bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Bupranolol, a New β-Blocker, on Platelet Functions of Rabbit and Human in Vitro
Authors: Umetsu Teruhiko , Sanai Kazuko , Kato Tadakatsu
Published: July 2018

The effects of bupranolol, a new β-blocker, on platelet functions were investigated in vitro in rabbits and humans as compared with propranolol, a well-known β-blocker. At first, the effect of adrenaline on ADP-induced rabbit platelet aggregation was studied because adrenaline alone induces little or no aggregation of rabbit platelets. Enhancement of ADP-induced rabbit platelet aggregation by adrenaline was confirmed, as previously reported by Sinakos and Caen (1967). In addition the degree of the enhancement was proved to be markedly affected by the concentration of ADP and to increase with decreasing concentration of ADP, although the maximum aggregation (percent) was decreased.

Bupranolol and propranolol inhibited the (adrenaline-ADP-)induced aggregation of rabbit platelets, bupranolol being approximately 2.4–3.2 times as effective as propranolol. Bupranolol stimulated the disaggregation of platelet aggregates induced by a combination of adrenaline and ADP, but propranolol did not. Platelet adhesion in rabbit was also inhibited by the β-blockers and bupranolol was more active than propranolol. With human platelets, aggregation induced by adrenaline was inhibited by bupranolol about 2.8–3.3 times as effectively as propranolol.

From these findings. We would suggest that bupranolol might be useful for prevention or treatment of thrombosis.
Inhibition of ADP-Induced Platelet Aggregation by Adenosine Tetraphosphate
Authors: Harrison Margaret J. , Brossmer R
Published: July 2018

In contrast to previous reports, highly purified adenosine tetraphosphate (AP4) does not induce the aggregation of platelets but inhibits the aggregation and release reaction in platelet-rich plasma promoted by ADP. The inhibitory action of AP4 on the aggregation by ADP is compared with that of AMP and ATP. The data presented suggest a competitive manner of inhibition of the ADP-induced aggregation by AP4.
Enhancement of Fibrinolytic and Thrombolytic Potential in the Rat by Treatment with an Anabolic Steroid, Furazabol
Authors: Kumada Toshihiko , Abiko Yasushi
Published: July 2018

The effect of long-term ingestion of an anabolic steroid, furazaboi, was studied on coagulo-fibrinolytic systems in the rat.

During the administration of furazabol at the daily dose of 0.04, 0.2 or 1 mg/rat for 3 months, the most remarkable changes were increase in the plasminogen activator activity in blood and the lung tissue and decrease in plasma fibrinogen level as well as decrease in plasma cholesterol. It was a very important finding that in most of the rats the furazabol treatment was effective in reducing susceptibility to lactic acidosis-induced pulmonary thrombosis. No meaningful changes were observed in other parameters tested including ADP-induced platelet aggregability, plasma recalcification time, plasma plasminogen, plasma antiplasmin activity, plasminogen activator content of tissues other than the lungs and the release of vascular activator induced by venous occlusion. One month after cessation of the furazabol treatment, these altered parameters tended to return to normal.

Independently on the furazabol treatment, highly significant positive correlation existed between the plasma activator activity and the pulmonary tissue activator content. This indicated that the major source of plasma activator in the rat was the lung tissue and that the furazabol treatment increased the circulating activator activity through enhancing activator content in the lungs.
Inactivation of α- and β-Thrombin by Antithrombin-III and Heparin
Authors: Machovich Raymund , Blaskó György , Borsodi Anna
Published: July 2018

Inactivation of α- and β-thrombin by antithrombin-III and heparin was studied, since it had been suggested that two forms of thrombin exist with respect to heparin sensitivity (Machovich 1975b).

It was found that the inactivation rates of α- and β-thrombin by antithrombin were different, namely α-thrombin was more sensitive to antithrombin than β-thrombin. Heparin facilitated the complex formation between α-thrombin and antithrombin-III, whereas β-thrombin inactivation was only slightly affected.

Furthermore, heparin protected α-thrombin against the inactivating effect of heat, while β-thrombin lost its activity during the heat treatment.

These findings suggest that the formation of β-thrombin in blood circulation may have an important role in thrombosis predisposition.
Demonstration of Reactivity to Airborne and Food Allergens in Cutaneous Vasculitis by Variations In Fibrinopeptide A and Other Blood Coagulation, Fibrinolysis and Complement Parameters
Authors: Theorell Henning , Blombäck Margareta , Kockum Carolina
Published: July 2018

In a 32-year-old woman and a 40-year-old man with cutaneous vasculitis, etiological allergic responses to foods and airborne allergens were found. During provocation tests, observations were made on blood levels of fibrinopeptide A( FPA) and coagulation factors, fibrinogen degradation products (FDP) and serum complement components. Skin biopsies were taken for microscopic and immunofluorescence analysis.

In case 1, anaphylactoid allergy to milk and reaginic and anaphylactoid hypersensitivity to grass pollens were found. Dermal provocations with grass pollens gave arthralgia, hematomas, serum C3 fluctuation, factor VII reduction and fibrinolysis. During peroral milk challenge, transient increases in FPA and FDP levels were observed before symptoms appeared.

In case 2, anaphylactoid hypersensitivity responses to bacteria, animal danders, foods and pollens were found. Two inhalations with sheep-wool extract resulted in a typical skin eruption. The first also gave an early reduction of C3 and then FPA liberation. Nasal birch-pollen test gave an increase of FPA in the latent period and then typical nodules. At least no low molecular weight FDP were detected during provocations.

In patients with vasculitis reactions to exogenous allergens, FPA and FDP estimations after provocations may discriminate harmful from innocuous allergens and reveal individual response patterns in coagulation and fibrinolysis systems.
Double-Blind, Randomized Comparison of Systemic Continuous Infusion of 0.25 Versus 0.50 mg/kg/24 h of Alteplase over 3 to 7 Days for Treatment of Deep Venous Thrombosis in Heparinized Patients: Results of the European Thrombolysis with rt-PA in Venous Thrombosis (ETTT) Trial
Authors: Bounameaux H , Banga J D , Bluhmki E , Coccheri S , Fiessinger J N , Haarmann W , Lockner D , Mahler F , Ninet J , Schneider P A , de Torrente A , van der Meer J , Verhaeghe and R
Published: July 2018

Thirty-two patients with acute, proximal-vein thrombosis were treated with heparin and alteplase (0.25 versus 0.5 mg/kg/24 h during 3-7 days) in a randomized, double-blind, multicenter, European (ETTT) trial. The treatment resulted in a decrease of the venographic Marder’s score from 18 (6-25) to 13 (2-24) units (median, range) in Group I (0.25 mg/kg/24 h, n = 15, median decrease 3.0, p = 0.32) and from 17.5 (3-33) to 15.5 (0-27) in Group II (0.5 mg/kg/24 h, n = 16, median decrease 4.0, p = 0.23). Comparison of the sequential venograms could be performed in 14 cases of Group I and in 15 cases in Group II. A minority of patients showed substantial partial recanalization of the initially obstructed veins on the control venogram (one in each treatment group) and most of the control venograms showed

Thus, the results of the ETTT trial show that the used low dosages of alteplase administered intravenously over 3-7 days in heparinized patients cannot be recommended as a treatment for patients with deep venous thrombosis of lower limbs and/or pelvis. Further studies are needed to define a more suitable dosage regimen of alteplase in this indication.
In Vitro Inhibition of Blood Coagulation by Tripeptide Aldehydes - A Retrospective Screening Study Focused on the Stable D-MePhe-Pro-Arg-H · H2SO4
Authors: Bágdy Daniel , Barabás Èva , Bajusz Sándor , Széll Erzsébet
Published: July 2018

A series of peptide aldehydes synthetized in our institute during the last 15 years were screened to detect their inhibitory effect on blood coagulation. Simple conventional clotting assays, platelet function tests and fibrinolytic methods were used to evaluate the inhibitory potency of the compounds in complex clotting systems as well as their supposed antifibrinolytic effect in vitro. Special attention was paid to the possible interactions with blood cells and plasma proteins, and to the functional stability of the inhibitors in several tissue homogenates. D-Phe-Pro-Arg-H (GYKI-14166, RGH-2958), Boc-D-Phe-Pro-Arg-H (GYKI-14451) and D-MePhe-Pro-Arg-H (GYKI-14766) were found to be the most potent inhibitors. The peptide aldehydes via formation of reversible complexes with thrombin impede the enzyme to react with the coagulation factors, platelet membrane and vessel wall. The compounds inhibit platelet aggregation induced by thrombin specifically without changing the sensitivity of platelets to other inducers. D-Phe-Pro-Arg-H and D-MePhe-Pro-Arg-H showed no antifibrinolytic effect. D-MePhe-Pro-Arg-H and Boc-D-Phe-Pro-Arg-H proved to be stable in dry state for years and in solution at room temperature for several days. The anticoagulant activity of the compounds was declared in NIH antithrombin units.
Availability of the Bβ(15-21) Epitope on Cross-Linked Human Fibrin and Its Plasmic Degradation Products
Authors: Chen Fabian , Haber Edgar , Matsueda Gray R
Published: July 2018

The binding of radiolabeled monoclonal antifibrin antibody 59D8 (specific for fibrin but not fibrinogen) to a series of degraded fibrin clots showed that the availability of the Bβ(15-21) epitope (against which 59D8 had been raised) was inversely proportional to the extent of clot lysis. Examination of digest supernatants revealed that the Bβ(15-21) epitope was released from clots as a high molecular weight degradation product in the presence of calcium ions but that the generation of low molecular weight peptides occurred in the absence of calcium ions. To address the question of epitope accessibility, we compared levels of fibrin clot binding among four radioactively labeled antibodies: antifibrin monoclonal antibody 59D8, two antifibrinogen monoclonal antibodies that cross-reacted with fibrin, and an affinity-purified polyclonal antifibrinogen antibody. We expected that the antifibrinogen antibodies would show enhanced binding to clots in comparison with the antifibrin antibody. However, the epitope accessibility experiments showed that all four antibody preparations bound fibrin clots at comparable levels. Taken together, these studies demonstrated that one fibrin-specific epitope, Bβ(15-21), remains available on clots as they undergo degradation by plasmin and, importantly, that the epitope is not solubilized at a rate faster than the rate at which the clot is itself solubilized. The availability of the Bβ(15-21) epitope during the course of plasminolysis assures the potential utility of antifibrin antibodies such as 59D8 for detecting thrombi and targeting plasminogen activators.
Toward Gene Therapy in Haemophilia A: Retrovirus-Mediated Transfer of a Factor VIII Gene into Murine Haematopoietic Progenitor Cells
Authors: Hoeben R C , Einerhand M P W , Briët E , van Ormondt H , Valerio D , van der Eb A J
Published: July 2018

To study and evaluate the potential of the haematopoietic system as a target for gene therapy in haemophilia A, we have infected murine bone-marrow cells with a recombinant retrovirus encoding blood-coagulation factor VIII and the bacterial enzyme neomycin-phosphotransferase. After transplantation of the infected bone marrow into lethally irradiated mice, the presence of intact vector could be demonstrated in DNA isolated from individual haematopoietic progenitor-cell-derived spleen colonies. About 8% of the spleen colonies were shown to contain the intact vector. Selection for resistance to the neomycin analogue G418 prior to transplantation specifically killed the uninfected bone-marrow cells and, as a result, over 90% of the spleen colonies contained the factor VIII vector. However, expression of factor VIII in vivo, either at the RNA or at the protein level could not be demonstrated. From these data we conclude that: 1) retroviral vectors can be used to transfer factor-VIII cDNA into haematopoietic progenitor cells; 2) the vector sequences are expressed immediately after integration; and 3) transcription of the vector is repressed in the progenitor-cell-derived cells.
Biochemical, Thrombolytic and Pharmacokinetic Properties of rt-PA P47G, K49N, a Substitution Variant of Human Tissue-Type Plasminogen Activator
Authors: Nelles L , Li X-K , Vanlinthout I , De Cock F , Lijnen H R , Collen D
Published: July 2018

rt-PA P47G, K49N, a substitution variant of recombinant human tissue-type plasminogen activator (rt-PA), in which proline at position 47 and lysine at position 49 were replaced by glycine and asparagine respectively, was previously described by Ahem et al. (J Biol Chem 1990; 265: 5540—5) to have an extended in vivo half-life with unaltered in vitro fibrinolytic properties. Because this variant might possess an increased in vivo thrombolytic potency, we have constructed its cDNA, expressed it in Chinese hamster ovary cells and determined its biochemical, thrombolytic and pharmacokinetic properties relative to those of home-made rt-PA and of alteplase (Actilyse®).

The specific fibrinolytic activities on fibrin plates were 160,000 ± 17,000, 210,000 ± 88,000 and 460,000 ± 72,000 IU/mg (mean ± SEM) for rt-PA P47G, K49N, rt-PA and alteplase, respectively, while the catalytic efficiencies for plasminogen activation (k
2
K
m) in the absence of fibrin were comparable (1.1 to 1.7 × 10-3 μM-1s-1). Fibrin enhanced the rate of plasminogen activation by rt-PA P47G, K49N 100-fold and by both wild-type molecules 390-fold. Binding of the variant rt-PA to fibrin was significantly reduced, but its affinity for lysine-Sepharose was unaltered. In an in vitro clot lysis system, consisting of a radiolabeled human plasma clot submersed in plasma, 50% clot lysis in 2 h required 0.67 ± 0.14 pg/ml rt-PA P47G, K49N, 0.36 ± 0.01 pg/ml rt-PA and 0.17 ± 0.01 pg/ml alteplase, respectively (mean ± SEM; n = 3 or 4). At these doses residual fibrinogen levels at 2 h were in excess of 80%.

The thrombolytic properties were examined in a hamster pulmonary embolism model. The thrombolytic potency, expressed as percent lysis per mg activator administered per kg body weight, was 160 ± 28 for rt-PA P47G, K49N, 150 ± 36 for wild-type rt-PA and 310 ± 42 for alteplase. The specific thrombolytic activity, expressed as percent lysis per pg steady-state t-PA-related antigen level per ml plasma, was 49 ± 18% for rt-PA P47G, K49N, 160 ± 49 for rt-PA and 500 ± 79 for alteplase. The clearance rates following bolus injection in hamsters were 0.18 ± 0.02, 1.9 ± 0.2 and 2.1 ± 0.1 ml/min respectively.

Thus, the substitution of only two residues in wild-type rt-PA markedly reduces its clearance, but it also significantly alters its specific thrombolytic activity, resulting in a virtually unaltered thrombolytic potency.
Lack of Stability of Aggregates after Thrombin-Induced Reaggregation of Thrombin-Degranulated Platelets
Authors: Kinlough-Rathbone Raelene L , Packham Marian A , Perry Dennis W , Mustard J Fraser , Cattaneo Marco
Published: July 2018

The stability of platelet aggregates is influenced by the extent of the release of granule contents; if release is extensive and aggregation is prolonged, deaggregation is difficult to achieve. The relative importance of the contributions of released substances to aggregate stability are not known, although stable thrombin-induced aggregates form in platelet-rich plasma from patients with barely detectable plasma or platelet fibrinogen, and ADP stabilizes thrombin-induced aggregates of platelets from patients with delta storage pool deficiency which otherwise deaggregate more readily than normal platelets. We degranulated platelets with thrombin (0.9 U/ml caused greater than 90% loss of delta and alpha granule contents) and recovered them as individual platelets in fresh medium. The degranulated platelets were reaggregated by thrombin (2 U/ml). To prevent continuing effects of thrombin, FPRCH2C1 was added when thrombin-induced aggregation of thrombin-degranulated platelets reached its maximum. EDTA (5 mM) or EGTA (5 mM) added at maximum aggregation did not deaggregate these platelets, indicating that the stability of these aggregates does not depend on Ca2+ in the medium. Whereas with control platelets a combination of PGE1 (10 μM) and chymotrypsin(10 U/ml) was required for deaggregation, with thrombin-degranulated platelets either PGE1 or chymo-trypsin alone caused extensive deaggregation. The rate and extent of deaggregation of thrombin-degranulated platelets by a combination of PGE1 and chymotrypsin was greater than with control platelets.

Electron microscope gold immunocytochemistry using antihuman fibrinogen IgG, anti-von Willebrand factor and anti-fibronectin showed a) that fibrinogen in the vacuoles of degranulated platelets was visible at focal points of platelet contact in the aggregates, but that large areas of platelet contact had no fibrinogen detectable between them; and b) in comparison to fibrinogen, little fibronectin or von Willebrand factor (vWf) was detectable in the platelets.

Since the linkages between thrombin-degranulated platelets reaggregated by thrombin can be disrupted either by raising cAMP (thus making glycoprotein IIb/IIIa unavailable) or by proteolysis, these linkages are less stable than those formed between normal platelets. It might therefore be expected that platelets that take part in thrombus formation and then recirculate are likely to form less stable thrombi than platelets that have not released their granule contents.
ADP Receptor Antagonists and Converting Enzyme Systems Reduce Platelet Deposition onto Collagen
Authors: Wagner William R , Hubbell Jeffrey A
Published: July 2018

The effect of the ADP receptor antagonists ATP and adenosine 5’-(β, γ-methylene)triphosphate (AMP-PCP), and the ADP-utilizing enzyme systems creatine phosphokinase/creatine phosphate (CPK/CP) and pyruvate kinase/phosphoenol pyruvate (PK/PEP) on platelet deposition onto type I collagen was examined. An in vitro perfusion system was used, which allowed continuous visualization of the deposition of fluorescently labelled platelets. This system also provided well-controlled rheology, precise quantification of deposition, and allowed the use of heparinized whole human blood (3 u/ml). Heparinization at this level permits the local generation of thrombin near surface platelet aggregates. The contribution of ADP is thus studied with the combined effects of thrombin, thromboxane A2, and other aggregating agents present. Results from these studies indicate that ATP was capable of inhibiting deposition by 60% at 1 pM and 90% at 5 μM (whole blood cone.). AMP-PCP inhibited deposition in a dose dependent manner with a Ki
of approximately 80 μM and a maximum inhibition of 60%. Inhibition by CPK/CP was measured at 20, 40, and 60 u/ml, with approximately 45% inhibition achieved for the latter two concentrations. PK/ PEP at 60 u/ml resulted in 70% inhibition. These results support a role for ADP in mediating platelet recruitment in thrombus growth on collagen. Previous work utilizing animal bleeding times supports this conclusion; the present study demonstrates that this role is not dependent upon endothelial or vasoconstrictive effects. Intraplatelet cAMP levels were raised with respect to controls upon exposure to ATP at 8.3 μM (p <0.025), and 15 μM (p <0.005), as well as AMP-PCP at 42-500 μM (p <0.005). Since increasing intraplatelet cAMP may affect platelet function, these results raise another possible interpretation for some of our platelet deposition data as well as that of previous experimenters. However, since ATP at 1 μM was not shown to affect intraplatelet cAMP, it is believed that at least part of the ATP-mediated inhibition was due to ADP receptor competition.
Induction of Tissue Factor Synthesis in Human Umbilical Vein Endothelial Cells Involves Protein Kinase C
Authors: Pettersen Kjell Sverre , Wiiger Merete Thune , Narahara Nobuhiro , Andoh Kiyoshi , Gaudernack Gustav , Prydz Hans
Published: July 2018

Incubation of human umbilical vein endothelial cells with one of the following compounds: endotoxin, recombinant interleukin-1β, recombinant tumor necrosis factor α, allogenic lymphocyte subpopulations or phorbol ester resulted in significant induction of tissue factor synthesis. Diacylglycerol had the same effect and also enhanced synergistically the induction caused by endotoxin and interleukin-1β. Two different inhibitors of protein kinase C, H7 and sphingosine, inhibited tissue factor synthesis at concentrations which did not depress protein synthesis in general, suggesting that protein kinase C is involved in the processes leading to tissue factor synthesis. Cells down-regulated for the tissue factor response to TPA responded essentially normally to endotoxin and interleukin-1 with regard to tissue factor synthesis.
On the Relationship Between Molecular Mass and Anticoagulant Activity in a Low Molecular Weight Heparin (Enoxaparin)
Authors: Bendetowicz Ana-Victoria , Pacaud Elisabeth , Béguin Suzette , Uzan André , Hemker H Coenraad
Published: July 2018

A low molecular weight heparin (enoxaparin, mean molecular weight ~ 4,400) was separated by gel chromatography into eight different fractions with a narrow distribution around the following mean molecular weights: 1,800, 2,400, 2,900, 4,200, 6,200, 8,600, 9,800 and 11,000. We compared the influence of enoxaparin on the generation of thrombin in plasma to that of the eight fractions.

We determined: a) the % of material with high affinity to antithrombin III (HAM) and the % of HAM above the critical chainlength necessary to allow for thrombin inhibition (ACLM), b) the specific catalytic activity on the decay of endogenous thrombin, and c) the inhibition of over-all thrombin formation in the extrinsic and the intrinsic pathway. From b and c we calculated the inhibition of prothrombin conversion in these pathways.

We found that a) there is a gradual decrease of the HAM fraction with decreasing molecular weight; b) the specific catalytic activity for the inactivation of thrombin does not vary significantly between the fractions when expressed in terms of ACLM; c) the potency to inhibit prothrombin conversion does not vary significantly between the fractions when expressed in terms of HAM.
Effects of Splenectomy on Immune Thrombocytopenic Purpura in (NZW x BXSB) F1 Mice: Analyses of Platelet Kinetics and Anti-Platelet Antibody Production
Authors: Mizutani Hajime , Furubayashi Takayasu , Kashiwagi Hirokazu , Honda Shigenori , Take Hironori , Kurata Yoshiyuki , Yonezawa Takeshi , Tarui Seiichiro , Ikehara Susumu
Published: July 2018

Effects of splenectomy on platelet kinetics and production of anti-platelet antibodies were studied in male (NZW × BXSB) F1 (W/B F1) mice, which are known as the animal model of immune thrombocytopenic purpura (ITP). Studies on organ localization of radiolabeled platelets revealed that splenic uptake significantly increases in W/B F1 mice in comparison with that of normal controls. W/B Fj mice showed a significant increase in platelet counts and, in contrast with sham-operated controls, high levels of platelet counts were maintained up to 6 weeks after splenectomy. Platelet lifespans (PLSs) did not reach normal levels, although prolonged PLSs were observed. In addition, platelet-associated antibody (PAA) values showed a tendency towards transient decrease, but there was no change in platelet-bindable serum antibodies (PBAs). These findings indicate that the suppression of anti-platelet antibody production is essential to the treatment of ITP; splenectomy may not be effective in treating severely affected ITP patients because, although the spleen is one of the major sites of platelet sequestration and antibody production, reticulo-endothelial systems (RESs) (liver, bone marrow, lymphnodes, etc.) other than the spleen are also responsible for the destruction of platelets. We therefore consider the W/B F1 mouse to be a useful model of human ITP, and believe that it provides valuable information for the development of new therapeutic agents in patients with ITP, especially those who do not respond to splenectomy.
Assessment of Lumiaggregometry for Research and Clinical Laboratories
Authors: White Melanie McCabe , Foust John T , Mauer Alvin M , Robertson James T , Jennings Lisa K
Published: July 2018

Platelet aggregometry is often used to help diagnose storage pool disease (SPD-reduced amounts of granule nucleotides) and release defects (abnormal release of granule nucleotides). The general assumption that normal aggregation patterns are sufficient to rule out the diagnosis of one of these disorders has been invalidated by the recent publication of two papers describing patients with clinical bleeding, prolonged bleeding times and normal aggregation patterns in spite of defective release. The lumiaggregometer provides a tool for measuring platelet release and aggregation simultaneously. This paper presents a standardized, reproducible method for the use of the lumiaggregometer based on a “standard curve”. Data obtained during the development of the procedure are presented including normal ranges of release at different concentrations of agonists, release measured in intrinsic disorders as well as in patients on aspirin, and values for release relative to varying platelet counts. A monoclonal antibody (anti-p24/CD9; MAb7) which activates platelets similarly to thrombin and may be a useful reagent for distinguishing SPD and release defects is also introduced.
Effects of Injected Antibody against the Platelet Glycoprotein llb/llla Complex on Monkey Platelet Fibrinogen
Authors: Suzuki Masahiko , Kawakatsu Toshihiro , Nagata Hirokazu , Hamamoto Kenjiro , Iwata Koji , Ohga Shigetoshi , Nomura Shosaku , Kokawa Terutoshi , Yasunaga Kojiro
Published: July 2018

Four monkeys were injected for a 10-day period with the Fab fragment of a murine monoclonal antibody (NNKY 1-32) which inhibits the binding of fibrinogen to the platelet glycoprotein (GP) Ilb/IIIa complex. Platelet fibrinogen levels were assessed quantitatively by electroimmuno assay and qualitatively by immunoelectron microscopy.

The platelet fibrinogen level fell to 9.0 ± 2.8% of the control level after antibody administration. Immunoelectron microscopy showed that the injected antibody was localized on the inner surface of the platelet a-granule membrane.

Our findings suggest that the GP Ilb/IIIa complex can be internalized by a-granules and that it may mediate the endocytosis of plasma fibrinogen by platelets.
Prevalence of Inhibitors in a Population of 3435 Hemophilia Patients in France
Authors: Sultan Y ,   French Hemophilia Study Group
Published: July 2018

A cooperative study between the 37 centers of the French Hemophilia Study Group was undertaken to establish the prevalence of inhibitor patients in the French hemophilia population. The prevalence reported in the literature varies widely from 3.6% to 17.5%. Some of the studies are dealing with a small number of patients and inhibitor patients are reported either to the total number of hemophiliacs or to the severely affected ones. The French study provided information concerning 3,435 hemophiliacs and showed a prevalence of 6.2% for the overall population. Prevalence of inhibitors was found to be 7% in the population of hemophilia A patients and 12.8% in the population of severely affected ones. The prevalence of inhibitors in the population of hemophilia B patients was 2% and 4% in the population of severely affected hemophilia B patients. The cooperative study also showed that 47.5% of inhibitors are detected before 10 years of age and that 82% of inhibitor patients are high responders. Analysis of inhibitor detection in patients under the age often showed that there was a peak in the population of 2 years old children. Although not comparable to the present study the high incidence of inhibitors with ultrapurified and recombinant FVIII reported in previously untransfused patient may be borne in mind.
Effects of Heparin and Endothelial Cell Growth Supplement on Haemostatic Functions of Vascular Endothelium
Authors: Minter Allison J , Dawes Joan , Chesterman Colin N
Published: July 2018

Heparin in combination with endothelial cell growth factor (ECGF) affects physiological responses and growth of human umbilical vein endothelial cells (HUVEC). We have examined the effect of heparin, crude ECGF (endothelial cell growth supplement [ECGS]), or both on the basal and thrombin challenged output of metabolites by HUVEC. The supernatant and/or cell lysate was assayed for released prostacyclin, von Willebrand factor, tissue plasminogen activator, plasminogen activator inhibitor and thrombospondin. Heparin modified release of all these metabolites when in combination with ECGS, and in general these responses were the opposite of those generated by inflammatory mediators such as interleukin-1. It has been postulated that heparin acts by potentiating the effect of ECGF, but heparin inhibited thrombospondin release and enhanced that of von Willebrand factor in the absence of ECGS, while ECGS alone inhibited release of plasminogen activator inhibitor. Thus, under our experimental conditions it would appear that heparin and crude ECGF can affect HUVEC independently of one another.
Influence of Cytotoxic Drugs on Platelet Functions and Coagulation in vitro.
Authors: Klener P , Kubisz P , Suranová J
Published: July 2018

Influence of melphalan on some platelet functions, plasmatic coagulation and fibrinolysis “in vitro” was investigated, using different concentrations of the drug (25, 50 and 250 μg/ml). The lowest concentration slightly inhibited adrenaline and/or collagen-induced platelet aggregation. Following the highest concentration of the drug, strong inhibition of aggregation was recorded, regardless of the inducer used. Melphalan was also shown to inhibit release of aggregating activity and release of platelet factor 4, as well as availibility of platelet factor 3 and platelet acid phosphatase. The intensity of inhibition depended on both, melphalan concentration and the time of preincubation. In contrast to this, adhesion of platelets to glass slide was not found to be influenced by melphalan. Similarly, melphalan did not induce (in any concentration) loss of LDH from platelet cytoplasma, while triton X-100 or freezing and thawing of platelets caused significant increase of LDH activity. From coagulation tests studied, only thrombin time and reptilase time was found to be moderately prolonged in the presence of melphalan.

Authors assumed that melphalan acts as a specific inhibitor of release reaction and can induce an acquired thrombocytopathy. The platelet membrane is not damaged by the drug, as was confirmed by the investigation of LDH activity. Influence on coagulation indicates some antithrombin effect of the drug. Although presented results were obtained in vitro, analogous changes in vivo could be suspected. Thus, impairement of platelet functions might play a part in haemorrhagic complications accompanying, in some cases, melphalan therapy.
Induction of the Platelet Release Reaction by 1.3-Dimethyl-5-Aminoadamantane, a New Adamantane Derivative
Authors: Haacke Uta , Wesemann Wolfgang
Published: July 2018

The amantadine derivative 1.3-dimethyl-5-aminoadamantane, D 145, induces in high concentrations of 2–10 mM the release reaction. Adenine nucleotides and 5-hydroxytryptamine (5-HT) are liberated to the same extent and in the same ratio as found after thrombin-induced release. The time course of release is very slow; maximal release is reached in 15–20 min. The process is temperature-dependent and dependent on energy derived from glycolysis and oxidative phosphorylation. Extracellular Ca++ does not promote the release process. D 145, in accordance with the mother-substance amantadine, inhibits 5-HT uptake non-competitively, KI = 0.15 mM. In concentrations of 0.1–1 mM D 145 triggers only the liberation of 5-HT, adenine nucleotides are not liberated. The ADP induced platelet aggregation is completely inhibited after preincubation with a 1 mM solution of D 145.
Comparison between British Comparative Thromboplastin (BCT) and a Factor II-VII-X Determination Method (Simplastin A) Based on Fresh Plasma Samples from Dicoumarol-Treated Patients
Authors: Korsan-Bengtsen K , Johsen M , Pehrsson N.-G
Published: July 2018

Comparisons between British Comparative Thromboplastin (BCT) and a factor II-VII-X determination method – Simplastin A – have been carried out monthly during the time from December 1973 to March 1976. On each occasion fresh plasma from 20 dicoumarol-treated patients was analyzed with the two methods. It was found that the linear regression model is well suited for the comparison, that the BCT thromboplastin did not change significantly during the study, and that the method of comparing has a coefficient of variation of approximately 6–8 %. This means, for instance, that a ratio of 3.8 with BCT corresponds to a ratio of 3.5 ± 0.26 with Simplastin A. Thus in one period you may have ratio 3 and in another ratio 4 as the lower limit of the therapeutic range although you use the same thromboplastin and the same method. The importance of having a stable reference thromboplastin and an established method of comparing thromboplastins is stressed.
Immunoelectrophoretic Studies on the Kinetics of Plasmic Degradation of Human Fibrinogen
Authors: Braude Roberto , Altman Raúl , Rouvier Jorge , Aguilar Nélida
Published: July 2018

In vitro degradation of fibrinogen results in the appearance of an early fragment, different in its electrophoretic mobility to that of fragments X, Y, D and E.

It reacts with antifibrinogen antiserum, does not react with anti-D antiserum and it is anti-genically related to fragment E.

Compared with terminal fragment E, it migrates more slowly towards the anode, it appears earlier and in a greater concentration at the beginning, decreasing and disappearing during degradation.

The antigen antibody crossed electrophoresis was used in these experiments, but to distinguish the fragments from already known X, Y or even fibrinogen, the applying of an intermediate gel with anti D was of great value.
The Stability of Glutardialdehyde-Stabilized 35-Heparinized Surfaces in Contact with Blood
Authors: Larsson R. L , Hjelte M.-B , Eriksson J. C , Lagergren H. R , Olsson P
Published: July 2018

Heparin can be bound to polymer surfaces by precipitation of an ionic heparin-amine complex which can be stabilized against dissolution by treatment with glutardialdehyde. The aim of this investigation was to study the degree and course of desorption of heparin from such a heparinized surface on contact with blood. This desorption must be considered when analysing the interaction between the heparinized surface and blood. Different heparinized surfaces were prepared by using 35S-labelled heparin, and the desorption of heparin on exposure in vitro to citrated plasma or heparinized blood as well as during exposure at in vivo conditions was quantified.

During in vitro experiments, the glutardialdehyde stabilized surface became stable with no further desorption of heparin after an initial loss of about 3% of the initial surface-bound heparin. Under in vivo conditions, there was an initial loss of about 12%. There was no further loss from surfaces inserted into the circulation of the dog for seven days as compared to those inserted for one hour.
Potentiation by α and Inhibition by β-Adrenergic Stimulations of Rat Platelet Aggregation
Authors: Yu S. K , Latour J. G
Published: July 2018

Epinephrine, known to potentiate and elicit aggregation of human platelets, was shown to inhibit thrombin-induced aggregation of rat platelets, delaying the onset of aggregation from 2 to 12 times. Incubation of rat platelet suspensions with propranolol (1.25–30 μM), inactive by itself, totally prevented the inhibitory effect of epinephrine and also permitted a potentiation effect to show up. On the contrary, phentolamine (1.25–30 μM) potentiated the inhibitory effect of epinephrine on rat platelets and unmasked an inhibitory effect on human platelets. Finally, isoproterenol (0.25–9 μM) produced a marked inhibition of aggregation induced by thrombin, ADP and collagen in the three species studied, but most particularly in the rat. From these results, we conclude that stimulation of the platelet adrenergic receptors may either result in promotion (α-stimulation) or inhibition (β-stimulation) of platelet aggregation. Furthermore, differences in the ratios or responses of α/β receptors may account for species variations in the platelet aggregation response to catecholamine challenge.
The Plasminogen Activator of the Arterial Wall
Authors: Donner L , Klener P , Roth Z
Published: July 2018

The plasminogen activator in 645 specimens of various human arteries – thoracic, abdominal aorta, carotic, pulmonary, renal, basilar, coronary – was studied using Todd’s histochemi-cal method. 92 cadavers were used, 1–18 hours post mortem from subjects aged from 272 days to 83 years. 45 specimens of pulmonary, renal and splenic arteries were obtained during surgery.

The greatest fibrinolytic activity was within the adventitia. Intima occasionally showed very little fibrinolytic activity, or none at all.

No statistically significant differences in plasminogen activator activity were found between the various arteries examined.

A statistically significant increase in fibrinolysis in adventitia of atherosclerotic arteries was established. No correlation was found between the fibrinolytic activity of the arteries and their alkaline phosphatase content.

Some properties of the plasminogen activator of the arterial vessel wall were evaluated. Influence of storage, inactivation with epsilonaminocaproic acid and extracted with potassium thiocyanate was studied.
Marathon Run II: Effects on Platelet Aggregation
Authors: Dimitriadou C , Dessypris A , Louizou C , Mandalaki T
Published: July 2018

Platelet count and aggregation were assessed in 9 Finnish amateur runners aged 34 to 48, and one 65-year old taking part in a non-competitive marathon race (42.2 km).

After the run the mean value of platelet count showed a very significant rise (p <0.001). The platelets were markedly more sensitive to both ADP and collagen-induced aggregation. A highly significant increase (p <0.001) was noted for both the intensity and velocity of platelet aggregation.

The finding of platelet hyperaggregability after prolonged strenuous exercise even in trained subjects is discussed. It is concluded that a thorough medical examination of the haemostatic balance is recommended before a marathon race.
Reduction of Mortality with Antithrombin III in Septicemic Rats: A Study of Klebsiella pneumoniae Induced Sepsis
Authors: Dickneite Gerhard , Pâques Eric-Paul
Published: July 2018

Experimental gram-negative sepsis was induced in the rat by Klebsiella pneumoniae. Although bacteria are susceptible to the treatment with the antibiotic Tobramycin, DIC could not be prevented. DIC was manifested by a leuko- and thrombocytopenia, decreases in fibrinogen and AT III and an increase of the aPTT. In this model the therapeutic treatment with human AT III was evaluated. To determine the optimal concentration of AT III a prestudy in a LPS induced DIC in the rat was performed. It was shown that a bolus i. v. injection of 500 U/kg improved survival and DIC, and was thus chosen for the Klebsiella sepsis model. The infectious load was adjusted to yield a mortality rate of 90-100% in the untreated Klebsiella group and a reduction to about 40-50% of the mortality rate by Tobramycin. It was found that AT III reduced mortality in the Klebsiella induced sepsis not only when given prophylactically but was effective even when administrated in a late stage of the DIC, i. e. 3 or 5 h post infection.
Therapeutic Effect of a Low Molecular Weight Dermatan Sulphate (Desmin 370) in Rat Venous Thrombosis - Evidence for an Anticoagulant-Independent Mechanism
Authors: Barbanti M , Calanni F , Milani M R , Marchi E , Semeraro N , Colucci M
Published: July 2018

We evaluated the capacity of a low molecular weight dermatan sulphate (D370) to prevent thrombus formation and to induce a reduction of a stabilized thrombus in a rat venous thrombosis model. Injection of D370, 10 min before induction of venous stasis (prevention model), prevented thrombus formation in a dose-dependent way (ED50: 2.3 mg/kg). When given to rats 6 h after induction of venous stasis (therapeutic model), D370 caused a time- and dose-dependent reduction in thrombus size (60% to 70% reduction 2 h after injection of 10 mg/kg). At comparable antithrombotic dosages (i.e. minimum dose giving complete inhibition of thrombus formation), heparin (0.5 mg/kg) only caused 40% reduction of a preformed thrombus while hirudin (1 mg/kg) was virtually ineffective (less than 10% reduction in weight). All three compounds inhibited 125I-fibrin(ogen) deposition on 6-h aged thrombi by more than 85%, suggesting that D370 and, to a lesser extent, heparin reduce thrombus size via mechanisms other than inhibition of thrombus accretion. The involvement of a fibrinolysis-mediated mechanism in the D370-induced effect is suggested by the following. EACA (1 g/kg), when given to thrombus-bearing control animals, did not influence thrombus weight. However, when administered before D370 treatment, it prevented the expected reduction in thrombus weight by more than 80%, without influencing the effect of D370 on 125I-fibrin(ogen) accumulation onto preexisting thrombi. D370 injection caused neither an enhancement of fibrinolytic activity nor a reduction of PAI in plasma. In vitro, D370 (200 μg/ml) was unable to potentiate the spontaneous or PA-induced lysis of 125I-fibrinogen labelled blood, plasma, or purified fibrin clots. It is suggested that prevention of thrombus formation by D370 is related mainly to inhibition of blood coagulation, whereas reduction of the weight of aged thrombi is primarily due to an anticoagulant-independent mechanism, most probably involving local enhancement of the fibrinolytic process. D370 may represent an alternative pharmacologic agent both in the prevention and in the therapy of venous thrombosis.
The Genetic Defect of Type I von Willebrand Disease “Vicenza” Is Linked to the von Willebrand Factor Gene
Authors: Randi Anna M , Sacchi Elisabetta , Castaman Gian Carlo , Rodeghiero Francesco , Mannucci Pier Mannuccio
Published: July 2018

Type I von Willebrand disease (vWD) Vicenza is a rare variant with autosomal dominant transmission, characterized by the presence of supranormal von Willebrand factor (vWF) multimers in plasma, similar to those normally found in endothelial cells and megakaryocytes. The patients have very low levels of plasma vWF contrasting with a mild bleeding tendency. The pathophysiology of this subtype is still unknown. The presence of supranormal multimers in the patients’ plasma could be due to a mutation in the vWF molecule which affects post-translational processing, or to a defect in the cells’ processing machinery, independent of the vWF molecule. In order to determne if type I vWD Vicenza is linked to the vWF gene, we studied six polymorphic systems identified within the vWF gene in two apparently unrelated families with type I vWD Vicenza. The results of this study indicate a linkage between vWF gene and the type I vWD Vicenza trait. This strongly suggests that type I vWD Vicenza is due to a mutation in one of the vWF alleles, which results in an abnormal vWF molecule that is processed to a lesser extent than normal vWF.
Pronounced Effects of the Combination of a New Thromboxane Antagonist (GR32191) and Heparin on Bleeding Time in Man
Authors: de Boer A , Kroon J M , Kroon C , van Vliet A , Schoemaker H C , Breimer D D , Donoghue S , Cohen A F
Published: July 2018

Potential pharmacokinetic and pharmacodynamic interactions between two oral doses of GR32191 (40 and 80 mg), a new thromboxane antagonist, and heparin (5,000 IU bolus + 1,000 IU/h for 3 h) were studied in eighteen healthy male volunteers using two separate double-blind, randomised, placebo-controlled, cross-over studies.

Mean (range) bleeding time values were 8.4 min (7.5–9.7) during heparin/placebo, 12.1 min (9.2–18.6) (GR32191/placebo) and 16.3 min (11.5–21.4) (GR32191/heparin) in the 40 mg study, while these values were 8.7 min (5.5–15.5), 16.0 min (9.3 – >36.0) and 23.8 min (10.7 – >36.0), respectively in the 80 mg study. Compared to screening values, the combination of 80 mg of GR 32191 and heparin had a greater effect on the bleeding time than the sum of the prolongations after the separate treatments (p = 0.05). In the 40 mg study this was not the case. Pharmacokinetics of heparin (as assessed by plasma anti-Xa and antithrombin activity) and GR32191 were unaltered during coadministration of the two drugs. GR32191 did not influence the effects of heparin on APTT. Heparin slightly diminished the inhibition of collagen induced platelet aggregation by 80 mg of GR32191 and the U-46619 (thromboxane A2-mimetic) induced platelet aggregation remained unchanged. Overall fibrinolytic activity (as evaluated by the fibrin plate test) was similar during all three treatments in the study with 80 mg.

The combination of 80 mg of GR32191 and heparin caused a prolongation of the bleeding time which was more than expected on the basis of their individual effects.
The Influence of Infusions of 1-Desamino-8-D-Arginine Vasopressin (DDAVP) In Vivo on Thrombin Generation In Vitro
Authors: Ibbotson S H , Davies J A , Grant P J
Published: July 2018

To investigate the effect of increasing FVIII:C in-vivo on coagulation ex-vivo, DDAVP was infused over 15 min in 10 volunteers and in-vitro thrombin generation measured. FVIII:C rose from 0.42 and 0.43 IU/ml before DDAVP to 1.38, 1.73 and 1.78 IU/ml at 15, 30 and 60 min respectively (p <0.001).

A computer-assisted thrombin generation test was performed in defibrinated plasma using chromogenic substrate, S2238. Time to reach 50% maximal thrombin activity (T50/s) and lag phase of thrombin generation (lag/s) were measured. Lag shortened from 75 and 60 s before to 45 s during and after infusion (p <0.001). T50 shortened from 78.5 and 76.0 to 62.5, 60.0 and 58.5 s at times 15 (p <0.01), 30 (p <0.001) and 60 (p <0.001) min. FVIII:C correlated inversely with lag and T50 (r = –0.847, p <0.001, r = –0.826, p <0.001, n = 10) respectively.

These findings show that acute elevations of FVIII:C in-vivo accelerate in-vitro thrombin production. This work suggests that elevated FVIII:C levels in-vivo may be important in thrombo-occlusive disease.