bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies on the Platelet Defect in Alcoholism
Authors: Cowan Dale H , Graham Richard C
Published: July 2018

Platelet ultrastructure, protein composition, and adenine nucleotide metabolism were studied in patients ingesting ethanol to elucidate the mechanism of ethanol-induced changes in platelet function and survival. Serial measurements were made in 2 patients who maintained blood ethanol levels in excess of 300 mg/100 ml for 3 to 4 weeks. No major changes in structure or metabolism were detected in platelets from the patient whose platelet counts remained stable during the ingestion period. By contrast, the development of thrombocytopenia in the other patient was associated with significantly reduced intracellular ADP, increased ATP/ADP ratio, decreased release of ADP, increased specific radioactivity of intracellular ATP and ADP, and increased formation of hypoxanthine. Additionally, platelets from this patient varied markedly in size, contained giant granules, and possessed a poorly defined micro-tubular system. After stimulation with ADP or collagen, centripetal granule migration was retarded, and the aggregates formed were small and loose. Several large proteins were absent from the supernatant fraction of sonicated platelets from the thrombocytopenic patient. Exposure of normal platelets to ethanol in vitro resulted in no detectable change in platelet ultrastructure. The data indicate that the ethanol-related abnormalities of platelet function are due in part to subnormal amounts of intracellular ADP and a deficit in the storage pool of ADP. Additionally, the results suggest that impairment in the release mechanism to the observed defect in the release reaction.
The Significance of Malate Dehydrogenase Isoenzymes in Human Blood Platelets
Authors: Schneider W , Gross R
Published: July 2018

Two MDH isoenzymes were detected in the homogenate of normal human blood platelets. According to their properties the cationic isoenzyme is compartmentalized in the mitochondria, the anionic one belongs to the cytoplasma. In spite of the few mitochondria in human blood platelets the proportion of the cationic enzyme is relatively high.

Both of these enzymes could belong to a transfer system for malate transport across the mitochondrial membrane. As human blood platelets do not contain creatine phosphate a system like that could be of significance for platelet function.
Platelet Adhesion, Release and Aggregation in Flowing Blood: Effects of Surface Properties and Platelet Function.
Authors: Baumgartner Hans R , Muggli Reto , Tschopp Thomas B , Turitto Vincent T
Published: July 2018

Platelet adhesion to natural and artificial surfaces and adhesion-induced aggregation were investigated in vitro using an annular perfusion chamber. The surfaces were exposed to anticoagulated blood under identical flow conditions (~ arterial shear rates). The initial attachment of platelets (contact) appeared less surface specific than spreading and release. Fibrillar collagen was the most powerful inducer of platelet degranulation whereas elastin, microfibrils and epon were virtually inactive. Fibrillar collagen caused release also in the absence of spreading. Surface coverage with platelets did not exceed 25 % unless spreading occurred. Perfusion with platelet-free plasma or platelet-poor blood did not remove adhering platelets. However, platelets were translocated from mural thrombi to the surface by such perfusion. In addition, platelets which detached from mural thrombi adhered more readily to elastin or microfibrils than platelets from the circulating blood. The initial attachment of platelets to subendothelium was inhibited in von Willebrand’s disease, the Bernard-Soulier syndrome and at high concentrations of dipyridamole; spreading was inhibited in storage pool disease of rats, at low temperature (20° C), with EDTA (3 mM) and Prostaglandin E1 (1 μM); and adhesion-induced aggregation was inhibited in thrombasthenia, storage pool disease and after ingestion of sulfinpyrazone or Aspirin.

It is concluded that the initial attachment (contact) of platelets, spreading and surface-induced release of platelet constituents are at least partially independent phenomena, the latter two being highly surface specific. At flow conditions which cause the disappearance of platelet thrombi, platelet adhesion appears as an irreversible process.
Fibrinogen and its Derivatives: Cofactors in the Intrinsic Generation of Thrombin
Authors: Triantaphyllopoulos D.C , Ryan L.T
Published: July 2018

The simultaneous addition of suboptimal concentrations of factor VIII and intact or plas-min-lysed fibrinogen into mixtures of the vitamin K dependent factors, phospholipids, adsorbed bovine serum (supplier of factor V) and calcium, increased the amount of thrombin which was generated three to twenty times over the sum of the amounts which were generated when factor VIII, or fibrinogen, or its derivatives were added separately into the thrombin generating mixture. When factor VIII was added together with both fibrinogen and its derivatives, the amount of thrombin generated was even greater, about 130% larger than the amount which was generated in the presence of equal concentrations of only intact fibrinogen plus factor VIII. Addition of albumin instead of fibrinogen or its derivatives has a similar but significantly lower effect on thrombin generation. It appears, therefore, that both intact fibrinogen and its plasminolytic derivatives, singly or in combination, and to a lesser extent albumin, act as cofactors in the reaction which is regulated by factor VIII.
Studies on Chemically Modified Fibrinogen
Authors: Kloczewiak M , Wegrzynowicz Z , Matthias F.R , Heene D.L , Zajdel Maria
Published: July 2018

Treatment of fibrinogen with maleic acid anhydride renders fibrinogen unclottable depending on the degree of modification of the molecule. According to radioactive studies the release of fibrinopeptides by thrombin or reptilase is undisturbed. The incoagulability is due to inhibition of the polymerization process of fibrinmonomers derived from modified fibrinogen, mainly caused by the increase of electronegative charges upon the fibrinogen molecule. According to discelectrophoretic analysis modified fibrinogen fails to produce fragments D and E following plasmic digestion, however, may be degraded to high molecular weight products. Modified fibrinogen reveals some similarities to abnormal fibrinogens in congenital dysfibrinogenemia with regard to its functional properties.
Enzymatic Removal of ADP from Plasma: Unaltered Platelet Adhesion but Reduced Aggregation on Subendothelium and Collagen Fibrils
Authors: Tschopp Th B. , Baumgartner H.R
Published: July 2018

Subendothelium of rabbit aorta and fibrillar collagen were exposed to citrated human or rabbit blood which was circulated through a perfusion chamber under flow conditions similar to those found in arteries. The resulting platelet adhesion and subsequent formation of platelet micro thrombi on the exposed surfaces were measured in 0.8 μm thick sections by a morphometry technique using light microscopy.

Removal of plasma ADP by the substrate-enzyme combination CP-CPK (creatine phosphate-creatine phosphokinase; 3 mM and 90 U/ml blood) did not affect the initial attachment and spreading of platelets on subendothelium, whereas platelet thrombus formation was strongly inhibited. On free collagen fibrils CP-CPK was much less inhibitory on platelet thrombus formation but platelet adhesion again was not affected. It is concluded that platelet aggregation induced by thrombogenic surfaces in the presence of arterial blood flow is at least partially governed by ADP released from adhering platelets. Platelet adhesion to the examined surfaces, however, does not seem to be mediated by plasma ADP.
The Effects of Therapy with Oestriol Succinate and Ethinyl Oestradiol on the Haemostatic Mechanism in Post-Menopausal Women
Authors: Davies Terence , Fieldhouse Gillian , McNicol George P.
Published: July 2018

The effects on the haemostatic mechanism of oestrogen therapy, given to prevent bone loss in post-menopausal women, have been investigated. Oestriol succinate was given orally to 10 women at a level of 2 mg/day for 1 month and for a further 3 months with incremental increase of 2 mg each month. 6 of the 10 women were subsequently treated with 25 μg/day orally of ethinyl oestradiol. Oestriol succinate therapy resulted in a small increase in the level of factor VII, a decrease in factor VIII concentration and increased sensitivity of platelets to aggregating agents. Ethinyl oestradiol treatment resulted in much more widespread changes with marked increases in coagulation factors VII, VIII, IX and X, decreased levels of antithrombin and dramatic increases in circulating plasminogen levels and euglobulin lysis activity. The data suggested that the nature of oestrogens employed therapeutically is important in determining the qualitative and quantitative effect of oestrogen therapy on components of the haemostatic mechanism.
Blood Coagulation and Fibrinolysis in Thyroid Disease
Authors: Farid Nadir R. , Griffiths Brian L. , Collins John R. , Marshall William H. , Ingram David Wallace
Published: July 2018

Fibrinolytic activity and factor VIII concentration were studied in 30 patients with moderate to minimal hypothyroidism and in 7 patients with hyperthyroidism. In the hypothyroid group, the results were related to serum thyroxine levels, HL-A phenotypes and thyroid autoantibody titres. As serum thyroxine decreased so did factor VIII concentration, however, euglobulin lysis time was correspondingly prolonged. Factor VIII appears to be the most sensitive among coagulation factors to the deterioration of thyroid function tests. There was a significant correlation between the reciprocal of thyroid antibody titres and fibrinolysis; however, there was no relationship between factor VIII concentration or fibrinolysis and a specific HL-A phenotype although the incidence of HL-A8 was increased in the group as a whole.

Euglobulin lysis time was prolonged in 6 out of 7 patients with Graves’ hyperthyroidism. Factor VIII was elevated in only 3 of these patients.
Comparative Sensitivity of Two Hemagglutination Inhibition Immunoassays for Fibrinolytic Degradation Products in Human Serum
Authors: Chi David S. , Chen James P.
Published: July 2018

A new modification of hemagglutination inhibition immunoassay for measuring fibrino-gen/fibrin degradation products (FDP) using glutaraldehyde as a coupling reagent was introduced. This method was compared with the tanned-red-cell hemagglutination inhibition immunoassay (TRCHII). Sheep erythrocytes (SRBC) were sensitized with fragment D or E, respectively. The procedure of sensitizing SRBC is simple and can be achieved within 2 hrs. The sensitized SRBC stored at 4° C for 6 months did not lose their activities. The sensitivities for assaying D and E relative FDP were 0.32 μg/ml, respectively, which were slightly better than those of TRCHII. This represents the first adaptation of glutaraldehyde erythrocyte hemagglutination inhibition immunoassay (GEHII) for the estimation of FDP employing either microtiter plate or test tube. The microtechnique and test tube method were found to be essentially equivalent. The FDP level in the sera of normal individuals and patients suspected of elevation of FDP were assayed by both TRCHII and GEHII. By linear least squares analysis, the results show a significant correlation between these two methods. These results indicate GEHII is a simple, rapid and sensitive immunoassay for FDP in human serum.
Studies on the Action Mechanism of the Antihemostatic Effect of lodopeptides
Authors: Golub A.L , Mende T.J
Published: July 2018

A synthetic iodopeptide having a glutamic acid-diiodotyrosine molar ratio of 1:1 has been shown to be an effective anticoagulant both in vivo and in vitro. Contrasted with heparin the following general conclusions may be made regarding its action. The iodopeptide does not act through the inactivation of thrombin in plasma. Iodopeptide does interact with fibrinogen to form a complex which, in vitro, is not soluble in buffered saline at physiological pH. At pH 8, iodopeptide interacts with fibrinogen to form a soluble complex in the presence of 0.9 % NaCl that is not coaguable either by thrombin or Crotalus venom enzymes. All the available evidence indicates that the fibrinogen to fibrin conversion is not inhibited under these conditions, but that fibrin, once formed, is not able to polymerize due to interference by iodopeptide. Similar results were obtained with heparin in vitro with thrombin-fibrinogen mixtures in the absence of NaCl.

Studies with Russell’s viper venom in native PRP strongly suggest that the iodopeptide also interferes with processes in the early coagulation pathway associated with prothrombin activation.
Alteration of Plasma Antithrombin III Levels in Ischemic Heart Disease
Authors: Yue Robert H. , Gertler Menard M. , Starr Toby , Koutrouby Russell
Published: July 2018

The amount of anti thrombin III in plasma was determined quantitatively in 218 males between 45–60 years of age. The mean antithrombin III value was found to be low in the group with low risk for ischemic heart disease, intermediate in the group with high risk for ischemic heart disease and highest in the group with acute myocardial infarction. Concomitant study of kaolin-activated partial thromboplastin time revealed a sharp decrease in its mean value in the group with acute myocardial infarction. The high correlation between antithrombin III and kaolin-activated partial thromboplastin time for the entire population suggests that the development of ischemic heart disease is a gradual process and that failure of the damping mechanism results as an acute event. These findings may be useful in the determination of the coagulation state of these patients.
Fibrinogen/Fibrin Degradation Products in Serum of Patients with Idiopathic Thrombocytopenic Purpura: Elevated Levels during Severe Thrombocytopenic Phase of the Disease
Authors: Nagasawa Toshiro , Kono Ichiro , Sakurai Tetsushi , Kashiwagi Heihachiro
Published: July 2018

Sera from 23 patients with idiopathic thrombocytopenic purpura (ITP), 14 patients with aplastic anemia with severe thrombocytopenia and healthy control subj ects were tested for the presence of fibrinogen/fibrin degradation products (FDP), using the tanned red cell hemagglutination inhibition immunoassay. The concentrations of circulating FDP of ITP patients (mean 12.01 μg/ml) were significantly higher than those of the patients with aplastic anemia (mean 4.01 μg/ml, p <0.05) or normal controls (mean 3.10 μg/ml, p <0.001). The patients with untreated ITP with very low platelet counts had higher levels of FDP than those of the treated group (p < 0.01). Serum FDP and a battery of other coagulation-fibrinolysis tests were serially carried out over a period of 10 weeks in two patients with ITP. The initially high FDP promptly decreased as circulating platelets increased in response to steroid in both patients, while plasma fibrinogen, euglobulin lysis time, prothrombin time and partial thromboplastin time remained essentially normal during the course of observation. The exact source of the increased serum FDP in ITP was not extablished, but a few possible mechanisms responsible for this abnormality were discussed.
On the Measurement of Spontaneous Platelet Aggregation. The Platelet Aggregation Test III.
Authors: Breddin K , Grun H , Krzywanek H. J , Schremmer W. P
Published: July 2018

A new measuring device was developed for the study of “spontaneous” aggregating activity of thrombocytes. In the photometric platelet aggregation test (PAT III) 0.6 ml of platelet-rich plasma (PRP) are rotated in a disc-shaped cuvette at 20 rpm and 37° C. Changes in optical density of PRP which are induced by the formation of platelet aggregates are continuously registered using a chart recorder. PAT III was developed for the detection of enhanced platelet aggregation, indicating a risk of thrombosis and thromboembolic complications.

In 146 healthy individuals a certain percentage showed slight primary aggregation (α1) which in some cases was followed by marked aggregation (α2) at a certain time (Tr) after the beginning of rotation. The percentage of individuals showing α2 increased with age. An increase of plasma pH in the rotating sample, which was caused by diffusion of CO2, was an important conditioning factor for aggregation. The test results depended on the platelet count in PRP. Aggregation curves were suppressed by admixture of erythrocytes and lipid turbidity. The tendency of platelets to aggregate increased within 60–90 min following blood sampling. During this period the interval to the onset of aggregation (Tr) became shorter and the maximum aggregation speed (α2) increased with time. PAT III yielded reproducible results when it was carried out more than 60 min after blood drawing.

In a group of 327 diabetic patients “spontaneous” aggregation occurred more frequently in all age groups as compared with the controls.

Additional equipment was available for the registration of ADP-, collagen-, or epine-phrine-induced aggregation similar to Born’s and O’Brien’s method. The device can easily be mounted on an Eppendorf photometer without further alterations.
Management of Haemophilia in Greece
Authors: Mandalaki T
Published: July 2018

A short review is given on several aspects of the management of haemophilia in Greece. A total number of 429 patients with congenital haemorrhagic disorders has been registered. The incidence of F. VIII deficiency is 85.9% and that of F. IX 12.9%. Information concerning distribution throughout the country, age, social standard, financial position and education is given too.

Haemophiliacs are systematically treated by Athens specialized Haemophilia Treatment Centres.

The most widely therapeutic material still used, is fresh frozen plasma. Lyophilized cryo-precipitate and F. IX (prothrombincomplex) concentrate are also prepared but they are not for every day use. Imported concentrates are used only for very serious cases.

Home therapy is attempted in limited scale and it is largely discussed.

Consultation, out-patients treatment and hospitalization, if needed, are free of charge for haemophiliacs regardless of their financial position or their national insurance.
Four Heparin Preparations: Anti-Xa Potentiating Effect of Heparin after Subcutaneous Injection
Authors: Johnson E. A , Kirkwood T. B. L , Stirling Yvonne , Perez-Requejo J. L , Ingram G. I. C , Bangham D. R , Brozović Milica
Published: July 2018

Four different heparin preparations - sodium and calcium salts of the same batch of heparin (mean molecular weight 15,000), low molecular weight sodium heparin (mean m. w. 9,000) and high molecular weight sodium heparin (mean m. w. 22,000) were injected subcutaneously on different days each into 6 healthy young volunteers in a randomized trial. Plasma heparin levels were measured using the anti-Xa assay at 1 hour, 3-4 hours and 6-7 hours after the injection. The highest anti-Xa potentiating effect was obtained after the injection of the low molecular weight sodium heparin (mean 0.381 i. u./ml) at 3-4 hours after the injection. With sodium heparin (m. w. 15,000) the highest values (0.135 i. u./ml) were found at 1 hour. Significantly lower anti-Xa potentiating effect was obtained 1 hour after the injection of calcium heparin and in particular after the injection of high molecular weight heparin (mean values 0. 072 i. u./ml and 0.043 i. u./ml respectively). Both these preparations showed an increase from 1 hour after injection to 3-4 hours after injection (mean values 0.082 i. u./ml and 0.057 1. u./ml at 3-4 hours after injection).

These results indicate that the salt and the molecular weight of the preparation may strongly influence the degree of anticoagulation achieved after subcutaneous injection.
Kinetic Characterization of Tissue-Type Plasminogen Activator (t-PA) and t-PA Deletion Mutants
Authors: Vries Carlie de , Veerman Harry , Nesheim Michael E , Pannekoek Hans
Published: July 2018

The binding of t-PA to fibrin is mediated both by its “finger” (F) and its “kringle 2” (K2) domain. In addition, these domains are involved in the stimulation of t-PA activity by fibrin. We analyzed the kinetic characteristics of Glu-plasminogen activation by t-PA and a set of t-PA deletion mutants in the absence and the presence of desA-fibrin. In the absence of desA-fibrin, the activity of t-PA (variants) is determined by the presence of the pro tease domain, irrespective of the composition of the amino-terminal heavy chain. In the presence of the cofactor desA-fibrin, the activity of t-PA (variants) is dependent on the domain composition of the heavy chain. The activity of t-PA is stimulated 2,400 fold by desA-fibrin, whereas the activity of the mutant lacking the K1 domain (del. Kl) increases 936 fold in the presence of this cofactor. Mutants lacking either the K2 domain (del. K2) or the F domain (del. F) exhibit an enhanced activity upon desA-fibrin addition of 200 and 210 fold, respectively. DesA-fibrin has no stimulatory effect on the activity of the mutant containing only the serine-protease domain (del.FE Kl K2) nor on the activity of the variant containing only the Kl domain and the serine-protease domain (del. FE K2). Furthermore, we determined the relative fibrin affinity of each t-PA variant, which is similarly dictated by the composition of the heavy chain.
Fibrinolysis and Coagulation in Patients with Infectious Disease and Sepsis
Authors: Philippé J , Offner F , Declerck P J , Leroux-Roels G , Vogelaers D , Baele G , Collen D
Published: July 2018

Sepsis is often associated with hemostatic dysfunction. This study aimed to relate changes in fibrinolysis and coagulation parameters to sepsis and sepsis outcome. Urokinase-type plasminogen activator (u-PA) antigen, tissue-type plasminogen activator (t-PA) antigen and activity, plasminogen activator inhibitor (PAI) type 1 antigen, PAI activity, antithrombin (AT) III activity, and protein C activity were measured in 24 patients suffering from sepsis or septic shock and the results were compared with those observed in 30 non-sepsis patients with severe infectious disease. The u-PA level was markedly increased in plasma of sepsis patients as compared to non-sepsis patients (11.5 ± 9.4 versus 1.6 ± 1.5 ng/ml, p <0.0001). PAI-1 antigen and t-PA activity showed a significant increase in sepsis patients (320 ± 390 ng/ml versus 120 ± 200 ng/ml, and 3.0 ± 3.6 IU/ml versus 1.0 ± 0.7 IU/ml, respectively, p <0.01). AT III was decreased in sepsis patients (58 ± 28% in sepsis versus 79 ± 26% in severe infectious disease, p <0.01) as was protein C (30 ± 18% versus 58 ± 27%, p <0.001). No significant difference was found for t-PA antigen nor for PAI activity. Nonsurvivors of sepsis were distinguished mainly by a high u-PA antigen level and increased t-PA activity. It is concluded that plasma u-PA antigen showed the strongest significant difference, among the parameters evaluated, between sepsis and severe infection. u-PA antigen may be of prognostic value in patients admitted to the medical intensive care unit for severe infectious disease.
Platelets of Habitual Smokers Have Reduced Susceptibility to Aggregating Agent
Authors: Foo L C , Roshidah I , Aimy M B
Published: July 2018

Platelet aggregation to collagen, and productions of 6-ketoprostagltandin-F1-alpha and tromboxane 82 during aggregation were measured after an overnight fast, involving both food and cigarettros, in 19 clinically healthy habitual smokers (10 or more cigarettes/day) and 23 non-smokers receiving the same diet. The subjects (all males; ages == 21-30 years) were residents of a school hostel. Mean platelet aggregation was significantly lower in smokerr; than non-smokers (23.2 ohms vs 3L.5 ohmS, p < 0.005). Non-smokers had significantly higher mean concentration of 6-keto-prostaglandin-F1-alpha than smokers (109.8 pmol/1 vs 92.3 pmol/1, p < 0.05). The level of thromboxane B2 did not differ significerntly between the two groups. These observations suggest that the role of smokittg as a risk factor for ischaemic heart disease is unlikely to be related to a direct enhancement of aggregation. On the contrary, the observations seem to suggest that habitual smoking may directly reduce platelet aggregability.
Effect of TRK-100, a Stable Orally Active Prostacyclin Analogue, on Platelet Function and Plaque Size in Atherothrombotic Strokes
Authors: Isaka Yoshinari , Handa Nobuo , Imaizumi Masatoshi , Kimura Kazufumi , Kamada Takenobu
Published: July 2018

Patients with carotid atheromatous lesions were prospectively studied with indium-111 platelet imaging, platelet aggregability and B-mode real-time ultrasound tests to determine the shortterm effects of orally active prostacyclin analogue TRK-100 (40 μg, three times daily for 4 weeks). To establish baseline values, all patients underwent indium-111 platelet imaging, platelet aggregation study and B-mode ultrasound. The results were positive for carotid plaque and platelet accumulation. Visual analysis showed repeated platelet scintigrams to be unchanged in five patients without antithrombotic therapy; repeated ultrasound studies showed no change in eight of ten plaques, while one showed progression and one regression of the plaque. In five TRK-100 treated patients, five of seven lesions with platelet accumulation at the baseline became negative, and two remained unchanged during the treatment; repeated B-mode ultrasound tests indicated eight of nine plaques remained unchanged, while one showed plaque size reduction. Quantitative analysis demonstrated that, TRK-L00 significantly reduced the ADP aggregation (1 μM) from 55.2 ± 21.3"/" to 24.0 ± 14.7% (± Sp; p <0.05) and the platelet accumulation index (25.7 ± 17.2% vs L0.4 ± 10.4%; p <0.05). However, there was no significant reduction in plaque scores during TRK-100 therapy compared with the baseline (2.70 ± 2.75 mm vs 2.51 ± 2.58 mm). The data obtained suggested that short-tenn TRK-L00 therapy has an inhibitory effect on platelet accumulation in carotid atheroma but does not cause significant changes in plaque size.
Experimental Studies on a Recombinant Hirudin, CGP 39393
Authors: Gray E , Watton J , Cesmeli S , Barrowcliffe T W , Thomas D P
Published: July 2018

The in vitro anticoagulant activities of recombinant desulphatohirudin (r-hirudin) were studied in the activated partial thromboplastin time (APTT) and the thrombin generation test : systems. In the APTT at concentrations below 5 μg/ml, r-hirudin showed a dose-response curye. At concentrations above 5 μg/ml, the plasma became unclottable, but in the thrombin generation test , at least 10 μg/ml of r-hirudin was required for full inhibition of thrombin generation. The antithrombotic effect was assessed using a rabbit venous stasis model; 150 μg/ml r-hirudin completely prevented thrombus formation at 10 and 20 min stasis. At antithrombotic dose, the mean bleeding time ratio measured in a rabbit ear template model, was not prolonged over control values. At higher doses, the bleeding time ratios were higher than those observed for the same dosage of heparin. These data indicate that while r-hirudin is an effective antithrombotic agent, antithrombotic doses have to be carefully titrated to avoid excessive bleeding.
Changes in Endothelial-Related Goagulation Proteins in Response to Venous Occlusion
Authors: Jennings I , Luddington R J , Harper P L
Published: July 2018

One hundred patients with a history of thrombophilia wer divided into two groups based on fibrinolytic response to venous occlusion. Good responders with a euglobulin clot lysis time ≤ 105 min showed significant release of tPA, PAI and vWE Of the poor responders with an ECIX > 105 min, 24% showed a subnormal increase in tPA, and a significant proportion of these also showed a reduced or absent rise in vWE

We have shown poor fibrinolytic response was related to either raised levels of PAI, poor release of both tPA and vWR or poor release of tPA or vWF alone suggesting different mechanisms of fibrinolytic impairment. Protein S levels were not significantly changed in either group following occlusion.
Stimulation of Plasmin Activity by Aspirin
Authors: Milwidsky Ariel , Finci-Yeheskel Zvendana , Mayer Michael
Published: July 2018

This study demonstrates an enhancing effect of aspirin on the amidolytic activity of plasmin. The stimulation of plasmin by aspirin was concentration-dependent and was attained at aspirin concentrations above 2 × 10−4 M. Aspirin produced a small, reproducible and statistically significant stimulation of the chromogenic activity of plasmin upon H-D-Valyl-L-Leucyl-L-Lysine-p-nitroanilide (3-2251) or pyro-Glu-Gly-Arg-p-nitroanilide (S-2444). Kinetic analysis demonstrated a slight decrease in the affinity of plasmin for substrate 3-2251 in the presence of aspirin, reflected by a change of the Km from 3.2 × 10-4 M to 3.8 × 10-4 M, and an increase of the Vm. The reciprocal Lineweaver-Burk curve indicated an uncompetitive type of stimulation. The stimulatory effect of aspirin was abolished by the lysine analogue α-aminohexanoic acid (AHA) but not by the α-amino acid glutamic acid. The effect of AHA suggests a specific involvement of lysine binding sites (LBS) on plasmin in the interaction of the enzyme with aspirin. Tlansient acidification of plasmin abolished its response to aspirin, to AHA and to their combination. The addition of aspirin to diluted human control or pregnancy plasma in vitro stimulated the plasma-mediated cleavage of the chromogenic substrate3-2251. In contrast to its effect on plasmin, aspirin failed to change the activity of tissue-type or urokinasetype plasminogen activators. It is conceivable that in addition to the antithrombotic effect of aspirin ascribed to its interaction with the platelets, aspirin also directly stimulates plasmin activity.
Role of α2-Antiplasmin in Fibrin-Specific Clot Lysis with Single-Chain Urokinase-Type Plasminogen Activator in Human Plasma
Authors: Declerck P J , Lijnen H R , Verstreken M , Collen D
Published: July 2018

The role of plasma α2-antiplasmin (α2-AP) in the fibrinspecificity of clot lysis by recombinant single-chain urokinase-type plasminogen activator (rscu-PA) and in the conversion of rscu-PA to its two-chain derivative (rtcu-PA, urokinase) was investigated in an in vitro human plasma clot lysis system. Fifty % lysis in 2 h of a 0.1 ml 125l-fibrin labeled human plasma clot immersed in 0.5 ml normal human plasma was obtained with 1.4 ± 0.15 µg/ml rscu-PA (mean ± SD, n = 8). This was associated with degradation of 23 ± 7% of fibrinogen and generation of 0.20 ± 0.09 µg/ml rtcu-PA. In α2-AP-depleted plasrna 50% clot lysis in 2 h required 2-fold less rscu-PA which was associated with 3-fold more extensive fibrinogen degradation and 2-fold more rtcu-PA generation. Fifty % lysis in? h, of a 0.1 ml α2-AP-depleted plasma clot, subriersed in 0.5 ml normal plasma, was obtained with 0.80 ± 0.05 µg/ml rscu-PA (n = 3, p <0.001 vs normal clot) and was associated with 17 ± 6% fibrinogen breakdown (p : 0.22 vs normal clot) and 0.08 ± 0.02 µg/ml rtcu-PA generation (p < 0.05 vs normal clot). In α2-AP-depleted plasma the equipotent rscu-PA concentration was 4-fold lower than in normal plasma and was associated with 3-fold more fibrinogen degradation and a similar extent of rtcu-PA generation

Thus, α2-AP in plasma contributes significantly to the fibrinspecificity of rscu-PA, primarily via prevention of conversion in plasma of rscu-PA to rtcu-PA. Clot associated α2-AP increases the resistance of the clot to lysis with rscu-PA, but plays an only minor role in the fibrin-specificity of clot lysis in normal plasma.
Microbubble-Induced Serotonin Secretion in Human Platelets
Authors: Malmgren R , Thorsen T , Lie R T , Holmsen H
Published: July 2018

The effect of nitrogen (N2) microbubbles on platelets resembles that of common platelet agonists with respect to aggregation (Thorsen T et al., Undersea Biomed Res 1986; 13:289-303). In the present study we examined the effect of microbubbles on platelet secretion of preloaded 14C-serotonin. We demonstrate that stirring of platelet-rich plasma with N2-microbubbles causes a loss of single platelets that is associated with secretion. However, secretion did not increase above baseline values until after 20 min of microbubble exposure, when platelet aggregation had reached 40%. After that time the secretion rate increased. There was no correlation between secreted serotonin and the degree of platelet aggregation. Although no 14C-serotonin secretion occurred in presence of acetylsalicylic acid (ASA), microbubble-induced platelet aggregation was only marginally reduced. Epinephrine alone caused significant platelet aggregation but no 14C-serotonin secretion and it enhanced N2-microbubble-induced platelet aggregation and secretion; ASA completely prevented secretion under these circumstances but failed to abolish the enhancement of aggregation compared with microbubbles alone. Earlier studies have shown that platelets adhere to the bubble surfaces (Thorsen T et al., Undersea Biomed Res 1987; 14:45-59). The results in the present study indicate that non-adhering platelets in the bulk phase are not activated by means of autocrine stimulation through dense granule material.
Characteristics of Thrombin-Degranulated Human Platelets: Development of a Method that Does not Use Proteolytic Enzymes for Deaggregation
Authors: Kinlough-Rathbone Raelene L , Packham Marian A , Guccione Maria A , Richardson Mary , Harfenist Elizabeth J , Mustard J Fraser
Published: July 2018

A method has been developed for preparing suspensions of washed human platelets that have lost as much as 90% of their dense granule and alpha granule contents as a result of stimulation by thrombin (0.9 U/ml for 3 min at 37° C), and recovering the platelets without using a proteolytic erwyme. Glycyl-Lprolyl-Larginyl-L-proline (GPRP) was used to prevent polymerization of released fibrinogen and arginyl-glycyl-aspartyl-serine (RGDS) to block the interaction of released fibrinogen, vWf or fibronectin with the glycoprotein IIb/IIIa complex. The thrombin used to degranulate the platelets was neutralized with D-phenylalanyl-Lprolyl-L-arginine chlororhethyl ketone (FPRCH2CI) and prostaglandin E1 was added to return the platelets towards a disc shape. The degranulated platelets aggregated in response to ADR platelet activating factor, arachidonate and the thromboxane 42 mimetic, U46619 in the presence of added fibrinogen; the platelets changed shape but did not aggregate in response to collagen. Thrombin and the calcium ionophore, A23L87, caused aggregation without added fibrinogen. Synergism between pairs of aggregating agents at low concentrations was observed. Little TXB2 was formed when the platelets were reaggregated by thrombin. RGDS and F(ab’)2 fragments of an antibody to fibrinogen inhibited reaggregation induced by thrombin and A23187 indicating that small amounts of fibrinogen at the platelet surface may support aggregation by strong agonists. Adherence of thrombin-degranulated platelets to a collagen-coated surface was less than for controls, but spreading was more extensive. Electron-microscopic immunogold cytochemistry with anti-human fibrinogen IgG showed numerous gold particles in platelet vacuoles. Thrombin-degranulated platelets can be used to study pathways involved in platelet aggregation without the complicating effects of released granule contents including ADR and to study indirectly the factors released from platelets that contribute to the stability of platelet aggregates.
A Novel Functional Assay of Protein C in Human Plasma and fts Comparison with Amidolytic and Anticoagulant Assays
Authors: Guglielmone H A , Vides M A
Published: July 2018

A simple and fast method for the quantitative determination of protein C activity in plasma is here described. The first step consists in the conversion of protein C in the test sample into activated protein C by means of an activator isolated from Southern Copperhead venom. Subsequently, the degradation of factor Va, in presence of protein C-deficient plasma, is measured by the prolongation of the prothrombin time which is proportional to the amount of protein C in the sample. The dose-response curve showed a linear relationship from 6 to 150% protein C activity and the inter- and intra-assay reproducibility was 3.5% and 5.6% respectively. In normal subjects, a mean of protein C level of 98 ± 15% of normal pooled plasma was found. Comparison with the anticoagulant assay in samples of patients with oral anticoagulant, liver cirrhosis, disseminated intravascular coagulation and severe preeclampsia revealed an excellent correlation (r = 0.94, p <0.001). Also, a similar correlation (r = 0.93, p <0.001) existed between amidolytic assay and the method here proposed for all the samples studied without including the oral anticoagulant group. These results allowed us to infer that this method evaluates the ability of protein C to interact with protein S, phospholipids, calcium ions and factor Va.
Modulation of Protein C Inhibitor Activity by Histidine-Rich Glycoprotein and Platelet Factor 4: Role of Zinc and Calcium Ions in the Heparin-Neutralizing Ability of Histidine-Rich Glycoprotein
Authors: Kazama Yoshiaki , Koide Takehiko
Published: July 2018

Effects of zinc and calcium ions on the heparin-neutralizing abilities of histidine-rich glycoprotein (HRG) and platelet factor 4 (PF4) were examined. Both HRG and PF4 effectively neutralized the ability of heparin to accelerate the activated protein C (APC) and the thrombin inhibitions by protein C inhibitor (PCI). The heparin-neutralizing ability of HRG in the APC inhibition by PCI, however, was decreased in a Ca2+-dependent manner and apparently lost at 1 mM Ca2+, while it was enhanced by Zn2+ regardless of the presence or absence of Ca2+. The heparinneutralizing ability of HRG in the thrombin inhibition by PCI was not affected by Ca2+.In contrast to HRG, there was no significant difference in the heparin-neutralizing ability of PF4 in the presence or absence of 1 mM Ca2+. These results strongly suggest additional physiological functions of HRG and PF4 as modulators of PCI.
In Vivo Characterization of a New Synthetic Thrombin Inhibitor
Authors: Knabb Robert M , Kettner Charles A , Timmermans Pieter B M W M , Reilly Thomas M
Published: July 2018

We have examined the in vivo pharmacology of DuP 714 (Ac-[D]-Phe-Pro-boroArginine), a representative of a new series of synthetic thrombin inhibitors which contain a boronic acid derivative of arginine. Intravenous bolus injections of DuP 714 in anesthetized rats and conscious rabbits produced transient elevations of clotting times. Clinically relevant prolongations of the APTT were also observed in rabbits after i. v. infusion of less than 0.1 mg kg-1 h-1. Efficacy against venous thrombosis was demonstrated in a rabbit model of stasis induced thrombosis. Clots formed in 100% of control animals and only 33% of animals treated with 0.5 mg/kg DuP 714, and were less severe in treated animals. In a rabbit arterial-venous shunt model mimicking arterial thrombosis, occlusion occurred within 30 min in 72% of control animals vs. 11% of animals treated with 0.1 mg kg-1 h-1 DuP 714. Results indicate that DuP 714 is a highly effective anticoagulant which should be useful for the prevention of both venous and arterial thrombotic diseases.
Daytime Fluctuations of Plasminogen Activator Inhibitor 1 (PAI-1) in Populations with High PAI-1 Levels
Authors: Juhan-Vague I , Alessi M C , Raccah D , Aillaud M F , Billerey M , Ansaldi J , Philip-Joet C , Vague P
Published: July 2018

The mechanism underlying diurnal variations in PAI-1 as well as the cellular origin of PAI-1 in subjects with high PAI-1 levels are unknown. We evaluated diurnal changes (8:00 am vs 4:00 pm) in PAI-1 (functional and immunological assays), t-PA Ag and t-PA/PAI-1 complex levels in controls and subjects with high PAI-1 levels. Three test groups were recruited among obese hyperinsulinmic subjects, emergency care unit patients with inflammatory syndrome or infection and pregnant women.

The classical afternoon decrease of PAI-1 level was observed in controls and obese subjects but its amplitude was greater in the latter. The decrease in t-PA Ag and t-PA/PAI-1 complex levels was the same in controls and in obese. As, in previous studies, elevated PAI-1 levels have been correlated with insulin resistance and a decrease in insulin sensibility has been described in the early morning, it is proposed that this “dawn phenomenon” could be implicated in the circadian variations of PAI-1 in controls and could be amplified in obese subjects. Great variability in PAI-1, t-PA Ag or t-PA/PAI-1 complex levels was observed in patients with acute inflammatory syndrome or infection for whom classical biorhythms are suppressed. No diurnal changes in PAI-1 and other fibrinolytic parameters were observed in patients with inflammatory syndrome or in pregnant women suggesting that other sources and/or other regulatory mechanisms of PAI-1 production are involved.
An Enzyme-Linked Immunosorbent Assay for Urokinase-Type Plasminogen Activator (u-PA) and Mutants and Chimeras Containing the Serine Protease Domain of u-PA
Authors: Declerck Paul J , Van Keer Leen , Verstreken Maria , Collen Désiré
Published: July 2018

An enzyme-linked immunosorbent assay (ELISA) for quantitation of natural and recombinant plasminogen activators containing the serine protease domain (B-chain) of urokinase-type plasminogen activator (u-PA) was developed, based on two murine monoclonal antibodies, MA-4D1E8 and MA-2L3, raised against u-PA and reacting with non-overlapping epitopes in the B-chain. MA-4D1E8 was coated on microtiter plates and bound antigen was quantitated with MA-2L3 conjugated with horseradish peroxidase. The intra-assay, inter-assay and inter-dilution coefficients of variation of the assay were 6%, 15% and 9%, respectively. Using recombinant single-chain u-PA (rscu-PA) as a standard, the u-PA-related antigen level in normal human plasma was 1.4 ± 0.6 ng/ml (mean ± SD, n = 27).

The ELISA recognized the following compounds with comparable sensitivity: intact scu-PA (amino acids, AA, 1 to 411), scu-PA-32k (AA 144 to 411), a truncated (thrombin-derived) scu-PA comprising A A 157 to 411, and chimeric t-PA/u-PA molecules including t-PA(AA1-263)/scu-PA(AA144-411), t-PA(AA1-274)/scu-PA(AA138-411) and t-PA(AA87-274)/scu-PA(AA138-411). Conversion of single-chain to two-chain forms of u-PA or inhibition of active two-chain forms with plasminogen activator inhibitor-1 or with the active site serine inhibitor phenyl-methyl-sulfonyl fluoride, did not alter the reactivity in the assay. In contrast, inactivation with α2-antiplasmin or with the active site histidine inhibitor Glu-Gly-Arg-CH2Cl resulted in a 3- to 5-fold reduction of the reactivity. When purified scu-PA-32k was added to pooled normal human plasma at final concentrations ranging from 20 to 1,000 ng/ml, recoveries in the ELISA were between 84 and 110%.

The assay was successfully applied for the quantitation of pharmacological levels of scu-PA and t-PA(AA87_274)/scu-PA(AA138-411) in plasma during experimental thrombolysis in baboons.

Thus the present ELISA, which is specifically dependent on the presence of the serine protease part of u-PA, is useful for measurement of a wide variety of variants and chimeras of u-PA which are presently being developed for improved thrombolytic therapy.
Analysis of the Defective Signal Transduction Mechanism through the Platelet Thromboxane A2 Receptor in a Patient with Polycythemia Vera
Authors: Ushikubi Fumitaka , lshibashi Takafumi , Narumiya Shu , Okuma Minoru
Published: July 2018

We previously reported a patient with polycythemia vera whose platelets showed subnormal responses to thromboxane A2 (TXA2) (Thromb Haemostas 1987; 57: 158-64). The patient’s platelets showed normal binding activity to TXA2 analogues but the generation of second messengers was defective. We further studied the mechanism of this dysfunction in this work. The coupling of TXA2 receptor with its relevant GTP binding protein was examined using STA2(a synthetic TXA2 mimetic)-induced augmentation of GTPase activity as its measure. Only subnormal increase in the GTPase activity of the patient’s platelet membrane was found after STA2 stimulation. Down regulation of TXA2 receptor of the patient’s platelet also showed abnormal behavior. These results suggested that the defective coupling of TXA2 receptor with GTP binding protein could be included as a cause of defective signal transduction in this patient’s platelets. This defect might be due to the abnormality of TXA2 receptor itself if the receptor was down regulated by its phosphorylation through agonist-induced conformational change.
Human Platelets Contain Scinderin, a Ca2+-Dependent Actin Filament-Severing Protein
Authors: Rodríguez Del Castillo A , Vitale M L , Tchakarov L , Trifaró J-M
Published: July 2018

A large body of biochemical and morphological evidence suggests that actin polymerizes in response to various stimuli which activate platelets. Previous work has shown the presence in platelets of gelsolin, a Ca2+-dependent regulator of actin filament length. This present work demonstrates that human platelets contain scinderin, another Ca2+-dependent actin filament-severing protein recently discovered in our laboratory. Extracts prepared from platelets were subjected to DNase-I-Sepharose 4B affinity chromatography. EGTA eluates from the affinity columns contained scinderin as demonstrated by mono and two-dimensional polyacrylamide gel electrophoresis and immunoblotting with scinderin antibodies. The concentration of scinderin in platelets was 75 fmol/mg total protein. This might represent 11% of the total actin filament-severing activity if both proteins are equally potent, on a molar basis, in severing actin filaments. Double staining immunocytochemical studies with antibodies against scinderin and rhodamine phalloidin, a probe for F-actin, also demonstrated the presence of scinderin in platelets. These findings suggest that scinderin may participate in the regulation of platelet actin networks.
Normalization of the Haemostatic Plugs of Dogs with Haemophilia A (Factor VIII Deficiency) Following the Infusion of a Combination of Factor Xa and Phosphatidylcholine/Phosphatidylserine Vesicles
Authors: Ni Hong yu , Giles Alan R
Published: July 2018

The bolus intravenous infusion of factor Xa in combination with phosphatidylcholine/phosphatidylserine (PCPS) vesicles, at a dose of 2.6 × 10-11 moles and 4.0 × 10-8 moles/kg body weight respectively, has previously been shown to correct the bleeding diathesis of haemophilic (factor VIII deficient) dogs (Br J Haematol 1988; 69: 491-7). The cuticle bleeding time (CBT) was used as the test to evaluate objectively this response. Transmission electron microscopy was performed to document the evolution of haemostatic plugs forming in the vascular bed of the injured cuticles of both normal and factor VIII deficient dogs with and without treatment with factor Xa/PCPS. The dosage previously shown to normalize the CBT in haemophilic and significantly shorten it in normal animals was used. Subjective and objective observations, using morphometric techniques, were made over a period of 25 min following injury induction. The administration of factor Xa/PCPS was associated with complete and sustained normalization of haemostatic plug development in the haemophiliacs including platelet recruitment, activation and compaction and subsequently fibrinous transformation. In the case of the normals, platelet activation, etc., was exaggerated and fibrinous transformation appeared to be accelerated. The haemostatic plugs forming in the treated haemophiliacs were indistinguishable from those forming in the normals and significantly different, with regard to all parameters measured, from the morphological appearances noted in the untreated haemophiliacs. These data suggest that the correction of the haemostatic defect previously observed results from the normalization of haemostatic plug formation and that this is sustained despite the promotion of systemic fibrinolysis that is also known to occur.