bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of Ibuprofen on Platelet Function in Normal Subjects and Hemophiliacs
Authors: McIntyre B.A. , Philp R.B. , Inwood M.J.
Published: May 2019

Most anti-inflammatory analgesics are contraindicated in hemophiliacs because of inhibition of platelets, erosion of gastric mucosa, and prolongation of bleeding time. New proprionic acid derivatives are claimed to have a lower incidence of gastrointestinal bleeding and less effect on the hemostatic system. One of these (ibuprofen, Motrin, Upjohn) was given (600 mg per os) to normal subjects and hemophiliacs on a random, double-blind basis (lactose placebo) and platelet adhesiveness and aggregation, platelet and red-cell counts, % packed cells, % hemoglobin and modified Ivy bleeding time were measured before and 2 and 24 hours (hr) after drug. Pre-drug and 24 hr post-drug values were normal but at 2 hr post-drug, ADP, adrenaline and collagen aggregations were inhibited and bleeding times slightly but significantly prolonged in the ibuprofen-treated normal subjects. Similar results were obtained in the ibuprofen-treated hemophiliacs but prolongation of bleeding time was not significant. In vitro studies with citrated platelet-rich plasma showed that ibuprofen inhibits platelet aggregation and synthesis of prostaglandins by platelets. Thus the results suggest that ibuprofen may be given to hemophiliacs rather than some of the older anti-inflammatory agents presently in use.
Autosomal Factor VIII Deficiency in Rabbits: Size Variations of Rabbit Factor VIII.
Authors: Benson R.E. , Dodds W.J.
Published: May 2019

Many rabbits from our Flemish Giant-Chinchilla colony have moderate to severely reduced levels of factor VIII coagulant activity (FVIII-C). Some have shown prolonged bleeding after venipunctures and gastrointestinal and intramuscular hemorrhages. Genetic studies indicate autosomal inheritance. Gel filtration of plasma from these rabbits by the method of Rick et al. (Blood, 49, 209, 1977) at 25°C, pH 6.8 revealed two distinct peaks of FVIII-C; the majority of activity eluting as high molecular weight (HMW) material at the void volume (V°) followed by a much smaller low molecular weight (LMW) peak eluting close to that of fibrinogen. By contrast, filtration of plasma from New Zealand (NZ) rabbits produced threefold greater protein at the V° and equal amounts of HMW and LMW FVIII-C. Increasing the pH to 7.4 had little effect on FVIII-C recovery, although filtration at 4°C virtually abolished the HMW FVIII-C peak of NZ plasma. Rat antiserum (AS) to rabbit HMW FVIII-C, absorbed with precipitate low in FVIII-C, detected precipitating antigen in both HMW and LMW fractions. After absorption with rabbit fibrinogen, the AS no longer detected HMW V° material. The antigenic relationship between HMW and LMW FVIII-C and fibrinogen thus remains unclear. The differences in amount of HMW protein and the ratio of HMW to LMW FVIII-C suggest that in comparison to NZ rabbits our animals have a variant factor VIII molecule as well as low FVIII-C.
The Role of the Consumer-Patient in the Growth of a Comprehensive Hemophilia Care Unit
Authors: Child Dorothy , Kopicki John , VanHarlingen David
Published: May 2019

The most efficient means for dealing with the myriad problems of hemophilia is through a comprehensive hemophilia care center involving medical and paramedical personnel, as well as numerous non-medical supportive services. As in all consumer services, however, problems arise when the care center team and the individuals who comprise it are unaware of the true needs of the patients they serve. To reduce such problems, an active and vocal group of hemophilia families and friends can provide insights otherwise unattainable. Working in cooperation with the center, they can provide guidance and seek funding to reduce the cost paid by the patient.

We describe the role of consumer-patients in the initiation and development of New Jersey’s first and only comprehensive care center for hemophilia. We also present a detailed examination of the kinds of assistance which has been provided and which is being provided in the growth and expansion of the center’s team and its activities. A plan for a statewide treatment network centered around this treatment facility will be presented.
An Evaluation of Hospital Facilities for Canadian Hemophiliacs
Authors: George R. , Inwood M.J.
Published: May 2019

Wide differences exist in the standard of facilities currently available to Canadian hemophiliacs. In order to categorize these facilities a survey was conducted by members of the Canadian Hemophilia Society of 106 hospitals currently treating hemophiliacs in the 10 provinces of Canada. Two of the provinces (Quebec and British Columbia) had incomplete surveys performed. A scoring system was devised to grade the hospitals from level I to level V, with the following results. Eleven level I (comprehensive treatment centres as defined by the World Federation of Hemophilia) were identified in seven provinces. Seventeen level II (facilities considered acceptable for regularly attending hemophiliacs) were present in 8 provinces. Thirty five level III hospitals (suitable for occasional and uncomplicated therapy) were situated in 8 provinces. Thirty five level IV facilities in 7 provinces and eight level V facilities in 4 provinces were considered to have inadequate facilities for routine or sporadic therapy. Only 63 of the 106 (level I and II and III) had suitable facilities for the reception of hemophiliacs. Of these 63 hospitals only 28 were providing materials and services for home infusion programs. In order that Canada can achieve a standard of facilities comparable to other countries actively involved in hemophilia care, hospitals in all provinces must be upgraded, along with an increased number and distribution of comprehensive treatment centres.
Hemophilia a and Variant of von Willebrand’s Disease in one Family
Authors: Sutor A.H. , Böttcher D. , Hasler K.
Published: May 2019

A 6 years old boy with the admitting diagnosis hemophilia A was transferred to our hospital because of persistent bleeding episodes inspite of adequate treatment with AHF. Laboratory data were as follow: F VIII coagulant activity 1.7%, inconstantly prolonged bleeding time (hemorrhagometry), inconstantly reduced platelet adhesiveness, normal IVY-bleeding time, normal platelet aggregation with ADP and collagen, reduced Ristocetin-induced aggregation with PRP, reduced Ristocetin-cofactor activity, normal F VIII associated antigen. An inhibitor against F VIII could be excluded. The bleeding stopped and the pathological coagulation findings were corrected after substitution therapy with isoagglutininfree cryoprecipitate. Because of these findings we made the diagnosis of variant of von Willebrand’s disease. His 8 years old brother had classical hemophilia A with a F VIII coagulant activity of 1.8% and all other coagulation parameters mentioned above within normal limits. In the mother a carrier state could not be detected. In the mothers and fathers family bleeders are not known.
Congenital Dysfibrinogenemia. (Fibrinogen OSLO II)
Authors: Godal H.C. , Brosstad F. , Kierulf P.
Published: May 2019

An autosomally inherited, qualitative fibrinogen defect, associated with prolonged thrombin clotting time, low plasma fibrinogen when assayed by a fibrin polymerization test and large amounts of fibrinogen antigen determinants in the supernatant after clotting, is presented. The plasma fibrinogen level was normal when assayed by an immunological technique or by quantitation of insoluble fibrin under conditions in which fibrin polymerization is enhanced. As judged from N-terminal amino acid analyses, fibrinopeptides were split off at normal speed, and the subunit chains of the fibrinogen appeared normal when examined on Polyacrylamide gels. The abnormality was not associated with bleeding tendency, and other routine coagulation tests gave normal results. The findings are in accordance with the concept of defective fibrin polymerization.
The Effect of Fibrinopeptide B Release on Temperature Dependent Fibrin Association
Authors: Edgar W. , Prentice C.R.M.
Published: May 2019

The effect of temperature on soluble fibrin complexes was studied using Biogel filtration and chromatography on fibrinogen-sepharose 4B at 20°C and 37°C. Complexes were formed by plasmin digestion of non-crosslinked fibrin produced by thrombin, ancrod, thrombin in 2M urea, or ancrod plus Agkistrodon contortrix venom. Thrombin removes fibrinopeptides A and B, ancrod removes fibrinopeptide A, while A. contortrix enzyme removes first fibrinopeptide B, followed by A. Complexes containing neither fibrinopeptide A or B, formed by digestion of fibrin produced by thrombin, or by ancrod plus Agkistrodon contortrix, were stable at 37°C. In contrast, complexes which retained fibrinopeptide B, formed from fibrin produced by ancrod or by thrombin in 2M urea, were unstable at 37°C. Fibrin polymerization was necessary for the stability of fibrin complexes. Complexes from plasmin digests of fibrin produced by ancrod plus A. contortrix enzyme in 2M urea, where no clot formation occurred, were unstable at 37°C. Using affinity chromatography, plasmin digests of thrombin-fibrin bound to fibrinogen-sepharose at 37°C, whereas those from ancrod-fibrin did not. A second set of polymerization sites in fibrinogen are proposed, distinct from the N-DSK and carboxyterminal sites. These are activated by removal of fibrinopeptide B and require clot formation.
Heparin Therapy in Ambulatory, Out-Patient Human Subjects by Continuous, Long Term, Intravenous Infusion Employing a Portable Pump
Authors: Samuels A.J. , Sullivan R.D.
Published: May 2019

A method is described for continuous, long term, ambulatory, out-patient, intravenous heparin therapy in human subjects. A portable light-weight pump originally designed for ambulatory, continuous intravascular chemotherapy in patients with malignant neoplastic disease was employed for this purpose. Six studies in four patients have been conducted to date. Diagnoses included: carcinoma of the breast, 65 year old female, thrombophlebitis of the iliofemoral veins and inferior vena cava thrombosis; carcinoma of the tonsil, 49 year old female, pulmonary emboli; carcinoma of the lung, 51 year old male; carcinoma of the lung, 47 year old female. In one patient, three individual courses of therapy were conducted for 56, 105 and 111 days. In the remainder, intravenous therapy was continuous for 58, 39 and 26 days respectively. The anticoagulant effect was almost continuously optimal and free of unusual, bleeding. There have been no adverse effects from the use of heparin in this fashion. Infection did not occur at the site of insertion of the intravenous catheter. The method is recommended for trial in selected patients with recurrent thrombotic disease and for experimental therapy in patients with malignant neoplastic disease where a trial of such adjuvant therapy may be indicated and feasible, on an ambulatory, out-patient basis.
The Activity of Heparin Preparations Studied by Amidolytic and Clotting Methods
Authors: Teien A.N. , Abildgaard U. , Höök M. , Lindahl U.
Published: May 2019

Two heparin standards, heparin isolated from human mastocytoma tissue, four commercial heparins and two heparin preparations separated by affinity chromatography (“High affinity heparin”=HAH and “Low affinity heparin”=LAH) were assayed by the activated partial thromboplastin time method (APTT), the calcium thrombin time method (CaTT) and two amidolytic methods (measuring the accelerating effect of heparin on the inactivation of thrombin or factor Xa by antithrombin III), with and without plasma in the test system. The specific activities of the various heparins were expressed relative to that of the 3rd. Int. Standard (=100). Found specific activities ranged 3 - 198 (LAH and HAH, respectively). In all assay systems HAH had the highest specific activity, followed by one of the commercial preparations and the 3rd Int. Standard. LAH and human heparin had very low specific activities, except in the APTT test system, an assay method which in addition mirrors other anticoagulant effects of heparin than the acceleration of antithrombin III. Apart from the higher effect of LAH and human heparin on the APTT, the difference in specific activities found for each individual heparin preparation with these various assay methods was slight.

In view of the reproducibility and simplicity of the amidolytic methods, it is suggested that they be adapted for heparin standardization.
The Arrangement of Glycolipids in the Human Platelet Plasma Membrane
Authors: Schick P.K. , MacDonald K.A.
Published: May 2019

Glycolipids present on the platelet surface were investigated with an external labeling technique. Washed platelets were incubated with galactose oxidase followed by reduction and labeling with 3H-sodium borohydride. Neither galactose oxidase nor sodium borohydride caused platelet leakage or release of platelet serotonin during the experimental procedure. Glycolipids were extracted, purified, separated by thin-layer chromatography, and quantitated by the fluorometric assay of sphingosine using fluorescamine. Platelet glycolipids represented 0.4% total platelet lipids. The following four platelet neutral glycolipids were identified and expressed as % of total platelet glycolipids: 41% trihexosylceramide, 29% globoside, 26% lactosylceramide, and 4% cerebroside. Only globoside and to a lesser extent trihexosylceramide were heavily labeled by the galactose oxidase technique. The study indicates that globoside and trihexosylceramide are located on the platelet surface. Glycolipids present on the platelet surface conceivably play an important role in platelet physiological activities.
Evidence for a Central Role for Calcium Ions in Regulation of Platelet Function
Authors: Detwiler T.C. , Charo I.F. , Feinman R.D.
Published: May 2019

It is generally believed that calcium ions play a key role in regulation of platelet function. This is based on 3 types of evidence.

1. Analogies with other cells. Calcium ions are known to trigger secretion and contraction in many cells, possibly reflecting a general role for calcium in all secretion and contraction.

2. Indirect evidence. Platelet aggregation and secretion are induced by divalent cation ionophores. The response to the ionophore A23187 is identical to that induced by other potent stimuli.

3. Direct evidence. Platelet activation can be blocked by drugs (e.g., certain local anaesthetics) that block release of calcium ions from sarcoplasmic reticulum; the inhibition can be overcome by addition of extracellular calcium in the presence of a calcium ionophore.

While this does not constitute definitive proof, the central role for calcium ions remains an attractive hypothesis that justifies attempts to further define calcium pools and fluxes in platelets.
The Role of Myosin Phosphorylation in Regulating Actin-Myosin Interaction in Human Blood Platelets
Authors: Adelstein R.S. , Barylko B. , Conti M.A.
Published: May 2019

Human blood platelets contain actin and myosin which are similar in many of their structural and biochemical properties to the contractile proteins found in muscle. Platelet myosin is composed of six polypeptide chains, two heavy chains of 200,000 daltons and two pair of light chains of 20,000 and 15,000 daltons. Platelets also contain a specific enzyme capable of phosphory-lating the 20,000 dalton light chain of platelet myosin. The kinase has been purified 600-fold and has been shown not to require Ca2+ or cyclic AMP for its activity (Daniel, J.L. & Adelstein, R.S., Biochemistry 15, 2370, 1976). Phosphorylation of platelet myosin results in a 7-10 fold increase in the actin-activated myosin ATPase activity but has no effect on the myosin K+-EDTA ATPase activity measured in 0.5 M KCl. The phosphatase responsible for dephosphorylation of platelet myosin has been partially purified. Dephosphorylation of platelet myosin results in a decrease in the actin-activated myosin ATPase activity, without affecting the myosin ATPase activity measured in the presence of K+-EDTA in 0.5 M KCl.

Thus, using the actin-activated ATPase activity as an indication of actin-myosin interaction, myosin phosphorylation-dephosphorylation appears to function as a reversible regulatory mechanism. That this type of regulation is not confined to platelets is indicated by finding a similar mechanism in proliferative rat myoblasts grown in culture (Scordilis, S.P., and Adelstein, R.S., Biophysical J., 17, 268a, 1977) and guinea pig vas deferens smooth muscle (Chacko, S., et al., PNAS. 74, 129, 1977).B.B. is on leave from the Nencki Institute, Warsaw, Poland.
Separation of Degranulated Platelets from Normal Platelets
Authors: Cieslar P. , Greenberg J.P. , Packham M.A. , Kinlough-Rathbone R.L. , Mustard J.F.
Published: May 2019

Platelets degranulated by thrombin (TDP) can be recovered, are effective in hemostasis and survive normally upon infusion into rabbits. Two approaches to determine whether platelets have been degranulated in vivo are: (1) measurement of circulating released materials; (2) detection of circulating degranulated platelets. We have used arabino-galactan (Stractan II) density gradient centrifugation to separate normal and degranulated platelets. The following distribution was obtained with washed rabbit platelets.

The serotonin, PF4 and adenine nucleotide contents of the TDP were less than those of normal platelets and the TDP in fraction I had the lowest amounts. When TDP were labeled with 51cr and mixed with equal numbers of normal platelets, 85% of the platelets in fraction I were found to be TDP. 51Cr-TDP were injected into normal rabbits and reharvested after 18 hours. The greatest proportion of TDP was isolated in fraction I. Thus this method may make it possible to separate platelets that have lost their granule contents during participation in reversible thrombus formation in vivo.

(* Visiting Fellow from the Faculty of Medicine, Charles University, Prague, Czechoslovakia.)
Identity and Kinetic Properties of a Protein (ACTIN) which Binds Metabolic Platelet ADP in an Ethanol-Insoluble Form
Authors: Daniel J.L. , Robkin L. , Holmsen H.
Published: May 2019

Thirty to fifty percent of the metabolic ADP in platelets is bound to protein and the complex is insoluble in ethanol, from which it can be isolated from platelets and subsequently extracted by perchloric acid. We have tentatively identified the protein as actin based on the following : 1) comparison of the amount of ethanol insoluble ADP and the actin content of the platelets and other nucleotide-binding proteins are not present in sufficient amounts; 2) its behavior on a gel filtration column; 3) its solubility properties; 4) the exchangeability of the bound ADP in platelet lysates. The kinetic behavior of the “actin”-bound nucleotide in theintactplatelet was studies in cells that had their (ethanol-soluble and-insoluble) metabolic pool preequilibrated with 14C-adenine. The change with time of the 3H/14C ratio in the pools after addition ofH-adenine was measured. In the resting platelet the 3H/14C ratio increased at the same rate in the ethanol soluble and-insoluble pool, indicating rapid equilibrium between the pools.However, when 3H-adenine was added to 14C-labelled platelets that had been incubated with antimycin and deoxyglucose, the 3H/14C ratio in the protein bound pool clearly lagged behind that of the soluble pool, the 3H/14C in the individual nucleotides suggested that soluble ATP was a precursor for protein-bound ADP. These results indicate that ATP is concumed to maintain a certain equilibrium between G-and F-actin in resting platelets. This technique 3H/14C ratio of studying the kinetic platelets parameters of “actin”-bound ADP may be of use for elucidating the role of contractile proteins in platelet functions.
Interaction of the First Component of Complement (CI) in Platelet Adhesion and Aggregation Induced by Collagen
Authors: Wautier J.L. , Souchon H. , Peltier A.P. , Caen J.P.
Published: May 2019

The attachment of CI to human platelets through Cls and the inhibition of platelet aggregation induced by collagen by purified Clq prompted us to investigate a possible interaction between platelet associated CI and collagen. Clq is a molecule composed of two moieties : a collagen like region and a globular region composed of polypeptides which may be isolated following hydrolysis by collagenase and pepsin respectively. Pure CI subcomponents (Clq, Clr, Cls), fractions of Clq (pepsin or collagenase treated) and specific antisera (anti Clq, anti Cls) were tested for their effect on collagen-induced aggregation and adhesion of gel filtered platelets. The pepsin derived fragment of Clq containing the collagen like region inhibited platelet aggregation and adhesion to collagen while collagenase treated Clq (globular moiety) did not modify the reaction. Preincubation of collagen with Cls inhibited platelet adhesion and aggregation. In other experiments platelets modified by anti Cls did not react with collagen while anti Clq, anti C3 or anti fibrinogen did not influence the reaction with collagen. These results suggest that the collagen like region of Clq binds to Cls with a possible reaction between Cls associated on the platelet or a platelet structure similar to Cls and collagen.
Participation of γ-Carboxyglutamic Acid Residues in Metal Binding: 13C nmr Studies of The Interaction of Lanthanide Ions with Bovine Prothrombin Fragment (12-44)
Authors: Furie B.C. , Blumenstein M. , Furie B.
Published: May 2019

Comparative studies of Ca(II) binding to bovine prothrombin and abnormal prothrombin have led to the hypothesis that γ-carboxyglutamic acid (Gla) residues either participate directly in metal liganding or stabilize the tertiary structure of the protein to facilitate metal binding, or both. Extending previous studies of the binding of lanthanide ions to factor X, prothrombin and prothrombin fragment 1, the interaction of lanthanide ions with prothrombin fragment (12-44), which contains eight Gla residues, has been examined by natural abundanceC NMR spectroscopy at 67.88 mHz. The relationship between bound metal ions and carbon atoms in fragment (12-44) was determined using paramagnetic Pr(III) and Eu(III) based upon the strategy that the magnitude of the change in the chemical shift of resonances of the carbon atoms induced by bound metals is related to the interatomic distance between bound metal and carbon nuclei. Assignments of the resonances in the 13C spectrum of fragment (12-44) were based upon our studies of the chemical shifts of carbon resonances in DL-Gla and known chemical shifts of carbon resonances of the other amino acid residues. Titration of fragment (12-44) with Pr(III) or Eu(III) was accompanied by a downfield or upfield shift, respectively, of the γ-carboxyl carbon resonances and no shifts were observed for other carbon resonances. The shift of resonances within the γ-carboxyl carbon envelope appeared selective, suggesting the non-equivalence of the relationship of the γ-carboxyl group of the eight Gla residues to the bound metal ions. These data offer direct evidence that some, but perhaps not all, Gla residues participate in metal liganding.
Effects of Infused Products of Tissue Injury on Generation of Circulating Fibrin Monomer, Condition of the Endothelium and Deposition of Blood Elements on Jugular Veins in the Dog
Authors: Stewart G.J. , Schaub R.G. , Niewiarowski S.
Published: May 2019

In previous studies we found that major surgery in dogs resulted in generation of fibrin monomer and adhesion of blood cells to veins. In this study we investigated the effects of a 4-hour intravenous infusion of products of tissue injury on early events in deposition of thrombotic material on veins. Substances infused were (per minute):histamine, 150 μg; serotonin, 200 yg; bradykinin, 6 μg; and, thromboplastin, 0.04 ml. Fibrin monomer was measured in pre- and post-infusion plasma. Jugular veins were prepared for scanning electron microscopy after removal of >90% of blood by whole body perfusion prior to asystole. No fibrin monomer was found in pre-infusion plasmas and in 1/4 post-infusion controls at low (1-20) dilution of protamine sulfate. However, monomer was found at higher dilution (1:80, 1:160) in post-infusion plasmas from all treatments. Even in the presence of circulating monomer no fibrin accumulated on the luminal surface of veins. Adhesion of blood cells to vessels was rare in dogs infused with Tyrode’s solution or thromboplastin but was common in dogs infused with histamine, serotonin or bradykinin. Adhering cells were mostly leukocytes (PMN, giant cells) and erythrocytes with few platelets. Cells adhered to grossly intact but altered endothelium and to areas of endothelial disruption at side branches and valves. These observations show that: (1) infusion of vasoactive substances caused generation of considerable monomer; (2) in vivo generation of monomer was not sufficient to lead to thrombosis of medium sized veins, (3) infusion of vasoactive substances caused leukocytes and erythrocytes to adhere to altered endothelium.
Evidence for the Presence in Human Plasma of a Streptokinase Co-Factor -Separable from Plasminogen
Authors: Korsan-Bengtsen K. , Johnsen M. , Gustavsson G.
Published: May 2019

Our original observation was that plasma made plasminogen-free by lysine sepharos chromatography accelerated the streptokinase activation of purified plasminogen when measured by means of a chromogenic peptide substrate. The substance with this property is preciptable from plasma with 3-5 % polyethylen-glycol (PEG) , it is eluted near the front on Sepharos 6 B gelfiltration and does not move on electrophoresis at pH 8.6. The activity is not lost on heating to 65°C for 10 min. Traces of immunoglobulins, C-3, C-4, alfa and betalipoproteins present in the purified co-factor preparations were removed by means of affinity chromatography without loss of co-factor activity. The co-factor does not in itself have any proteolytic activity and it cannot be activated with streptokinase or urokinase to give proteolytic activity. It is a potent accelerator of the streptokinase plasminogen activation. One of the two tested commercial streptokinase preparations was found to be influenced more than the other by the co-factor. When this preparation was run on a Sephadex 200 column a certain fraction with a high co-factor “sensitivity” was identified. We have not been able to separate two distinct activities from the streptokinase preparation.
Thrombokinetic Study to Predict Epidodes of Thrombogenesis
Authors: Koganemaru S. , Taketomi Y. , Kuramoto A. , Okamoto K.
Published: May 2019

To evaluate platelet participation in the development of cerebrovascular lesion;cerebral infarction and / or bleeding in animal model, platelet survival and thrombopoiesis were measured by 75 Se-selenomethionine incorporation, using spontaneously hypertensive rat-stroke prone(SHR-SP. Okamoto-Yamori Strain). Our study on this newly established strain, was focused on the early period of life(5 and 10 weeks), when their blood pressure begin to increase above normal and spontaneous hypertension is established. Maximum peak of 75 Se-selenomethionine uptake into the platelets in circulation came on 3rd day after intravenous injection in SHR-SP as well as in controls(SHR-stroke resistant and normotensive Wistar rat). The value of incorporated activity in SHR-SP of 10 weeks with developing high blood pressure was significantly higher than those of 5 weeks and the controls. Expected survival obtained from the results by 75 Se-selenomethionine, suggested short survival of SHR-SP in 10 weeks of age, compared with the control. Short survival with increased turnover indicated increased consumption due to extrinsic moments, which might be brought about by vascular damage associated with the development of hypertension.
The Effect of a Combination of Sulfinpyrazone (SUL) & Aspirin (ASA) on Platelet Survival & Thrombus Size in an Experimental Thrombosis Model
Authors: Giles A.R. , Buchanan M.R. , Hirsh J.
Published: May 2019

Platelet survival (PS) may be decreased in some thromboembolic disorders. Treatment of patients with drugs that suppress platelet function may or may not be associated with normalisation of PS. It is uncertain, however, whether a lack of influence on PS necessarily indicates lack of an antithrombotic effect. We have examined this problem in an arterial thrombosis model in rabbits. A non-occlusive mural thrombus was produced in the aortic arch and PS studied using 51Cr-labelled homologous platelets. Thrombus size was assessed by measuring 51Cr accretion. Three groups of 15 animals were studied as follows:group A - thrombus induced 30 min prior to start of PS, group B- as for group A but animals treated with SUL (25 mg/Kg) and ASA (25 mg/Kg) prior to thrombus induction and 12 hrly thereafter and group C- sham operated controls. Animals in group B showed a 45% reduction in thrombus size when compared with group A (p<0.05). The platelet Tl/2 in groups A, B, and C were 25.7, 25.6, and 33.8 hrs respectively. The difference between groups A and C and between groups B and C was significant (p<0.01).Thus, treatment with SUL and ASA reduced thrombus size but did not modify the change in PS associated with thrombus induction. Re-examination of PS data by 7 different analytical methods established that all were equally sensitive in discriminating thrombosis but all failed to detect a drug effect. It is concluded that failure to modify platelet survival does not necessarily exclude an antithrombotic effect and that this apparent lack of sensitivity does not relate to the method of data analysis used.
Procoagulant Activity of Rabbit Peritoneal WBC: Effects of Cortisone and Endotoxin
Authors: Robinson A.J. , Rapaport S.I. , Brown S.F.
Published: May 2019

This study tested the hypothesis that cortisone increases the procoagulant activity of WBC of rabbits injected with endotoxin. Rabbits were given either saline (control) or cortisone acetate, 25 mg daily for 5 days intramuscularly. On day 5, animals received intravenously either 1 ml of saline or of E. coli endotoxin, 100 μg/kg. Peritoneal leukocytes were collected 1 hour (h) later. Two types of suspensions were prepared following intraperitoneal starch:primarily heterophils (16 h after starch), primarily macrophages (72 h after starch). Suspensions were washed, counted, and examined for clotting activity in a one-stage thromboplastin time assay. Activity was expressed in units (U) from a reference curve made with rabbit brain thromboplastin (clotting time of 1/10 dilution, 12.6 sec = 100 U). Heterophil suspensions were tested at approximately 60,000 WBC per mm3. After incubation in Hank’s solution at 37° for 30 min they gave the following results:saline-saline, 1.8 U;cortisone-saline, 0.6 U;saline-endotoxin, 7.8 U;cortisone-endotoxin, 5.3 U. Sonicated suspensions were also tested and possessed markedly decreased activity. Incubated macrophage suspensions were tested at different concentrations:saline-saline (30,000 per mm3) 3.1 U;cortisone-saline (15,000 per mm3) 0.7U; saline-endotoxin (14,000 per mm3) 51.8 U; cortisone-endotoxin (6,000 per mm3) 82.2 U. Sonicated macrophages from endotoxin-treated animals retained potent procoagulant activity.Thus, endotoxin induces WBC procoagulant activity primarily in macrophages rather than heterophils. Activity is greatest in macrophage suspensions from cortisone-treated animals.
The Therapeutic Range of Oral Anticoagulation, A Comparison of 14 Different Thromboplastins
Authors: Puckert F. , Marbet G.A. , Christeller S. , Airenne H. , Krajnc V. , Morniroli I. , Müller L. , Fassier H. , Morger T.
Published: May 2019

Success and side effects of oral anticoagulation depend on the degree of anticoagulation achieved in the patients. Many physicians are not aware that the therapeutic ranges are different with different thromboplastins. We have compared 13 commercial thromboplastins and a human brain preparation on 100 patients on long term phenprocoumon therapy. Simultaneous sample collection and assays in triplicate for all methods followed exactly the manufacturers′ recommendations. Six comparison systems were basedon percentage values derived from both manufacturers’ and our own standard curves, on clotting time ratios and on the established reference ranges, 15 to 25% with human brain and 5 to 10/2 with Thrombotest. The statistical analysis included variations of the reference methods and models for linear structural relationships. The data show that the lower limit of the therapeutic range varies widely between 5 and 25% and the upper limit between 10 and 25% for the commercial preparations tested. This study offers the great advantage of a direct comparison of a large number of widely used preparations and allows a much better estimation of the error of each method. It is pointed out that the analysis is only valid for a narrow range where the relationships are truly linear.
Coagulation and Immunological Investigations in Combined Factor V and Factor VIII Deficiency
Authors: Giddings J.C. , Seligsohn U. , Bloom A.L.
Published: May 2019

Plasma samples were obtained from patients of two families with inherited combined deficiency of factor V and factor VIII and examined by coagulation and immunological methods for the presence of factor V and factor VIII related antigens. A deficiency of procoagulant factor V was confirmed in all patients although a factor V related antigen was consistently demonstrated in all plasma samples by an inhibitor neutralisation technique using a non precipitating rabbit antibody. A deficiency of procoagulant factor VIII was also confirmed in all patients and factor VIII related antigens were detected by inhibitor neutralisation using human antibody and by electroimmunoassay using a precipitating rabbit antibody. The amounts of factor V and factor VIII related antigens present in the patient’s plasma samples were similar to those found in normal human plasma. Ristocetin co-factor assay using washed, fixed human platelets was normal in all patients. The results suggest that inactive antigenic determinants of procoagulant factor V and factor VIII are present in the plasma of these patients and are consistent with the possibility that a common precursor of factor V and factor VIII is defective in these families.
In Vitro Thrombogenicity Test for Clinical Factor IX Concentrates Using Synthetic Substrates
Authors: Miller-Andersson M. , Seghatchian M.J.
Published: May 2019

Venous thromboses have been reported following the clinical use of some factor IX preparations. This may be due to several causes as for instance traces of FXa or thrombin formed during processing or presence of surface activation products involved in the early stages of the coagulation pathway. There is an urgent need for simple and reliable thrombogenecity tests. Activation by Ca2+ should preferentially be avoided, as Ca2+ by itself is capable of activating the prothrombin complex. All clotting methods are affected by the heparin content (4-5 u/ml) and excess amount of sodium citrate present in the F IX concentrates. We have therefore developed a colourimetric method using synthetic substrates. The assay is performed in the absence of precence of known proteolytic inhibitors in order to improve the specificity of the test system and identify the nature of the thrombogenicity. Using this method, various concentrates can be classified in three groups according to the rate of cleavage of the substrates. Hot materials have been titrated with several inhibitors. The contaminating enzymes have also been isolated and characterized. The results clearly indicate the presence of two entirely different types of hot material. One group contains mainly FXa and thrombin and can be neutralized by AT III. The other group probably contains large amounts of surface products. The “thrombogenicity” of this group can be mimiced by kaolin activation of plasma and inhibited by Trasylol.
Characterization of Purified Urinary Trypsin Inhibitors
Authors: Takada A. , Takada Y. , Minakata K. , Sumi H.
Published: May 2019

Human urine was passed through Celite 545 column, and the non adsorbed eluate was adsorbed to Bentnite, then eluted with 2 % pyridine. The eluate was precipitated with ammonia sulfate, and the precipitate was dissolved, then passed through DEAE-Sephadex A-50, trypsin-Sepharose and Sephadex G-200. There were three types of urinary trypsin inhibitors (UTI) of m.w. of 69,000, 45,000, and 25,000 respectively. No difference in inhibitory spectra was observed among these three UTI’s. Purified UTI inhibited trypsin, chymotrypsin, and kallikrein. The inhibition of chymotrypsin did not exceed 50even at the high concentration of UTI. UTI inhibited plasmin to a lesser extent but no inhibition of thrombin, Cls, Clr or urokinase was observed.UTI had 7% carbohydrate and no HIS, PRO, or TRP was found in aminoacid analysis. UTI was not identical to α1A but related to inter α- trypsin inhibitor immunologically. Some persons excreted both UTI and α1A in the urine although most people did only UTI. Since α1A is not found in the urine of most people, UTI may not be the same as inter α-trypsin inhibitor which is filtered into the urine.
The Functional Activities of the Human Plasmin-Derived Light(B) Chain.
Authors: Robbins K.C. , Summaria L. , Wohl R.C.
Published: May 2019

A human plasmin-derived light(B) chain has been isolated and found to be functionally active. It is both a proteolytic and an esterolytic enzyme, but it has no plasminogen activator activity It reacts with streptokinase to form an equimolar complex which has both human and bovine plasminogen activator activities. The binding site for streptokinase is located on the light(B) chain of plasmin. The light(B) chain, with N -Cbz-L-lysine-p-nitrophenyl ester as the substrate had aof 83.0 μM and a kcat of 2.08/sec whereas the light(B) chain·streptokinase complex had a Km of 71.4 μM and a kcat of 9.00/sec, indicating an increased catalytic efficiency of the active site in the activator complex. The human Lys-plasminogen activator activity of the complex, when measured by the esterase activity of the plasmin being generated, showed a K value of 1.65 μM and a kcat of 43.2/sec, a two-fold increase in catalytic rate over streptokinase. Fragmentation of streptokinase beyond SKI, in the complex, results in decreased activator catalytic rates. The light(B) chain·streptokinase complex was an effective activator in in-vitro clot lysis systems using cross-linked fibrin clots. The light(B) chain adsorbs to fibrin clots resulting in accelerated lysis of these clots by activators.
The Immunosuppressive Activity of Plasmic Degradation Products of Human Fibrinogen
Authors: Edgington T.S. , Curtiss L.K. , Plow E.F.
Published: May 2019

Plasmic cleavage of human fibrinogen leads to generation of immunosuppressive activity not expressed by the intact molecule, and which is demonstrable in vitro and in vivo. This activity is not associated with the high molecular weight derivatives X, Y, D and E, but is present in the small dialyzable peptide fraction obtained from plasmic digestion. The peptides inhibit in a non-toxic fashion, the stimulation of 3H-thymidine uptake and blastogenesis of lymphocytes by phytohemagglutinin (PHA) and allogeneic cells (MLC) under conditions of both macrophage dependence and macrophage independence. The peptides also suppress the plaque-forming cell response of mice to sheep red blood cells in vivo. Approximately 30 μg peptides/culture leads to a 50% inhibition of the PHA and MLC systems, and approximately 400 μg/mouse produces a 50% suppression of the plaque-forming cell response. Intact fibrinogen chains exhibit negligible activity, but plasmic digests of Aα chain are suppressive. Consistent with derivation from the Aa chain was the demonstration that the activity was generated from limited plasmic digest of fibrinogen which produced fragment X, and this activity was soluble at 80°C for 10 minutes. The release of the active peptide by limited plasmic degradation, and the activity of these peptides at physiologic concentrations suggests that this system may be of importance in vivo in association with local fibrinogenolysis or fibrinolysis at sites of thrombosis. This has been in part substantiated by the experimental initiation of fibrinogenolysis in vivo with streptokinase.
Platelet Factor 4 and β-Thromboglobulin
Authors: Pepper D.S.
Published: May 2019

If the prethrombotic state exists and the platelet release reaction is involved, a radioimmunoassay of any platelet specific substance which is released by physiologic stimuli would be helpful. For technical reasons we have chosen β-thromboqlobulin, a protein of MW 36.000, which at present has no known function. In comparison with βTG, the PF4-RIA is technically less satisfactory, the observed levels of PF4 are 2-3 fold lower than BTG and the halflife appears to be similar. Several factors limit this general approach and are not readily calculated other than from experimental measurements. These factors include(a) the ratio of concentrations in plasma and serum(b) the halflife of the marker in vivo(c) the sensitivity and precision of the RIA. Our experience with BTG and PF4 indicates that the major limitation is the halflife (both approximately 20 minutes) and that other platelet specific substances (for instance the high molecular weight glycoprotein TSP) should be investigated to find a marker with a halflife of hours or days. The BTG test is technically trouble free and has been used by us for 3 years on patients samples. It must finally be tested however in a properly designed clinical trial, and in the case of ‘prethrombotic states’ this presents considerable problems of patient numbers in terms of the incidence and detection of thrombotic disease. In practice, ‘false positive’ elevation of the released substances can occur, apart from known coagulation mechanisms, we have observed elevated levels of BTG in patients having uraemia and bacterial infections. Nevertheless, taking all factors into consideration, the BTG/PF4-RIA tests represent a major advance in our ability to assess platelet function and we are confident that a better understanding of platelet involvement in thrombotic disease and normal processes will result.
Cultured Normal and von Willebrand Porcine Aortic Endothelial Cells-An in Vitro Model for Studying the Nature and Site of Platelet-Endothelial Component Interaction
Authors: Booyse F.M. , Fass D.N. , Bowie E.J.W.
Published: May 2019

Normal and von Willebrand(vWd) porcine aortic endothelial cells(EC) have been maintained and subcultured for10-12 months(14-18 passages) without any apparent change in characteristic growth morphology or ultrastructure. Immunofluorescence staining ofthese EC for ristocetin-Willebrand factor(RWF), using monospecific rabbit antiporcine RWF(aRWF), showed differences in the extent and nature of intercellular staining and the apparent absence of extracellular EC associated RWF-containing material(filaments)in vWd cells. Platelet-endothelial(damaged) interaction was decreased in normal EC cultures by pretreatment of the cultures with aRWF, no platelet interaction was seen with untreated vWd EC. These cultures were used as an in vitro model system(optimal pH, divalent cations, protein, exposure time and rotation speed)for studying the nature, extent and differencesin the platelet-microfi1ament interaction in normal and vWd EC. Preliminary data on the microfilament site of attachment of washed platelets or antibody-coupled beads, the interaction of platelets with matrix-bound purified RWF and the presence of immunologically identifiable RWF-containing filaments in normal and vWd EC suggest a possible role for endothelial-bound RWF in platelet-vessel wall interaction.
A Bypass Mechanism for Activating Human Factor IX with Human Factor VII and Thromboplastin
Authors: Østerud B. , Rapaport S.I.
Published: May 2019

The data reported herein were obtained in a search for mechanisms triggering intrinsic clotting which bypass the contact activation factors. An assay system was designed which permitted us to examine the effect of thromboplastin and Factor VII (F.V11) upon F.IX under conditions which produced minimal direct effect of thromboplastin and F.VII on F.X. Purified F.IX was incubated with purified F.VII, brain thromboplastin and Ca ions.Then, a mixture of purified bovine F.VIII (activated with thrombin), phospholipid, Ca ions and F.X was added. The reaction was stopped at 3 min with EDTA, and a subsample assayed for F. Xa with the chromogenic substrate S-2222. When 10 μl of F.IX (15.4 U/ml) was incubated with 10 μl of F.VII (0.2 U/ml), 5 jul brain thromboplastin, and 3 Jul 50 mM CaCl2 for 10 min at 370, F.IXa was formed as shown by generation of 0.830 U/ml of F.Xa. When 10 μl of F.XIa (0.24 U/ml) was substituted for the F.VII and thromboplastin, 1.040 U/ml of F.Xa was generated. Both the thromboplastin-F.VII generation of F.IXa and the F.XIa generation of F.IXa was time-dependent. Only minimal F.Xa was generated in this assay system when the first incubation mixture contained thromboplastin and F.VII without F.IX (0.088 U/ml), thromboplastin and F.IX without F.VII (0.002 U/ml), or F.VII and F.IX without thromboplastin (0.000 U/ml). Only minimal F.Xa was generated when F.VIII was left out of the second incubation mixture. These data establish that thromboplastin and F.VII can activate F.IX in a reaction bypassing F.XIa.
Demonstration of Transglutaminase Activity in Limulus Lysate
Authors: Chung S.I. , Seid R.C. , Liu T.-Y
Published: May 2019

When purified coagulogen (Mol. wt. 24,500) is acted upon by the Limulus clotting enzyme (Tai, J.Y., and Liu, T.-Y., (1977) J. Biol. Chem. in press), a soluble C-peptide (Mol. wt. 6,000) and an insoluble coagulin (Mol. wt. 20,000) are formed. The latter interacts in a non-covalent fashion to form the soft-clot (Tai, J.Y. et al, (1977) J. Biol. Chem. in press). The possibility of stabilizing the coagulin by a cross-linking process similar to that in the mammalian system has been examined. Transglutaminase activity was demonstrated in the freshly prepared Limulus lysate by its ability to incorporate primary amines into peptide-and protein-bound glutamine residues of B-chain of oxidized insulin, casein and limulus coagulin. The enzyme was activated by Ca2+, >> by Mn 2+ or Sr2+ and totally inhibited by sulfhydryl reagents such as iodoacet-amide, mercury benzoate and S-methyl-methyl sulfonate, but not by inhibitors to serine proteinases. Dansylcadaverine was incorporated into several proteins with differing molecular weights during the gelation of the whole lysate. Part of the clots formed were insoluble in 30% acetic acid. A high concentration of amines inhibited the formation of insoluble clot. Examination of the insoluble clot by SDS-gel electrophoresis, as well as by exclusion chromatography in 6M guanidine after reduction and carboxymethylation revealed the presence of high molecular weight polypeptide materials (>200,000). These results are strongly indicative of transglutaminase catalyzed covalent cross-linking of polypeptide chains during clot formation.
The Structure of Soluble Fibrin Complexes and Fdp After Defibrination by Echis Carinatus Bite
Authors: Edgar W. , Warrell M.J. , Warrell P.A. , Prentice C.R.M.
Published: May 2019

Studies on fibrinogen, fibrinogen-fibrin soluble complexes and FDP were made on eleven patients in Nigeria who had defibrination following Echis carinatus bite. On admission, before treatment with antivenom, all patients had non-clotting blood, reduced or zero fibrinogen levels, low factor Xtll levels and increased concentrations of soluble complexes and FDP, The fibrin component of the soluble complexes, separated by fibrinogen-sepharose chromatography, consisted of both intact fibrin and fibrin degraded at the ∞ chain. After isolation by Biogel chromatography the soluble complexes were also found to contain γ dimer chains. The FDP consisted of several X species, Y, D and D-dimer, and fragment E. The major fragment in all patients was D, but a few samples contained significant quantities of D-dimer, indicating in vivo activation of factor XIII. Fibrinopeptide A studies showed degraded fibrinogen, as well as fibrin, in the soluble complexes and degradation products, suggesting that fibrinogenolysis, in addition to fibrinolysis, had occurred, probably as a result of secondary activation of the fibrinolytic system in response to defibrination.