bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies Of Factor VIII Inhibitor Bypassing Activity (FEIBA)
Authors: Barrowcliffe T W , Gray E , Kemball-Cook G
Published: July 2018

The activities of an activated Factor IX concentrate (FEIBA, Immuno AG) were studied by two in vitro assays: a one-stage method using VUI-deficient plasma as substrate, and a two-stage assay based on the thrombin generation test. The nature of the active principle was explored by measuring the reduction in activity when FEIBA was incubated with specific antibodies.

Incubation of FEIBA with human antibodies to Factor VIII reduced its activity by about 30% in the one-stage assay, and about 50% in the two-stage assay, suggesting that FEIBA contains Factor VIII procoagulant activity. Inactivation of the Factor VIII in FEIBA was somewhat slower than that of normal Factor VIII, indicating partial protection from inhibition. Human antibodies to Factor IX inhibited the one stage activity by about 30%, and incubation with both antibodies also produced a 30% reduction in activity. The remaining procoagulant activity decayed only slowly when incubated with non-inhibitor plasma. In contrast, purified human Factor Xa lost activity rapidly on incubation in normal plasma, as did a purified fraction from a Factor IX concentrate, which had high activity in the one-stage assay.

These results suggest that the in vitro activity of FEIBA is due to at least two components. One component appears to be dependent on both Factors VIII and IX and may be a complex of VIII and IXa. The other component acts later than Factors VIII and IX in the coagulation cascade but, unlike purified Factor Xa, is relatively resistant to inactivation by plasma inhibitors such as antithrombin III. FEIBA was also found to contain phospholipid, and it may be that the phospholipid protects both the Factor VIII and activated enzymes from their inhibitors.
Postoperative Hypercoagulability, Detection And Measurement Using A Modified Recalcification Time System, Effects Of Low Dose Heparin And Types Of Surgery
Authors: Lundquist P B , Swedenborg J
Published: July 2018

The purpose of the study was to demonstrate postoperative hypercoagulability and its possible prevention by low dose heparin (LDH). Healthy volunteers (with LDH), patients undergoing cholecystectomy (with & without LDH) and patients undergoing arterial reconstructive surgery with synthetic grafts (with LDH), were tested. All were tested 1, 3 & 5 hrs after LDH.

Overall coagulability was determined by using platelet free plasma and a modified recalcification time system with a nefelometer to detect first fibrin formation. Recalcification times were measured before (T0) and after (TA) plasma activation against glass, in vitro. Heparin levels were determined with the Factor Xa inhibition test using a chromogenic substrate (CoatestR, Kabi).

T0 and TA were prolonged after LDH in healthy volunteers. Cholecystectomy caused shortening of T0 and TA. This could be prevented by LDH, raising T0 to level recorded after LDH in normals. Patients receiving synthetic arterial grafts showed no prolongation of T0 and TA after surgery with LDH, but rather a shortening. These patients showed hypercoagulability in spite of LDH.

It is concluded that postoperative hypercoagulability can be traced with the presented method and counteracted by LDH in patients undergoing cholecystectomy but not in patients receiving synthetic arterial grafts. All patients on LDH had similar heparin levels, determined with the Factor Xa inhibition test. Vascular surgery with synthetic grafting and Cholecystectomy seem to induce two different kinds of hypercoagulability, where the former is thought to be induced by the foreign surface. Subcutaneous heparin therapy (LDH) results in poor inhibition of surface induced coagulation, in vivo.
Stimulation Of Prostacyclin Production In Aortic Endothelial Cells By A Non-Dialysable Serum Factor
Authors: Hall E R , Rafelson M , Wu K
Published: July 2018

The production of prostacyclin (PGI2) by vascular endothelial cells is thought to be of primary importance in maintaining normal hemostasis. We have investigated the production of prostacyclin in bovine arterial endothelial cells maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 30% fetal calf serum. Intact, confluent monolayers of endothelial cells (3x106 cells) in passages 2 through 6 were used. The growth medium was removed and the cells were washed in DMEM that did not contain serum. 3 mls of medium alone or containing normal plasma or serum was then added and incubated at 37°C for 15 min. Then, 1 mg of arachidonic acid was added and the cells incubated for an additional hour. The test medium was removed, centrifuged to remove any loose cells and stored at -70°C. To determine the production of PGI2 by the endothelial cells, the medium was assayed for 6-keto-PGF1α, the stable metabolite of PGI2, by radioimmunoassay. The synthesis of prostacyclin by bovine aortic endothelial cells was significantly increased in a concentration dependent manner by both normal platelet poor plasma and normal serum. This increase in prostacyclin production was inhibited by both aspirin and indomethacin, indicating an increase in synthesis rather than the release of PGI2. Furthermore, this increase could be demonstrated in the presence or absence of added arachidonic acid. The active component in plasma and serum was non-dialysable, eliminating the possibility of a small compound such as bradykinin or angiotensin II. This active factor was present after freezing and thawing the plasma and serum and was heat stable (60°C, 5 min). The presence of an endogenous prostacyclin stimulating factor may be significant in the in vivo regulation of prostacyclin production.
Prostacyclin Release From Ex Vivo Vascular Endothelium: Comparative Studies With Ionophore A23187, Arachidonic Acid, Bradykinin, And Thrombin
Authors: Goldsmith J C , Jafvert C T
Published: July 2018

Prostacyclin (PGI2) release from systemic bovine, sheep, canine, and fetal lamb arterial segments has been evaluated in a template device and in cultured cell monolayers employing a radioimmunoassay for 6-keto-PGF1α.

Canine segments released the greatest concentration of PGI2 and fetal sheep the lowest under unstimulated conditions. After exposure of the various vascular segments to the ionophore A23187, PGI2 release was increased 5-20 fold over baseline. Similar responses were observed following stimulation with arachidonic acid. Bradykinin induced a dose-dependent release of PGI2 from bovine vascular segments. Pulmonary arterial segments were more responsive than aortic. When exposed to thrombin, all tissues examined except canine did not demonstrate an increase in PGI2 release over baseline. After thrombin stimulation, canine segments slightly increased their PGI2 release from 91.4 ± 15.7 pmoles/ml/15 min to 152.7 ± 21.8 pmoles/ml/15 min. The other fetal and adult systemic vascular segments were unresponsive to thrombin as a releasing agent for PGI2 as were cultured monolayers of bovine and sheep systemic endothelium. This feature contrasted to prior studies with cultured human umbilical venous endothelial cell monolayers in which thrombin released PGI2. Cultured bovine endothelial cell monolayers were employed to examine thrombin-cell interactions more extensively. High affinity binding sites for thrombin were demonstrated on cultured bovine aortic and pulmonary endothelium. Examination of vascular tissue from diverse species has confirmed the presence of PGI2 in super- natents obtained from these vessel segments. Studies with the template model suggest that the vasodilator effects of bradykinin could be a consequence of the release of PGI2 from the endothelium. Our results also suggest that either thrombin binding is causally unrelated to PGI2 release from systemic animal endothelium or that this endothelium lacks a mediator required for thrombin to exert its effect.
Single Oral Dose Administration Of A Specific Thromboxane Synthetase Inhibitor To Normal Volunteers - A Double Blind Placebo Controlled Study
Authors: Kakkar V V , Westwick J , Zahavi J , Tyler H
Published: July 2018

A specific thromboxane synthetase inhibitor (UK-37248-01) is a new imidazole derivative synthesised by Pfizer Central Research. In vitro and animal experiments showed the compound to be a potent, selective and orally active one.

Six healthy male volunteers received a single dose of 100mg of a specific thromboxane synthetase inhibitor or a matching placebo in a double blind cross-over study, there being a 2 week interval between treatments. Production of thromboxane B2 in serum was markedly reduced 1 hour after the drug administration, with about 50% inhibition still evident at 6 hours. The drug had no effect no circulating β-thromboglobulin and platelet factor 4 antigen levels, or on platelet aggregation to ADP or collagen, though release of 5HT was reduced. Increase in bleeding time (2-3 minutes) was recorded in 3 volunteers 1 hour after the drug administration.

Chemical assay of the compound showed that up to 45% of the oral dose was excreted in the urine within 24 hours; 90% of this was excreted within the first 4 hours. Routine haematology and clinical chemistry tests repeated 24 hours and 4 days after dosing gave no indication of drug related toxicity.
A Microelectrophysiologic Basis For The Fibrillation Retardant Effects Of Sulfinpyrazone And Salicylate
Authors: Iansmith D H S , Nash C B , Bandura J P
Published: July 2018

The antagonism of platelet function has been suggested as a mode of action for sudden death prophylaxis by sulfinpyrazone (S) and salicylates (A). To evaluate an antiarrhythmic means for such action, 5.0 to 20.0 mg/Kg A or S was administered i.v. to open-chest, intermittently cardiac paced canines in which arterial pressure and EKG were monitored. Fibrillation thresholds (FT) were measured before and after coronary arterial ligation and drug administration, by extrastimuli of increasing current strength during the vulnerable period of the T wave. Acutely both A and S increased FT 2 to 3 times that of post-infarcted values. To elucidate the mechanism for this response Purkinje fibers were isolated from the left ventricles of adult canines. These were pinned to the paraffin floor of a lucite isolation well, affixed with stimulating and surface recording electrodes, and partitioned with a plastic collar coated with vegetable fat to provide a fluid seal. Separate inflow and outflow ports allowed for individualized microenvironments for each chamber. After 60 min of tissue equilibration with well oxygenated, 36°C normal Tyrode Solution (TS), the fluid of the chamber distal to the stimulus source was altered to include 2-35 mg% A or 2-50 ugm/ml S, with or without CO2 substitution for O2 (pH 6.4) or HC1 (pH 6.4). Action potentials (AP) from each chamber were monitored during 60 min of altered TS and then 60 min of normal TS reperfusion. In a dose-dependent manner A depressed AP parameters to cause automaticity and inactivity, while S was without effect. Whereas S was able to prolong tissue survival and support electrical activity in a situation of local acidosis (if absolute hypoxia were not superimposed), A was not. Such information suggests that although both A and S are arrhythmia retardant in the postinfarction period, their modes of action may differ. Both might act as “membrane stabilizers”; however, it appears that electrogenic aberration is necessary before S manifests an effect.
Sulphinpyrazone Does Not Influence Rejection Of Renal Transplants
Authors: Turney J H , Bewick M , Weston M J
Published: July 2018

Platelet-mediated intravascular fibrin deposition may be the distal arm of the transplant rejection process. There have been reports that anti pi ate let drugs may prevent ameliorate, or even reverse renal transplant rejection. 50 consecutive cadaveric renal transplant recipients were randomly allocated to receive sulphinpyrazone 400 mg twice daily or no treatment additional to conventional immunosuppressive therapy. Patients were followed for 6 months, at which time they were assessed for graft survival and function, and for the number of rejection episodes. Rejection was defined as a rise in serum creatinine for which no other explanation was identified. Results were analysed by the Chi-squared test with Yates’ correction. Results at 6 months: There were no differences between the two groups, neither did the function of the surviving grafts differ.

We conclude that, in this study, antiplatelet therapy with sulphinpyrazone does not affect the outcome of cadaveric renal transplantation.
Endotoxin Inhibits Prostacyclin Release From Rabbit Aorta
Authors: Zahavi J , Honey A C , Westwick J , Kakkar V V
Published: July 2018

Released prostacyclin (PGI2) activity has been studied in aortic rings of 19 New Zealand white female rabbits. These rings produced a potent inhibitor of platelet aggregation, identified as PGI2. All the rabbits were anaesthetized with pentobarbital and thereafter a solution of endotoxin (E. Coli, 0111, B4, Difco Lab.) was injected intravenously to 7 rabbits (304μg/kg every 15 min during 1 hour to achieve an estimated plasma level of 1-2 μg/ml). Another 5 rabbits served as controls and were injected with saline. After 1 hour the aorta was rapidly excised, cleaned, cut into small rings and the released PGI2 activity studied at various time intervals (5-30 min) at 37°C. The mean release of PGI2 (in pg/mg wet tissue) in the control rabbits was 201 (range 50-443). It decreased significantly to 104 (range 0-237) after 30 min. In the endotoxaemic rabbits, the initial PGI2 release was only 73 (range 0-329) (p<0,008 compared to control rabbits). This level did not change with time and was 71.9 (range 0-261) after 30 min suggesting that the “endotoxinemic” vessels were initially relatively exhausted and were not able to release PGI2. In the remaining 7 rabbits the aorta was removed immediately after anaesthesia and aortic rings incubated for 5-30 min Krebs-Henleit buffer or endotoxin 0.2-10 μg/ml and the released PGI2 activity studied. There was a dose dependent inhibition which was more pronounced after 30 min incubation.

The decrease in PGI2 release from rabbit aorta following endotoxaemia removes the inhibitory effect on platelet aggregation of the arterial vessel wall and consequently may contribute to the development of a thrombogenic state.
The Effect Of Angiotensin II On PGI2 Production By Endothelial Cells In Culture
Authors: Lonchampt M O , Romquin N , Bonne C , Regnault F
Published: July 2018

Angiotensin II, a vasoconstrictor peptide, is also a very potent stimulator of PGI2 synthesis in blood vessels. In this study we have investigated its direct effect in endothelial cells in culture.

Bovine aorta endothelial cells were used in primary culture. Angiotensin II was added in Tris buffer for 3 min. PGI2 was measured by radioimmunoassay of 6-keto PGF1α , the degradation product of PGI2 in this system. Angiotensin II was shown to stimulate PGI2 synthesis at concentration as low as 0.5nM. At 5nM the PGI2 production was five-fold increased as compared to controls. A synthetic peptide Sar1-Ile 8-Angiotensin II, known as a specific inhibitor of the hormone in other tissues, was a powerful agonist at the doses of 0.2 to 20nM and exhibited an antagonistic activity at higher doses (1μM).

These results suggest that endothelial cells are target cells for angiotensin II as well as smooth muscle cells in blood vessels. Since PGI2 is a potent vasodilator, its production induced by angiotensin II could act as a modulator of the vasoconstrictor effect of the peptide.
Studies On The Dynamics Of Antithrombin III During Heparin Infusion By Radioimmunoassay
Authors: Urano S , Nakagawa M , Kitani T , Maeda Y , Watada M , Kanayama S , Ijicihi H
Published: July 2018

A radioimmunoassay method for antithrombin III (ATIII) was developed in order to detect the AT III levels correctly in plasma and tissues and the effect of heparin infusion was investigated on rat using this method and 125I labeled ATIII. Rat AT III was purified from rat defibrinated plasma by heparin sepharose affinity chromatography and gel filtrations. This purified AT III was used for the preparation of specific AT III antiserum. Labeling of AT III with 125I was performed according to the method by Hunter and Greenwood. Plasma level of AT III were significantly decreased in the treated group with heparin for 6 hours, although significant difference was not observed in AT III contents in various organs. The behavior of i.v. injected AT III laveled with 125I in the normal control and treated groups proved the difference on the half life of AT III. Control group gave 52 hours and it was shortened in the treated group. The percent radioactivity per ml plasma after 6 hours of heparin infusion was 1.16±0.51, and 2.01±0.38 in the control group, and significant difference was observed (p < 0.05). On the contrary the percent dose radioactivity per g tissue wet weight was significantly increased in the liver, lungs, and large intestine on the heparin treated group. The decreased amount of the intravenously injected laveled AT III appears to be trapped and metabolized in the various organs mainly in the liver during heparin infusion. The decrease of plasma AT III levels on the patients treated with heparin may be explained from these experimental results.
Inhibition Of Platelet-Associated Factor Xa By Heparin-Antithrombin
Authors: Teitel J M , Rosenberg R D
Published: July 2018

The binding of Factor Xa (FXa) in conjunction with activated Factor V to the platelet surface greatly accelerates the rate at which prothrombin (P) is converted to thrombin. It has been shown that bound FXa is protected from inhibition by heparin-antithrombin (HAT). We examined the interaction of FXa with HAT in blood samples by quantitating the rate of the FXa induced cleavage of the arg 155-ser 156 bond of the P molecule. This was accomplished by measuring two of the species released by this cleavage, F2 and F1+2, by means of a new RIA. Aliquots of blood, constantly stirred at 20°, were mixed at intervals with H, final concentration 2 u/ml, or buffer. Aliquots were then sampled at intervals for measurement of F2/F1+2. Secretion of platelet releasable proteins was monitored for PF-4 by RIA. The calculated bond cleavage rate showed a linear dependence on the proportion of P activated at the time of H addition (r= 0.73). Thus, at 0.5% zymogen conversion, the mean rate was 0.72 nmol/l/min (±se 0.37); at 9.7-25.2% conversion, it was 84.8 nmol/l/min (±se 16.9). Since the concentration of H added exceeded the maximum PF-4 values by 113x on a molar basis, it is unlikely that H had been neutralized by a secreted platelet protein. Identical experiments were performed in which platelet release was suppressed (mean 2.4x) by PGI2. Under these conditions, the dependence of the rate on prior P activation was reduced. At 8.6-36.2% activation, the mean rate was 40.3 nmol/l/min (±se 20.5). Similarly low rates were seen in a system using recalcified platelet poor plasma in place of whole blood. Thus, FXa generated in blood is progressively inaccessible to inhibition by HAT as increasing amounts of P are cleaved. This protection is in turn a function of the presence of platelets, and their ability to undergo the release reaction. These data suggest that therapeutic heparinization may be difficult to achieve in a setting of extensive localized release of platelet proteins.
Salicylate Kinetics In The Rat: Relationship To Antithrombotic Activity
Authors: Philp R B , Francey I
Published: July 2018

Recently we reported that acetylsalicylic acid (ASA) 100 mg/kg i.v. had no antithrombotic effect in a rat model of arterial thrombosis whereas 200 mg/kg i.v. had significant antithrombotic activity. The present study investigates salicylate kinetics at the non-protective level. Carotid arteries of urethane-anesthetized rats were injured electrically (1 mA, DC, for 1 min) and thrombogenesis measured by recording downstream temperature. Right and left side injuries were compared before and after ASA, 100 mg/kg i.v. Blood samples were collected at various times post injection and serum assayed for salicylate content by Trinder’s method. All salicylate levels are reported as mg/dL ± SEM. Control injuries produced a temperature fall of 1.4°C ± 0.08 (SEM) and post ASA injuries a fall of 1.8°C ± 0.17 (SEM) (P < .1 > .05). Serum salicylate was 20 mg ± 4.58 1 min post injection and the calculated lh was 40 min with decay following first order kinetics. The intraperitoneal injection of ASA 200 mg/kg produced a serum salicylate level of 8.1 ± 1.55 5 min post injection with a peak of 19 ± 2.03 30 min post injection. Both absorption and elimination followed first order kinetics with Th values of 10 and 44 min respectively. Urine collected 40 min post injection contained 9.4 ± 3.38 free salicylate which increased to about 40 following chemical digestion, indicating the presence of metabolites. The results indicate that serum salicylate levels < 20 do not offer antithrombotic protection in this model and that this level is achieved only briefly following ASA 100 mg/kg i.v. or 200 mg/kg i.p. The brief half-life of ASA in the rat has implications for the interpretation of data concerning the inhibition of prostacyclin synthesis by vessel wall. The wide variation in effective ASA doses reported for various animal models of thrombosis is probably a function of the nature of the model rather than of efficient prostacyclin synthesis.
Gel Chromatographic Amd Electrophoretic Evidence For The Existence Of Soluble 125-I Fibrinogen-131-I-Fibrin-Des AA (AABB) Complexes In Plasma Containing Ca++ At 37°C
Authors: Kierulf P , Brosstad F , Godal H C
Published: July 2018

Material and methods: Ca++-boosted (2.5 nmol) protrombin adsorbed plasma (=Ca++-plasma) was prepared by adsorbing thrice with Ba(SO4)2 and dialysing against Ca++-containing Ringer-solution. Ca++-plasma showed negligible thrombin generation as evidenced by Fibrinopeptide A apparance (RIA-FPA), even when continously chromatographed at 20°C for 48h (FPA <15 ng/ml). Plasma containing 125-I fibrinogen and/or 131-I-fibrin Des AA (or Des AABB) monomers (Brosstad et al, Hemostasis 6,225,1977) was subjected to either gel chromato-graphy or to enzyme-fixation. Gel chromatography at 37°C was performed on Sepharose CL-6B, freeze-dried, reconstituted in Ca++-plasma, and developed with Ca++-plasma or Ca++- plasma with EDTA Enzyme-fixation was done by adding thrombin-activated (removable, insolubilized thrombin) Factor XIII (XIIIX). Subsequently XIIIX- activity was quenched with EDTA Enzyme-fixed, radiolabelled complexes were either harvested by thrombin-clotting in the presence of EDTA or chromatographed on Sepharose CL-6B (0.15 M Tris-HCl pH 7.0, 0.15 M NaCl, 2.5 M urea, 10mmol EDTA). Harvested clots were mer-captolysed and SDS-gel electrophoresed. Gel chronatographed specimens were radioprofiled and freeze-dried.

Results: In Ca++-plasma both Des AA and Des AABB 131-I fibrin eluted in front of the 125-I fibrinogen, whereas in the presence of EDTA 131-I fibrin Des AA eluted ocnpletely within the 125-I fibrinogen peak. SDS-gel elec trophoresed, mercaptolysed, clots, containing enzyme-fixed,radiolabelled, fibrinogen derivatives,demonstrated 125-I and 131-I activity in γ-γ-dimers and to lesser extent in α-oligemers. Gel chromatographed, enzyme-fixed specimens, demonstrated radiolabelled dimerized fibrinogen derivatives (125-I and 131-I activity) . RIA-FPA of such dimers demonstrated approx. 2 moles FPA/mol dimer.

Conclusion: Soluble,covalently-linkable largely dimeric fibrinogen fibrin cenplexes exist in Ca++-plasma at 37°C.
Diabetic Retinopathy And Thromboxane Like Substance
Authors: Lazzari M A , Gimeno M , Sutton N M , Lopez J R
Published: July 2018

Diabetes Mellitus (DM) is a risk factor in the development of vasculopathies and its complications. It produces also its own microangiopathy. Evidence was reported of increased platelet activity in DM in different assays. Platelets aggregation and the arachidonic cycle could play a key role in the increased tendency to thrombosis. A disorder of ratio TXA2/PGI2, two opposing prostaglandin derivatives, could be the initial step. We intended to evaluate a thromboxane like substance (TLS) produced from platelet rich plasma (PRP) and to compare between normals and diabetic retinopathy (DR) patients. TLS was measured in 16 controls and 16 patients. Assay was done with the aggregating activity developed in PRP (considered TLS) after addition of arachidonic acid (f.c. 2 mM). The supernatant of the PRP (100 μl) was taken 40 sec. after the aggregation started and were added to a normal PRP treated with aspirin (f.c. 40 μl/ml) adjusted to 250.000 - 300.000 pl/μl and the degree of platelet aggregation measured in a Chrono Log Aggregometer. TLS was inactivated after its incubation during 2 min. at 37°C. This finding suggests this activity is due to TXA2.

The results obtained (expressed in % of platelet aggregation) were: controls x 16.37% ± 6.28 and DR x 36.00% ± 9.72.

The increase detected in the DR group supports previous experimental reports suggesting the role of the thromboxane A2 in vaso occlusive complication of diabetes mellitus.
Coagulation Defects Due To Inactivation Of Plasma Inhibitors By Granulocytic Elastase
Authors: Jochum M , Lander St , Fritz H
Published: July 2018

Neutral proteinases such as elastase and cathepsin G are liberated from secretory granules of PMN leukocytes in the course of diseases like septicemia, acute myelocytic leukemia or emphysema. Despite the presence of a high antiproteinase potential (α1-antitrypsin, α2-macroglobulin, anti-thrombin III, α2-antiplasmin, α1-antichymotrypsin) in plasma these proteinases are able to degrade clotting and other plasma factors unspecifically thus contributing to severe coagulation disorders. Evidence is presented that the plasma inhibitors (antithrombin III, α2-anti- plasmin and α1-antichymotrypsin) which are not able to inhibit granulocytic elastase, are rapidly inactivated by this proteinase and may thereby intensify consumption of clotting and other plasma proteins during inflammation.
Plasminogen Activator (PA) Activity In Metastatic Cells: An Amidolytic Approach
Authors: Coen D , Bini A , Balconi G , Delaini F , Mussoni L , Donati M B
Published: July 2018

It has been proposed that fibrinolytic activity can play an important role in the process of metastasis formation. Nevertheless, it is not yet clear in which phase of the tumor growth and dissemination this activity is involved. We measured the fibrinolytic activity of cells from primary tumor and metastatic nodules of 3LL, an i.m. implanted murine tumor which selectively metastasizes to the lungs. Tumor cells have been studied both immediately after mechanical disruption of tumor tissue and after in vitro culturing to confluence. Their P.A. activity was tested by an amidolytic assay in which cells were incubated with purified plasminogen (3CU/ml) and 4mM S-2251 (Kabi Diagnostica, Stockholm, Sweden), a plasmin specific chromogenic substrate. After 3 hour incubation at 37°C, the reaction was stopped with acetic acid and absorbance read at 405 nm.

Cells from the primary tumor and metastatic nodules showed a similar fibrinolytic activity, which was in both cases in- increased 3 to 4 fold in cell extracts obtained after preincubation with TRITON X-100. A dose-response curve plotted with increasing urokinase concentrations showed a parallel course. This data suggests that, in the 3LL model, PA activity is not one of the properties characterizing the selection of metastatic cells.

On the other hand,cultured cells presented consistently higher levels of PA than their native counterparts, suggesting that adhesion of cells in culture may stimulate PA production or, alternatively, that cultured cells are a selected population in comparison to the overall number of native cells.
Vascular And Platelet Effects Of Synthetic Analogues Of Prostacyclin
Authors: Pareti F I , Mannucci P M , Vanasia S , Gandolfi C , Mongielli N , Mills D C B
Published: July 2018

A series of synthetic analogues of prostacyclin (PGI2) have been compared for their ability to increase the formation of cyclic AMP and to inhibit the aggregation of human platelets, and for relaxation of isolated bovine coronary arteries and lowering of the blood pressure of concious rats. The most active inhibitors of aggregation were derivatives of PGI2 with a triple bond at C14:15. There was good aggreement between the inhibition of platelet aggregation and elevation of cyclic AMP in platelets, and both effects were enhanced by phosphodiesterase inhibitors. The most active compounds of the series were K13415 (14,15-didehydro 20-methyl PGI2), and a carbocyclic analog, FCE21258 (5e-14,15-didehydro carbo PGI2) which were both more active than PGI2 itself. In addition, the carbocyclic compounds were stable at neutral pH. Compounds K13817 (14,15-didehydro 16-methyl PGI2) and FCE21292 (5e-14,15 didehydro 20-methyl carbo PGI2) were similar in activity to PGI2 as platelet inhibitors, but had less vasodilator and depressor activity. The results show considerable differences in the structural specificities of the platelet and vascular activities of PGI2.
Effects Of Fibrin, Fibrinogen And Fibrin(-Ogen) Degradation Products On The Growth Of Aortic Smooth Muscle Cells In Culture
Authors: Tanaka K , Ishida T , Watanabe K
Published: July 2018

Importance of smooth muscle cell proliferation has been emphasized as a key event in atherogenesis. A number of factors have been shown to stimulate cell proliferation in vitro. These include platelet, lipoprotein, insulin and so on. We studied the effects of fibrin, fibrinogen and fibrinogen degradation products on the growth of aortic smooth muscle cells in culture. Smooth muscle cells in the 7th-15th subculture were grown from the explants of the media of rabbit thoracic aortas.

Growth behavior of the cultured smooth muscle cells in response to fibrin clot was observed with phase-contrast microscope. Fibrin clot stimulated the cell proliferation during the period of 24 hours cultivation.

Effects of fibrin,fibrinogen and fibrinogen degradation products on the incorporation of [3H] thymidine by the cultured smooth muscle cells were studied in replicate culture method. Both fibrin and fibrinogen stimulated the incorporation of [3H] thymidine during the period of 24 hours cultivation. But they inhibited the incorporation after 48 hours, and smooth muscle cells were degenerated and detached from the tissue culture dish. Fibrinogen degradation products inhibited the incorporation of [3H] thymidine by smooth muscle cells.

The cultured smooth muscle cells showed fibrinolytic activity by Todd’s fibrinolysis autography.

These results suggest that both fibrin and fibrinogen have the stimulatory effect, while fibrinogen degradation products have the inhibitory effect on the proliferation of smooth muscle cells, and that fibrinolytic activity of arterial wall might play a role in smooth muscle cell proliferation that leads to the development of atherosclerotic lesions.
The Use Of Proteolytic Probes To Identify Platelet Membrane Receptors
Authors: Bemdt Michael C , Phillips David R
Published: July 2018

One approach to the study of structure-function relationships with plasma membrane glycoproteins is to use proteases whose substrate specificity permit cleavage of only a single glycoprotein. We have employed this approach using two proteases, human platelet calcium-dependent protease and thrombin, as probes to investigate the identity of the platelet membrane von Willebrand Factor and thrombin receptors, respectively. Extensively purified calcium-dependent protease added exogenously to washed human platelets catalyzed the hydrolysis of only one membrane glycoprotein, glycoprotein Ib. The hydrolytic product of glycoprotein Ib as determined by immunoprecipitation and other criteria was glycocalicin. Correlation of the level of intact glycoprotein Ib with maximal agglutination rate to and binding of Factor VIII/von Willebrand Factor confirms previous observations consistent with glycoprotein Ib being the von Willebrand Factor receptor on human platelets.

Since the catalytic integrity of thrombin is requisite for platelet response, we have sought to identify a thrombin substrate(s) on the platelet membrane surface. Glycoprotein V was the sole apparent thrombin substrate evident on O’Farrell gels of periodate-labeled control and thrombin-treated washed human platelets. We have recently purified this glycoprotein to homogeneity and confirmed it to be a thrombin substrate (Bemdt and Phillips (1981) J. Biol. Chem., 256: 59-65). Several lines of evidence will be discussed that are consistent with glycoprotein V being the probable functional thrombin receptor on human platelets.
A Variant of Factor X That Is Defective Only In Extrinsic Coagulation
Authors: Bertina R M , Alderkamp G J H , de Nooy E
Published: July 2018

Screening - prior to liver biopsy - of the blood of a 48- year-old patient with a negative anamnesis for bleeding complications, revealed a strongly prolonged prothrombin time (PT), while the APTT was normal. The PT prolongation was not the consequence of an antithromboplastin, vitamin K deficiency, oral anticoagulation or factor VII deficiency (FVII 0.5 U/ml). A more detailed analysis demonstrated the presence of an abnormal factor X molecule with the following properties: coagulation assays: 0.016 U/ml (thromboplastin), 0.43 U/ml (RVV/cephalin), 0.44 U/ml (kaolin/cephalin); spectrophoto- metric assay (RVV-X/S2337), 0.47 U/ml; immunological assay (Laurell), 0.50 U/ml. The low reactivity towards the extrinsic activator depends on the thromboplastin used: human brain, 0.016 U/ml; bovin brain, 0.11 U/ml.

The FX-variant has a normal electrophoretic mobility (crossed immunoelectrophoresis) and can be absorbed from plasma by Al(OH)3 The variant has been purified from the patients plasma using affinity chromatography on anti-factor X Sepharose, followed by DEAE-Sephadex gradient elution.

The isolated variant shows the same low reactivity towards extrinsic activation. Comparison with isolated normal FX showed that it has the same molecular weight (NR 70,000; R 51,000 and 19,000). The heavy chain stains positive with the Schiff’s reagent. Variant FX can be activated by RVV-X under formation of αXa (51,000), which in the presence of phospholipid is degraded to αβXa (48,000) and αγX (35,000).

Analysis of the kinetics of activation of normal and abnormal FX by FIXa, FIXa plus FVIII, or RVV-X revealed no essential differences. Only the activation by FVII-thrombo- plastin showed to be abnormal.

The complete absence of bleeding complications in this particular patient, might indicate that in vivo the extrinsic activation of factor X is only of minor importance.
Improved Purification Method Of Human Plasma Factor V And Applications Of A Rabbit Anti-Human Factor V Antibody
Authors: Breederveld C , Bruin T , Sturk A , Hakvoort Th , Cate J W ten
Published: July 2018

Human factor V, purified from citrated plasma by a method recently described by us (Bolnuis et al 1979) was found to be slightly contaminated with α2-macroglobulin. A rabbit antiserum to this preparation, obtained by frequent subcutaneous immunization, showed factor V procoagulant neutralizing activity. Immune electrophoresis demonstrated precipitation lines with α2-macroglobulin. This contamination was succesfully removed from the factor V preparation by affinity chromatography with specific raboit anti human α2-macroglobulin bound to Sepharose 2B. The antiserum to the original, impure factor V preparation showed no more precipitation arcs after absorption with 3% normal plasma and ammonium sulphate precipitation of the complexes. Factor V procoagulant neutralizing activity titre was estimated 1: 8 (50% neutralization after 1 hour incubation at 37°C.) This antiserum was used for the following studies: 1. An inhibitor neutralization assay was developed for measuring antigenic activities of the factor V molecule. Antigen concentration in a range from 12.5-100% could be reliably measured. 2. Fab-fragments, obtained by papain digestion of the purified IgG (by protein A SePharose chromatography) were labeled with 125I by an immobilized lactoperoxidase metnod. These Fab-fragments fully retained their factor V procoagulant neutralizing activity. 125I-Fab-frag ments were used in specific binding studies to both non-activated and ADP and thrombin-activated human blood platelets.
The Effect Of Piracetam In Preventing Recurrent Deep Venous Thrombosis
Authors: Bick R , Nix M , Skondia V
Published: July 2018

Piracetam, 2-Oxy-1-pyrrolidine Acetamide is a cyclic derivative of gamma-aminobutyric acid. This agent has recently been shown to have activity in humans as a platelet suppressant. For the prophylaxis of recurrent deep venous thrombosis (RDVT), many clinicians are now resorting to antiplatelet agents, rather than vitamin K antagonist because of the questionable efficacy and severe bleeding associated with these latter agents. The most common antiplatelet agents used for prophylaxis of RDVT are ASA, dipyridamole, and sulfinpyrazone given alone or in various combinations. The purpose of this study was to assess the efficacy of Piracetam as an antiplatelet agent for the prophylaxis of RDVT. Twenty patients with a history of RDVT or with active DVT were entered into the trial; 10 of these were randomized to a combination of ASA (600 mg. BID) + Dipyridamole (50 mg QID) and 10 were randomized to Piracetam at 9.6 gm/day as 2.4 gm QID. Pre-ingestion evaluation included a complete history and physical and complete evaluation of hemostasis including PT, PTT, FDP, PSO4, AT- III, platelet adhesion, platelet aggregation, PF-4, and platelet survival by sizing. Follow-up evaluation was performed at 6 weeks, 3 mo., and 6 mo. All patients demonstrated a laboratory response to antiplatelet therapy as manifest by aggregation and PF-4 studies, all patients on Piracetam and most patients in the other group demonstrated improvement in platelet survival studies. During the six month observation, no patients ingesting Piracetam developed rethrombosis; however, two patients ingesting ASA/Dipyridamole relapsed. These results suggest Piracetam to be equal to and possibly superior to ASA/Dipyridamole in preventing RDVT. In view of this efficacy together with lack of toxicity Piracetam may prove highly useful as a clinically effective antiplatelet agent in the prophylaxis of RDVT.
A Novel Method For The Direct Determination Of Platelet-Associated IgG (PAIgG) Employing Rocket Immunoelectrophoresis (Laurell)
Authors: Kunicki T J , Kristopeit S M , Aster R H
Published: July 2018

The diagnosis and management of immune thrombocytopenia requires an effective method for the quantitation of platelet- associated IgG (PAIgG). Although several methods have been recently devised for this purpose, the search continues for a method which will prove to be accurate and reproducible, and yet simple enough to be utilized by the general clinical hemostasis laboratory. We now describe the development of an assay for PAIgG which we believe satisfies these criteria.

Human platelets were isolated by differential centrifugation, washed five times in Tris-HCl pH 7.4 or phosphate buffered saline pH 7.4, both containing 5mM EDTA, and disrupted in 0.1M sodium acetate buffer pH 5.0 containing 1% (v/v) Triton X-100. Rabbit antibody specific for γ chains of human IgG (purified IgG fraction from DAKO) was carbamylated such that its isoelectric point was decreased to roughly 5.0. Total PAIgG was measured by electrophoresis at pH 5.0 of Triton X-100 disrupted washed platelets against the carbamylated rabbit anti-human IgG (γ) in 1% agarose gels containing 0.5% Triton X-100.

The mean total PAIgG for 8 normals, was 5.7 fg/platelet (range 4.9 to 7.0). This technique is applicable to the quantitation of alloantibodies bound to normal platelets (indirect assay) as well as the quantitation of PAIgG on platelets of individuals with immune thrombocytopenic states, such as ITP.
Factors Affecting Thrombus Formation On Surfaces
Authors: Schultz J S , Lindenauer S M , Penner J
Published: July 2018

An ex-vivo test device consisting of a flow-through cylinder with a rotatable central rod has been developed for evaluating thrombus formation on surfaces. The rod can be coated with polymers, or segments of blood vessels can be placed on the rod. Blood access to the device is obtained by means of a A-V shunt, using dogs as the test animal. Measurement of platelet, fibrinogen, and red cell content characterizes thrombus composition on the rod and provides several indicies of the coagulation systems involved in thrombus development.

Tests of more than a dozen biomaterials showed that thrombus amount and composition on surfaces differ many fold under similar flow conditions. Increases in shear from 100 sec-l to 200 sec-l on the rod surface, by increasing the rotation rate of the rod, resulted in virtually complete reduction of rbc attachment, corresponding to a 75% inhibition of fibrinogen deposition while platelet attachment was only reduced by 50%. This demonstrates that shear has a differential effect on the various components of the clotting system.

The use of systemic drugs like aspirin and heparin showed that the effects on thrombus formation are quite different for materials with various degrees of thrombo-genicity. Also it was demonstrated that when the same polymer material was cast from two different solvents to obtain variations in surface morphology the thrombogenicity was quite different.

A direct comparison of the thrombogenicity of biomaterials and vascular endothelium is possible by placing an everted piece of vein on the coated rod.

Tests showed that silastic and vein endothelium were similar with respect to platelet and frbrinogen deposition. This ex-vivo procedure provides an opportunity to assess the antithrombogenic effect of drugs applied directly to the surface of the excised vessel segment.
Calcium Ion Binding Within Fibrinogen Fragment D
Authors: Britton David W , Lawrie Jan S , Kemp Graham D
Published: July 2018

Fibrinogen contains a number of strongly bound calcium. ions and a considerable body of evidence new exists to show that the plasmin degradation product fragment D contains one strongly bound calciumion. It is also established that this calcium ion has a notable effect on the plasmin resistance of the molecule. Previous work from this laboratory strongly suggests that the binding site is located towards the C-terminus of the γ chain. We have also investigated the influence of calcium. ions on the conformation of fragment D by ultracentrifugation and chemical cross-linking. In the presence of calcium ions there is a preponderance of intra-molecular cross-linking even at high concentrations of fragment D using bisimidates such as dimethyl suberimidate and dimethyl adipimidate. In the absence of calcium. ions there is an increase in the extent of inter-molecular cross-linking. From such evidence we would propose that calcium ions stabilise a compact conformation within fragment D. The presence of calcium ions also affects the stability of the D:E complex.
Comparison Of The Major Membrane Glycoproteins Of Human, Rabbit And Rat Platelets
Authors: Toor B , McGregor J L , Clemetson K J , McGregor L , Dechavanne M , Lüscher E F
Published: July 2018

Rabbit and rat platelets have been extensively investigated under in vitro or in vivo conditions to try to understand the pathology of thrombosis in man. Here, surface-labelling techniques have been used to find out if the platelet surface has a similar composition in these two animals and in man or not. Human, rabbit and rat platelets were isolated, washed and surface-labelled by techniques specific for protein or for sugars (sialic acid or penultimate galactose/N-acetyl galactosamine residues). Labelled platelets were solubilized in sodium dodecyl sulphate and separated under reducing conditions on 7.5 % Laemmli polyacrylamide gels. Dried gels were exposed to film by fluorography or indirect autoradiography. Terminal Gal/Gal NAc residues (no neuraminidase treatment) were strongly labelled with rat and rabbit platelets compared to human platelets which labelled very poorly. Terminal sialic acid labelling with rat and rabbit platelets showed a weak labelling of a glycoprotein (GP) with the same M.Wt. as GPIb which is the most intensely labelled GP in man. However two GP (with rabbits) and one GP (in rats) were intensely labelled at a M.Wt. similar to that of GPIa in man. These GP had a different M.Wt. with terminal Gal/Gal NAc labelling. Bands with a similar M.Wt. to GPIIb and IIIa in man were strongly iodinated with rabbit platelets but with rat platelets only a single band at the position of GPIIb was strongly iodinated. These results strongly indicate that there are considerable differences in surface composition between rabbit, rat and human platelets.
The Lack Of Relationship Between Anti-Xa Activity And Antithrombotic Activity Of Low Molecular Weight Heparin
Authors: Ockelford P A , Carter C J , Hirsh J
Published: July 2018

The effectiveness of low dose heparin in the prevention of venous thrombosis and pulmonary embolism has been attributed to its enhancement of plasma anti-Xa activity. Low molecular weight (MW) heparin fractions have a relatively high anti-Xa activity, both in vitro and ex vivo, and it has been assumed that this high anti-Xa activity correlates with antithrombotic activity.

We have compared the antithrombotic effect of a very low MW heparin fraction (mean MW 3,000) with standard heparin (mean MW 15,000) using a rabbit prophylaxis model and thromboplastin as the thrombogenic stimulus. The antithrombotic effect of these heparins was related to ex vivo anti-Xa heparin activity. Both heparins showed a dose-dependent antithrombotic effect, enabling the relative antithrombotic and anti-Xa activities to be compared over a dosage range. Even when the ex vivo anti-Xa activity achieved with the low MW heparin was significantly greater than for the standard heparin (0.41 and 0.16 anti-Xa units per ml; p < 0.01), significantly less inhibition of thrombosis was observed with the low MW heparin fraction (24% and 88%; p < 0.02). For an equivalent antithrombotic effect (50% inhibition of thrombosis) an ex vivo anti-Xa activity in excess of five fold was required in the low MW heparin treated animals. These findings are in contrast with our previous experience using a low MW fraction (mean MW 4,600) and suggest that the relationship between anti-Xa activity and antithrombotic effect may be MW dependent.
Association Between Plasma Concentration Of Factor VIII Related Antigen, ABO Blood Type And Lewis Blood Type
Authors: Orstavik K H , Magnus P
Published: July 2018

Individuals with blood type 0 have a lower plasma concentration of factor VIII than individuals with blood type A. Since the Lewis blood type system and the ABO blood type system are related, we looked for a possible association between the plasma concentration of factor VIII and Lewis blood type.

Plasma concentration of factor VIII related antigen (FVIIIR:Ag) was determined by the Laurell electroimmunoassay in 333 individuals. These individuals were identical and fraternal twins who had been bled as part of a twin study on coagulation factors. The association between factor VIII and ABO blood type was confirmed since a significantly lower concentration of FVIIIR:Ag was found in individuals with blood type 0 (77%) than in individuals with blood type A (106%). Lewis blood type had no significant effect on the concentration of FVIIIR:Ag when the whole material was examined. Within individuals with blood type 0, a much lower concentration of FVIIIR:Ag was found in individuals with Lewis blood type Le(a-b-) (52%) compared to individuals with Lewis blood type Le(a-b+) (80%) or Le(a+b-) (88%).

The possibility that individuals with blood type 0 and no Lewis antigens have a low plasma concentration of factor VIII may have implications for the detection of carriers of hemophilia A.