bluuebunny/crossref_metadata_2025 huggingface dataset:
Altered Platelet Factor X Activating Activity in Myeloproliferative Disorders
Authors: Cortellazzo S. , Colucci M. , Barbui T. , Semeraro N.
Published: May 2019

The pathogenesis of the haemostatic disorders in patients with myeloproliferative diseases has not yet been completely elucidated. Several platelet function abnormalities have been reported but they do not always correlate with the clinical manifestations. In this report a recently described platelet coagulant activity (factor X activating activity) was studied in 2 patients with essential thrombocythacmia, 10 with polycythaemia vera, 4 with chronic myeloid leukaemia and 2 with reactive thrombocytosis. It was significantly reduced in the 5 patients with bleeding tendency, and was also reduced in one patient with a history of peripheral ischaemia and in 3 patients with no haemostatic complications. It was either normal or increased in the remaining patients who had no haemostatic complications. In the two subjects with lowest activity (5 and 131) and most serious bleeding, chemotherapy resulted in normalisation of the platelet count and of platelet coagulant activity and in disappearance of the bleeding episodes. Our results suggest an association between bleeding tendency and reduced platelet coagulant activity. These findings may be of relevance to 1) the pathogenesis of abnormal bleeding in patients with myeloproliferative disorders and 2) the understanding of the role of platelet coagulant activities otter than pLatelet factor 3 in disturbed haemostasis.

Supported by CNR (Italy).
Morphological and Funcitional Stdies of Platelet-Vessel Wall Interaction in Myeidproliferative Disease, (MPD).
Authors: Sacher R.A. , Jacobson R.J. , McGill M.
Published: May 2019

In an attempt to identify the defects(s) in platelets of patients with MPD, citrated peripheral blood fron 4 patients were studied using a technique that measures platelet function under simulated blood flew conditions (Baumgartner H. Microvasc. Res. 5:167, 1973). In 2 patients with thrcirtocytosis (1 with chronic myelocytic leukemia, CML, and platelet count of 7.03 × 105/μ1 and 1 with primary thranbocythemia, PT and platelet count 1.14 × 106/μl the platelet aggregate per unit length of vessel wall determinations were 224% and 165% of normal values respectively. In contrast in 2 patients with CML and normal platelet count (1.67 × 105 and 2.10 × 105/μl), these values were 24% and 77% of normal controls respectively. Electron microscopy (EM) of aggregates on the vessel wall in the patient with CML arid thranbocytosis showed these aggregates to be composed of cellular debris, lysed platelets and platelet membrane ghosts. Direct EM examination of unstimulated peripheral blood platelets frcm 2 additional patients (1 with CML and 1 with PT) revealed normal morphology. These studies shew that in patients with MPD, a major influencing factor prorating platelet-vessel wall interaction is the nimber of circulating platelets and furthermore the aggregates so formed appear to consist of morphologically abnormal platelets.
A Comparison of Two Assays for Platelet Antibodies
Authors: Kelton J.G. , Giles A. , Neame P.B. , Blajchman M. , Hirah J.
Published: May 2019

The optimal method for assaying platelet bound antibody is uncertain. We have compared a fluorescent assay (FA) with a (quantitative) antiglobulin consumption assay for platelet associated IgG (PAIgG) in normals and thrombocytopenics with ITP, SLE, non-immune and drug-induced thrombocytopenia. Forty-eight of 49 hospitalized and healthy controls had normal PAIgG levels by the antiglobulin consumption assay (2.6 ± 0.2 fg IgG/platetet,

± SE, normal 0-5 fg) and the FA was negative in 41. The PAIgG level was elevated (20.0 ± 1.9 fq IgG/platdet) in 42 of 45 determinations on ITP patients. The FA was positive in 21. Positivity in the FA test did not relate closely to PAIgG level. The PAlqG was elevated (29.0 - 7.3) in 14 of 15 assays in thrombocytopenic SLE patients. The FA was positive in 3. The PAIgG level was elevated in all 9 patients with drug-induced thrombocytopenia (42.4 ± 23.2) without addition of the drug to the test system. The FA was positive in 5. In 2 of 12 patients with non-immune thrombocytopenia the PAIgG level was slightly elevated (both patients had multiple myeloma) and the FA was positive in 5. The results suggest that the quantitative antiglobulin consumption assay is more sensitive than the fluorescent assay in the diagnosis of immune mediated throinbocytopenia. The significance of the lack of correlation between positivity in the fluorescence test and the PAIgG levels found in patients with ITP is uncertain.
Investigation of the Catalytic Properties of the Dysthrombin, Thrombin Quick
Authors: Henriksen R.A. , Owen W.G. , Nesheim M.E. , Mann K.G.
Published: May 2019

Thrombin Quick (TQ) may be isolated following treatment of Prothrombin Quick [Owen, et al, Mayo Clinic Proceedings, 53: 29-33, (1978)] with Taipan venom, phospholipid and Ca2+. The clotting activity of TQ with fibrinogen is 1/200 that of normal thrombin (T) The activation of Factors V and VIII, and the aggregation of platelets by TQ occurs with an effectiveness of about 1/50 that of thrombin. When incubated with antithrombin III, both T and TQ form inhibitor complexes as determined by dodecylsulfate gel electropheresis. Titration of T and TQ with the fluorescent inhibitor dansylarginine-4-ethylpiperidine amide indicates an equivalent number of active sites based on protein absorption at 280 nm. However, the two enzymes may be distinguished by the decreased fluorescence enhancement observed with TQ relative to T, indicating an increased polarity in the inhibitor binding site of TQ. With the substrate benzoylarginine ethylester, TQ has a Km = 4.5 x 10-5M and kcat - 6.93 compared to Km = 4.0 × 10-5M and kcat = 17.7 for T. This indicates that the defect in TQ esterase activity is in the catalytic mechanism itself and not in substrate binding. The rate of inhibition of TQ by diisopropylphosphofluoridate is decreased. Decreased acylation and deacylation rates for TQ relative to T are observed for tydrolysis of the active site titrant 4-methyumbel1 i feryl-p-guanidlnobenzoate.
Action and Interaction of Sulfinpyrazone in Anticoagulated Patients
Authors: Walter E. , Hodok-Laibach P. , Zimmermann R. , Weber E.
Published: May 2019

For 8 weeks 29 anticoagulated patients with elevated serum uric acid levels were controlled with 14 days intervals. From week 3 till the end of 6 they received 600 mg sulfinpyrazone p. d. Because of excellent compliance the uric acid decreased from 7.0±0.2 mg% to 3.7±0.2 and raised to 7.1±0.3 (x±x) after omitting the drug. In contrary to this collagen-induced aggregation (5ug/ml, Vmax = ΔE/min.) decreased during the first 14 days of therapy from 0.388±0.048 to 0.228±0.045 (p<0.02) and reached 0.166±0.031 (p<0.01) at the end of the therapy, but with O.269±0.040 did not return to the starting point two weeks after stopping the drug. The increasing effect of therapy and the long lasting effect point to an active metabolite of sulfinpyrazone, which la slowly eliminated. The interaction of sulfinpyrazone with phenproeoumon consisted in a di - placement of the anticoagulant from protein-binding sites, resulting in a fall of the Thrombotest value from 11.2% to 9.0% (p<0,0l). During sulfinpyrazone therapy the GGT increased significantly, indicating an induction of drug metabolizing enzymes in the liver, which had to be counteracted with a higher phenproeoumon dosis for therapeutic anticoagulation.
Regular Haemodialysis Therapy (RDT) Induces A Prothrombotic State
Authors: Turney J. , Woods H.F. , Weston M.J.
Published: May 2019

To determine whether activation of coagulation factors and platelets, reported during haemodialysis results in a prothrombotic state we have studied indices of coagulability in 15 undialysed and 32 dialysed uraemic patients. Mean plasma fibrinogen (g/1) in uraemic patients (3.53) was significantly higher than in controls (2.45) and rose further in dialysed patients (3.81). Factor VIII coagulant activity (CA) was greater in dialysed (202 ± 15.9%) than in undialysed uraemic patients (184 ± 14.2%) both being significantly greater than control (84 ± 3.9). Similarly Factor VIII related antigen (RA) was higher in dialysed patients (216 ± 18.5%) than uraemics (156 ± 9.6%) (control = 94 ± 5.6%). The mean RA/CA ratio and the frequency of a high RA/CA ratio (< 1.5), an index of thrombin induced consumption of Factor VIII-CA and intravascular coagulation, were higher in dialysed than nondialysed patients.

Despite the higher circulating levels of procoagulants and the evidence of thrombin activation the availability of heparin cofactor-antithrombin III (AT III) was lower in dialysed (154%) than in uraemic patients (159%) though in both groups AT III levels were higher than in controls (100%).

These results indicate that RDT is associated with a prothrombotic state which is not completely compensated for by an increase in antithrombin III.
Anti-A Content of Intermediate-Purity Concentrates of Factor VIII
Authors: Smith J.K. , Bowell P.J. , Gunson H.H. , Snape T.J.
Published: May 2019

Haemolysis may be induced in haemophiliacs of blood group A if they receive very large doses of concentrates containing high levels of anti-A. The anti-A content of factor VIII concentrates made at PFL has been, measured by an automated haemagglutination method. The standard was a pool of 500 sera of random blood groups, assigned a value of 100 units per ml. 23 batches of concentrate prepared from 5 1 packs of FFP, each containing about 24 donations of random blood groups, contained 23.7 ± 4.1 (mean ± S.D.) units anti-A/iu factor VIII.

It was predicted that, if 5 l pools were replaced by single donations of FFP which were not mixed until thawing and collection of the cryoprecipitate, there would be less neutralisation of anti-A by group A donations and the anti-A level in the concentrate would be increased. Unexpectedly, the anti-A content of 9 batches prepared from single donations of random blood groups was 5.8 ± 1.5 units/iu factor VIII, significantly lower than for batches made from 5 1 packs. It is postulated that free anti-A is less susceptible to cryoprecipitation than is anti-A completed with A substance during pooling of group O and A plasmas at 20°. After re-solution of the cryoprecipitated complex, equilibrium may be re-established to give free anti-A.
Sequential Platelet and Fibrinogen Turnover Studies Associated with left Ventricular Assist Device (LVAD) Implantation
Authors: Rodvien R. , Dalal K.B. , Mitchell R. , Litwak P. , Bosak J. , Baleix J. , Hill J.D.
Published: May 2019

Quantitation of thromboembolism is necessary to guide polymer fabrication and anticoagulant use for artificial organ implantation. Therefore as part of the LVAD program we have solid-phase radioiodinated bovine fibrinogen and developed an autologous 111In technique to quantitate platelet survival. Mean platelet survival time(n/a) by the method of Murphy and Francis was 130±20 hrs for 10 animals; platelet aggregation and serotonin release were unaffected by the labelling procedure, n/a for homologous fibrinogen was 55.0±7.3 hrs. for 11 cows; clottability was 93%.

LVAD implantation of the Thoratec Model VIC devices with textured surfaces produced both shortened platelet (47+4.4 hrs.) and fibrinogen survival (30±4.6 hrs.) at 24 hrs. despite coumadin, aspirin and persantin. Survival curves changed from linear to curvilinear, suggesting consumption. Sequential studies at 1, 2 and 4 weeks showed continued platelet and fibrinogen consumption though to lesser degrees than at 24 hrs. Despite coumadin, aspirin and persantin, on-going consumption is occurring and can be quantitated, thereby providing a laboratory monitor to guide future development and clinical use of the LVAD.
Inhibition of Leukocyte Adhesion and Migration in Canine Jugular Veins by a Slowly Metabolized Derivative of Lidocaine (Tocanide).
Authors: Stewart Gwendolyn J. , Knight Linda C.
Published: May 2019

Local dissection followed by brief stasis caused massive adhesion to and invasion of medium sized veins of dogs, rabbits and humans by polymorphonuclear neutrophils (PMN). In dogs both were inhibited by intravenous lidocaine (Am. J. Pathol., 74:507). Inhibition of PMN adhesion and invasion by tocanide, a primary amine derivative of lidocaine (2-amino-2,6-propionoxylidide, Astra Pharmaceutical Co.), with a biological half life of 10-12 hours was quantitated with IIIIn-labeled leukocytes. Canine jugular veins were freed from surrounding tissue and side branches ligated. After 3-hour intravenous infusion of saline (control) or tocanide (0.7 mg/kg/minute for 20 minutes followed by 0.07 mg/kg/minute for 2 hours 40 minutes) jugular were occluded for 5 minutes, cannulated and washed free of nonadhering blood elements before removal for counting of radioactivity and examination of scanning electron microscopy. In one group tocanide was started before, and the other group after neck dissection and Injection of IIIIn-leukocytes (dissection and stasis provided stimulus for attraction of PMN). Vessel and blood counts were standardized to give blood equivalents of PMN per gram of vessel. Results were: Controls, 0.92 ml/gm; tocanide started after dissection, 0.54 ml/gm; and tocanide started before dissection, 0.12 ml/gm. SEM showed typical adhesion and invasion by PMN. Plasma levels of toeanide were 2-4 μM. Thus, tocanide inhibited the PMN component of inflammation by 46-87% depending on regimen.
Identification and Distribution of the Calcium-Dependent Regulator Protein in Human Platelet Preparations
Authors: Levine S.N. , Steiner A.L. , White G.C.
Published: May 2019

Calcium-dependent regulator protein (CDR) is a 17,000 molecular weight polypeptide which has been demonstrated in a number of mammalian cells and has been shown to 1) modulate activation of adenylate cyclase activity, 2) stimulate erythrocyte membrane (Ca++/Mg++) ATPase activity, 3) stimulate cardiac microsomal calcium uptake, 4) activate smooth muscle myosin light chain kinase activity, and 5) regulate microtubule assembly and disassembly. Because of the role of CDR in calcium metabolism and contractile protein function, we have examined the presence and subcellular distribution of this protein in preparations of human platelets. CDR was quantified using a two-step phosphodiesterase assay (Kakiuchi et al, PNAS 70:3526, 1973). Bovine brain CDR which was homogeneous by SDS polyacrylamide gel electrophoresis was used as a standard. Whole platelets contained approximately 4.4 ug CDR/mg platelet protein. When platelets were fractionated by glycerol osmoticlysis followed by separation on 27% sucrose, the soluble portion had the highest specific activity and the membrane fraction, the lowest. These studies demonstrate the presence of CDR in platelets and indicate subcellular compart-mentalization of the protein. The role of CDR in calcium metabolism and contractile protein function in platelets is currently under study, (AHA 78-1215/NIH AM17438, DE02668)
Attempted Determination of Endogenous Heparin in Blood
Authors: Jacobsson K.-G. , Lindahl U.
Published: May 2019

A method was developed for the micro-scale determination of heparin in blood plasma. After extraction with water-saturated phenol glycosaminoglycans were recovered from the aqueous phase by ion-exchange chromatography on DEAE-cellulose. The heparin content of the product was determined by a colorimetric antithrombin-activation assay. Recoveries of 40-60% of both radioactivity and biological activity were obtained when the method was applied to the reisolation of 3H-labeled heparin, added at a concentration of 20 ng (about 0,003 unit) per ml blood. In the absence of exogenous heparin no antithrombin-activating material could be detected. The amount of endogenous heparin, if any, in human blood was calculated to less than 2 ng (about 0.0003 unit) per ml.

(Supported by grant No. 2309 from the Swedish Medical Research Council)
Quantitation of Platelet Adherence to Rabbit Aortae and Platelet Survival After two Injuries with a Balloon Catheter
Authors: Kinlough-Rathbone R.L. , Groves H.M. , Maric S. , Packham M.A. , Mustard J.F.
Published: May 2019

Following a single balloon catheter injury to a rabbit aorta (INJ 1) a monolayer of platelets covers the subendothelium within 10 min, the surface becomes relatively non-reactive to further platelet accumulation and platelet survival is not altered. We have now studied whether a second similar injury (INJ 2) of the non-reactive, smooth muscle cell-rich neointima 7 days after INJ 1 makes the surface of the neointima reactive to platelets or alters platelet survival. 51Cr-platelet adherence to the neointima of aortae subjected to INJ 2 in vitro 7 days after an initial in vivo injury was not significantly different from the adherence following a single in vitro injury (16,600 ± 3100 platelets/mm2 and 27,600 ± 4000 respectively, ρ > 0.2). In vivo adherence of 51Cr-platelets to the surface of rabbit aortae was similar following INJ 1 (0.084 ± 0.009% of the circulate, platelets) and INJ 2 (0.130 ± 0.03%, p > 0.2). Platelet survival after injury to the neointima was not significantly different in animals with an undamaged aortic endothelium (74.6 ± 5.9 hr and 80.2 ± 4.3 hr respectively, ρ > 0.5). Thus, a second injury involving the smooth’ muscle cell-rich neointima that forms after removal of the endothelium with a balloon catheter does not cause more platelets to accumulate than the initial injury, nor shorten platelet survival.
On the Release Function of Adhering Platelets
Authors: Malmgren R. , Larsson R. , Olsson P. , Tornling G. , Unge G.
Published: May 2019

A Method for measuring the continuous uptake and release of serotonin during 60 minutes by platelets adhering to a polymer surface is presented.

Heparinised human blood with autologous 51Cr and 14C-serotonin labelled platelets is exposed to the polymer in Chandler loops rotated slowly for different periods. Initially the adhering platelets take up serotonin from non-adhering aggregating platelets by an active process and then slowly begin to release serotonin. Acetylsalicylic acid treated platelets adhere and take up serotonin to the same extent as untreated platelets, but do not undergo a release reaction.

Coating of the polymer surface with fibrinogen prior to blood exposure or with fibrin (achieved by thrombin treatment of the fibrarinogen adsorbate) does not affect the degree of adhesion and serotonin uptake, but inhibits the release reaction. When only fibrino-peptide A is split off with Defibrase from the fibrinogen layer the release function of the adhering platelets reappears.

It is concluded that the release mechanism of adhering platelets is not only dependent of the functional state of the platelet itself, but also on the ultimate composition of the protein layer to which the platelets adhere.
Effects of Aspirin and Sulfinpyrazone on Thromboxane and Prostacyclin Synthesis in Rabbits
Authors: McDonald J.W.D. , Cerskus A.L. , Ali M.
Published: May 2019

Arachidonic acid (AA) or collagen were infused into rabbits causing intravascular platelet aggregation with thrombocytopenia, hypotension and death. Thromboxane and prostacyclin synthesis were measured by radioimmunoassay of plasma TXB2 and 6-keto-PCF1α. The effects of pretreatment with aspirin (ASA) or sulfinpyrazone (SPZ) were assessed.

Death in drug-treated rabbits was always associated with elevations of plasma TXB2 (1-40 ng/ml) and of 6-keto-PGF1α (1-20 ng/ml). Collagen produced only small elevations of plasma TXB2 compared to AA but protection by ASA correlated better with inhibition of TXB2 and 6-keto-PGF1α synthesis than with inhibition of aggregation. Low dose ASA produced less inhibition of prostacyclin synthesis than high dose ASA but was less effective in preventing thromboxane synthesis and death.
Biphasic Prolonged Inhibition of Platelet Prostaglandin Biosynthesis Induced by Sulphinpyrazone
Authors: Butler K.D. , Pay G.F. , Wallis R.B. , White A.M.
Published: May 2019

In rabbits, high doses of sulphinpyrazone (S) result in prolonged inhibition of in vivo collagen-induced thrombocytopenia and platelet MDA production (Buchanan et al., Thromb. Res. 1978, 13, 883). After confirming these results we have shown in guinea pigs that 7 h after oral administration of S there was greater inhibition of AA-induced platelet aggregation than 1 h afterwards, i.e. at the peak plasma S concentration. Thus the concentration of AA needed to induce 50% maximal platelet aggregation at 7 h was raised 2.36 times and 4.73 times after 3 mg/kg and 10 mg/kg of S respectively. When in vivo thrombocytopenia was induced with the Arthus reaction the second phase of inhibition occurred down to a dose of 10 mg/kg p.o. with the first phase seen at higher doses. The inhibitory activity is associated with a high M.W. fraction of plasma proteins [50,000-100,000 Daltons). Phenylbutazone, aspirin and ticlopidine did not induce the plasma-borne effect.
Inhibition of the Activities of α2-Plasmin Inhibitor (PI) and CĪ Inhibitor (CĪ-Inh by the Synthetic Fibrinolytic Agent, 3-Hydroxypropyl Flufenamide (Flu-HPA)
Authors: Miles L.A. , Burnier J. , Verlander M. , Goodman M. , Kleiss A.J. , Griffin J.H.
Published: May 2019

Flu-HPA is one of a series of flufenamic acid derivatives that enhances plasminogen-de-pendent clot lysis in vitro. Studies of possible mechanisms of action of Flu-HPA were undertaken. The influence of Flu-HPA on the inhibition of purified plasmin by purified PI was studied. PI activity was assessed by its inhibition of the cleavage of the tripeptide S-2251(H-D-Val-Leu-Lys-pNA) by plasmin. Flu-HPA was dissolved in DMF or in n-ethanol and preincubated with PI before addition of plasmin. At FIu-HPA concentrations greater than ImM and up to 60mM, the inhibitory activity of PI was totally lost. The inhibitory effect of normal human plasma on plasmin was also completely abolished at concentrations of Flu-HPA between 2.5 and 40mM. The effect of Flu-HPA on the inhibition of purified plasma kallikrein by purified CĪ-Inh was also studied. CĪ-Inh activity was measured by its inhibition of cleavage of the tripeptide Bz-Pro-Phe-Arg-pNA by kallikrein. When FIu-HPA, dissolved in DMF or in methanol, was preincubated with CĪ-lnh, a concentration dependent inhibition of CĪ-Inh activity was observed. CĪ-Inh activity was abolished by concentrations of Flu-HPA greater than ImM. Flu-HPA also inhibited the activity of CĪ-Inh on purified Factor XIIa. These observations suggest that this flufenamic acid derivative may enhance fibrinolysis not only by inhabiting PI activity but also by decreasing the inactivation of plasminogen activators by CĪ-Inh.
Red Cell Filterability and Cardiovascular Risk Factors
Authors: Lagrue G. , Marcel G. A. , Faucher G. , Grumel J.M. , Beiler D. , Branellec A.
Published: May 2019

Red cell deformability, which allows cells of 7 μ diameter to flow through capillaries not larger than 3 μ, can be approached by the measure of blood filterability on Nuclepore 5 u filters (Reid H.L. et al. J. Clin. Pathol. 1976; 29: 855). Filterability seems reduced in certain states predisposing to thrombosis.

We have, in 72 patients, correlated red cell filterability (using two batches of filters) expressed in μl/s, with the number of cardiovascular risk factors present (high blood pressure, overweight, diabetes, hyperuricemia, hyperlipemia, smoking). Statistical analysis has been performed using regression analysis for each batch of filters, and both significance levels have been combined using Fisher’s method. The table illustrates the relationship between the number of risk factors and filterability. There is a statistical difference between groups as a whole (p<0.0l). The difference is highly significant between 0 and 4 risk factors (P<0.0005). Filterability decrease is also directly correlated with the number of cigarettes smoked per day (p<0.05). This is significant innen (p<0.05), but not in women.
Doppler Angiography for the Non-Invasive Investigation of the Carotid Artery in the Neck
Authors: Warlow Charles , Fish Peter
Published: May 2019

The accuracy, and possible clinical application of non-invasive ultra sound imaging of the cervical carotid, artery in patients with cerebrovascular disease has been investigated by comparing a pulsed multi-channel directional Doppler imaging devise (“MAVIS” - GEC Medical) with conventional carotid angiography. Of 23 normal or minimally diseased internal carotid arteries only 4 appeared as stenosed on Doppler angiography, the other 19 were normal; of 14 stenosed arteries only 1 appeared normal on imaging,1 appeared occluded end 12 showed clear images of stenosis; all 6 occluded arteries appeared occluded on ultra sound imaging. It was difficult to obtain satisfactory images of the external carotid arteries in all cases. The additional facility to display a velocity flow profile at preselected points along the arterialimage should increase the usefulness of this technique, particularly when there is only minor atheromatous irregularity or ulceration, and such a device will be briefly disrussed.
Soluble Fibrin in Plasma at 37°C Exists as DES-A Fibrin in Monomeric Form
Authors: Müller-Berghaus G. , Mahn I. , Krell W. , Kröhnke I.
Published: May 2019

We intended to find out in which state fibrin exists in plasma at 37°C. In order to generate fibrin, small amounts of thrombin were added to human plasma containing 125I-fibrinogen, Thereafter, thrombin was quenched with hirudin and “31I-fibrinogen added to observe possible 125I-fibrin-131I-fibrinogen complex formation. Gel filtration of this mixture on sepharose columns equilibrated with plasma revealed 2 peaks. At 20°C, but not at 37°C, peak A eluted in front of the fibrinogen peak contained fibrin-fibrinogen complexes. If isolated fractions of peak A eluted at 20°C were rechromatographed at 37°C, the material was mainly eluted in peak B (= fibrinogen peak). If isolated fractions of peak B eluted at 37°C were rechromatographed at 20°C, part of the 125I-radioactivity representing fibrin was eluted in peak A. As purified des-AB fibrin is eluted in polymeric form at 37°C and as purified des-A fibrin is eluted only in monomeric form at 37°C, we conclude from our experiments that fibrin genera, ting in plasma at 37°C exists in monomeric form. Furthermore, the experiments demonstrate that des-A fibrin does not form complexes with fibrinogen or other proteins at 37°C (Supported by the DFG).
Production by Thrombin of a Proteolytically Modified form of Antithrombin and Release of the Same form from the Antithrombin-Thrombin Complex
Authors: Fish W. , Björk I.
Published: May 2019

In addition to the in vitro formation of an inactive, stable complex (ATT) between thrombin (T) and the plasma serine protease inhibitor, antithrombin (AT), a sizeable proportion of AT is converted by T to a form which has lost the ability to inhibit T and which exhibits a reduced affinity for heparin. Under non-denaturing conditions, the isolated modified AT (ATm) could not be differentiated from native AT by hydrodynamic, electrophoretic or immunological analyses. Under reducing/denaturing conditions, ATm yielded two polypeptides, one of about 50000 and one of about 5000 daltons. Preliminary studies indicate that these are the result of a proteolytic cleavage by T of an arg-ser bond in the C-terminal end of the single-chain AT molecule. Dissociation of ATT by prolonged treatment in hot SDS or by treatment with hydoxylamine or ammonia in SDS produced only T and ATm. Under nondenaturing conditions, dissociation by the two latter agents released ATm and up to 40% of the potential T activity from ATT. However, under no conditions tested could intact AT be shown to be produced from dissociation of ATT. These results suggest that scission by T of a specific arg-ser bond in the active site region of AT, or, alternatively, conversion of this bond to a tetrahedral intermediate state, occurs during the formation of the inactive enzyme-inhibitor complex.
Observations on a New Variant of Hereditary Dysfibrinogenaemia (“Homburg”?) with Delayed Fibrin Polymerization and Without Clinical Abnormality
Authors: Dumitrescu K. , Taubert W. , Nienhaus Kh , Wenzel E.
Published: May 2019

31 families with delayed fibrin polymerization were described recently (Flute, Brit. Med. Bull. 33, 253, 1977). 14 of these showed no clinical abnormality. An abnormally low prothrombin time (28%) as well as low concentrations of clottable fibrinogen (Clauss:58mg%) were observed in a 21-year-old sportsman. Normal concentrations of factor II - XIII were quantitated. Platelet function (aggregation, morphology) as well as platelet count and bleeding time (Duke) were found to be normal. Neither biochemical nor clinical parameters (Aethanol gelation test, Antithrombin III) indicating DIC could be observed. Thrombincoagulase (TC) time (30.4"), reptilase (R) time (70) and plasmathrombin (PT) time (56) were found to be significantly prolonged, when purified human fibrinogen had been added to the patients’ plasma, the prothrombin time was found to be normal (65%), the TC, R, PT-times were significantly shortened. By heat precipitation (280 mg/100 ml) and immunological techniques, high fibrinogen concentrations were measured (Laurell-technique). When the plasma was incubated (37°C, 60 min.) with high amounts of thrombin calcium (50 NIH), no more than 60 mg fibrinogen/100 ml were removed. No increased risk of thromboembolism or hemorrhage in the family (N = 7) was observed. The fibrin formed in the patients’ blood is stabilized normally by F.-XIIIa.
Platelet Involvement After Myocardial Infarction
Authors: O’Brien J.R. , Handin R.I. , Etherington M.D. , Shuttleworth R.D. , Calwell W.
Published: May 2019

After myocardial Infarction (MI) the Heparin Thrombin Clotting Time (HTCT) of platelet (plt) poor plasma is short, indicating an increase in Heparin Neutralizing Activity (HNA). Plt. Factor 4 (PF4) released in vitro also neutralizes heparin. Does the plasma HTCT in MI reflect PF4 released in vivo? Fifteen patients with MI were compared with 23 controls. The mean HTCT was 43.9 sees, in controls and 14.8 sees, in patients. Plasma PF4 measured by RIA was abnormal in 3 patients but strictly normal in the other 12 (n = 12, mean 3.96 ng/ml; controls n = 22, mean 3.54). There was no correlation between the plasma PF4 and the HTCT. The plts, were frozen and thawed and the patients’ plts, released less HNA (0.17 units/109 plts.) relative to the controls (0.70 units/109plts.) and there was a tight inverse correlation between the plasma HTCT and the intra-plt. HNA. Plts, were isolated and stimulated maximally with thrombin; then malondialdehyde (MDA) production reflecting PG synthesis was monitored fluorometrically. Patients liberated less MDA. (415 ng/109plts.) than the controls (911 ng/plts.). All differences are significant, except the PF4. Plasma fibrinogen and α1 acid glycoprotein were also measured. Thus after an MI and presumably as a result of it, plts, are damaged or “exhausted” as reflected both by a decrease in an enzymatic process - PG synthesis and by a decrease in the content of HNA (? PF4). This interim report also clearly demonstrates that the plasma HTCT does not reflect the same attribute as plasma PF4 detected by RIA.
Platelet and Fibrinogen Production: Effect of Lipid a and Liposomes
Authors: Ramsey R.B. , Evatt B.L. , Alving B.M. , Alving C.R. , Hamner M.B.
Published: May 2019

Previous studies have demonstrated that endotoxin administered in a single intravenous dose produced increased platelet and fibrinogen production. To determine if the lipid moiety of the endotoxin was implicated, we studied the effect of intact endotoxin (E. coli 026:B6), lipid A, lipid A incorporated into liposomes, and lipid A solubilized in triethylamine (TEA) on platelet and fibrinogen production in male New Zealand rabbits. Animals received these preparations by single intravenous 1 h infusions of 1.0, 5.0, 10.0, 25.0, or 50.0 μg/kg body weight. Selenomethionine-75Se was injected 18 h after infusion, and the percentages of incorporation into platelet and fibrinogen were used to measure thrombopoiesis and fibrinogen synthesis. All 4 types of infusions increased both platelet production and fibrinogen synthesis and a dose-response relationship was observed; however, the threshold dose for stimulation varied with the type of material infused. More lipid A (by weight) was required to stimulate either platelets or fibrinogen than any of the other materials infused. When incorporated into liposomes or solubilized with TEA, lipid A produced a response similar to that produced by intact endotoxin. These data suggest that the lipid A moiety itself can stimulate platelet and fibrinogen production.
Plasminogen Activator and Factor VIII in Cirrhosis Following Infusion of Lysine Vasopressin and Triglycyl Lysine Vasopressin
Authors: Prowse C.V. , Douglas J.G. , Cash J.D.
Published: May 2019

While lysine vasopressin (LVP) has been used for many years for the treatment of cirrhotic patients with bleeding oesophageal varices, on the basis of its ability to cause splanchnic vasoconstriction, its use remains contraversial. Previous studies have shown that infusion of this drug to normal subjects results in a rapid increase in plasminogen activator and factor VIII. When infused into cirrhotic patients with varices we found that LVP caused a large increase in the, already raised, level of plasminogen activator (and factor VIII). This increase in fibrinolytic potential might contribute to poor deposition of fibrin in bleeding patients. .

In contrast infusion of the analogue triglycyl lysine vasopressin (GVP) in the same patients produced no change in plasminogen activator or factor VIII. GVP retains the vasoconstrictive properties of LVP and has a considerably longer half-life. In view of these advantages clinical trials of the use of GVP in gastrointestinal bleeding are warranted.
A Kinetic Study of Platelet Malondialdehyde (MDA) Formation and its Pharmacological Inhibition in rat Platelet-Rich Plasma (PRP)
Authors: Rajtar G. , Livio M. , Merino J. , Gaetano de G.
Published: May 2019

Experimental conditions were defined for kinetic analysis of MDA formation and inhibition in PRP prepared from citrated blood of male CD rats. Portions of PRP were stirred at 37°C with different concentrations of thrombin or arachidonic acid (AA) and the reaction was stopped by TCA. The rate of MDA generation (measured by a thiobarbiturate reaction) increased linearly during the first few min, reaching a plateau within 5 min. In all subsequent experiments PRP was incubated with thrombin for 2 ½ min or AA for 2 min. Km for thrombin was about 10 NIH U/ml and for AA 0.35 mM. Vmax were 0.27 and 0.40 nmol. MDA/1.4 × 109 platelets/min. Acetylsalicylic acid (ASA) or indomethacin (I) were incubated with PRP at 37°C for either 1 or 10 min before addition of thrombin or AA. Both drugs inhibited MDA formation in an apparently competitive way. Ki values were 10-30 times lower for I than for ASA. Ki values Of both drugs were about 20 times lower after 10 than after 1 min incubation. The inhibitory effect of either drug could be decreased by diluting the PRP samples, and appeared to be additive. These data suggest that: 1) rat platelets kept in their own plasma are a suitable model for studying the kinetics of MDA generation; 2) in rat platelets ASA and I act on the same enzymatic intracellular mechanism; 3) irreversible acetylation of platelet cyclo-oxygenase does not account for ASA’s initial inhibitory effect on platelet MDA generation.
Kinetic Study of Thrombogenesis on Stroke-Prone Spontaneously Hypertensive Rats (SHRSP). The Effect of Platelet-Inhibiting Drug and Vitamin E.
Authors: Kuramoto A. , Okita H. , Koganemaru S.
Published: May 2019

The increase of platelet production was demonstrated in stroke-prone SHR(SHRSP) after 10 weeks of age, probably due to increased platelet consumption compared with those in stroke-resistant SHR(SHRSR)(Koganemaru, S., et al. Thrombus. Haemostas.(Stuttgart)38:139, 1977). We extended our study to see the effect of aging, blood pressure, platelet-inhibiting drug and vitamin E(Vit.E) on this kinetic change. SHRSP(A1-sbF37-39) and SHRSR (BlF36-38) with matched age (5 weeks, 10 weeks and 9-12 months) and sex were used. The maximum uptake of 75se-methionine(75SeM) into platelets was not affected in both salt loading group and Clonidine group in SHRSP. A significant decrease was observed in dipyridamole group (0.096 ± 0.020, p<0.05). The platelet survival time was shortened in Vit. E deficient group of SHRSR; 3.0 days at 9-10 weeks. In Vit.E sufficient group of SHRSP. the platelet survival time was corrected to normal. The maximum uptake of 75SeM increased in both Vit.E deficient group of SHRSP (0.119 ± 0.010 vs 0.192 ± 0.030, p<0.01) and SHRSR (0.071 ± 0.016 vs 0.119 ± 0.018, p<0.01). The increased platelet production was not affected by blood pressure but it can be suppressed by treating with dipyridamole or giving Vit.E before 10 weeks of age.
Effect of Indobufen on Platelet Functions in Angina Patitents at Rest and During Exercise
Authors: Pogliani E. , Fantasia R. , Polli E.
Published: May 2019

Platelet aggregation induced by 3 concentrations of ADP and collagen was assessed in 30 patients with stable angina before and after exercise with bicyle ergometer. The patients received a single Oral 200 mg dose of indobufen and placebo according to a cross-over design in double-blind conditions. An increase in platelet sensitivity to both aggregating agents occurred when exercise was carried out after placebo, whereas indobufen markedly inhibited ADP- and collagen-induced aggregation. Circulating platelet aggregates increased in some patients during exercise after placebo but not after indobufen. In 20 patients 14C-serotonin release induced by collagen in PRP was investigated. The release was increased when placebo was administered before exercise but it was inhibited when indobufen was given before exercise. In the same patients MDA levels after thrombin-induced stimulation of platelets in PRP increased during exercise after placebo but decreased during exercise after indobufen. These results suggest that effort may be an important factor in activation of platelet functions and that the use of drugs blocking the arachidonate pathway and the release reaction may be appropriate in patients with angina.
Small Doses of Subcutaneous Heparin, Plasma Free-Fatty-Acids, and the Incidence of Ventricular Arrhythmias in Patients with Acute Myocardial Infarction
Authors: Arnesen H. , Skjæggestad Ø. , Wik N.
Published: May 2019

The frequency of deep vein thrombosis (DVT) diagnosed with the 125I-fibrinogen technique in patients with acute myocardial infarction (AMI) has been found to be reduced frcm about 20% without anticoagulation to about 5% with warfarin or small doses of subcutaneous heparin.

A somewhat higher incidence of ventricular tachycardia in patients with AMI treated with small doses of subcutaneous heparin has been reported. A possible mechanism might be heparin-induced activation of lipoprotein lipase with consequent increase of plasma free-fatty-acids (FFA), which have been found to be arrhythmogenic in patients with AMI.

In the present prospective trial, 99 patients with AMI and a history of less than 12 hours were allocated at random to treatment with subcutaneous heparin 5000 IU twice daily, or warfarin. In a randomized subsample of 21 patients fasting FFA analyses were performed before and 2 hours after the administration of anticoagulants on day 1 and 2.

No measurable increase in FFA concentrations was demonstrated in the heparin-treated patients, in spite of a significant influence on the thrombin clotting time.

The frequency of ventricular arrhythmias as detected by continous tape recordings was equal in the two treatment groups.

It is concluded that subcutaneous heparin 5 000 IU every 12 hours seems to be a safe measures of prophylaxis against venous thromboembolic ccmplications in patients with AMI.
Anti-Platelet and Vasodilatory Action of Prostacyclin /PGI2/ in Healthy Men and in Patients with Peripheral Artery Disease
Authors: Szczeklik A. , Gryglewski R.J. , Nizankowski R. , Szczeklik J. , Skawinski S. , Musial J.
Published: May 2019

Intravenous infusion of PGI2/2-20 ng/kg/min/ into 10 healthy men caused vasodilatation of skin vessels, moderate decrease in blood pressure, suppression of platelet aggregability to ADP, dispersion of circulating platelet aggregates, but no distinct changes in plasma and fibrinolytic clotting factors. These results prompted us to treat with PGI2, six patients with far-advanced arteriosclerosis obliterans. A cooled solution of PGI2/5 ng/kg/min/ in alkaline buffer was administered for 72-96 hrs into femoral artery, leading to warmness and rubor of extremity, inhibition of platelet aggregation, and increase in blood flow in peripheral muscles and skin as measured with radioactive xenon. The clinical results were very good as manifested by relieve of pain, healing of chronic ulcers and resolution of deep necrosis. PGI2 due to its strong anti-platelet and vasodilatory properties seems a promising agent in therapy of peripheral artery disease.
Requirement of Factor VIII for the Generation of Xa in a Purified System and in Plasma.
Authors: Ofosu F. , Giles A. , Hirsh J. , Blajchman M.
Published: May 2019

The requirements of purified Factors IXa, VIII and X for Factor Xa generation were studied by quantitating the Factor Xa produced using the amidolytic action of Xa on the synthetic chromogenic substrate S-2222. The rate of Factor Xa generation was found to be dependent on the initial concentrations of Factors VIII, IXa and X. The exposure of Factor VIII to thrombin (0.001 U/ml) for up to 10 minutes increased the rate of Factor Xa generation tenfold. Longer exposures (>15 minutes) led to inactivation of Factor VIII. Factor Xa production was demonstrably slower in plasma, even under conditions where the Factor IXa inhibitory activity of plasma had been neutralized by excess Factor IXa. Using purified clotting factors, calcium and phospholipid, no Factor Xa was formed in the absence of Factor VIII. In contrast, the addition of Factor IXa to plasma obtained cither from severe hemophiliacs or normal plasma which had been immunodepleted of coagulant and antigenic Factor VIII and had no demonstrable Factor VIII coagulant activity resulted in the generation of Factor Xa. These findings suggest that Factor VIII is required for the activation of Factor X by Factor IXa in the purified system but that mechanisms appear to be present in plasma which enable Factor IXa to participate in the activation of Factor X even when Factor VIII is absein.
Prevention of Venous Thromboembolism
Authors: Salzman E.W.
Published: May 2019

Prospective clinical trials have shown that reduction in frequency of deep vein thrombosis (DVT) and pulmonary embolism (PE) can be achieved by physical preventive methods or by administration of antithrombotic drugs. There is however confusion concerning the best form of prophylaxis for different patient groups, difficulty in translating results of clinical trials directly into practice, and ignorance of the dollar cost of the various regimes. Critical analysis of the literature permits prediction of expected frequencies of fatal PE and of DVT and complications of therapy, but questions remain. The prevalence of silent PE in different populations is not established. Definition of a high risk group is imprecise, so patients at lesser risk are exposed to the side effects of antithrombotic prophylaxis with little to gain. Since the natural history of DVT is poorly understood, side effects of anticoagulant therapy are also inflicted on many with little likelihood of PE for the benefit of a few susceptible patients. Apparent differences in rate of occurrance of DVT on the two sides of the Atlantic are suggested but unproven and not explained.

Despite these uncertainties, determination of cost/effectiveness of preventive programs is possible and can provide a workable guide to clinical practice.
Prostacyclin (PGI2) and Platelet Adherence to Vascular Cells
Authors: Hoak J.C. , Smith J.B. , Fry G.L. , Czervionke R.D. , Haycraft D.L.
Published: May 2019

Platelet adherence and PGI2 release were studied using cultures of endothelial cells (EN) and fibroblasts (F) from vessels of human umbilical cords, and mouse hemangioendothelioma (MHE) cells. MHE cells form vascular channels in-vivo which develop thrombi as part of a consumption coagulopathy. A radioimmunoassay for 6-keto-PGF1α was used to measure PGI2 released from cell monolayers incubated with 0.5U bovine thrombin or incubation medium control (IM). For platelet adherence studies, 51Cr-labeled labeled platelets were added to untreated or 1 mM aspirin (ASA)-treated monolayers after IM or 0.5U thrombin. In additional adherence studies, exogenous PGI2 was added before thrombin and Platelets. Concentrations of 6-keto-PGF1α (nM) after thrombin were: 62.5 ± 8.8 venous. EN, <9.2 ± 2.4 arterial EN, and <3.0 for F, MHE. and ASA-treated venous EN. Incubation with 150 nM exogenous PGI2 decreased thromhin-induced platelet adherence to ASA-treated venous EN (52 to 4%), arterial EN (61 to 10%), MHE (65 to 3%), F (75 to 24%) and to empty culture dish controls (81 to 43%). The failure of PGI2 to produce more effective inhibition of platelet adherence to non-endothelial surfaces suggests that the endothelium may possess additional nonthrombogenic components.
The Role of Phospholipids and Factor Va in the Mechanism of Prothrombin Activation
Authors: Rosing J. , Tans G. , Govers-Riemslag J.W.P. , Zwaal R.F.A. , Hemker H.C.
Published: May 2019

The kinetic parameters of the conversion of prothrombin into thrombin by activated clotting factor X (factor Xa) have been determined in the absence and presence of Ca2+, phospholipid (phosphatidyl serine/phosphatidylcholine vesicles) and activated blood clotting factor V (factor Va). In free solution the Km for prothrombin is 298 μM which is well above its plasma concentration of 4μM. Under these conditions the Vmax of thrombin formation is 1.25 Moles min-1 Mole Xa
-1. When phospholipid is present the km for prothrombin drops to 0.1μM while the Vmax is only slightly affected (3 Moles min-1 Mo Le Xa
-1). For the complete prothrombin activating complex consisting of factor Xa, factor Va, Ca2+ and phospholipids the kinetic constants greatly favour thrombin formation. A for prothrombin of 0.26μM and a Vmax of 2130 Moles min-1 Mole xa
-1 are measured under these conditions. These results help to elucidate the role of phospholipid and factor Va in prothrombin activation. The earlier observed rate enhancements caused by phospholipid and factor Va are explained as effects on the Km for prothrombin and the Vmax of thrombin formation, respectively. The changes of the kinetic parameters for prothrombinase complexes of various composition will be considered with respect to the function of the accessory components in the mechanism of prothrombin activation. Implications of these data for in vivo blood coagulation will be discussed.
Immunochemical Studies on Antithrombin III
Authors: McKay E.J.
Published: May 2019

Depressed Antithrombin III (AT) levels Increase thrombic tendency in man, therefore value in assaying this protein has been established. Immunochemical analysis of AT in clinical disease has however proved controversial, consequently systematic studies were undertaken to rationalize the requirements necessary to optimise these methods in particular electro-Immunoassay. The known binding affinity of AT for heparin has been exploited to differentiate high affinity AT from its inhibitor - protease complexes and has resulted in reports stating that heparin added to the agar gel prior to electrophoresis significantly reduces the time required for completion of antigen/antibody complexes. Our studies however have demonstrated that the antibody required for quantitative analysis must be capable of not only reacting with “native” antigenic determinants of AT but also with “neo” antigens that are exposed when inhibitor-protease complexes are formed. Heparin should not be used in the test protocol, for it has a paradoxical effect on Immunopreclpltation in gels, masking some antigenic determinants of unbound - high affinity AT on one hand, and appear to disrupt the Immunoprecipitin “rocket” formed with the inhibitor-protease complexes during electrophoresis on the other.