bluuebunny/crossref_metadata_2025 huggingface dataset:
Monoclonal Antibodies to Tissue-Type Plasminogen Activator which Prolong its Clearance In Vivo
Authors: Reilly Thomas M , Knabb Robert M , Chiu Andrew T , Bradfute David L , Timmermans Pieter B M W M
Published: June 2018

Three murine monoclonal antibodies to tissue-type plasminogen activator (t-PA) were evaluated for their effects on the binding of iodinated t-PA to cultured human hepatoma cells (Hep G2), and on extending the half-life of t-PA injected into rabbits. Two of the antibodies, AE5 and EG2, significantly inhibited t-PA binding in vitro, and extended the in vivo half-life of t-PA four to five-fold. A third antibody, BA10, which had a much smaller inhibitory effect on t-PA binding, had no influence in extending t-PA’s half-life. MOPC-21, a control antibody not directed to t-PA, had no effect on either test. Our results are the first to correlate different compounds’ effects on t-PA binding with their ability to retard t-PA clearance in vivo, and provide additional evidence for the importance of a liver cell receptor in the t-PA clearance process.
Tissue-Type Plasminogen Activator (t-PA) and Dilute Blood Clot Lysis Time in Nephrotic Patients
Authors: Cucuianu Mircea P , Rus Horea G , Roman Stefan , Mărcuşu Codruţa , Spînu Constantin , Manasia Mihai , Niculescu Florin
Published: June 2018

When compared to normal weight normolipidemic control subjects, dilute blood clot lysis time was found to be obviously (p <0.001) prolonged in hypertriglyceridemic patients without proteinuria and slightly (p <0.05) accelerated in hyperlipidemic nephrotic patients in spite of their very high levels of plasma fibrinogen. As a result the ratio plasma fibrinogen (mg/dl) per clot lysis time (minutes) was 1.241 ± 0.08 (X ± SEM) in control subjects, 0.574 ± 0.07 in hypertriglyceridemic patients and 2.69 ± 0.172 in nephrotic patients. This finding suggesting that a larger amount of fibrin is rather readily dispersed from dilute blood clots of nephrotic patients was associated with higher levels of plasma t-PA: Ag (9.45 ng/ml ± 1.18 in nephrotic patients versus 5.8 ng/ml ± 1.23 in controls before venous occlusion and respectively 33.1 ng/ml ± 3.83 versus 20.3 ± 3.40 in controls after venous occlusion). Plasminogen activator activity of the euglobulins as assessed by the bovine fibrin-agarose plate was significantly higher in nephrotic patients only after venous occlusion. Plasma samples of nephrotic patients exerted a more potent inhibition of fibrinolysis in a urokinase activated system. This effect was, however, mainly due to the high levels of α2 macroglobulin in nephrotic plasma which apparently have little influence on dilute blood clot lysis time.
Fibrinolytic Profiles in Local Low-Dose Thrombolysis with Streptokinase and Recombinant Tissue Plasminogen Activator
Authors: Berridge D C , Earnshawl J J , Westby J C , Makin G S , Hopkinson B R
Published: June 2018

Fibrinolytic parameters have been monitored in 44 patients undergoing local low-dose intra-arterial thrombolysis for acute peripheral arterial ischaemia. Streptokinase (Sk), at a dose of 5,000 units/hr with 250 units/hr heparin, was used in 23 patients and recombinant tissue plasminogen activator (r-tPA) at a dose of 0.5 mg/hr was used in 21 patients. Successful lysis was seen in 18 (86%) patients following r-tPA and in 15 (65%) patients following streptokinase. There were 4 minor haematomas in each group usually at the catheter entry site. Both agents produced a systemic effect, which was still seen 12 hours post-infusion. However, that produced by r-tPA was delayed and significantly reduced compared to that produced by Sk.

These results confirm the relative fibrin specificity of r-tPA. When used as a continuous low-dose intra-arterial infusion, r-tPA offers a significantly lower, potentially safer, systemic effect than conventional therapy with streptokinase.
Binding Properties of Monoclonal Antibodies against Human Fragment D-Dimer of Cross-Linked Fibrin to Human Plasma Clots in an In Vivo Model in Rabbits
Authors: Holvoet P , Stassen J M , Hashimoto Y , Spriggs D , Devos P , Collen D
Published: June 2018

Two (MA-15C5 and MA-8D3) out of approximately 500 monoclonal antibodies, obtained by fusion of P3X63-Ag8-6.5.3 myeloma cells with spleen cells of mice immunized with purified fragment D-dimer from human fibrin, demonstrated a more than 1,000-fold higher affinity for fragment D-dimer than for native fibrinogen. MA-15C5 was directed against a neoantigenic determinant only expressed in fragment D-dimer. MA-8D3 reacted equally well with fragment D-dimer of crosslinked fibrin and with fragment D of non-crosslinked fibrin but not with fragment D of fibrinogen. Both monoclonal antibodies did not crossreact with rabbit fibrin and its degradation products.

The binding of 125I-labeled Fab fragments to human plasma clots, introduced and aged for 1 hr in the jugular vein of heparinized rabbits was studied. Following injection of an equimolar mixture of Fab fragments derived from MA-15C5 and MA-8D3, the clot to blood ratios of radioactivity increased from 3.2 ± 1.2(mean ± SD) at 4 hr to 7.2 ± 1.4 at 17 hr. The binding of Fab fragments of MA-15C5 and MA-8D3 was independent of the age (1 to 72hrs) of the clot and of heparin anticoagulation and was only slightly decreased (by 20%) in the presence of circulating human fibrinogen (90 mg/kg body weight) and of human crosslinked fibrin degradation products at a plasma concentration of 10 pg/ml. The binding of Fab fragments of MA-15C5 and MA-8D3 to occlusive human plasma clots in the femoral artery of rabbits was comparable to that of the non-occlusive human plasma clots in the jugular vein. The Fab fragments of MA-15C5 and MA-8D3, labeled with 123I to a specific activity of 10 μCi/pg were injected intravenously (3 μg/kg) in 72 rabbits with a nonocclusive 0.2 ml human plasma clot in the jugular vein and in 7 control rabbits that underwent the surgical procedure without clot formation. Total body scans performed at hourly intervals revealed a higher relative increase in gamma counts over the thrombus region in the group with thrombus as compared to controls: at 6 hr 54 ± 18 vs 16 ± 13% (mean ± SEM, p <0.1) and at12 hrs 35 ± 11 vs –7 ± 12 (p <0.05). The vein segment to blood ratios of 123I at 24 hrs were 6.6 ± 2.4 in the group with clot and 1.5 to 0.7 in the control group (p <0.01). We conclude that these Fab fragments may have a sufficiently high fibrin-affinity to allow in vivo thrombus localization by external scanning.
Synergistic Effect of Low Doses of Tumor Necrosis Factor and Sera from Patients with Systemic Lupus Erythematosus on the Expression of Procoagulant Activity by Cultured Endothelial Cells
Authors: Hasselaar Paula , Derksen Ronald H W M , Oosting Janine D , Blokzijl Laya , de Groot Philip G
Published: June 2018

The effect of 15 antiphospholipid antibody (APA) positive SLE sera, 13 APA negative SLE sera, 10 APA negative sera from patients with other connective tissue diseases (CTD) and 15 control sera on the expression of endothelial procoagulant activity (PCA) was studied. Endothelial cells (EC) were stimulated with tumor necrosis factor (TNF) and 20% serum for 4 hr and the surface PC A expression was measured. Without TNF, none of the sera stimulated PC A expression. With subop timal TNF stimulation, 14/15 APA positive SLE sera, 7/13 APA negative SLE sera, 2/10 CTD sera and 2/15 control sera enhanced PC A expression. This stimulating effect resided in the IgG fraction and was associated with the presence of APA, but not with a history of thrombosis. Purified APA had no PC A stimulating activity. PC A expression was inhibited by cycloheximide and heat treatment (30 min, 56° C) of serum.

In conclusion, 21/28 (75%) SLE sera increase the TNF-induced endothelial PC A expression. Although this effect predominantly occurs with APA positive serum, a causative role of APA was not demonstrated.
Is It Possible to Reduce the Risk of Cardiovascular Events in Subjects Suffering from Intermittent Claudication of the Lower Limbs?
Authors: Boissel J P , Peyrieux J C , Destors J M
Published: June 2018

This study meta-analysed randomized, double-blind, placebo controlled trials in patients with intermittent claudication of the lower limbs comparing ticlopidine to placebo in order to test the hypothesis that the drug, a pure antiplatelet agent, is able to reduce the incidence of thrombotic cardio-vascular events on atherosclerotic arteries in these patients. A highly significant reduction, from 9% to 3% (p ranging from 0.006 to 0.002), was observed for fatal or non-fatal cardio-vascular events in a total of 611 patients (301 with ticlopidine, 310 with placebo). The duration of follow-up ranged from 6 to 12 months. Side-effects, defined as withdrawal from study medication for any reason but death, cardio-vascular events or cancer, were 2.4 times more frequent in the ticlopidine treated patients as compared to placebo. We concluded that in this high risk population, prevention of cardio-vascular events is likely to be effective.
Platelet Aggregation Is Stimulated by Lactose-lnhibitable Snake Venom Lectins
Authors: Ogilvie Mary L , Byl JoAnn Wilson , Gartner T Kent
Published: June 2018

Five lactose-specific lectins from snake venoms were tested for the ability to stimulate the aggregation of human platelets. Three of the lectins, bushmaster (Lachesis muta), cottonmouth (Aricistrodon piscivorous leukostoma) and rattlesnake (Crotalus atrox) lectins, consistently stimulated secretion and aggregation. Thrombolectin (Bothrops atrox) occasionally caused aggregation. Copperhead (Agkistrodon contortrix contortrix) lectin did not by itself cause platelet aggregation. Lactose, a specific inhibitor of hemagglutination mediated by these lectins was a potent inhibitor of lectin-induced aggregation. Antiserum specific for bushmaster lectin inhibited aggregation by bushmaster lectin. In contrast, the same antiserum and anti-cottonmouth lectin serum enhanced aggregation by low levels of the other lectins.

A variety of substances were assayed in the aggregometer for the ability to inhibit aggregation in response to these lectins. Both secretion and aggregation were inhibited by PGI2 and PGEx. Furthermore, lectin-induced aggregation was completely blocked by trifluoperazine and partially blocked by indomethacin. Monoclonal antibodies specific for GP IIb/IIIa (AP2, A2A9, LJP5, LJCP8) but not monoclonals directed against other platelet membrane proteins (API and AP3) inhibited lectin-induced aggregation. The peptide Arg-Gly-Asp-Ser but not Arg-Ala-Asp-Ser was a potent inhibitor of aggregation.
Platelet Size Affects both Micro- and Macro-Aggregation: Contributions of Platelet Number, Volume Fraction and Cell Surface
Authors: Wong Truman , Pedvis Lloyd , Frojmovic Mony
Published: June 2018

The relationship between platelet size (v), platelet number (N0), platelet volume fraction (φ) and platelet aggregation was evaluated in human platelet subpopulations separated on the basis of volume using counterflow centrifugation. The original platelet population and three size-dependent platelet fractions were concentrated and resuspended in autologous citrated platelet-poor plasma at varying N0. Micro-aggregation (PA) and macro-aggregation (TA) were determined respectively by electronic particle counting and light transmission (turbidometry). At similar N0, large platelets were about two-fold more sensitive and more rapidly recruited into both micro (PA) and macro (TA) aggregates in response to ADP than the small platelets (v were respectively 7.3 ± 0.2 and 4.2 ± 0.2 fl, each 16 ± 4% of the total population). At volume fractions favouring the small platelets, however, the above differences persisted for PA, but not for TA. Similar size-dependent differences were observed for two other receptor-mediated platelet activators, namely a stable thromboxane A2 analogue (U46619) and platelet activating factor (PAF), but not for ristocetin-induced agglutination. Increasing platelet size appears to optimize platelet aggregation due to (i) the simple geometric advantage of large-sized platelets associated with any given N0, seen for both PA und TA, and (ii) intrinsic differences in size-dependent subpopulations which favour more efficient platelet membrane surface changes associated with receptor-mediated micro-aggregation (PA), for which a likely model is proposed.
Characterization of Monoclonal Antibodies to Human Tissue-Type Plasminogen Activator: Catalytic Inhibition and One-Two Chain Discriminatory Reactivities
Authors: Stigbrand Torgny , Frängsmyr Lars , Bergsdorf Nils , Wallen Per
Published: June 2018

A new set of monoclonal antibodies was generated against the tissue plasminogen activator (t-PA). One of the antibodies, 1:3 C5, was found to be able to distinguish between the one- and two-chain form of t-PA and also exerted significant amidolytic inhibitory activity. Several of the antibodies, as judged from their binding properties in immunosorbent tests, were found to be suitable for immunoaffinity purification purposes, i.e. 1:3C5, 1:3 G5, and 1:2 B9. Three of the mabs, 1:2 B9, 1:3 G5 and 2:2 BIO, were selectively reactive with the A-chain of t-PA, whereas indirect evidences indicated 1:3 C5 to be reactive with a conformational epitope on the B-chain. Three of the antibodies were reactive with porcine t-PA. This new set of antibodies should prove useful for structure-function investigations of t-PA.
Familial Dysfunction of Protein S
Authors: Mannucci P M , Valsecchi C , Krachmalnicoff A , Faioni E M , Tripodi A
Published: June 2018

We describe a previously unreported defect of protein S characterized by low levels of cofactor activity for activated protein C contrasting with low normal levels of total and free protein S antigen. The distribution of protein S between the free form and the form complexed with the complement component C4b-binding protein was normal on two-dimensional immunoelectrophoresis. The proband developed juvenile deep-vein thrombosis while taking oral contraceptives. Her defect was transmitted in an autosomal dominant fashion from her asymptomatic mother. Other relatives carrying the same laboratory abnormality (mother, maternal uncle, two sisters and one brother) were also asymptomatic. We postulate that the defect is due to a dysfunctional protein S present in plasma in normal amounts and with normal proportions of the free and complexed forms of the protein.
Anti-Hypertensive Drugs Non-Specifically Reduce “Spontaneous” Activation of Blood Platelets
Authors: Wilson J , Orchard M A , Spencer A A , Davies J A , Prentice C R M
Published: June 2018

Abnormal activation of blood platelets may be a contributory factor in the accelerated vascular disease which occurs in hypertension. We investigated the effects of lowering blood pressure in 12 patients with mild hypertension on several aspects of platelet function, initially in a placebo-controlled, double-blind, crossover study with nisoldipine, and subsequently in the same patients comparing nisoldipine with the patients’ usual anti-hypertensive therapy. Values were compared with those from an age, sex-matched control population. Seven hypertensive patients with renal failure were also studied. Administration of nisoldipine reduced ex vivo “spontaneous” aggregation of blood platelets significantly, and a similar significant effect was seen when blood pressure was lowered by the patients usual anti-hypertensive therapy. “Spontaneous” aggregation occurring in the control population was similar to that in the treated hypertensives. Blood platelet count, and aggregation in response to ADP and adrenalin were unaffected by treatment. Median plasma beta thromboglobulin levels were significantly higher in the untreated hypertensive patients (43 ng ml-1) than in the controls (30 ng ml-1), and there was a trend to reduced values for beta thromboglobulin on treatment of the hypertension. These results indicate that blood platelet activity is enhanced in hypertension and that function returns towards normal when blood pressure is lowered by treatment.
von Willebrand Factor Multimer Analysis Using a Sensitive Peroxidase Staining Method
Authors: Tomita Yukiharu , Harrison Janet , Abildgaard Charles F
Published: June 2018

A peroxidase staining method for von Willebrand factor (vWF) multimer analysis was modified for comparison with an autoradiographic method. This method consists of SDS-agarose electrophoresis followed by blotting to a nitrocellulose membrane and a sensitive peroxidase staining method. The resolution of vWF multimers on the nitrocellulose membrane is comparable to that on the conventional autoradiography. Results can be obtained in 3 days. This nonradioisotopic method will be useful for the determination of the type of von Willebrand’s disease in clinical laboratories.
Effect of Propranolol, Barbiturate Anesthesia and Splenectomy on Factor VIII: C and Plasminogen Activator Release Induced by DDAVP
Authors: Pina-Cabral J M , Cunha-Monteiro A , Sousa-Dias M C , Andrade J A
Published: June 2018

After the injection of DDAVP in 39 non-anaesthezised dogs (0.4 μg/kg) factor VIII: C activity rose to 145% of baseline values (p <0.0001) and the fibrinolytic potential of euglobulin precipitate rose to 196% (p <0.0001). The injection of DDAVP was repeated three times in each dog of a group of good responder animals at weekly intervals, but after: A) Pentobarbital anesthesia (30 mg/kg) - the increase of factor VIII: C was reduced from 164% to 116% (n = 11; p <0.0005) and the increase in fibrinolytic activity was reduced from 270% to 192% (n = 11; p <0.05). B) Injection of propranolol (1 mg/kg) - the increase of factor VIII :C was reduced from 167% to 110% (n = 13; p <0.0005) and there was no significant decrease of fibrinolytic activity (n = 13; n.s.). C) Splenectomy - the increase of factor VIII: C was reduced from 166% to 122% (n = 10; p <0.0005) and fibrinolytic activity was not significantly changed from 196% to 256% (n = 9; n.s.). There were no statistically significant differences in the increases of factor VIII :C and fibrinolytic potential of euglobulin precipitate after repeating only the injection of DDAVP three times in the same animal at weekly intervals (n = 5; n.s.). We conclude that the increases in factor VIII: C and fibrinolytic activities observed after DDAVP infusion in the dog are due to different mechanisms of action. Pentobarbital anesthesia reduced the increase of factor VIII: C and fibrinolytic activity, but on the other hand, beta-blockade and splenectomy influenced differently the behaviour of both biological activities.

Injection of the synthetic analogue of vasopressin 1-deamino-8-d-arginine vasopressin (DDAVP) increases the plasma levels of factor VIII procoagulant activity (factor VIII: C) and plasminogen activator both in man and dog (1, 2, 3, 4).

The mechanism of action of DDAVP in determining these responses is not well known. In this work, after having confirmed that DDAVP increases factor VIII: C and fibrinolytic activity in the blood of the unanaesthezised dog, and in order to clarify the mechanisms involved in these pharmacological effects, we repeated the DDAVP injection in animals previously submitted to propranolol, pentobarbital anesthesia and splenectomy.
The Relative Importance of the Factors II, VII, IX and X for the Prothrombinase Activity in Plasma of Orally Anticoagulated Patients
Authors: Xi Ma , Béguin S , Hemker H C
Published: June 2018

The individual importance of each of the four vitamin Independent clotting factors on the generation of prothrombinase activity in the plasma of orally anticoagulated patients has been investigated.

Addition of purified factors VII, IX or X to plasma from deeply anticoagulated patients (International Normalized Ratio 2.8-4.8) did not influence the amount of prothrombinase activity or the amount of thrombin formed. Only the prothrombin level in the plasma determines the course of thrombin generation. Addition of increasing amounts of purified factor II, VII, IX or X to plasmas deficient in respectively factor II, VII, IX or X showed that the prothrombinase activity increases linearily with the concentration of factor II added and that the concentration below which the factors VII, IX and X start to have a measurable effect on prothrombinase activity are 5%, 20% and 30%, respectively. Half maximal prothrombinase activity was found at about 1% factor VII, 5% factor IX and 8% factor X respectively.

From these observations we conclude that primarily the variation in factor II level determines thrombin generation and hence presumably the antithrombotic effect of oral anticoagulant therapy. It therefore seems likely that, for the control of oral anticoagulant therapy, tests that reflect factor II activity would be suitable.
Quantitation of Plasma Levels of Tetranectin - Effects of Oral Contraceptives, Pregnancy, Treatment with L-Asparaginase and Liver Cirrhosis
Authors: Kluft C , Los P , Clemmensen I , Brommer E J P , Gevers Leuven J A , Boks A L , Vellenga E
Published: June 2018

Tetranectin is a tetrameric protein that binds to kringle 4 of plasminogen. Increase of electrophoretic mobility of the otherwise slowly migrating tetranectin in the presence of ethylenediaminetetraacetate was used to develop a reproducible electroimmunoassay to quantify plasma levels. Plasma levels in normals were found within narrow limits of 100 ± 16 (SD)%, (100% =0.15 pmol/1). There was no difference between males and females, smokers and non-smokers, and there were no significant changes with age from 20 to 49 years.

Patients with severe liver cirrhosis showed a large variation in plasma tetranectin levels but no systematic or average reduction, in contrast to strong reductions in plasma levels of other proteins. Patients treated with L-asparaginase showed a gradual reduction in time in plasma levels of various proteins, though tetranectin showed no significant reduction.

It is concluded that tetranectin can be assayed reproducibly in plasma and has a well regulated plasma level. This level is not sensitive to conditions with reductions in synthesis of many proteins, such as during cirrhosis of the liver and during L-asparaginase therapy. The reductions in plasma levels during the use of oral contraceptives and pregnancy indicate involvement of sex steroids in the metabolism of tetranectin.
Platelet Adhesion to Subendothelial Structures under Flow Conditions: No Effect of the Lipoxygenase Product 13-HODE
Authors: de Graaf Jacques C , Bult Hidde , de Meyer Guido R Y , Sixma Jan J , de Groot Philip G
Published: June 2018

It has been shown that endothelial cells can convert linoleic acid to 13-hydroxyoctadecadienoic acid (13-HODE) and it has been suggested that 13-HODE has non-thrombogenic properties. However, no direct evidence has been presented that indicates that 13-HODE indeed modulates platelet-vessel wall interaction.

In this study we have bound a purified 13-HODE to a thrombogenic surface and its effect on platelet adhesion was studied and compared to the effects of an analogous hydroxy fatty acid, 15-hydroxyeicosatetraenoic acid (15-HETE). The effect of 13-HODE on platelet adhesion was studied both under static and flow conditions. In this report we show that binding of up to 40 times the physiological concentration to a thrombogenic surface has no inhibitory effect on platelet adhesion under static or flow conditions. We conclude that 13-HODE is not an important regulatory substance in platelet-subendothelium interaction, although this does not exclude it has a putative anti-adhesive role on intact endothelium.
Age Dependence of Blood Fibrinolytic Components and the Effects of Low-Dose Oral Contraceptives on Coagulation and Fibrinolysis in Teenagers
Authors: Siegbahn A , Ruusuvaara L
Published: June 2018

Basal t-PA antigen concentration, PAI-I activity and tibrinolytic capacity was studied in plasma from 20 healthy teenager girls (age 15.9 ± 1.3 years) and two groups of older healthy volunteers, consisting of L7 women (age 32 ± 8 years, group 1) and 35 men (age 34 ± 8 years, group 2). Basal t-PA antigen concentrations in plasma were found to be highly age-dependent with higher values with increasing age. The teenager girls had significantly lower values compared with the two groups of elderly volunteers. PAI-I levels were significantly higher in plasma from the teenager girls and the fibrinolytic capacity after 15 min of venous occlusion was significantly lower.

In this study we also determined the effect of low-dose oral contraceptives (OC) on coagulation and fibrinolysis in the teenager group. Each teenager served as her own control with samples drawn before and on OC after 4 months of use. The coagulation parameters, factor VIII activity, AT III, protein C and platelet counts were all within reference values before and on OC. The fibrinolytic activity in plasma after venous occlusion (15 min) increased significantly when the teenagers had used OC for 4 months. This phenomenon was explained by significantly decreased PAI levels and also by significantly increased t-PA antigen release from the vessel wall after venous occlusion.
Effect of Thromboxane Synthetase lnhibition on Platelet Function and Morphology During Ovine Pregnancy-Induced Hypertension
Authors: Miller Kevin W m , Keith James C
Published: June 2018

Arterial blood pressure, serum fibrir/fibrinogen degratory products, plasma thromboxane B2, in vitro platelet aggregation, and platelet ultrastructure were studied in ten gravid ewes during fast-triggered ovine pregnancy-induced hypertension and subsequent administration of the thromboxane synthetase inhibitors CGS13080 and CGS12970. During the hypertensive period, blood pressure (p <0.005) and plasma thromboxane B2 levels (p <0.005) were significantly altered. Collagen-induced in vitro platelet aggregation lag times increased (p <0.01), and percent aggregation (p <0.05), primary (p <0.01), and secondary (p <0.005) aggregatory slopes decreased. Collagen also failed to induce aggregation in some ewes. Primary slopes of ADP-induced in vitro platelet aggregation decreased (p <0.01) during hypertension. Degranulation and open canalicular tubule system swelling were observed in platelets which produced abnormal or no aggregation response. However, these ultrastructural abnormalities did not necessarily correspond to hypertensive periods.

Thromboxane synthetase inhibitor administration lowered blood pressure (p <0.005) and plasma thromboxane B2 levels (p <0.005). Abnormalities in collagen and ADP-induced platelet aggregation curves were also corrected, and ultrastructural abnormalities were not detected. Marked elevations in plasma thromboxane levels during ovine pregnancy-induced hypertension may have had an “exhaustive” effect on thrombocytes which was reversed by thromboxane synthetase inhibition.
Monitoring of Heparin and Low Molecular Weight Heparin with Capillary and Venous Whole Blood
Authors: Harenberg J , Giese Ch , Dempfle C E , Stehle G , Heene D L
Published: June 2018

A method for determination of antifactor Xa-like activity in capillary whole blood obtained from the fingertip is described, which employs the Heptest coagulation assay. Values obtained with capillary whole blood are compared to values of corresponding plasma and venous whole blood samples. Normal values in plasma, venous whole blood, and capillary blood from the fingertip were 17.1 ± 2.1, 10.0 ± 1.3 and 10.4 ± 1.3 sec, respectively. The intraindividual coefficients of variations range from 0.4 to 1.8% in all assays. The day to day coefficient of variation of normal values ranged between 0.8 and 2.0% for all assays. The within assay coefficients of variation ranged from 3.0 to 7.7% for whole blood samples and from 1.5 to 2.2% for plasma samples after addition of no, 0.2 or 1.0 units of normal or LMW heparin to the samples. After administration of heparin or LMW heparin in healthy persons the coagulation values of the different coagulation assay systems displayed coefficients of correlation betweell r = 0.87 and r = 0.95. Correlation coefficients between the coagulation tests and the S 2222 chromogenic substrate method ranged from r = 0.77 to r = 0.92. In patients, who received LMW heparin for prophylaxis of thromboembolism the coagulation assay correlated between r = 0.78 and 0.92. The coagulation assays and the S 2222 method displayed coefficients of correlation between r = 0.74 and r = 0.83. The data indicate that Heptest sensitively measures antifactor Xa-like activity in capillary whole blood as well as venous whole blood samples containing low quantities of heparin or LMW heparin.
Growth and Stability of Thrombi in Flowing Citrated Blood: Assessment of Platelet-Surface Interactions with Computer-Assisted Morphometry
Authors: Sakariassen Kjell S , Kuhn Herbert , Muggli Reto , Baumgartner Hans R
Published: June 2018

The differential quantitation of platelet deposition in perfusion
studies is a major problem. We report on methods to prepare semithin sections of platelet deposits on collagen coated on glass and plastic cover slips, to study growth and stability of thrombi in three dimensions, and the development of a computer-assisted differential quantitation of platelet-collagen interactions. The interactions were quantified as percentage of the surface covered with platelets (platelet adhesion), thrombus height, thrombus density and thrombus area per unit sectional length, respectively.

Cover slips coated with fibrillar equine collagen in parallelplate
perfusion chambers were exposed to flowing citrated blood at shear rates ranging from 200 to 2,600 s−1. Thrombi, partially
enmeshed in the collagen meshwork, prevailed on the surface at all shear rates. Maximal platelet adhesion and thrombus density were seen at >5 μg/cm2 collagen, while thrombus area and height were maximal at >10 μg/cm2. The volume of the thrombi appeared correlated to the number of deposited platelets (r = 0.92). En face preparations showed deposits of platelet islands
which grew in diameter with time, particularly in the direction of the blood flow, becoming progressively confluent. Sections cut parallel to the direction of the blood stream indicated that this growth pattern was at least partially caused by thrombi bent in the direction of the blood flow. This view is consistent with data from corresponding sections cut perpendicular to the direction of the blood flow showing that the initial thrombus growth at 2 min is isotropic, while anisotropic growth, characterized with decreased growth in height, is observed at 5 and 10 min.

Our three-dimensional analysis suggests that the growth occurs mainly in height, and that blood shear forces may bend the thrombi toward the surface resulting in platelet thrombi preferentially elongated in the direction of the blood flow.
Modulation of Heparin Cofactor II Function by S Protein (Vitronectin) and Formation of a Ternary S Protein-Thrombin-Heparin Cofactor II Complex
Authors: Preissner Klaus T , Sié Pierre
Published: June 2018

The complement inhibitor S protein, which is identical to the adhesive protein vitronectin, functions as heparin-neutralizing factor by protecting thrombin as well as factor Xa against fast inactivation by antithrombin III. The interference of S protein with glycosaminoglycan-catalyzed inhibition of thrombin by heparin cofactor II was investigated in these studies. S protein significantly counteracted the anticoagulant activity of heparin and pentosan polysulfate but not of dermatan sulfate. In the presence of 0.3 μg/ml heparin, 0.5 μg/ml pentosan polysulfate, or 2 μg/ml dermatan sulfate, S protein induced a concentrationdependent
reduction of the inhibition rate of thrombin by heparin cofactor II. This resulted in a decrease of the apparent pseudo first-order rate constants by about 17-fold (heparin), or about 7-fold (pentosan polysulfate), whereas no neutralization of dermatan
sulfate was demonstrable at a physiological ratio of S protein to heparin cofactor II. Exposure of the glycosaminoglycan-binding region of S protein by reduction and carboxymethylation of the protein increased the neutralizing activity of S protein towards heparin and pentosan polysulfate. The results of these functional experiments correlated well with the demonstration of direct binding of S protein to both polysaccharides but not to
dermatan sulfate. While reduced/carboxymethylated S protein remained also ineffective in neutralizing other dermatan sulfate compounds with varying degree of sulfation, a synthetic highly basic tridecapeptide, representing a portion of the glycosaminoglycan-binding domain of S protein, counteracted their anticoagulant activity. Independent on the polysaccharide used, S protein was found incorporated within a ternary complex with thrombin and heparin cofactor II during the inhibition reaction as judged by crossed immunoelectrophoresis, ultracentrifugation as well as ELISA analysis, emphazising the function of S protein as scavenger protein for enzyme-inhibitor complexes of the coagulation system. These findings demonstrate the role of S protein as effective neutralising plasma protein of the anticoagulant activity of various glycosaminoglycans also with respect to heparin cofactor II. Although the glycosaminoglycan-binding domain of S protein readily neutralized different dermatan sulfate compounds, physiological modulation of heparin cofactor-II-dependent
inhibition of thrombin by native S protein appears to be restricted to the vascular compartments, where other glycosaminoglycans than dermatan sulfate appear to be operative.
Modified Crossed Immunoelectrophoresis to Study with Whole Plasma the Reversible Complex Formation of Histidine-Rich Glycoprotein with Plasminogen
Authors: Kluft C , Los P
Published: June 2018

To study the reversible complex formation between the plasma protein histidine-rich glycoprotein (HRG) and plasminogen, crossed immunoelectrophoresis of HRG was modified. In the modification, purified plasminogen was introduced into the gel of the first dimension electrophoresis.

Two molecular forms of plasminogen, Glu- and Lys-plasmino-gen, induced a dose-dependent reduction of the electrophoretic mobility of HRG, with a half maximal retardation for both plasminogens at 0.50–0.55 μM of added plasminogen to the agarose gel. HRG in plasma behaved as a uniform fraction with respect to plasminogen binding. In contrast, with the same modified technique another plasma protein, β2-antiplasmin, separated into a retarded plasminogen-binding form and a nonretarded non-plasminogen-binding form.

The method can be used to assess several aspects of reversible complex formation between plasma proteins, as demonstrated for plasminogen binding of HRG and α2-antiplasmin in whole plasma.
A Monoclonal Antibody-Based Qunatitative Enzyme Immunoassay for the Determination of Plasma Fibrinogen Concentartions
Authors: Hoegee-de Nobel E , Voskuilen M , Briët E , Brommer E J P , Nieuwenhuizen W
Published: June 2018

The most commonly used fibrinogen assays in the clinic are clotting rate assays, e.g. the Clauss method. Such functional assays may be disturbed by e.g. heparin, anticoagulant fibrinogen degradation products (FgDP) and in the case of a dysfi-brinogenemia. Immunological methods would not suffer from these interferences. However, immunological assays for fibrinogen, which do not measure FgDPs, do not exist.

To set up such an enzyme immunoassay (EIA) we developed two monoclonal antibodies. The first monoclonal antibody (G8) has its epitope in the carboxyl-terminal 150 amino acid stretches of the fibrinogen Aa-chains. G8 is used to coat the wells of microtitration plates, and is the capture antibody in this EIA. The second antibody (Y18) has been described by us previously (Blood 1985; 66: 503). It is directed against fibrinopeptide A, covalently bound to the ±-chains i.e. against the amino-terminal stretches of the A±-chains. Y18 is conjugated with horse-radish peroxidase, and used as tagging antibody.

The EIA does not react with, and is not interfered by FgDP such as purified fragments X and Y, up to a concentration of 800 μg/ml. An FgDP mixture such as generated by Streptokinase treatment of plasma does not respond. Fibrin degradation products (whole blood lysate) up to 800 μg/ml do not interfere nor do heparin, EDTA or oxalate. The time-to-result of the EIA is only 45 minutes. Some patient plasmas yielded dose-response curves which are not parallel with the calibration curve of the EIA. An explanation for this phenomenon could not be given. Our fibrinogen EIA may be especially suitable to monitor patients with conditions which favour proteolytic damage to fibrinogen such as thrombolytic therapies.
Characterization of the Binding of Bovine Thrombin to Isolated Rat Hepatocytes
Authors: Weyer Britta , Petersen Torben E , Sonne Ole
Published: June 2018

Isolated rat hepatocytes possess per cell 4,500 high-affinity binding sites for thrombin with a Kd of 30-40 pM, and 2.8 × 105 low-affinity sites with a Kd of 30 nM. These binding sites are highly specific for thrombin. Half-maximal binding of 125I-labelled thrombin is achieved after 3 min at 37¸ C and 7 min at 4¸ C. The reversibly bound fraction of the ligand dissociates according to a biexponential time course with the rate constants 1—2 × 10−2 s−1 and 3—4 × 10−4 s−1. Part of the tracer remains cell-associated even after prolonged incubation, but all cell-associated radioactivity migrates as intact thrombin upon sodium dodecyl sulphate polyacrylamide gel electrophoresis. The bound thrombin is minimally endocytosed as judged by the resistance to pH 3-treatment. Cell-associated radioactivity dissociated from the cells binds just aswell in a receptor assay as tracer incubated in a conditioned medium under the same conditions, indicating the absence of a quantitatively important receptor-mediated degradation ofthe ligand.
Fibrin Metabolism in Patients with Acute Myocardial Infarction During and After Treatment with Tissue-Type Plasminogen Activator
Authors: Ring Michael E , Butman Samuel M , Bruck Denise C , Feinberg William M , Corrigan James J
Published: June 2018

In order to define some of the determinants of successful thrombolysis and reocclusion during fibrinolytic therapy for acute myocardial infarction (AMI), specific molecular markers of fibrin metabolism were serially measured in 15 patients with AMI treated with tissue-type plasminogen activator (t-PA). Fibrin formation was assessed by measurement of fibrinopeptide A (FpA) and fibrinolysis by assay of B-P peptides 1—42 and 15—42 and crosslinked fibrin degradation products (XDP). At baseline, FpA levels were high while markers of fibrinolysis were near normal. Following a 90-minute infusion of t-PA (0.5—1.1 mg kg−1 hr−1), all markers of fibrinolysis increased. Levels of FpA remained elevated despite heparin at the initiation of cardiac catheterization. None of these markers discriminated between patients with successful reperfusion from those without. At 4 hours, B-β 15—42 peptide and XDP levels remained elevated suggesting persistence of fibrinolysis beyond the short circulatory half-life of t-PA. FpA levels at 4 hours were lower in patients who underwent acute coronary angioplasty compared to those who received additional low dose t-PA (12.3 ± 4.5 vs. 30.4 ± 5.5 ng/ ml, p <0.05). By 48 hours, markers of fibrinolysis had returned toward normal except in 2 patients with persistently elevated B-P 15—42 peptide levels who suffered reocclusion on days 5 and 6 (75 and 44 vs. 29 ± 3 nM, p <0.005). In conclusion, molecular markers of fibrin metabolism during fibrinolytic therapy may provide clinically relevant data.
Commercial Immunodepleted Deficient Plasmas Contain Cleaved High Molecular Weight Kininogen (HK)
Authors: Mannhalter C , Lang H
Published: June 2018

Comparative analysis of high molecular weight kininogen (HK) in various commercial congenital and immunodepleted deficiency plasmas was performed by immunoblotting of HK. It was found, that some artificially depleted deficiency plasmas contained proteolytically cleaved, kinin-free kininogen. In contrast, in all congenitally deficient plasmas, HK was present in the intact, single chain form. Thus, cleavage of kininogen could have been triggered by or during the immunodepletion procedure. It was seen, that the degree of proteolytic cleavage and degradation of HK in depleted plasmas differed among various manufacturers. E.g. depleted products of one company contained only trace amounts of cleaved HK, in contrast to products of another one, in which HK was completely degraded. The immunoblot analysis of HK reflects the occurrence of proteolytic events during the production of artificially deficient plasmas and can therefore serve as a quality control method.
Clot Lysis Mediated by Cultured Human Microvascular Endothelial Cells
Authors: Speiser Wolfgang , Anders Elisabeth , Binder Bernd R , Müller-Berghaus Gert
Published: June 2018

The lysis of fibrin clots on the surface of cultured human omental tissue microvascular endothelial cells (HOTMEC) and cultured human umbilical vein endothelial cells (HUVEC) was studied. Fibrin clots were made by mixing fibrinogen, plasminogen and thrombin on the surface of both cell types. Clot lysis was seen only on the surface of HOTMEC, which were found to synthesize about 100-fold more tissue plasminogen activator (tPA) antigen than HUVEC. Clot lysis of HOTMEC could be blocked by anti-tPA IgG but was not affected by the incorporation of exogenous plasminogen activator (PAI) into the clot in concentrations (75 arbitrary units) exceeding the tPA activity (21 ± 2.5 IU) of the cells. Thus, it is likely that tPA secreted by HOTMEC is protected from inhibition by PAI in the presence of fibrin and endothelial cells. The stimulation of EC to release an excess of tPA over PAI, in contrast to the secretion of an excess of PAI over tPA found in unstimulated cells in the absence of fibrin, is obviously no prerequisite for the initiation of fibrinolysis on the surface of HOTMEC. As thrombin was used for clot formation, its influence on tPA and PAI synthesis of both cell types was investigated. In contrast to HOTMEC, which were not affected by Α-thrombin, HUVEC revealed a dose-dependent increase in tPA and PAI synthesis upon incubation with the enzyme. This increase in tPA production by HUVEC was not sufficient to lyse the clots within 48 hours. Furthermore, HUVEC. behaved differently towards thrombin as these cells in contrast to HOTMEC revealed the typical shape change reaction upon incubation with the enzyme
Tissue-Type Plasminogen Activator in Patients with Intracranial Meningiomas
Authors: Tsuda Hiroko , Oka Kazunari , Noutsuka Youko , Sueishi Katsuo
Published: June 2018

Three of 13 patients with intracranial meningiomas showed the pre- and postoperative elevation of tissue-type plasminogen activator (t-PA) related fibrinolytic activity in euglobulin fractions (EFA). During operation, two of these three patients showed a significant elevation of the level of fibrin(ogen) degradation products and oozing in the operating field. However, oozing was not observed in the third patient who had been given tranexamic acid preoperatively. Fibrin autography revealed that a broad lytic band of mol wt 50–60 kDa, probably free t-PA, appeared in the plasma obtained from two of the three patients after operation when EFA elevated significantly. In all patients studied, the t-PA antigen levels were normal preoperatively but increased both during and after operation, and correlated mainly with the intensities of a lytic band of mol wt 110 kDa, probably t-PA complexed with its major inhibitor (PAI–1). These results suggest that the excessive fibrinolysis can induce the local hemorrhagic diathesis during operation and may be related to t-PA function in plasma
Antithrombin III Molecular Variants with Defective Binding to Heparin or to Serine Proteases: Evidence of Two Different Abnormal Patterns Identified by Crossed Immunoelectrofocusing
Authors: Leone G , Stefano V De , Ferrelli R , Teofili L , Tengborn L , Vahtera E , Bizzi B
Published: June 2018

Molecular heterogeneity of antithrombin III (AT III) was investigated by a technique of crossed immunoelectrofocusing (CIEF) in plasma samples of patients from 16 families with AT III congenital defect, including 8 AT III molecular variants. The AT III CIEF pattern was normal in all the patients with AT III quantitative deficiency, showing a balanced decrease of all the peaks. Out of the 8 AT III variants investigated, 6 had an abnormal pattern: the three variants with defective binding to heparin (AT III Roma, AT III Barcelona, AT III Malmö) shared a similar abnormal pattern; three variants with defective binding to serine proteases (AT III Pescara, AT III Milano, AT III Tampere) had a common abnormal pattern clearly different from the first one, whereas the other two variants deficient in the inactivation of the serine proteases (AT III Chicago, AT III Milano 2) showed a normal pattern. The first type of pathological pattern (type Roma) was characterized by the presence of an abnormal peak overlapping the normal isoforms present at pH 4.8-4.6 and by an additional peak at pH 4.5. The second type of pattern (type Pescara) showed an additional peak at pH 4.5 and an abnormal quantitative distribution of the isoantithrombins all throughout the pH range (5.2-4.6). In order to separate the abnormal AT III fraction from the normal one, plasma of a patient with Roma defect and serum of a patient with Pescara defect were passed throughout an heparin-ultrogel column. The CIEF of the different AT III Roma and AT III Pescara fractions obtained by affinity chromatography confirmed that the abnormalities found in the corresponding patterns of the native samples were related to the pathological isoantithrombins only. It was concluded that the CIEF can be an useful tool to characterize abnormal antithrombins and can reveal close affinities among AT III molecular variants belonging to different subgroups according to the conventional tests.
Further Evidence of von Willebrand Factor Involvement in Thrombotic Thrombocytopenia Purpura
Authors: Chediak J , Eldridge J , Bergmann F , Sobel D , Baron J , Maxey B , Telfer M C
Published: June 2018

Fourteen patients diagnosed as having thrombotic thrombocytopenia purpura (TTP) were studied. Those who survived have been followed during a 1 to 7 year period. The clinical diagnosis was based on changing neurological findings, thrombocytopenia and evidence of microangiopathic hemolytic anemia. Laboratory tests included the determination of von Willebrand factor antigen (VWF:Ag), ristocetin cofactor (RiCof) and the electrophoretic mobility of von Willebrand factor (CIE VWF:Ag). The ratio of RiCof to VWF:Ag was then calculated. Control individuals included healthy subjects and patients with thrombocytopenia of several etiologies. Statistical differences between the values of RiCof, the ratio of RiCof : VWF: Ag and the CIE of VWF: Ag were found for samples comparing active disease and remission phase. The recovery from thrombocytopenia paralleled the correction of abnormal parameters. Similarly, significant differences were found when above parameters were compared between thrombocytopenia of TTP with other thrombocytopenic states. We suggest that these abnormal tests could be useful in distinguishing TTP from other disorders, and may have prognostic significance in patients already diagnosed as having TTP.
A Simple Nomogram for the Derivation of International Normalised Ratios for the Standardisation of Prothrombin Times
Authors: Poller L
Published: June 2018

The WHO international scale of reporting prothrombin time results is based on the calibration of local and commercial thromboplastin reagents used in the test against an international reference preparation to derive an International Sensitivity Index (ISI) for each batch of local reagent. This quantifies an individual reagent’s responsiveness to the coumarin induced defect.

Once the ISI has been assigned, the derivation of International Normalised Ratios (INR) for reporting results depends on the use of a scientific calculator by which the local ratio is raised to the power of the ISI of the local reagent or use of an alternative formula and logarithms.

A system for interpretation of INR, without the need for calculations, is presented for the known range of thromboplastins between 1.0 and 3.0 ISI in nomogram form. This chart may facilitate the general application of the INR system.