bluuebunny/crossref_metadata_2025 huggingface dataset:
Application Of High Performance Liquid Chromatography (HPLC) To The Study Of Platelet Phospholipid Metabolism
Authors: Alam I , Smith J B , Silver M J
Published: May 2019

A method for the separation of phospholipids using HPLC has been developed. Different radioactive prostaglandins and phospholipids are detected by passing a portion of the effluent from the HPLC column directly through a Radio Flow-1 detector. The remainder of the effluent is collected for phosphate and fatty acid determinations using a fraction collector. This system eliminates the solventquenching limitation associated with U.V. detection and offers many advantages over thin layer chromatography (TLC) which frequently requires two dimensional development and scraping of zones from the TLC plate. Separations of phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylcholine, spingomyelin and platelet activating factor (PAF) have been achieved. The system has been applied to the study of thrombin-induced changes in the phospholipids of human platelets prelabeled with 14C-arachidonic acid. After incubation of prelabeled platelets for 10 min. at 37° with 5 U/ml thrombin, a 30% decrease in radioactive phosphatidylcholine was detected.
Loss Of Platelet Membrane Glycoproteins Or Terminal Sialic Acid Detected By Fitc-Lectins
Authors: McGregor L , McGregor J L , Clemetson K J , Dechavanne M , Lüscher E F
Published: May 2019

Pre-thrombic conditions in certain individuals resulting from enhanced platelet-vessel wall or platelet-platelet interactions are perhaps characterized by a reduction in certain membrane glycoproteins or loss of terminal sialic acid. In order to investigate if such changes are detectable, the binding of FITC-lectins to human platelets treated under in vitro conditions with certain proteases to mimic possible in vivo changes occuring on the platelet surface, has been examined. Human platelets were isolated, washed and either treated with neuraminidase (10 U) or plasmin (1 CU) before fixing with formaldehyde. Binding studies were performed by the method of Monsigny et al. using FITC labelled wheat germ agglutinin (WGA), Lens culinaris lectin (LCL), Ricinus communis agglutinin (RCA) and concanavalin A (ConA). The number of lectin-binding sites (n) and the dissociation constant (Kd) were obtained by Steck and Wallach reciprocal plots. After neuraminidase or plasmin treatment n was reduced but Kd remained approximately the same with WGA. FITC-RCA-60 gave a slight fluorescence with untreated and very strong fluorescence with neuraminidase treated platelets. Platelet glycoproteins separated by 2-dimensional gel electrophoresis were identified by binding of fluorescent lectins. Plasmin decreased the intensity of GP Ib and IIb and removed Ia completely. Neuraminidase decreased the labelling of Ib by WGA. These techniques show promise as methods of detecting pre-thrombotic conditions.
The Values Of Platelet Factor 4 In Diabetic Patients Before And After The Isometric Exercise
Authors: Lučić A , Lepšanović L , Kulauzov M , Bukvić R
Published: May 2019

In order to evaluate the level of platelet activation in patients with the high risk for developing the thromboembolic complications and influence of isometric exercise on this process, the estimations of platelet factor 4 (PF 4) by the radioimmunoassay have been done in 20 diabetic patients. The half of examined patients had the diabetic microangiopathy as well as macroangiopathy (subgroup A) and the other half (subgroup B) had no clinical
sign of macroangipathy. The control group comprises 10 healthy volunteers. The estimations of PF 4 have been done at rest and af ter 2 minutes isometric exercise. The mean values of PF 4 in both subgroups at the rest were significantly elevated in relation to the values in control group (sbg A = 92,7 ng/ml, sbg B = 44,05 ng/ml and control 7,91 ng/ml). After the isometric exercise the mean values of PF 4 in diabetics have been moderately decreased (sbg A = 69,01 ng/ml, sbg B = 39,2 ng/ml), but increase in relation to mean values in control group (24,2 ng/ml). The results indicate the high degree of in vivo platelet activation in diabetic patients, especially in those with macroangiopathic changes. The isometric exercise in the mean time, did not effect the platelet release reaction in examined patients.
The effect of antihuman platelet antibodies on human platelets
Authors: Ali-Briggs E , van Leeuwen E , von dem Borne A , Jenkin C
Published: May 2019

The influence of an heterologous antihuman platelet serum (AS) was examined an immunological approach to investigate platelet function. AS induces aggregation and release from platelets in citrate plasma. Neither thrombin nor complement were requirements, and inhibitors of ADP-(adenosine) and collagen-induced (aspirin) aggregation were only slightly inhibitory. EDTA-F and four-times washed platelets were also aggregated by AS. These data sugg AS-induced aggregation proceeds, at least in part, by a mechanism independen of ADP and does not involve platelet prostaglandin synthesis. The active principle of the AS was found to be associated with the IgG fraction. AS fo immunoprecipitates (by immunodiffusion) with platelets, fibrinogen and facto VIII; absorbtion with cryoprecipitate did not influence the aggregation resp Since platelets have common antigenic determinants with other blood cells, absorbtion studies with such cells were performed. Absorbtion of AS with red cells (until negative in the indirect antiglobulin test) had little effect o AS-induced aggregation and release. Following absorbtion with leucocytes, immunofluorescence studies revealed that lymphocyte reactive antibodies were almost totally removed while those of the granulocyte and platelet were stil present. This absorbed AS retained the ability to induce both platelet aggregation and release.
Fluorogenic Peptide Substrates For Proteases In Blood Coagulation, Kallikrein-kinin And Fibrinolysis Systems: Substrate For Plasmin And Factor XIa
Authors: Iwanaga S , Kato H , Maruyama I , Adachi N , Ohno Y , Takada K , Kimura T , Sakakibara S
Published: May 2019

Fluorogenic peptide substrates, peptidyl-4-methylcoumarin amides (MCA), are now commercially available and proved to be useful for the assay of α-thrombin, Factor Xa, urokinase, Limulus clotting enzyme, plasma and urinary kallikreins. We have newly synthesized the fluorogenic substrates for plasmin and Factor XIa and tested for their possible use as specific substrates for these enzymes. Among six peptidyl-Lys-MCA, Boc-Glu-Lys-Lys-MCA and Boc-Val-Leu-Lys-MCA were good substrates for bovine and human plasmins, which were activated by urokinase. Streptokinase-activated plasmin complex (molar ratio with 1 to 1) hydrolyzed the former but not the latter. Using Boc-Phe-Ser-Arg-MCA and Boc-Leu-Thr-Arg-MCA, a specific assay method for Factor XIa activated with Factor XHa in the presence of HMW kininogen and kaolin, was developed. The kinetic parameters of various proteinases towards the specific substrates were as follows: α-Thrombin (Boc-Val-Pro-Arg-MCA, Km=2.1x10-5M), Factor Xa (Boc-Ile-Glu-Gly-Arg-MCA, 1.6x10-4), Plasma kallikrein (Z-Phe-Arg-MCA, 2.4xl0-4M), Pancreatic kallikrein (Pro-Phe-Arg-MCA, 1.6x10-4M), Urinary kallikrein (Pro-Phe-Arg-MCA, 2.2x10-4M), Urokinase (Glutaryl-Gly-Arg-MCA, 4.4xl0-5M), Limulus clotting enzyme (Boc-Leu-Gly-Arg-MCA, 2.7X10-5M), Plasmin (Boc-Glu-Lys-Lys-MCA, 6.7xl0-4M and Boc-Val-Leu-Lys-MCA, 2.5x10-4M).
Kaolin-Activated Euglobulin Lysis Time (KELT)
Authors: Greig H
Published: May 2019

The most commonly used test for clinical assessment of fibrinolytic activity is the Euglobulin Lysis Time (ELT). However the normal range is very wide, the long times are inconvenient and detection of inhibition is impossible. An attempt has been made to utilise the acceleration of the ELT when kaolin is present, to devise a test with shorter times, a narrower normal range, and better precision. The Euglobulin lysis time was carried out by a modification of the method of NILSSON and OLOW, after precipitation of the euglobulin in the absence of kaolin (ELT) and in the presence of 1 mg. kaolin/ml. plasma (KELT). In 14 control subjects the mean, SD, and range for the ELT were 168.6’, 54.6’, 84-290’; the corresponding values for the KELT were 60.3’, 8.3’ and 46-74’. However, it was found that there was no correlation between the ELT value and the corresponding KELT (’r’ = -0.021); on the contrary, the longer the ELT, the greater the shortening produced by kaolin and there is a direct correlation between the ELT and the shortening of the lysis time by kaolin; ’r’ = 0.988. It is concluded that the KELT has no value as a clinical measure of fibrinolytic activity; further, the results suggest that kaolin may remove an inhibitor(s) of plasminogen activation as well as initiating Factor XII - mediated plasminogen activation.
Levels of platelet factor 3 in citrate plasma and in whole blood as determined by a chromogenic peptide substrate assay
Authors: Sandberg H , Gellerbring A.-K. , Andersson L.-O.
Published: May 2019

A newly developed sensitive method for determination of platelet factor 3 (PF 3) using a chromogenic substrate (S 2238) (Sandberg and Andersson, Thromb. Res., in press) was used for comparison of the PF 3 levels in platelet-rich plasma (PRP) in citrate with the levels in PRP in the anticoagulant EDTA/citrate/PGE 1/theophylline. It was shown that in citrate PRP, release of PF 3 was started after about 20 minutes from blood sampling. Release of β-thromboglobulin (β-tg) was found to occur simultaneously with the release of PF 3. No release of PF 3 or β-tg could be detected within 3 hours from blood sampling in PRP in the EDTA/citrate/PGE 1/theophylline anticoagulant.

The PF 3 level in whole blood was measured by a method which was a modification of the method used for plasma samples. It was found that, using the same donor, the PF 3 level of plasma and blood in the EDTA/citrate/PGE 1/theophylline anticoagulant was essentially the same. The levels of PF 3 found in blood upon standing in citrate or in EDTA/citrate/PGE 1/theophylline anticoagulant were in accordance with the levels found in plasma. Experiments with whole blood without any anticoagulant showed that release of PF 3 begin to occur simultaneously with release of β-tg, about 5 minutes before clotting. Thus the data show that PF 3 and β-tg are released in parallel.
Human Prothrombin Activation: Determination of Plasma and Serum Levels of Prothrombin Fragment 3 by Radioimmunoassay
Authors: Walz Daniel , Brown Thomas
Published: May 2019

Human prothrombin activation is unique in that, in addition to the release of fragment 1.2 (FI.2) from the NH-terminus of prothrombin by factor Xa during the generation of thrombin, an additional 13 residue polypeptide, fragment 3 (F3), is autocatalytically removed from the amino-terminus of the thrombin A chain. We have developed a rapid radioimmunoassay for human F3 which incorporates short incubation times and the use of a preprecipitated second antibody; the assay can be performed in three hours. Specificity studies in buffer systems show prothrombin and prethrombin 1 cross-reacting at a level of 0.001; purified thrombin does not cross-react. In the presence of 5% BSA, prothrombin displays considerably less cross-reactivity. No immunoreactive material to F3 antibodies could be detected in 400 μL of plasma. Serum, obtained from whole blood clotting, contained measurable quantities of F3 (40-100 ng/mL). This amount in serum represents only 5-10% of the theoretical amount available should all of the fragment be hydrolytically cleaved during the conversion of prothrombin to thrombin. This assay procedure is currently being utilized to monitor the activation of purified human prothrombin in the absence and presence of selected plasma inhibitors. (Supported in part by NIH 05384-17 and the Michigan Heart Association).
Modification of the Outcome of Coronary Thrombo-Embolism by Low and High-Dose Aspirin
Authors: Moore S , Belbeck L , Pineau S
Published: May 2019

Previous work in this laboratory has shown that an occlusive thrombus (O.T.) induced in the left anterior descending coronary artery (L.A.D.) of dogs, by the placement of a segment of magnesium-aluminum wire (M.A.W.) in the lumen of the artery, is associated, with the development of full thickness infarct (F.T.I.). Incomplete (mural) thrombus is associated with focal areas of ischaemic damage, mainly sub-endocardial, in the myocardium of the antero-lateral left ventricle. A 1.0 cm. length of M.A.W., shaped in the form of a tennis racquet, was placed by cardiac catheterization in the proximal L.A.D. of 30 mongrel dogs. Ten dogs received low dose aspirin (L.D.A.); (14 or 29 mgm/Kg daily in a single dose). Ten dogs received high dose aspirin (H.D.A.); (100 mgm/Kg daily in divided doses). Treatment was begun one day before operation and continued until death or killing. Ten control dogs received no drug. All control dogs died within 3 days of operation. Five had O.T. and F.T.I. The remainder showed focal ischaemic myocardial damage. None of the L.D.A. group died. At autopsy 3 or 4 days after operation, none had O.T. or F.T.I. In the H.D.A. group 3 died; 4 had O.T. and F.T.I. Thus L.D.A. provided significant protection from death, O.T., and F.T.I. H.D.A. provided some (P<0.005) but less protection from death but did not significantly affect the incidence of O.T. or F.T.I Supported by Ontario Heart Foundation Grant T15-7.
Aspirin Dosage and Antithrombotic Effect
Authors: Clagett G , Russo Marianne , Hufnagel Howard
Published: May 2019

It is generally accepeted that there is no correlation between aspirin (ASA) dosage and antithrombotic effect. This hypothesis was tested by devising an animal model of thrombosis consisting of an A-V shunt with flow rate controlled at 200 ml/sec. The occlusion time (T-O) was found to be dependent upon formation of a platelet rich thrombus confirmed by electron microscopy. Three groups of dogs (4 in each group) were treated with 3 dosages of ASA (160, 325, and 650 mg BID for 4 days) and mean T-O under treatment was divided by mean T-O during a control period to obtain an index of prolongation of T-O by ASA in each animal. The mean indexes of prolongation for the groups were 1.31±0.14 (160 mg), 1.41±0.12 (325 mg), and 2.67±0.32 (650 mg), with the last being significantly greater (p <.01) than the others. This paralleled the salicylate levels: 1.0±0.37 (160 mg), 1.75±0.31 (325 mg), and 4.98±0.53 mg/dl (650 mg). The index of prolongation T-O for each animal was correlated with ASA dosage in mg/kg (r=0.86 p <.001) and salicylate levels (r=0.77, p <.01). To confirm a dosage effect, 3 animals were treated with low (160 mg BID) and high (650 mg BID) dose ASA. In each animal, T-O (secs.) was significantly longer (p <.001) with the higher dose. ASA in all doses produced equivalent suppression of collagen-induced aggregation, 14C-serotonin release, and platelet prostaglandin synthesis as measured by thrombin-stimulated malonyldialdehyde production. These results suggest an antithrombotic dose dependency of ASA not apparent from in vitro platelet function tests.
Evaluation of Trials of Antithrombotic Agents in Acute Myocardial Infarction
Authors: Blackburn B , Chalmers T , Smith H
Published: May 2019

Forty randomized control trials dealing with antithrombotic therapy in the treatment and secondary prevention of myocardial infarction were evaluated for quality of trial design, implementation and analysis. To reduce bias, methods sections were differentially xeroxed before evaluation. Two quality scores were obtained: 1) one reflects the authors’ description of patients and treatments; blinding of randomization and of patients, physicians and observers; measurements of efficacy of randomization, blinding, and compliance; and prior estimate of appropriate sample sizes. 2) The other reflects appropriateness and adequacy of statistical analysis. Higher quality scores were obtained by large multi-centered trials (P=.03); those with credit given to a biostatistician (P=.02); and those done recently (P=.04). Decrease in control CFR was found across time (P=.03). Results obtained for anticoagulant treatment showed 8 of the 11 trials with reduction in CFR, 2 with no difference and one with an increase in CFR. 5 of the 10 trials of fibrinolysins were positive. 5 of 9 secondary anticoagulant trials were positive. Two large trials with a trend in favor of aspirin plus a positive sulfinpyrazone (P<.02) trial suggest that antiplatelet agents may be prophylactic. No association between above results and the quality score of the trials was found. This study indicates that anticoagulants but not fibrinolysins reduce acute mortality, and antiplatelet agents are potentially useful in prophylaxis.
The Trinodular Structure of Fibrinogen: Confirmation by Both Shadowing and Negative Stain Electron Microscopy
Authors: Ericksen H , Fowler W
Published: May 2019

Fibrinogen molecules sprayed on mica, dried in vacuo and shadowed with platinum appear as trinodular rods. We describe here the flotation technique of negative staining by which we were able to visualize individual fibrinogen molecules. The fibrinogen molecules in our negatively stained preparations have a trinodular structure identical to that of shadowed molecules. The molecules are 45 nm long, the two outer nodules are 6-7 nm in diameter, and the middle nodule is 4-5 nm in diameter. We believe that the 20 nm diameter globules seen in previous negative staining studies are aggregates of these molecules.
Arrangement Of Monomeric Units In Fibrin
Authors: Hermans Jan
Published: May 2019

Measurements of light scattering have given much information about formation and properties of fibrin. These studies have determined mass-length ratio of linear polymers (protofibrils) and of fibers, kinetics of polymerization and of lateral association and volume-mass ratio of thick fibers. This ratio is 5 to 1. On the one hand, this high value suggests that the fiber contains channels that allow the diffusion of enzymes such as Factor XHIa and plasmin; on the other hand, the high value appears paradoxical for a stiff fiber made up of elongated units (fibrin monomers) arranged in parallel. Such a high fiber volume is a property of only a small set out of many high-symmetry models of fibrin, which may be constructed from overlapping three-domain monomers which are arranged into strands, are aligned nearly parallel to the fiber axis and make adequate longitudinal and lateral contacts. These models contain helical protofibrils related to each other by rotation axes parallel to the fiber axis. The protofibrils may contain 2, 3 or 4 monomers per helical turn and there are four possible symmetries. A large specific volume is achieved if the ends of each monomer are slightly displaced from the protofibril axis, either by a shift or by a tilt of the monomer. The fiber containing tilted monomers is more highly interconnected; the two ends of a tilted monomer form lateral contacts with different adjacent protofibrils, whereas the two ends of a non-tilted monomer contact the same adjacent protofibril(s).
Automated Microdensitometry And Protein Assays As Measures For Platelet Adhesion And Aggregation On Collagen Coated Slides Under Controlled Flow Conditions
Authors: Muggli R , Baumgartner H , Tschopp Th
Published: May 2019

Microscope slides were homogeneously coated over a length of 2 cm with a mixture of soluble and fibrillar collagen and exposed at 37°C and under laminar flow to citrated whole rabbit blood at a flow-rate of 100 ml/min. Surface coverage with platelets (adhesion) and platelet accumulations higher than about 5 μm in height (aggregation) were determined by automated microdensitometry of fuchsine stained ‘en face’ preparations. The platelet mass per unit surface was measured with a modified Lowry technique whose sensitivity was equivalent to 5×l05platelets. Platelet number, amount of protein and surface coverage with platelet accumulations correlated. After a perfusion time of 10 min thrombi up to 30 μm in height and oriented in the direction of flow had developed on the collagen coated area. Surface coverage with platelets was 75% and the amount of deposited protein 1.4 μg/mm2(2×l06platelets/mm2). On the uncoated surface single platelets predominated; the surface coverage was 20% and the density of platelets 8×104/mm2. Acetyl- salicylic acid at 100 μm decreased platelet aggregation by about 80% without affecting adhesion.

The new parallel plate perfusion system offers rapid quantitation of platelet-surface and platelet-platelet interaction after exposure to flowing blood and iftay also be diagnostically useful.
Immunochemical Studies of Antisera to Human Fibrinopeptide B
Authors: Butler V , Weber D , Nossel H , LaGamma K , Canfield R
Published: May 2019

Specific immunochemical measurement of fibrinopeptide B (FPB) in clinical blood samples is made difficult: (1) by the presence, in some samples, of larger fibrinogen-derived fragments which contain the FPB sequence and crossreact with anti-FPB serum, thus contributing to immunoreactive plasma FPB; and, (2) by the presence of carboxypeptidase B, which is capable of rapid in vitro cleavage of the carboxyl-terminal arginine, thus lowering immunoreactive plasma FPB. Since accurate FPB assay requires immunological distinction from larger FPB-containing peptides and from desarginyl-FPB, the immunochemical specificity of 7 rabbit anti-FPB sera has been characterized in detail. Antisera have been identified which react with FPB 20 times more effectively than they react with fibrinogen, Bβl(Pyr)-l18(Met), or plasmin-cleaved Bβl(Pyr)-42(Arg); using such antisera, larger FPB-containing peptides have been identified by increases in FPB immunoreactivity following thrombin treatment of plasma dialysates. One antiserum has been identified which reacts with desarginyl-FPB almost as effectively as it reacts with FPB. Using appropriate antisera, it is now possible to identify the presence of larger FPB-containing peptides and of carboxypeptidase B-cleaved desarginyl-FPB. The selective use of immunochemically characterized anti-FPB sera should facilitate the more precise quantification of FPB in human plasma.
Isolation of Fibrinogen Protomer by Partial Reduction of Disulfide Bonds in Concentrated Urea
Authors: Albert A , Cacho F , Saiz J
Published: May 2019

Partial reduction of human and bovine fibrinogen (F). recently dissolved in 6 and 8M deionized urea, with different amounts of dithioeritritol (DTE) (10 to 60:100 DTE:F, w/w) has been carried out in nitrogen atmosphere. Disc gel electrophoresis in 0.1% SDS shows changing amounts of six molecular species depending on the reaction time and DTE:F ratio. Three bands are identical with those obtained by total reduction of fibrinogen, one with unreduced material, the remaining two show mobilities corresponding to molecular weights of 170.000-190.000 (band H, with yields up to 40-50%) and 130.000-140.000 (band N).

Total reduction of the isolated species H shows identical composition in fibrinogen chains as the original material. Stabilization by alkylation of this soecies is only successful in 0.1% SDS. Comparative ultracentrifugation studies show sedimentation coefficients of 6.8 S for fibrinogen incubated with SDS and 4.1 S for the H Species, which agree with a ratio 2:1 for their molecular weights.

These results confirm the existence of two protomer entities easily cleaved by partial reduction but the need of high concentration of urea to separate the protomer, suggests the existence of additional strong non-covalent interactions between the two hemimolecules.
Conformational Alterations of α2-Antiplasmin Induced By Complex Formation With Plasmin
Authors: Plow E , Wiman B , Collen D
Published: May 2019

The conformational and structural changes induced in the α2-antiplasmin (AP) molecule by complex formation with plasmin have been analyzed utilizing quantitative radioimmuno-chemical analysis. Complexes prepared in plasmin excess (PAP-P) and therefore subjected to limited proteolysis and complexes prepared in AP excess (PAP-A) have been compared with free AP. With AP antiserum. PAP-A, PAP-P and AP yielded reactions of complete identity by immunodiffusion analysis. In radioimmunoassay, however, these were clearly distinguished, and four distinct sets of antigenic determinants were delineated. Set I determinants were expressed equivalently by PAP-P, PAP-A and AP and were, therefore, not altered by complex formation. This set was recognized by 90% of the antibodies, and the determinants Were all included within a large fragment of Mr 60,000 derived from the NH2-terminal region of AP. The other three sets of determinants were modulated by complex formation. Set II was expressed by PAP-A and AP but not by PAP-P, and these were sensitive to proteolysis by plasmin. Set III determinants were expressed only by AP and were localized to a peptide of Mr 8,000 derived from the COOH-terminal region of AP. Set IV determinants were also present only on AP but were not present in the peptide and required an intact reactive site in AP for expression. Thus, there is evidence for multiple conformational modulations in AP induced by complex formation, and these modulations can be pinpointed to specific loci within the AP molecule.
Red Cell Deformability and Risk Factors in One Hundred Patients with Cerebrovascular Thrombosis
Authors: Boisseau M , Lorient M , Bricaud H
Published: May 2019

The rheological importance of red cell deformability on thrombotic states has recently been established. The aim of the present study was to demonstrate the relationship between the loss of erythrocyte flexibility and vascular risk factors in one hundred patients presenting with cerebrovascular accidents (CVA).

Erythrocyte deformability measured by Nucleopore filtration method was significantly (p 0.001) decreased in patients with CVA (filtration time (FT) = 57 ± 28 s. x ml-1) compared with two hundred heathly subjects (FT = 33 ± 6 s. x ml-1). Among the risk factors erythrocyte deformability mainly depended upon the gravity of Hypertension (FT = 77 ± 35 s. x ml-1; p 0.001). Additionnal risk factors such as hyperlipemia, diabetes were less effective.

In all cases high FT were observed in patients with widespread atherosclerotic lesions (carotid stenosis, arteritis).

Finally in thrombotic states, as CVA, the decrease of red cell deformability appears to be strongly correlated to vascular diseases and risk factors.
Pathways of Stimulation of Tissue Factor Activity Generation by Leukocytes
Authors: Niemetz J , Muhlfelder T
Published: May 2019

Leukocytes may generate a potent procoagulant thromboplastic (tissue factor) activity (TFa) if appropriately stimulated. Among the stimulants endowing leukocytes with TFa are endotoxins (lipopolysaccharides:LPS), antigen-antibody, complexes and a complement component: the chemotactic fragment of C5. (C5fr) which is a particularly potent activator. As LPS and antigen-antibody complexes can activate complement with ensuing generation of C5fr, the question arose whether C5fr is the sole terminal pathway for activation of leukocyte TFa. Studies of the amount of activity generated with the LPS and C5fr, showed that normal leukocytes stimulated with C5fr produce significantly more TFa than leukocytes stimulated with LPS. In contrast, leukocytes from patients with paroxysmal nocturnal hemoglobinuria which respond normally to LPS, generate considarably less TFa when stimulated with C5fr. Furthermore, the TFa in normal leukocytes - appears sooner following LPS stimulation than following C5fr stimulation. These results suggest that at least in vitro C5fr need not be the sole external stimulant for the generation of TFa.
Measurement of Endotoxin Levels in Plasma Using Limulus Amoebocyte Lysate and Chromogenic Substrate, S2222
Authors: Clark S , Scully M , Webb P , Kakkar V
Published: May 2019

A method has been devised for the measurement of endotoxin in plasma using the chromogenic substrate, S2222, a substrate which has been shown to be particularly sensitive to the Limulus Lysate. Time curves of the rate of release of the chromogen in mixtures in which procoagulase activation was concurrent, were complex with a lag phase which was shortened by increasing endotoxin concentrations. At a final concentration of 0.5ng/ml and 370 activation was complete within 60 minutes. The enzyme was inhibited by soya bean trypsin inhibitor but not by pancreatic trypsin inhibitor or hirudin. In the method finally adopted the lysate (25µl) was incubated with endotoxin (E.coli 026.86 Difco) and magnesium chloride (final concentration 33mM) in a total volume of 225µl. After 12 minutes preincubation 165µl of S2222(0.4mM) was added and the increase in abdorbance at 405nm over two minutes measured using an Abbott Biochromatic Analyser 100. Linear assay curves were obtained with final concentration of 0.2 to 2.0ngs endotoxin/ml with ΔOD 405/min of 0.35 at 2.0ng endotoxin/ml of final incubation mixture. ΔOD /min in control tubes were of the order of 0.02. For measurement from plasma samples, endotoxin was first extracted with chloroform. Linear calibration curves were achieved at a concentration of endotoxin of 1 to 5ng/ml of whole blood with a net OD/min at the highest concentration of 0.25.
The Relation Between VIIIR:AG and VIII:C Studied with Two Antisera
Authors: Muller H. P. , van Tilburg N. H. , Bertina R. M. , Veltkamp J. J.
Published: May 2019

FVIII was separated into low molecular weight FVIII (LMW FVIII) and high molecular weight FVIII (HMW FVIII) by gel chromatography in the presence of high salt concentration or by high salt elution of LMW FVIII from FVIII bound to anti HMW FVII-Sepharose. Specific antibodies were raised in rabbits against HMW FVIII and LMW FVIII. After removal of the contaminating anti HMW activities the rabbit anti LMW FVIII was still able to neutralize the FVIII coagulant activity of normal plasma and of IMW FVIII with canparable efficiency and it had no effect on the VIIIR:WF of FVIII in normal plasma or in HMW FVIII. Anti LMW FVIII does not bind to HMW FVIII and does not precipitate FVIII as tested by counter immunoelectrophoresis. Rabbit anti HMW FVIII precipitates FVIII in normal plasma, inhibits VIIIR:WF activity, while it has no effect on the FVIII coagulant activity of LMW FVIII. The coagulant activity of FVIII in normal plasma is slightly inhibited by anti HMW FVIII presumably by non-specific effects (sterical hindrance). It is concluded that inhibitory antibodies against VIII:C raised in rabbits recognize antigenic structures only present on LMW FVIII. Antibodies against HMW FVIII raised in rabbits appears to recognize structures only present on HMW FVIII.
Separation of factor VIII by Ion Exchange Chromatography
Authors: Austen D. E. G.
Published: May 2019

Using a chemically treated agarose, it has been possible to separate factor VIII clotting activity from factor VIII related antigen and ristocetin co-factor in human factor concentrates, or alternatively from antigen and platelet aggregating factor in porcine materials. It appears that components are separated by an ion-exchange mechanism although hydrophobic interactions and steric influences cannot be ruled out. A column technique is employed and products appear to be stable. The latter is an important feature since factor VIII which has been exposed to charged surfaces is often unstable. However, part of this good stability may be attributable to the lysine-containing buffer which is employed. Factor VIII clotting activity has a normal antibody reaction after separation and the antigen is of normal mobility. The factor is recovered from the column at relatively low ionic strength (0.5 molar sodium chloride) and this might suggest that the components are separated without factor VIII being disaggregated into sub-units. Unlike some ion-exchange separations of factor VIII it is not necessary to purify the starting materials to a higher quality than that of a washed and adsorbed cryoprecipitate and one passage of such a material through the column results in high purification.
Blood Viscosity and Platelet Aggregation in Male and Female Patients with Transient Ischaemic Attacks (TIA)
Authors: Palareti G. , Poggi M. , Fortunato G. , Coccheri S.
Published: May 2019

A series of 40 patients with TIA (25 males and 15 females) was thoroughly investigated by means of angiography and computerized tomography, and divided into a group (A) of 15 “sine materia”, and a group (B) of 25 with direct or indirect evidence of vascular occlusive or stenotic changes. Blood viscosity at 230 sec-1 37° was cp 4.2 ± 0.3 in the controls, cp 4.7 ± 0.7 in all patients (p < 0.05) cp 4.98 ± 0.7 in all male patients (p < 0.01 versus male controls), and cp 4.75 ± 0.8 in group B (p < 0.02). Haematocrit and Fibrinogen were also significantly increased in all male patients and in group B. Circulating platelet aggregates (CPA) were increased in 40% of the patients. Almost all patients with elevated CPA were males, with a slight prevalence in group B. Changes in blood viscosity parameters and in platelet aggregation in TIA patients were therefore related both to evidence of vascular lesions, and to sex, since they were found to prevail in male patients of both groups.
Platelet Aggregation and Circulating Fibrinogen Complexes in Patients with TIA
Authors: Abbate R. , Gensini G. F. , Neri Serneri G. G. , Inzitari D. , Taiuti R. , Amaducci L.
Published: May 2019

In the Atherosclerosis Project of the National Research Council we investigated 31 controls and 25 patients with transient ischaemic attacks (TIA) in whom an angiographic study of the carotid arteries and of the aortic arc has been performed. Circulating platelets aggregates (Wu and Hoak 1974) and circulating high molecular weight fibrinogen complexes (HMWFC) by gel-fil. tration of plasma beta-alanine precipitate (Graeff and von Hugo 1974), fibrinolytic activity (ELT) and antithrombin III (Howie et al.1973) were determined. The patients who had suffered from TIA showed an increased platelet aggregation (17/25, P<0.001) and an increased concentration of HMWFC (15/25,P<0.001).Fibrinolytic activity and AT III activity showed no significant differences between the two groups.The angiographic study showed in 11 patients identifiable atherosclerotic lesions (IAL), but no correlation between arteriographie lesions and increase of platelet aggregation and HMWFC concentrations was found.
Thrombotic Thrombocytopenic Purpura: Mechanism for Effectiveness of Plasmapheresis
Authors: Kelton J. G. , Neame P. B. , Walker I. , Turpie A. G. , McBride J. , Han P. , Nicholson W. , Hirsh J.
Published: May 2019

Thrombotic thrombocytopenic purpura (TTP) is a rare but serious illness of unknown etiology. Treatment by plasmapheresis has been reported to be effective but the mechanism for benefit is unknown. We have investigated the effect of plasmapheresis in 2 patients with TTP by quantitating platelet associated IgG (PAIgG) levels prior to and following plasmapheresis. Both patients had very high levels of PAIgG at presentation (90 and A8 fg IgG/platelet respectively, normal 0-5). in both, the PAIgG levels progressively fell to within the normal range and the platelet count rose following plasmapheresis. One patient remained in remission with normal platelet counts and PAIgG levels. The other relapsed after plasmapheresis and the PAIgG level rose prior to the fall in platelet count. Plasmapheresis was repeated and resulted in normalization of both the platelet count and PAIgG level. It is suggested that plasmapheresis removes antiplatelet antibody or immune complexes which may be of etiological importance in this illness.
Severe Acute Disseminated Intravascular Coagulation (DIC): Clinical Imporiance; Heparin Therapy; and Etiology
Authors: Mant M. J. , King E. G.
Published: May 2019

We have reviewed 47 patients (pts.) with severe acute DIC (mean fibrinogen 100.5 mg/dl, platelets 45,700/μl, prothrombin time 19.4 sec, partial thromboplastin time 86.5 sec). Most pts. were critically ill. Predisposing conditions included infection (32), shock (32), and surgery or trauma (21). Routine treatment included aggressive therapy for underlying diseases and metabolic disturbances, vitamin K, folate, and blood conponents Twelve pts. were treated with heparin and 10 (83%) died. Heparin apparently benefited only 1 pt. and in 7 bleeding increased. Thirty (86%) of 35 pts. who did not receive heparin died. Analysis suggested that heparin would not have affected the outcome in any of them. in all pts. who died multiple potential causes were present. Thrombosis, ischemia, or hemorrhage were the apparent inmediate cause of death in only 2 pts. but were contributory causes in others. in most pts. with significant oonplications potentially attributable to DIC other causative pathology was present. Twenty-five pts. had autopsies. Microvascular thrombi were not found but large vessel thrombi were relatively common. VJe conclude that 1) Severe DIC is usually a preterminal event in critically ill pts. 2) Other pathology may be more important than DIC when death or significant ' thrombosis or bleeding occur. 3) Heparin is rarely beneficial and often increases bleeding. 4) Microvascular thrombi may not be the only mechanism responsible for DIC.
Zonal Immobilization Procedure for Chromatographic Analysis of Fibrin Complexes
Authors: Shainoff John , Dardik Beatriz
Published: May 2019

Conventional gel filtration methods for detection of fibrin complexes depend on fraction collection and multiple analyses of the eluted protein to characterize the chromatogram. A new approach has been made possible through invention of a system for attaching the separated protein to the gel and staining it with either fluorescent or radioiodinated antibody. in this system, glyoxyl agarose (agarose-0-CH2-CH0) is used as the gel nitration medium to separate the proteins, and is subsequently activated to immobilize tnem in their separated positions by electrophoresing alkaline cyanoborohydride into the suspending medium. When used with small disposable columns this zonal immobilization an direct staining procedure provides a speedy and inexpensive alternative to fraction collecting. Further, it has been found that the dissociable complexes that are formed by reversible interaction of fibrin monomer with fibrinogen are not separable from fibrinogen under conditions commonly used for gel filtration. That is because solubility or fully polymerizable fibrin depends on its interaction with fibrinogen. to detect such fibrin in plasma, we equilibrate columns with buffer containing rabbit fibrinogen as a carrier to maintain solubility. Studies on patients indicate that the stability of fibrin complexes in blood may vary in characteristic ways depending on underlying disease processes. (Supported by USPHS grants HL-16361 and HL-19767)
High Fibrinogen (Fg) Content in Plasma Clots Harvested At Arrested, Incipient Coagulation
Authors: Godal H. C. , Brosstad F.
Published: May 2019

Incomplete transformation of Fg to fibrin may occur in vivo and in vitro,especially in connection with the use of heparin. We have studied to what extent unproteolyzed Fg is incorporated in plasma clots harvested at early and late arrest in Fg to fibrin conversion. Since des-AA fibrin will predominate in clots harvested at incipient gelation whilst des- AABB fibrin will be characteristic of clots harvested at late stages in Fg to fibrin conversion, preformed, enzyme-free des-AA or des-AABB fibrin monomers were added in varying amounts to Fg-boosted, inactivated serum containing varying Fg concentrations. (Fg + fibrin= 2 g/1 in all samples) . Labelled monomers or labelled Fg were used. The clots were harvested, squeezed, washed and Fg/fibrin ratio of clots calculated on the basis of recorded radioactivity. All clots contained Fg,but substantially more when formed at high Fg/fibrin rations (incipient gelation) than at low (late stages in Fg to fibrin conversion). Thus, clots formed at Fg/fibrin ratio=9/l contained 3 times as much Fg as fibrin, whilst clots formed at a Fg/fibrin ratio=l/9 contained Fg/fibrin=l/20.Presence of preactivated FSF(activated by insolubilized thrombin) did not influence the Fg/fibrin ratio of clots. Streptokinase-induced intraclot lysis rate of clots containing Fg were substantially higher than of clots containing fibrin only, a finding especially noticeable in clots with a high Fg content, and might explain the low thromboembolic complication rate seen in arvin/reptilase therapy. Finally, it should be stressed that clots harvested at incipient gelation contain large amounts of Fg, which may lead to misinterpretation as to the amount of fibrin actually formed.
A Novel Method for the Rapid Purification of Human and Rat Fibrin(OGEN) Degradation Products in High Yields
Authors: Nieuwenhuizen W. , van Ruijven-Vermeer I. A. M. , Haverkate F. , Timan G.
Published: May 2019

A novel method will be described for the preparation and purification of fibrin(ogen) degradation products in high yields. The high yields are due to two factors. on the one hand an improved preparation method in which the size heterogeneity of the degradation products D is strongly reduced by plasmin digestion at well-controlled calcium concentrations. At calcium concentrations of 2mM exclusively D fragments, M.W.= 93-000 (Dcate) were formed; in the presence of 1OmM EGTA only fragments M.W.= 80.000 (D EGTA) were formed as described. on the other hand a new purification method, which includes Sephadex G-200 filtration to purify the D:E complexes and separation of the D and E fragments by a 16 hrs. preparative isoelectric focussing. The latter step gives a complete separation of D (fragments) (pH = 6.5) and E fragments (at pH = 4.5) without any overlap, thus allowing a nearly 100% recovery in this step. The overall recoveries are around 75% of the theoretical values. These recoveries are superior to those of existing procedures. Moreover the conditions of this purification procedure are very mild and probably do not affect the native configuration of the products. Amino-terminal amino acids of human Dcate, D EGTA and D-dimer are identical i.e. val, asx and ser. in the ratgly, asx and ser were found. E 1% for rat Dcate=17-8 for rat D EGTA=16.2 and for rat D- dimer=l8.3. for the corresponding human fragments, these values were all 20.0 ± 0.2.
Purification and Properties of Rat α2 Acute-Phase Macroglobulin (α2M)
Authors: Nieuwenhuizen W. , Emeis J. J. , Hemmink J.
Published: May 2019

For our studies on the relation between blood fibrinolytic activity and repair of mechanically damaged arteries in our rat model we need a specific and sensitive assay for α2M. in the rat α2M is an acute-phase protein of which the level in blood is normally near zero but increases as a result of the damage. Moreover α2M is known to inhibit proteases involved in the fibrinolytic system. We developed a new purification procedure in which, conditions known to be harmful to the functionality of α2M were avoided. α2M was purified from plasma of turpentine-treated rats and proteolytic activities were suppressed throughout the purification procedure. The purification scheme successively involves: rivanol precipitation, Con A-Sepharose chromatography and DEAE-cellulose chromatography. Thus 48 mg of α2M was obtained from 100 ml rat plasma i.e. 20% recovery. The preparations were biochemically and immunologically pure. Amino acid and carbohydrate compositions were determined. The molecular weight is 760.000. The molecule consists of 4 subunits, M.W. = 190.000. A 1%1cm = 8.8 and p1 = 4.8. It binds 1 mole of trypsin or plasmin per mole. Bound proteases were only active on low molecular weight substrates such as BAEE and BOC-L-val-gly-L-arg βNA. Kinetic data of the bound enzymes (pH-optimas, Km and Vmax) indicate that factors other than steric hindrance are involved in the inhibitory action of α2M.
Production By Thrombin of A Proteolytically Modified Form of Antithrombin and Release of The Same Form from the Antithrombin-Thrombin Complex
Authors: Fish W. , Björk I.
Published: May 2019

In addition to the in vitro formation of an inactive, stable complex (ATT) between thrombin (T) and the plasma serine protease inhibitor, antithrombin (AT), a sizeable proportion of AT is converted by T to a form which has lost the ability to inhibit T and which exhibits a reduced affinity for heparin. Under non-denaturing conditions, the isolated modified AT (ATm) could not be differentiated from native AT by hydrodynamic, electrophoretic or immunological analyses. Under reducing/denaturing conditions, ATm yielded two polypeptides, one of about 50000 and one of about 5000 daltons. Preliminary studies indicate that these are the result of a proteolytic cleavage by T of an arg-ser bond in the C-terminal end of the single-chain AT molecule. Dissociation of ATT by prolonged treatment in hot SDS or by treatment with hydoxylamine or ammonia in SDS produced only T and ATm. Under nondenaturing conditions, dissociation by the two latter agents released ATm and up to 40% of the potential T activity from ATT. However, under no conditions tested could intact AT be shown to be produced from dissociation of ATT. These results suggest that scission by T of a specific arg-ser bond in the active site region of AT, or, alternatively, conversion of this bond to a tetrahedral intermediate state, occurs during the formation of the inactive enzyme-inhibitor complex.
Type of Agitation and Platelet Viability and Function After 22°C Storage: Correlation with Maintenance of Discoid Shape
Authors: Holme S. , Vaidja K. , Murphy S.
Published: May 2019

Platelet viability as measured by in vivo 51cr recovery, platelet morphology, and in vitro aggregation with ADP and thrombin were studied with platelet concentrates (PC) stored for transfusion under carefully controlled conditions. The PC were prepared from whole blood with citrate-dextrose-phosphate as anticoagulant. The platelet count was kept between 0.8 − 1.6 × 106 platelets per mm3 in a volume of 50 ml. The PC were stored in containers constructed of polyethylene (PE) or Polyvinylchloride (PVC) at 22°C for 72 hours. The bags were placed on a horizontal shaker or a ferris wheel type of apparatus during storage. No significant changes in pH or platelet count were observed during storage. PC stored on the wheel showed a moderate loss of viability and marked loss of aggregation response compared to PC on the shaker. Under optimal conditions with PC in PE on the shaker maximal rate of aggregation was reduced only 26% compared to fresh PC. PC stored in PVC showed a statistically significant greater decrease. A good correlation was observed between the percentage of discoid platelets present in the PC as judged by phase microscopy and the extent of platelet shape change response to ADP. Both parameters correlated positively with in vivo 51Cr recovery. We conclude: 1) The type of agitation used significantly effects maintenance of platelet viability and function during storage. 2) Under the conditions used, in vitro function was better preserved in PE than in PVC bags. 3) In vitro function is not inevitably lost during storage at 22°C, but is critically dependent on storage conditions. 4) In vivo platelet viability (51Cr recovery) correlates with maintenance of disc shape. The latter can be quantitated by the shape change response with ADP.