bluuebunny/crossref_metadata_2025 huggingface dataset:
Plasma from Patients with Hepatic or Renal Failure Stimulates Prostacyclin Release from “Exhausted” Rat Aorta Slices
Authors: Hoof Van A. , Chamone D.A.F. , Vermylen J.
Published: May 2019

Slices of rat aorta (1.5 to 2 mg wet weight) were washed several times in tris buffer until no further release of anti-aggregating activity could be detected. These “exhausted” aorta fragments were then incubated for 60 to 120 min. at room temperature with 250 μl platelet-poor plasma from either a healthy subject or from a patient with hepatic or renal failure. 100 ul of supernatant plasma were subsequently added to 300 ul normal human platelet-rich plasma; the mixture was challenged one minute later with ADP (2 to 4 μM final concentration) and platelet aggregation recorded.

The plasmas from 6/6 uraemic patients undergoing maintenance haemodialysis stimulated prostacyclin release more than the simultaneously studied normal plasmas.

The 17 patients with liver disease were classified using Child’s criteria for hepatic functional reserve. Stimulation of prostacyclin release was found with the plasma of 4/4 patients with advanced hepatic dysfunction, 4/6 patients with moderately impaired hepatic function and 4/7 patients with good hepatic function.

Enhanced prostacyclin release may play a role in the pathogenesis of the haemorrhage diathesis of patients with renal or hepatic failure.
Photometric Measurement of Fibrinolytic Activities During Urokinase Therapy
Authors: Nienhaus Kh. , Wenzel E. , Jäger H. , Pfordt L. , Biewer I.
Published: May 2019

The biochemical reactions on administered urokinase (N = 12 patients suffering from deep vein thrombosis; N = 3 patients with DIC; N = 5 patients with acute arterial stenosis) were tested by evaluating amidolytic activities photometrically using chromogenic substrates [chromozym UK resp. chromosym PL, Pentapharm Basel, S 2227 Kabi). In addition, conventional coagulation tests were performed, and the bleeding time, platelet count and volume distribution of platelets were controlled. A significant drop in plasminogen in all of the UK-treated patients proved that the administered UK produced plasminogen activation, but did not necessarily result in severe fibrinolysis as indicated by the conventional coagulation parameters. In correlation with these findings, the biochemical data seemed to show that plasmin activities increased parallel with the decreasing concentrations of plasminogen (Mancini). When the initial dose of UK was administered (150, 000CTA/30 Bin.), a rapid increase in plasmin-activity as well as in UK-activity followed. Bott activities decreased (8–12 h), when UK was infused continuously (75, 000/h/70 kg b.w.). An increase in platelet count during therapy was proven statistically. This phenomenon was also observed in patients suffering from DIC. No typical changes in the platelet volume were observed in patients suffering from acute chronic deep vein thrombosis during the UK-therapy.
Laser Raman Spectroscopy of Fibrinogen and Fibrin
Authors: Marx J. , Hudry-Clergeon G. , Bernard L.
Published: May 2019

Raman spectroscopy is now a useful mean of estimating the secondary structure of proteins. The study of the Amide I and Amide III bands revealed an important increase in the β-sheet form of fibrin, compared with that of fibrinogen. This effect is accompanied by significant variations in the bands characteristic of aromatic chromophores. These observations favour the hypothesis (Hudry-Clergeon et al.1975, Thromb. Res. 6, 533) that important structural events occur during the fibrinogenfibrin transition.
Platelet Factor 3 Availability During Platelet Clumping - Studied with a Method Based on a Chromogenic Peptide Substrate
Authors: Korsan-Bengtsen K. , Christenson B. , Andersson I. , Johnsen M.
Published: May 2019

Platelet factor 3 (Pf3) together with factor V accelerates the conversion of prothrombin to thrombin by factor X a. Our method to determine platelet factor 3 activity is based on the measurement of thrombin formation with the substrate H-D-Phe-Pip-Arg-pNA in a system containing normal platelet poor plasma and Russells’ viper venom. There is a linear relationship between the rate of thrombin formation and the concentration of Pf3. The method is very sensitive. Thus 10 μl of frozen platelet rich plasma(PRP) or a lipid emulsion prepared from brain give absor-bances > 2.0. When PRP is aggregated with ADP or collagen in concentrations which give maximal aggregation, no Pf3 activity whatsoever can be demonstrated. Treatment with kaolin or simply prolonged shaking of the PRP on the other hand increase the Pf3 activity extensively. Our conclusion is that aggregation of platelets per se do not make Pf3 available.
Prothrombin Metz: Purification and Characterization of a Variant of Human Prothrombin
Authors: Rabiet M.J. , Elion J. , Labie D. , Josso F.
Published: May 2019

For the purification of the abnormal prothrombin (Pt Metz), advantage has been taken of the existence in the family of three siblings who, being double heterozygotes for Pt Metz and a hypoprothrombinemia, have no normal Pt. Purification procedures included barium citrate adsorption and chromatography on DEAE Sephadex as for normal Pt. As opposed to some other variants (Pt Barcelona and Madrid), Pt Metz elutes as a single symetrical peak. By SDS Polyacrylamide gel electrophoresis, this material is homogeneous and appears to have the same molecular weight as normal Pt. Comigration of normal and abnormal Pt in the absence of SDS, shows a double band suggesting an abnormal charge for the variant. Pt Metz exhibits an identity reaction with the control by double immunodiffusion.

Upon activation by factor Xa, Pt Metz can generate amydolytic activity on Bz-Phe-Va1-Arg-pNa (S2160), but only a very low clotting activity. Clear abnormalities are observed in the cleavage pattern of Pt Metz when monitored by SDS gel electrophoresis. The main feature are the accumulation of prethrombin 1 (P1) and the appearance of abnormal intermediates migra-ti ng faster than P1.
Effect of Suloctidil on Shortened Platelet Regeneration Time in Cardiovascular Patients
Authors: Deperon R. , Roncucci R. , Lambelin G. , Lansen J. , Van Stalle F.
Published: May 2019

Previous investigations based on the measurement of platelet regeneration time (PRT) rather than platelet survival time (PST) have suggested that this parameter is significantly reduced in patients bearing vascular prosthetic material and in a number of patients with occlusive vascular disease (1). Since it is well-known that shortened PST can be normalised by antiplatelet drugs, it seemed interesting to investigate whether the measurement of PRT could also detect this effect. Suloctidil (S), a new vasoactive agent, has been chosen for this study which was perforad in parallel double-blind conditions vs placebo. 32 patient. (24 males and 8 females) with either prosthetic heart valves, aortoiliac grafts or occlusive arterial disease were investigated [mean PRT t/2 value before treatment for the S group (n=16), 2.8·.1 days]. They were given S at a dose of 600 mg, daily during 3 to 4 weeks. The results obtained indicate that S treatment significantly increases shortened PRT t/2 values (mean PRT t/2 value after S treatment : 3.8 ± 0.2; p < .001). Placebo treatment had no effect in a similar group of patients (n=16; mean PRT t/2 value before treatment : 3.0±0.1 days; after treatment : 3.2±0.2 days). (1) R. Roncucci et al., Thromb. Res., accepted for publication.
The “Dead Digit, Palpable Pulse” Syndrome: A Preliminary Study of the Value of Antiplatelet Drugs in this Disorder
Authors: Morris-Jones W. , Preston F.E.
Published: May 2019

We report here the results of a pilot study to investigate the value of antiplatelet drugs in patients with pregangrene of one or both feet associated with palpable peripheral pulses but without clinical or laboratory evidence of a myeloproliferative disorder in diabetes mellitus. 32 patients were studied. For each patient pregangrene of one or both feet had been present for a minimum period of six weeks. All patients had failed to respond to medical therapy. Platelet aggregation, using ADP, collagen and adrenaline was assessed before therapy. PRP was also tested for spontaneous platelet aggregation (SPA). BTG assays and platelet aggregate ratios were also performed. Aspirin, 325mgs t.d.s. and persantin 100mgs t.d.s. produced significant and maintained clinical improvement in 5 of the 7 patients with SPA- 7 of 12 patients with hypersensitive platelets and 3 of 13 patients with normo-or hypo-sensitive platelets. Sympathectomy was performed in patients who failed to respond to drug therapy. The mean PAR was 0.94 for the non-responder group and 0.78 for the responder group. The mean BTG for non-responders was 44ng/ml (normal 50). The mean BTG for the responder group was 59.lng/ml. The results suggest that antiplatelet drugs may be of therapeutic value in patients with the “dead-digit palpable pulse” syndrome arid that controlled clinical trials are indicated.
Sulfinpyrazone or Acetylsalicylic acid to Prevent Postoperative Thrombus Formation in Arteriovenous Fistulas of Haemodialysis Patients
Authors: Albert F.W. , Schmidt U.
Published: May 2019

The effect of sulfinpyrazone (200 mg three times a day) and acetylsalicylic acid (500 mg three times a day) on the incidence of thrombosis of arteriovenous shunts was investigated in a controlled clinical trial. In 36 patients with chronic renal failure scheduled to begin haemodialysis the same operating team constructed a subcutaneous fistula in the distal forearm. During the first six weeks after the operation the antithrombotic efficacy proved to be good for both substances. No differences of thrombotic events between the two treatment groups were statistically significant. But in contrast to acetylsalicylic acid sulfinpyrazone made no significant inhibition of platelet - aggregation; sulfinpyrazone probably will prevent the clot formation by prolonging the shortened platelet survival in uraemic patients. In a high rate of patients given ace-tylsalicylic acid (10 out of 17) there were local bleeding and gastrointestinal side effects. In consequence we should prefer sulfinpyrazone, because in the sulfinpyrazone group side effects were minimal and in none patient withdrawal from the study was necessitated.
Evidence for the Presence of FVIII Subunits in Plasma and Clinical FVIII Concentrates Using Quantitative Electrophoresis
Authors: Seghatchian M.J. , Hirst K.
Published: May 2019

X-Electrophoresis of normal plasma and low purity FVIII concentrates indicated two major peaks of cross reacting FVIIIR:Ag, extending from the front of fibrinogen to the α2 region. In high purity concentrates (Hemofil and Koate) smaller aggregates, with faster mobility predominated, confirming the heterogeneous nature of FVIII concentrate preparations.

Analysis of the gel content for other FVIII related activities revealed that FVIIIR:RCF was associated only with larger aggregates, while FVIII:C as determined by both chromogenic and clotting techniques were mainly associated with the smaller forms. The coexistence of FVIII subunits, with an immunological identity line, is brought about by using high concentrations of FVIII. The presence of such an entity in plasma was confirmed by in vitro addition of high purity concentrate to plasma, FVIII:C neutralization activity and reduction with Dithiothereitol (D.T.T.). The subunits found after D.T.T. treatment gave a symetrical peak with similar molecular distribution of the fast moving peak, present in high purity concentrate.

It is concluded that each population of FVIII aggregates would consist of the subunits cross-linked by disulphide bonds. The relative concentration of these aggregates, present in differing amounts in various preparations, could be determined quantitatively: Thus an extremely useful method for the identification of the native fonos of FVIII and the quality control of concentrate preparation is provided.
Actin Exposure on the Surface of Normal Human Platelets and Thrombasthenic Platelets Following Thrombin-Induced Secretion: Mediation by Platelet Membrane α-Actinin
Authors: George James N. , Morgan Rebecca K.
Published: May 2019

To define membrane changes which may relate to platelet function during hemostasis, we studied platelet membrane proteins following thrombin-Induced secretion. Washed platelets In Tyrode’s buffer with 4 mM EDTA were treated with human thrombin (2 U/ml) to stimulate secretion without aggregation. Then platelets were labeled with (125I)-diazo-tized diiodosulfanilic acid (125I) and analyzed by SDS-polyacrylamide gel electrophoresis. Thrombin caused secretion of 61% of the granule glycoprotein (GP-G) but lysis, determined by 51Cr release, was only 0.3% in both thrombin-treated and control platelets. In control platelets 125I labeled only the membrane glycoproteins (MCP). After thrombin 125I labeling of the MGP was unchanged but GP-G and actin were also labeled. 125I-actin was identified by elution into 0.3 mM phosphate and co-migration with actin in isoelectric focusing and 3 SDS-gel systems.125I-actin, quantified as cpm/MGP cpm, was 2.12 ± 0.15 (SD)-times greater after thrombin than in control platelets in 5 normal subjects. Thrombin-induccd actin exposure was less in 2 patients with Glanzmann’s thrombasthenia: 125I actin was only 1.25 and 1.44-times greater after thrombin. Since platelet membrane α-actinin (MCP III) is deficient in thrombasthenic platelets, it may be important in the surface exposure of actin during platelet secretion.
Effects of Receptor Mobility and Association on Thrombin Binding in Platelets
Authors: Jung S. M. , Jamieson G. A.
Published: May 2019

We have studied the effects on thrombin binding of factors influencing membrane fluidity and receptor association. Platelets fixed with glutaraldehyde were able to bind thrombin but quantitative data could not be obtained due to agglutination. Increases in membrane microviscosity by incubation with cholesterol-rich liposomes resulted in an increase in ability to bind thrombin at the high affinity sites but no change in low affinity binding, hile decreases in membrane microviscosity were without effect. Lectins were also used to link the carbohydrate portions of glycocalicin, the putative receptor, without affecting access of thrombin to the receptor in the peptide “tail”. Con A, WGA and Lens culinaris (40 ug/ml) caused a 10-fold increase (Kd 4 nM to 0.4 nM) in the affinity of binding to formalinized platelets but a 75% decrease (2400 to 600 sites) in the amount of thrombin bound in the high affinity range while peanut agglutinin and Ricinus communis lectin had the same affinity as the control but with a doubling of the number of sites (2400 to 4200) in the latter case: low affinity binding could not be measured because of platelet agglutination in the presence of the lectins at high thrombin concentration. These results suggest that factors affecting membrane mobility and receptor cross-linklng can modulate the ability of platelets to bind thrombin.
The Effect of Carbenicillin and Aspirin in the Prevention of Experimental Sudden Death in the Dog
Authors: Johnson G.J. , Heckel R. , Franciosa J.A.
Published: May 2019

Carbenicillin (C) and aspirin (A) inhibit platelet (P) aggregation (AG) and prolong the bleeding time. Previous studies in our laboratory demonstrated a 55% incidence of sudden death (SD) in dogs following acute coronary embolization. Since P AG may contribute to myocardial ischemia in this model, we studied the effect of C and A-induced P dysfunction on the incidence of SD. 40 dogs were randomized to treatment with saline or C 750mg/Kg/day subqx5, and 40 dogs ware randomized to placebo or A 600mg p.o./day × 5. 17 other dogs were treated with estradiol cypionate (E) lmg/Kg/TM. P AG was inhibited in A and C treated animals, but normal in control dogs. Thrombocytopenia (P < 15,000/dl) was induced in all E treated dogs. Glass bead coronary artery embolization was performed in anesthetized dogs. 19 of 38 control dogs died within 15 min after embolization while only 2/22 C treated dogs, 5/20 A treated dogs and 0/17 E treated dogs died in 15 min. Survival of C and E-treated dogs was significantly greater (p<0.01) than controls, while survival of A-treated dogs was not significantly better (p<0.25) than placebo. These studies demonstrate that SD following glass bead coronary embolization can be prevented by pretreatment with C. This protective effect is probably related to C-induced P dysfunction since marked thrombocytopenia offers similar protection. Further studies of the role of P AG and P-inhibitory drugs in SD are indicated.
A Novel Dysfibrinogenemia with Abnormal γ-Chain(Fibrinogen Nagoya).
Authors: Takamatsu Junki , Oqata Kanji , Kamiya Tadashi , Koie Katsuo , Takashi Takagi , Iwanaga Sadaaki
Published: May 2019

Six individuals in 3 generations of Japanese family had prolonged thrombin clotting time, but no history of hemorrhagic or thrombotic disease. Very low fibrinogen levels were obtained by thrombin clottable protein, while immunological procedures gave normal values of fibrinogen. The serum contained 40-80μg/ml of unclottable fibrinogen related antigens. The patients’ plasma had an inhibitory effect on the fibrin formation in normal plasma. Major defect of this fibrinogen was a delayed aggregation of fibrin monomers.

On CM-chromatography (CM-52) of the S-carboxymethylated fibrinogen, three different γ-chains, named γx, γL and γR, were separated. They did not differ in their electrophoretic mobilities in SDS-PAGE, but were distinguishable in PAGE containing 8M urea. Moreover, the amino acid compositions and tryptic peptide mappings of each chain revealed a little difference from those of normal fibrinogen γ chains, suggesting the difference in amino acid substitution or oligosaccharide chain structure.

Based on these findings, we designated this fibrinogen as fibrinogen Nagoya; its possible identity without other dysfibrinogenemia has not been excluded.
The Role of Mitochondria in the Regulation of the Cytosolic Ca2+ levels in Human Blood Platelets
Authors: Scharf R.E. , Lüscher E.F.
Published: May 2019

Mitochondria isolated by density gradient centrifugation from blood platelets show active Ca2+ transport. The required energy can be provided either by oxidation of succinate or by added ATP. The highest amounts of Ca2+ (30-32 nmoles Ca2+/mg protein) are taken up when both, succinate and ATP, are available. In the presence of selective inhibitors of mitochondrial metabolism such as antimycin A, oligomycin or with uncoupling agents only insignificant amounts of Ca2+ are accumulated. Strong inhibition is also exerted by ruthenium red and by Mg2+, whereas Pi acts as a stimulator of Ca2+ uptake. Basal mitochondrial respiratory activity is stimulated by Ca2+ (as well as by ADP).

This suggests that mitochondria, besides other specialized structures, play a role in removing Ca2+ from the cytosol of blood platelets. Activation of platelets is linked to an increase in respiratory activity; therefore, mitochondria not only may be involved in the regulation of Ca2+ levels in the resting state, but may also contribute to the reversibility of Ca2+ dependent alterations which accompany the activated state.
Effect of Heparin on Aggregation, Release Reaction and Thromboxane A2synthesis in Human Platelets
Authors: Chuang H.Y.K. , Mohammad S.F. , Mason R.G.
Published: May 2019

Studies on the effect of heparin on platelet functions have resulted in conflicting observations: heparin has been reported to cause aggregation of platelets, potentiate aggregation induced by various aggregating agents, or cause inhibition of aggregation. Using paritally purified heparin (beef lung or porcine mucosa) we observed that addition of heparin to citrated platelet rich plasma(C-PRP)potentiated the aggregation of platelets induced by ADP, epinephrine, or arachidonic acid. Presence of heparin in C-PRP results in complete inhibition of thrombin induced effects and partial inhibition of platelet aggregation induced by collagen. Presence of heparin in C-PRP also resulted in release of significantly higher concentrations of 14C-serotonin when platelets were challenged by appropriate aggregating agents. Those concentrations of heparin that resulted in potentiation of aggregation had no appreciable effect on c-AiMP or c-GMP levels of platelets. However, the presence of heparin results in a significant elevation of thromboxane A2 as determined by contraction of rabbit aorta or after conversion to thromboxane B2 by thin layer chromatography. These observations are of interest since increased production of thromboxane A2 in the presence of heparin may explain in part, the potentiation of platelet aggregation in vitro or thrombocytopenia observed frequently in patients receiving heparin intravenously Supported in part by grants HL22583 & 20679 from NHLBI of NIH.
Electron Microscopy of Metal-Shadowed Fibrinogen Molecules Deposited on Carbon Films at Different Concentrations
Authors: Mosesson Michael W. , Escaig Jacques , Feldmann Gérard
Published: May 2019

The hypothesis that fibrinogen (ϕ) molecules are elongated (47.5 nm) trinodular structures is based upon electron microscopy (EM) of metal-shadowed preparations in which collections of three discrete linearly aligned particles were commonly observed and assumed to represent an intact molecule. In our study, human ϕ at cone, ranging from 0.1 to 150 μg/ml in 0.1% NH4-acetate buffer. pH 9.3 ± 0.1, was deposited on carbon films, shadow with platinum, and then examined. At a ϕ cone, of 10 μg/ml or higher, most images showed considerable polydispersity, evidently due to overloading, that was characterized by multinodular shapes; discrete particulate forms were also commonly observed. At a cone, of ~ 1 μg/ml, the predominant form was a discrete particle with a mean diameter and height of 13.7 ± 1.4 nm and 5.4 ± 0.8 nm, respectively; only rarely were 3 such particles aligned in a manner consistent with a trinodular structure. At a ϕ cone, of - 0.1 μg/ml, the number of particles per unit of surface was considerably diminished, but virtually all such particles were well separated (> 20 nm) from one another. The particle volume corresponds to that of a single anhydrous ϕ molecule. These findings do not support the conclusion that ϕ molecules are elongated trinodular structures but suggest instead that the monodisperse form of ϕ is reflected by an individual particle and that multinodular forms represent collections of more than one ϕ molecule.
Immunochemical Studies on Fibrinogen
Authors: Gollwitzer R. , Tanswell P. , Lotter H. , Henschen A. , Timpl R.
Published: May 2019

The immunochemistry of fibrinogen has been subject of concise studies during the last years. Fibrinogen is a strong immunogen, based on a large variety of antigenic determinants located at different sites in the molecule. By using purified antibodies several of the surface and masked antigenic determinants on the individual chains and the large disulfide bonded CNBr-peptides were identified. According to these investigations the γ-chain is partially buried in the native molecule, whereas the antigenic determinants of a CNBr-peptide covering the C-terminal half of the α-chain are located on the surface of the protein. The structure of the antigenic determinants of this peptide which exhibits an extreme species variability has been more precisely defined in both bovine and human fibrinogen. A distinct structural variability was also observed for different antigenic determinants located in fi-brinopeptides A and B.

Supported by the Deutsche Forschungsgemeinschaft, SFB 51.
Vitamin K-Dependent Carboxylase: Purification and Properties
Authors: Suttie J. W.
Published: May 2019

The vitamin K-dependent plasma clotting factors are synthesized as inactive precursors and converted to active forms by the carboxylation of specific glutamyl residues to γ-carboxyglutamyl residues. A stable preparation (complex A) of the rat liver microsomal vitamin K-dependent carboxylase has been obtained by selectïve extraction of other proteins from the membrane. Treatment of crude microsomes with dilute nonionic detergent, chaotropic agents, and sodium cholate yields a preparation that is 150-fold purified over the starting microsomes and is substantially increased in total activity. This lipoprotein complex has a high requirement for vitamin KH2 and is not active with [vitamin K + NADH]. ‘Organic hydroperoxides will drive carboxylation in the absence of vitamin K in crude microsomes but not in complex A. In many other respects, complex A has properties similar to detergent-solubilized microsomes. The microsomal carboxylase culverts Phe-Leu-Glu-Glu-Leu predominately to two forms of Pho-Leu-Gla-Glu-Leu which can oe separated by ion exchange chromatography. The more acinic of these two forms can be metabolized to the more basic form by crude microsomes, but complex A has lost the ability to carry out this conversion. About half of the carboxylase activity of complex A can be solubilized in a high concentration of Triton X-100, and this material can be further purified by affinity chromatography.
Conformation of the γ-Carboxyglutamic Acid-Rich Region of Prothrombin: Conformation-Specific Antibodies as Structural Probes
Authors: Furie B. , Blanchard R.A. , Tai M. , Provost K. , Furie B.C.
Published: May 2019

Stabilization of the tertiary structure of bovine prothrombin (PT) by Ca(II) has been evaluated immunochemically using conformation-specific antibodies directed against limited regions of the protein. Antisera raised against native PT or des-γ-carboxyPT (AbPT) were fractionated by sequential immunabsorption using affinity chromatography. Three antibody subpopulations, specific for different determinants on the protein, were isolated and evaluated using RIA: (1) Anti-(12—44), directed against the Ola-rich region 12-44, requires Ca(II) or other metals to interact strongly with PT. Anti-(12-44) cross-reacts poorly with AbPT, factor IX, factor X, and human PT; (2) Anti-PT-Ca(II) is specific for the binary PT-Ca(II) complex and does not interact with PT. Anti-PT-Ca(II) is directed against multiple antigenic determinants in the fragment 1 region; (3) Anti-AbPT is specific for AbPT and does not cross-react with PT. We conclude that occupancy of the high affinity metal binding site by metal ions is associated with a significant change in the conformation of the 12-44 region and other aspects of the N-terminal portion of PT. These antigenic surfaces are not present in AbPT. Conversely, some determinants on AbPT are not observed on PT. These results are consistent with our current model of the role of Gla in metal-dependent intramolecular and intermolecular bridging in the vitarnin-K-dependent proteins.
Immunochemical Studies of Antisera to Human Fibrinopeptide B
Authors: Butler V.P. , Weber D.A. , Nossel H.L. , LaGamma K.S. , Canfield R.E.
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 inmuroreactive 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)-118(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 Bonos in Concentrated Urea
Authors: Albert A. , Cacho F. , Saiz J. L.
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 timeand 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).

Totalreduction of the isolated species H shows identical composition in fibrinogen chains as the original material. Stabilization by alkylation of this species 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.
Functional Properties of Two Different Molecular Forms of α2-Plasmin Inhibitor
Authors: Clemmeneon I. , Thorsen S. , Müllertz S.
Published: May 2019

The α2-plasmin inhibitor in plasma exists in at least two molecular forms (1). One form (PB) binds to plasminogen-Sepharose. The other form (NPB) comprising around 0.4 of the total amount of the inhibitor in plasma does not bind to plasminogen-Sepharose. PB was purified by affinity chromatography (1) while NPB was partially purified by conventional methods (2). Both inhibitor forms inhibited plasmin in a fast reaction and the change in catalytic concentration of plasmin with increasing concentrations of both forms was a linear function. At identical active site concentrations of the two forms (titrated with pNPGB-titrated plasmin) PB produced a more marked inhibition of urokinase-induced fibrinolysis. Similarily PB produced a more marked decrease of the binding af Lys-plasminogen to fibrin than NPB. The results indicate that an equilibrium between the 2 forms may be important for the biological regulation of the inhibition of plasmin.

1: I. Clemmensen, The Physiological Inhibitors of Coagulation and Fibrinolysis; D. Collen, B. Wiman, M. Verstraete, eds., p. 131-136, Amsterdam 1978.

2: U.Christansen & I. Clemmensen, Biochem. J., 175:635-641, 1978.
Red Cell Deformability and Risk Factors in one Hundred Patients with Cerebrovascular Thrombosis
Authors: Boisseau M. R. , Lorient M. F. , Bricaud H.
Published: May 2019

Erythrocyte deformability measured by Nucleopore filtration method was significantly (p 0.001) decreased in patients with CVA (filtration time (FT) = 57 ± 28 s. × ml-1) compared with two hundred heathly subjects (FT = 33 ± 6 s. × ml-1). Among the risk factors erythrocyte deformability mainly depended upon the gravity of Hypertension (FT = 77 ± 35 s. × ml-1; p 0.001), Additionnai 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.
A New Fluorogenic Substrate Method for Assay of Bacterial Endotoxins Using Limulus Hemocyte Lysate
Authors: Harada T. , Ohki M. , Niwa M. , Iwanaga S.
Published: May 2019

Limulus hemocyte lysate contains a proclotting enzyme., which is transformed to the active clotting enzyme in the presence of gram-negative bacterial endotoxins. The clotting enzyme coagulates a clottable protein, named coagulogen, contained also in the lysate. This gelation reaction of the lysate, named Limulus test, has been widely employed as a simple and very sensitive assay method for endotoxins. We developed a new fluorogenic substrate, Boc-Leu-Cly-Arg-4-methyLcoumarin amide, for Limulus clotting enzyme and established an enzymatic assay method for endotoxins, using the substrate. Because the endotoxin mediates the activation of proclotting enzyme in the lysate, the measurement of amidasc activity could be applicable for quantitation of the endotoxins. In fact, the amidase activity determined fluorometrically increased by increasing concentration of E. coli 0111: B4 endotoxin added to the lysate, and a linear relationship between the toxin concentration and the activity was observed in the range of 5×10-6 to 5×10-2 ug endotoxin. The method was a fifty times more sensitive than that of the Limulus test and was very reproducible. However, the method was not directly applicable for the assay of endotoxins in circulating blood, as the amidase activity was strongly inhibited by antithrombin III and α2-plasmin inhibitor. Thus, some pretreatment with heat or chloroform on plasma samples before the assay was required.
Potential Sources of Emboli in Transient Cererral and Retinal Ischaemia
Authors: de Bono D. , Warlow C. , Hyman N.
Published: May 2019

91 consecutive patients with transient ischaemic attacks (TIAs) were examined for potential source of emboli using echo cardiographyand ambulatory ECG monitoring in addition to clinical examination and angiography. Patients were divided into those with amaurosis fugax and/or carotid TIAs (45), “hemiphenomena” TIAs <26) and vertebrobasilar TIAs (20).

“Structural” cardiac defects (of which mitral valve prolapse accounted for just under half) occurred in 34% of patients, significantly more than in age-matched controls. 40% of patients with amaurosis fugax and/or carotid TIAs had carotid artery lesions, as did 30% of those with “hemiphenomena” attacks.6 patients had both cardiac and carotid lesions. Arrhythmias were detected in 15% of patients and a similar proportion of controls. Hypertension and ischaemic heart disease were more common in the patients.

Structural cardiac abnormalities, which may not be haemodynamically significant, might account for cerebral and retinal emboli in about a third of patients presenting with TIAs. The natural history and the most effective therapy of this subgroup require evaluation independently of patients with predominantly carotid atheromatous lesions.
High Fibrinogen (Fg) Content in Plasma Clots Harvested at Arrested, Incipient Coagulation
Authors: Godal H.C. , Brosstad F.
Published: May 2019

Inccrnplete transformation of Fq to fibrin may occur in vivo and in vitro, especially in connection with the use of heparin. We have studied to what extent unproteolyzed Fq 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 Eg to fibrin conversion, pre formed, enzyme- free des-AA or des-AABB fibrin monomers were added in varying amounts to Fg-boosted, inactivated serum containing varying Fq concentrations. (Fg + fibrin 2 g/1 in all samples). Labelled monomers or labelled Fg vero 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 lowdate 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=1/9 contained Fg/fibrin=1/20. Presence of preactivated FSF(activated by insolubilized thrombin) did not influence the Fg/fibrin ratio of clots. Streptokinase-induced intraclot lysis rate of clots containinq 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 trironboembolic 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-control 1ed calcium concentrations. At calcium concentrations of 2mM exclusively D fragments, M.W.= 93.000 (Dcate) were formed; in the presence of 10mM 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.
Determination of α1-Antitrypsin with a Chromogenic Peptide Substrate Methodology and Clinical Application
Authors: Witt I. , Lill H. , Karitzky D. , Flad H.
Published: May 2019

α1-Antitrypsin (α1-AT) is a relatively unspecific inhibitor of proteinases . The co-dominant hereditary defect of the protein can be associated with obstructive pulmonary emphysema in adulthood or with juvenile hepatocirrhosis. A rapid and reliable method for the determination of α1-AT activity with the chromogenic substrate Z-Val-Gly-Arg-pNA has been developed: a standardized trypsin solution of known activity is allowed to react with the plasma or serum sample and the residual trypsin activity determined after incubation. The difference between the initial and residual trypsin activities corresponds to the α1-AT activity. The assay is carried out in tris/HCl-buffer at pH 8.0 (ionic strength = 0.15) and 25°C; measurements are taken at 405 nm, Both kinetic and 2 point measurement is possible. The coefficient of variation for day-to-day precision is<5%.

The following guide values were determined on 15 healthy probands: 141 ± 15 inhibitor U/ml for plasma, and 130 ± 25 inhibitor U/ml for serum. The minute volume of plasma or serum used precludes non-specific reactions of the substrate with other proteases. Homozygous carriers of this protein defect can be accurately identified by means of this test.
Abnormal Ristocetin-Aggregation of Platelets in Uraemia and on Haemodialysis Therapy
Authors: Woods H.F. , Turney J. , Weston M.J.
Published: May 2019

Impaired platelet aggregation to ADP and collagen is recognised in uraemia and the loss of normal platelet function contributes to the prolonged capillary bleeding time observed in uraemia. In von Willebrand’s disease prolongation of capillary bleeding time is associated with impaired platelet aggregation to ristocetin because of a relative or absolute deficiency of the Factor VIII-Ristocetin Cofactor. We have studied platelet aggregation to ristocetin and Factor VIII coagulant activity (CA) and related antigen (RA) in 14 uraemic and 28 dialysed patients. Ristocetin aggregation (delta-0.D/min) was significantly less in uraemia (50 ± 21: m ± S.D) and in dialysis patients (38 ± 18) than in controls (78 + 20). Factor VIII CA and RA were significantly higher in uraemic and dialysed patients than in controls.

In crossover studies plasma from uraemic patients either enhanced or did not change ristocetin aggregation of normal platelets but plasma from dialysed patients impaired ristocetin aggregation of normal platelets. Plasma from dialysed patients was not contaminated by heparin. Thus in uraemia impaired platelet aggregation to ristocetin is unlikely to be due to a Factor VIII deficiency and may be due to a membrane defect as in Bernard-Soulier disease. In dialysed patients’ plasma there appears to be an additional circulating inhibitor of platelet-ristocetin interaction.
Changes Induced by Venous Stasis on Factor VIII Activities (VIII:C; VIIIR.AG; VIIIR:WF) in Different Clinical Conditions
Authors: Ponari O. , Pini M. , Manotti C. , Dettori A.G.
Published: May 2019

F.VIII related activities (coagulant-VIII:C, antigen-VIIIR:AG, v.Willebrand factor-VIIIR.WF) before and after venous occlusion (VO) were investigated in pregnant women, in women on the pill, and in men with atherosclerosis obliterans, “ean basal values of atherosclerotic patients were not different from healthy male’controls. A concordant rise in all the three activities was seen after VO in both groups without any change in the ratios between them. Basal values of women on long-term hormonal contraception were also comparable with those of a control group, while a group of third trimester pregnant women showed high values for all components, and very high ones for VIIIR:AG, with striking increases in AG/C and AG/WF ratios. Following VO, strictly concordant variations in the three activities were observed in all cases, so that ratios remained always unchanged, at their different levels.

The results are consistent with the hypothesis of a “complete” F.VIII released activity, induced by venous stasis in these conditions. Pregnancy has quite different effects on F.VIII in comparison with estro—progestinic treatment. No variations seem to be associate! with occlusive arterial disease.
Isolation and Partial Characterization of Equine Antithrombin III.
Authors: Estry D.W. , Bell T.G. , Tishkoff G.H. , Mattson J.C. , Estry S.C.
Published: May 2019

A protein analogous to human antithrombin III was isolated from fresh horse plasma. The procedure for purification was a modification of Thaler and Schmer’s two-step isolation procedure. The horse protein was homogeneous on 7.5% SDS-PAGE gels and had a molecular weight of 62,000 to 64,000 daltons in both reducing and non-reducing systems (human; 62,300). Rabbit anti-human antithrombin III was used to demonstrate a line of partial identity by Immunoelectrophoresis between the horse and human protein. The horse protein rapidly neutralizes human thrombin (34,000 daltons) and the reaction appears to be greatly potentiated by heparin. In order to establish the formation of 1:1 covalent stoichiometric complex between horse AT III and thrombin (IIa), time studies were run in the presence and absence of heparin. AT III (62,000) at 15 seconds, 2, 5, 10 and 60 minutes formed a stable complex with thrombin (32,000) having a molecular weight of 86,000 daltons. Additional bands developing with time are due to the autolytic capabilities of the uncomplexed IIa. The major autolytic band had a molecular weight of 70,000 daltons. Addition of heparin potentiated the interaction although it did not change the stoichio-metry of the complexes formed. The data accumulated to date demonstrates the similarities between the human and horse protein and the possibilities of using the horse as a model system for the evaluation of AT III replacement therapy in vivo.
Heparin and Platelet During Haemoperfusion of Patients with Acute Liver Disease
Authors: Hughes R.D. , Lane D.A. , Cella G. , Clark S.E. , Kakkar V.V. , Ton H.Y. , Langley P.G. , Williams Roger
Published: May 2019

A study has been made of the factors involved in the haemostatic problems associated with the haemoperfusion of patients with acute liver failure to remove toxic metabolites. Five hsemoperfusions were performed using polymer coated charcoal columns and five perfusions with an albumin coated resin column. The following haemostatic parameters were measured prior to and during a 4 hour period of haemoperfusion; plasma heparin levels, β-TG, PF4, platelet aggregation, platelet count and Swank screen filtration pressure. In eight of the ten patients haemoperfusion was clinically well tolerated with no significant change in blood pressure, screen filtration pressure, or haemostatic parameters, though in one patient, subclinical changes in βTG (840 ng/ml) and PF4 (>500ng/ml) were observed. Two other patients became severely hypotensive requiring discontinuation of haemoperfusion. In one of these a high screen filtration pressure (205 mm Hg at 10s) and high βTG (860 ng/ml) were measured with a heparin level exceeding 2 u/ml. In the other a rapid consumption of heparin was observed without a significant rise in screen filtration pressure or βTG. It is suggested that haemoperfusion in acute liver failure in most cases is free from adverse platelet reactions but complications can arise relating to an increased consumption of heparin and/or platelet aggregation.
Platelet and Fibrinogen Kinetics in Patients with Aorto-Femoral Dacron Grafts: Evaluation of Period of Maximum Risk from Thrombosis
Authors: Kester R.C. , Rajah S.M. , McCollum C.N. , Learoyd P. , Pepper M.
Published: May 2019

The thrombogenicity of Dacron arterial grafts may lead to eventual closure, although Dacron aorto-femoral grafts rarely thrombose because of wide calibre and rapid blood flow. The less favourable outcome of Dacron grafts with narrow diameter or suboptimal flow may be improved by antithrombotic therapy during the “thrombogenic period” of graft maturation. To evaluate this period, platelet and fibrinogen kinetics using, Chromium 51 and Iodine 125 respectively, were measured pre-operation and at 3, 6 and 9 months, in 10 patients after aorto-femoral Dacron bypass. Six age-matched volunteers were simultaneously studied. Platelet survival time was reduced 8.8 to 7.4 days (p<0.01) and platelet turnover increased at 3 months post-operation compared with pre-operative levels 39 to <47/103/days. Similarly, fibrinogen T½ life was decreased 3.7 to 3.4 days and fractional catabolic rate increased at 3 months 0.27 to 0.34 (p< 0.01). These indices of thrombogenic activity returned to pre-operative levels by 9 months. We suggest that Dacron aorto-femoral grafts remain thrombogenically active for about 9 months. Where blood flow conditions are suboptimal or graft diameters are small, it may be prudent to use antithrombotic therpy to protect patency.