Blache D , Nargeot J , Nerbonne J M
Published: July 2018

An increase in platelet cyclic AMP (cAMP) via stimulation of adenylate cyclase is thought to be the underlying mechanism by which potent prostaglandins i.e. PGD2 PGI2 inhibit platelet functions. We report here new and direct evidence for the inhibitory effects of cAMP on platelet aggregation and serotonin release. Washed platelets from rat were incubated with a new photoactivatable cAMP analogue (4,5-dimethoxy-2-nitrobenzyl ester); this compound is almost physiologically inert before irradiation and liberates free cAMP (“cAMP jumps”) following light flashes. A single flash, delivered after 2 min incubation in 100-200 μM of the analogue, dramatically inhibited thrombin-induced aggregation, as compared with controls. Endogenous serotonin release, measured in the same samples by means of an electrochemically treated carbon electrode was undetectable after the cAMP jump. Pre-irradiated solutions added to platelets had no effect. The kinetics of the flash-induced effects were also studied. From these results we can conclude that: i) the photoactivatable cAMP derivative has to permeate through the platelet membrane; ii) the analogue remains photolabile; and, iii) intracellular cAMP, resulting from photolysis dramatically inhibits platelet aggregation and serotonin release. It is possible that cAMP exerts its effects by regulating cytoplasmic free calcium concentration and/or other actions affecting platelet activation.
Platelet Aggregation and Plasma Lipoproteins in Alcoholics During Alcohol Withdrawal
Authors: Desai K , Owen J S , Wilson D T , Hutton R A
Published: July 2018

Platelet aggregation, platelet lipid composition and plasma lipoprotein concentrations were measured each week in a group of seventeen alcoholics, without overt liver disease, for one month, following acute, total alcohol withdrawal. The platelets were initially hypoaggregable but, within 1-2 weeks of cessation of drinking, they became hyperaggregable and then gradually returned towards normal values. Hyperaggregability could not be explained by increases in either the cholesterol or the arachidonic acid content of the platelets. Plasma very-low-density lipoprotein cholesterol levels remained high throughout the study, but the initially raised levels of high-density lipoprotein (HDL) cholesterol fell by 26%. Low-density lipoprotein (LDL) cholesterol concentration rose by 10% after two weeks of withdrawal but then returned to about the starting level. The resulting changes in the plasma LDL-cholesterol: HDL-cholesterol ratio, which had increased by more than 50% after two weeks of abstinence, essentially paralleled the time course of enhanced platelet reactivity in all but four of the alcoholics. These findings suggest that alterations in plasma lipoprotein concentrations during acute alcohol withdrawal may be a contributory factor to the haemostatic disorders present in such patients.
Immunological Relationship Between the Fast-Acting Plasminogen Activator Inhibitors from Plasma, Blood Platelets and Endothelial Cells Demonstrated with a Monoclonal Antibody Against an Inhibitor from Placenta
Authors: Philips M , Juul Anne-Grethe , Thorsen S , Selmer J , Zeuthen J
Published: July 2018

Two plasminogen activator inhibitors (I and II) were demonstrated in human placenta. The complex between inhibitor I and tissue-type plasminogen activator was purified by immunoadsorption to solid-phase anti-activator antibodies. The purified complex (Mr 95.000) was used for immunization of mice and subsequent production of monoclonal antibodies. One antibody (F37), which reacted with both free and complex-bound inhibitor I, was used for further study by a method involving binding of the antibody to protein A-Sepharose, immunoadsorp-tion of antigen and analysis of the resulting supernatant by SDS-polyacrylamide gel electrophoresis and enzymography. The analysis showed that F37 reacted with the fast-acting plasminogen activator inhibitors recently demonstrated in plasma, blood platelets and endothelial cells, indicating that these inhibitors and inhibitor I share a common epitope. Inhibitor II did not react with F37. Inhibitor II is identical to the placenta inhibitor previously described by others. It reacted selectively with polyclonal antibodies against that inhibitor.
Vitamin K Antagonism of Coumarin Intoxication in the Rat
Authors: Wallin R , Susan D , Patrick D , Ballard J O
Published: July 2018

An in vitro system which expresses all enzyme activities related to vitamin K-dependent carboxylation of blood clotting factors was prepared from livers of rats overdosed with warfarin, difenacoum and dicumarol respectively. In this system, the activities of the two pathways that are known to produce active reduced vitamin K1 cofactor for the carboxylation reaction were measured. Also the ability of high concentrations of vitamin Kx to overcome inhibition of clotting factor synthesis was studied. In the systems prepared from livers of warfarin and difenacoum intoxicated rats, pathway I was inactive. Vitamin K epoxide reductase was also inactive which strongly suggests that this enzyme catalyzes the activity of pathway I in vivo. Reduction of vitamin by pathway II bypassed the inactive pathway I and resulted in carboxylation activity. This pathway therefore mediates the antidotic effect of vitamin K1 in the coumarin intoxicated liver. In the in vitro system prepared from dicumarol intoxicated livers the activity of pathway I was not significantly affected. Dicumarol however was a strong inhibitor when added to liver microsomes in vitro.
Electroblot and Immunoperoxidase Staining for Rapid Screening of the Abnormalities of the Multimeric Structure of von Willebrand Factor in von Willebrand’s Disease
Authors: Lombardi Rossana , Gelfi Cecilia , Righetti P G , Lattuada Antonella , Mannucci P M
Published: July 2018

A new electrophoretic method is described for rapid screening of abnormalities of the multimeric structure of von Willebrand factor in von Willebrand’s disease. The method is based on the transfer of the separated proteins from agarose gels onto nitrocellulose foils followed by immunoperoxidase staining. It has the advantage of not requiring radio-iodinated antibodies and reduces the working time for the entire procedure from 5-6 days to 3 days. Electroblotting followed by immunoperoxidase staining differentiates patients with intact multimeric structure from those without intermediate and/or large multimers. The more subtle defects of the inner structure of the smallest multimers found in patients with type II von Willebrand’s disease can also be identified. A potential disadvantage of electroblotting and immunoperoxidase staining is the lesser sensitivity of this technique, which results in the detection of a smaller number of multimers (11-12 bands) than by autoradiography without transfer onto nitrocellulose (16-17 bands).
Visualization of von Willebrand Factor Multimers by Immunoenzymatic Stain Using Avidin-Biotin Peroxidase Complex
Authors: Aihara M , Sawada Y , Ueno K , Morimoto S , Yoshida Y , Serres M de , Copper H A , Wegner R H
Published: July 2018

A technique for the detection of von Willebrand factor multimers separated by discontinuous SDS agarose electrophoresis has been developed using non-radioactive com-v pounds. The multimeric patterns were visualized by monospecific anti-human vWF:Ag followed by incubation with biotinylated antibody. After addition of avidin-biotin-peroxidase complex, the peroxidase activitiy was detected by 4-chloro-l-naphthol, giving sharp bands with a clear background.

By this method, the differences of vWF : Ag multimers could be easily observed between normal plasma and the plasmas from variant type vWD (IIA, IIB, platelet-type). Large and intermediate multimers were absent in the plasma with vWD type IIA, while only large multimers were absent in the plasma with vWD IIB and platelet-type. The absence of large multimers was also observed in two commercial F VIII preparations having the ratio of vWF/vWF : Ag 0.18 and 0.63. The preparation with the ratio of 0.63 showed the presence of larger intermediate multimers.

Electrophoresis in SDS 1.5% agarose gel revealed triplet structure of each small multimer, and a relative increase of the smallest subband was observed in vWD IIA plasma, platelet-type vWD plasma and commercial F VIII preparations.

The procedures described are easy and safe to perform and are useful for screening or classifying cases with vWD in general laboratories.
Blood Platelet Plasminogen Activator Inhibitor: Two Different Pools of Endothelial Cell Type Plasminogen Activator Inhibitor in Human Blood
Authors: Sprengers E D , Akkerman J W N , Jansen B G
Published: July 2018

An assay for plasminogen activator inhibitor in human platelets is described. With this assay we find an average value of 6.8 × 10−8 IU/platelet (S.D. = 3.0 × 10−8; n = 20) in a healthy population. We characterized the PA-inhibitor from platelets and identified it as endothelial cell type plasminogen activator inhibitor, by its immunologic and functional properties. Besides the plasma pool of plasminogen activator inhibitor with a very high turnover rate, platelets constitute a second pool of plasminogen activator inhibitor in the circulation of the same order of magnitude. The two different pools of plasminogen activator inhibitor might have a different physiologic function.
The von Willebrand Factor in Myocardial Infarction and Unstable Angina: A Kinetic Study
Authors: Margulis Tamara , David Miriam , Maor N , Soff G A , Grenadier E , Palant A , Aghai Esther
Published: July 2018

Recent studies have demonstrated elevations of von Willebrand Factor following acute myocardial infarction (AMI). In order to determine if this parameter may serve as a marker for AMI, we tested the blood levels of vWF and Factor VIII :C in 28 patients with AMI, 9 patients with unstable angina, 7 patients with atypical chest pain, and 25 healthy volunteers. The level of ristocetin cofactor activity of vWF was between 70 and 144% in the control group. In patients with AMI, the mean level of this activity was 175% on the first day following infarction, rose to a peak of 270% on the fifth and sixth days, and was still significantly greater than normal in all patients on the 14th day. The vWF: Ag level closely paralleled the rise of ristocetin cofactor activity of vWF, with a peak of 336% on day 5. FVIII :C was not significantly changed. No significant elevation of vWF was observed in patients with unstable angina. The ristocetin cofactor activity of vWF and vWF: Ag thus are sensitive biochemical indicators for recent AMI, and may serve as useful markers for up to 14 days following infarction, when the traditional enzymes have returned to normal levels.
α2-Antiplasmin: Functional Characterization and Metabolism in a Heterozygote Deficient Patient
Authors: Knot E A R , ten Cate J W , Lamping R J , Kian Gie Liem
Published: July 2018

An 81-year-old male with a mild life-long bleeding history and an α2-antiplasmin (α2-AP) plasma level of 55% biological activity and 41% antigen activity (normal range 80-140%) was studied. The ratio of plasminogen binding (PB): non-plasminogen binding (NPB) α2-AP assayed by modified crossed immunoelectrophoresis (CIE) was 7.3/2.7 (controls 6.3 ± 0.49 SD/3.7 ± 0.49 SD). The patient’s α2-AP showed decreased affinity for fibrin, i. e. 8.3% versus 32.4% of normal control α2-AP associated with fibrin during clotting of plasma. A metabolic study performed with human purified 125I-α2-AP(PB/NPB 7.7/2.3) showed a plasma radioactivity disappearance half-life of 72.9 h (n 60.1 ± 5.3 h) with a normal fractional catabolic rate and a reduced absolute catabolic (synthetic) rate of 0.70 mg/kg/day (n 2.10 ± 0.60 mg/kg/day). The exchange between the central and third compartment was increased. The increased α2-AP PB form and the increased plasma radioactivity disappearance half-life are suggestive of a slower conversion of the PB form into the NPB form and/or slower degradation of the PB form. The bleeding tendency in this patient could be explained by decreased synthesis of α2-AP and decreased binding to fibrin.
Calibration of BCT/441, the ICSH Reference Preparation for Thromboplastin
Authors: Thomson Jean M , Darby K V , Poller L
Published: July 2018

An international collaborative exercise has been undertaken to calibrate a secondary international reference preparation (IRP) of thromboplastin on behalf of the International Committee for Standardization in Haematology (ICSH). This preparation of British Comparative Thromboplastin (BCT/441) is required because supplies of the WHO primary IRP (BCT/253) are necessarily limited.

The calibration was performed at seven centres with only a small degree of interlaboratory variation. As a result of this study an ISI value of 1.04 has been assigned to the preparation.

Opportunity was also taken to assess the reliability of a simplified calibration based on lyophilised plasmas. The results of the latter appeared reliable.

BCT/441 will be available to officially designated National Control Laboratories for calibration of local thromboplastins to promote prothrombin time standardization in oral anticoagulant control.
Monoclonal Antibodies Towards the Fast Inhibitor of Tissue Plasminogen Activator from Human Plasma and Serum
Authors: Urdén G , Johansson Ulla , Chmielewska Joanna , Brandt J , Wiman B
Published: July 2018

Hybridoma cells were produced by fusing mouse myeloma cells (SP 2/0 - Ag 14) with spleen cells from a Balb/c mouse, previously immunized with the partially purified complex between tissue plasminogen activator (t-PA) and its fast inhibitor from human plasma (serum). Screening with a radioimmunoassay revealed a number of hybridomas secreting antibodies directed towards the complex. Of these, about 1/3 reacted both with the complex and t-PA, whereas about 2/3 reacted only with the complex. Three of the latter hybridomas, producing antibodies directed towards the inhibitor-moiety in the complex have been cloned and the antibodies were studied in detail. PA-inhibitor activity in plasma or serum and t-PA/PA-inhibitor complex could be specifically adsorbed on all three insolubilized monoclonal antibodies (MCI, MC2 and MC3). None of the antibodies seems to be directed against structures of vital importance for the functional activity of the PA-inhibitor. In accordance with this finding the antibody with the highest avidity (MCI) reacts equally well with the PA-inhibitor alone or in complex with t-PA. A radioimmunoassay was devloped with this antibody and significant displacement was obtained with samples with PA-inhibitor concentrations above 2 AU/mL. In 13 plasma samples with different levels of PA-inhibitory activity a significant correlation was obtained when comparing this activity with the PA-inhibitor antigen as measured with the radioimmunoassay (r = 0.88).
Investigation of a Congenital Abnormal Plasminogen, Frankfurt I, and Its Relationship to Thrombosis
Authors: Scharrer I M , Wohl R C , Hach V , Sinio L , Boreisha I , Robbins K C
Published: July 2018

A new abnormal plasminogen, Frankfurt I, has been identified in the plasma of a 42 year-old male patient who has recurrent deep vein thrombosis. Clinical laboratory data showed normal hemostasis test results. Since plasma plasmin generation rates gave low values, the fibrinolytic system was analyzed for a possible fibrinolytic system defect. Functional and antigen plasminogen concentrations both in the plasma and with the isolated, purified plasminogen showed that only 49% of the antigen concentration had potential functional active sites. Also, a reduced antigen concentration was found in both the propositus, and his mother (46% active sites).

Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the purified Frankfurt I plasminogen showed a normal native Glu-plasminogen band. Crossed-immunoelectrophoresis revealed a peak with normal size and shape, but displaced with respect to normal Glu-plasminogen toward the anode, i. e., was, as a whole, more negatively charged. Isoelectric focusing followed by zymog-raphy on a agarose-fibrin plate proved this observation, but did not indicate a separation of the normal from the abnormal plasminogen molecular species, also, fewer bands were found in the abnormal plasminogen isozyme pattern.

Kinetic studies of Frankfurt I Glu-plasminogen and plasmin led to the conclusion that most of the functional abnormality is related to absence of active sites in half of the molecules. The plasmin generated was very unstable in the absence of stabilizing ligands and/or substrates. After reduction, the plasmin was completely converted to the typical two plasmin chains, A and B.
Absence of Transplacental Passage of Pentosan Polysulfate During Mid Trimester of Pregnancy
Authors: Forestier F , Fischer A M , Daffos F , Beguin S , Diner H
Published: July 2018

Eight women who were going to have an abortion between the 18th and 23rd week of gestation for chromosomal abnormalities or haemoglobinopathies received intravenously 50 mg of pentosan polysulfate (PSP). Maternal results of haemostasis prior and after the injection of the drug were compared. Fetal coagulation parameters were tested on samples obtained by direct puncture of the umbilical cord under ultrasound guidance, 30 min after injection. Results were compared to those of normal fetuses at the same stage of gestation, obtained in the same conditions. In mothers’ plasma, 30 min after injection, APTT was prolonged, factor Xa generation was markedly impaired, and factor V level was deeply decreased. By contrast, no modifications of these parameters were observed in fetal plasma, 30 min after the injection of PSP to their related mothers when compared to control fetuses. Thus the absence of biological modifications induced by PSP injection could demonstrate that this drug does not cross through the placenta.
Hepatitis-Free Interval After Clotting Factor Therapy in First Infused Haemophiliacs
Authors: Morfini M , Rafanelli D , Longo G , Messori A , Rossi Ferrini P
Published: July 2018

Post-infusion hepatitis is known to occur very frequently in haemophiliacs after treatment with unheated commercial clotting factor concentrates, obtained from large plasma donation pool. On the contrary, single-donor cryoprecipitate is likely to carry a lower risk of transmitting hepatitis.

To evaluate this hypothesis, we retrospectively reviewed the medical records of 25 first infused haemophiliacs (from 1981 to 1984) treated with unheated commercial clotting factor concentrates (n = 19) or cryoprecipitate (n = 6).

The hepatitis-free interval after the beginning of therapy was expressed as exposure days. The end point of each patient, i.e. the hepatitis occurrence, was defined as an increase of aminotransferases (ALT and AST) and/or the seroconversion of HBV-markers, which were checked every three months.

The life-table method and log-rank test showed that cryo-precipitates had a significantly longer hepatitis-free interval (p = 0.0131, log-rank test) and a lower risk of transmitting hepatitis (p = 0.01-0.05, life-table method) than the commercial concentrates. However, the safety of cryoprecipitate therapy was shown to cover only a few exposure days, and so the real advantage of this product depends on the bleeding frequency of the patient concerned.

We believe that these methods and our findings may be useful to assess and compare the safety of the new “heat-treated” clotting factor concentrates.
Bioavailability in Rats of Human Recombinant Tissue Plasminogen Activator After Subcutaneous and Intramuscular Injection
Authors: Garcia Frade L J , Poole S , Hanley S , Creighton L J , Curtis A D , Gaffney P J
Published: July 2018

The bioavailability of human recombinant tissue plasminogen activator (rt-PA) in rats was measured after subcutaneous (s.c.) and intramuscular (i.m.) injection. Rt-PA was absorbed after both i.m. and s.c. injection, giving peak plasma concentrations within 30 min and 1 h, respectively, with detectable concentrations up to 6 h. These peak values of bioavailable t-PA were obtained in a functional fibrin plate assay of euglobulin precipitates and expressed as +88% and +243% (for s.c. and i.m. routes respectively) above basal rat fibrinolytic activity. Prior injection of rt-PA, s.c. or i.m., significantly reduced the weights of thrombi induced in the inferior vena cava after injection.
Quantitative Immunoblotting of Plasma and Platelet Protein S
Authors: Schwarz H Peter , Heeb Mary Jo , Lämmle Bernhard , Berrettini Mauro , Griffin John H
Published: July 2018

Protein S, the activated protein C cofactor, was measured in plasma and in platelets by a quantitative immunoblotting assay using a double antibody technique. Either whole plasma or platelet lysates were electrophoresed on sodium dodecyl sulfate polyacrylamide gels and transferred to nitrocellulose membranes by electroblotting. Based on the demonstration of proportional transfer of protein S from the gel to the nitrocellulose membrane, a reproducible and sensitive quantitative assay for protein S antigen (∼ 70,000 MW) was developed that correlated well with the Laurell rocket assay for plasma protein S. Pooled normal plasma contains 22 μg/ml protein S. Total platelet protein S antigen at ∼ 70,000 MW was determined in gel filtered platelets of ten healthy adults (mean, 163 ng per 1088 platelets). In the supernatants of thrombin-stimulated platelets, 63% of the total platelet protein S antigen was measured. Thus, quantitative immunoblotting is a useful method to detect low levels of protein S in platelets or in plasma with the advantage of giving qualitative information, i.e. apparent MW, of protein S.
Factor VIII: C (FVIII: C) Recovery and Half-Life After Infusion of Steam-Treated High Purity Factor VIII Concentrate in Severe Hemophilia A - Comparison of One-Stage Assay, Two-Stage Assay and a Chromogenic Substrate Assay
Authors: Hellstern Peter , Kiehl Ralf , Miyashita Chieko , Schwerdt Holger , von Blohn Gerhard , Köhler Michael , Büttner Maria , Wenzel Ernst
Published: July 2018

Factor VIII :C recovery and half-life was measured in 16 hemophilia A patients under comprehensively standardized conditions. Each patient received the same lot of a steam-treated high purity FVIII concentrate at a dose of 19-33 U/kg body weight. A comparison was made between the one-stage assay, the two-stage assay and a chromogenic substrate test for FVIII :C determination using a FXa-sensitive chromogenic substrate. Factor VIII :C potency of the administered FVIII concentrate was measured using calibration curves derived from a concentrate standard and FVIII: C plasma levels were read from calibration curves derived from a plasma standard. The chromogenic assay showed a good reproducibility at FVIII: C levels between 0.015 and 0.50 U/ml. The FVIII :C recoveries calculated from the results of the one-stage assay, the two-stage assay and the chromogenic substrate test were 109 ± 20, 92 ± 14 and 81 ± 11% (x ± SD), respectively. The elimination half-lives of FVIII :C were calculated by non-linear least square analysis using a modified computerized Gauss-Newton algorithm. The half-lives calculated from the FVIII: C plasma levels measured by the one-stage assay, the two-stage assay and the chromogenic test were 23.8 ± 6.4, 22.2 ± 5.7 and 17.1 ± 4.8 h (x ± SD), respectively. No previous study has reported such long half-life values. Our findings indicate that measurements of recoveries and half-lives by the chromogenic FVIII :C assay and by computerized nonlinear least square analysis allow the possibility of individualized FVIII replacement therapy.
Piretanide: A New Synthetic Fibrinolytic and Anti-Platelet Agent
Authors: Chohan I S
Published: July 2018

Piretanide, 4-phenoxy-3-(l-pyrrolidinyl)-5-sulfamoyl benzoic acid, a potent diuretic, enhances endogenous plasma fibrinolytic activity after a single dose of 6 mg administered orally. Acceleration of fibrinolytic activity becomes manifest within 1 h, reaches its peak in 3 h and is associated with little change in fibrinogen, however, it is accompanied with diminished urokinase excretion initially. Piretanide does not cause lysis of fibrin in vitro in any concentration. Primary platelet aggregation, induced by adenosine-diphosphate, is inhibited by piretanide, in vivo. In in vitro experiments piretanide leads to effective inhibition of ADP-induced platelet aggregation with complete inhibition at 5 mM concentration. Piretanide, after ingestion of a single dose of 6 mg, causes highly significant decrease of platelet factor-4 release.
Three Abnormal Fibrinogen Variants with the Same Amino Acid Substitution (γ 275 Arg → His): Fibrinogens Bergamo II, Essen and Perugia
Authors: Reber P , Furlan M , Henschen A , Kaudewitz H , Barbui T , Hilgard P , Nenci G G , Berrettini M , Beck E A
Published: July 2018

We report on three unrelated individuals with the same uncommon type of dysfibrinogenemia, originating from Bergamo, Essen and Perugia. None of them showed bleeding symptoms while the Bergamo patient and members of her family presented with a thrombotic tendency. The presence of a defective fibrinogen was suggested by prolonged thrombin and rep-tilase times. Furthermore, fibrinogen concentrations of less than 0.28 g/L were determined by the functional assay whereas values of 1.5-2.4 g/L were measured by heat precipitation or electroimmunoassay. Fibrinogen was isolated by affinity chromatography on insoluble fibrin monomer. The rate of fibrinopeptide release by thrombin was normal while the fibrin polymerization reaction was strongly delayed. An abnormal peptide (γ265-310) was isolated by high-performance liquid chromatography after cyanogen bromide cleavage of the purified γ-chain of fibrinogen Bergamo II and Essen. The same peptide was also isolated following cyanogen bromide treatment of the intact fibrinogen Perugia. Sequence analyses of these peptides demonstrated the same amino acid exchange in all three fibrinogens: γ275 arginine → histidine. The described fibrinogen variants appear to possess a molecular defect which has thus far only been observed in fibrinogen Haifa.
Behaviour and Quantitation of Extrinsic (Tissue-Type) Plasminogen Activator in Human Blood
Authors: Kluft C , Jie A F H , Allen R A
Published: July 2018

Functional assay of extrinsic (tissue-type) plasminogen activator (EPA) in plasma on fibrin plates was evaluated. Using specific quenching antibodies, we demonstrated the method to be specific for EPA under all conditions tested. Contributions of urokinases and intrinsic activators were excluded. The quantity of EPA in blood samples, as compared with purified uterine tissue activator, shows 1 blood activator unit (BAU) to be comparable to 0.93 ng.

The median values for EPA activity for healthy volunteers were: baseline, 1.9 BAU/ml (n = 123); diurnal, 5.5 BAU/ml (n = 12); DDAVP administration, 11.7 BAU/ml (n = 39); exhaustive exercise, 25 BAU/ml (n = 24); venous occlusion (15 min), 35 BAU/ml (n = 61). A large inter-individual variation in EPA activity was found, while individual baseline values tended to be constant for periods of weeks.

In vitro in blood EPA activity shows a disappearance of 50% in about 90 min at 37° C; EPA activity in euglobulin fractions is stable for ≤2 hr at 37° C.

A rapid decrease in EPA activity occurs in vivo, as noted after extracorporal circulation and exercise stimulation (t½ decay, 2-5 min).
Inactivation of Hepatitis B and Non-A, Non-B Viruses by Combined Use of Tween 80®, β-Propiolactone, and Ultraviolet Irradiation
Authors: Prince A M , Stephan W , Kotitschke R , Brotman B
Published: July 2018

To assess the sterilization efficacy of a combined Tween 80®, β-propiolactone and ultraviolet irradiation procedure applied to a FVIII preparation to which an estimated 105.9 chimpanzee infectious doses (CID50) of hepatitis B virus had been added per ml, two chimpanzees were inoculated with 10 ml each of treated and untreated preparations. The untreated preparation, which was obtained from donors with normal alanine aminotransferase (ALT) levels, induced non-A, non-B hepatitis in both recipient animals, and delayed hepatitis B infection in one of these. Neither animal receiving the treated preparation developed either type of hepatitis. When subsequently challenged with the untreated material, both of the latter animals developed non-A, non-B and hepatitis B infection, proving their susceptibility to both types of infection.

It was concluded that the combined procedure inactivated an estimated 106.9 CID50 of hepatitis B virus and an unknown quantity of a non-A, non-B virus.

The finding of non-A, non-B virus infectivity in a pooled FVIII preparation despite careful ALT screening of plasma donors emphasizes the necessity of subjecting such preparations to sterilization procedures.
Verapamil and Collagen-Induced Platelet Reactions- Evidence for a Role for Intracellular Calcium in Platelet Activation
Authors: Han P , Boatwright C , Ardlie N G
Published: July 2018

Calcium is considered to have an essential role in various platelet reactions. Using platelets preincubated with Chlortetracycline, a fluorescent divalent cation indicator, and suspended in a calcium free medium, it was shown that collagen-induced intracellular calcium redistribution occurred before the platelet shape change, the release reaction and thromboxane B2 formation. Verapamil, at concentrations which affect intracellular calcium movements, inhibited intracellular calcium redistribution in platelets and the subsequent collagen-induced platelet reactions. Low concentrations of the ionophore A23187 overcame the inhibitory effect of verapamil. These experiments provide evidence that intracellular calcium mobilization is involved in the activation of platelets by collagen. Furthermore, calcium may be released from different cellular pools since platelet secretion, aggregation and thromboxane B2 formation were inhibited at lower concentrations of verapamil than was the platelet shape change.
The Effect of N-6 and N-3 Polyunsaturated Fatty Acids on Hemostasis, Blood Lipids and Blood Pressure
Authors: Mortensen J Z , Schmidt E B , Nielsen A H , Dyerberg J
Published: July 2018

Diverging results from studies of marine oil supplementation to western diets initiated the undertaking of a double-blind crossover study, with administration to healthy volunteers for 4 weeks of either 10 g of fish oil or 10 g of vegetable oil. Each oil containing approx. 40% of n-3 and n-6 polyunsaturated fatty acids (PUFA) respectively. During the n-3 PUFA period, systolic blood pressure, plasma total lipids, triglycerides and VLDL concentrations fell significantly whereas plasma antithrombin-III (AT-III) rose. Cutaneous bleeding time increased significantly. In contrast only AT-III rose during the n-6 PUFA feeding, however, more marked than during the n-3 oil period. It is concluded that a n-3 PUFA oil supplement to the western diet exerts an effect that generally is considered as beneficial in terms of the risk of developing cardiovascular diseases. It is in this respect superior to that of n-6 PUFA, stressing the necessity of a more differentiated approach to advices on dietary PUFA enrichment than presently is exerted.
Interaction of Heparin with Histidine-Rich Glycoprotein and with Antithrombin III
Authors: Lijnen H R , van Hoef B , Collen D
Published: July 2018

The interaction between heparin, histidine-rich glycoprotein and antithrombin III was studied in purified systems. Histidine- rich glycoprotein binds heparin and thereby interferes with its interaction with antithrombin III, resulting in neutralization of the anticoagulant activity. This interaction occurs with clinical grade heparin as well as with high affinity (for antithrombin III) heparin and with a high affinity heparin fragment with Mr. 4,300.

Low affinity heparin competes with high affinity heparin for the binding to histidine-rich glycoprotein which results in an apparent increase of the anticoagulant activity of high affinity heparin.

The interaction between heparin and histidine-rich glycoprotein is counteracted by Ca2+-binding anticoagulants, indicating that it is dependent on the presence of divalent metal ions. Ethylenediaminetetraacetate is a much more potent inhibitor of the interaction between heparin and histidine-rich glycoprotein than citrate.
The Effect of Thrombin on Platelet Accumulation on the Vessel Wall - Influence of Heparin and Aspirin
Authors: Dejan E , Cazenave J-P , Hatton M W C , Richardson M , Groves H M , Kinlough-Rathbone R L , Packham M A , Mustard J F
Published: July 2018

Rabbit aortae were removed from exsanguinated rabbits, washed, everted on probes, treated with thrombin, washed to remove unbound thrombin and used to measure the accumulation of 51Cr-labeled platelets in vitro. Thrombin pretreatment of normal rabbit aortae did not cause platelet accumulation on the endothelium; platelets appeared to accumulate only at sites where the subendothelium had been exposed. The quantitative data obtained with 51Cr-labelled platelets was reinforced by observations by scanning electron microscopy. 125I-labelled thrombin became associated with the endothelium and also with de- endothelialized vessels, and some of it could be displaced by high concentrations of heparin. Exposure of vessels to heparin after thrombin treatment eliminated the enhanced platelet accumulation caused by the thrombin treatment, probably because heparin displaced thrombin from the aortae, as demonstrated in experiments with 125I-thrombin. Inhibition of PGI2 production by aspirin treatment of the vessels did not enhance platelet accumulation on normal or thrombin-treated aortae. Thus, although thrombin treatment of the endothelium does not cause platelets to adhere to it, thrombin does cause increased platelet accumulation on the areas where the subendothelium is exposed or where endothelial cells are damaged.
Enkephalins Modify Granulocyte-Endothelial Interactions by Stimulating Prostacyclin Production
Authors: Boogaerts M A , Vermylen J , Deckmyn H , Roelant C , Verwilghen R L , Jacob H S , Moldow C F
Published: July 2018

Granulocyte (PMN)-endothehal interactions have been implicated in the primary events of vascular injury and atherogenesis. We now present data which show that endogenous opioid peptides, e.g. enkephalins (ENK), dampen immune-triggered, granulocyte-induced endothelial damage by enhancing prostacyclin production. Concurrent exposure of human umbilical vein endothelial cells (HUEC) to Met55-enkephalin increased arachidonic acid (AA, 20 uM) and thrombin (T, 10 U/ml) induced 6-keto-PGF1α-release to respectively 197.2 ± 28.1% and 204.1 ± 17.8% (mean ± SEM) of base line stimulation (p < 0.025). The increases noted were significant at ENK-concentrations as low as 10-12M. Simultaneous addition of naloxone with ENK completely abolished the enhanced 6-keto-PGF1α- release. Addition of a protease resistant enkephalin analogue significantly (p <0.01 over several different concentrations) reduced PMN adherence to HUEC; concomitantly 51Cr-leakage from HUEC that had been exposed to PMN plus activated serum complement was decreased. The even further enhanced 51Cr- leakage that occurs when platelet release products (e.g. serotonin) are included is also decreased by added enkephalin. These data suggest that endogenous neurotransmitters may affect endothelial prostaglandin metabolism, and by so doing provide a protective effect during in vitro, and perhaps in vivo, PMN mediated endothelial injury. This link between neurohumoral and inflammatory systems might enhance our understanding of stress- related phenomena in inflammation and vascular diseases.