bluuebunny/crossref_metadata_2025 huggingface dataset:
Dose Finding Study of a Low Molecular Weight Heparin, Innohep, in Haemodialysis
Authors: Ryan K E , Lane D A , Flynn A , Shepperd J , Ireland H A , Curtis J R
Published: July 2018

A pilot investigation was performed with Innohep, a low molecular weight (LMWH) preparation (peak maximum molecular mass 3,000-6,000), to determine possible dose regimens for patients undergoing regular maintenance haemodialysis for chronic renal failure. Results from this study suggested that suppression of macroscopic clot formation and fibrinopeptide A (FPA), a marker of fibrin formation, could be achieved following bolus injections rather than bolus injections and an infusion. On the basis of these preliminary findings, a randomised crossover study was performed in eight patients undergoing regular maintenance haemodialysis for 5-7 h to determine the effective antithrombotic dose of this LMWH. Single i.v. bolus doses of 1,250 AFXa u, 2,500 AFXa u and 5,000 AFXa u (n = 7-8) were compared to an UFH regime of 5,000 iu + 1,500 iu/h. Excessive clot formation in the dialyser bubble trap, necessitating additional UFH to enable completion of a prolonged (up to 7 h) dialysis, was observed in all patients on the 1,250 AFXa u dose (mean duration of dialysis prior to UFH, 3 h) but in a single patient only receiving the other LMWH doses. A dose-related response in the AFXa activity, measured by chromogenic substrate (CS) assay was seen in the three LMWH groups, with levels declining significantly (p <0.05) from 1-7 h. This contrasted with the constant levels maintained during dialysis with UFH. FPA levels were significantly elevated after 2 h following the 1,250 AFXa u bolus and after 4 h following the 2,500 AFXa u bolus. There was no significant difference in FPA levels between the 5,000 AFXa u bolus and UFH. β-thromboglobulin (pTG) levels rose significantly towards the end of dialysis in all LMWH groups and, at 5 h, were significantly increased following the 5,000 AFXa u and 2,500 AFXa u doses when compared to the UFH regime. AFXa levels correlated negatively with FPA levels (r = -0.62; p <0.01). In conclusion, for administration of Innohep, a bolus dose of 2,500 AFXa u would appear to be sufficient for dialyses of short duration (up to 4 h), whilst a 5,000 AFXa u bolus is as effective as UFH for a 6 h dialysis. AFXa activity measured by CS assay is related to inhibition of fibrin formation and can be used for monitoring purposes.
Effect of the Hirudin Carboxy-Terminal Peptide 54-65 on the Interaction of Thrombin with Platelets
Authors: Jandrot-Perrus Martine , Huisse Marie-Geneviève , Krstenansky John L , Bezeaud Annie , Guillin Marie-Claude
Published: July 2018

The carboxy-terminal region of hirudin (residues 54-65) has previously been shown to inhibit thrombin clotting activity without binding to the catalytic site of the enzyme. In the present study, the effect of hirudin 54-65 on thrombin interaction with specified platelet proteins has been investigated. Hirudin 54-65 was found to inhibit thrombin-induced platelet aggregation and secretion in a dose-dependent manner. Substitution of either Phe56, Glu57, Ile59, Pro60 or Leu64 showed that these residues were critical for inhibition of thrombin-induced platelet activation whereas sulfation of Tyr63 increased the inhibitory potency of the peptide. Hydrolysis of glycoprotein V, a platelet membrane substrate for thrombin, was only partially inhibited by hirudin 54-65. Although hirudin 54-65 did not decrease the amount of thrombin bound to platelets during cross-linking experiments, it was found to inhibit the specific binding of thrombin to platelet glycoprotein Ib. Since the carboxy-terminal region of hirudin has previously been reported to bind near the trypsin-catalyzed β cleavage site, we have analyzed the consequences of a to β-thrombin conversion on both thrombin-hirudin 54-65 interaction and thrombin activity toward platelets. The β cleavage induced a decrease in the affinity of thrombin for both glycoprotein Ib and hirudin 54-65. Altogether, our results indicate that thrombin recognition sites for hirudin 54-65 and platelet membrane glycoprotein Ib share common structures located near the β cleavage site at Arg 73 on the thrombin B chain.
Rapid Aequorin Loading into Platelets in the Presence of DMSO - Characteristics of the Responses (Changes in Light Transmission and in Calcium) to Various Agonists
Authors: Potevin F , Lecompte T , Favier R , Samama M
Published: July 2018

We have looked at different parameters which could modify platelet behaviour during and after aequorin loading in the presence of DMSO. There is a decreased platelet reactivity in response to ADP, PAF and A-23,187 which appears to be mainly due to the exposure to EGTA during washing and loading and the 1 ml volume of the test suspension. All the studied agonists (including PMA) which elicit aggregation are able to induce an intracellular Ca2+ change detected with the aequorin probe. By contrast, epinephrine alone induces neither aggregation nor Ca2+ rise, but potentiates the responses to ADP. Different consecutive phases in Ca2+ changes after stimulation with ADP, PMA and A-23,187 can be evidenced. In the presence of external Ca2+, the second component of the Ca2+ change evoked with ADP is dependent on aggregation and the subsequent TXA2 synthesis. When the external medium is Ca2+ depleted, the two Ca2+ peaks induced by ADP disappear whereas a Ca2+ rise persists (endogenous mobilization) with the other agonists, being independent of TXA2 and ADP release. Ca2+ mobilization parallels activation with A-23,187 but not with low concentrations of thrombin.
Platelet Survival in Patients with Dilated Cardiomyopathy
Authors: Weidinger Franz , Glogar Dietmar , Sochor Heinz , Sinzinger Helmut
Published: July 2018

Dilated cardiomyopathy is associated with thromboembolic complications, which correlate poorly, however, with a visible left ventricular thrombus. Therefore, this study was performed to assess whether an abnormality of platelet function in vivo can be detected in patients with dilated cardiomyopathy. Platelet survival was measured after autologous labeling with indium-111 oxine in 28 patients with dilated cardiomyopathy and angiographically normal coronary arteries (mean ejection fraction 21 ± 9% [standard deviation], range 4 to 39%) and in nine patients with coronary artery disease and similar left ventricular dysfunction (mean ejection fraction 21 ± 10%). Plasma levels of beta-thromboglobulin and platelet factor 4 were measured in patients with idiopathic cardiomyopathy (n = 15) and platelet scintigraphic images of the heart (n = 24) were obtained in subsets of both patient groups. Platelet survival was significantly and similarly shortened in patients with idiopathic and ischemic cardiomyopathy (67 ± 34 and 55 ± 24 h, respectively) compared to controls (209 ± 9 h, n = 12; p <0.001). Of the two platelet-specific proteins, beta-thromboglobulin was increased in the patients compared with controls (42 ± 17 versus 22 ± 6 ng/ml, p <0.001). Platelet scintigraphy 24 h (n = 24) and/or 48 h (n = 9) after labeling showed a diffuse pattern of enhanced platelet uptake over the heart which varied in intensity among patients, but which was never seen in controls (n = 6). This increased platelet uptake was similar in patients with idiopathic and ischemic cardiomyopathy but did not correlate with either ejection fraction or cardiothoracic ratio. Patients taking Coumadin (n = 9) had lower values of quantitative and qualitative scintigraphic platelet uptake than patients without Coumadin (n = 14; p <0.05).

Thus, platelet survival is markedly shortened and cardiac platelet uptake diffusely enhanced in patients with dilated cardiomyopathy of either idiopathic or ischemic origin. Whether enhanced platelet activation per se contributes to the risk of systemic embolization in this patient population requires further investigation.
Are Haemostatic and Fibrinolytic Parameters Predictors of Preeclampsia in Pregnancy-Associated Hypertension?
Authors: Caron C , Goudemand J , Marey A , Beague D , Ducroux G , Drouvin F
Published: July 2018

The plasma levels of several haemostatic and fibrinolytic parameters were measured before and after delivery in 61 hypertensive pregnant women of whom 22 developed preeclampsia, and compared to the results obtained in 42 normal pregnant women. In the two last weeks before delivery (D ≤ –15) tPA antigen, PAI-1 activity, vWF: Ag/FVIII: C ratio, ATIII activity and platelet count were found to be significantly different in the hypertensive pregnant women with and without preeclampsia. Combined all together, an association of three of these five parameters were found to be pathological (i.e.: tPA: Ag ≥19 ng/ml, PAI-1 activity ≥58 IU/ml, vWF: Ag/FVIII: C ratio 5≥2.6, ATIII activity ≤73%) in none of the hypertensive women without preeclampsia and in only 35% of the preeclamptic group. A positive correlation was demonstrated between vWF:Ag/FVIII:C ratio and tPA:antigen levels suggesting that both tPA and vWF: Ag could be considered as early indicators of a possible micro angiopathy occurring in preeclampsia. However, due to the high dispersion of the results, it appears that the investigated haemostatic and/or fibrinolytic criteria give only presumptive arguments before assigning risk for preeclampsia development among hypertensive pregnant women.
A Method for Measuring Activated Factor VIII in Plasma
Authors: Kessels H , Béguin S , Wagenvoord R , Hemker H C
Published: July 2018

A method is described which enables a quantitative measurement of the concentration of activated factor VIII (VIIIa) in plasma. Based on the ability of factor VIIIa to accelerate the activation of factor X by factor IXa, phospholipid and calcium ions, the course of factor X activation in time is measured using a chromogenic substrate. Free factor Xa is able to activate non-activated factor VIII present in a plasma sample, which increases the factor X activation velocity, and thus disturbs the measurement of factor VIIIa. Furthermore, factor Xa was found to be inactivated by serine protease inhibitors from the plasma sample. By adding surplus chromogenic substrate these reactions of factor Xa are inhibited and at the same time the rate of substrate conversion is a measure of the amount of factor Xa present. Factor X activation and amidolysis of chromogenic substrate then take place simultaneously. It is shown that under proper conditions the factor X activation velocity is linearly proportional to the factor VIIIa concentration. This causes the optical density to increase as a parabolic function of time. The concentration of factor VIIIa can be obtained from the quadratic coefficient of the equation describing the parabola. The method is specific for factor VIIIa in that the extrinsic factor X activator is shown to have no influence on the measurement of factor VIIIa in thromboplastin activated plasma. We conclude that a sensitive and reliable method for assessing factor VIIIa concentrations in plasma has been developed on the basis of simultaneous inhibition and measurement of factor Xa by a high concentration of chromogenic substrate.
Determination of the Mean Normal Prothrombin Time for Assessment of International Normalized Ratios
Authors: Peters R H M , van den Besselaar A M H P , Olthuis F M F G
Published: July 2018

To report a Prothrombin Time (PT) as International Normalized Ratio in controlling oral anticoagulant therapy, the Mean Normal PT (MNPT) is required. To correct for methodological differences in performing the PT test, each laboratory should determine its own MNPT for each batch of reagent using fresh blood samples from a large number of normal individuals. This would be a laborious procedure. Two models for simplified assessment of MNPT were investigated by two laboratories in a collaborative study. According to the models, the MNPT of a new batch of reagent is calculated, using the PT of a lyophilized control plasma measured with the new batch and a reference batch, as well as the MNPT of the reference batch obtained with fresh samples. Experimental results were obtained with 19 batches of bovine thromboplastin, 4 lyophilized normal control plasmas and fresh blood samples of 40 normal individuals. The PTs of the 4 lyophilized normal control plasmas were not identical to the MNPT of the fresh normal samples and also different from each other. Therefore, the uncorrected PTs of these control plasmas cannot be used as MNPT.

In general, there was good agreement between measured and calculated MNPT, although some control plasmas gave better results than others. There were no significant differences between the results obtained by both calculation models.
The Autoactivation of Factor XII in the Presence of Long-Chain Saturated Fatty Acids – A Comparison with the Potency of Sulphatides and Dextran Sulphate
Authors: Mitropoulos K A , Esnouf M P
Published: July 2018

The incubation of purified human factor XII (Hageman factor [HF]) in the presence of long-chain saturated fatty acids (FA) like stearate (C-18) or behenate (C-22) resulted in a time-dependent increase of amidolytic activity. The HF autoactivation progress curves were sigmoidal. The first order rate for the initial period was constant; this was followed by a period of decreasing rate and a plateau of zero rate. These progress curves were similar to those obtained on the incubation of HF in the presence of sulphatide vesicles or dextran sulphate. The initial rate of autoactivation of HF was dependent on the FA concentration of contact surface and increased with increasing concentration of HF. At constant concentration of contact surface and varying concentration of HF, autoactivation rates in the presence of behenate, sulphatide vesicles or dextran sulphate followed Michaelis-Menten kinetics. The Km values for all three contact surfaces were above the physiological plasma concentration of HF whereas the catalytic efficiency in the presence of behenate (0.034 εM-1s-1) was about % of that in the presence of sulphatide vesicles (0.053 εM-1s-1) and considerably higher than that in the presence of dextran sulphate (0.004 εM-1s-1). Long-chain saturated FA bound to human serum albumin at the high- or low-affinity sites are ineffective, whereas the crystalline non-bound stearate or behenate provided a potent contact surface. Since oleic acid or other unsaturated fatty acids did not promote autoactivation it is suggested that the requirement for an effective contact surface is one containing immobile negatively charged groups with a critical charge density such as that found in miscelles composed of FA in the crystalline phase. It is suggested that saturated FA on triglyceride-rich lipoproteins resulting from the action of lipoprotein lipase could provide these requirements.
Inhibition of Extrinsic Pathway Inhibitor Shortens the Coagulation Time of Normal Plasma and of Hemophilia Plasma
Authors: Nordfang Ole , Valentin Sanne , Beck Thomas C , Hedner Ulla
Published: July 2018

An increasing amount of evidence suggests that coagulation factors VIII and IX play a role not only in the intrinsic but also in the extrinsic pathway of coagulation. In this context the influence of the Extrinsic Pathway Inhibitor (EPI) on the coagulation time of hemophilia plasma lacking FVIII or FIX has been investigated. The coagulation time was measured in a dilute thromboplastin assay. Addition of recombinant EPI (rEPI) prolonged the coagulation time of normal plasma while the addition of an inhibitory antibody against EPI shortened the coagulation time. At low concentrations of thromboplastin the coagulation time of hemophilia plasma was prolonged and at all dilutions of thromboplastin, addition of anti-EPI IgG normalized the coagulation time of a hemophilia plasma. Analysis of 10 individual donor plasma samples and 8 individual hemophilia samples showed that addition of anti-EPI IgG shortened the coagulation time more in hemophilia plasma than in normal plasma. This illustrates the importance of a powerful extrinsic FVII dependent pathway to achieve hemostasis in the case of FVIII or FIX deficiency (hemophilia A and B).
Antithrombotic Effect of MCI-9042, a New Antiplatelet Agent on Experimental Thrombosis Models
Authors: Hara H , Kitajima A , Shimada H , Tamao Y
Published: July 2018

The antithrombotic effect of MCI-9042, (±)-2-(dimethylamino)-1-[[o-(m-methoxyphenethyl)phenoxy] methyl] ethyl hydrogen succinate hydrochloride was investigated in three different experimental thrombosis models in animals. Simultaneous injection of serotonin and collagen into the tail vein in mice induced acute pulmonary thromboembolic death. MCI-9042 reduced the mortality in a dose dependent manner and its ED50 value was 1.9 mg/kg po. Ticlopidine (TCP) which is a positive reference compound as an antithrombotic drug reduced the mortality at doses of 10 mg/kg po and above. Cyproheptadine (CPH) and ketanserin (KTS) which are S2-serotonergic antagonists were also effective on the reduction of mortality. In mesenteric arterial thrombosis induced by electric stimulation in mice, MCI-9042 prolonged the occlusion time resulted from platelet thrombus formation (PD50: 23 mg/kg po). CPH and KTS prolonged the occlusion time as potent as MCI-9042, but TCP prolonged the occlusion time only at the high doses of 100 mg/kg po and above. In experimental arterial thrombosis which generated in implanted polyethylene tubing, MCI-9042 reduced the incidence of thrombus formation in the tubing and its ED50 value was 18 mg/kg po. TCP was also effective in this model with an ED50 of 170 mg/kg po. The present results lead to the consideration that serotonin plays a more important role in thrombus formation than that conjectured formerly, and suggest that MCI-9042 becomes a new kind of antithrombotic drug with S2-serotonergic receptor antagonism.
A Monoclonal Antibody, VM64, Reacts with a 130 kDa Glycoprotein Common to Platelets and Endothelial Cells: Heterogeneity in Antibody Binding to Human Aortic Endothelial Cells
Authors: Mazurov Alexey V , Vinogradov Dimitry V , Kabaeva Naile V , Antonova Galina N , Romanov Yuri A , VIasik Tatjana N , Antonov Alexander S , Smirnov Vladimir N
Published: July 2018

A new monoclonal antibody (mAb), VM64, reacts with a common antigen on the surface of human platelets and vascular endothelial cells (EC). Under nonreduced conditions it recognized in immunoblotting a protein of 130 kDa both in platelets and EC. VM64 precipitated the same 130 kDa protein from the lysate of surface radioiodinated platelets. Electrophoretic mobility of this protein was not altered by reduction and differed from the bands precipitated by reference mAb against platelet glycoproteins (GP) Ia–IIa, Ib, IIb–IIIa and GMP130. VM64 binding to platelets and EC was specific and saturable. The number of binding sites on platelets was 9.9 ± 3.5 × 103 per platelet and on the surface of EC monolayer – 2.40 ± 0.32 × 106 per cell. VM64 also binds to platelets from Glanzmann's thrombasthenia patients which lack GPIIb–IIIa. VM64 did not affect platelet aggregation induced by ADP, collagen, thrombin and ristocetin. In the monolayers of EC from umbilical vein and human aorta, VM64 stained the area at the periphery of the cells adjacent to the cell-cell boundaries. In preconfluent cultures preferential staining was observed at the active leading margins of the cells. Unlike EC cultures from umbilical vein, where all cells were positively stained, in aortic EC cultures some unstained or poorly stained cells were constantly present, indicating a heterogeneity of EC population related to the expression of VM64 antigen. The biochemical characteristics of VM64 antigen, its presence both on platelets and EC and typical distribution on the surface of EC suggested that this antigen is identical to PECAM (CD31) protein.
The Relationship of Antiphospholipid Antibodies to Thromboembolic Disease in Systemic Lupus Erythematosus: A Cross-Sectional Study
Authors: Long A A , Ginsberg J S , Brill-Edwards P , Johnston M , Turner C , Denburg J A , Bensen W G , Cividino A , Andrew M , Hirsh J
Published: July 2018

In order to determine whether an association exists between antiphospholipid antibodies (APLA) and thromboembolic events in patients with systemic lupus erythematosus (SLE), we performed a cross-sectional study of consecutive unselected SLE patients. The occurrence of previous thromboembolic events was determined by investigators blinded to the APLA status of the patients by critical review of objective tests that had been performed at the time of symptomatic presentation and by performing venous Doppler ultrasound of the legs to elicit venous reflux as an indication of previous venous thrombosis. The presence of APLA was determined by coagulation assays for the lupus anticoagulant (LA) using five tests with well-defined control ranges and by ELISA assay for anticardiolipin antibodies (ACLA). These tests were measured on two separate occasions. The results of the study demonstrate a statistically significant association between persistently abnormal ACLA assays and thromboembolic events and a non-significant trend between persistently abnormal LA and thromboembolic events. Transient abnormalities of LA and ACLA were less strongly associated with thromboembolic events. We conclude that in patients with SLE, there is a significant association between thromboembolism and APLA.
Blood Lead and Platelet Aggregation – Evidence for a Causal Association
Authors: Sharp Dan S , Beswick Andrew , Renaud Serge , Toothill Colin , Elwood Peter C
Published: July 2018

Adenosine diphosphate (ADP) and thrombin are agonists of platelet aggregation in which intracellular calcium plays a significant role as a signal transducer. Lead is a well established toxicological agent affecting intracellular mechanisms controlling free ionized calcium concentration in a number of cell lines. This epidemiological study is the first demonstration of a significant relationship between the extent of primary ADP-induced platelet aggregation measured by optical densitometry in platelet rich plasma (PRP) and blood lead concentration. High blood lead levels are associated with decreased aggregation in this population of 2,150 men aged 49-65 years. This is reflected by a negative regression relationship of –0.19% of maximal extent (PRP vs platelet poor plasma) of aggregation per εg of Pb/dl (T = –3.82, p <0.001). In contrast, no relationship is noted between thrombin-induced aggregation and blood lead concentration. Smoking behaviour represents a potential confounder which may be the explanation for the observed relationship. However, because smoking status is simultaneously related to both ADP- and thrombin-induced aggregation, but is simultaneously related to ADP-induced aggregation and blood lead concentration in a different way, the observed relation is likely to be causal. The mechanisms by which ADP and thrombin effect intracellular calcium transduction signals appear to be distinctively different. The findings in this population-based study are not inconsistent with this difference.
Comparison of High Purity Factor IX Concentrates and a Prothrombin Complex Concentrate in a Canine Model of Thrombogenicity
Authors: MacGregor I R , Ferguson J M , McLaughlin L F , Burnouf T , Prowse C V
Published: July 2018

A non-stasis canine model of thrombogenicity has been used to evaluate batches of high purity factor IX concentrates from 4 manufacturers and a conventional prothrombin complex concentrate (PCC). Platelets, activated partial thromboplastin time (APTT), fibrinogen, fibrin(ogen) degradation products and fibrinopeptide A (FPA) were monitored before and after infusion of concentrate. Changes in FPA were found to be the most sensitive and reproducible indicator of thrombogenicity after infusion of batches of the PCC at doses of between 60 and 180 IU/kg, with a dose related delayed increase in FPA occurring. Total FPA generated after 100-120 IU/kg of 3 batches of PCC over the 3 h time course was 9-12 times that generated after albumin infusion. In contrast the amounts of FPA generated after 200 IU/kg of the 4 high purity factor IX products were in all cases similar to albumin infusion. It was noted that some batches of high purity concentrates had short NAPTTs indicating that current in vitro tests for potential thrombogenicity may be misleading in predicting the effects of these concentrates in vivo.
Sustained Bleeding after a leech Bite in the Apparent Absence of Hirudin
Authors: Munro R , Hechtel F O P , Sawyer R T
Published: July 2018

The bite of the medicinal leech bleeds for many hours. For decades it has been assumed that the remarkably prolonged bleeding time of a leech bite wound is due to hirudin, a specific anti-thrombin secreted by the leech during feeding. By measuring haematological parameters of blood oozing from a leech bite wound on 15 different occasions in 7 human volunteers, we demonstrate that the hirudin-sensitive coagulation parameters, including thrombin-induced platelet aggregation, are prolonged for only 15 min, after which they return to normal. This suggests that excess hirudin secreted by the leech is washed out during this period. However, bleeding from the leech bite wound persists for a mean of 10 h. Platelets in smears of exuding blood show no evidence of spontaneous aggregation, but in vitro platelet aggregation can be induced by exogenous collagen at any time. In view of sustained bleeding in the apparent absence of hirudin, attention is focussed onto an unsuspected factor or factors which may better explain the prolonged bleeding phenomenon.
Increased Plasminogen Activator Inhibitor Activity in Non Insulin Dependent Diabetic Patients – Relationship with Plasma Insulin
Authors: Juhan-Vague I , Roul C , Alessi M C , Ardissone J P , Heim M , Vague P
Published: July 2018

Type 2 diabetic patients are known to frequently have a high insulin level and were recently described as having high plasminogen activator inhibitor (PAI) activity, compared to normal controls. As we have shown in several clinical conditions (normal subjects, obese patients, angina pectoris patients) that plasma PAI activity was linked with plasma insulin, we have studied in 38 type 2 diabetic patients the relationship between PAI activity, insulin and other parameters. Patients showed higher level of PAI activity, as well as plasma glucose, insulin, triglyceride, cholesterol and Apolipoprotein B levels than normal controls; highest values were observed with diabetic patients also affected by coronary artery disease. A significant correlation was found between PAI activity and insulin (r = 0.60, p <0.001), body mass index (r = 0.32, p <0.05) and Apolipoprotein B (r = 0.33, p <0.05). The two latter correlations disappeared after adjustment for insulin.

These results are in agreement with our previous report showing an in vitro effect of insulin on the synthesis of PAI by a hepatocellular cell line. Hyperinsulinemia presented by type 2 diabetic patients may increase the hepatic synthesis of PAI, inducing an hypofibrinolysis, which could play a role in the development of the vascular complications.

Attempts to reduce hyperinsulinemia could have a favorable effect by lowering PAI activity.
Platelets as Scavengers of Neutrophil-Derived Oxidants: A Possible Defence Mechanism at Sites of Vascular Injury
Authors: Dallegri Franco , Ballestrero Alberto , Ottonello Luciano , Patrone Franco
Published: July 2018

Platelets (PLTs) were found to inhibit the chemiluminescence (CL) response of neutrophils (neutrophilic polymorphonuclear leukocytes, PMNs) activated with phorbol myristate acetate. The inhibition of the PMN CL response could be efficiently prevented by pulsing PLTs with carmustine (BCNU) to block their glutathione cycle. In ancillary experiments, the CL response of PMNs was inhibited by catalase (H2O2-scavenger), azide (myeloperoxidase-MPO-inhibitor), taurine (hypoclorous acid- HOCL-scavenger) and chloride ion omission. These data suggest that the PMN CL response requires the HOCL production by the following pathway: H2O2 + Cl- HOCL + H2O. Therefore, the BCNU-preventable PLT-induced inhibition of CL may reflect the consumption of PMN-derived H2O2by the PLT glutathione cycle with a consequent impairment of the HOCL production. Consistent with such a possibility, PLTs lowered the H2O2 and HOCL recovery from PMNs via a BCNU-inhibitable process. Based on these results, we suggest that PLTs have the capacity of limiting the oxidant production by PMNs. This PLT capacity may represent a natural device for the protection of vascular structures from PMN-mediated oxidative stresses.
Expression of Fibrinogen Receptors in Platelets of Migraine Patients - Correlation with Platelet GPIIb Content and Plasma Cholesterol
Authors: Walkowiak Bogdan , Kozubski Wojciech , Pawlowska Zofia , Prusinski Antoni , Cierniewski Czeslaw S
Published: July 2018

Binding of fibrinogen to platelets washed from blood of migraine patients (n = 30) and control donors (n = 24) was compared. In addition, contents of platelet glycoprotein IIb and platelet fibrinogen were determined in both groups by radioimmunoassay. The receptor capacity for fibrinogen in platelets activated by ADP was significantly higher (p <0.01) in migraine patients (52,505 α 4,925) than in controls (33,881 λ 3,965). The mean contents of GPIIb (3.51 α 0.34 μg/108 platelets) and fibrinogen (37 .26 α 4.05 μg/108 platelets) in migraine platelets were also markedly increased (p <0.01 and p <0.001, respectively) when compared to controls (2.21 α 0.18 μg of GPIIb ano 18.75 λ 2.29 μE of fibrinogen per 108 platelets, respectively). There was a high correlation between the number of fibrinogen receptors exposed by ADP and the total amount of platelet GPIIb both in migraineurs (R = 0.69, p <0.01) and controls (R = 0.62, p <0.01,), as well as plasma cholesterol in the case of migraine patients (R = 0.82, p <0.001)
Platelet Aggregation in Whole Blood - Studies with a Platelet Counting Technique - Methodological Aspects and Some Applications
Authors: Falcon C , Arnout J , Vermylen J
Published: July 2018

We describe a method for measuring platelet aggregation in whole blood by single platelet counting. The importance of a low stirring speed (100 rpm) to obtain agonist-specific aggregation is stressed. Despite this low stirring speed, the sensitivity to agonists equals that of the turbidometric technique in platelet-rich plasma. The optimal concentration of formaldehyde for fixing the aggregates, the effects of storage times and anticoagulant are studied. Applicability to the study of platelet function inhibitors or of inherited platelet function disorders is illustrated. It is concluded that this technique, used under the appropriate conditions, combines the advantage of measuring platelet aggregation in a more physiologtc environment with the advantages of the turbidometric technique such as high sensitivity.
Isolation and Structural Charaderization of a Potent Inhibitor of Coagulation Factor Xa from the Leech Haementeria ghilianii
Authors: Condra Cindra , Nutt Elka , Petroski Christopher J , Simpson Ellen , Friedman P A , Jacobs J W
Published: July 2018

The present work reports the discovery and charactenzation of an anticoagulant protein in the salivary gland of the giant bloodsucking leech, H. ghilianii, which is a specific and potent inhibitor of coagulation factor Xa. The inhibitor, purified to homogeneity, displayed subnanomolar inhibition of bovine factor Xa and had a molecular weight of approximately 15,000 as deduced by denaturing SDS-PAGE. The amino acid sequence of the first 43 residues of the H. ghilianii derived inhibitor displayed a striking homology to antistasin, the recently described subnanomolar inhibitor of factor Xa isolated from the Mexican leech, H. officinalis. Antisera prepared to antistasin cross-reacted with the H. ghilianii protein in Western Blot analysis. These data indicate that the giant Amazonian leech, H. ghilianii, and the smaller Mexican leech, H. officinalrs, have similar proteins which disrupt the normal hemostatic clotting mechanisms in their mammalian host’s blood.
Isolation and Characterization of Human Blood Platelet mRNA and Construction of a cDNA Library in λgt11
Authors: Wicki Andreas N , Walz Alfred , Gerber-Huber Susan N , Wenger Roland H , Vornhagen Rolf , Clemetson Kenneth J
Published: July 2018

We have developed a purification method for the isolation of platelet-specific poly (A+) RNA and demonstrated that human blood platelets, despite the absence of a nucleus, contain stable mRNA. The poly (A+) RNA was used to construct a platelet- specific cDNA expression library in λgtll. The platelet derivation of the purified mRNA was confirmed by identification of membrane glycoprotein Ib (GPIb) message by immunoprecipitation of rabbit reticulocyte lysate translation products with poly- and monoclonal antibodies against GPIbα and by sequencing of a GPIbα cDNA clone
Functional and Immunologic Protein S in Normal Pregnant Women and in Full-Term Newborns
Authors: Fernández José A , Estellés Amparo , Gilabert Juan , España Francisco , Aznar Justo
Published: July 2018

Total and free protein S antigen and C4b-binding protein (C4bp) were determined by rocket immuno-electrophoresis, and functional protein S was assayed by a coagulation method, throughout pregnancy and normal puerperium and in a group of normal full-term newborns (FTN). The functional protein S assay is based on a modification of the APTT, using a mixture of test sample, protein S deficient plasma, activated protein C, phospholipids and calcium. This protein S functional assay is specific for protein S since the APTT prolongation by normal plasma was abolished by incubation of plasma with monospecific, rabbit antiprotein S IgG. The ratios of functional protein S/free protein S antigen in healthy men (n = 13) and women (n = 14) were 1.0 ± 0.13 (mean ± SD) and 1.03 ± 0.20, respectively. During pregnancy there is a decrease in functional protein S and a progressive decrease in total and free protein S antigen, with a functional/free protein S ratio of 0.75 ± 0.28 in the third trimester of pregnancy (n = 16). In early puerperium the functional protein S level was lower than the free protein S antigen level (ratio about 0.5). In the FTN group, the free protein S level was 39% and protein S activity was about 70% that of adults, with a functional/free protein S ratio of 1.84 ± 0.31. C4bp values were 23.5 ± 10.3% in the FTN group, and crossed immunoelectrophoresis showed that in this group the major protein S peak corresponded to free protein S. These results indicate that both in early puerperium and in FTN group, free protein S antigen may not be an adequate parameter for estimating of functional protein S activity. The decrease in functional protein S activity during early puerperium may be connected with the risk of developing thrombotic episodes during the postpartum period.
Additive Effect of dDAVP and Standard Heparin inIncreasing Plasma t-PA
Authors: Agnelli Giancarlo , Levi Marcel , Cosmi Benilde , ten Cate Jan Wouter , Nenci Giuseppe G
Published: July 2018

The aim of this study was to evaluate the existence of an additive effect between standard heparin and dDAVP in the enhancement of endogenous fibrinolysis. Eight healthy volunteers were studied in a randomized double blind placebo controlled cross-over trial. The volunteers were treated i. v. with dDAVP, 0.4 μg/kg, over 15 minutes followed by an i. v. bolus dose of either standard heparin, 5,000 I.U., or saline. A 48 hour wash-out period was adopted. The infusion of dDAVP followed by standard heparin resulted in a higher increase in plasma t-PA actiyity, t-PA antigen, circulating t-PA specific activity and FPLA when compared with dDAVP followed by saline. The difference was already statistically significant at 15 minutes after the infusion of dDAVP and lasted for up to 60 minutes after the end of the infusion of dDAVP. Plasma PAI 1 showed a slightly higher decrease after dDAVP plus standard heparin than after dDAVP plus saline but this difference was not statistically significant. No statistically significant changes of fibrinogen and α2-antiplasmin levels were observed. As expected, the infusion of standard heparin resulted in an increase in plasma anti-Xa activity and in a prolongation of aPTT Our results demonstrated an additive effect of dDAVP and standard heparin on the increase in circulating t-PA, the effect of dDAVP being potentiated and prolonged by heparin. This observation could prospect a combined use of dDAVP and standard heparin in the prophylaxis and treatment of thromboembolic diseases.
Plasma D Dimer: A Useful Marker of Fibrin Breakdown in Renal Failure
Authors: Gordge M P , Faint R W , Rylance P B , Ireland H , Lane D A , Neild G H
Published: July 2018

D dimer and other large fragments produced during the breakdown of crosslinked fibrin may be measured by enzyme immunoassay using monoclonal antibodies. In 91 patients with renal disease and varying degrees of renal dysfunction, plasma D dimer showed no correlation with renal function, whereas FgE antigen, a fibrinogen derivative which is known to be cleared in part by the kidney, showed a significant negative correlation with creatinine clearance. Plasma concentrations of D dimer were, however, increased in patients with chronic renal failure (244 ± 3l ng/ml) (mean ± SEM) and diabetic nephropathy (308 ± 74 ng/ml), when compared with healthy controls (96 ± 13 ng/ml), and grossly elevated in patients with acute renal failure (2,451 ± 1,007 ng/ml). The results indicate an increase in fibrin formation and lysis, and not simply reduced elimination of D dimer by the kidneys, and are further evidence of activated coagulation in renal disease. D dimer appears to be a useful marker of fibrin breakdown in renal failure.
Recombinant Human Erythropoietin Increases Blood Pressure, Platelet Aggregability and Platelet Free Calcium Mobilisation in Uraemic Children: A Possible Link?
Authors: Van Geet Chris , Damme-Lombaerts Rita Van , Vanrusselt Marleen , MOl An de , Proesmans Willy , Vermylen Jos
Published: July 2018

Recombinant human erythropoietin was administered to 10 uraemic children on chronic haemodialysis, all of whom responded by correcting their haemoglobin. In addition, they showed an increase in blood pressure; platelet aggregations, subnormal before therapy, improved during treatment. The intracellular free calcium concentration in platelets after thrombin stimulation also increased significantly during erythropoietin administration. We hypothesize that the effect of erythropoietin on platelet aggregability and on blood pressure may be due to an increase in the intracellular free calcium mobilisation in platelets and possibly in smooth muscle cells respectively.
Clinical Significance of Various ELISA Assays for Detecting Antiphospholipid Antibodies
Authors: Falcón C R , Hoffer A M , Forastiera R R , Carreras L O
Published: July 2018

We have compared the prevalence of antiphospholipid antibodies (APA) measured by enzyme-linked immunosorbent assay (ELISA), in 119 selected patients using five different antigens: bovine cardiolipin, phosphatidylserine, phosphatidylinositol, bovine partial thromboplastin and human brain partial thromboplastin. All the plasmas have been evaluated for the presence of lupus anticoagulant (LA) activity by clotting techniques. We found a significant association between the incidence of LA and APA (p <0.001), only moderate agreement between the prolongation of the activated partial thromboplastin time (APTT) and ELISAs (r around 0.50) and a good agreement between the ELISAs (r around 0.80). The combination of antibodies against cardiolipin (ACA) and human brain partial thromboplastin (AHPTA) allowed the detection of antibodies in most of the LA positive cases. ACA, AHPTA and antiphosphatidylinositol antibodies detected all the positive samples. Six patients (5%) had a single APA detected. The clinical associations of APA according to phospholipid specificity, immunoglobulin isotype and titer are shown.
Plasminogen Activation at Low Temperatures in Plasma Samples Gontaining Therapeutic Concentrations of Tissue-Type Plasm i nogen Activator or Other Thrombolytic Agents
Authors: Rijken D C , Seifried E , Barrett-Bergshoeffi M M , Dooijewaard G
Published: July 2018

It is known that in vitro plasminogen activation in blood samples taken during thrombolytic therapy with tissue-type plasminogen activator (t-PA) may lead to artefactually low fibrinogen and α2-antiplasmin values. To mimic this phenomenon, pooled normal plasma was supplemented with 2.5 μg/ml t-PA and incubated at various temperatures. The rates of fibrinogen degradation and α2-antiplasmin consumption were most pronounced at 37° C, were less pronounced at 25° C, but surprisingly, did not further decrease at 10° C, 0° C or −8° C. In contrast, when plasma was supplemented with 10° IU/ml urokinase or 30 IU/ml streptokinase, the rates of fibrinogen degradation and α2-antiplasmin consumption gradually decreased with incubation temperature and were negligible at 10° C and lower temperatures. The rate of plasminogen activation also decreased gradually with temperature in mixtures of purified fibrinogen, plasminogeo, α2- antiplasmin and t-PA. These results imply that, in a plasma milieu, additional factors with a stimulatory activity are involved in t-PA-induced plasminogen activation at around 0° C. The abnormally high reaction rate at low temperatures explains in vitro plasminogen activation observed during the processing of t-PA-containing blood samples.

In contrast to the activation of plasminogen by t-PA, the slow inhibition of t-PA (2.5 μg/ml) by proteinase inhibitors in plasma could be minimized to a negligible level by keeping the plasma samples at 0° C. This makes it possible to reliably monitor t-PA activity during thrombolytic therapy
Effect of Fibrin-Like Stimulators on the Activation of Plasminogen by Tissue-Type Plasminogen Activator (t-PA) - Studies with Active Site Mutagenized Plasminogen and Plasmin Resistant t-PA
Authors: Lijnen H R , Van Hoet B , De Cock F , Collen D
Published: July 2018

The activation of plasminogen by t-PA was measured in the presence and absence of fibrin stimulation, using natural human plasminogen (nPlg) and rPlg-Ala740, a recombinant plasminogen with the active site Ser740 mutagenaed to Ala. Recombinant wild type t-PA (rt-PA) was used as well as rt-PA -Glul275, a recombinant single chain t-PA in which the Arg of the plasmin sensitiv e Arg275- Ile276 peptide bond was substituted with Glu. Conversion of 125I-labeled single chain plasminogen to two-chain plasmin by wild-type or mutant t-PA, was quantitated by SDS gel electrophoresis and radioisotope counting of gel slices, and expressed as initial activation rates (v0 in pM s−1) per 1 μM enzyme. In the absence of fibrin stimulation, the vs for the activation of nPlg and rPlg-Ala740 with the single chain forms of both t-PAs were comparable (0.6 to 2.7 pM s−1) but were lower than with the corresponding two-chain forms (5.3 to 23 pM s−1). In the presence of 1 μM soluble fibrin monomer (desAAfibrin), the v0 for nPlg and rPlg-Ala740 by single chain rt-PA was also comparable (24 and, 33 pM s-1 respectively), whereas with 1 pM CNBr-digested fibrinogen, the vs for nPlg with single chain rt-PA was about 20-fold higher than that of rPlg-Ala740 (135 and 7.5 pM s−1 respectively). In contrast, the vs for nPlg and rPlg-Ala740 by single chain rt-PA- G1u275, two-chain rt-PA-G1u275 or two-chain rt-PA were comparable in the presence of either desAAfibrin or CNBr-digested fibrinogen.

These findings confirm and establish: 1) that single chain t-PA is an active enzyme both in the presence and absence of fibrin stimulator; 2) that, in a system devoid of plasmin activity (rPlg- Ala740), the two-chain form of t-PA is about L5 times more active than the single chain form in the absence of fibrin but equipotent in the presence of desAAfibrin; and 3) that the mechanism of stimulation of plasminogen activation with single chain t-PA by CNBr-digested fibrinogen is different from that by soluble fibrin.
Binding of Plasma von Wllebrand Factor by Arterial Microfibrils
Authors: Fauvel-Lafève Françoise , Legrand Yves J
Published: July 2018

We developed an ELISA test to measure the binding of plasma von Willebrand factor (vWF) to arterial microfibrils and compared this interaction to the binding of vWF to collagen under the same conditions. We found that vWF binds to microfibrils in a similar manner as it binds to collagen: the binding was independent of the presence of cations, and temperature of incubation and was displaced by 1 M NaCl. Using purified 125I-vwF we showed that the binding was saturable and could be displaced by cold vWF in excess. Using immunoblotting we showed that vWF binds to a 97 kDa protein present in the microfibrils different from the 128 kDa thrombospondin-like structure (GP 128) which in microfibrils is known to interact with blood platelets. These results indicate that in the subendothelium, microfibrils bind plasma vWF and this reinforces the thrombogenic role of these structures.
The Action of Ticlopidine on Human Platelets
Authors: Hardisty R M , Powling M J , Nokes T J C
Published: July 2018

The effects on platelet function of a 5-day course of Ticlopidine (Tel) have been studied in two groups of volunteers receiving different dosage schedules. Tel had a relatively greater inhibitory effect on aggregation induced by ADP than by other agonists, and a greater effect, in contrast to that of an ADP receptor antagonist, on the second phase than on the initial rate of aggregation. Tel inhibited ATP secretion in response to ADP and 0.05 u/ml thrombin, but not to higher concentrations of thrombin or to calcium ionophores. No inhibitory effect was observed on Ca2+ influx or intracellular mobilization, on the binding of monoclonal antibodies to the glycoprotein IIb-IIIa complex or on the state of association of the complex. We suggest that Tel neither inhibits the binding of ADP to its receptor nor acts directly on the fibrinogen binding site, but that it may inhibit a step in signal transduction between these two events.
Modification of Na+/H+ Exchanger in Human Platelets by 1,2-Dioctanoylglycerol, a Protein Kinase C Activator
Authors: Ozaki Yukio , Mastsumoto Yuki , Yatomi Yutaka , Higashihara Masaaki
Published: July 2018

Protein kinase C activation in human platelets has a modulatory role in maintaining intracellular pH (pHi), by adjusting pHi at a particular value (7.22). Changes in pHi induced by protein kinase C appeared to be dependent upon the difference between H+ efflux catalyzed by the Na+/H+ exchanger and H+ production. The pHi recovery after acid loading was significantly facilitated by protein kinase C activation. Analysis of the rate constant for pHi recovery suggested that the turnover rate or the apparent affinity of the Na+/H+ exchanger for H+ was increased. Protein kinase C also decreased the Km value of the Na+/H+ exchanger for extracellular Na+. Thus, it is suggested that the role of protein kinase C in platelet pHi regulation is dual, adjusting the pHi value at a certain setpoint on the one hand, and increasing the rate constant of the Na+/H+ exchanger on the other.
D-Dimer in Local Thrombolytic Therapy with Low Doses of Recombinant Human Tissue-Type Plasminogen Activator (rt-PA) in Patients with Peripheral Arterial Occlusive Disease
Authors: Koppensteiner Renate , Speiser Wolfgang , Minar Erich , Ahmadi Ramazan , Ehringer Herbert
Published: July 2018

The generation of D-dimer was studied in the course of local thrombolytic therapy of peripheral arterial occlusions with low doses of recombinant human tissue-type plasminogen activator (rt-PA) in 7 patients. Intermittent local application of rt-PA resulted in a marked increase in D-dimer exceeding values usually seen after intravenous application of manifold higher doses used in myocardial infarction. The increase in D-dimer was related to the estimated thrombus size (length of the occlusion) and the total dose of rt-PA applied. During local rt-PA infusion 6 of 7 patients maintained plasminogen activator inhibitor capacity (PAI-cap) between 34% and 79% of their corresponding pre-treatment levels; detectable levels of PAI-cap in circulating blood during the procedure did not interfere with the success of therapy.