bluuebunny/crossref_metadata_2025 huggingface dataset:
The Relative Frequency of Hereditary Thrombotic Disorders Among 107 Patients with Thrombophilia in Israel
Authors: Ben-Tal O , Zivelin A , Seligsohn U
Published: July 2018

Since most patients with thrombophilia in Israel are referred for diagnosis to our center, it was possible to estimate the relative frequency of the hereditary disorders leading to thrombophilia. 107 unrelated patients were evaluated over 4 years. Diagnoses were established in 23 patients (21.5%) while in 84 (78.5%) no abnormality was detected. Antithrombin III deficiency was found in 8 patients (7.5%), dominant protein C deficiency in 6 (5.6%), recessive homozygous protein C deficiency in 1, protein S deficiency in 3 (2.8%) and dysfibrinogenemia in 1. Four additional patients (3.7%) had a lupus anticoagulant. The frequency of deep vein thrombosis and pulmonary embolism was similar in patients with and without a definite diagnosis. Thrombosis of visceral or cerebral vessels and a positive family history were more frequent among patients in whom a definite diagnosis was made. In both groups there was a substantial lag between the time of presentation of the first thrombotic episode and the time of evaluation. Since the number of referred patients with thrombophilia has gradually increased over the period of the study, it is at present impossible to establish the prevalence of the various hereditary disorders leading to thrombophilia in the population.
Low Molecular Weight Heparin Novo (LHN-1) Does Not Cross the Placenta During the Second Trimester of Pregnancy
Authors: Omri A , Delaloye J F , Andersen H , Bachmann F
Published: July 2018

Unfractionated heparin (UF-H) has been the drug of choice for the treatment of thromboembolic disorders during pregnancy. Low molecular weight heparin (LMW-H) preparations may present some advantages over UF-H. They have longer half-lives and a better bioavailability after subcutaneous (s. c.) injection and may cause less bleeding. It has not yet been established whether LMW-H Novo (LHN-1) crosses the placenta. 17 women admitted for abortion during the second trimester of pregnancy (induced by application of prostaglandine PGE2 gel at a concentration of 0.25 mg/ml into the cervix) were given s. c. 35 anti-Xa units per kg of body weight of LHN-1 (Novo). 10 patients not receiving LHN- 1 and their fetuses served as a control group. 7 women in whom the time interval between injection of LHN-1 and expulsion of the fetus was less than 3 h or more than 7 h were excluded from further study. In one fetus blood collection failed. Anti-Xa and anti-IIa levels increased approximately ten-fold in women receiving LHN-1 [anti-Xa units/ml from 0.02 ± 0.01 (mean ± SD) to 0.17 ± 0.01, p <0.001; anti-IIa units/ml from less than 0.01 ± 0.01 to 0.07 ± 0.03], but remained below the detection limit in their fetuses as well as in the women and fetuses of the control group.

We conclude that LHN-1 at these doses does not cross the placenta during the second trimester of pregnancy to suggest that LHN-1 may be a safe alternative to heparin in the management of the thromboembolic complications during pregnancy.
A Comparison between Effects of Estradiol Valerate and Low Dose Ethinyl Estradiol on Haemostasis Parameters
Authors: Lindberg U B , Crona N , Stigendal L , Teger-Nilsson A C , Silfverstolpe G
Published: July 2018

Ethinyl estradiol (EE) is still used to some extent as hormonal replacement therapy (HRT) in the climacteric period. As regards oral contraception, it is well known that the induced increase in cardiovascular disease is related to the estrogen component (invariably EE) in a dose-related fashion. Considerably lower doses of EE are needed in HRT compared to oral contraception.

To delineate and compare effects of EE and estradiol valerate (E2V) in doses needed in HRT on haemostasis parameters, 24 postmenopausal women were engaged in a study with an open cross-over design. The doses compared (10 μg EE and 2 mg E2V daily) are the lowest which eliminate climacteric symptoms in a majority of women. Unlike E2V, EE caused increased levels of factor VII: Ag, factor VIII: C and β-thromboglobulin, which may be changes towards hypercoagulability. Both estrogens decreased the AT III activity. Long-term administration (6 + 12 w) of the estrogens induced further changes in haemostatic parameters. 10 μg EE increased factor VII: Ag in contrast to 2 mg E2V. Furthermore both estrogens increased factor VIII :C and factor II-VII-X. A decrease in platelet count was induced by both EE and E2V.

Oral contraception and adjuvant estrogen therapy in men with prostatic carcinoma are known to imply an increased cardiovascular risk. It is noteworthy that the pattern of changes in haemostatic parameters induced by as little as 10 μg of EE is the same as seen after the administration of combined oral contraceptives or the substantially higher doses of EE given as adjuvant therapy to men with prostatic carcinoma.
Recombinant Desulphatohirudin (CGP 39393) Anticoagulant and Antithrombotic Properties In Vivo
Authors: Talbot M D , Ambler J , Butler K D , Findlay V S , Mitchell K A , Peters R F , Tweed M F , Wallis R B
Published: July 2018

The effects of the newly available biotechnology product, recombinant desulphatohirudin (CGP 39393) have been investigated in rats. This highly potent and selective thrombin inhibitor exhibited marked anticoagulant properties with controllable titration of anticoagulant effect, as measured by activated partial thromboplastin time (APTT), up to nearly four times control values. Furthermore, CGP 39393 exhibited impressive antithrombotic activity in vivo. In an arteriovenous shunt model of thrombus formation on a cotton-thread, the compound was capable of complete inhibition of thrombus development (ED50 = 0.3 mg/kg i.v. and 1.0 mg/kg s.c.). Venous stasis thrombosis was also highly susceptible to inhibition by CGP 39393 (ED50 = 0.01 mg/kg i.v. and 0.45 mg/kg s.c.). Comparison of the anticoagulant and antithrombotic activities of the compound shows that potent antithrombotic effects (83-97% inhibition in the rat shunt model) are achieved within the generally acceptable range of anticoagulation. These results suggest a clear potential for this new agent in the clinical treatment of thrombotic disease.
Functional Assays for Protein C Activity and Protein C Inhibitor Activity in Plasma
Authors: Geiger Margarethe , White Teresa M , Griffin John H
Published: July 2018

An assay system for protein C (PC) activity and PC-inhibitor in plasma was developed. The assay was based on: (1) binding of PC to wells of a microtiter plate coated with a murine monoclonal anti-PC antibody (C3) that did not interfere with the activity or activation of PC; (2) activation of immobilized PC with Protac C; (3) incubation with or without a source of activated PC inhibitor; and (4) measurement of amidolytic activity using the substrate S-2366. The activity assay was specific for PC and sensitive to <1 μl of plasma or 4 ng PC. Inhibition of activated PC by plasma followed pseudo first order kinetics. Heparin caused a dose dependent increase in the inhibition rate with half maximal stimulation at approximately 3 U/ml and maximal stimulation at heparin concentrations ≥10 U/ml. This assay is suitable not only for determination of functional plasma levels of PC and PC inhibitor activities but also for kinetic studies of inhibition of activated PC in complex systems, such as plasma. Studies showed that urokinase interfered with the inhibition of APC by plasma inhibitor(s).
Endothelial Prostacyclin Release in Systemic Lupus Erythematosus
Authors: Coade Stephen B , Haaren Els van , Loizou S , Walport Mark J , Denman A Michael , Pearson Jeremy D
Published: July 2018

The ability of sera from patients with SLE to stimulate endothelial cell prostacyclin production was studied using a standardized assay system for testing the effects of serum on cultured human endothelial cell monolayers. The effects of 20 normal and 32 SLE sera on endothelial prostacyclin production were measured. No differences between the rates of prostacyclin production were seen between the two groups, either basally or when prostacyclin release was stimulated with thrombin or bradykinin. In the SLE samples there was no correlation between anticardiolipin IgG or IgM titres and their ability to stimulate basal or agonist-induced prostacyclin release. These results suggest that the elevated risk of thrombosis in SLE patients is not associated with inhibition of endothelial cell prostacyclin synthesis.
Thromboxane A2 and Prostacyclin Generation in the Microvasculature of Patients with Atherosclerosis – Effect of Low-Dose Aspirin
Authors: Kyrle P A , Minar E , Brenner B , Eichler H G , Heistinger M , Marosi L , Lechner K
Published: July 2018

Generation of thromboxane A2 (TxA2) and prostacyclin (PGI2) at the site of platelet-vessel wall interaction, i.e. in blood emerging from a standardized injury of the microvasculature made to determine skin bleeding time was investigated in 7 patients with atherosclerosis (angiographically verified obstructions of the femoral arteries) and in 7 normal control subjects apparently free of atherosclerotic lesions. Similar amounts of TxA2 (measured as thromboxane B2, TxB2) were generated at the site of plug formation in the patients with peripheral vascular disease (PVD) and in the control subjects. Significantly lower levels of PGI2 (measured as 6-keto-prostaglandin F1α, 6-keto- PGF1α) were found in blood from an injury of the microvasculature in the patients compared with the controls. These data do not suggest a major role of the platelet prostaglandin metabolism in the development of atherosclerosis. However, decreased synthesis of PGI2 by endothelial cells might contribute to the development and/or progression of atherosclerotic lesions. In the patients with PVD, low-dose aspirin (50 mg/day for 7 days) resulted in a >90% inhibition of the TxB2 production at the site of plug formation. Following low-dose aspirin 6-keto-PcF1α levels were below 20 pg/ml (limit of sensitivity of our radioimmunoassay procedure) in the majority of the samples.

We therefore conclude that in patients with PVD a decreased synthesis of PGI2 by endothelial cells might contribute to the progression of atherosclerosis. Furthermore, low-dose aspirin treatment results in a similar inhibition of the platelet prostaglandin generation as recently observed in healthy subjects.
Pharmacokinetics and Haemostatic Status During Consecutive Infusions of Recom binant Tissue-Type Plasminogen Activator in Patients with Acute Myocardial Infarction
Authors: Seifried E , Tanswell P , Ellbrück D , Haerer W , Schmidt A
Published: July 2018

Pharmacokinetics and systemic effects of recombinant tissue type plasminogen activator (rt-PA) were determined during coronary thrombolysis in 12 acute myocardial infarction patients using a consecutive intravenous infusion regimen. Ten mg rt-PA were infused in 2 minutes resulting in a peak plasma concentration (mean ±SD) of 3310±950 ng/ml, followed by 50 mg in 1 h and 30 mg in 1.5 h yielding steady state plasma levels of. 2210±470 nglml and 930±200 ng/ml, respectively. All patients received intravenous heparin. Total clearance of rt-PA was 380±74 ml/min, t,½α was 3.6±0.9 min and t,½β was 16±5.4 min.

After 90 min, in plasma samples containing anti-rt-PA-IgG to inhibit in vitro effects, fibrinogen was decreased to 54%, plasminogen to 52%, α2-antiplasmin to 25%, α2-macroglobulin to 90% and antithrombin III to 85% of initial values. Coagulation times were prolonged and fibrin D-dimer concentrations increased from 0.40 to 2.7 μg/ml. It is concluded that pharmacokinetics of rt-PA show low interpatient variability and that its short mean residence time in plasma allows precise control of therapy. Apart from its moderate effect on the haemostatic system, rt-PA appears to lyse a fibrin pool in addition to the coronary thrombus.
Indirect Activation of Blood Coagulation in Colon Cancer
Authors: Wojtukiewicz Marek Z , Zacharski Leo R , Memoli Vincent A , Kisiel Walter , Kudryk Bohdan J , Rousseau Sandra M , Stump David C
Published: July 2018

Systemic activation of the coagulation mechanism is known to exist in patients with colon cancer. The mechanism of such activation was investigated using immunohistochemical techniques applied to fresh frozen sections of resected primary colon cancer specimens. Tumor cells stained for tissue factor, factor V, and urokinase-type plasminogen activator. Perivascular and intercellular areas stained for fibrinogen and the “a” subunit of factor XIII. Staining was minimal or absent for protein C, protein S, plasminogen activator inhibitors 1-3, factor VII, factor X, and fibrin (the antigenic site on the amino-terminal portion of Bβ chain that is exposed following thrombin cleavage of fibrinopeptide B was not detected). The lack of an intact thrombin- generating pathway in situ associated with viable colon cancer cells is consistent with the findings of others that coagulation activation in colon cancer may be triggered by a soluble tumor product that exerts its effect at sites distant from the tumor. These results may explain the absence of clinical responsiveness of colon cancer to antithrombotic drug therapy and may clarify therapeutic strategies for this common tumor.
Effect of Retraction on the Lysis of Human Clots with Fibrin Specific and Non-Fibrin Specific Plasminogen Activators
Authors: Sabovic M , Lijnen H R , Keber D , Collen D
Published: July 2018

The effect of the serum content of human clots on their sensitivity to lysis with plasminogen activators was studied in a system composed of 125I-fibrin labeled clots immersed in buffer or in citrated plasma. The effect was studied with plasma clots before or after mechanical compression and with whole blood clots before or after retraction, using either the fibrin specific plasminogen activators recombinant tissue-type plasminogen activator (rt-PA) or recombinant single chain urokinase-type plasminogen activator (rscu-PA), and the non-fibrin specific activators recombinant two chain urokinase-type plasminogen activator (rtcu-PA), or streptokinase (SK).

In a buffer milieu, all plasminogen activators had a similar fibrinolytic potency towards serum-rich clots (non-compressed plasma clots or non-retracted blood clots): 50% clot lysis in 4 h required 50 to 85 ng plasminogen activator per ml. Serum-poor clots (compressed plasma clots or retracted blood clots) were resistant to lysis in a buffer milieu but became sensitive to lysis following preincubation in plasma for 48 h. These findings indicate that plasma proteins entrapped in clots contribute significantly to their sensitivity to lysis and suggest that the amount of bound or entrapped plasminogen may be a limiting factor. In a plasma milieu, all plasminogen activators lysed serum- rich plasma or blood clots, albeit at higher concentrations (3 to 40 times higher than in the buffer milieu) and with different efficiencies: 50% clot lysis in 4 h required approximately 600 ng/ ml of rtcu-PA but 1,500 to 2,000 ng/ml of rscu-PA. These findings suggest that components of plasma are responsible for increased resistance of clots towards lysis and that the effect is variable for different plasminogen activators. Serum-poor plasma or blood clots were very resistant to lysis with non-fibrin specific agents, but became more sensitive after preincubation in plasma. However, serum-poor plasma or blood clots were sensitive to lysis with fibrin specific plasminogen activators, suggesting that during clot lysis with fibrin specific agents, plasminogen recruited from surrounding plasma may contribute significantly to clot lysis. The concentration of plasminogen activator required to obtain 50% clot lysis in a plasma milieu of compressed plasma clots or retracted blood clots was 390 and 1,600 ng/ml respectively for rt-PA and 1,100 and 3,200 ng/ml respectively for rscu-PA. These data suggest that in a plasma milieu retracted blood clots are more sensitive to lysis with fibrin specific plasminogen activators than with non-fibrin specific agents.
Time Dependence of Aequorin-lndicated Calcium Levels in Stimulated and Unstimulated Platelets: Evidence for Multiple Aequorin Environments in Platelets
Authors: Lages Bruce , Weiss Harvey J
Published: July 2018

Aequorin-induced calcium signals were examined in human unstimulated platelets and platelets stimulated with various agonists as a function of time. The total aequorin response in unstimulated platelets, obtained by Triton x-100 lysis in the presence of 1 mM Ca, decreased in a distinctly non-linear manner over 20-60 min. This decrease was slightly, but significantly, greater in platelets maintained in the continuous presence of 1 mM Ca than in platelets maintained without external Ca, and could not be accounted for completely by leakage of aequorin from the cells. Basal Ca levels in unstimulated platelets also decreased in a non-linear manner, with a similar sensitivity to the contmuous presence or absence of external Ca. These observed changes in aequorin response thus appear to be at least partially due to an mtracellular discharge of aequorin, and are therefore consistent with the view that aequorin in platelets is heterogeneously d1stnbuted among localized environments differing in Ca concentration. The aequorin signals observed initially in platelets stimulated by ADP or epinephrine were lost completely over a penod of 30-60 min in almost all cases studied, while initial rates of aggregation were either unchanged (epinephrine) or only partially decreased (ADP) over this same time period. In contrast, thrombin- and A23187-induced aequorin signals were virtualy unchanged over periods up to 90 min. Minimal changes with time also occurred in the aequorin signals induced by phorbol ester or by collagen in the presence of indomethacin. These differences in time dependence suggest that the signals generated by ADP and epinephrine may derive from different sources of aequorin than those associated with the signals induced by other agonists. Thus, differences in indicator localization may indeed contnbute to the observation of aequorin, but not quin 2/fura-2, signals following epinephrine stimulation as previously suggested, but this may not be the sole explanation for the discrepancies between these two Ca indicators seen with other agonists.
Platelet Mobilization Induced by PAF and Its Role in the Thrombocytosis Triggered by Adrenaline in Rats
Authors: Castro-Faria-Neto Hugo C , Bozza Patricia T , Martins Marco A , Dias Paulo M F L , Silva Patricia M R , Cordeiro Renato S B
Published: July 2018

The injection of PAP (6 μg/kg, i. v.) induced, in rats, haemoconcentration accompanied by an increase in the platelet number, as attested by the counts of platelets in blood samples diluted in formalin-free EDTA solution. This increase was significant at 15 min, peaked from 1 to 4 h and returned to basal levels 24 h after the lipid administration. The release of platelets induced by PAP was inhibited dose-dependently by specific PAP receptor antagonists such as WEB 2086 (0.5-2 mg/kg), BN 52021 and 48740 RP (5-25 mg/kg). Furthermore, platelet mobilization was clearly impaired in splenectomized animals stimulated by PAP, whereas thrombocytopenia and haemoconcentration by the same stimulus were intact. It was also noted that a second injection of PAP, 24 h after the initial stimulation with the lipid, failed to induce an increase in platelet counts, indicating autodesensitization. Desensitization to PAP or pretreatment with PAP antagonists clearly prevented the increase in the platelet counts after stimulation by adrenaline (15 μg/kg). These findings suggest that, in rats, PAP can induce release of platelets by a spleen-dependent mechanism and that this lipid may be relevant to the thrombocytosis triggered by adrenaline.
A Randomised Trial of Subcutaneous Low Molecular Weight Heparin (CY 216) Compared with lntravenous Unfractionated Heparin in the Treatment of Deep Vein Thrombosis
Authors: Ninet J , Bachet Ph , Prandoni P , Ruol A , Vigo M , Barret A , Mericq O , Boneu B , Janvier G , Duroux P , Girard Ph , Laprevote-Heully M C , Sourou P , Robert D , Chagny M , Nenci G , Agnelli G , d’Addato M , Palumbo H , Bensaid J , Gouffault J , Leborgne P , Hellocco A Le , Ducreux J C , Tempelhoff G , Sala-Planell E , Rosendo-Carrera A , Torres-Gomez A , Blettery B , Bachmann F , Gaux J C , Muntlak H , Caulin Ch , Bergmann J F ,   A Collaborative European Multicentre Study
Published: July 2018

The standard treatment of deep vein thrombosis is given by continuous intravenous infusion of unfractionated heparin. This entails hospitalisation, nursing care, immobility and repeated laboratory tests (e.g. activated partial thromboplastin time [APTT], platelet count). In addition approximately 10% of patients suffer major haemorrhages. The potential advantages of a low molecular weight heparin (CY 216) given subcutaneously were explored in a randomised trial with blind quantitative evaluation of venograms. The study included 166 patients and both “therapeutic efficacy” and “intention to-treat” analyses showed that subcutaneous CY 216 in fixed doses based only on body weight was more effective on the Arnesen and Marder phlebographic scores than continuous i. v. standard heparin with daily dose adjustment according to results of coagulation tests. There was no increase in the risks of pulmonary embolism, haemorrhage or clot extension.
Decreased Fibrinolysis in Reperfused Ischaemic Tissue
Authors: Cherry G. W , Ryan T. J , Ellis J. E
Published: July 2018

The fibrinolytic activity of skin flaps subjected to ischaemia and reperfusion was studied in five pigs. Total interference with the blood supply up to 24 hours did not in itself lead to changes in fibrinolytic activity. However, reperfusion of the skin after six hours of ischaemia caused a dramatic reduction in fibrinolytic activity measured 18 hours later.

Similar studies on reperfused femoral artery and vein after six hours of ischaemia showed marked reduction in fibrinolytic activity of the vessels.

It is suggested that reperfusion of tissue made ischaemic for six hours is damaging and might be a contributory factor to vascular pathology.
Vessel Wall and Thrombogenesis - Endotoxin
Authors: McKay Donald G
Published: July 2018

The generalized Shwartzman reaction is a definitive disease process which is the result of two minor episodes of acute inflammation of the circulating blood. The identifying feature of the reaction is thrombosis of the micro circulation, specifically of glomerular capillary thrombosis which persists long enough to cause bilateral renal cortical necrosis. The reaction is the result of an interplay between leukocytes, platelets, complement, the contact system, the extrinsic prothrombin activator system, adrenal glucocorticoids, adrenal catecholamines, alpha - adrenergic receptor sites and the fibrinolytic enzyme system. The reaction occurs only when these systems are affected within narrow quantitative limits. It is a model of the effects of bacterial endotoxin on the blood-vascular system and has important implications for acute inflammatory episodes of the circulating blood induced by other inflammatory agents such as antigen-antibody complex and particulate matter.
Studies on Thrombolysis with Streptokinase
Authors: Gottlob R , El Nashef B , Donas P , Piza F , Kolb R
Published: July 2018

The patterns of fibrin and the contents of plasminogen, of α1 antitrypsin and of a2-macroglobulin were studied in whole blood clots before and after retraction and in arterial and venous thrombi of various age by an indirect immunofluorescent method.

By reaction with antifibrinogen a network of fibrin fibers was displayed in the first week. The fibers were condensed to form trabecula in the first months. After several months the reaction with antifibrinogen was reduced or abolished even in non organized thrombi.

In clots and thrombi the plasminogen content is lost during retraction, However, a considerable increase is observed in thrombi up to several months old.

The plasma plasmin inhibitors are only partially expressed during retraction. Later a considerable increase may occur.

The relevance of these findings for thrombolysis is discussed.
Enhancement of ADP-induced Platelet Aggregation by Adrenaline in Vivo and Its Prevention
Authors: Yamazaki Hiroh , Kobayashi Itsuro , Sano Tadahiro , Shimamoto Takio
Published: July 2018

The authors previously reported a transient decrease in adhesive platelet count and an enhancement of blood coagulability after administration of a small amount of adrenaline (0.1-1 µg per Kg, i. v.) in man and rabbit. In such circumstances, the sensitivity of platelets to aggregation induced by ADP was studied by an optical density method. Five minutes after i. v. injection of 1 µg per Kg of adrenaline in 10 rabbits, intensity of platelet aggregation increased to 115.1 ± 4.9% (mean ± S. E.) by 10∼5 molar, 121.8 ± 7.8% by 3 × 10-6 molar and 129.4 ± 12.8% of the value before the injection by 10”6 molar ADP. The difference was statistically significant (P<0.01-0.05). The above change was not observed in each group of rabbits injected with saline, 1 µg per Kg of 1-noradrenaline or 0.1 and 10 µg per Kg of adrenaline. Also, it was prevented by oral administration of 10 mg per Kg of phenoxybenzamine or propranolol or aspirin or pyridinolcarbamate 3 hours before the challenge. On the other hand, the enhancement of ADP-induced platelet aggregation was not observed in vitro, when 10-5 or 3 × 10-6 molar and 129.4 ± 12.8% of the value before 10∼6 molar ADP was added to citrated platelet rich plasma (CPRP) of rabbit after incubation at 37°C for 30 second with 0.01, 0.1, 1, 10 or 100 µg per ml of adrenaline or noradrenaline. These results suggest an important interaction between endothelial surface and platelets in connection with the enhancement of ADP-induced platelet aggregation by adrenaline in vivo.
Potentially Thrombogenic Materials in Factor IX Concentrates
Authors: Kingdon H. S , Lundblad R. L , Veltkamp J. J , Aronson D. L
Published: July 2018

Factor IX concentrates manufactured from human plasma and intended for therapeutic infusion in man have been suspected for some time of being potentially thrombogenic. In the current studies, assays were carried out in vitro and in vivo for potentially thrombogenic materials. It was possible to rank the various materials tested according to the amount of thrombogenic material detected. For concentrates not containing heparin, there was substantial agreement between the in vivo and in vitro assays, with a coefficient of correlation of 0.77. There was no correlation between the assays for thrombogenicity and the antithrombin III content. We conclude that many presently available concentrates of Factor IX contain substantial amounts of potentially thrombogenic enzymes, and that this fact must be considered in arriving at the decision whether or not to use them therapeutically.
The Role of Vitamins in Hemostasis
Authors: Quick Armand J
Published: July 2018

The physiological mechanism to prevent and control abnormal bleeding is dependent on three vitamins (C, K, and Q). Two of these are unequivocally established as essential for hemostasis while the existence of the third (Q) is supported by experimental evidence and by clinical and therapeutic observations (Quick 1972; Quick 1974). The interrelationship of these three vitamins has remained moot except for clue observations. Both vitamins C and K have a key structure in their molecules which supplies a redox mechanism, ascorbic acid and 2-methyl, 1,4-naphthoquinone, respectively. Both vitamins are concerned with growth. Lack of vitamin C, which clinically is the basic defect in scurvy, does not appear to cause a defect in blood coagulation while vitamin K affects the clotting mechanism by being essential for the production of four distinct clotting factors: prothrombin, factors VII, IX and X.

In this presentation an attempt is made to correlate the action of the vitamin K-dependent clotting factors grouping them in a diagram to show how two systems of thrombin formation exist, one being essentially intrinsic, the second extrinsic requiring tissue thromboplastin and factor VII. The possible interlocking of vitamin Q in this mechanism is presented.
On the Reliability of Plasminogen Measurement Employing the Proactivator-Activator Converting Method
Authors: Martin M , Auel Miss H ,   Technical Assistance
Published: July 2018

A simple plasminogen determination method is presented. It is based upon the conversion of plasminogen into activator by large and constant amounts of streptokinase. The activator contained in a standard coagulum consisting of bovine fibrin, streptokinase, and a 1:40 dilution of human plasma converts the plasminogen adsorbed on bovine fibrin into plasmin. Lysis of the test coagulum is hereby induced. The speed of such lysis is limited by the concentration of the activator incorporated in the test coagulum. The variable component of the activator being human plasminogen, the speed of lysis is directly dependent upon the concentration of human plasminogen in the standard coagulum. Using the thromboelastograph according to Hartert in recording the test clot lysis times, this method of plasminogen determination was shown to be a simple and quick procedure. The standard deviation ranged from ± 13.2 to 68 %, depending upon the plasminogen value to be measured (lower rates of error were attached to high, and higher rates of error to low, plasminogen concentrations). The biological variation of plasminogen values in a group of 26 men aged from 40 to 65 years was calculated to be ±21 %. Both plasminogen and plasmin, its activated form, were exchangeable in the test, i.e. plasminogen determinations performed by activator assay did not differentiate between plasminogen and plasmin. There was no influence by varying anti-SK titers in the plasma up to a circulating antibody content of 2 million. Furthermore, plasma antiplasmins did not affect the plasminogen measuring system. Plasminogen tested by activator assay displayed values closely related to those achieved by immunochemical methods. Plasminogen measurements were performed in patients undergoing streptokinase and urokinase infusion treatment. 5,000 u streptokinase per hour, as well as 270,000 CTA-u urokinase per hour, infused over a period of 2 days produced a fall in plasminogen down to 30-60% of normal. In contrast, 100,000 u streptokinase per hour lowered the plasminogen concentration down to values of below 1%. The foregoing data indicate that plasminogen measurement, according to the principles outlined here (activator assay), may be regarded as a valuable and rehable method for the routine control of streptokinase and urokinase therapy.
Retention of Platelet Fibrin Stabilizing Factor during the Platelet Release Reaction and Clot Retraction
Authors: Joist J Heinz , Niewiarowski Stefan
Published: July 2018

Platelet FSF was found to be retained by rabbit, pig or human platelets during the release reaction induced by thrombin, collagen or plasmin and during a 2 hours incubation of platelet rich fibrin clots.
Lipid Infusions in Man
Authors: Grøttum K. A , Nordøy A , Hellem A
Published: July 2018

The soybean lipid emulsion Intralipid was infused during two hours in 13 volunteer individuals. Biochemically, lipid infusions increased the platelet content of triglycerides, but did not cause significant platelet aggregation, and the platelet counts and survival time were unchanged. However, the lipid infusions reduced platelet and erythrocyte electrophoric mobility, reduced the availability of platelet factor 3 by ADP or kaolin and reduced platelet adhesiveness. A coating of parts of the platelet surface by lipid particles or some other changes in the platelet membrane could explain the effects on platelet electrophoretic mobility, platelet factor 3, and adhesiveness.
Platelet Coagulant Activities and Clinical Severity in Haemophilia
Authors: Walshi Peter N. , Rainsford S. G , Biggs Rosemary
Published: July 2018

Assays for platelet coagulant activities concerned with the initiation and progress of intrinsic coagulation were done pair-wise in two groups of 16 haemophiliacs without detectable factor VIII. The patients in the ‘severe’ group had experienced 7.9-22.8 (mean 13.7) spontaneous haemorrhages per 100 days during an average of 431 days of observation, whereas those in the ‘mild’ group had bled 0-5.5 (mean 3.2) times per 100 days during an average of 282 days of observation. Contact product forming activity, collagen-induced coagulant activity and intrinsic factor-Xa forming activity were decreased in the severely-affected group and increased in the mildly affected group, whereas there was no great difference in platelet factor 3 activity between the two groups. Variations of platelet coagulant activities concerned with the initiation and early stages of intrinsic coagulation may influence clinical severity in haemophilia.
Variation in the Promoter Region of the β Fibrinogen Gene Is Associated with Plasma Fibrinogen Levels in Smokers and Non-Smokers
Authors: Thomas A E , Green F R , Kelleher C H , Wilkes H C , Brennan P J , Meade T W , Humphries S E
Published: July 2018

We investigated the association between fibrinogen levels and a HaeIII restriction fragment length polymorphism located at −453 bp from the start of transcription of the β fibrinogen gene. 292 healthy men aged 45 to 69 years, recruited from general practices throughout Britain, were studied. None had a history of ischaemic heart disease. 41.1% (120) were smokers and fibrinogen levels were higher in this group. The frequency of the noncutting allele (designated H2) was 0.19 and was the same in smokers and non-smokers. The H2 allele was associated with elevated levels of fibrinogen in both smokers and non-smokers and the effect of genotype was similar in both groups. After smoking, HaeIII genotype was the strongest predictor of fibrinogen levels and explained 3.1% of the variance in fibrinogen levels. These results confirm earlier studies that variation at the fibrinogen locus contributes to the between-individual differences in plasma fibrinogen level.
A Frequent Thrombomodulin Amino Acid Dimorphism Is not Associated with Thrombophilia
Authors: van der Velden P A , Krommenhoek-Van Es T , Allaart C F , Bertina R M , Reitsma P H
Published: July 2018

We report a C/T dimorphism in the thrombomodulin (TM) gene that predicts an Ala455 → Val replacement in the sixth EGF-like domain of TM. This dimorphism has allelic frequencies of 82 (Ala) and 18% (Val) in a normal population. In a group of protein C deficient patients and in a group of subjects with unexplained thrombophilia the allelic frequencies were found to be the same as in the normal population. This indicates that with respect to thrombophilia the dimorphism is essentially neutral.
High Level Expression of Recombinant Human Tissue Factor in Chinese Hamster Ovary Cells as a Human Thromboplastin
Authors: Rehemtulla Alnawaz , Pepe Michael , Edgington Thomas S
Published: July 2018

Tissue factor (TF) is the high affinity transmembrane receptor and cofactor for cellular initiation of the plasma coagulation protease cascades by factor VIIa. We describe the synthesis of recombinant huTF by stably transfected CHO cell lines carrying integrated huTF DNA, and the isolation of huTF glycoprotein with specific functional activity equivalent to natural huTF, The expression vector (pCDM8), carrying the cytomegalovirus promoter to drive transcription of a partial cDNA construct encoding the complete huTF protein chain, was cotransfected with a plasmid containing the neomycin resistance gene for selection. These clones were further selected for level of expression of huTF protein. Optimal expression compatible with stability and cell growth was approximately 13.5 x 106 molecules per cell. To our knowledge, this is one of the highest levels of expression described for a recombinant transmembrane receptor in mammalian cells. Recombinant huTF protein was obtained by single-step immuno-affinity purification, and exhibits heterogeneity due to N-linked glycosylation. The protein was indistinguishable from natural huTF based on functional properties of the glycoprotein reconstituted in lipid vesicles, and expression of conformational epitopes. Large scale production of recombinant huTF is feasible to permit basic studies of protein structure as well as for design of huTF thromboplastin reagents.
Inhibition of Recombinant Human Blood Coagulation Factor VIIa Amidolytic and Proteolytic Activity by Zinc Ions
Authors: Pedersen Anders H , Lund-Hansen Torben , Komiyama Yutaka , Petersen Lars C , Oestergård Per B , Kisiel Walter
Published: July 2018

Although it is well established that calcium is an essential cofactor in blood coagulation, recent experimental evidence suggests that zinc may also play an important role in hemostasis. In the present study, we have examined the effects of zinc ions on the amidolytic and proteolytic activity of recombinant factor VIIa in the presence of physiological levels of calcium ions. The amidolytic activity of factor VIIa was inhibited half-maximally by 20 ΜM zinc. The amidolytic activity of a derivative of factor VIIa lacking the Γ-carboxyglutamic acid domain was also inhibited half-maximally by 20 ΜM zinc, suggesting that the mechanism of zinc inhibition of factor VIIa amidolytic activity did not involve its Γ-carboxyglutamic acid residues. The amidolytic activity of a complex of recombinant tissue factor and factor VIIa was inhibited half-maximally by 70 ΜM zinc. In contrast to the results obtained with factor VIIa, the amidolytic activities of other human vitamin K-dependent coagulation proteases including factor Xa, thrombin and activated protein C were not appreciably affected by 50-100 ΜM zinc. The proteolytic activation of factor X by a complex of factor VIIa and relipidated tissue factor apoprotein was inhibited half-maximally by 40 ΜM zinc, whereas activation of factor IX in this system was inhibited half-maximally by 70 ΜM zinc ions. Considerably higher levels of zinc (∽100 ΜM) were required to inhibit half-maximally the rate of factor X activation by a complex of factor VIIa and functional tissue factor on the surface of either a human bladder carcinoma cell line, J82, or stimulated human umbilical vein endothelial cells. Activation of factor IX by factor VIIa and tissue factor on the surface of J82 cells was not influenced by zinc. However, the activation rate of factor IX on human umbilical vein endothelial cells was inhibited half-maximally at 100 ΜM zinc. The activation of factor X by factor VIIa in the presence of small unilamellar phospholipid vesicles was inhibited half-maximally by 20 ΜM zinc whereas factor IX activation by factor VIIa was not appreciably influenced by 10-100 ΜM zinc. Our data demonstrate that plasma levels of zinc ions inhibit the amidolytic and proteolytic activities of factor VIIa. The mechanism of this inhibition, as well as its possible physiological relevance, is unknown.
Standard and Low Molecular Weight Heparin Have no Effect on Tissue Plasminogen Activator Induced Plasma Clot Lysis or Fibrinogenolysis
Authors: Weitz Jeffrey I , Kuint Jacob , Leslie Beverly , Hirsh Jack
Published: July 2018

Although heparin is often given as an adjunct to tissue plasminogen activator (t-PA), the effect of heparin on t-PA induced fibrin(ogen)olysis is controversial. To address this controversy, we examined the effects of standard and low molecular weight heparin (enoxaparine) on both t-PA induced clot lysis and t-PA mediated fibrinogenolysis in a human plasma system. Accordingly, 125I-labeled fibrin clots were incubated in t-PA containing citrated plasma in the presence or absence of these glycosaminoglycans, and the extent of thrombolysis was determined by measuring residual radioactivity of the clots, while Bβ1–42 levels were used as a specific index of fibrinogenolysis. Over a wide range of t-PA concentrations (0.1 to 1.6 μg/ml), neither heparin nor enoxaparine influences either t-PA induced clot lysis or t-PA mediated Bβ1–42 generation. These findings suggest that either agent could be used as an adjunct to t-PA without compromising either the thrombolytic potential of t-PA or its clotselectivity
The Influence of Transport Parameters and Enzyme Kinetics of the Fibrinolytic System on Thrombolysis: Mathematical Modelling of Two Idealised Cases
Authors: Zidanšek A , Blinc A
Published: July 2018

Experimental data obtained by magnetic resonance imaging and photographing clot dissolution in vitro have shown that whole blood clots dissolve almost two orders of magnitude faster when urokinase is introduced into the clot by pressure induced permeation than when its access is limited to diffusion. In view of these findings, two mathematical models have been developed that quantitatively link the enzymatic and transport properties of the fibrinolytic system to the velocity of thrombolysis. Without a pressure gradient across the thrombus, the plasminogen activator molecules diffuse into the thrombus through the blood-thrombus boundary plane. The blood-thrombus boundary slowly moves inwards due to thrombolysis that is spatially restricted to a relatively narrow zone. The velocity of thrombolysis is primarily limited by the diffusion constants of the plasminogen activator and plasmin. In contrast, when plasminogen activator is rapidly distributed along the thrombus by pressure induced bulk flow, lysis occurs at each segment of the thrombus after a lag period that is due to plasmin activation and sufficient fibrin degradation. The lag time is determined primarily by the catalytical properties of the plasminogen activator and plasmin. The mathematical models with the observations of the clot boundaries during lysis permit the characterization of plasmin action on the fibrin network.
Stimulation of Tissue-Type Plasminogen Activator Synthesis by Retinoids in Cultured Human Endothelial Cells and Rat Tissues In Vivo
Authors: Kooistra Teake , Opdenberg Jeannette P , Toet Karin , Hendriks Henk F J , van den Hoogen Ria M , Emeis Jef J
Published: July 2018

Tissue-type plasminogen activator (t-PA) and its inhibitor, plasminogen activator inhibitor 1 (PAI-1), play an important role in regulating the fibrinolytic capacity of plasma. Both t-PA and PAI-1 are synthesized by the endothelium. We report that retinoic acid (vitamin A acid) and other retinoids rather specifically stimulate the production of t-PA by cultured human umbilical vein endothelial cells. Effective retinoids induced a dose-dependent (range: 0.01-50 μM) increase in the production of t-PA of maximally about six-fold, while simultaneously causing no or only a small increase (less than two-fold) of PAI-1. The effects on t-PA synthesis were apparent by 4-8 h, and reached maximal values after about 24-48 h of incubation with retinoid. The retinoid effect on t-PA production was accompanied by increased t-PA mRNA levels, without any parallel change in PAI-1 or GAPDH mRNA concentrations. The study also shows that modifications at the carboxyl group of retinoic acid are associated with a decrease in stimulatory potency. The stimulatory pathway appears to be identical for all retinoids but distinct from a pathway by which another strong inducer, sodium butyrate, induces t-PA synthesis in endothelial cells. The induction of t-PA by retinoids might involve protein kinase C (PKC) as judged by an experiment using a specific PKC inhibitor.

The effect of retinoids on the fibrinolytic system in vivo was assessed by feeding rats with a vitamin A deficient diet or a diet with excess of vitamin A or other retinoids. The activity and antigen levels for t-PA in plasma and tissue samples were strongly decreased in vitamin A-starved animals, and enhanced in retinoid-fed animals. The recognition of a class of compounds, the retinoids, that can increase t-PA synthesis in cultured human endothelial cells and in rat tissues in vivo, may be of importance in designing an effective method to enhance plasma fibrinolytic activity, and thereby in preventing thrombotic phenomena.
Platelets Adhere to Sulfatides by von Willebrand Factor Dependent and Independent Mechanisms
Authors: Data Richard E , Williams Sybil B , Roberts David D , Gralnick Harvey R
Published: July 2018

Unstimulated human platelets from normal volunteers adhere to sulfatides (galactosylceramide-I3-sulfate) as single cells but do not adhere appreciably to other lipids including gangliosides, neutral glycolipids, phospholipids or cholesterol-3-SO4. Platelet adhesion to sulfatide is saturable and dose-dependent, reaches maximal levels in 90 to 120 min, and is not divalent cation-dependent. Because sulfatides bind von Willebrand factor (vWf) with specificity and high affinity and platelet adhesion to structurally related sulfated glycolipids is approximately proportionate to their ability to bind vWf, we examined whether vWf mediates platelet adhesion to sulfatides. Platelets from a patient with severe Type I von Willebrand’s disease adhere poorly to sulfatides. However, adhesion to levels seen with normal platelets is restored by the addition of vWf. Adhesion of normal platelets can be partially inhibited by a monospecific antibody to vWf. Normal platelet adhesion to sulfatides, however, is not increased following preincubation with vWf. Both vWf binding and platelet adhesion to sulfatides can be inhibited by the sulfated polysaccharide dextran sulfate at low concentration, fucoidan at high concentrations, but not by heparin, fibrinogen, fibronectin, or the synthetic peptides Gly-Arg-Gly-Asp-Ser-Pro or Gly-Arg-Gly-Glu-Ser-Pro. Thus, adhesion to sulfatides appears to be of two types; vWf dependent (50-75%) and vWf independent (25-50%).
The Use of Laser-Light Scattering and Controlled Shear in Platelet Aggregometry
Authors: Hubbell Jeffrey A , Pohl Phillip I , Wagner William R
Published: July 2018

Laser-light scattering was used to observe and quantify the dynamics of human blood platelet aggregation in platelet-rich plasma (PRP). Aggregation was performed in a controlled shear environment by placing the PRP in the annular space between a rotating cylindrical rod and a stationary cylindrical tube. The instrument was capable of very sensitive continuous semi-quantitative measurements of chemically-induced microaggregation. As a demonstration of the technique, results are presented for ADP-induced aggregation at doses of 10, 1, and 0.1 μM and collagen-induced aggregation at a dose of 5 μg/ml, each at shear rates of 1,000 s-1 and 500 s-1. Extensive aggregation was observed in response to ADP at even the low dose of 0.1 μM, indicating a high sensitivity to microaggregates. The sensitivity of the ultimate size of the ADP-induced aggregates to ADP concentration was shear dependent. The formation of microaggregates by collagen stimulation was shown to be almost immediate, as contrasted with a 10-20 s typical lag when observed turbidometrically. Disaggregation was observed with 1 μM ADP, but this was only partial, as contrasted with the complete recovery of transmittance observed in the turbidometric technique. Electronic particle sizing and counting was employed to semiquantitatively verify the aggregate size distributions found from mathematical conversion of the laser-light scattering data.