bluuebunny/crossref_metadata_2025 huggingface dataset:
Potentiation of the Antithrombotic Action of Dermatan Sulphate by Small Amounts of Heparin
Authors: Thomas D P , Gray E , Merton R E
Published: July 2018

We have examined the effect in impairing thrombus formation of a preparation of dermatan sulphate (DS) alone and DS plus small amounts of unfractionated heparin (UFH). In rabbits given a dose of 150 μg/kg of DS alone, there was minimal reduction in serum-induced stasis thrombosis (Wessler model), with an overall score of 92.5% (100% = no impairment of thrombus formation). When the same dose of DS containing UFH was given (two different subgroups given DS containing 0.25 and 2.5% heparin by dry weight, respectively), the overall thrombus score was reduced to 60% (P <0.003), with no significant difference between the two subgroups. To achieve a comparable result with DS alone, a dose of 1 mg/kg was required. We conclude that very small amounts of UFH significantly enhance the antithrombotic action of DS, by a mechanism that has yet to be determined.
In Vitro Effects of Urokinase — Prevention by Different Inhibitors
Authors: Oethinger M D , Seifried E
Published: July 2018

The present in vitro study investigated dose-, time- and temperature-dependent effects of two-chain urokinase plasminogen activato(u-PA, urokinase) on normal citrated plasma. When 10 μg/ml u-PA wereadded to pooled normal plasma and incubated for 30 min at an ambient temperature (25° C), α2-antiplas-min decreased to 8% of the control value. Incubation on ice yielded a decrease to 45% of control,whereas α2-antiplasmin was fully consumed at 37° C. Fibrinogen and plasminogen fell to 46% and 39%, respectively, after a 30 min incubation at 25° C. Thrombin time prolonged to 190% of control.

Various inhibitors were studied with respect to their suitability and efficacy to prevent these in vitro effects. Aprotinin exhibited a good protective effect on fibrinogen at concentrations exceeding 500 KlU/ml plasma. Its use, however, was limited due to interferences with some haemostatic assays. We could demonstrate that L-Glutamyl-L-Glycyl-L-Arginyl chloromethyl ketone (GGACK) and a specific polyclonal anti-u-PA-antibody (anti-u-PA-IgG) effectively inhibited urokinase-induced plasmin generation without interfering with haemostatic assays. The anti-u-PA-antibody afforded full protection ofα2-antiplasmin at therapeutic levels of u-PA.

It is concluded that u-PA in plasma samples from patients during thrombolytic therapy may induce in vitro effects which should be prevented by the use of a suitable inhibitor such as GGACK or specific anti-u-PA-antibody.
Heparin Stimulates Fibrinolysis in Mesothelial Cells by Selective lnduction of Tissue-Plasminogen Activator but not Plasminogen Activator Inhibitor-l Synthesis
Authors: Grulich-Henn Jürgen , Preissner Klaus T , Müller-Berghaus Gert
Published: July 2018

The regulation of tissue-plasminogen activator (tPA) and plasminogen activator inhibitor 1 (PAI-l) synthesis was studied in cultured human mesothelial cells derived from omentum (HOMC). Heparin (100 U/ml) as well as pentosan polysulfate (300 pg/mt) stimulated tPA synthesis by HOMC 2.9-4.5-fold. Heparin-induced tPA production was dose- and time-dependent and was inhibited by cycloheximide. tPA production by HOMC was also stimulated by phorbol 12-myristrate l3-acetat (2.6-fold), fibrin clots (1.9-fold), or batroxobin (1.9-fold).Heparin and pentosan polysulfate did not stimulate PAI-1 production by HOMC, while phorbol l2-myristrate 13-acetat (100 nM) increased the concentration of PAI-1 in the conditioned medium by 2.6-fold over 24 h. The interaction of heparin with HOMC was studied by direct binding experiments. Dose-dependent specific binding of biotinylated heparin to HOMC was saturable at about 10 pg/ml, the Kp was estimated to about 0.15 pM. Biotinylated heparin bound rapidly to HOMC and reached a plateau within 60 min. Unlabeled heparin as well as pentosan polysulfate inhibited binding of biotinylated heparin in a dose-dependent fashion. These data demonstrate that heparin interacts with HOMC, and increases the fibrinolytic capacity in these cells by selectively increasing the production of tPA.
A Precise and Rapid Microtitre Plate Clot Lysis Assay: Methodology, Kinetic Modeling and Measurement of Catalytic Constants for Plasminogen Activation during Fibrinolysis
Authors: Jones A J S , Meunier A M
Published: July 2018

A rapid and precise turbidimetric clot lysis assay employing a microtitre plate reader and personal computer is described in detail. The use of such widely available instrumentation, the convenience and rapid throughput suggest the assay could be developed as a reference method with which to measure the potency of tissue plasminogen activator (t-PA) in conjunction with the WHO reference preparation. The method has been used to investigate molecular parameters involved in fibrinolysis. Aggregation status of the fibrin does not appear toinfluence the mechanism of plasminogen activation and clot lysis by plasmin. High ratios of plasminogen to fibrin resulted in a change in clot turbidity and in a change in the lysis profile of turbidity versus time. This is probably the result of plasminogen binding to fibrin and consequent restriction of the access of plasmin to its sites of cleavage in the fibrin. A simple model is proposed, and equations have been derived, for the kinetics of lysis which adequately describe the mechanism and which are confirmed by experimental data. This model results in estimates of the Km and kcat for the activation of plasminogen by t-PA during clot lysis of approximately 150 nM and0.1 s-1, respectively, in excellent agreement with published values The assay should therefore prove useful in quantitative evaluationsof the molecular phenomena occurring during fibrinolysis. The more rapid activation of lys-plasminogen than glu-plasminogen by t-PA was confirmed. However, evidence was obtained that the lys-form binds more tightly to fibrin by the same factor. This observation suggested that the appropriate substrate in the kinetic model is fibrin-bound plasminogen. When the data were re-analysed using published values ofthe affinities of the two forms, t-PA was found to activate both forms indistinguishably on the fibrin surface, consistent with suggestions that the conformation of glu-plasminogen changes to one more like that of lys-plasminogen upon binding to fibrin.
The Platelet Count in Carcinoma of the Lung and Colon
Authors: Costantini Vincenzo , Zacharski Leo R , Moritz Thomas E , Edwards Richard L
Published: July 2018

Platelet counts were evaluated in 714 patients with advanced non-small cell lung cancer (N-SCLC), small cell carcinoma of the lung (SCCL), and colon cancer entered to a clinical trial. Patients had not received prior chemotherapy. Platelet counts were not different in patients who had received radiation therapy prior to entry to the study. In comparison to the other tumor types, patients with N-SCLC demonstrated an increased prevalence of thrombocytosis (counts greater than 400,000/mm3), higher platelet counts at the time of entry to the study, higher over all mean platelet counts, relative preservation of high platelet levels during disease progression, and no relationship between platelet numbers and the amount of chemotherapy given. By contrast, platelet counts in patients with SCCL were negatively correlated with the absolute amount of cyclophosphamide and adriamycin given, and declined most dramatically with disease progression and death. Platelet numbers did not correlate with fibrinopep-tide A or fibrin split product levels suggesting that disseminated intravascular coagulation or fibrinolysis may have had less influence on platelet numbers than certain other factors. By contrast, significant correlations were found for all three tumor types between platelet numbers and other indicators of bone marrow function including anemia, total leukocyte count, and absolute neutrophil count; and the fibrinogen level. Based upon these findings, we postulate that the host response to malignancy, possibly in the form of production of bone marrow-stimulating cytokines, may play a prominent role in regulation of platelet counts in these (and perhaps other) neoplasms, and that a particularly prominent and persistent degree of marrow stimulation exists in patients with N-SCLC.
Effects of Fixed Minidose Warfarin on Coagulation and Fibrinolysis Following Major Gynaecological Surgery
Authors: MacCallum P K , Thomson J M , Poller L
Published: July 2018

Coagulation and fibrinolytic studies have been performed in patients who were undergoing major gynaecological surgery and randomised to either fixed minidose warfarin (1 mg daily) or matched placebo. With warfarin, a prolongation of the prothrombin time was observed on day 2 which persisted for at least 5 days and was greater than with placebo. The maximal postoperative mean INR was, however, only 1.2 which is considerably less than the target value for prophylaxis of deep vein thrombosis with full dose warfarin. The warfarin group showed two unexpected findings: significantly elevated fibrin specific degradation products throughout the postoperative period compared with placebo and absence of the expected rise of PAI, the major fibrinolytic inhibitor, on the first day after surgery. Levels of fibrinogen degradation products and FI + 2 prothrombin fragments rose significantly and progressively in both groups in the postoperative period. With placebo, FI + 2 showed an apparent higher percentage increase on each post-operative day but the differences between the groups were not significant. Increased fibrinolysis may be one of the mechanisms for the protective action of minidose warfarin in prophylaxis of DVT after major surgery.
Impact of Smoking, Physical Training and Weight Reduction on FVII, PAM and Hemostatic Markers in Sedentary Men
Authors: Gris Jean-Christophe , Schved Jean-François , Feugeas Olivier , Aguilar-Martinez Patricia , Arnaud Anne , Sanchez Nicole , Sarlat Christian
Published: July 2018

The variations of FVII, PAI-1, TAT complexes, fibrinopeptide A, D-Dimers and beta thromboglobulin plasma levels were studied on 30 sedentary men, smokers and non-smokers, who were admitted to a 6 months’ program of physical training and smoking cessation. After 3 months of intervention, sustained physical training was associated with the decrease of FVII and PAI-1 levels. Mild exercise performed during a second 3-month period could maintain normal FVII and PAI-1 activities but participants who stopped the training increased their FVII and PAI-1 plasma levels. FVII was not influenced by smoking habits. Smoking cessation seemed to slightly potentiate the decrease of PAI-1 levels associated with mild exercise. Overweight, FVII and PAI-1 levels were correlated and the weight reduction induced by training was related to the changes in the factors. In smokers, physical exercise was associated with a significant increase of hemostatic markers. This exercise-induced variation disappeared after 3 months of intervention in participants who stopped smoking and reappeared in those who smoked again after 6 months of intervention. This finding was not influenced by the physical training program.
Evaluation of International Normalized Ratios by a Controlled Field Survey with 4 Different Thromboplastin Reagents
Authors: Kazama Mutsuyoshi , Suzuki Setsuko , Abe Takeshi , Tahara Chieko , Shimazu Chisato , Akiyama Yoshiko , Higashi Katsumi , Ishiguro Izumi , Kimura Toshiyuki , Motoi Shigemi , Tsukai Kazuo , Kobayashi Masaki , Niwa Yoshimasa , Makita Hisao , Yasumuro Yoko , Nakatake Toshihiko
Published: July 2018

A nationwide survey has been performed in Japan involving 75 laboratories to assess the relative reliability of different methods of reporting prothrombin time results in anticoagulant control. The interchangeability of results using prothrombin time, prothrombin activity percentage, prothrombin ratio and international normalized ratios (INR) were compared with four different thromboplastin reagents and a range of coagulometers. A secondary batch of reference thromboplastin of human brain origin (BCT/454) was used to calibrate the local thromboplastins and for comparison of methods of reporting. The study revealed the closest agreement of the results between BCT and the other reagents, and the regression lines of these reagents were almost identical, when the results were reported as INR. Box-Whisker plot analysis showed that the distribution of the results was large with the more deficient plasmas with all methods of reporting. It was found by this analysis that the interchangeability of the results was greatest when the results were expressed by INR, because the mean values obtained of each plasma using different thromboplastin reagents gave the lowest CV and the frequency of the far-out data was least, compared with the other methods of expression. On the other hand, the type of coagulometer had almost as much effect as the thromboplastin reagent on the prothrombin time, even if INR was used. Interchangeability of INR would be further improved by providing ISI values for each reagent/ instrument combination.
Relationship of Major Histocompatibility Complex Class II Genes to Inhibitor Antibody Formation in Hemophilia A
Authors: Lippert Lloyd E , Fisher Lyman Mc A , Schook Lawrence B
Published: July 2018

Approximately 14% of transfused hemophiliacs develop an anti-factor VIII inhibitory antibody which specifically neutralizes factor VIII procoagulant activity. In this study an association of the major histocompatibility complex (MHC) with inhibitor antibody formation was evaluated by restriction fragment length polymorphism (RFLP) analysis using BamHI, EcoRI, HindII, PstI, PvuII and TaqI digested genomic DNA probed with DP beta, DQ alpha, DQ beta and DR beta class II MHC gene probes. The RFLP patterns for 16 non-inhibitor and 11 inhibitor hemophiliac patients were analyzed. These 24 enzyme:probe combinations generated 231 fragments. Fifteen (15) fragments associated with the inhibitor phenotype; odds ratios ranged from 5.1 to 45 and lower bounds of 95% confidence intervals were > 1.000 for all 15 fragments. Five (5) fragments associated with non-inhibitors, with odds ratios ranging from 6.4 to 51.7. This report establishes a MHC related genetic basis for the inhibitor phenotype. No statistically significant differences in the distribution of serologically defined HLA-DR phenotypes were observed between the inhibitor and non-inhibitor groups.
Prevention of Canine Coronary Artery Thrombosis with Echistatin, a Potent Inhibitor of Platelet Aggregation from the Venom of the Viper, Echis carmatus
Authors: Shebuski Ronald J , Ramjit Denise R , Sitko Gary R , Lumma Patricia K , Garsky Victor M
Published: July 2018

A model of acute, platelet-dependent canine coronary artery thrombosis was utilized to assess the antithrombotic effect of a synthetic, RGD-containing 49-residue protein termed echistatin. This protein is derived from the venom of the viper, Echis carinatus. In vitro, echistatin inhibited ADP (10 ΜM)-induced platelet aggregation with IC50 values in human and canine platelet-rich plasma of 101 ± 4 and 127 ± 32 nM, respectively. In vivo, in the dog, infusion of echistatin for 30 min at 20 pg kg−1 min−1 or 2.6 nM kg−1 min−1 resulted in total abolition of acute platelet-dependent coronary thrombus formation in all dogs tested (n = 5). Infusion of a lower dose (10 pg kg−1 min−1) was not effective in prevention of thrombus formation. Blood samples were taken before and after infusion of echistatin in order to determine ex vivo platelet aggregatory responses. Echistatin (20 pg kg−1 min−1, i.v.) attenuated ex vivo platelet aggregation elicited by ADP, U-46619 and collagen and increased bleeding time by 2.9 ± 0.5-fold over control. Thus, in the dog, echistatin is an effective antithrombotic agent inhibiting both platelet aggregation in vivo in the coronary artery as well as ex vivo with a concomitant increase in bleeding time. Furthermore, the effects of echistatin on platelet aggregation and bleeding time are reversible with restoration to control levels occurring 30-60 min after termination of the infusion.
Comparative Effects of Proteinase Inhibitors, Plasminogen Antiactivators, Heparin and Acetylsalicylic Acid on the Experimental Disseminated Intravascular Coagulation Induced by Thrombin
Authors: Moriau M , Rodhain J , Noel H , De Beys-Col C , Masure R
Published: July 2018

In an experimental study in the rabbit, the modifications of some haemostasis parameters (platelet count, platelet retention and aggregation, platelet factors 3 and 4, platelet and plasma plasmin inhibiting activities, fibrinogen and other plasma factor levels, FDP), and histological findings are compared in both the normal animal and the animal with disseminated intravascular coagulation (DIC) induced by thrombin perfusion after administration of fibrinolytic inhibitors (plasminogen antiactivators and proteinase inhibitors).

In the normal animal, the administration of fibrinolytic inhibitors is followed by haemostatic changes similar to those found in thrombophilic states. The modifications are more pronounced with plasminogen antiactivators than with proteinase inhibitors.

In the animal with DIC, the administration of fibrinolytic inhibitors enhances the haemostatic and the biological disorders produced by thrombin perfusion. The effect of the plasminogen antiactivators is even more evident.

The preventive administration of heparin reduces or abolishes the biological and histological disorders induced by thrombin; its beneficial effect is considerably reduced when thrombin is combined with fibrinolytic inhibitors.

The administration of acetylsalicylic acid appears to be ineffective for the prevention of haemostatic and histological changes induced by thrombin perfusion.
Rapid Detection and Quantitation of Precipitating Streptokinase-Antibodies
Authors: Spöttl F , Kaiser R
Published: July 2018

Streptokinase-therapy is regularly followed by a marked increase of precipitating streptokinase-antibodies. These antibodies cause a significant inhibition of streptokinase-induced plasminogen activation. Counterelectrophoresis is a useful method of tracing the antibodies in serum. For quantitative estimation a modified radial immunodiffusion method is presented. The antibodies were identified as IgG.
Clinical Manifestations and Management of Inherited Thrombophilia: Retrospective Analysis and Follow-up after Diagnosis of 238 Patients with Congenital Deficiency of Antithrombin III, Protein C, Protein S
Authors: De Stefano V , Leone G , Mastrangelo S , Tripodi A , Rodeghiero F , Castaman G , Barbui T , Finazzi G , Bizzi B , Mannucci P M
Published: July 2018

The clinical history of 238 patients with inherited thrombophilia (AT III = 94, PC = 103, PS = 41) was analyzed retrospectively at diagnosis and in the follow-up period after diagnosis. At diagnosis 129 patients (54%) had suffered from thrombosis, with a recurrence rate of 48%. The most frequent onset manifestation was deep vein thrombosis of lower limbs (58%). Thrombotic history started before 40 in 80% of the cases. Forty-nine percent of the venous thromboses were preceded by a triggering event, in most cases pregnancy (17%) and surgery (12%). After diagnosis, follow-up lasted a total of 1,113 pt-years. A policy of short-term prophylaxis during risk situations for all patients and long-term prophylaxis in symptomatic patients failed to prevent venous thrombotic episodes (diagnosed by objective methods) in 4 previously asymptomatic subjects and recurrence in 7 previously symptomatic subjects. After knowledge of the patients’ diagnosis the incidence of venous thrombosis/100 pt-years was reduced as compared before diagnosis as total episodes (onset + recurrencies) (1.0 vs 1.9), onset episodes (0.7 vs 1.3) and recurrent episodes (1.3 vs 4.8), even though the differences were not statistically significant. However most of the venous thromboses occurred at a more advanced age (67% after 40 years) and without any apparent cause (83%), at significant variance with the period preceding the diagnosis; in particular the incidence of venous thrombotic onset in patients younger than 40 passed from 1.3/100 pt-years to 0.2/100 pt-years. In 6 recurrences after diagnosis a poor compliance for antithrombotic treatment was recognized. We conclude that a policy based on knowledge of diagnosis and on the implementation of antithrombotic treatment during risk situations appears to modify the clinical outcome of deficiencies of natural anticoagulants even in the absence of long-term antithrombotic prophylaxis; in particular morbidity of young patients is significantly reduced.
Effects of the Synthetic Thrombin Inhibitor Argatroban on Fibrin- or Clot-Incorporated Thrombin: Comparison with Heparin and Recombinant Hirudin
Authors: Berry Christopher N , Girardot Christine , Lecoffreo Catherine , Lunven Catherine
Published: July 2018

The inhibitory effects of argatroban on clot- or fibrin-bound human thrombin were studied using the thrombin-specific chromogenic substrate S2238 (200 μM). These effects were compared to those of recombinant hirudin (rHV2 Lys 47) and the heparin/antithrombin III complex. Argatroban concentration-dependently inhibited the cleavage of S2238 by a thrombin solution, which had been titrated to give the same change in OD405 nm as fibrin-bound thrombin, with an IC50 of 1.1 μM with 90% inhibition at 8 μM. rHV2 Lys 47 and heparin had IC50 values of 1.2 nM and 0.003 U/ml respectively under these conditions. However, when the compounds were tested against fibrin-bound thrombin, argatroban had an IC50 of 2.8 μM with 65% inhibiton at 8 μM, whereas rHV2 Lys 47 had an IC50 of 23 nM (with only 56% inhibition at 200 nM), and heparin had an IC50 of 0.5 α 0.38 U/ml (with only 58% inhibition at 5 U/ml); i. e. the two compounds were 19 and 168 times less active against fibrin-bound thrombin than against thrombin in solution. The differences between the inhibitory effects of the compounds against thrombin bound to a plasma clot were even more striking in that the IC50 of argatroban was increased from 1.1 (vs. thrombin in solution) to 2.7 μM, while, although rHV2 Lys 47 and heparin had IC50 values of 2.8 nM and 0.004 U/ml against thrombin in solution, they had little (32% inhibition by 4 pM rHV2 Lys 47) or no effect (even at 5.0 U/ml heparin) against the amidolytic activity of a plasma clot. We conclude that argatroban could present advantages over hirudin and heparin in the treatment of pathologies where the enzymatic activity of clot-bound thrombin may play a significant role.
Surface Independent Factor XI Activation by Thrombin in the Presence of High Molecular Weight Kininogen
Authors: von dem Borne Peter A Kr , Koppelman Stefan J , Bouma Bonno N , Meijers Joost C M
Published: July 2018

A deficiency of one of the proteins of the contact system of blood coagulation does not result in a bleeding disorder. For this reason activation of blood coagulation via this system is believed to be an in vitro artefact. However, patients deficient in factor XI do suffer from variable bleeding abnormalities. Recently, an alternative pathway for factor XI activation has been described. Factor XI was found to be activated by thrombin in the presence of dextran sulfate as a surface. However, high molecular weight kininogen (HK), to which factor XI is bound in plasma, and fibrinogen were shown to block this activation suggesting it to be an in vitro phenomenon. We investigated the thrombin-mediated factor XI activation using an amplified detection system consisting of factors IX, VIII and X, which was shown to be very sensitive for factor XIa activity. This assay is approximately 4 to 5 orders of magnitude more sensitive than the normal factor XIa activity assay using a chromogenic substrate. With this assay we found that factor XI activation by thrombin could take place in the absence of dextran sulfate. The initial activation rate was approximately 0.3 pM/min (using 25 nM factor XI and 10 nM thrombin). The presence of dextran sulfate enhanced this rate about 8500-fold. A very rapid and complete factor X activation was observed in the presence of dextran sulfate. Although only minute amounts of factor XIa were formed in the absence of dextran sulfate, significant activation of factor X was detected in the amplification assay within a few minutes. HK inhibited the activation of factor XI by thrombin strongly in the presence, yet only slightly in the absence of dextran sulfate (26 and 1.2 times, respectively). Despite the strong inhibition of HK on the activation of factor XI by thrombin in the presence of dextran sulfate, HK had only a minor effect on the factor Xa generation.

We conclude that activation of factor XI by thrombin can take place regardless of the presence of a surface or HK. This activation might therefore be physiologically relevant. The inhibitory effect of HK on the thrombin-mediated factor XI activation is largely dextran sulfate dependent. Due to the amplification in the intrinsic system, trace amounts of factor XIa might generate physiological sufficient amounts of factor Xa for an adequate haemostatic response.
A Pilot Study of Pro-Urokinase in the Treatment of Deep Vein Thrombosis
Authors: Mola Marco , Mannucci Pier Mannuccio , Pini Marlo , Prandoni Paolo , Gurewich Victor
Published: July 2018

Safety and efficacy of the thrombolytic agent pro-urokinase (pro-UK) in the treatment of deep vein thrombosis of the lower limbs (DVT) have been investigated in an open, uncontrolled, pilot study. Fifteen patients were infused with 800.000 IU (5 mg)/h of pro-UK over 24 h (120 mg), together with unfractionated heparin adjusted to maintain the activated partial thromboplastin time between 1.5 and 2.5 times the basal value. Efficacy was assessed comparing venographic changes in the 11 evaluable limbs before and after pro-UK infusion. The Marder score decreased from a median pre-thrombolysis value of 28 (range 4-40) to 16 (3-38) (p <0.05). One major hemorrhagic event (retroperitoneal bleeding 4 days after the end of the pro-UK infusion) occurred. Fibrinogen, alpha2-antiplasmin and plasminogen significantly decreased from baseline values after 12 and 24 h, fibrin(ogen) degradation products significantly increased. Changes in hemostasis parameters were unrelated to thrombolytic efficacy. The results of this pilot study indicate that pro-UK is thrombolytic in DVT and that it can be administered simultaneously with conventional heparin treatment.
A Standard Nomenclature for Factor VIII and Factor IX Gene Mutations and Associated Amino Acid Alterations
Authors: Peake Ian , Tuddenham Edward
Published: July 2018

The exponential increase in reported mutations within the factor VIII and IX genes in patients with haemophilia A and B has resulted in the use of several different systems for reporting such mutations. The Factor VIII/IX Subcommittee believe that a standardised reporting system will be beneficial to all interested in the field of haemophilia genetics. This article contains proposals for a mutation nomenclature based on general working practices and standardised to the present state of our knowledge of the sequence and structure of the factor VIII and IX genes.
The Presence of Glycogen Synthase in Preparations of Platelet Plasma Membranes
Authors: Greenberg J. H , Fletcher A. P , Jamieson G. A
Published: July 2018

Glycogen synthase and glycogen (63 ± 35 μg/unit) have been identified in partially-purified preparations of platelet membranes isolated by density-step centrifugation following glycerol-lysis; this combination is responsible for the endogenous (collagen-independent) activity measured during the assay of collagen: glucosyltransferase. Glycogen synthesized in this way may be complexed with protein since it is precip-itable with trichloracetic acid. High and low density membranes prepared by continuous density gradient centrifugation are devoid of glycogen synthase activity.
Effect of Experimental Coronary Thrombosis upon Platelet Kinetics
Authors: Moschos Christos B. , Lahiri Kamalesh , Manskopf Gisbert , Oldewurtel Henry A. , Regan Timothy J.
Published: July 2018

A study was designed to determine the effect of experimental coronary thrombosis upon quantitative changes of platelets in the coronary sinus blood and in the systemic circulation. In addition, using 51Cr-labelled platelets, their distribution in the organ primarily affected by the presence of thrombus, namely the heart, and organs known to be involved in platelet kinetics such as liver, spleen and lungs, was also examined. Following coronary occlusion, a consistent pattern of progressive decrease in platelet counts in the effluent of the infarcted area (coronary sinus) as well as in the systemic blood, maintained for at least 18-20 hours following the thrombotic event, was observed. There was a significant increase in platelet sequestration by spleen, liver and lung and presence of platelet thrombi in the microcirculation of the coronary arterial system. The results suggest that a thrombotic process in a medium size artery induces a more generalized involvement of the platelet population through mechanisms which are not clear at this time. The changes in platelet counts, following coronary thrombosis, obtained as described in this study offer diagnostic possibilities in cases where an active thrombotic process is suspected. However, further studies are necessary before the value of this diagnostic approach is established.
Energy Production and Utilization by Human Platelets in the Presence of Some Guanidines and Phenols (Uremic Toxins) that Inhibit Aggregation
Authors: Carroll Hugh J
Published: July 2018

Platelet aggregation induced by ADP can be inhibited by plasma from uremic patients or by toxins isolated from their plasma, e. g. guanidinosuccinic acid, methylguanidine, phenol and hydroxyphenylacetic acids. Since these chemical substances can interfere with energy metabolism in tissues other than platelets and since ATP production is needed for ADP-induced aggregation, alterations in platelet energy metabolism could underlie excessive bleeding in uremic patients. Platelets incubated with idioacetate and deprived of anaerobic glycolysis produced the same quantity of ATP through respiration in the presence of all the uremic toxins studied as in their absence. Similarly, platelets incubated with cyanide and deprived of the oxidative pathway utilized anaerobic glycolysis to produce normal quantities of ATP in the presence of all the uremic toxins. The utilization of ATP, as indicated by active transmembrane potassium transport, was also unaffected by the above listed guanidines and phenols. It is concluded that the in vitro inhibition of ADP-induced platelet aggregation by the guanidines and phenols studied is not due to inhibition of production or utilization of ATP.
A Study of the Use of the Thromboxane A2 Antagonist, Sulotroban, in Combination with Streptokinase for Local Thrombolysis in Patients with Recent Peripheral Arterial Occlusions: Clinical Effects, Platelet Function and Fibrinolytic Parameters
Authors: Lonsdale Robert J , Heptinstall Stan , Westby John C , Berridge David C , Wenham Peter W , Hopkinson Brian R , Makin Geoffrey S
Published: July 2018

In peripheral thrombolysis adjuvant anti-platelet therapy may help to lyse otherwise resistant thrombus, thereby increasing the number of patients successfully treated and reducing the “time to lysis”. If continued after lysis it may help to prevent early rethrombosis. In this pilot study 21 patients undergoing peripheral thrombolysis with streptokinase were randomised to receive the thromboxane receptor antagonist sulotroban or placebo. The dose of sulotroban given was 2 mg/min (four patients), 4 mg/min (five patients) or 8 mg/min (four patients), eight patients received placebo. The clinical and laboratory effects of the treatment were monitored.

Thrombolysis was achieved more quickly in patients receiving sulotroban, however, there was no difference between groups in the number of patients in whom recanalisation was achieved (six of eight receiving placebo and eight of 13 receiving sulotroban) or in the number of cases of early rethrombosis. During lysis there was an increase in plasma beta-thromboglobulin with similar levels being found in patients receiving sulotroban and streptokinase and those receiving streptokinase alone. No other major changes in platelet function during lysis were seen in patients receiving streptokinase alone. Sulotroban significantly reduced platelet aggregtion and 14C-5HT release in response to several platelet agonists. With the thromboxane mimetic U46619 the degree of inhibition of aggregation and 14C-5HT release depended on the dose of sulotroban used. High levels of inhibition were associated with an excess of haemorrhagic complications especially in combination with a low plasma fibrinogen level.

We conclude that the use of low dose sulotroban in combination with streptokinase merits further study and may have a role in accelerating lysis. However, the combination of high dose sulotroban with streptokinase causes an increased incidence of bleeding. Monitoring platelet function during such therapy may identify patients likely to bleed.
Lack of Association Between Antiphospholipid Antibodies and Migraine
Authors: Tsakiris D A , Kappos L , Reber G , Marbet G A , Le Floch-Rohr J , Roux E , de Moerloose P
Published: July 2018

We prospectively studied the prevalence of lupus anticoagulant, anticardiolipin antibodies (aCL) and various haemostatic parameters in 71 patients with migraine and compared the results with a control group of 32 subjects with back pain never having experienced migraine. The patients with migraine were divided into two groups: group I with migraine without (n = 18) and with aura lasting less than 60 min (n = 24) and group II with migraine with prolonged aura or migrainous infarction (complicated migraine, n = 29). The following results were obtained: a) no difference in aCL positivity was noted between migrainous patients and controls and between common migraine and complicated migraine patients and b) no statistically significant difference in haemostatic parameters (except for thrombin-antithrombin III complexes) was found between the two groups of migraine and between aCL positive and negative migrainous patients. These data suggest that anticardiolipin antibodies are not involved in the pathogenesis of migraine complications.
Self-Association of Antithrombin III Relates to Multimer Formation of Thrombin-Antithrombin III Complexes
Authors: Preissner Klaus T
Published: July 2018

During the reaction of antithrombin III (AT III) with target proteases the inhibitor serves as pseudo-substrate and undergoes profound conformational changes, becomes incorporated into a covalent stoichiometric enzyme-inhibitor complex which is, in contrast to native AT III, recognized by monoclonal antibody 4C9. In the absence of the target enzyme thrombin, incubation of AT III with 1–2 M guanidine, 0.6% deoxycholate, heating to 56° C, or buffer at pH 4 resulted in inactivation of the inhibitor with concomitant exposure of the epitope for 4C9 and formation of AT III multimers (from 3.9S to 7.1–7.4 S). Loss of activity, formation of multimers and exposure of neoepitope(s) of AT III occurred in a concerted fashion and followed second order kinetics with an activation energy of E
a = 31 kcal/mol. AT III-multimerization induced by treatment with 1 M guanidine (mainly AT III-tetramers with M
r of 250,000) and formation of the binary AT III-thrombin complex revealed similar self-association patterns as judged by gel electrophoresis under non-denaturing conditions. In the presence of heparin, even higher multimers of AT III-thrombin complexes were noted. Moreover, self-association products of the ternary vitronectin-thrombin-AT III complex, which is the ultimate reaction product following thrombin inhibition in the circulation, could be recognized and quantitated due to exposure of the 4C9 epitope on AT III, indicating that AT III exists in multimeric forms within binary and ternary complexes. It is proposed that the ability of these complexes to form high M
r association products is at least in part mediated by the propensity of AT III to multimerize and that multimeric forms of AT III may be involved during clearance of AT III-enzyme complexes.
Heparin Binding Assay of von Willebrand Factor (vWF) in Plasma Milieu – Evidence of the Importance of the Multimerization Degree of vWF
Authors: de Romeuf Christophe , Mazurier Claudine
Published: July 2018

We developed a simple and fast method for studying the heparin binding of von Willebrand factor (vWF) in the plasma milieu. Using plasma from patients with von Willebrand disease (vWD) subtype II, we found that the heparin binding was impaired when compared with a normal plasma control. Further experiments performed with purified vWF of various multimeric composition, obtained either by gradual reduction or gel filtration, confirmed that heparin binding is dependent on the multimerization of vWF and that high molecular weight (HMW) multimers of vWF are required for normal heparin binding. After reduction of plasma vWF by 1.5 mM DTT, the vWF monomer still binds to heparin but to a lower extent. Under these conditions, no significant differences were obtained between control and patients showing that the heparin binding domain located on the vWF subunit is not altered in the subtypes IIA, IIB and IIC studied.
Double Strand Conformation Polymorphism (DSCP) Detects Two Point Mutations at Codon 280 (AAC→ATC) and at Codon 431 (TAC→AAC) of the Blood Coagulation Factor VIII Gene
Authors: Pieneman W C , Reitsma P H , Briët E
Published: July 2018

Hemophilia A is a hereditary, X-linked, bleeding disorder that is caused by a defect in the factor VIII gene. Here, we report two novel point mutations in the factor VIII gene that result in an aberrant electrophoretic mobility of double strand PCR fragments (double strand conformation polymorphism, DSCP). In exon 9 a TAC→AAC mutation at codon 431, replacing Tyr by Asn, was observed in a family (A211) with moderately severe hemophilia A. A family with mild hemophilia A revealed an A→T mutation in codon 280 (exon 7) that results in the replacement of Asn by Ile. One of these two mutations was not detected in an analysis based on single strand conformation polymorphisms (SSCP).

At present we have no explanation for the effect of the nucleotide changes on the electrophoretic mobility of double strand DNA. Although DSCP is not able to detect all mutations the combination of DSCP analysis with SSCP analysis increases the sensitivity in a screening for factor VIII mutations.
Effects of Hybrid Peptide Analogs to Receptor Recognition Domains on α- and γ-Chains of Human Fibrinogen on Fibrinogen Binding to Platelets
Authors: Mohri Hiroshi , Ohkubo Takao
Published: July 2018

We synthesized a series of hybrid peptides that correspond to the γ-chain dodecapeptide (400-411), variable numbers of glycine residues, and the RGDS peptide [Y-HHLGGAK-QAGDV(G)
n
RGDS] to investigate the relationship of these receptor recognition domains of fibrinogen to platelet membrane glycoprotein IIb/IIIa. The tetrapeptide RGDS, the GRGDSPA peptide and the dodecapeptide inhibited binding of fibrinogen to GPIIb/IIIa by 50% (IC50) at concentrations of 17 ± 1.6 μM, 15 ± 2.1 μM, and 87 ± 6.8 μM, respectively. The inhibitory effect of hybrid peptides increased as the number of glycine residues increased, plateauing with 9-11 glycine residues in hybrid peptide analogs, which had an IC50 of 0.68 ± 0.14 μM. These hybrid peptides completely inhibited the binding of fibrinogen to activated platelets when used in sufficient concentrations. The peptide Y-HHLGGAKQAGDV(G)9RGDS blocked ADP-induced aggregation in citrated platelet-rich plasma with IC50 of 3.5 ± 0.6 μM. When the peptide Y-HHLGGAK-QAGDV(G)9RGDS was labeled with 125I to quantify its binding to platelets, maximal binding was observed within 30 min. The binding sites of the hybrid peptide were 43,600 molecules/platelet (K
d = 3.1 ± 0.5 × 10-7 M) to stimulated platelets and 12,500 molecules/platelet (K
d = 1.4 ± 0.2 × 10-7 M) to nonstimulated platelets. The hybrid peptides had the same binding affinity to platelets as fibrinogen and inhibited platelet function. Moreover, anti-GPIIb/IIIa antibody inhibited the binding of the labeled hybrid peptide to stimulated platelets. These results indicate that in the native fibrinogen molecule the presence of both RGD sequence or γ-chain domain at optimal distances increased the binding affinity to GPIIb/IIIa. These domains may be the source of hybrid peptide, expanding a new class of platelet inhibitors that act at membrane receptors for adhesive proteins.
Effects of Antithrombotic Drugs in a Rat Model of Aspirin-Insensitive Arterial Thrombosis
Authors: Schumacher W A , Steinbacher T E , Heran C L , Megill J R , Durham S K
Published: July 2018

These studies describe experimental conditions where aspirin is less effective than other antiplatelet and anticoagulant drugs in inhibiting acute arterial thrombosis. External electrolytic injury of the rat carotid artery was used to induce occlusive thrombi in 97% of vehicle-treated rats. Thrombi were revealed by light and electron microscopy to be comprised primarily of platelets enmeshed in a fibrin network. The thrombin inhibitor D-phenylalanyl-L-prolyl-L-arginyl chloromethy ketone (PPACK; 6 mg/kg, i. v.) decreased thrombus weight by 90%. Aspirin alone (1, 10 and 30 mg/kg, i. v.), dipyridamole alone (5 mg/kg i. v.) and aspirin (1 and 10 mg/kg, i. v.) in combination with dipyridamole (5 mg/kg, i. v.) did not inhibit thrombosis. The platelet-activating factor (PAF) antagonist, WEB 2086, (1 mg/kg i. v.) was also ineffective. Other drugs had intermediate activity. Thrombi were decreased 56% by the thromboxane receptor antagonist, BMS 180,291, either alone (5.8 mg/kg i.v.) or in combination with aspirin (10 mg/kg, i.v.). Heparin (900 U/kg, i.v.), warfarin (0.25 mg/kg, p.o. once daily for 3 days) and ticlopidine (200 mg/ kg, p.o. once daily for 3 days) reduced thrombus weight by 63, 73 and 43% respectively. Reductions in thrombus weight were always associated with improvements in either average blood flow or vessel patency.
Experimental Thrombosis on a Collagen Coated Arterioarterial Shunt in Rats: A Pharmacological Model to Study Antithrombotic Agents inhibiting Thrombin Formation and Platelet Deposition
Authors: Freund Monique , Mantz Françoise , Nicolini Philippe , Gachet Christian , Mulvihill Juliette , Meyer Laurent , Beretz Alain , Cazenave Jean-Pierre
Published: July 2018

A rat thrombosis model was developed to assess the efficacity of antithrombotic drugs. It had the following characteristics: controlled hemodynamic and rheological conditions corresponding to arterial flow, a collagen coated surface as a relevant thrombogenic stimulus, a method of measurement allowing dynamic monitoring of thrombus formation and the possibility to assess the thrombus structure. A shunt composed of polyethylene and silicone catheters, including in the middle of the shunt a collagen coated glass capillary, was inserted between the two primitive carotids of the rat. The duration of patency of the shunt was recorded using a thermic probe fixed on its central part. In this model, the patency of the shunt was 539 ± 55 s. Platelet and fibrinogen-fibrin accumulation in successive one centimeter segments along the shunt were measured using 111In labeled platelets and 125I labeled fibrinogen. Platelet accumulation occurred on the collagen coated surface and at the junctions between the different components of the shunt, where flow was disturbed. The effects of four antithrombotic agents were measured: aspirin, clopidogrel, heparin and r-hirudin. Clopidogrel, heparin and hirudin significantly prolonged patency duration of the shunt, whereas aspirin was inactive. Aspirin did not reduce platelet or fibrinogen-fibrin accumulation on the collagen coated surface. Platelet accumulation on the collagen surface was significantly lower in the clopidogrel group (50 mg/kg) than in the group treated with heparin (500 U/kg), demonstrating the direct antiplatelet effect of clopidogrel. Hirudin at doses giving similar values of APTT as heparin (500 U/kg) prolonged the occlusion time to over 2 h while the heparin occlusion time was only 20 min. We conclude that this experimental arterioarterial thrombosis model is sensitive to both antiplatelet and anticoagulant agents, allowing discrimination between antiplatelet and anticoagulant mechanisms of thrombosis prevention. The technique is easily performed in a small animal, fast and reproducible, and thus comparable to primate models for use in the screening of new antithrombotic drugs.
In Vitro Characterization Of Low Molecular Weight Fractions Of Heparin
Authors: Fareed Jawed , Messmore Harry L , Walz Daniel A , Choay Jean , Lormeau J C
Published: July 2018

Numerous extraction, chromatographic (ion exchange, gel, and affinity), chemical and enzymatic degradation methods have been employed to obtain heparin fractions. The present assays to evaluate potency (e.g. pharmacopeial and coagulant) do not truly reflect the antithrombotic properties of these fractions. In addition, the synthetic peptide substrate based assays to measure the anti Xa activity do not correlate with the coagulant anti Xa assays. We have developed an in vitro test battery to evaluate low molecular weight heparin fractions. Porcine mucosal heparin fractions are assayed for anti Xa activity in coagulant and amidolytic assays and the results are expressed as a ratio. The effect of these fractions on coagulant assays such as prothrombin time (PT), partial thromboplastin time (PTT), thrombin time (TT), Stypven time (ST) on freshly prepared normal human plasma (NHP) is determined The retention characteristics of these fractions on platelet factor 4 and AT-III bound sepharose columns were also determined. We have compared the extracted and chemically depolymerized heparin fractions and found that the anti Xa activity doesn’t always correlate with the other parameters studied. The extracted fractions were slightly stronger in the USP assays and showed a biphasic retention on the PF-4 column whereas the chemically depolymerized product showed only one peak. On the other hand, on the AT-III column both fractions showed similar elution patterns. Our studies suggest that heparin and its fractions exhibit differential behavior on various assays and a specific test may not be used as an index of the potency of their antithrombotic effects. Furthermore, the potency of these fractions should be stated on a weight basis when evaluated in the in vivo animal models rather than in terms of a specific test (e.g. anti Xa activity and US Pharmacopeial assays).