bluuebunny/crossref_metadata_2025 huggingface dataset:
Platelet Aggregation Induced In Vivo By Heparin And Heparin Fractions
Authors: Silane M , Lindon J N , Ransil B J , Rosenberg R D , Salzman E W
Published: July 2018

As we have reported, heparin-induced platelet aggregation in vitro varies among heparin subfractions, being generally less with lower molecular weights and having a reciprocal relationship with antithrombin affinity.

We now have studied heparin-induced platelet aggregates in vivo by the technique of Wu and Hoak using arterial blood from unanesthetized rabbits. Porcine mucosal heparin was fractionated by gel filtration into high molecular weight (ave. 15,000 Daltons) or low molecular weight (ave. 6,000 Daltons) preparations. IV administration of commercial porcine mucosal heparin (spec. act. 150 u/mg) or high (spec. act. 183 u/mg) or low (spec. act. 208 u/mg) molecular weight fractions was followed by an increase in the platelet aggregate ratio compared with preinjection control values. The rise in platelet aggregate ratio with heparin was significantly different from the effect of a saline placebo (n=8) but was not significantly different among rabbits receiving the commercial heparin (n=9) or the high (n=8) or low (n=8) molecular weight preparations. Peak rise in circulating aggregate ratio occurred 2 minutes after the injection, and values returned to control levels within 15 to 30 minutes. There was no change in platelet count in blood collected in EDTA, suggesting that the aggregates were not removed from the circulation in vivo.

Heparin fractions of low molecular weight were further separated according to antithrombin affinity by an antithrombin binding technique. In 8 rabbits low molecular weight/high antithrombin affinity heparin (spec. act. 480 u/mg) did not cause formation of platelet aggregates. The results were significantly different from those with commercial heparin (p=0.05) or with the other heparin fractions (p=0.06).

Clinical use of low molecular weight heparin of high antithrombin affinity may lead to fewer heparin-induced platelet effects and to an improvement in anticoagulant therapy.
Is Phosphorylation Of Adp Required For The Induction Of Shape Change And Aggregation?
Authors: Mills D C B , Lipson C
Published: July 2018

Observations on the activity of nucleoside diphosphokinase on the external surface of washed rabbit platelets have suggested that phosphorylation of ADP at the expense of intracellular ATP occurs in association with aggregation. We have studied aggregation and shape change of washed human platelets labelled in vitro with 32Pi, using ADP or an analogue, 2-methylthio ADP, to induce aggregation in the presence of fibrinogen. EDTA was added during (30sec) or after (2min) aggregation, and the platelets were removed gently by centrifugation through silicone oil. Nucleotides were concentrated from the medium by absorption on, and elution from charcoal-coated beads, and were analysed by paper electrophoresis and HPLC. ATP (50-1OOuM) was added to some samples to trap any labelled ATP formed. This reduced the rate of aggregation but did not stop it. Less than 0.1% of the intracellular radioactivity was recovered as extracellular ATP and this amount was not signfleantly increased by ADP. Under these conditions less than 2000 molecules of ADP were phosphorylated for the aggregation of each platelet. This suggests that phosphorylation of ADP is not required for aggregation or for shape change.
Induction And Potentiation Of Platelet Aggregation By Clonidine And 7ρ-Aminoclonidine, Partial Agonists Of The α2-Receptor Which Regulates Platelet Adenylate Cyclase
Authors: Stump David C , Macfarlane Donald E
Published: July 2018

Epinephrine induces platelet aggregation, potentiates aggregation by other agents, and blocks the stimulation of the adenylate cyclase by prostaglandins. Synthetic α-adrenergic agents have not been shown to induce aggregation. The effects of clonidine, an α2-agonist, and ρ-aminoclonidine on platelets were examined. Clonidine potentiated aggregation induced by 0.5μM ADP by 1.4-fold (1/2 max 0.5μM). It did not induce significant aggregation itself, and it inhibited aggregation induced by 5μM epinephrine (1/2 max lμM). It inhibited cyclic AMP accumulation induced by PGE1 by a maximum of 25% (1/2 max O.lμM) and it blocked inhibition by epinephrine. No significant specific binding of [3H] clonidine was observed to intact platelets. ρ-Aminoclonidine induced aggregation with delayed second phase (1/2 max 0.2μM), and potentiated ADP aggregation by 2-fold (1/2 max 0.2μM). Aggregation induced by epinephrine was more rapid, and was partially inhibited by ρ-aminoclonidine. It inhibited cyclic AMP accumulation by 50% max (1/2 max O.lμM) and attenuated epinephrine’s effect to the same level. The direct effects of ρ-aminoclonidine were blocked by lμM yohimbine, a selective α2-antagonist. Both clonidine and ρ—aminoclonidine blocked the specific binding of [3H]yohimbine (1/2 max 0.5μM). These results suggest that the platelet bears an α2-receptor with affinity for epinephrine, ρ-aminoclonidine and clonidine as agonists but that these agents display differing intrinsic activity and/or receptor reserve.
Effect Of Prostaglandin E1 On “Inflammatory” Or “Ischemic” Skin Ulcer In The Patients With Peripheral Vascular Disease
Authors: Tohjima T , Uehara N , Sawada K , Tsuda Y , Sakai K , Funayama H , Shiokawa Y
Published: July 2018

Clinical, physiological and biochemical studies of PG E1 treatment were done, in a series of 7 collagen disease patients with “inflammatory” skin ulcer and 5 diabetics with ischemic ulcers or apical gangrene of toes. Intravenous infusions of prostaglandin (PG)EL was given continuously in the dose of lng/kg/min for 72 hours. Blood samples were collected from cubital vein, before, during, right after, and at 7 days after PG E1 therapy. Platelet aggregations induced by ADP collagen epinephrine were evaluated by light transmittance. Platelet iPG E(immuno-reactive PG E like material) levels were measured by radioimmunoassay. Essential fatty acid compositions of plasma, platelet, and red cell were analyzed by gas chromatography.

Results were as follows: 1. In all of cases, complete or almost complete healing of skin ulcers or abolition of the pain were noted. 2. Skin temperature was elevated during PG E1 treatment. 3. The platelet basal iPG E levels were significantly decreased by PG E1 treatment(p<0.025). 4. The plasma and platelet linoleic acid levels were significantly higher than before the treatment plasma: p<0.05, platelet: p<0.025). 5. In most cases, platelet aggregation was increased during PG E1 treatment than before.

Conclusion: Dramatic therapeutic effects of PG E1 were observed on “inflammatory” or “ischemic” skin ulcer in patients with peripheral vascular disease. This effect might be resulted from improvement of PG metabolism abnormality in the platelet etc. Platelet aggregation in vitro may dissociate from the results in vivo.
Sulfinpyrazone Improves Myocardial Blood Flow And Inhibits Platelet Release During Exercise In Coronary Disease
Authors: Steele P , Gold F , Sklar J
Published: July 2018

Exercise (EX) is associated with activation of the platelet release reaction (REL), and REL during EX is exaggerated in men with coronary disease (CAD). Sulfinpyrazone (SFP) and aspirin (ASA) inhibit REL at rest and during EX. Sixteen men with CAD underwent treadmill EX with measurement of β-thromboglobulin (β-Th) and thromboxane B2 (TBX) (radioimmunoassay) at rest and just after anginalimited EX. Eight men were randomly assigned to SFP (200 mg po QID) and eight to ASA (300 mg po BID) and EX repeated 7 days later. Placebos were given for 7 days and EX repeated (double blind, cross-over). Myocardial blood flow distribution (MBFD) was measured during EX (201Thal1ium; 7-pinhole tomographic image acquisition and analysis). β-Th was elevated at rest (40.±5. ng/ml; N=16; AVE±SEM; normal 18.±2. ng/ml; N=22; p<0.001) and during EX (120.±8. ng/ml; normal 28.±4. ng/ml; p<0.001). TBX was not detected in venous blood at rest, but was present in 14 men during EX (15.±3. pg/ml; N=16; normal 0. pg/ml; N=22). SFP decreased β-Th at rest (control 33.±4. ng/ml; SFP 17.±3. ng/ml; N=8; p<0.001) and during EX (control 121.±14. ng/ml; SFP 46.±7. ng/ml; p<0.001). ASA also decreased β-Th at rest (control 46.±3. ng/ml; ASA 28.±7. ng/ml; N=8; p<0.001) and during EX (control 118.±9. ng/ml; ASA 44.±7. ng/ml; p<0.001). TBX was not detected in any man either at rest or during EX during treatment with SFP and ASA. All men had abnormal MBFD during control EX. SFP improved MBFD (+364.±62. normalized counts; N=8; integrated count rate difference between control and EX with SFP; control vs placebo +17. normalized counts). ASA did not alter MBFD (+140.±29. normalized counts; ASA vs placebo +7 normalized counts). Neither SFP nor ASA altered heart rate, systolic blood pressure or ST segment depression during EX. Results suggest that REL is activated by EX in men with CAD and that SFP and ASA inhibit EX REL, including TBX. SFP has a greater effect on MBFD during EX than ASA.
Betathromboglobulin And Fibrinopeptide A In Exercise-Induced Myocardial Ischemia
Authors: Turpie A G G , de Boer A C , Sealey B J , Butt R , Genton E
Published: July 2018

To investigate the role of platelet release and thrombin activity in exercise-induced myocardial ischemia, plasma betathromboglobulin (BTG) (mean ± SD, ng/ml) and plasma fibrinopeptide A (FpA) (pmol/ml) were measured before and after treadmill testing using the Bruce Protocol in normal subjects and 77 patients with suspected coronary artery disease (CAD). In normals, there was no difference between pre and post-exercise BTG (31 ± 9 v 27 ± 9) nor FpA (1.0 ± 0.8 v 0.8 ± 1.3). In 31 patients with a negative exercise test there was no difference in mean pre and postexercise BTG conc (36 ± 14 v 41 ± 33; p>0.1), but there was an increase in mean FpA. Ten of the patients with negative exercise tests had documented CAD by coronary angiography and their FpA rose from 2.1 ± 1.8 to 4.3 ± 4.4 (p < 0.05) after treadmill testing; 6 had a significant increase in FpA and 4 in BTG. In 46 patients with an abnormal exercise test (>1 mm ST-segment depression) there was an increase in mean BTG post-exercise (pre 42 ± 17 v post 60 ± 60; (p<0.005) and the increase was significant in 32%. The increase was more common in those patients who developed chest pain during the test. Plasma FpA increased from 1.6 ± 1.2 to 4.1 ± 4.6 in the patients with a positive test (p<0.005) with an increase in 33% of patients. These data indicate that exercise-induced myocardial ischemia is associated with platelet release and thrombin activity in patients with CAD. Increase in BTG after treadmill testing occurred mainly in patients with a positive test who developed chest pain. The FpA in patients with CAD increased in patients with either positive or negative treadmill tests. Increase in FpA post-exercise appears to be a more sensitive indicator of CAD than BTG.
Production Of Thromboxane B2 By Platelets Is Related To Their Density
Authors: Martin J F , Shaw T , Jakubowski J , Penington D G , Martin T J
Published: July 2018

Platelets of different densities are probably produced from megakaryocytes of different ploidy. Thromboxane B2 (TxB2) production was studied in density dependent subfractions of the total platelet population.

Platelets were isolated by a new technique using velocity sedimentation into a polyvinylpyrrolidone-coated colloidal silica (Percoll) gradient followed by equilibrium centrifugation through a continuous linear Percoll gradient. Fractionation of the second gradient yielded density-dependent platelet subpopulations which were functionally competent and showed minimal leukocyte and plasma protein contamination. By pooling fractions and adding buffer, suspensions containing similar concentrations of platelets representative of low, intermediate and high density subpopulations were prepared, each containing 20–30% of the total population. Aliquots of each suspension were stirred in an aggregometer and thrombin or arachidonic acid (AA) were added to induce aggregation. After three minutes the reaction was arrested by addition of ice cold ethanol and TxB2 measured by radioimmunoassay.

When 0.5 mM AA (final concentration) was the aggregating agent, for every 108 platelet the least dense subpopulations produced 10.2 ng TxB2, and the most dense subpopulations 22.4 ng TxB2,(means of four experiments), whilst the intermediate density subpopulations gave intermediate values. However, thrombin at 0.5 or 1.0 units/ml produced similar amounts of TxB2 in all subpopulations. In a second series of experiments, AA-stimulated serotonin secretion in density dependent platelet subpopulations correlated closely with platelet number.

These observations imply functional differences between platelet subpopulations.
The Relationship Between The Multimeric Structure Of F VIII-VWF And The Facilitation Of Platelet Adhesion To Human Subendothelium (Von Willebrand Factor Activity VWF-A)
Authors: Sixma Jan J , Sakariassen Kjell S , Bolhuis Piet A
Published: July 2018

The relation between the VWF-A of F VIII-VWF and its multimeric structure was studied in an annular perfusion chamber according to Baumgartner, with a steady flow system.

51Cr-labelled aspirin treated human platelets and human post mortem renal arteries were employed. F VIII-VWF was purified from cryoprecipitate by agarose gel chromatography and radiolabelled by the lactoperoxydase method. The multimeric distribution was determined by SDS-agarose electrophoresis. Five different commercial high purity concentrates contained multimers between 0.5 and 3.5 x 106 mol wt. None of these concentrates had VWF-A at ristocetin cofactor activities (RIC0F-A) of 1.0 u/ml. A low potency concentrate with mul- timers in the same mol wt range had normal VWF-A. Mild reduction (dithioerythritol-DTE ≤ 2mM) caused a shift in , mol wt range from 3.0 - 20.0 × 106 towards 0.5 - 2.0 × 106 with little decrease in RIC0F-A and VWF-A. Reduction with 10 mM DTE produced multimers of 1.5 and 0.5 × 106 without RICOF-A and VWF-A, but binding normally to the vessel wall. The void volume peak of 125I-VIII-VWF was rechromatographed on Sepharose-2B and arbitrarily divided in fractions A: mol wt 8,0 - 18.0 × 106 ;B: mol wt 4.5 - 11.0 × 106 ; and C: mol wt 2.5 - 6.5 × 106 . Binding of 125I-VIII-VWF to the subendothelium was highest for A, intermediate for B and lowest for C. Correction for mean mol wt showed that almost equal numbers of molecules bound from all three fractions. When the quantity of bound VIII-VWF was thus expressed, all fractions had a similar relative VWF-A.

These data indicate that high purity concentrates do not correct the bleeding time at normal RICOF-levels, because they lack VWF-A. Multimers of high mol wt normally carry both RICOF-A and VWF-A, but the high mol wt is no absolute requirement. These data are in agreement with the notion that VWF-A resides on a specific polypeptide chain in the molecule.
Microfibrils (MF) Platelet Interaction: Requirement Of Von Willebrand Factor In Platelet Adhesion To A Placenta Microfibrillar Component (PMC)
Authors: Fauvel F , Legrand Y J , Gutman N , Muh J P , Tobelem G , Souchon H , Karniguian A , Caen J P
Published: July 2018

It has been shown that collagenase resistant arterial microfibrils (MF) are able to interact with platelets and therefore represents, besides collagen, a second thrombogenic structure in the vessel wall. In vitro observation using a PMC purified from the villosities of human placenta by a mechanical non denaturing procedure confirm this interaction between platelets and MF. PMC was homogenous under electron microscope (feltwork of MF with a mean diameter of 120 – 130 A) and was glycoproteic in nature. PMC were able to induce an aggregation of human platelets only if the platelets were in plasma. The role of Von Willebrand factor (F VIII/WF) as a cofactor of the aggregation of platelets by MF has been postulated from the fact that twice washed platelets from normal subject resuspended in PPP obtained from a severe Von Willebrand deficient patient were not aggregated by the PMC. Furthermore, aggregation was restored after resuspension of the same platelets in the PPP of the same patient 30 and 120 minutes after perfusion of cryoprecipitate (40 units F VIII/RA per kg).

F VIII/WF mediates platelet adhesion after binding to subendothelium of human artery. Our observation strongly supports the idea that MF are the subendothelial components to which F VIII/WF binds, thus promoting an adhesion of platelets.
Following Mild Carotid Endothelial Injury, Platelet-Arterial Wall Interaction Is Impaired In Pigs With Von Willebrand'S Disease
Authors: Fuster V , Dewanjee M K , Kaye M P , Fass D N , White J G , Bowie E J W
Published: July 2018

Platelet-arterial wall interaction appears to be important in the genesis of atherosclerosis. Pigs with homozygous von Willebrand’s disease (vWd) appear resistant to atherosclerosis. We investigated whether there is impairment in platelet-arterial wall interaction in porcine vWd. Superficial endothelial injury was produced in a 4 cm segment of carotid artery by drying for 3 minutes in a stream of air which entered one end (100 ml/min) and exited through a small hole in the other end. This procedure was performed in 9 normal and 6 vWd pigs 24 hours after the I.V. infusion of III-Indium-labeled platelets. Carotid blood flow was re-established and the pigs were sacrificed 24 hours later and the carotids fixed in glutaraldehyde.

The ratio of radioactivity (cpm/g) of injured/noninjured carotid arteries was markedly decreased in the vWd pigs. In addition, transmission and scanning electron microscopy confirmed superficial endothelial damage in all injured segments, but only significant platelet deposition in the normal pig injured segments.

After a mild endothelial injury, this “in vivo” evidence of impaired platelet-arterial wall interaction in vWd may be related to the resistance to atherosclerosis in these animals.
Comparison Of The Effects Of Aspirin And Heparin On Platelet Accumulation On The Injured Neointima Of Rabbit Aortae
Authors: Groves H M , Kinlough-Rathbone R L , Mustard J F
Published: July 2018

We have previously shown in rabbits that treatment with aspirin (ASA) in doses that inhibit PGI2 production by the aorta does not cause platelet accumulation on the endothelium or cause increased accumulation on the subendothelium when the endothelium has been removed with a balloon catheter. We have also shown that, in contrast to the single layer of platelets that adheres to the subendothelium, extensive microthrombus formation occurs on the surface of the injured neointima. The objective of the present experiments was to compare the effects of ASA and heparin on platelet accumulation on the injured neointima. The endothelium was removed from rabbit aortae with a balloon catheter and at 7 days, when a substantial neointima had formed and was essentially non-reactive to the circulating platelets, the aortae were again injured by the passage of a balloon catheter. Platelet accumulation (measured with 53-Cr- labeled platelets) was approx. 50,000 per mm2 whereas on the uninjured neointima at 7 days it was 1,200 per mm2. Administration of ASA (in a dose that inhibits platelet function and PGI2 production by normal vessels, 100 mg/kg I.V.) 10 min before the neointima was injured did not cause a significant increase or decrease in the number of platelets that accumulated on the injured neointima (53,200 ± 11,200 platelets per mm2 without ASA; 48,100 ± 7,900 platelets per mm2 for ASA-treated rabbits). In contrast to the effects of ASA, heparin (500 U/kg), as described previously, caused a significant reduction in the number of platelets that accumulated on the injured neointima (50,000 ± 8,800 platelets per mm2 without heparin; 25,400 ± 5,500 platelets per mm2 for heparin-treated animals (p<0.05). The results from these experiments indicate that ASA inhibition of platelet function and PGI2 production by the vessel wall does not change platelet accumulation that occurs upon injury of the neointima of a large artery. However, the results with heparin indicate that heparin changes the extent of platelet accumulation on the injured neointima.
Thrombin Induced Contraction Of Isolated Smooth Muscles
Authors: Fareed Jawed , Messmore Harry L , Kindel G , Karczmar A G , Fenton J W
Published: July 2018

During the clotting of whole human blood or plasma, we have observed the generation of vasospasmodic activity with isolated smooth muscle preparations. Since kallikreins reportedly have musculotropic effects, such effects of related serine proteases were examined on isolated guinea pig ileum and rat uterus smooth muscle preparations. Like glandular kallikrein, human α-thrombin caused pronounced muscular contractions of these preparations and did so more effectively than trypsin and much more so than plasmin, activated coagulation factor X or XII, or noncoagulant β- and γ-thrombins. Levels as low as 0.1 to 1 clotting (NIH) units/ml final bath concentration of α-thrombin caused such contractions(5 units/ml grossly contracted muscle preparations) and the contractile activity was inhibited by either hirudin or antithrombin III plus heparin, indicating the requirement for the catalytically active enzyme. These contractions were not blocked by antihistamic (tripell ana- mine), antimuscarinic (atropine), or antiganglionic (mecamylamine) agents, further suggesting that the contractions were not mediated by an autonomic mechanism but rather were attributable to a direct function of the enzyme. Because physiologic concentrations of less than 5 units/ml were effective, our data imply that vasospasmodic responses may not only result from thromboxanes and platelet release products (e.g. serotonin and ADP) but also to direct α-thrombin induced contractile effects on smooth muscles exposed at sites of vascular injury. Such smooth muscle contractions may facilitate in the prevention of blood loss or may contribute to pathologic states (e.g. ischemia, stroke) impeding blood circulation.
Purification And Characterization Of Calmodulin From Bovine Platelet
Authors: Kambayashi J , Sakon M , Kōsaki G , Sobue K , Kakiuchi S
Published: July 2018

Calmodulin, which was originally reported as a Ca2+ dependent activator of phosphodiesterase, mediates a number of actions of Ca2+ as a second messenger for stimulus linked cellular responces in eukaryotic cells. In blood platelets, the availability of Ca2+is a prerequisite for steps of platelet reaction leading to the formation of hemostatic plug. The presence of this protein in platelets has been reported. In the present study, calmodulin was purified from bovine platelets and was extensively characterized.

The identification of the protein was based on the activity to activate the brain calmodulin deficient phosphodiesterase. Calmodulin was purified 600 fold from bovine platelet pellet by a 3-step purification method consisted of trichloroacetic acid treatment, DEAE-cellulose column chromatography and phenothiazine affinity chromatography. The recovery rate was more than 70% and the purified protein was homogenous on polyacrylamide gel electrophoresis. The mobility of the purified material upon polyacrylamide gel electrophoresis was identical with that of brain calmodulin in the presence of either Ca2+or EGTA. In the quantitative analysis of the phosphodiesterase activation, the behaviour of the purified protein was also identical with brain calmodulin.

From these strict characterization, it may be concluded that the purified protein is indistinguishable from brain calmodulin. The amount of the protein in bovine platelet was estimated to be 63 micrograms per wet gram, which is about one sixth of the amount in brain tissue.
Binding And Metabolisation Of PGI2 By Erythrocytes
Authors: Willems Ch , van Mourik J A , Stel H V , van Aken W G
Published: July 2018

Prostacyclin (PGI2) is rapidly hydrolysed in aqueous solution at neutral pH. Previously we have reported that PGI2 is stabilized by plasma components; yet PGI2 is rapidly metabolized in vivo. These findings prompted us to study the fate of PGI2 upon incubation in whole blood. The data on the distribution of PGI2 between blood cells and plasma indicate that PGI2 not only binds to platelets but also to erythrocytes. The Kinetics of binding were studied in more detail by incubating [3H] PGI2 at 37°C with washed erythrocytes resuspended in autologous plasma. Binding of [3H] PGI2 plateaued within 2 min. and was concentration dependant. The binding of [3H] PGI2 was not influenced by PGE1 or 6 keto PGF1α. [3H] 6 keto PGF1α showed no substantial binding to erythrocytes when compared with [3H] PGI2. Upon repeated incubation of [3H] PGI2 with erythrocytes less binding occured than would have Been expected from time and concentration dependancy. The latter finding is explained by the demonstration of breakdown of [3H] PGI2 - most likely into [3H] 6 keto-PGF1α - and by measurements of the biological activity of PGI2 Although metabol isation of PGI2 complicates the evaluation of binding kinetics it coufd be shown, by using inhibition of platelet serotonin release, that erythrocytes and platelets compete for PGI2. Binding of PGI2 to erythrocytes and subsequent metabolisation explains the apparent lability of PGI2 in whole blood. It is to expected that under physiological conditions erythrocytes suppress the effectiveness of PGI2 to act as a circulating platelet inhibitor.
13-Azaprostanoic Acid Potentiates Prostacyclin Induced Platelet Deaggregation
Authors: Parise L V , Venton D , Le Breton G C
Published: July 2018

13-Azaprostanoic acid (13-APA), a specific thromboxane/endoperoxide receptor antagonist, reverses platelet aggregation stimulated by the endoperoxide analog U46619. The present report demonstrates that 13-APA also potentiates prostacyclin (PGI2) reversal of U46619-induced aggregation. Human platelet rich plasma was aggregated with 3 × 106M U46619. Deaggregation was induced 2 min. sitosequent to the addition of aggregating agent and was measured over a 3 min. period. Concentrations of 13-APA (4 × 10-4M) and PGI2(4 × 10-9M) were chosen such that each agent individually induced approximately 20% deaggregation. Addition of half of the above concentrations of these agents i.e. 2 × 10-4M 13-APA plus 2 × 10-9M PGI2resulted in 62% deaggregation, demonstrating that the observed response was supraadditive. Only 8% deaggregation was induced by 2 × 10-4M 13-APA alone and 0% by 2 × 10-9M PGI2 alone. PGI2 causes platelet deaggregation presumably through elevation of cAMP. 13-APA, however, did not increase cAMP levels even at concentrations of 13-APA as high as 1.2 × 10-3M i.e. 9.8 ± 1.3 pmoles/ml for control and 10.8 ± 1.2 for 13-APA. Nevertheless it is possible that the observed potentiation of deaggregation was the result of 13-APA facilitating PGI2 stimulation of adenylate cyclase. Measurement of cAMP during deaggregation, however, showed no significant difference between treatment with PGI2 alone and treatment with PGI2 plus 13-APA i.e. 11.3 ± 0.4 pmoles/ml for control, 11.4 ± 0.3 pmoles/ml for 13-APA, 16.1 ± 0.5 pmoles/ml for PGI2 and 16.5 ± 0.8 pmoles/ml for PGI2 plus 13-APA. These results clearly establish that 13-APA and PGI2deaggregate platelets by distinctly separate mechanisms. In this regard we propose that PGI2 causes platelet deaggregation by stimulating intraplatelet calcium sequestration through a cAMP dependent process. 13-APA, on the other hand, blocks the ability of U46619 to mobilize intraplatelet calcium. The combination of these two mechanisms presumably results in the observed potentiation of deaggregation.
13-Azaprostanoic Acid Or Indomethacin Inhibit TXA2/PGH2 Stimulated Release Of Calcium From Isolated Platelet Vesicles
Authors: Rybicki J P , Venton D L , Le Breton G C
Published: July 2018

13-azaprostanoic acid (13-APA) acts as a direct antagonist of the human blood platelet thromboxane/endoperoxide receptor. Previous studies have suggested that arachidonic acid (AA) metabolites e.g. thromboxane A2 (TXA2) and prostaglandin H2 (PGH2) can mobilize Ca2+ from Ca2+ accumulating vesicles derived from the platelet dense tubular system. In the present study we investigated the effect of 13-APA on this Ca2+ mobilization process. Platelet vesicles were prepared as previously described with the exception that oxalate was not incorporated into the vesicles as a precipitating anion. 45Ca uptake was determined by millipore filtration and isotope counting of the filter paper. 3H-sucrose was used as an index of extravesicle Ca2+ . TXA2 production by the vesicles was measured by radioimmunoassay of TXB2. Vesicles were incubated with 5 × 10-5M Ca2+ before addition of AA. Ca2+ uptake and TXB2 production were determined before and at 3, 9, and 16 mm following the addition of AA. It was found that the vesicles accumulated 10 nmoles of Ca2+ per mg of protein under steady state conditions. Addition of A A (2.5 × 10-5 M) resulted in the release of 19 ± 6.2%, 34 ± 4.8% and 42 ± 5.1% of accumulated Ca2+ at 3, 9 and 16 min respectively. Pretreatment of the vesicles with indomethacin (4 × 10-6M) completely inhibited AA- induced release and reduced TXB2 production by 82%. Pretreatment of the vesicles with 13-APA (2 × 10-5M) also completely inhibited Ca2+ release . In this case, however, there was no inhibition of TXA? synthesis, i.e. 56 ± 9 ng TXB? per mg protein in the control ana 64 ± 12 ng TXB2 per mg protein with 13-APA. These results confirm previous studies that AA must be metabolized to TXA2/PGH2 in order to release Ca2+ from isolated membrane vesicles. The finding that 13-APA blocks TXA2/ PGH2 stimulated release of Ca2+ suggests that this release process is mediated through a specific receptor interaction. (Supported by a grant in aid AHA 79895.)
Arachidonic Acid-Induced Platelet Aggregation Independent Of Cyclooxygenase And Lipoxygenase
Authors: Maclouf J , Levy-Toledano S , de la Baume H , Hardisty R M , Caen J P
Published: July 2018

It is generally admitted that arachidonic acid (20:4 W6) can stimulate platelet aggregation after transformation into prostaglandin G2 and H2 further converted in thromboxane A2. This action is prevented by cyclooxygenase inhibitors. Washed platelets were isolated on metrizamide gradient and resuspended in a Ca2+ free buffer. Their stimulation by C20:4W6 was followed by (14C)-serotonin (5HT) release, thromboxane (TX) synthesis and a change of light transmission, not dependent on aggregation, accompanied by a slight lysis (14%). The addition of external Ca2+ suppressed lysis and allowed the formation of aggregates. Under these conditions, ccyclooxygenase inhibitors like acetyl salicylic acid, indomethacin or flurbiprofen totally suppressed TX synthesis without preventing platelet aggregation or (14C)- 5HT release. Other C 20 polyunsaturated fatty acids could not substitute for C20 : 4W6-induced aggregation and Ca2+ was found to be the most effective divalent cation for protection of the cell against lysis as well as in allowing the platelet to aggregate in the absence of TX formation. The use of the lipoxygenase inhibitor BW 755C did not prevent C20: 4W6 - induced aggregation of aspirin-treated platelets, suggesting that the phenomenon was independent of this pathway also. The total suppression of oxidative metabolism with these inhibitors was verified by the analysis of eicosanoids using glass capillary column gas chromatography.

It is suggested that under these conditions, C20 : 4 W6-induced platelet aggregation might be due to an ionophoric action of C20 : 4W6 in the absence of oxidation.
Lack Of Inhibition Of Thromboxane Production Despite Inhibition Of Platelet Function By 1,3-Bis(2 Chloroethyl)-1-Nitrosourea (BCNU)
Authors: McKenna R , Ahmad T , Prancan A , Simon D , Frischer H
Published: July 2018

We have previously shown that BCNU inhibits human platelet glutathione reductase (GSSG-R) prior to inhibiting platelet function; since thromboxane production is important in platelet function, we evaluated the effect of BCNU induced inhibition of GSSG-R on platelet thromboxane production.

Control platelet GSSG-R activity was 0.091 ]jmoles NAD(P)H oxidized min-1lmg-1 protein at 37°C (±0.015 S.D.; n=9); inhibition was detectable at 10-7M% BCNU (70% of control) with a >90% inhibition at and above 10-5M BCNU. Platelet aggregation in response to 1.5×10-3M Arachidonic acid (AA), 10 μM epinephrine, 6 μg/ml equine collagen and 3 μM ADP were inhibited at 10-5M BCNU and abolished at 10-4 BCNU.

BCNU (10-3M) did not affect the increase in oxygen consumption induced by AA. Using the rabbit aorta superfusion bioassay for thromboxane A2 (TXA2), threshold concentrations of AA in 10-5 and 10-4 BCNU platelets resulted in an increased measure of aortic tension 13.5 ± 9.4 mm S.D. (n=6) and 23.2 ± 9.5 mm respectively, compared with control values of 4.5 ± 2.4. Acetylsalicylic acid (5 × l0-4M) inhibited the contraction: 1.7 ± 1.1 (n=5). The conversion of 14C AA to thromboxane B2 (TXB2) and PGE2, as measured by radio TLC, was not decreased in BCNU treated platelets. There is a significant increase in TXB2 (p<0.05;n=4) and in the ratio of TXB2:PGE2 in platelets treated with 10-4M BCNU and 10-3M imidazole when compared to platelets treated with imidazole alone.

In conclusion BCNU induced inhibition of platelet GSSG-R and platelet function occurs despite preservation of thromboxane production
Human Platelet Aggregation Without Thromboxane (Tx) Formation
Authors: Westwick J , Smith J B , Kakkar V V
Published: July 2018

It is not known whether PG endoperoxides have to be converted to TxA2 in order to induce aggregation and secretion We have examined this crucial question by measuring human platelet aggregation, 14C-5HT release and TxB2 formation induced by collagen, arachidonic acid (AA), adrenaline, U46619 and PGH2 in presence of either 1) 1-2-(4-carboxy-phenoxy) ethyl imidazole hydrochloride, a potent and selective thromboxane synthesis inhibitor (TSI) or a carbocyclic thromboxane A2 (CTA) - a so called thromboxane receptor antagonist. TSI, 1.5 to 75μM produced a dose related inhibition (IC50 5μM n=5) of Tx and elevation of PGE2 synthesis in adrenaline (8μM) and collagen (1.5μg/ml) stimulated platelets. When 14C-SHT labelled platelets were stimulated with 4.5μM adrenaline in the presence of 0, 2.5, 15 and 300μM TSI a 35±2.8, 27±0.5, 24.5±5.0% release of 14C-5HT release resulted and a 10014, 9612, 9711 and 9114% of control aggregation occurred. Similarly when human platelets were stimulated with 0.8mMAA a 51±1.4% (mean ±SE) release of 14C-5HT occurred, while in the presence of 15, 75 and 300μM of UK, a 42±0.4, 32±1, 33±3% of 14C-5HT release resulted. Aggregation induced by the PG endoperoxide analogue U46619 (lμM) was not inhibited by 300μM TSI or l00μM indomethacin, although 99.5% inhibition of Tx formation resulted, and 50 to 60% inhibition of 14C-5HT release was produced both by TSI and indomethacin. However CTA (l-3±M) produced a dose related inhibition of both aggregation and release induced by U46619. CTA (l-10μM) was found to produce superimposable dose related inhibition of collagen induced aggregation and secretion of platelets, whether the platelets were pretreated with 300μM TSI, or not.

These results suggest that Tx formation is not necessary for human platelet aggregation, although is contributory to 14C-5HT release induced by collagen, adrenaline, AA and U46619.

Also caution must be employed when CTA is used to elucidate the role of TxA2, as it appears to be an effective PG endoperoxide antagonist.
Thrombolytic Properties Of Purified Human Tissue Plasminogen Activator In A Dog Femoral Vein Thrombosis Model
Authors: Korninger C , Matsuo O , Suy R , Stassen J M , Collen D
Published: July 2018

Experimental thrombosis was produced in an isolated 4 cm section of the femoral vein in the groin region of beagle dogs using a mixture of 125I-labeled human fibrinogen (6 × 106 cpm), dog blood, thromboplastin and thrombin. Saline, 1,000,000 IU of urokinase or 100,000 Units (1 mg) of highly purified tissue plasminogen activator (isolated from the culture fluid of human melanoma cells) was infused intravenously over four hours. The extent of thrombolysis was calculated from the difference between the radioactivity introduced in the clot and that in the recovered clot six hours after the start of the infusion.

In four control animals thrombolysis was 31.0 ± 3.0% (mean ± S.D.). Infusion of urokinase in 3 dogs resulted in 42.7 ± 14.3% lysis. In 5 dogs receiving plasminogen activator thrombolysis was 65.6 ± 21.6%. During infusion the blood radioactivity rose significantly (from 2.5 to 6.6% of the total) in the tissue plasminogen activator group; only a slight rise was observed in the urokinase group and a progressive decline in the control group.

Neither in the controls nor in the tissue plasminogen activator treated animals significant activation of plasminogen, consumption of α-antiplasmin or fibrinogen breakdown occurred and no major bleeding was noted. Infusion of urokinase, however, resulted in systemic activation of the fibrinolytic system with extensive fibrinogen breakdown causing a significant bleeding tendency.

It is concluded that in the experimental model used human tissue plasminogen activator has higher specific thrombolytic and a lower systemic fibrinogenolytic effect than urokinase.
Characterization Of The Active Site Of Urokinase With A Series Of Potent Inhibitory Amidines
Authors: Geratz J D , Shaver S R , Tidwell R R
Published: July 2018

Twenty amidine-substituted indole-like heterocycles were synthesized and examined for their blocking effect against human urokinase and a number of related arginine- or lysine- directed proteases. Kinetic analyses were carried out with the help of peptide anilide substrates and revealed a reversible competitive inhibitory pattern with each compound. The Ki values were therefore interpreted to reflect binding conditions at the active site of the enzymes.

A highly potent inhibitor of urokinase was discovered in 5-amidino-l-(4-amidinobenzyl)indole which proved to be 18 times more effective on a molar basis than p-aminobenzamidine and 150 times more effective than benzamidine. The Ki value at 37°C and pH 8.3 was determined as 3.2 × 10-6 M. In striking contrast to the findings with the other proteases studied, urokinase was very sensitive to inhibition by 6-amidinoindoline (Ki 1.8 × 10-5 M), yet was much less susceptible to inhibition by the fully unsaturated analog 6-amidinoindole. Steric factors resulting from the difference in planarity between the two compounds are held responsible for this observation. In plasminogen activation assays the antiurokinase effect of the heterocycles mirrored their potency in the assays employing the synthetic urokinase substrate.

The significant differences in the inhibitory activities of amidines against urokinase, on the one hand, and plasmin, thrombin and factor Xa, on the other hand, will be useful for experiments where selective inhibition of plasminogen activation is to be achieved. The compounds will also be of help in characterizing other tissue activators with respect to urokinase.
The Thrombolytic Activities In A Rabbit Model Of Venous Thrombosis Of An Acyl Human Plasmin Derivative (BRL 26920) And An Acyl Streptokinase Human Plasminogen Activator Complex (Brl 26921)
Authors: Dupe R J , English P D , Green J , Smith R A G
Published: July 2018

The structures of the novel acyl-enzyme fibrinolytic agents BRL 26920 and BRL 26921 are given. A model of venous thrombosis in which thromboplastin-induced thrombi are formed in the inferior vena cava of the rabbit is described. Thrombi were held in place by an implanted woollen thread and thrombolysis was measured by release of 125I-FDP into the circulation and by radiochemical quantitation of residual clots. BRL 26920 was compared with the same dose of activator-free human plasmin in the model. The acyl form of the enzyme was found to be significantly more active and caused significantly less disturbance of haemostasis than the free enzyme. Release of radioactivity from labelled thrombi suggested that the duration of fibrinolysis following a bolus of BRL 26920 was at least 4 hours.

Similar comparisons of BRL 26921 with unmodified streptokinase human plasminogen activator complexes were made in this model. Although neither agent caused large haemostatic changes the acyl-enzyme was significantly more active.
Subcutaneous Heparin Therapy In Renal Insufficiency
Authors: Salzmann W , Andrassy K , Ritz E , Koderisch J
Published: July 2018

It is often necessary to treat uremic patients with low dose heparin therapy,e.g. for prophylaxis of Cimino-fistula thrombosis. However, little information is available on heparin pharmacokinetics and heparin action in uremic patients. In the present study, plasma activity time profiles during low dose heparin therapy were investigated in control individuals (CO) and in uremic patients (UP). Patients and methods: Heparin levels 0;3;8;10;12;24;27;32;34 and 36 h after s.c. injection of various doses of heparin (2×7.500 IU/day for 2 days; 3×5.000 IU and 2×5.000 IU each for 2 days) were measured in 9 control individuals and in 11 uremic patients (Ccr < 10 ml/min.). Heparin activity was determined by measurements of (1) neutralisation of factor Xa-activity (Denson and Bonnar); (2) neutralisation of Xa measuring amidolytic activity (Teien and Lie); (3) PTT and thrombin time. Heparin cofactor concentrations were measured with immuno-diffusion and by measuring amidolytic activity. Results: With the dose of 2×5.000 IU heparin s.c. no difference between CO and UP was found; in contrast, peak concentrations of heparin were significantly lower in UP after 7.500 IU heparin s.c. With the dose of 3×5.000 IU heparin s.c.there was a significant (p<0.05) difference of heparin levels between CO and UP. This difference was even more pronounced after repeated administration of heparin; heparin levels in UP were markedly lower than in controls. The antithrombin III levels did not change significantly during the study. Conclusion : The results show that in order to reach a given profile of heparin activity, higher s.c. doses of heparin must be administered in uremic patients than in non-uremic controls.
Heparin And Antithrombin III.Levels During Cardiopulmonary Bypass Surgery-A Pilot Study
Authors: OBrien P F , Savidge G F , Williams B
Published: July 2018

Heparin and antithrombin III (AT III) levels were followed in ten patients during cardiopulmonary bypass surgery in a pilot study designed to assess the adequacy of the local conventional protocol for heparinization and reversal. Heparin was administered according to the local standard procedure on the basis of body surface area and was neutralized by doses of protamine sulphate calculated on the basis of the initial loading dose of heparin. A fluorometric method was employed to assay plasma heparin and AT III concentrations using the synthetic fluorescent tripeptide, H-D-phe-pror-arg-5-amidoisophthalic acid, dimethyl ester, as the thrombin substrate. Heparin levels were in the range of 2.0 to 6.5 IU/ml during bypass with significant differences between and within patients in response to periodic additional injections of heparin, due to neutralization of heparin by PF4 released during bypass. All patients showed a sudden drop in the level of AT III of 15 - 35 IU/dl after the initial dose of heparin given before the start of surgery. The AT III levels varied greatly during bypass both between and within patients, falling to as low as 11 IU/dl in one patient whose pre-heparin level was below the normal range, and to zero in another patient. AT III levels rose after administration of whole blood or frwh frozen plasma, and most patients recovered to within 30 IU/dl of original AT III concentration. In all cases the amount of protamine sulphate administered to neutralize heparin was excessive. The diversity of observed patient responses to heparin, heparin neutralization by PF4 release, half-life of heparin, and AT III levels demonstrates the requirement for monitoring heparin and AT III levels before, during and after cardiopulmonary bypass surgery to maintain heparin concentration within a specified range, to facilitate the exact neutralization of heparin at the conclusion of bypass and to assure adequate levels of endogenous AT III prior to surgery and to maintain sufficient concentration of AT III during bypass to ensure the desired anticoagulant effect.
Studies On The Mechanism Of Heparin Induced Thrombocytopenia
Authors: Messmore Harry L , Fareed Jawed , Parvez Zaheer , Kniffin Judith M , Squillaci Grace , Hixon Barbara , Moncivaiz Eunice
Published: July 2018

Although heparin-induced thrombocytopenia is a well recognized complication of heparin therapy (low dose or therapeutic), the mechanisms involved remain to be clarified. We have studied the serum (heated to 56°C for 30min) and platelets of patients who became thrombocytopenic while receiving treatment with beef lung heparin. The in-vitro aggregation of these platelets in their own platelet rich plasma (PRP) occurred equally well with porcine mucosal (Na+ and Ca++ salts), sheep mucosal (Na+ salt) and beef lung heparins. In-vitro aggregation also occurred with low molecular weight (4-8 × 103 daltons) porcine heparin fractions. Normal PRP aggregated readily with each of these heparins when patient serum was present. We also found that the release reaction occurs as measured by luciferase coupled luminescense aggregometry. This reaction was only slightly less when aspirin-treated platelets were used. Electron microscopy of platelets aggregated with patient serum and heparin revealed intact ghosts which showed morphology identical to collagen-treated platelets after release has occurred. This activity can be removed from the patient serum with protein A-sepharose column, and can be eluted off the column with 1M acetic acid. Heparin fragments (MW 1-2 × 103 daltons) retain potent anti-Xa activity mediated through antithrombin-III. These fragments do not aggregate the platelets of PRP in the presence of patient or control serum. This suggests the possibility that ultralow MW heparin would not cause thrombocytopenia in a patient who developed it while on high MW heparin, but this remains to be proven, and individual reactions may vary.
Factor VIII Related Activities In Various Species
Authors: Rotblat F , Hawkey C , O’Brien D P , Tuddenham E G D
Published: July 2018

VIII coagulant antigen (VIII:CAg) and VIII related antigen (VIIIR:Ag) have been studied in various species using citrated plasma obtained from animals at the London Zoological Society Gardens.

VIII:CAg was measured by a one-site fluid phase immunoradiometric assay using an antibody to factor VIII arising in a patient with haemophilia. Fab' fragments were prepared and labelled with 125I . A pool of 20 normal human plasmas was used as the standard. Three dilutions of each citrated animal plasma were tested in this assay. Bound antibody was separated from free by precipitation at 38% saturated ammonium sulphate (SAS).

VIIIR:Ag was measured by a similar one-site fluid phase immunoradiometric assay utilising a rabbit antihuman VI11R:Ag antibody (Behring). IgG purified from this antibody was labelled with 125I and free antibody was separated at 25% SAS.

All the animals tested who had cross-reacting material present showed parallel dose response curves in the VIII:CAg assay - All species of monkeys had VIII:CAg levels within the normal human range (50-200 u/dl).Other mammals had detectable but lower levels - 8-47 u/dl with the exception of Guinea pig which had 111 u/dl. No detectable VIII:CAg was present in wild boar, red deer, goat, guanaco, onager or chicken plasma.

VIIIR:Ag activity was present in all animals tested except ferret and chicken, although in all cases the assay curves were non-parallel to the human standard plasma. The slopes of these curves were calculated and compared to the human plasma slope. As with VIII:CAg the closest values are seen in monkeys and apes.

Chicken plasma has no VIII:CAg or VIIIR:Ag and this or another avian plasma may be suitable for use as a carrier in immunoradiometric assays of human coagulation factors.