bluuebunny/crossref_metadata_2025 huggingface dataset:
Abnormal Blood Rheology in Young Male Diabetics with and without Retinopathy
Authors: Lowe G.D.O. , Drummond M.M. , Belch J.J.F. , Lowe J.M. , MacCuish A.C. , Manderson W.G.
Published: May 2019

We compared red cell deformability (filtration rate through 5 μ sieves), blood viscosity (rotational viscometer), haematocrit, plasma fibrinogen and plasma viscosity in young male diabetics (age <50 years) and normal controls matched for age and smoking habit. diabetics with no retinopathy or other vascular complications (n = 20) had normal red cell deformability, but increased blood viscosity at shear rates of 100s-1 (p<0.05) and is-1 (p<0. 01), due in part to moderate elevations of haematocrit, fibrinogen and plasma viscosity. Diabetics with retinopathy (n = 10) had a more marked increase in viscosity and also reduced red cell deformability (p<0.05). Increased blood viscosity is present prior to the onset of detectable vascular complications in male diabetics, while reduced red cell deformability is associated with complications.
Biochemical and Immunochemical Characterization of the in vitro Thromboplastin Generation by Human Monocytes
Authors: van Ginkel C.J.W. , van Mourik J.A. , Oh J.I.H. , Vreeken J. , van Aken W.U.
Published: May 2019

During short-terra culture monocytes (M) only, not granulocytes or lymphocytes, generate thromboplastin (TP) activity which is absent Immediately after isolation. To clarify the mechanism whereby M generate TP, J approaches were applied: I. To investigate the presence of hidden” TP, freshly Isolated M were subjected either to mechanical disruption, to enzymatic stripping or to dellpldation, followed by reconstitution of the lipoproteins with exogenous phospholipids. No TP activity could be detected after any of these treatments. II. Using an antiserum against human brain TP apoprotein which cross-reacted with monocyte TP, it was shown that intact and disrupted freshly Isolated M lack TP related antigen whereas cultured M display binding of anti-TP antibodies. However, M cultured in the presence of protein synthesis Inhibitors and cultured lymphocytes, lacked TP antigen. III. Using several agents known to affect cellular processes, TP generation was shown to be dependent on;protein and RNA synthesis, energy metabolism, cAMP metabolism, assemblage of microtubules and divalent cations. Inhibition of prostaglandin synthesis by indomethacln did not arrect TP generation. M from 3 patients with COD displayed normal TP generation. These two observations suggest that prostaglandin synthesis and oxygen radicals are not involved Is TP generation. In conelusion, our data indicates that de novo synthesis of TP apoprotein Is responsible for the in vitro generation of TP activity by monocytes.
Increased Leukocyte Thromboplastin Activity by Sensitized Lymphocytes: A Link Between Immunology and Blood Coagulation
Authors: van Aken W.G. , Zeylemaker W.P. , Stricker E.A.M. , Oh J.I.H. , van Ginkel C.J.W.
Published: May 2019

Fibrin deposition and infiltration of mononuclear leukocytes (MN) frequently occurs concomitantly at the site of an Immune reaction. As human MN can produce thromboplastin (TP) we have investigated whether immune stimulation of MN can trigger the production of TP by these cells. Both specific antigen (tuberculin PPD) and allogeneic lymphocytes were used for stimulation. MN from l4 donors with a high response to PPD and from 11 donors with a low response to PPD (based on the incorporation of 3H-thymidine) were cultured for 7 days in the presence or absence of PPD and the TP activity of the whole cell suspension was determined. When PPD was absent, TP levels in MN from low and high responders were not different (113±47 and 197±111 u/ml resp. p>0.10). With PPD, however, MN from high responders produced 1470±413 u TP/ml compared to 126±49 u TP/ml produced by MN from low responders (p<0.005). Similar results were obtained when MN were kept in culture for either 4 or 11 days. However, after 24 hours in culture» TP synthesis was not different in the MN of the 2 groups. When MN were cultured in a mixed leukocyte reaction, allogeneic MN produced more TP (405±151 u/ml) than control cultures (95±21 u/ml) (p<0.025;n=22). These data indicate that stimulated lymphocytes can enhance TP actiyity of leukocytes. It is postulated that fibrin formation In immune reactions such as delayed hypersensitivity and rejection of kidney allografts Is mediated by immunologically stimulated MN.
Ultramicroscopio and Coagulation-Fibrinolytic Findings Induced by Tissue Thromboplastin
Authors: Nagata H. , Seya T. , Oguma Y. , Yamauchi M. , Murakoshi T. , Hasegawa H.
Published: May 2019

We have studied the ulerastructures of t issue thromboplast in (T. Tbp) to demonst rate how It changes during coagulation.

[Haterials and Methods]T. Tbp from lungs of rabbits was used for these studies. It was injected into ear veins of rabbits. Lungs were resected at several seconds, 10sec, 1 min, 5 min, 24 hrs or 48 hrs after the injection. Tljey were examined by transmission electron microscope.

[Results] Concentrically arranged membrane structures of the injected T. Tbp disappeared in extremely short time after the injection.1 min after the injection, fibrin fibers were seen between single sheet of membrane and endothelial cells of capillaries. In the rabbit which had died suddenly after the injection of T. Tbp, multiple pulmonary thrombi made of fibrin and platelets were seen In capillaries. The endothelial cells of capillaries were destroyed and interstitial tissues were edematous.

The hypercoagulable state was seen 10-30sec after the start of the Injection, indicating the shortening of r of TEG. Then, It gradually returned the level before injection. Moreover, changes of the measurements of fibrinogen, antiplasmin and prekallikrein were also seen after the injection.
The Presence of Antithrombin Activity in Platelets
Authors: Czapek E.E. , Kwaan H.C.
Published: May 2019

The platelet has been implicated as the nidus around which clot formation occurs. We therefore decided to investigate whether any antithrombotic activity was associated with platelets. Thrombin activity was measured using the chromogenic substrate S-2238. Kreshly prepared human platelet rich plasma (PRP) and platelet poor plasma (PPP) were found to have similar amounts of antithrombin activity. This antithrombin activity could be easily removed from a platelet pellet by repeated washing. However, sonicating PRP resulted in the appearance of additional antithrombin activity. After 6 minutes of sonlcation, the antithrombin activity of PRP decreased to 35% of presonication levels, whereas the antithrombin activity of PPP remained 100%. When subjected to Laurell immunoelectrophoresis using an antibody to antithrombin III(AT-III), a greater amount of antigen was detected in PRP than in PPP. We conclude that platelets contain antithrombin material which is probably AT-III.
F.VIII R.Ag in Hepatitis
Authors: Kotitschke R. , Scharrer J.
Published: May 2019

F.VIII R:Ag was determined by quantitative immunelectrophoresis (I.E.) with a pre-fabricated system. The prefabricated system consists of a monospecific f.VIII rabbit antiserum in agarose on a plastic plate for the one-and two-dimensional immunelectrophoresis. The lognormal distribution of the f.VIII R:Ag concentration in the normal population was confirmed (for n=70 the f.VIII R:Ag in ± of normal is = 95.4 ± 31.9). Among the normal population there was no significant difference between blood donors (one blood donation in 8 weeks; for n=43 the f.VIII R:Ag in % of normal is = 95.9 ± 34.0) and non blood donors (n=27;f.VIII R:Ag = 94.6 ± 28.4 %). The f.VIII R:Ag concentration in acute hepatitis B ranged from normal to raised values (for n=10, a factor of 1.8 times of normal was found) and was normal again after health recovery (n=10, the factor was 1.0). In chronic hepatitis the f.VIII R:Ag concentration was raised in the majority of the’ cases (for n=10, the factor was 3.8). Out of 22 carrier sera 20 showed reduced, 2 elevated levels of the f.VIII R:Ag concentration. In 5 sera no f.VIII R:Ag could be demonstrated. The f.VIII R:Ag concentration was normal for n=10, reduced for n=20 and elevated for n=6 in non A-non B hepatitis (n=36). Contrary to results found in the literature no difference in the electrophoretic mobility of the f.VIII R:Ag was found between hepatitis-patients sera and normal sera.
Endotoxinaemia, Granulocytic Elastase Release (ELP), Coagulation Disorders and a ANtiprotease Behaviour in Cercopithecus Aethiops and Patients with Septicemia
Authors: Hofmann A. , Hofmann U. , Weinmann E. , Egbring R. , Havemann K.
Published: May 2019

A.)As Aasen (1978) demonstrated, a rapid increase of granulocytic elastase in plasma occurs in dogs following the slow infusion of endotoxin. Working with green monkeys, we did not find severe thrombopenia, leucopenia, coagulation defects and ELP until after two high doses of E. coli endotoxines. Using two-dimensional-immunelectrophoresis ELP could be detected as a complex with α1AT. The coagulation defects occurring in both type’s of animals may be due to DIC and/or to direct proteolysis caused by released ELP, since i.v. administered ELP also leads to an increased consumption of coagulation factors.

B.)Out of 80 patients with either proven or supposed septicemia, we found 40 with possitive endotoxine test results, half of whom had α1C -AT-ELP-complexes im plasma, as proven in one-dimensional-immunelectrophoresis. As expected, the coagulation defects were most distinctly reduced in these 20 patients, while α1AT arise α2M are found to be reduced.
Aspirin as a Potlwial Antithrombotic Drug: An Experimental Approach for more Rational Clinical Use
Authors: Livio M. , Villa S. , Gaetano de G.
Published: May 2019

Blood platelets and vascular walls generate prostaglandin (PG) derivatives with oppening effects. Since aspirin (ASA) inhibits synthesis of common precursors of these derivatives, the rationale for its clinical use in thrombosis prevention trials has been questioned. This is an experimental approach to this important therapeutic dilemma. Male CD rats (250-350g) were given a single i.p. dose of ASA (0.5-200mg/kg) and killed from 1 to 120 h thereafter. Platelet PG production was measured by a thiobarbiturate assay of malondialdehyde (MDA) under thrombin (25u/ml) stimulation. Vascular PG activity released from rings of abdominal aorta and inferior vena cava was determined as platelet aggregation inhibitory potency and characterized as PGI2 by standard criteria. The inhibitory effect of ASA lasted longer in platelets (96-120 h) than in venous (24-48 h) or arterial tissues (<24 h). Platelets were more sensitive to ASA (ID50=3.6mg/kg) than arterial tissues (ID50=25mg/kg) but as sensitive as venous tissues (ID50=2.3mg/kg) when PG synthesis was measured 1 h after treatment. The proposed unique sensitivity of platelet cyclo-oxygenase to ASA therefore needs reconsidering. ASA’s effect on various systems might be better dissociated based on the different duration of inhibition. We suggest that the lowest single dose of ASA totally inhibiting platelet PG synthesis should be given at the longest possible intervals.
Blood Platelet Count and Blood Platelet Survival in Male Stroke Prone, Spontaneously Hypertensive Rats During Ageing
Authors: Meuleman D.G. , Vogel G.M.T. , Delft van A.M.L.
Published: May 2019

Blood platelets play probably an important role in the occurrence of stroke. We determined the survival of radiolabelled blood platelets, the blood platelet count and blood pressure in male stroke prone, spontaneously hypertensive rats during ageing. Our investigations revealed that 1) blood pressure starts to rise at an early age (8 weeks) reaching a steady state (approx. 250 mmHg) at 15-20 weeks of age, 3) blood platelet count is steadily decreasing from 9 weeks of age on, reaching a steady state (approx. 50% of the original platelet count) at 20 weeks of age, 3) blood platelet survival is gradually shortening from 12 weeks of age on, reaching a steady shortened half-life of 18 hours (normal value approx. 42 hours). It is concluded that the rise in blood pressure induces at first instance a moderately increased platelet consumption, which is not compensated by an increased platelet production and which is not detectable by platelet survival measurement. In a later phase, when the blood pressure is in a steady state, platelet consumption is further increased, leading to significantly shortened platelet survivals. This process is then compensated by increased platelet production leading to a steady state thrombocytopenia.
Amino acid Sequence of a Peptide from Type III Collagen Involved in Platelet Adhesion
Authors: Fauvel F. , Legrand Y.J. , Bentz H. , Pignaud G. , Kühn K. , Caen J.P.
Published: May 2019

The probable importance of the role of type III collagen in the initiation of thrombosis is due to its localization in the subendothelial layers of the vessel wall.

The adhesion of platelets to type III has been therefore quantified by a method based on the filtration of non-adhesive 14C 5HT-labe11ed platelets through a Sepharose 2B column.

Type III collagen was purified from calf skin by pepsin extraction and salt precipitation. Type III collagen was cleaved by cyanogen bromide and the adhesion induced by the resulting peptides was measured. The activity was attached to the central alpha l(III) CB4 peptide which was further cleaved by hydroxy lamine, chymotrypsin and trypsin. An adhesive potency was linked to three fragments (HA 1 obtained by hydroxylamine, C2 by chymotrypsin and T2 by trypsin) which possess a common portion of 9 amino-acids, localized in the central part of the alpha l(III) CB4 peptide and of the entire alpha 1(III) Chains, which probably represents the active part of type III collagen. Its activity could be due to a particular sterical conformation linked to the presence of 3 imino acid residues in the sequence Gly-Lys-Hyp-Gly-Glu-Hyp-Gly-Pro-Lys of this fragment.
Antithrombin III Affinity for Thrombin, Trypsin and α-Chymotrypsin
Authors: Marciniak E.
Published: May 2019

The relative affinity of human AT III for thrombin, trypsin and α-chymotrypsin was evaluated tn reactions proceeding in the molar excess of inhibitor, to reflect more closely natural conditions occurring in plasma. Residual AT III was titrated as total factor Xa-binding capacity independent of the velocity of inhibition. The decrease in binding properties of purified AT III reacting with thrombin or trypsin was consistent with formation of enzyme-inhibitor complexes at 1:1 molar ratio and was associated with a partial obliteration of antigenic determinants. Purified α1-trypsin inhibitor competed with AT III but not for thrombin. α-chymotrypsin caused an excessive loss ot AT III binding properties; once the enzyme-inhibitor ratio exceeded 1:5 proteolysis of AT III occurred with formation of multiple protein fragments evident in gel filtration in fibrinogen-depleted human plasma where the overall binding capacity for both trypsin and thrombin exceeded that of AT III, a marked difference was seen in the rate of AT III utilization by each of these enzymes. Every μM of trypsin gradually added utilized on the average only 2% of AT III capacity, whereas the amount of thrombin capable of binding 1 μM of purified AT III obliterated of AT III capacity in plasma. Neutralization of thrombin proceeded after AT III was totally depleted. These results contribute to the notion that AT III is the primary inhibitor of thrombin with an unique affinitv for this enzyme in blood.
Prothrombin Activation on the platelet surface
Authors: Miletich J.P. , Kane W. , Majerus P.W. , Lindhout M.J. , Jackson C.M.
Published: May 2019

Previously reported high affinity receptors for Factor Xa which exist on the platelet surface have been identified as Factor V (more accurately Factor Va). Approximately 200 Factor Xa receptors exist per normal platelet and the rate of prothrombin activation is correlated with the extent to which these receptors are occupied by Factor Xa. Platelets from congenitally Factor V deficient patients possess reduced numbers of Factor Xa receptors and proportionally reduced rates of prothrombin activation. The role ot phospholipid, in addition to that of Factor Va in mediating the association ot Factor Xa and prothrombin with the platelet surface has been assessed by employing structurally modified forms of Factor Xa and prothrombin. Possible explanations for the comparatively greater rate of prothrombin activation observed with platelets than with model system ca. 20-fold, will be discussed by comparing the platelet and model systems. (Supported by US NHLBI, SCOR in Thrombosis (HL14147) and HL12820).
A Clinically Silent Antithrombin III Defect IN an ann Arbor Family
Authors: Penner J.A. , Hassouna H. , Hunter M.J. , Chockiey M.
Published: May 2019

A familial decrease in activity in plasma protease inhibitor, Antithrombin III (AT III «as observed in three generations. This autosomal trait is not associated with thrombsis. It is a benign abnormality despite AT III functional values of 23% of normal in heparin co-factor assay. The low activity AT III was purified from fresh plasma on a heparm affinity column. Its functional, biochemical and immunochemical properties were studied. In the progressive AT III assay, both proteins behaved similarly. Binding of low activity I125 AT III to α1, thrombin or heparin column was identical Purified and plasma AT III from normal and propositus had the same electrophoretic mobilit on Polyacrylamide gel and by Immunoelectrophoresis (IEP) in 1.5K agar gelsHowever plasma and purified low activity AT III when analyzed by two dimensional (IEP) with heparin placed in the first gel showed one major immunoprecipltate and a much smaller more electronegative one against AT III antiserum. Under identical conditions, purifi normal AT III showed a reversed pattern. On electrofocusing and by immunodiffusion against antiserum to thrombin, the major immunoprecipltate was identified as an AT III thrombin comply. A variant of the propositus’ AT III favors rapid binding of thrombi without need of heparin.
A New Variant of Antithrombin III. Study of three Related Cases.
Authors: Wolf M. , Boyer C. , Lavergne J.M. , Larrieu M.J.
Published: May 2019

A qualitative abnormality of antithrombin III (AT III) was demonstrated in three member from the same family. The mother (58 y.) had recurrent episodes of venous thrombosis an pulmonary embolism whereas the affected daughters (20 and 29 y.) are asymptomatic. AT III was investigated in presence and absence Of heparin hy enzymatic assays using chromogenic substrates (S-2238 and S-2160) and by rocket - and crossed - immunoelectro phoresis using two different monospecific anti-AT III antisera. In the three patients the qualitative abnormality of AT III was suggested by the discrepancy between a norml level of AT III antigen and decreased heparin-cofactor activity (49, 51, 54%). By crossed immunoe1ectrophoresis, patients’ AT III migrated as a single peak in absence of heparin, with the same electrophoretic mobility as that of the control. In presence of heparin (25 u/ml), crossed immunoelectrophoresis demonstrated the presence of two peaks, one with a normal, and the other with a decreased electrophoretic mobility. A double peak was also observed by rocket immunoelectrophoresis in presence of heparin. In this family with a variant of AT III, the three heterozygous affected cases demonstrate two popu1ations of AT III, with a different affinity for heparin.
Toxicity in Mice of Factor IX Concentrates
Authors: Magner A. , Aronson D.L.
Published: May 2019

The use of Factor IX concentrates has been associated with thrombo-hemorrhagic problems presumed to be due to activated coagulants. High doses of Factor IX (2000 μ/kg) injected IP into mice result In death between 4 and 24 hours. Reduction with dithiothreitoi removes the toxic effect but no reduction IX toxicity is seen with enzyme inhibitors (DFP, soybean trvpsin inhibitor or heparin). Activation of the Factor IX concentrate by insoluhilized Kussells Viper Venom and Ca# yielded a decreased mortality despite a non-activated partial thromboplastln time of 20" at a 1/5000 dilution.

These results implicate components other than activated components of the Vitamin K dependent enzymes In the toxicity of Factor IX concentrates.
Characterization of PIG Platelet Granule Proteins
Authors: Fukami M.H. , Daniel J.L. , Bauer J.S.
Published: May 2019

Platelet granules contain glycoproteins similar to those found in platelet membranes (Hagen et al , BBA , 445, 21 4 , 1 976 ). Pig platelet granule fractions enriched in mitochondria, α-granules or dense granules were analyzed by SDS Polyacrylamide gel electrophoresis to determine if there are differences among the organelles. In a reduced system (5Ϊ OTT) the proteins of the ï-granules and dense granules showed staining patterns with Coomassie blue that were distinctly different from whole platelets, isolated membranes or mitochondria. In the granules about 10 to 12 bands with less mobility than actin were visualized. Staining with PAS was obtained in bands with apparent molecular weights of 250, 225, 185, 170, 150, 120, 55, 4B and 40 K. The 185 K band appeared to be the same as “thrombin sensitive protein”. The mobility of the 55 and 48 K hands were identical with the B (B) and γ-bands of bovine fibrinogen. The PAS staining of the granule components was more intense than that of whole platelets for the same amount of protein, indicating that granule membranes may be as rich in glycoproteins as external plasma membranes. With both PAS and Coomassie blue, the a-granule and dense granule staining patterns were almost identical. This observation may be relevant to recent studies which showed that both granule types exhibited similar release characteristics, suggesting that they share a common release mechanism. NIH-JSPHS Grant No. 14217
Platelet α-Granules: Content and Characteristics of Secretion
Authors: Kaplan Karen , Broekman Johan M. , Chernoff Arthur , Linder Barbara , Goodman DeWitt
Published: May 2019

Platelet α-granule contents and secretion were studied using specific radioimmunoas says and cell culture assays. Washed human platelets were disrupted by nitrogen cavitation and subcellular fractions were separated by ultracentrifugation through sucrose density gradients. α-Granules were found to contain platelet factor 4 (PF4), 8-thromboglobulin (8TG) fibrinogen, and the platelet-derived growth factor: their contents were distinct from those of dense granules and of lysos omes. A distinct pattern of release of the four agranule components was ob served. Release of α- and dense-granule components was induced by ADP and epinephrine; α-granule release was, however, more sensitive than dense granule to low levels of thrombin, collagen, and the endoperoxide analogue (15S)-hydroxy-IIα, 9α (epoxymethano)prosta-5Z, l3E-dienoic acid. Studies with cyclooxygenase (CO) inhibitors suggested that α-granule release in vitro can be mediated both by the CO pathway and by other mechanisms. Thus, aspirin and indomethacin inhibited collagen and thrombin induced release, but “the inhibitory effects were progressively overcome with higher levels of these agents. Studies in vivo showed that normal plasma levels of PF4 and 8TG were only slightly reduced by aspirin, whereas the elevated basal levels of these components found in patients with arterial disease were reduced by aspirin to a greater extent. The in viv data thus support the in vitro findings that both CO mediated and alternative pathways are involved in release of α-granule components.
Post-Transfusion Purpura (PTP) - A Multifaceted Enigma
Authors: Hoak J. C. , Aster R. H. , Fry G. L. , Blaschke J.W. , Thompson J. S. , Parsons T.J.
Published: May 2019

A 61 yr. old woman presented with thrombocytopenia and diffuse bleeding which developed the days after, a colon resection and transfusion of 6 units of blood. Her serum contained IgG antibody (AB) which aggregated platelets from 60% of normal donors. Using the 51Cr-release technique and indirect immunofluorescence, strongly positive reactions were serum at a serum dilution of 1:100 with 50% of platelets from a panel and with 90% at a 1:25 conclution. Strongly positive reactions were seen with 96% of cells from a lymphocyte panel Unlike classical PTP, her serum reacted strongly with platelets from 3 of 5 P1A1 negative donors. In 3 donors, platelets, which failed to aggregate with pt. serum, aggregated with serum known to contain AB to PlAl platelets. Inhibition of platelet aggregation (PA) resulted when anti-IgG was incubated with pt. serum. Prostacyclin, (PGI2), 0.025 to 1 μM, also inhibited PA by pt. serum, A cytotoxic AB, requiring complement, was demonsrated against cultured human endothelial cells using fluorescence microscopy with the vital dye diacetyl fluorescein and ethidium bromide. Thus, PTP may involve platelet antigens other than PIA1, and multiple antibodies may operate in its pathogenesis. Of particular importance was the demonstration of an AB against endothelium which may have been involved in the production of early, lethal hemorrhage.
Vital Microskopic Observation of Fluorescent Labelled Platelets
Authors: WeBelmarm G. , Kortenhaus H. , Schröer H.
Published: May 2019

Platelets from platelet rich plasma (PRP) of Wistar rats were labelled by incubation with fluoroisothiocyanate (FITC) in Ringer-citrate-dextrose solution. Labelled platelets were reinjected into a tail vein of a rat previously prepared for vital-microscopic observation of the mesenteric vasculature. Using a low-level-light camera, labelled platelets could be observed by a fluorescence technique as distinct luminescent particles moving with the blood stream, and their rheological behaviour could be observed.

Results: Platelets moved with different velocities in the same vessel. Single platelets stuck temporarily to the vessel wall but no aggregation was observed. Extensive sticking has been observed in places of leukocyte-rolling. Using the described method it should be possible to alter platelets in vitro and to observe their behaviour in vivo after reinjection.
Identification of Crosslinked Fibrin Degradation Products in Plasma of Patients During Fibrinolytic Therapy Using a Heat Extraction/SDS-Polyacrylamide Gradient Gel Electrophoretic Technique
Authors: Francis C.W. , Marder V. J. , Martin S.E.
Published: May 2019

To quantitate plasmic degradation products of crosslinked fibrin in plasma, a technique has been developed which employs heat precipitation, SDS-polyacrylamide gradient gel electrophoresis of the dissolved, reduced heat precipitate, and quantitation by densitometrie analysis of γ-γ derivatives identified in the stained get. When studied with this sensitive electrophoretic technique, plasmic digests of purified crosslinked fibrin were found to contain a heterogeneous group of γ-γ chain derivatives with molecular weights between 76,000 and 100,000 daltons. In samples of normal plasma to which digests of crosslinked fibrin had been added, this heat extraction/ge1 electrophoretic technigue allowed the detection of γ-γ derivatives with a sensitivity of 20 µg/ml. Derivatives of γ-γ chains with molecular weights of 82,000 and 86,000 daltons have been identified in the plasma of patients with DIC and during fibrinolytic therapy but were not found in normal plasma or in normal plasma treated in vitro with urokinase. This quantitative assay can be performed in 24 hours and appears to be of value in judging the efficacy of thrombolytic therapy.
The Influence of Antiplatelet Drugs on Platelet Survival After Aortic Damage or Implantation of a Dacron Arterial Prosthesis
Authors: Wilkinson A.R. , Hawker R.J. , Hawker L.M.
Published: May 2019

The effect of antiplatelet drugs on platelet survival, as measured by indium-III labelled platelets was studied in two groups of surgically treated animals. In the dog, platelet survival and loss were measured following surgery and implantation of a dacron arterial prosthesis. These same parameters were measured after aortic damage in the rat. Animals treated with the antiplatelet drugs, sulphinpyrazone and ticlopidine were compared with controls. In the dog, the sulphinpyrazone treated animals had a significantly increased platelet survival and decreased platelet loss following surgery (p<0.05). In the rat both ticlopidine and sulphinpyrazone showed significantly increased platelet survival and decreased loss of platelets following surgery when compared with controls (p< 0.001).

We conclude that both ticlopidine and sulphinpyrazone merit evaluation as potential agents for reducing the risk of thrombotic occlusion in peripheral vascular disease and also in patients with arterial prostheses.
Nephrotic Syndrome (NS): Evidence for Increased Platelet Prostaglandin Synthesis.
Authors: Stuart M. , Spitzer R. , Nelson D. , Sills R.
Published: May 2019

NS is associated with an increased incidence of arterial and venous thrombosis. Hemostasia was evaluated in 11 children with NS (6 with active disease Grp I, and 5 in remission Grp II). Studies included assays for fibrinogen, FDP, Antithrombin III (AT III), platelet count and aggregations. Platelet malonyldialdehyde (MDA) in the presence of N-ethyl maleimlde (NEM) or thrombin was used as an indicator of endoperoxide formation, and platelet survivals were performed in 3/11. Platelet hyperaggregability was present in Grp I and was associated with significantly increased platelet MDA in the presence of both NEM (4.0 ± 0.29), or thrombin (1.77 ± 0.32) compared to normal controls (3.20 ± 0.26; 1.26 ± 0.18). Other evidence for a “hypercoagulable” state included a marked reduction in plasma AT III levels to 9.4 ± 3.8 (controls 24 ± 3 mg/100 ml), and a reduction in platelet life-span in both children in Grp I in whom this study was performed (T½ of 2.1 and 2.5 days). Grp II patients did not demonstrate platelet hyperaggregability and platelet MM was normal (3.21 ± 0.4; 1.13 ± 0.19). AT III levels were normal at 26.5 ± 4.8 mg/100 ml, and platelet life-span was normal in 2/2 children (T½ of 3.6 and 4.4 days). The normal half-life of 4.4 days was obtained in the same child in whom a T½ of 2.5 days was present during active disease. Since a reduction in platelet survival is associated with an increased risk of thromboembolism in a number of pathological states, this finding is of clinical significance and may identify the patient with NS who is at risk. Platelet hyperaggregability in this syndrome is prostaglandin related, and appears to be due to an increase in platelet endoperoxide formation.
Evidence for Dependance of Beta Thromboglobulin Levels on Renal Function
Authors: Deppermann D. , Andrassy K. , Seelig H. , Ritz E. , Post D.
Published: May 2019

Beta-Thromboglobulin (TG) is a thrombocytic protein which is released into the circulation upon lysis of thrombocytes. Its serum concentration is thought to be a sensitive indicator of thrombocyte consumption. Although a relation of TG platelet consumption could be observed by us in patients with normal renal function, no such relation was found in pat. with renal failure (RF). Therefore we examined whether TG, similar to beta-2-microglobulin (BMG), behaves like a tubular protein. - Patients and methods - 90 pat. with RF were examined. None of the pat. had diseases with known platelet consumption or medication which interferes with platelets. TG and BMG were measured by RIA. - Results - There was a highly significant correlation between TG, serum creat. (r=0,78) and Ccr (r=0,73) and BMG. The normal range (x ±SD) of TG was 33,9± 9,7 ng/ml and values consistantly about 50 ng/ml were observed at Ccr < 50 ml/l,73 m2. TG was found in tubular proteinuria. - Conclusion - TG is a tubular protein the concentration of which rises in patients with renal failure. This finding invalidates the use of TG for detection of thrombosis in patients with impaired renal function.
In Vitro Anticoagulant Activity of Esters of Heparin.
Authors: Mardiguian J. , Trillou M. , Marin J.
Published: May 2019

Chemical modification of Heparin by esterification of its hydroxyl groups has been carried out in order to study its influence on the in vitro anticoagulant activity. Beef and pig mucosal Heparins have been fractionated by gel filtration and by ion-exchange chromatography to yield fractions which have been esterified.

Two types of esters have been prepared : 4-chlorophenoxy-acetic and 4-chlorophenoxy-isobutyric.

The anticoagulant activity of the these esters has been evaluated in vitro (using U.S.P. method, anti-Xa and anti-thrombin assays) and correlated with their chlorine content and U.V. absorption.

This study shows that the in vitro anticoagulant activity of heparin is decreased by esterification of its OH groups, to an extent depending upon the type of ester, the molecular weight and the degree of substitution.

The results of these experiments will be reported and discussed in detail.
On the Membrane Association of Platelet Glycocalicin.
Authors: Solum N.O. , Hagen I. , Filion-Myklebust C. , Stabæk T.
Published: May 2019

More observations indicate that the hydrophilic protein glycocalicin found in soluble fractions after homogenization of platelets in the presence of calcium ions, exists in a different molecular form on the intact platelet. Thus, SDS-PAGE of SDS-dissolved whole platelets show glycocalicin to be derived from a more complex protein than the solubilized protein itself. Crossed immunoelectrophoresis of Triton x-100 extracts of platelets revealed anti-glycocalicin-reactive material existing as two components with different eloctrophoretic mobilities and with the fast-moving one corresponding to highly purified water-soluble glycocalicin. Crossed hydrophobic interaction immunoelectrophoresis confirmed the hydrophilic nature of this component, whereas the slow-moving component showed the characteristic retardation of an amphiphilic membrane protein. Glycocalicin is solubilized during freezing and thawing of platelets in buffer, immunoquantitation (Laurell) of solubilized glycocalicin showed that EDTA(3,6mM), NEM (4.0mM) and IAA(4.0mM)reduced solubilization by 87%, 65% and 57%, respectively, and also inhibited calcium-dependent protease of platelet homogenatos indicating that the solubilization may be enzyme-mediated.
Fluid-Phase Immunoradiometric Assay of F. VIII/WF Demonstrating Abnormal Antigenic Reactivity in Variants of Von Willebrand’s Disease
Authors: Girma J.P. , Ardaillou N. , Meyer D. , Lavergne J. M. , Larrieu M.J.
Published: May 2019

Antigenic reactivity of F. VIII/MF was studied in cryo-supernatant (n = 3) and in plasma from variants of vWd (n = 4) by fluid phase IRMA. Specific anti-human F. VIII/WF rabbit or goat 125-I-Fab fraqments were used as antibody. The slopes of the dose-response curves of cryo-supernatant and plasma from variants were significantly lower (p<0.001) than those of normal (n = 7), classical vWd (n = 2) or hemophilia A (n = 2) plasma and normal cryoprecipitate (n = 5). The ratio of the slopes of variant to control plasma was 0.60, 0.76, 0.74 and 0.86 using rabbit Fab fragments and 0.43, 0.51, 0.73 and 0.80 using goat Fab fragments (normal plasma : 0.98 ± 0.01 and 1.01 ± 0.04 respectively). Antigen-antibody binding was studied according to Scatchard by reacting a constant amount of VIIIR:Ag with serial dilutions of antibody. In control plasma and cryoprecipitate, at least two groups of binding sites were demonstrated, of high (K1 = 13.2) and low (K2 = 1.7) affinity. In variants of vWd and cryo-supernatant, weak binding sites observed at high antibody concentration were normal. At low antibody concentration, the strong binding sites differed from normal, the curves demonstrating a positive cooperativity. This abnormal antigenic reactivity is associated in cryo-supernatant and variants of vWd with a lack of VIIIR:RCo and of the large, slow-moving, forms of VIIIR:Ag by electrophoresis. Such data suggest an abnormality of aggregation of F. VIII/WF in variants of vWd.
Abnormal Factor VIII Molecule in Mild Von Willebrand’s Disease
Authors: Castillo R. , Maragall S. , Labal F. , Monteagudo J. , Ordinas A.
Published: May 2019

In 22 cases with normal levels of the three entitles of factor VIII (VIII:C, VIIIR:AG and VIIIR:WF), FVIIIR:AG showed afaster anodal mobility and a lower concentration measured by immunoradiometric assay than that found with Laurell’s immunoelectrophoresis. Moreover, the factor VIII protein elution by agarose gel filtration of plasma and cryoprecipitate was delayed. Seven of those cases belong to a family in whom one of the member showed frequent bleeding episodes. In these individuals the FVIIIR;AG precipitation with Concanavalin A was determined and appeared decreased.

On the other hand in 19 out of 20 subjects with classic moderate von willebrand’s disease (in whom the three entities of factor VIII were occasio nally reduced in parallele), FVIIIR:AG also showed a faster anodal uob{ 1-ty and the factor Vln protein elution by agarose gel filtration was delay ed.

All these results suggest that patients with moderate von Willebrand’s disease show a qualitatively deficient factor VIII molecule synthesis, while the mildest cases may have normal levels of the three entities of the factor VIII.
Human Platelet Aggregation Induced by the Synthetic Endoperoxide Analogue U-46619 is Independent From Secretion, Prostaglandin Production and Extracellular Calcium
Authors: Di Minno G. , Bianchi L. , de Gaetano G. , Silver M.J.
Published: May 2019

U-46619 is a stable analogue of cyclic prostaglandin endoperoxides which induces human platelet aggregation independently from nucleotide secretion. We studied platelet aggregation, 14C-5 HT release and malondialdehyde (MDA) production induced by this compound in stirred or unstirred platelet-rich plasma (PRP) samples from 11 healthy volunteers. Each subject was tested both before and 90 min after aspirin ingestion (500 mg). The threshold aggregating concentration (TAC) of U-466l9 ranged between 240 and 900 nM. Aggregation was maximal between 30 and 60 min after venepuncture and was concentration-dependent (60-7, 200nM). At concentrations below the TAC, U-466l9 induced primary reversible aggregation without detectable l4C-5 HT release. At TAC or higher concentrations aggregation and release proceeded as parallel events. Neither MOA production nor intracellular LDH loss could be detected in any of the tested situations. Aspirin ingestion did not modify the above pattern of platelet responses. In unstirred samples l4C - 5 HT release occurred to the same extent as in stirred platelet suspensions. Addition to citrated PRP of Na2 - EDTA did not affect either aggregation or release. It is suggested that aggregation and secretion may be independent, parallel responses of platelet activation by U-466l9 and do not require either extracellular calcium or activation of endogenous arachidonic acid metabolism. (Supported by the Italian CNR and NIH).
The Effect of Contrast Media, 125Iodine-Labelled Fibrinogen, Heparin and Aspirin on Plasma BTG Concentration
Authors: Turpie A.G.G. , Han P. , Genton E. , Butt R. , Zielinsky A.
Published: May 2019

Betathromboglobulin, a platelet-specific protein, released on activation of platelets is being evaluated as a marker of active thrombosis. Theoretically, several diagnostic and therapeutic procedures used in the thrombotic disorders might affect BTG by inducing platelet release or by interfering with the radioimmunoassay (RIA) method. This study determined the effect on plasma BTG concentration of contrast media used in venography, I125-labelled fibrinogen for leg scanning and heparin or aspirin used in the management of venous thrombosis. Mean plasma BTG in normals was 28 ± 8 ng/ml. (n = 70), and do not fluctuate. Plasma BTG, in 10 patients with normal venogram measured before and 30 minutes post-venorrraphiv was 23 ± 10 ng/ml, and 26 ± 15 ng/ml, respectively (p > 0.1). 125I fibrinogen in plasma by, conrecinitating in the BTG/antibody complex, elevates bound radioactivity in the RIA and results in spuriously low levels of BTG and may proclude its use as a diagnostic test in patients having leg scanning. In-vitro, henarin (> 50 units/ml.) or aspirin (> 40 mg/100 ml.) affect the 1 hour incubation assay and give falsely high BTG levels but do not affect an optimized assay with 24 hours incubation. This study indicates that diagnostic and therapeutic measures in venous thrombosis patients may influence BTG, measurement and caution is necessary to avoid confusing the results.
Differential Reactivity of Three Specific Anti-VIII:C Antibodies in Haemophilia a Patients
Authors: Lavergne J.M. , Girma J.P. , Meyer D.
Published: May 2019

Specific anti:VIII:C antibodies in three haemophilia A patients (high responders, cases 1-3) were purified by two proceduses. In a solid phase system, 125-1 IgG containing antiVIII:C antibodies was incubated with purified Factor VIII coupled to CNBr-activated Sepharase 2B. Specific antibodies were eluted using HCl-glycine buffer, pH 2.4 or 3.5.

In a liquid phase system, 125-I IgG was incubated with Factor VIII; following precipitation with 3.5% PEG, the specific immune complexes were recovered by gel filtration.

Anti-VIII : C antibodies were isolated by issociation at pH 2.4 or 3.5, followerd by Sephade x G 200 gel flltration. Using antibodies from case 1 (titre 1,000 Oxford IJ./ml), obtained by both procedures: a two-site IRMA demonstrated no VIII:C-Ag in twelve severe haemophllla A patlents and a normal amount in two haemophilia A+ patients. Cases 2 and 3 had a lower anti-VIII:C titre (100 anrl 150 U./ml) but their antibodies showed, in plasma, a hlgher affinlty for VIII:C-Ag than those of case 1. The affinity was estimated by displacement of the bound radioactivity in IRMA by competitive inhibition. Antibodies from case. 2 failed to dissociate from VIII :C-Ag at pH 3.5 or 2.4. Antihodies from case 3 did not dissoclate at pH 3.5 and only at pH 2.4 but were ineffective in the two-site IRMA. These results suggest that 1) there is no relationship between titre and affinity of anti-VIII:C antibodies; and 2) with the procedures of purification available only those antibodies with a high titre (≥1,000 U./ml) may be of use in IPMA.
Identification of Heterozygotes for Hemophilia B and VWD by Discriminant Analysis
Authors: Graham J.B. , Elston R.C. , Miller C.H. , Goldin L.R. , Kasper C.K.
Published: May 2019

Published data from California (Blood 50:351, 1977) and unpublished data from N.C. on carriers of hemophilia B were examined statistically to determine the optimal methods of identifying the carriers. Factor IX:C assays were available on both groups, and IX:Ag assays were also available on the California women. Linear and quadratic discriminant analysis, univariate and bivariate, with and without the “jack-knife” procedure were utilized. The carriers from California were divided into 29 CRM- and 18 non-CRM-(CRM+) from the phenotypes of their male relatives. It was found that univariate linear discriminant analysis of IX:Ag assays was optimal for CRM- carriers, and l inear bi variate ana lysis of IX:C and IX:Ag assays for CRM+ carriers.

Identifying heterozygotes for vWd was done most efficiently (81%) by trivariate linear I discriminant ana lysis utilizing assays for VIII:C, VIIIR:Ag, and VIIIR:WF. This was superior to: i) any single test alone (VIII:C; 15%, BT;17%, VIIIR:Ag;27%, VIIIR:W F;42%), ii) abnormal ratios (23%), and iii) one ab normal test of any type (58%). It was possible to identify asymtomatic “transmitters” as heterozygotes, even some who had all tests in the normal range.
Fibrin Formation in Shear Flow: A and B Peptide Release
Authors: Sellers J.P. , Puryear H.A. , Clark H.G.
Published: May 2019

Fibrinogen reacted with thrombin at pH 6.3 in shear flow fields varying from 0 to 150 sec-1 gives insoluble fibrin at differing ratio, with a minimum rate near 50 sec-1. The relative rate of release of A and B peptides is also altered by the shear flow field. The release or B peptide is faster in stagnant solutions than in the higher shear flow.
Isolation of a Specific Procoagulant Activity in Human Lung Carcinoma Cell Lines
Authors: Mohammad S.F. , Moffler M. , Mason R.G.
Published: May 2019

Although increased risk of thrombosis in patients with certain malignancies is recognized, carefully planned studies have not been carried out to examine the role of malignant cells as initiators or supporters of thromboembolic disorders.- Using the following brief experimental protocol we carried out a series of experiments to Study the interaction of malignant eel Is with platelets or blood coagulation system, i) Cultured human normal or malignant cells were detached from substratum by various techniques and added to human platelet rich or platelet poor plasma (PRP or PPP). ii)Disrupted cells or their subcellular fractions were added to PRP or PPP, After addition of intact cells or subcellular fraction, their effect on platelet aggregation (in PRP} or blood coagulation system (in PPP; recalcification and partial thromboplastin time} was examined. We observed that three lung carcinoma cell lines (LCCL) possessed a distinct procoagulant activity. Intact LCCL cells initiated rapid clot formation when added to heparinized PPP also. The procoagulant activity was present largely in soluble fraction. This activity is labile and appears to have a strong affinity for glass and plastic surfaces. Rapid loss of procoagulant activity is observed at 56°C or alkaline pH (above 3.5). This activity was found to be specific for LCCL since a careful evaluation of 20 other cell lines showed no such procoagulant activity when examined under identical conditions. (Supported in part by grant PDT-7g from American Cancer Society)
Diagnosis of the Prethrombotic State: The Role of Platelet Evaluation
Authors: Colman R.W.
Published: May 2019

Arterial thrombosis is believed to involve the initial formation of a platelet aggregate. This diagnosis of a putative prethrombotic state should logically involve evaluation of the function of platelets in persons prone to clinical arterial thrombotic disorders. Examples of assays used to measure in vivo platelet behavior are platelet survival measurement, detection of circulating platelet aggregates, and measurement of platelet specific proteins released into plasma. Studies reflecting platelet reactivity in vitro include quantification of the threshold for platelet aggregation and release by physiologic agonists, measurement of spontaneous platelet aggregation, evaluation of platelet coagulant activities, and determination of platelet size. Each of these tests have been applied in many of the following clinical conditions:transient cerebral, is chemic attacks, stroke, anginapectoris, coronary insufficiency, myocardial infarction, homocy-stinuria, hyperlipoproteinemia, paroxysmal noctural hemoglobinuria, prosthetic heart valves, arteriovenous shunts and renal dialysis. Abnormalities in one or more of these tests have been recorded but whether these changes are a cause or effect of the thrombotic conditions has not been established. Nevertheless, discovery of subpopulations with changes in platelet reactivity nay facilitate the identification of individuals who may benefit from antiplatelet drug prophylaxis or therapy.