bluuebunny/crossref_metadata_2025 huggingface dataset:
Assay And Occurrence Of Two Molecular Forms Of α2-Anti- Plasmin In Plasma
Authors: Kluft C , Los P , Jie A F H
Published: July 2018

A procedure to assay a plasminogen-binding (PB) and a non-plasminogen-binding (NPB) form of α2-antiplasmin in plasma has been developed. The procedure involves agarose electrophoresis in a medium containing lys-plasminogen resulting in specific retardation of the PB form. Both forms are quantified by crossed immuno-electrophoretic techniques.

The presence of both forms in vitro appears not to be an artefact as evidenced by its normal presence irrespective of the use of an inhibitor cocktail, different anticoagulants, treatment with various enzymes or aging of plasma.

In a normal plasma pool 65% of α2-antiplasmin-related-antigen appears as the PB form. In the thirteen individuals constituting the pool, values range from 61-71%. In a case of homozygous α2-antiplasmin deficiency both forms were absent; in heterozygotes with approximately half normal plasma levels both forms were found in the normal ratio.

On coagulation of blood part of α2-antiplasmin becomes irreversibly bound to fibrin by the action of factor XIIIa. In the remaining serum the amount of the PB form in particular is reduced, suggesting that this form is preferentially bound to fibrin. Associated with the consequent change in ratio between PB and NPB forms in serum, compared to plasma, a clear discrepancy between immunochemical assay and functional (immediate plasmin inhibition) assay is evoked. This discrepancy can be explained by assuming different expression of both forms in the two assays.

It is concluded that the study and assay of α2-antiplasmin in blood has to take into account the occurrence of this inhibitor in two forms, which apparently behave differently in reactivity towards plasmin and in binding to fibrin. (Supported by Praeventiefonds, projectnumber 28-443).
Changes In The Primary Fibrinolysis Inhibitor (α2-Antiplasmin) After Cardio-Pulmonary Bypass
Authors: Bagge L , Nyström S-O , Saldeen T
Published: July 2018

The blood level of the primary fibrinolysis inhibitor (PFI) has been reported to be relatively unchanged in most clinical conditions. However, we have found a strong increase 2-7 days after trauma in the form of this inhibitor (PFIα) with high affinity to plasminogen whereas the form with no or weak affinity to plasminogen (PFIβ) is not increased. The increase of PFIα may be due to increased synthesis in the liver. The method used for the measurement of the two forms is a two-step affinity chromatography using lysine- and plasminogen-Sepharose, respectively.

In the present investigation we have studied the serum level of the two forms before and after the cardio-puhnonary bypass in 14 patients operated upon for cardiac surgery. Four patients had a blood loss of more than 1000 ml during the first 16 hours after bypass (group 1)6 patients lost between 500-1000 ml (group II) and 4 patients lost less than 500 ml (group III).

PFIα was significantly lower and PFIβ was significantly higher in group I than in groups II and III. One patient with the largest blood loss showed the strongest decrease (30 % of normal value) in PFIα in the whole material and the strongest increase in PFIβ (160 %). This patient also had the lowest level of plasminogen activators and plasminogen.

The reason for the decreased level of PFIα in the patients with heavy bleeding may be a consumption and the increase in PFIβ suggests that this form may be a degradation product of PFIα.
Biochemical Characteristics And Inactivation Studies Of An Inhibitor Of Urokinase, From Leucocytes
Authors: Kopitar M , Drobnič-Košorok M , Babnik J , Turk V
Published: July 2018

Leucocyte cells are known to contain the acid, thiol and many types of neutral proteinases; among the latter also plasminogen activator. During the last five years appeared also many reports on the endogenous inhibitors of proteinases. In the preceeding publications we reported the isolation and characterization of two inhibitors of proteinases, from leucocytes (of thiol proteinases and of serino proteinases).

In the present work we present the biochemical characteristics as well as the inactivation (by cathepsin D) of an urokinase inhibitor isolated from pig blood.

Leucocytes were isolated from the peripheral pig blood by dextran procedure. Inhibitor of urokinase could be isolated either from cytoplasm (post granular supernatant) or from the extract of nuclei. Further purification of inhibitor was performed by cation exchange chromatography, by gel filtration on Sephadex G-100 and G-150 and by affinity chromatography on urokinase-Sepharose 4B and on antibody (of urokinase inhibitor )-Sepharose 4B. Inhibitor was isolated in the homogenous form, in a single polypeptide chain with molecular weight of 68 000, designated (leucocyte) Inhibitor-3. This inhibitor has an isoelectric point from 4.4-4.5 and is stable in buffer solutions from pH 3-8, and belongs to the type of so called fast reacting inhibitors. We also established that cathepsin D inactivates this inhibitor by hydrolysis of the inhibitor molecule. The conversion of active inhibitor into inactive protein proceeds catalytically. These last inactivation studies suggest a new pathway by which the active urokinase inhibitor in leucocyte cells may be inactivated.
Changes In The Antithrombin III Activity At The Interface Plasma-Phospholipids
Authors: Josso F , Béguin S
Published: July 2018

The role of antithrombin III (AT III) on kinetics of thrombin activity was studied in an inhibitor free coagulation system in which purified AT III was added at various concentrations. Coagulation was initiated either by kaolin or by Stypven in the presence of phospholipids.
Kinetics of the respective activities of factor Xa and thrombin were followed on chromogenic substrates S-2222 and S-2238 and on fibrinogen for the latter.

These experiments gave the following results :
(i) kinetics of thrombin formation as well as the peak level of thrombin activity were strongly dependent on AT III concentration ; (ii) Xa formation was unaffected by AT III level ; moreover the decrease of Xa activity was much slower than that of thrombin and was negatively correlated with phospholipid concentration.

These results illustrate the impact on blood coagulation of minor deviations from normal of AT III level. This assumption was confirmed by AT III infusion to normal subjects. On the other hand our findings cast doubt on the physiological importance of the inhibitory activity of AT III on serine-proteases other than thrombin. At the interface piasma/phospholipids, when complexed with phospholipids and protein cofactors (i.e. factors V and VIII) the affinity of the enzymes for the inhibitor becomes negligible compared to their affinity towards their respective zymogen substrates. In a complete coagulation system, inhibition of thrombin formation by AT III seems to result mainly from the inhibition of the cooperative action of thrombin on its own formation.
Serial Platelet Survival Half-Life (PS) In Peripheral Vascular Disease And Diabetes Mellitus
Authors: Kazmier F J , Fuster V , Chesebro J H , O’Fallon J H , Palumbo P J
Published: July 2018

Mayo Clinic, Mayo Foundation, Rochester, Minnesota

Platelets are believed to play an important role in the pathogenesis of atherosclerosis and thrombotic occlusive arterial disease. As part of a prospective study of peripheral occlusive arterial disease in diabetes mellitus, platelet survival half-life (PS) was determined in years 1 and 2 from the disappearance pattern of 51cr-labeled platelets obtained by measuring radioactivity in serial samples over 8 days in four groups of patients age 50-70. Using computer assisted least-square analysis, a single exponent is fitted to the data and half-life determined.

Groups included (1) normal subjects (NC); (2) subjects with clinical and objective evidence (transcutaneous doppler ultrasound and treadmill exercise) of peripheral arterial occlusive disease (AS0); (3) subjects with diabetes mellitus without AS0 (DM); (A) subjects with both diabetes mellitus and AS0 (DM+AS0). 112 subjects were tested in year 1 and 75 of these were available for retesting in year 2.

In year 1, there is a difference in the distribution of platelet survival (PS) for the four groups (p=.002). PS is significantly short in groups AS0 and DM+AS0 compared with controls (NC), Year 2 values for PS (75 subjects) were compared to year 1. Using the t test on mean differences PS for each group was not significantly different year 2 vs year 1; mean percent differences year 2 vs year 1 were 1.2% (NC), 0.2% (ASO), 3.2% (DM), and -9.3% (9-3% lower than year 1 for DM+ ASO).

Platelet survival half-life (PS) does distinguish popula tion groups and is reproducible when repeated.
Systemic Thromboemboli In Mitral Starr-Edwards Prosthesis: A Long-Term Followup (10-19 Years)
Authors: Fuster V , Chesebro J H
Published: July 2018

The study comprised 170 consecutive patients (pts) seen at Mayo Clinic between 1962 and 1970 who underwent mitral valve replacement with Starr-Edwards prosthesis (model 6000 in 35% of pts, model 6120 in 65% of pts) because of significant chronic mitral incompetence (functional Class III-IV). All patients were on oral anticoagulants and were followed until January 1980 with a followup of 10 to 19 years (yrs) (median 15 yrs). We analyzed (1) the cumulative incidence of systemic emboli, their location and whether or not they left residual deficit; and (2) possible risk factors that might have been predictive, including in the analysis: age, sex, functional class, presence or absence of atrial fibrillation, degree of cardiomegaly, type of prosthesis and degree of anticoagulation.

At 12 yrs of followup, 48% of pts had at least one episode of emboli (among these pts, 12% had two episodes); the rate of emboli was maximal within 1 yr postoperatively but persisted over the total followup at a rate of about 3% per yr. Of all emboli, 94% occurred in the cerebral circulation and 66% left neurological deficit. Regarding predictive factors, the incidence of emboli was slightly higher in pts with the prosthesis model 6000 (p<0.05) and in pts considered to be poorly anticoagulated (p<0.05).

This unique long-term followup study on pts with mitral Starr-Edwards prosthesis reveals that systemic emboli is a persistent and significant problem. Preliminary data indicates that the addition of a platelet inhibitor to the oral anticoagulant may be beneficial.
Vessel Injury, Thrombosis And Platelet Survival
Authors: Winocour P D , Cattaneo M , Kinlough-Rathbone R L , Mustard J F
Published: July 2018

In vascular disease it is unclear whether shortened platelet survival (PS) primarily reflects vessel injury or thrombosis. In rabbits and rats (4-6 animals per group) we examined the relations among experimental thrombosis, vessel wall injury and platelet survival and turnover. In rabbits preinjected with autologous 51Cr-platelets, a 20 cm intra-aortic-catheter in situ for 4 days resulted in a thrombus (mean wt. 23.4 mg). A significant amount of 51Cr was associated with the aorta (0.53 ± 0.13% of total 51cr circulating before surgery). PS was reduced (no catheter 62.4 ± 8.8; catheter 37.0 ± 5.6 hr, p<0.05). With a 10 cm catheter, thrombus wt. was similar (mean 24.0 mg). 51cr associated with the aorta was 0.25% ± 0.06% but PS was unaffected (no catheter 55.4 ± 6.8; catheter 53.8 ± 3.6 hr). In other experiments, sham-operated controls were compared with rabbits with 20 cm catheters. Mean thrombus wt. was 30.2 mg and 0.53 ± 0.11% of the 51cr was associated with the aorta. PS was significantly shorter in the catheter rabbits (36.7 ± 2.8 hr) vs sham-operated controls (68.0 ± 10.4 hr, pp<0.02); platelet turnover was significantly increased (14,500 ± 970 vs 9,950 ± 1,020 per mm3/hr, pp<0.01). Three groups of rats preinjected with homologous 51Icr- platelets were studied: a) sham-operated, b) aortic catheter 7.5 cm, c) aortic catheter 12.5 cm. No macroscopic thrombi were observed at any time during the 4 days that catheters were in situ. Mean PS was: a) 97.7 ± 2.4, b) 88.5 ± 2.6 and c) 63.7 ± 5.1 hr (pp<0.001, a vs c). Platelet turnover was a) 8,400 ± 720, b) 8,900 ± 870 and c) 11,000 ± 1,150 per mm3/hr. 51Cr associated with the aortae was a) 0.004 ± 0.001, b) 0.013 ± 0.006 and c) 0.027 ± 0.011% of total (pp<0.05, a vs c). Thus in rats, catheters shorten PS without thrombosis. Therefore, with catheter-induced vessel injury PS appears directly related to length of catheter and extent of injury. Shortened PS can occur without thrombus formation and thrombus formation can occur without changing PS or platelet turnover.
Stimulation Of Human Platelets By Lysophosphatidic Acids
Authors: MacIntyre D E
Published: July 2018

Production of lysophosphatidic acids (LPA) by “activated” cells may be important in stimulus-response coupling. Using human citrated platelet rich plasma (PRP) we examined the effects of Oleoyl (0), Palmitoyl (P) and Decanoyl (D) LPAS on platelet function. DLPA (≤300 μM) did not affect platelet function, and OLPA and PLPA (≤200 μM) did not inhibit uptake of 3H-5HT (0.3 μM). However OLPA and PLPA (1-30 μM) induced reversable aggregation in the absence of 5HT release (primary aggregation), and higher concentrations (≤300 μM) induced irreversible aggregation and TxB production and release of ≤79% of 5HT, 89% of βTG and ≤73% of β -N-acetyl glucosaminidase that was released by thrombin (1U/ml). LPA-induced release of platelet granule constituents was associated with <5% release of cytoplasmic markers LDH or 3H-adenine. OLPA or PLPA (300 μM) suppressed the elevation of platelet cyclic AMP induced by PGE1. Primary and secondary aggregation induced by LPA were suppressed by PGE1 (1 μ M) and the intracellular Ca antagonist, TMB-8 (200 μM). The Ca transport inhibitor verapamil (100 μM) was ineffective. Indomethacin (30 μM) and the ADP antagonist, β,γ-methylene-ATP (30 μM) inhibited only secondary aggregation. PRP exposed to OLPA or PLPA (30 μM) for greater than two minutes at 37°C failed to respond to subsequent addition of LPA but showed augmented responses to ADP, U 46619, vasopressin and 5HT. OLPA and PLPA resemble the more powerful platelet agonists (e.g., collagen, thrombin) in that they induce secretion of βTG, 5HT and lysosomal hydrolases and cause aggregation that is independent of ADP, 5HT or arachidonate metabolites. Endogenous LPA may function as a mediator of platelet aggregation and degranulation. Alternatively LPA may serve as a mimic of related platelet agonists (acetyl- glyceryl-ether-phosphorylcholine [AGEPC], or PAF-acether).
Endothelial Alterations During The Platelet Activating Factor (PAF) Respiratory Distress Syndrome
Authors: Lewis J C , O’Flaherty J T , McCall C M , Wykle R L
Published: July 2018

PAF (l-0-alkyl-2-0-acetyl-sn-gylcero-3-phosphocholine) induces polymorphonucelar leukocyte and platelet stasis in rabbit lung capillaries in vitro and produces a model of acute respiratory disease. Since PAF mediates anaphylactic reactions, a study was done to determine the ultrastructural effects of PAF treatment. Mature rabbits were treated by intravenous administration of either PAF (0.15-10 μg/kg: 8 animals) or BSA (the PAF carrier: 3 animals) prior to sacrifice and intraventricular perfusion with 0.1 M phosphate buffered (pH 7.2) glutaraldehyde (2.5%). Animals (n=5) injected with a high concentration of PAF (3-10 μg/kg) and sacrificed within 15 minutes of PAF administration had grossly contracted lungs, the vasculature of which (as observed by scanning electron microscopy) contained numerous marginated leukocytes and platelet aggregates. Animals (n=3) given PAF in the concentration range 0.15-2.4 μg/kg had less consistent lung contraction and fewer platelet aggregates within capillaries. Luminal surfaces of capillary endothelial cells in all PAF treated animals (when observed by transmission electron microscopy [TEM]) were dramatically altered. In contrast to the uniformly smooth surfaces in control animals, vessels in the PAF treated animals had tortuous surfaces with plasma membrane discontinuities and protrusion of plasma membrane fragments into the capillary lumen. Morphometric analysis of TEM micrographs substantiated statistically significant (p<0.01) increases in the number and size of plasmalemmal vesicles.

These observations clearly document a cytotoxic effect for capillary endothelium. This cytotoxic action may in part explain the clinical effect of PAF.
Group Analysis Of Abnormal Factor X Molecules
Authors: Fair D S , Edgington T S
Published: July 2018

Factor X lies central in blood coagulation being the point of convergence of the intrinsic and extrinsic pathways and in its activated form is the initial enzyme in the final stages of clot formation. The interaction of Factor X with its activators, cofactors and substrates suggest that there are multiple structural loci present on the surface of this molecule available for these molecular associations. Hence, abnormal molecules may reflect different specific structural defects resulting in decreased function. We analyzed 28 abnormal Factor X plasmas by four parameters: 1) antigen (Ag) measured in radioimmunoassay and activity as measured by: 2) prothrombin time (PT), 3) Russell’s viper venom time (RVV), and 4) activated partial thromboplastin time (APTT). Abnormal Factor X plasmas were from congenital (16), transient (2) and amyloid associated (10) deficiencies. In order to objectively determine groups of abnormal molecules we evaluated their distributions in the following manner: (1) PT activity vs Ag concentration; (2) RVV activity vs PT/Ag; and (3)APTT activity vs RVV/(PT/Ag).

A non-random distribution of plasmas was observed which could be grouped into ten classes and subclasses of abnormal Factor X molecules. These data suggest that the groups of abnormal molecules may reflect common structural defects shared among its members located at specific structural loci involved in either the activation or the function of these Factor X molecules.
Sialic Acid Content And The Biological Activity Of Stuart Factor (F X)
Authors: Engel A M
Published: July 2018

Substantial release of sialic acid (SA) from purified F X was obtained with sialidase from Clostridium perfringens. The total SA content of F X was found to be 1.7%, in agreement with previous reports. After almost complete removal of SA, aliquots were examined by polyacrilamide disc electrophoresis and by polyacrilamide in sodium dodecy1 sulphate (SDS) and the gels were stained for proteins and for glycoproteins. When SA was progressively removed from F X its clotting activity was slightly increased while its electrophoretic mobility was retarded, as expected. The tryptic activation of F X was affected by the removal of 60-70% of SA, yielding more clotting activity than the native F X. No difference was observed between the esterase activity of regular F X and asialo-X as measured by the hydrolysis of N-alpha-acetyl glycyl-L-lysy1-methyl ester acetate (AGLMe).

When asialo-X was interacted with 25% sodium citrate the kinetics were accelerated and the maximal clotting and esterase activities were obtained at 6 hours of activation while the native F X reached the peack of both activities at 24 hours. Both activation mixtures were analyzed, at the period of maximal activity, for liberation of peptidic material by thin-layer electrophoresis (TLE). TLE patterns of asialo-X varied according to the activation system used; they also differed from the ones obtained with native F X.

The data indicate that SA influences the kinetic of activation of F X and the nature and distribution of the peptidic material released during activation.
Interaction Of Platelets With Soluble, Polymerizing And Polymerized Fibrin In Human Plasma
Authors: Brosstad F , Kierulf P , Godal H C , Eika C
Published: July 2018

Previous studies on platele/fibrin interactions find polymerizing fibrin capable of inducing platelet aggregation but are controversial as to the same ability of soluble fibrin. It has also been controversial wether the fibrin-induced platelet aggregation is mediated via the platelet release or not. Since previous studies have utilized fibrin prepared With soluble phase thrombin or reptilase, it was of interest to reinvestigate the problem with fibrins prepared with insoluble phase reptilase (des-AA) and thrcmbin (des-AABB). Urea-solubilized des-AA or des-AABB fibrin monomers were added to hunan, citrated PRP (ci-PRP) in the aggregcmeter either in soluble anoints (8% of ci-PRP fibrinogen cone) or in amounts resulting in the formation of visible fibrin (50% of the ci-PRP fibrinogen level, or added as polymerized fibrin which had been homogenized.

The increase in light transmission and beta-trcmboglobulin releas (β-TG) were recorded. Soluble or polymerized, homogenized fibrin had no effect on either parameter, whilst fibrin added in amounts resulting in visible fibrin formation (polymerizing fibrin) caused both platelet aggregation and β-TG release comparable to that of collagen (100 ug/ml) Fibrin monomer solvent (urea) had no such effects. In experiments with EDTA-PRP, visible aggregation was not observed but βTG confirmed that platelet release had taken place . With KCN-PRP and Acetylosalicylic-PRP,neither aggregation, norβ-TG ,was observed. It is concluded that: 1. Soluble or polymerized fibrin does not aggregate platelets. 2. Polymerizing fibrin aggregates platelets. 3. Platelet aggregation observed with polymerizing fibrin is mediated via the platelet release reaction. 4. Des-AA and des-AABB fibrin are equally potent in inducing platelet aggregation.
Receptor Mediated Uptake Of Fibrin Monomer
Authors: Shainoff J R , Dardik B N , Gonda S R
Published: July 2018

Electron microscopy of macrophages exposed to fibrinogen solutions containing dissolved fibrin that was labelled with heme-octapeptide for cytochemical detection indicated that 1) the fibrin became adsorbed in a monomeric rather than aggregated form at saturating levels on the cell surface over a 2-5 min. period, 2) the adsorbed fibrin was fully internalized by vesicular uptake and fused with lysosomal bodies over a subsequent 30-60 min, and 3) a loss of adsorptive capacity of the cell surface for uptake of more fibrin accompanied internalization of fibrin. The uptake of fibrin in monomeric form despite its existence in aggregated but dissociable form in solution indicated that the fibrin was being adsorbed by a receptor with affinity for the same sites that participate in aggregation, sites that are exposed by release of fibrinopeptide A. Removal of fibrinopeptide B itself does not induce high affinity uptake. By analogy to the loss of adsorptive capacity accompanying internalization of the fibrin by macrophages, clearance of monomer from the circulation also appears to depend on a consumable receptor in that initial rates of clearance of 125I-labelled monomer at high dosages (>1 mg/kg) follow kinetics of a 2nd order chemical reaction, while pseudo-first order kinetics apply with low dosages (<0.3 mg/kg). A requirement of dissociability of monomer from soluble complexes for rapid uptake is indicated by observation that the poorly dissociable form of fibrin lacking both fibrinopeptides A and B is cleared from the circulation at a very slow rate compared to fibrin lacking A alone. Importance of dissociability is indicated also by observation that injection of Factor XIIIa inhibits uptake to a degree comparable to removal of both A and B. The demonstration of monomer uptake indicates that clearance of fibrin may precede its deposition in aggregated form. It is suggested that the rapid but saturable uptake of monomer may proceed independently of phagocytosis of macroaggregates.
Platelet-Associated IgG Quantitation By A Simplified Rapid Nephelometric Technique
Authors: Morse B S , Giuliani D , Nussbaum M
Published: July 2018

The complexity of tests used to measure PAIgG has diminished its effectiveness as a measure of platelet bound antibody. We recently reported a simplified radial immunodiffusion technique to quantify PAIgG using washed solubilized platelets and low level IgG immunoplates (Behring-Calbiochem). Three days were required for diffusion to reach completion. Normal values ranged from 1.6 - 7.0 fg IgG/platelet. 92% of patients with immune thrombocytopenia (ITP) had values which exceeded the mean by greater than 2 S.D. units. To further simplify and hasten the measurement, we have developed a nephelometric method. 0.25 × 108 platelets, obtained from 10-30 ml of blood collected in EDTA are solubilized using 1% triton X-100 plus sonication. Nephelometric buffer containing polyethylene glycol is added followed by anti-human IgG. Relative light scattering units thus generated can be converted to ng of IgG by comparison with standards. Results of PAIgG levels can thus be made available within several hours of blood collection.

These results are in excellent agreement with PAIgG determined by radial immunodiffusion. Our cumulative experience indicates a mean normal control value of 3.9 ± 1.9 fg IgG/platelet. 90% (40/45) of patients with immune thrombocytopenia exceed the mean by greater than 2 S.D. units. Elevated values are also seen in SLE patients with (50%) or without (25%) thrombocytopenia, 50% of patients with sepsis and thrombocytopenia, 80% of patients with IgG myeloma, and 20% of patients with thrombocytopenias of diverse etiologies.
The Inhibitory Effect Of Dilazep On Adhesion And Aggregation Of Platelet Following Aortic Intimal Injury In Rabbit
Authors: Sumiyoshi A , Hayashi T , Fujii M
Published: July 2018

The inhibitory effect of dilazep and aspirin on in vivo platelet adhesion and aggregation in rabbit aorta subjected to endothelial injury was investigated. Endothelial injury was induced by insertion of polyethylene tubing from the femoral artery into the aorta. In the beginning before surgery, experimental animals were intravenously given sufficient drug to inhibit platelet aggregation in vitro in response to ADP and collagen. For a quantitative analysis of platelet accumulation on the damaged aortas, 51Cr-labeled platelets were used. For morphological study, the aortas were fixed by perfusion at one hour after injury and examined by light and scanning electron microscopy for platelet adhesion and aggregation in injured area.

Radioactivity of damaged aortas in rabbits administered dilazep (50 or 100 μg/kg) or aspirin (10 mg/kg) was significantly lower than in rabbits untreated by drug. Dilazep and aspirin did not prevent completely the adherence of platelets on injured area of the aorta, but inhibited considerably the platelet aggregation to form raised platelet thrombus.
Clinical Evaluation Of Wave Conformation Of Photo- Pletismograph And Doppler In Distal Angiopathies
Authors: Pavlovsky M , Meschengieser S
Published: July 2018

Objective studies and interpretation of circulatory problems in hands and feet are difficult. Several non invasive techniques for vascular diseases developed in recent years may have great application in small vessels.

We have studied 280 patients with distal circulatory problems;recording(graph)the flow patterns obtained in a) distal arteries of the arm and leg with a bidirectional Doppler of 9.4 MH,and b) the skin arterioles at the tips of fingers and toes with a photopletismograph EP 100 (PP). The general patterns obtained comparing the waves at the graph recorded with the PP and Doppler were: A) a similar shape wave;B) a slight atenuation of the PP waves;C) a decrease of PP waves;D) an increase in PP waves. Patterns A and B are consider ed normal;Patterns C-D are abnormal.

In vasospastic disorders great modifications of PP waves could be observed,with changes from patterns C to D or viceversa with the effect of stress-temperature and drugs. Degenerative arterio-pathies of big and small vessel could be distinguished evaluating the relation of the waves of both systems. Measurements of distal segmental pressures and the recovery time of pulse in fingers of toes with both systems add valuable information. We observed differences in wave types in some individuals, between Doppler and PP which are difficult to explain with our actual knowledge.

We have found that the combination of both methods is an excellent objective system for the better study and drug therapy control of different small vessel diseases.
Experimental Thrombogenesis By A Non-Invasive, Non-Traumatic Method
Authors: Hamer J D , Malone P C , Sevitt S
Published: July 2018

A method is described for producing thrombi in the femoral veins of dogs by non-invasive, non-traumatic means.

Previous studies have shown a decline in venous PO2 in the depths of valve pockets during non-pulsatile flow, which may be reflected in progressive endothelial hypoxic injury. It has also been shown that healthy well oxygenated platelets and leucocytes attach themselves to such damaged endothelium.

Dogs anaesthetised by Sagatal (Nembutal) were kept in the supine position with extended legs for 6 hours; some were not allowed to move or be moved for the whole experimental period; others had their legs moved passively at 20 minute intervals - these two groups were “motionless” and “frequently moved” controls. The third (experimental) group were kept motionless for 2½ hours, and allowed to wake slightly as the first anaesthetic dose wore off; during this time they began to shiver. This movement effects pulsatile venous flow and vibrates the valve cusps in the deep vein. Venography was employed to identify the presence of thrombi which were checked at autopsy.

Results showed numerous (70%) “classical”.thrombi among 12 limbs in the experimental group. Of the 18 control limbs only one showed a thrombus, this being in the frequently moved group. No thrombi were found in the 10 limbs left motionless.

The hypothesis that endothelial hypoxia is the primary instigator of deep venous thrombosis, therefore appears to be supported. Thrombosis develops when pulsatile flow is restored. The animal model described is the subject of developmental studies, and is available for further investigation of interaction between blood elements and venous endothelium, together with prophylactic techniques.
Role Of Prothrombin And Factor IX In The Initiation Of Thrombus Formation
Authors: Kennedy F , Hassouna H I , Penner J A , Schultz J
Published: July 2018

This study was designed to investigate the role of plasma clotting factors in the initiation of thrombus formation on materials intended for prolonged blood contact. Prothrombin was isolated from dog plasma using BaC03 adsorption, citrate elution and chromatography on dextran sulfate. Antibodies against various fractions were purified and tested for their ability to inhibit clot formation in normal dog plasma. Prothrombin time was not significantly affected by antibodies, but recalcification time was more than doubled. This recalcification time was not corrected by adding BaC03 adsorbed plasma back to normal dog plasma incubated with antibody. These results led to the conclusion that these antibodies were specifically inhibiting prothrombin and factor IX.

These purified antibodies were tested in preconditioned male dogs to specifically block prothrombin from taking part in coagulation. Dogs had previously been surgically fitted with arteriovenous shunts between the carotid artery and the internal jugular vein, and were given autologous platelets labelled with Cr and 125I-labelled human fibrinogen prior to testing. The arteriovenous shunt was opened into the flow chamber for one hour, during which time a dual channel scintillation counter was positioned over the test chamber to monitor fibrinogen and platelet deposition at 5 minute intervals. After one hour the chamber was removed and the size of the resulting thrombus was measured by total weight, fibrinogen, red cell, and platelet content. In dogs previously given antibodies against prothrombin, thrombus was not found on the various biomaterials applied to the rod within the flow chamber. We conclude that prothrombin is of major importance in the initiation of clot formation on artificial surfaces.
Phospholipid Topography Coagulation
Authors: Hemker H C , Zwaal R F A , Rosing J , van Dieyen G , Bevers E
Published: July 2018

Phospholipids play a role in thrombin generation because the constitute the support on which both the factor X activating enzyme (“tenase”) and the prothrombin activating enzyme (proth rcmbinase) are built.

In model systems with pure synthetic phospholipids it can be shown that the binding of the coagulation factors to a phospholipid surface is a function of both phospholipid charge and membrane fluidity. A relatively high molefraction of phosphatidyl serine (PS) is a prerequisite for optimal binding of the vitamin K dependant factors. Precise measurements on the binding constant and the number of binding sites of factor X show that at molefractions of PS between 0.01 and 0.25 aproximately one PS binds per Gla-residue. Above 0.25% of PS vesicle agregation occurs.

The normal intact circulating platelet shows a molefraction of PS of ̴ 0.03 in the outer leaf let of its plasma membrane and therefore hardly if at all stimulates thrombinformation.

Upon activation by thrombin (2nM) together with collagen, (10μg/ml) the outer leaflet aquires increasing amounts of PS without platelet disruption taking place.

This suggests a flip-flop movement of phospholipids across the membrane as part of the activation reaction of platelets. It can be shown that optimal “tenase” supporting phospholipid compositions appear before prothrombine supporting ones. The activation by thrombin and collagen is only partly inhibited by prostacyclin even at high concentrations.
New Developments In Interactions Of Blood With Artificial Surfaces
Authors: Salzman E W
Published: July 2018

There are major new developments concerning interactions of blood with artificial surfaces. There is new understanding of mechanisms: the nature of the plasma protein film adsorbed on artificial surfaces, the interaction of HMW kininogen with adsorbed fibrinogen, the significance of adsorbed antithrombin to the behavior of heparinized surfaces, and the lack of prostacyclin production and ADPase activity. There are new in vivo diagnostic methods: measurement of labelled platelet and fibrinogen survival, assay of plasma levels of products of activated coagulation or platelets (FPA, thrombin/antithrombin complexes, β - thromboglobulin, platelet factor 4 thromboxane B2), and in vivo detection of thrombi with 125I-fibrinogen or 1 Inplatelets. New methods also exist for characterization of surfaces: ESCA, SEM, TIRIS, TIRFS. New materials are available with different modes of thromboresistance: bland polymers such as polyurethanes and siloxanes, modified collagens, heparinized surfaces, porous materials, and aldehyde treated materials of animal origin. There are new drugs for protection of hemostatic elements during extracorporeal circulation: PGI2, PGE1, ibuprofen. New applications for prosthetic devices have become practical: peritoneo-jugular shunts for treatment of ascites, left ventricular assist devices, intra-aortic balloon pumps, total artificial hearts.

Such developments raise important new questions: the relation of short-term tests of thromboresistance to longterm behavior of devices in vivo, the problem of thrombi at the junction of prosthetic device with the host, calcification in long-term applications, and new approaches to management of traditional clinical problems (e.g., continuous treatment with portable miniaturized hemodialysers, small vessel prostheses, extracorporeal cardiopulmonary support without anticoagulation).

Subsequent speakers will consider these topics more fully.
Multiplicities In The Interpretation Of Clinical Trials
Authors: Meier P
Published: July 2018

The use of conventional statistical inference methods to assess the differences between treatments is appropriate when a single endpoint (e.g. total mortality) is considered and the groups to be compared are well defined (e.g. all those randomly allocated to treatment). In practice there are many endpoints (e.g. coronary events, cardiovascular mortality, total mortality), many subgroups (males, females, those under forty years of age), and multiple times at which the data may be examined. The meaning of conventional statistical tests is greatly altered in the face of such multiplicities. Examples illustrating the problem and recommendations for keeping errors of inference under control are given.
Amino Acid And Peptide Thioeters: New Sensitive Substrates For Bovine Factors IXa , Xa , XIa , XIIa , Thrombin, Plasma Kallikrein And Trypsin
Authors: Powers James C , McRae Brian J
Published: July 2018

A series of amino acid and peptide thioesters have been synthesized as sensitive assay substrates for the serine proteases involved in blood coagulation. Each substrate had a P1 Arg residue. The thiol leaving group (P1
') and the P2 amino acid residues were varied. The thioesters have much higher Kcat/KM values than the corresponding amides such as 4-nitroanilides and 7-amino-4-methylcou- marin amides. In addition, the thiol released upon enzymatic hydrolysis of the substrates, can be measured chromogenically by use of a thiol reagent such as 4,4'- dithiodipyridine contained in the assay mixture. Thioesters are among the most sensitive assay substrates for coagulation serine proteases due to the combination of a high turnover rate with the ease of detection of the hydrolysis product. In addition they can be utilized with enzymes such as factor IXa for which no other suitable synthetic substrate is currently available.
Hydrodynamic Studies Of Von Willebrand Protein And Factor VIIIc Using The Partition Cells In The Analytical Ultracentrifuge
Authors: Barlow Grant H , Martin Eric S , Marder Victor J
Published: July 2018

In an attempt to determine the functional hydrodynamic properties of von Willebrand and Factor VIIIc proteins studies were made using the moving and fixed partition cells in the analytical ultracentrifuge. Experiments using a formalin fixed platelet ristocetin assay as the functional assay showed a non parallel response when the data was plotted as the rate of agglutination vs. the dilution after partition sedimentation. The slope of this plot diminished as a function of the centrifuge time. These observations negate calculations of the sedimentation parameters. It is suggested that this non parallel response in activity is attributable to the different specific activities of the different polymers. It is postulated that hydrodynamic calculations can be made based on the slope of the rate of agglutination vs. dilution curve. On the other hand when functional assays involve the determination of Factor VIIIc activity a normal parallel response is obtained following partitioning. This allows for the calculation of sedimentation coefficients from the activity data. Values ranging from about 12S to 25S are obtained. These differences show that the non parallel response by von Willebrand protein is not anomalous and further indicates that current techniques for quantitating von Willebrand protein should be revised.
Combined Factor VIII And Plasma Exchange Therapy In The Management Of Patients With Factor VIII Inhibitors
Authors: Wensley R T , Stevens R F , Burn A M
Published: July 2018

Five patients with Haemophilia A and Factor VIII inhibitors and three patients with spontaneous Factor VIII inhibitors presented with severe or life-threatening haemorrhage. All, except one, received intermittent high dose human Factor VIII therapy combined with one or more plasma exchanges. The exception was a patient with spontaneous Factor VIII inhibitors who was plasma-exchanged without receiving Factor VIII. The five Haemophilia A patients showed a uniformly good clinical response to treatment with complete resolution of their bleeding episodes. Their inhibitors were reduced to a level approaching zero and adequate plasma Factor VIII levels were achieved. In contrast, the three patients with spontaneous Factor VIII inhibitors failed to show any clinical response to therapy. They had measurable plasma Factor VIII levels before the Factor VIII therapy, but the administered Factor VIII produced no additional increment. Their inhibitor levels were only minimally altered by therapy. Studies of the inhibitors from the haemophiliacs showed complete Factor VIII neutralisation in incubation mixtures, but inhibitor plasmas from the spontaneous cases failed to completely neutralise the admixed Factor VIII. It is postulated that in haemophiliacs with antibodies, replacement therapy is associated with the formation of stable imnune complexes which remain in the intravascular space and are removed at subsequent plasma exchange. These complexes do not show coagulant or anticoagulant activity. However, in non-haemophiliacs with acquired Factor VIII inhibitors, weaker association of Factor VIII and antibody in the immune complexes may account for the measurable plasma Factor VIII activity, and also enable the dissociated antibody to diffuse out of the intravascular space and hence be unavailable for removal by plasma exchange.
Thrombogenicity Of Antihemophiliac Preparations With Factor VIII Inhibitor Bypassing Activity
Authors: Melewski D J , Ambrus J L , Ambrus C M , Tourbaf K
Published: July 2018

In hemophilia A patients with inhibitor to Factor VIII, prothrombin complex, concentrates were found effective in treating hemorrhagic episodes. However, in several patients DIC or thromboembolic complications developed. Some of the manufacturers have altered production methods eliminating certain activated coagulation factors from the preparations. After these modifications some of these preparations were found to be less effective clinically. In the first study, we compared potential thrombogenicity of two preparations: Autoplex and FEIBA and as control prothrombin complex preparation with no appreciable activated factor content (Prothromplex) for the ability to induce thrombosis in an isolated segment of the renal vein of C57BL6(J), ICR/HA male mice, and Sprague-Dawley male rats. The minimum thrombosis inducing dose was 200 prothrombin complex units per kilogram of Prothromplex, 25 FEIBA units of FEIBA and 0.45 FEIBA units of Autoplex. The fact that Autoplex is approximately 51 times more active them FEIBA can probably be explained by the fact that the latter contains more factor IXa and Xa activity than the former.
Relationship Between Factor VIIIR:Ag, Factor VIII C And Glycoemic Control And Vascular Complications In Insulin-Dependent Diabetics
Authors: Tantalo V , Rossi E , Cossu M M , Migliavacca P , Saibene V
Published: July 2018

Increased levels of Factor VIII are usually re ported in diabets mellitus, even if contradictory relationships with the vascular complications have been observed,due to unselection of patients. We have measu red Factor VIIIR:Ag and Factor VIII C(two stages method) in 60 insulin dependent diabetics (IDD), aged between 7 and 78, compared to 25 sex and age matched normal controls (NC).The presence of retinopathy (r) or macroangiopathy (m) was assessed by fIuoroangiography and EKG plus oscillography, respectively.

No significant correlations were found between FVIIIR:Ag, FVIIIC and concomitant blood glucose, glycosylated hemoglobin, GH, and the duration of diabetes. A progressive increase in FVIIIR:Ag according to the extent of the vascular damage is evident. An enhancement of the level of FVIIIC, greater than that of FV IIIR:Ag, index of enhanced thrombin production, is already present even in absence of vascular damage in IDD, being more pronounced in patients with vascuIar complications.
The Use Of Factor VIII:C Depleted Normal Plasiia In The Factor VIII:C Assay
Authors: Pancham N , Bogdanoff D , Mintz C , Wada S , Coan M
Published: July 2018

Normal plasma was made deficient in functionally active FVIII:C. This plasma supplemented with bovine FV was used as a substrate and compared to hemophilic plasma in the FVIII:C assays. The assays were performed on mechanical as well as optical clot detecting systems using the one-stage procedure.

Using a statistically valid experimental design, the plasma artificially-deficient in FVIII:C was shown to be comparable to hemophilic plasma, when both FVIII concentrates and pooled normal plasma samples were assayed. The plasma artificially deficient in FVIII:C was stable upon freezing and thawing and upon reconstitution after freezedrying. Batch-to-batch variation was not significant. The introduction of this plasma as a reagent eliminates the need for collecting hemophilic plasma which may contain inhibitors to FVIII.

The main conclusions of the analyses are that artificially deficient FVIII substrate is fully comparable to naturally deficient human substrate in both accuracy and precision.
Characterisation Of Active Components Of Fix Concentrates Used In FVIII Inhibitor Patients
Authors: Seghatchian M J , Mackie I J
Published: July 2018

It is well recognised that haemostasis may be achieved in patients with FVIII inhibitors using FIX concentrates (PTX). However, it has not been possible to correlate the clinical response with enzymicity, FVIII levels and other in vitro results. This is partially due to the presence of variable amounts of inhibitors and stabilizers such as: citrate, heparin, and anti proteases in different preparations. Agarose gel electrophoresis was performed in an attempt to separate these inhibitors so that the various activities may be measured relative to one another. PTX from 4 sources were used in this study,(Kabi Vitrum, Immuno, Oxford and Edinburgh fractionation centres); FVIII was measured by two stage clotting(FVIII:C)and amidolytic (CAM) assays, and its electrophoretic distribution was followed by preincubation with 125I-anti VIII:C (VIII:CAg). Enzymicity was measured with various substrates (S2160, S2222, S2302, S2238, S2288, S2251). All concentrates showed a fast moving peak of FVIII:C and VIII:CAm activity, in amounts in excess of that detected before electrophoresis. This peak correlated well with the distribution of bound radiolabel (VIII:CAg). Preincubation of PTX with FVIII concentrate caused the VIII:CAg peak to migrate more slowly, in its normal α 2 position. FVIII:RAg was present in much lower amounts than FVIII:C. When a twin- peak of VIII:CAm activity was sometimes seen, the slower peak was associated with S2288 activity, which may represent the action of activated factors VII or IX: other substrates showed variable patterns of enzymicity but the strongest activity was with S2160 and S2302 from the well to the β region, and this could be completely inhibited by addition of anti Kallikrein. The presence of an abnormal molecular form of FVIII, and the discrepancy between pre and post electrophoresis FVIII levels, indicate that FVIII could play a major role in the action of PTX. This may involve 1) a complex with phospholipid, FIX and FX making it potentially more effective; 2) enhancement of VIII:C activity in vivo by binding to FVIII:RAg; 3) altered antigenic sites on the FVIII, making it unrecognisable to inhibitor.