bluuebunny/crossref_metadata_2025 huggingface dataset:
Binding of Thrombin to Fibrin
Authors: Róka L. , Rademacher F. G.
Published: May 2019

After clot formation using bovine thrombin and bovine fibrinogen, one can detect no longer the total amount of thrombin. The amount missing in the supernatant is trapped in the clot and can be removed in active state after lysing the clot. The capacity for trapping thrombin by a constant amount of fibrin depends on the structure of the fibrinpolymer. This can be shown by producing different kinds of clots by variation of pH or monomer concentration during polymerisation or using Arvinmonomer insted of Thrombinmonomer. The trapped thrombin shows no clotting activity but some activity with chromogenic substrats remains. to neutralize trapped thrombin about 5 times more antithrombin III is necessary than for the same amount of free thrombin. The trapped thrombin diffuses out of the clot indicating the reversibility of the binding of thrombin to fribin.
Evidence of Abnormal Factor VIII Related Protein in Diabetes Mellitus
Authors: Cacciola E. , Giustolisi R. , Lombardo T. , Musso R. , Russo M. , Sortino G.
Published: May 2019

Biochemical and functional properties of Factor VIII related protein (VIII:RP) have been studied in the plasma from two groups of diabetic patients with or without vascular complications compared to normal subjects.

The Factor VIII related antigen (VIIIR:AG) was performed by Laurell’s electroimmunoassay (LA) and electrophoretic mobility (EM) by two dimensional crossed immunoelectrophoresis (2 DCIE). Biophysical properties were evaluated by cryoprecipitation and concanavalin A (Con A) treatment at different concentrations. Biological functions were determined using the ristocetin cofactor (RiCoF) assay on fixed-washed platelets and procoagulant activity (FVIII : C).

In diabetic patients, particularly in the ones at higher thrombogenic risk, the evaluation of VI I I :C/VIII R:AG and VIIl:vWF/VI I I R:AG ratios showed a significant(P<0.00Decreased VIII R:AG with a higher co-ristocetin activity than the procoagulant one. Furthermore, biophysical and bio chemical modifications of diabetic Factor VII IR:AG, mainly a lower EM on 2 DC I E, were found. These results suggest that the Factor VI I IR:AG abnormality, detectable in diabetic plasma, can be related with an abnormal glycoprotein biosynthesis.
Changes Induced Bt 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. Mean 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 VIII R: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.
Reversible Factor VIII-Abnormalities in Reye’s Syndrome
Authors: Joist J. H. , Vargo J. , Haymond M. W. , Keating J. P. , DeVivo D. C.
Published: May 2019

Reye’s syndrome, an acute and frequently fatal viral infection with severe metabolic disturbances is frequently associated with marked hemostatic alterations. Hypopro-thrombinemia and deficiencies of other clotting factor activities as well as findings suggestive of disseminated intravascular coagulation have been reported. This report deals with detailed sequential qualitative and quantitative studies of blood coagulation factors in four children with this disorder. Depression of factors IX, X, VII, V and II and marked disproportionate elevation of VIIIAHF, VIIIAGN and VIIIvWF associated with a loss of the slow moving component of VIIIAGU on crossed immunoelectro phoresis were consistently observed during the initial phase of the illness. in three of the four children who survived, the changes were fully reversible. Since laboratory evidence for DIC was lacking (normal fibrinogen, consistently negative protamine sulfate precipitation test) the findings seem to indicate that Reye’s syndrome is associated with an acute, transient stimulation of synthesis of normal and abnormal factor VIII as well as decreased synthesis or synthesis of functionally abnormal clotting factors by the liver.
Factor VIII and Ristocetin-Induced Aggregation in Sickle Cell Disease
Authors: Mackie I. J. , Bull H. , Walford D. , Brozovic M.
Published: May 2019

Haemostatic variables were studied in 23 patients with sickle cell disease.(SCD) who were followed over a year. Investigations included measurements of factor VIIIc, VIIIAg, antithroabin III biological and antigen levels, fibrinogen and platelet aggregation to ADP, collagen, porcine AHG and ristocetin. All eight SS patients in the steady state had normal levels of F VIIIc and high levels of F VIIIAg with narked discordance between the two values (mean VIIIc:VIIIAg ratio 0.3). By contrast, 15 patients with SC disease or S thalassaemia tended to show less narked discordance between levels of F VIIIc and F VIIIAg(mean VIIIc:VIIIAg0-7). Patients in the steady state showed normal platelet aggregation to ADP, collagen and porcine AHG, but ristocetin induced aggregation was absent or markedly reduced. Platelet aggregation to ristocetin is absent in most individuals of Afro-Carribean origin and the ristocetin co-factor activity nay be difficult to assess in this ethnic group.

During crisis both factor VIIIc and factor VIIIAg invariably high. Factor VIIIAg was studied using crossed isnunoelectrophoresis in 5 patients. in one patient two peaks were found and in the others the F VIIIAg had more anodal nobility than normal. These findings are suggestive of the presence of lower molecular weight fragments of factor VIIIAg and may account for the apparent very high levels as measured by Laurell rocket technique.
Antithrombins in Childhood Malnutrition
Authors: Jiménez R. A. , Ingram G. I. C. , Vargas W.
Published: May 2019

Antithrombin activities in 30 severely malnourished children and 40 normal children were estimated in clotting tests by thrombin neutralization, as antiXa and by a heparin antithrombin assay; and by immunodiffusion as α2-globulin and α1-antitrypsin. The patients’ mean α2-globulin was severely depressed, and there were less marked depletions in mean values for thrombin neutralization, antiXa and in the heparin antithrombin assay (which showed the flat curve thought to reflect a thrombotic tendency). The α1-antitrypsin values were normal. The findings support the concept of antithrombin as the summation of α2-globulin and α1-antitrypsin (with α2-macroglobulin); and may be related to the high incidence of thrombosis reported in childhood malnutrition
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 stoichiometry 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.
Storage Pool Deficiency in Neonatal Platelets
Authors: Stuart Marie J.
Published: May 2019

It is recognized that newborns exhibit a transient thrombocytopathy. Some investigators have described this as an “aspirin-like” defect, while others have ascribed it to a “storage-pool” abnormality. In an attempt to resolve this discrepancy platelets from 10 normal newborns, 10 normal adults and 10 aspirin treated adults were studied. None of the mothers of the newborn infants had ingested drugs known to interfere with platelet function. Platelet aggregations with ADP (3uM), epinephrine (5uM) and collagen demonstrated abnormal 2nd phase aggregation in both the newborns and in the aspirin treated adults. When mixtures of equal volumes of platelets from newborn and aspirin treated controls were tested, normal, irreversible aggregation was observed. Mutual correction was not observed on preincubation of newborn platelets with aspirin prior to mixing. When platelet malonaldehyde production was measured as an index of prostaglandin synthesis, in the presence of thrombin or NEM, no statistically different differences were observed between normal adults and newborn platelets. Malonaldehyde production was markedly reduced in the aspirin treated controls. Storage-pool deficient platelets, when combined with aspirinized platelets, prove mutually corrective in aggregation studies since storage pool deficient platelets overcome the abnormality in prostaglandin synthesis of the aspirin treated cells. These latter cells in turn provide the necessary storage-pool nucleotides to cause mutual correction and irreversible aggregation of the mixture. Our findings indicate that newborn platelets have a “storage-pool” defect.
Radio Immunoassay of Thromboxane B2
Authors: Ferraris V. , Smith J. B. , Silver M. J.
Published: May 2019

Thromboxane B2 (TxB2) is one of the major end products of the cyclooxygenation of arachidonic acid (AA) in blood platelets, lung and certain other tissues. The immediate precursor of TxB2 is the unstable thromboxane A2 which has been reported to cause platelet aggregation and to constrict coronary arteries as well as the respiratory tree. We report here on the development of a novel radioimmunoassay for TxB2 in human plasma. Radioactive TxB2 was prepared by incubating 3H-AA3 (sp. act. = 64 Ci/mM) with a suspension of washed rabbit platelets for 10 min. at 37°C. The 3H-TxB2 in the lipid extract was subsequently purified by silicic acid column chromatography. Antibodies to TxB2 were generated by immunizing rabbits with TxB2 which had been coupled to albumin using 1,1’ – carbonyldimidazole. The binding of 3H-TxB2 to diluted plasma (final dilution – 1 /1500) from immunized rabbits was inhibited by authentic TxB2 (Nelson et al, Tetrahedron Letters No. 37, 1976) but not by prostaglandins. Thus, 1.5 picomoles of cold TxB2 caused 50% inhibition of the binding of 3H-TxB2 while no cross reactivity was observed with PGD2, PGE2 or PGF2α. Small amounts of TxB2 (10–50nM) were found in normal human plasma but large amounts (~500 nM) were found in sera obtained from normal human blood clotted at 37°C. The assay should be useful for determining plasma levels of TxB2 in various disease states. The work supported in part by Grant HL-14890 from the NIH. V.F. was funded by the U. S. Army (AR601–112).
Chromogenic Peptide Substrates for Kallikreins and Urokinase
Authors: Claeson G. , Gustavsson S. , Aurell L. , Karlsson G. , Friberger P.
Published: May 2019

A chromogenic substrate for plasma kallikrein has been modelled after the amino acid sequence preceding one of the scissile bonds of kininogen, that is, the C-terminal part of bradykinin. Similar attempts to use the natural substrates as models for designing synthetic substrates for glandular kallikrein and urokinase have not been successful. However, by means of trial and error (variations of the amino acids in positions P1, P2 and P3) we have obtained correlations between substrate structure and activity. These correlations were then used to construct chromogenic peptide substrates for glandular kallikrein and urokinase.

The new substrates have been utilized in methods for the determination of prekallikrein in plasma, kallikrein in urine and urokinase in urine respectively.
Differentiation and Stabilization of AHF Separated from vWF by Polyelectrolytes
Authors: Macdonald V. E. , Semar M. , Johnson A. J. , Brind J.
Published: May 2019

Low-charge density, solid-phase ethylene maleic anhydride polyelectrolytes (PES) have been used to quantitatively separate factor VIII-antihemophilic factor (AHF) from factor-VIII von Willebrand factor (vWF). Under these circumstances, the AHF is adsorbed on the resin while the vWF remains in the supernatant plasma. Addition of large amounts of proteolytic inhibitors does not influence the separation. The vWF can be purified further by subsequent adsorption on high-charge density PEs. the PE-adsorbed AHF and vWF show marked differences in elution patterns and stability. The AHF is eluted in high salt while the vWF is eluted at lower salt concentration. Addition of certain amino acids to the eluting buffers results in extremely stable AHF preparations, whereas the same amino acids have the opposite effect on the vWF eluates. further purification by selective PEG precipitation results in 100-fold purification of both materials. These intermediate-purity concentrates, prior to gel chromatography, were found to contain complement components not previously reported. comparison of several commercial factor VIII preparations demonstrates that this is a common contaminant in all preparations examined thus far. The differences in properties of the separated AHF and vWF seen during fractionation and purification by these methods strongly suggest that they are two independent molecules.
Platelet Function and Blood Loss after Cardiopulmonary Bypass
Authors: Rosario M. D. , Leinberger H. , Suehiro A. , Zeitlin S. , Moore G. , Barker B. , Mamiya R. T. , McNamara J. J.
Published: May 2019

The relationship between platelet function and bleeding tendency was studied in 42 patients undergoing open heart surgery. Template bleeding time (TBT) and hourly chest tube outflow were measured as indices of bleeding tendency. Platelet function tests included platelet count (PC), aggregation with epinephrine, ADP, and collagen, and a modified platelet slide adhesion test (PSAT) which allowed us to quantitate in vivo the relative amount of more adhesive platelets, i. e., the “spread” forms, as compared to the number of smaller non-adhesive platelets. Post operatively the PC decreased 36% (from 214.1 ± 11.4 K to 136.3 ± 6.8 K, p<0.001) in all patients irrespective of the amount of blood loss. Using the PSAT, the product of the % “spread” forms and the PC showed a significant 57% decrease (from 110.5 ± 7.4 K to 47.4 ± 4.6 K, p<0.001) which was 19% more than the PC decrease implying that the more adhesive platelets had been expended. TBT increased significantly (7.7 ± 0.5 min. to 17.7 ± 1.3 min., p<0.001) and aggregation to epinephrine decreased significantly (60.9 ± 4.8% to 36.0 ± 3.4%, p<0.001), but no significant change was noted with ADP or collagen. There was no significant difference in the above parameters between patients bleeding more than 113 cc/hr. from 1 to 5 hrs. post op (N=14) and those bleeding less (N=28). A second study of 16 patients with bleeders having >1000 cc output in the first 4 hrs. post op showed results similar to the first group comparing pre vs. post and bleeders (N=8) vs. non-bleeders. This study indicates that after cardiopulmonary bypass surgery platelet function is frequently compromised and this dysfunction though probably contributing to a bleeding tendency did not correlate well with the magnitude of blood loss.
Further Evidence of the Involvement of Thrombin in Platelet Release by ADP and Other Agents
Authors: Ardlie N. G. , Akbar Huzoor-
Published: May 2019

There is evidence of the involvement of coagulation factors in platelet aggregation and the release reaction caused by ADP and collagen but this has been challenged. This report concerns further experiments which can explain the apparently conflicting observations of various laboratories and which provide additional evidence of the involvement of coagulation factors and thrombin in the platelet release reaction caused by ADP and other agents.

Washed platelets suspended in a buffer solution responded poorly to ADP with no second phase aggregation or release of [3H]5HT. In contrast, washed platelets suspended in dialysed plasma underwent second phase aggregation and released radioactivity. This response depended on calcium. Dialysed plasma deficient in factors XI or X did not restore second phase aggregation or the release reaction. Hirudin and heparin inhibited second phase aggregation and release by ADP and epinephrine. However, the inhibitory effects of heparin and hirudin on ADP,epinephrine and collagen were not observed when citrate was present. To explore the possibility that a reduction of the free calcium concentration accounts for the inhibition of action of these antithrombin agents by citrate experiments with EGTA were carried out. This alternative chelating agent also prevented the inhibitory actions of heparin and hirudin.

These observations support the view that platelets and clotting function cooperatively in platelet reactions involved in haemostasis. We suggest that small amounts of thrombin formed prior to fibrin clotting mediate platelet reactions in haemostasis initiated by collagen and ADP and that secondary aggregation does not represent a citrate artifact.
The Role of Fibrinogen in the Interaction of Platelets with Particles
Authors: Pfueller Sharron L. , Firkin B. G.
Published: May 2019

The initial interaction of polystyrene latex particles with platelets has been studied by means of their agglutination with platelets measured nephelometrically .2 to 10 min. after addition of latex particles to citrated PRP at 37°C slow agglutination occurs. When latex is preincubated with PPP for 5 to 40 mins at 37°C, sedimented and resuspended in buffer it is “activated” and produces an immediate and rapid agglutination when added to PRP. This “activation” like activation of zymosan particles (Z) occurs in plasma deficient in Factor VIII and/or von Willebrand factor, is not prevented by hirudin, and does not occur in plasma heated at 56°C for 10 min or in recalcified PPP; addition of fibrinogen restores activity to these treated plasmas. Unlike Z activation, it is not inhibited by heparin, EDTA, or destruction of Complement. Activation can be mimicked by preincubation with fibrinogen alone, but not with IgG or albumin. In PRP, in contrast to washed platelets, latex preincubated with IgG caused less agglutination than when pretreated with fibrinogen or buffer. Agglutination by activated latex in PRP, unlike aggregation by activated Z and other aggregating agents, causes only slight 14C-serotonin release and is not inhibited by aspirin, indomethacin or adenosine,but, like the reaction with Z, is inhibited by EDTA, chlorpromazine, cocaine-HCl, N-ethylmaleimide and iodoacetate. Platelets from patients with thrombasthenia show a defective response to latex activated with either normal PPP or fibrinogen. Particles of inulin which activate C in the same way as Z, but which absorb much less fibrinogen, cause no response of either normal PRP or washed platelets. Thus normal platelets, but not those from thrombasthenics, appears to possess a receptor for fibrinogen which is important in their interaction with particulate material.
Effects of Modification of the Platelet Surface on Receptors for Aggregated IgG and Ristocetin-Von Willebrand Factor
Authors: Pfueller S. L. , Jenkins C. S. P. , Lüscher E. F.
Published: May 2019

Modification of the platelet surface was used to study the receptors for ristocetin-von Willebrand factor and aggregated IgG (an Fc receptor). Expression of these receptors was measured by agglutination of platelets to ristocetin in the presence of von Willebrand factor and by binding of aggregated IgG coupled to [3H] labelled diazobenzene. Under conditions where the release reaction was inhibited, treatment of platelets with chymotrypsin, trypsin, pronase or papain caused loss of the expression of the receptor for ristocetin-von Willebrand factor and an enhancement of that for aggregated IgG. Membrane alterations produced by ADP or ionophore A23187 also abolished agglutination to ristocetin-von Willebrand factor but did not affect the receptor for aggregated IgG. Treatment of formaldehyde-fixed platelets with trypsin, pronase or papain destroyed their ability to agglutinate to ristocetin-von Willebrand factor but did not alter binding of aggregated IgG. Therefore the disappearance of receptor activity for ristocetin-von Willebrand factor following proteolysis appears due to removal of protein and glycoprotein rather than to alterations in membrane configuration. The Fc receptor is not sensitive to proteolysis but is partially masked by protease-sensitive material.
Isolation of Specific Homologous Antibodies to Factor VIII by Immunoadsorption
Authors: Lavergne J.-M. , Meyer D. , Koutts J. , Ardaillou N. , Girma J. P. , Larrieu M. J.
Published: May 2019

Current immunological studies of Factor VIII use heterologous antibodies which predominantly measure Willebrand Factor (WF) and give little information on Factor VIII procoagulant activity (VIII:C). Purification of homologous antibodies specific for VIII:C has been hampered by the fact that they do not form immune precipitates. We have attempted to isolate such antibodies by solid phase immunoadsorption and subsequent elution. Human Factor VIII was specifically bound to goat anti-human Factor VIII IgG previously immobilized onto Sepharose 6 B beads. IgG isolated from a Haemophiliac with a high titer anti-VIII:C antibody (700 Oxford U/ml) was labeled with 125-I and reacted for 72 hours with these beads. The column was then washed with 0.1 M glycine, 0.5 M NaCl, pH 10 buffer to remove non specifically adsorbed material. Specifically adsorbed material was then eluted with 2.5 M MgCl2, pH 7.3, and the peak of radioactivity was filtered on Biogel A-5 m. Both anti-VIII:C and radioactivity were recovered in three distinct peaks. The third peak, corresponding to IgG, contained anti-VIII:C activity with a 10 fold purification as estimated by specific radioactivity. The second peak, eluting just in front of the IgG, had half the specific radioactivity of the third peak. The first peak, corresponding to the Vo, contained Factor VIII related antigen and very little anti-VIII:C activity. The pattern by SDS-polyacrylamide gel electrophoresis is compatible with the existence of Factor VIII (WF-VIII:C)-anti-VIII:C complexes in the first peak; VIII:C-anti-VIII:C complexes in the second, and free anti-VIII:C IgG in the third one. Thus the method leads to the formation of stable VIII:C-anti-VIII:C complexes, allowing the purification of specific human anti-VIII:C antibodies.
Hereditary Antithrombin III Deficiency. Evidence of Increased Thrombin Formation in the Early Stage of Hemostasis
Authors: Josso F. , Aurousseau M. H. , Fischer A. M. , Dautzenberg M. D. , Beguin S.
Published: May 2019

High incidence of thromboembolism in observed in families with antithrombin III deficiency, despite the rather moderate defect (25 to 50 per cent) observed in the affected subjects, considered as heterozygous for the disease. The mechanism by which such a mild deficiency may promote thrombosis still remains unknown.

A new family with antirhrombin III deficiency is reported, including three affected generations. Three young male adults of a same sibship died from massive pulmonary embolism aged from 22 to 28 years. In four living members of the family, three of which had antecedents of thrombophlebitis, antithrombin III level was close to 50 per cent by all assay methods used. Electro-phoretic and immunochemical studies failed to demonstrate any qualitative defect of the antithrombin III molecule.

In these patients the only abnormalities of hemostasis lied in the early stage of the hemostatic pathway. In vitro, thrombin formation occured very rapidly after initiation of coagulation, as demonstrated by the kinetics of thrombin generation and factors V and VIII activation. In vivo, factors V and VIII were activated during bleeding much more rapidly than in controls and this activation was not suppressed by low doses of heparin (10 u./kg) as in the controls.

These findings indicate that antithrombin III acts chiefly in the regulation balance of the early stages of hemostasis and thrombosis.
Attempts to Define a Platelet ADP Receptor with 203Hg-p-Mercuribenzene Sulphonate (MBS)
Authors: Mills D. C. B. , Macfarlane D. E.
Published: May 2019

ADP inhibits stimulation of platelet adenylate cyclase through a receptor with specificity and affinity indistinguishable from those of the ADP receptor mediating aggregation. The former, but not the latter effect is blocked by MBS (0.3–0.6 μM in PRP) indicating involvement of an externally oriented thiol. To remove effects of plasma thiols, aspirin treated platelets, labelled with 14C-adenine, were washed successively by centrifugation into albumin and passage through sepharose 2B into Ca-free Tyrode’s solution; this preserved their responses to PGE1 and ADP. Platelets incubated with 203Hg-MBS were pelleted through a layer of radiographic contrast medium. MBS binding showed a saturable high affinity component (ca. 250,000 sites/cell: 1/2 saturated at 8–10 μM). Excess cysteine reduced but did not eliminate non-saturable binding. Treatment of the platelets with Ellmans reagent (DTNB) reduced the apparent number of high affinity sites to ca. 100,000 and increased the apparent affinity of binding to ca. 2.5 μM. Effects of MBS on adenyl cyclase, monitored in the same experiment, correlated closely with high affinity binding. SDS electrophoresis of unreduced platelets on gradient Polyacrylamide gel slabs showed preferential labelling of several proteins by MBS, but the results suggested some redistribution of the label during extraction and separation. The number of high affinity sites for MBS found indicates the maximum number of thiols involved in ADP effects on adenylate cyclase. The true number is probably much smaller as MBS will label all externally available thiols.
Increased Heparin Clearance in Experimental Pulmonary Embolism
Authors: van Aken W. G. , Chiu H. , Hirsh J. , Regoeczi E. , Horner A. A.
Published: May 2019

Patients with pulmonary embolism (PE) have been reported to have a shortened heparin half-life (t½). Heparin clearance was investigated in rabbits to explore the possible contribution of platelets in increased heparin clearance in acute RE. Heparin clearance was measured by a protamine titration (heparin activity) assay and by 35S radioactivity. Both assay methods gave comparable results and showed a shortened heparin t½ in PE. Heparin t½ was significantly shorter in PE (12.0 min) than in venous thrombosis (VT) (18.2 min) and in control rabbits (20.0 min) when a dose of heparin 50 U/kg was injected. Heparin t½ was not reduced in animals with PE that were treated with heparin 200 U/kg and in animals that were embolized with polyether urethane coated-glass pellets. No significant absorbance of 35S heparin to the surface of PE or VT was found. The heparin t½ was shortened to the same extent in severely thrombocytopenic animals (platelet count <30,000/mm3) with PE as in non-thrombocytopenic animals with PE (12.8 min compared to 12.0 min) and was significantly shorter than in thrombocytopenic, VT and control animals (p<0.005). In addition, platelet survival was uninfluenced by PE and there was no increase in antiheparin activity in plasma after PE. It is concluded that the mechanism for the shortened heparin t½ is not platelet mediated, that it is influenced by the nature of thromboembolic material and that this effect on heparin t½ appears to be dose dependent.
Reduced Antigenic Reactivity in “Variants” of von Willebrand’s Disease
Authors: Ardaillou N. , Girma J. P. , Shoa’i I. , Lavergne J. -M. , Meyer D. , Larrieu M. J.
Published: May 2019

Factor VIII related antigen (VIIIR:AG) was measured in 49 cases of von Willebrand’s disease (vWd) by an immunoradiometric assay (coated tube system), using purified IgG prepared from antisera raised in both a rabbit and a goat. The control dose-response curve showed a linear relationship between bound radioactivity and log plasma dilutions between 1:25,000 and 1:400. At higher concentrations of plasma (i. e. lower dilutions), the percentage of bound radioactivity reached a plateau. In severe cases of vWd, no measurable VIIIR:AG (<1.10-4 u/ml) was detected. Most of the other patients had a normal response pattern with reduced levels of antigen. This contrasted with 13 patients who showed an abnormal dose-response curve. In all 13 cases, the maximal percentage of bound radioactivity (plateau) was lower than in the normal. These patients had a 20–30% absolute reduction of antibody binding capacity. This phenomenon was obtained with both rabbit and goat antibody. In addition, 4 of these patients (from 2 families) showed a lack of parallelism to the normal in the linear portion of their dose-response curve. This lack of parallelism only occurred with the rabbit and not with the goat antibody. All of these 13 patients fulfilled the criteria for “variants” of vWd, i. e. abnormal electrophoretic mobility and a disproportionately reduced Willebrand Factor activity in comparison to VIIIR:AG. These abnormalities of the immunoradiometric response are further evidence that an abnormal protein with reduced antigenic reactivity is present in “variants” of von Willebrand’s disease.
Soluble Fibrin Monomer Complexes in Patients with Renal Cell Carcinoma
Authors: Andrassy K. , Gärtner R. , Riedasch G. , Zimmermann R. , Ritz E. , Hafter R.
Published: May 2019

Patients with renal cell carcinoma (r. c.) are known to exhibit retention of bromsulphalein, increase in alkaline serum phosphatase (a. p.) and diminution of prothrombin time (Stauffer-syndrome). These abnormalities were thought to reflect impaired liver function. However, the increase in a. p. was shown to be due to appearance of Regan’s isoenzyme. The present study was designed to test the hypothesis that coagulation abnormalities in the patients result from low grade (intratumoral) intravascular coagulation. 26 patients with r. c. with/without metastases were studied preoperatively: Assay of coagulation tests: soluble fibrin monomer complexes (SFMC) by etha-nol gelation and by precipitation with β-alanin and subsequent agarose chromatography according to Hafter and Graeff. – Thrombin coagulase time (t. c.) was abnormal in virtually all patients (23/26); in addition reptilase time (r. t.) (15/26), FDP as meassured by TRCHII (8/26) and staph, clump-test (4/26) and normotest (6/26) were abnormal. T. c. and r. t. were particularly abnormal in 12/26 patients in whom increased cone, of SFMC were found. It is concluded that low grade intravascular ‘coagulation, possibly within the vascular bed of the highly vascular tumor, and to a lesser extent fibrinolysis accounted for the coagulation abnormalities in (non)-metastasing renal cell carcinoma.
An Investigation of Thrombasthenic Platelets with Selected Lectins
Authors: Firkin B.G. , Stott L.
Published: May 2019

Recent studies have identified a glycoprotein abnormality in platelet membrane preparations from patients with thrombasthenia. Whole blood was collected into iso-osmolar buffered formalin (0.1%) – the platelet rich plasma separated and stored at 4°C for 24 hours to ensure fixation with abolition of the release reaction as a possible factor in lectin aggregation. The platelets were washed 3 times and resuspended in Tris saline buffer pH 7.4. The cells were reacted with the lectins E-PHA, Ricinis communis 1 (RC-1) and Con A at varying concentrations in a Born aggregometer. Normal formalinised platelets (NFP) were strongly agglutinated by E-PHA and RC-1 at concentrations of 50 and 15 μg/ml respectively. Con A at concentrations of 125-250 μg/ml also gave agglutination of NFP, but only to 30-40% of that with the other two lectins. Formalinised platelets prepared from 4 thrombasthenics all reacted normally with E-PHA and RC-1 but were agglutinated minimally by Con A. Con A was labelled with II125 and binding studies undertaken. A marked reduction of Con A I125 uptake was observed with thrombasthenics, such that at saturation the binding sites of the lectin were approximately 25% that of NFP. Since trypsin digestion of platelet membranes results in an overall reduction of platelet glycoprotein, NFP were treated with trypsin. NFP treated in this fashion showed enhanced agglutination with E-PHA and RC-1 and no discernable difference with Con A agglutination and binding of Con A I125 was only slightly reduced. Con A may identify an important structural site in the core of the glycoprotein involved in platelet aggregation.
Clinical and Experimental Studies on the Bleeding Tendency During Defibrinogenation Therapy
Authors: Ishimaru S. , Furukawa K. , Takahashi M. , Fujimaki M. , Fukutake K.
Published: May 2019

Defibrinogenation with thrombin-like enzyme is expected to be a new type of anticoagulant therapy without serious bleeding complications. However, it appears to be dangerous to perform surgical procedure in defibrinogenated state. In order to investigate the causes of the bleeding tendency which occurs during defibrinogenation therapy, the following clinical and experimental studies were undertaken. Ten mongrel dogs were used, the superior caval vein was replaced by expanded polytetrafluoroethylene graft. 50 to 100 μl/kg of batroxobin was administered 2 days prior to the operation. 5 out of 10 dogs died due to bleeding postoperativelly, and the causes of the bleeding seemed to be the decreased fibrinogen concentration and the reduced platelet function. Fifteen patients suffering from peripheral vascular thrombosis were treated with 30 to 60 μl/kg of batroxobin. In 9 patients, urokinase was administered combined with batroxobin. No bleeding complications were observed in these patients. In another 6 patients, surgical procedure was performed after batroxobin administration. Postoperative bleeding was observed in one out of these 6 patients. This patient was operated on after the initial administration of batroxobin. FDP level was 512 μg/ml and ADP induced platelet aggregation was within normal limits, but fibrinogen concentration was unmeasurable at the time of operation. The cause of the bleeding seemed to be the decreased fibrinogen concentration. From these clinical experiences, it is suggested that platelet adhesion to glass and ADP induced platelet aggregation are not influenced by the decreased fibrinogen concentration nor the increased level of FDP during the defibrinogenation therapy with batroxobin. These results indicate that an adequate level of fibrinogen is needed for certain hemostasis besides normal platelet function.
Platelet Microtubule Subunit Proteins
Authors: Crawford N.
Published: May 2019

Microtubule subunit proteins have been isolated and purified from pig platelet homogenates and reassembled in vitro to form structures similar to those seen in the intact platelet. The major subunit protein tubulin has many of the properties of neurotubulin; it is a 6S dimer protein which binds colchicine and can be split into two non-identical monomers [α and β] in discontinuous alkaline SDS Polyacrylamide gel electrophoresis. Present also in the preparation are two/three high molecular weight proteins [8–12% of the total protein] which are similar to those seen in brain tubulin preparations and appear to be essential components in the reassembly process. An antibody has been raised to platelet tubulin and used for the fluorescent visualisation of cell microtubules. Their morphological distribution has been contrasted with that of structures stained with anti-actin antisera and the effect of colchicine, cytochalasin B and low temperatures on the specificity of these antibodies has been investigated. Some views will be presented on the equilibrium which exists in platelets between microtubular subunit proteins and the fully formed microtubules. Because of the possible cytoskeletal importance of the microtubules in shape change phenomena and their role in controlling membrane properties, factors which directionally influence this equilibrium may be important sites of action for antithrombotic drugs.
Studies on the Polymerization Sites in Fibrinogen
Authors: York J.L. , Blombäck B. , Hessel B.
Published: May 2019

A microcolumn assay has been developed for the quantitation of the affinity of various soluble fragments of fibrin and fibrinogen for fibrinmonomer-Sepharose. The assay involves a study of the concentration dependence of the substance in question on the inhibition of the binding of thrombin activated 125I-N-DSK (amino terminal disulfide knot) to the fibrinmonomer-Sepharose (Fbm-Seph). Presumably the same surface orientated sites involved in the affinity binding are also involved in polymerization; consequently, this approach provides a method for evaluating specific regions of the fibrin molecule as possible polymerization sites. Thrombin activated N-DSK (N-DSKa
X) binds to Fbm-Seph. at one non-interacting site, but unactivated N-DSK does not. Io-dination of N-DSK prevents binding. Specifically, increased iodination of Bβ Tyr117 of N-DSK is correlated with a decreased affinity for Fbm-Seph. On the other hand, γ chain tyrosines 18 and 32 are not susceptible to iodination and therefore cannot play a role in polymerization. Amidination of the lysines of activated N-DSK does not inhibit binding to Fbm-Seph. The isolated chains of N-DSK were poor inhibitors of 125I-N-N-DSKa binding as were the fibrinopeptides. N-DSK activated with Reptilase and thrombin activated plasmin fragment E had only half the affinity of thrombin activated N-DSK. The tryptic core, DS-4 of N-DSK was a poor inhibitor of N-N-DSKa
X binding. Iodinated fibrinogen, although 98% clottable, forms a very weak clot and iodinated N-N-DSKa
X isolated from this iodinated fibrinogen has poor affinity for the Fbm-Seph. These results imply that one or more major polymerization sites occur in the N-DSK portion of the fibrin molecule.
A Bleeding Diathesis Associated with a Platelet Storage Pool Deficiency Acquired During Cardiopulmonary Bypass Surgery
Authors: Harbury C.B. , Galvan C.A.
Published: May 2019

The main purpose of this study was to assess whether patients undergoing cardiopulmonary bypass surgery (CPBS) acquire a temporary platelet storage pool deficiency (PSPD). Pre- and postoperative total and releasable platelet ADP and ATP were measured. The lucerifase assay for ATP was used (Holmsen). Platelets were counted and sized using a Coulter H3 system. Bleeding was assessed by chest tube drainage (CTD), cc per 6 hours. Sixty-six patients were studied. None received platelet transfusion. There was a significant decrease in total and releasable ADP and ATP, paired t test p <0.001 in each case. Pre vs postoperative releasable ADP in nano-moles/108 platelets: 2.15 to 1.51, ATP: 1.38 to 1.0, total ADP: 3 to 2.5, total ATP: 5.5 to 4.9. Mean platelet volume pre- vs postoperatively was not significantly different (paired t test) CTD above 400 cc was significantly correlated with PSPD of 0.5 nm or more decrease in releasable ADP (X2 = 5.79, p<0.02). If patients with platelet counts below 100,000 are included in platelet injury group, X2 = 8.2, p<0.01. A decrease of 1 nm/103 platelets or more of releasable ADP was significantly correlated with duration of CPB. If bypasses above and below 90 minutes are compared, X2 = 7.97, p <0.01, above and below 105 minutes X2 = 9.05, p< 0.01. There is a significant drop in platelet count during bypass surgery, but there is no significant change in platelet size. Thus, a large granule rich platelet population was not removed. Acquisition of a platelet storage pool deficiency was significantly associated with a bleeding diathesis postoperatively. The acquisition of a platelet storage pool deficiency was significantly associated with duration of cardiopulmonary bypass.
Hemostasia in the Oral Cavity
Authors: Tomic D.
Published: May 2019

Various factors and reasons can be responsible for acute, severe or protracted hemorrhages in the oral cavity. Hemostasis with conventional methods frequently poses a difficult, dangerous and involved problem. A special method is developed utilizing the chemical and physical properties of inorganic and organic compounds, and this is found to be efficient for rapid and safe hemostasis. It acts on the blood coagulation process by lowering the pH value of blood, as well as by its astringent and osmotic properties. Altogether, the result is hemostasis and favorable wound healing. With the use of bone cement, the initial clot is physically protected, thus contributing more to reliable hemostasis, particularly after dental extraction. The method used, that is, bone cement in combination with the developed hemostatic agent, can be used without additional treatment for severe primary, as well as secondary hemorrhages. Patients suffering from hemophilia and other disorders of blood coagulation were treated successfully.
Diagnosis and Treatment/Control of Consumption Coagulopathy Using Thrombelastography (TEG)
Authors: Raby C.
Published: May 2019

The amplitude (Emx) of a whole-blood TEG (thrombelastogram) tracing stems from three factors : platelets, fibrinogen and factor XIII. These factors are the first defective in a consumption coagulopathy process. Consequently the amplitude is reduced. In contrast, because of a circulating thromboplastin activity which triggers this process, the “r” parameter is modified much more slowly. Such a paradoxical dissociation of the tracings’ elements characterizes an early stage of DIC. In a later stage “r” increases and the tracings indicate that of a mixed hypocoagulability without any specific character. A transfer test in which the “r” parameter of an isogroup normal plasma is compared with that (r’) of a mixture in equal parts of the normal and the patient’s plasma will enable to make a decision. A r’/r ratio lower than 1 reflects a procoagulant activity masked by the consumption process in the patient’s blood, and will confirm the exact nature of the process. The ideal dose of heparin to be administered is that which will be able to reverse this ratio and to permanently maintain it between 1 and 1.5.
The Preparation of Platelet-Rich Plasma
Authors: Clarke Noel , McCabe Vincent
Published: May 2019

Different gravity forces and times have a marked effect on the yield of platelets and plasma obtained from the centrifugation of whole blood (Woods et al. Thrombosis & Haemostasis, 36, 302-310, 1976).

The sedimentation of red cells and harvesting of platelets and plasma by centrifugation with step change in the gravity field was modelled in a computer programme, and the effect of the reflux of plasma and platelets was taken into account (methods in reference). The optimum method (at present) was based on a high centrifugal field (Gay = 1740g; Gmax = 1992g; 3000 rpm; rav = 173 mm; rmax = 198 mm) for a short period (75 seconds) followed by a low centrifugation field (Gav = 48g; Gmax = 55g; 500 rpm) for about 3 minutes. During the first stage platelets refluxed to the plasma zone, and during the second stage the red cell zone moves outwards faster than the platelets. The recovery of platelets was 12.6 ± 3.6% higher than that obtained with the optimum single stage centrifugation method using ethylenediamine tetra-acetic acid (EDTA). It is not yet known how representative the recovered platelet populations are in regard to size. The platelet recovery is lower with citrate than EDTA using both methods. These findings were confirmed by optically monitoring the plasma zone in a circular transparent rotor.

The centrifugal platelet and plasma yield is also influenced for each individual blood by unknown intrinsic sedimentation properties, e.g. erythrocyte sedimentation rate. The fitting of more precise information to the computer programme is required before the ideal method can be achieved.
Computerized Multi-Channel Thrombelastograph
Authors: Kanda Yozo , Takada Akikazu
Published: May 2019

Widely used Thrombelastographs(TEG) made by Heilige Co. measure the coagulation and fibrinolysis of blood by an optical method. The light rays scan on the film according to the viscoelasticity of the blood. The result cannot be known until the film is developed at the end of the experiment. It is troublesome to develop and replace the film. The purpose of the present work is to overwhelm the inconvenience. A silicon photo diodes array is used as a photo sensor and placed in the position where the light hits. The array includes 128 diodes, each of which has a corresponding memory (128 bit shift resistor). These memories read in what parts of these diodes are exposed to the light when the rotarysockets in the main instrument change the direction. These memories are read out by synchronizing clock-pulse and counted in binary code. The signals in binary code are converted into analog signals which correspond to the envelopes of TEG patterns. A microcomputer is added in order to calculate the maximum amplitudes of the envelopes. These components are assembled in the same space as the recording cassette. Therefore, by replacing the recording cassette by the newly made recording system and connecting to a dot printing recorder, we can get directly printed envelopes of TEG patterns. We can also get the digital values of reaction times, coagulation times and maximum amplitudes by connecting the recording system to a digital printer. The newly developed recording system could overwhelm the inconvenience of TEG currently used and save time. (This work was supported in part by a grant (149011) from the Ministry of Education, Japan)
Thrombolytic Therapy with Porcine Plasmin: Biochemical and Clinical Results
Authors: Asbeck F. , van de Loo J.
Published: May 2019

Preliminary results are presented on the systemic action of porcine plasmin which was used as a thrombolytic agent. Patients suffering from acute arterial thromboembolism (2) or deep venous thrombosis (4) received porcine plasmin (pp) in the following dose schedule: 2000 U given as an initial dose within the first 60 minutes, and 2000 U given as a maintenance dose by continuous infusion within the following 8 hours. The maintenance dose was repeated at each of the 2, 3, or 4 consecutive days.

Investigations of the plasmas showed a rapid drop of fibrinogen to 50% of the initial value. FDP raised up to 300 μg/ml. There was no change of the mean plasminogen concentration. Systemic fibrinolytic activity was very low and could only be demonstrated in traces during all stages of the therapy. Analysis of the inhibitors showed a continuous drop of the alpha2-macroglobulin to levels of 50-100 mg/dl. Plasmin-antiplasmin complexes were detected in considerable amounts.

The treatment was well tolerated by all patients. In one patient, a complete recanalization of a teflon bypass of an iliac artery could be achieved. In the other patients, only partial recanalizations could be demonstrated. A combination of porcine plasmin with streptokinase therapy is possible.