bluuebunny/crossref_metadata_2025 huggingface dataset:
Large Scale Plasma Thawing for Factor VIII Recovery
Authors: Foster P. R. , Hunter J. , Dickson A. J. , White B. J.
Published: May 2019

Freeze dried factor VIII concentrate of intermediate purity is widely accepted as the product of choice in the treatment of haemophilia A, yet supply of this material from National resources is inadequate. Substantial loss of factor VIII can occur during the production process and a point of major loss exists when plasma is thawed for removal of cryoprecipitate. The scale of operation is important since little or no loss occurs on small-volume thawing (i.e. 1-2ml) due to the shorter time of exposure to damaging conditions such as extremes of pH and ionic strength or enzyme attack. To overcome this the rate of thaw must be maximised within the constraint provided by factor VIII solubility, which is temperature dependent.

To design a satisfactory process we have used pilot-scale equipment to study continuous thawing, utilising fluid removal for temperature control. For batch thawing of crushed plasma, at a rate of 20 1/hr, plasma temperatures ranged up to 7°C but for continuou thawing remained below 1.2°C. A thawing rate of 80 1/hr has been achieved with the temperature held below 2°C throughout. Plasma pools of up to 150 litres in volume have been handled in this way with a mean residence time of 12 minutes and this has encouraged us to design prototype equipment for use with fresh frozen plasma.
Comparative in Vitro and in Vivo Study of Various Factor VIII Preparations
Authors: Allain J.P. , Verroust F. , Soulier J.P.
Published: May 2019

A comparison of nine commercial and non-commercial Factor VIII preparations was made. They consisted of llyophilized cryoprecipitate, 4 intermediate and 4 high purity concentrates. Protein, Fibrinogen, Factor VIII complex, IgG, anti-A and anti-B antibody levels were measured. Factor VIII:C content varied from 4-7 u/ml in cryoprecipitate, 12-31 u/ml in intermediate and 21-40 u/ml in high purity concentrates. These three categories of Factor VIII preparations can be better defined by 2 ratios: u FVIII/mg proteins and u F VIII/mg fibrinogen. They were respectively < 0.5 and < 1 in cryo, 0.5-1 and 1-3 in intermediate purity concentrates, > 1 and > 3 in high purity concentrates. The F VIII :C/F VIII : AG ratio ranged from 0.3 to 0.6 in any preparation. The F VIII:C/F VIII :VWF ratio was always lower than 1.

Each preparation was injected to several classic hemophilia A patients for treatment of minor hemorrhages. The peak of activity was always found 1 hour post-injection and the recovery ranged from 80 to 105%. The Factor VIII half-life ranged from 10 to 12.5 hours. No significant differences in half-life or recovery was found, and the clinical efficacy was similar. With the exception of fibrinogen load, all products carry similar risk for hepatitis, anti-IgG immunization and hemolysis. The differences lie in the ease of injection, the price and the yield of Factor VIII from starting plasma. Nevertheless, high purity concentrates should be used when high doses are reauired for surgery or treatment of patients with inhibitor.
Absence of Myosin on Platelet Membranes Supported by Membrane Iodination, Immuno-Fluorescence and Functional Studies
Authors: Mattson J.C. , Esselman W.J. , Schwarz M.L. , Zuiches C.A.
Published: May 2019

Immunofluorescence and functional studies were performed utilizing the immunoglobulin fraction of antisera raised against chromatographically purified human platelet myosin. When non-permeable, formalin fixed platelets were used, no FITC staining of the platelet membrane occurred indicating the myosin is not present on the platelet surface. When similar studies were performed on formalin fixed platelets using antibodies raised against the contractile protein complex, thrombosthenin, membrane fluorescence occurred. Autoradiographs of SDS-PAGE gels of the immune precipitate produced by reacting 125I labeled human platelet thrombosthenin with antithrombosthenin demonstrated that anti-thrombosthenin antisera was capable of reacting with at least three iodinated proteins In addition to myosin. 125I lactoperoxidase catalyzed labeling of the external membrane proteins of resting and ADP stimulated platelets indicated no external labeling of myosin although actin appeared to be labeled. In functional studies, incubation of human platelets with antimyosin did not produce aggregation nor did it Inhibit subsequent aggregation by ADP, collagen or epinephrine. Similarly, treatment of intact platelets with antimyosin did not cause Inhibition of clot retraction. These studies support the thesis that myosin is not localized on the external platelet membrane.
An Improved Method of Making Factor VIII Concentrates
Authors: Rock G. , Palmer D.S. , Tackaberry E.S. , Wickerhauser M.
Published: May 2019

The yields from batch preparation of Factor VIII concentrates can be substantially improved by collecting the blood into heparin rather than into CPD as anticoagulant. The resultant cryoprecipitate contains 78 ± 9% of the original plasma activity if 20 mls of supernatant per litre of starting plasma are left with the cryoprecipitate to maintain heparin levels. This cryoprecipitate was further purified by solubilization at 37°C for 5 minutes using 40 cc of saline per litre of starting plasma. This preparation was adjusted to pH 6.3 and 4.5% PEG 4000. Then, after removal of the precipitate by centrifugaron, the 4.5% PEG supernatant is adjusted to pH 6.0 and 11% PEG. The 11% PEG precipitate obtained after centrifugation is resolubilized in 1/100th the original plasma volume with buffer (0.1 M glycine, 20 mM citrate, 0.15 H saline) containing 1 unit of heparin per ml. Experiments using plasma pools containing 1-15 donor units gave yields ranging from 390-490 plasma Factor VIII equivalents per litre of the starting plasma. The final product retains an average of 90% of the initial Factor VIII activity after 24 hours at 22°C. It is believed that the present method could substantially reduce the cost of producing Factor VIII concentrates.
DDAVP Factor VIII Concentrate and its Properties in VIVO and in VITRO
Authors: Nilsson I-M. , Mikaelsson M. , Vilhardt H. , Wiechel B.
Published: May 2019

DDAVP fraction I-0 was prepared from a plasma pool derived from 80 blood donors who had received DDAVP and tranexamic acid. A control fraction I-0 was prepared from a plasma pool from the same group of blood donors under identical conditions except for the drug treatment.

The DDAVP plasma pool as well as the DDAVP fraction I-0 contained 2-3 times as much VIII:C as the controls. VIIIR:Ag increased to a lesser extent. No difference in stability of VIII:C was seen.

In vivo studies showed that infusion of the DDAVP fraction I-0 to 3 patients with severe haemophilia A caused a 2-3 times higher increase in VIII:C than after infusion of the same volume of the control fraction I-0. No difference in disappearance rate of VIII:C was seen.

Both factor VIII concentrates normalised the defect in VWD.

It is concluded that administration of DDAVP to blood donors prior to collection of blood increases the content of VIII:C in the ensuing factor VIII concentrate and secondly that such stimulation has no demonstrable qualitative effect on the factor VIII obtained.
ß Thromboglobulin (βTG), Platelet Factor 4 (PF4), and Fibrinopeptide A (FPA) in Patients with Porcine (PO) and Prosthetic (PR) Heart Valves
Authors: Dudczak R. , Niessner H. , Thaler E. , Lechner K. , Kletter K. , Frischauf H. , Damanig E. , Aicher H.
Published: May 2019

βTG, PF4, FPA (all measured by RIA), platelet counts and LDH were determined in 17 patients with PO bioprostheses and 23 patients with PR heart valves. All patients with PR valves and 4 with PO were under oral anticoagulant treatment. ßTG and PF4 were significantly (p<0.001) elevated in both patient groups (Table).

A strong positive correlation of βTG and PF4 (R=0.8660) was found in the patient group well as in controls. βTG and PF4 were higher in patients with PR valves than with PO valves, the difference however was not significant. NO higher levels were found in patients with a history of thramboembolism. FPA was normal in PO and PR patients. LDH levels were significantly (p<0.001) higher and platelet count lower in patients with PR valves. The data indicate that bioprostheses do not cause hemolysis or activation of the coagulation system. Elevation of platelet specific proteins support the assumption that both types of valves cause platelet damage.
Platelet - Serotonin Dynamics in Animals with Prosthetic Arterial Crafts
Authors: Clagett Patrick G. , Russo Marianne , Hufnagel Howard
Published: May 2019

In dogs with prosthetic aortic grafts, we have documented chronic changes in circulating platelets (Circulation 58: II-225, 1978). Following placement of grafts, serotonin (5HT) levels decreased from 2.01±0.22 to 1.37±0.17 mg/10 platelets (p < .005). Serotonin levels remained low for up to 6 months after graft placement and returned to normal when platelet survival time (PST) began to normalize. Maximum in vitro uptake of 14C-5HT was depressed after graft placement (89.0±0.71 before vs 72.0±1.11% after, p <.001), but time to achieve maximum uptake was much less (16.5±2.4 before vs 5.0±2.8 sec. after, p <.001). In vivo release of 5HT was assessed by labelling autologous platelets with 51Cr and 14C-5HT. In animals with grafts, PST was shortened but 14C label survived 1.55±0.10 times longer than 14Cr label. This was significantly greater (p <.02) than control animals with, normal PST in whom 14C label survived 1.21±0.05 times longer than 51Cr. The ratio of 14C/51Cr activity over 5 days following infusion of double labelled platelets increased from 3.16±0.52 (day 1) to 5.85±1.18 (day 5), p <. 001. Among controls, this ratio increased only slightly (1.99±0.34 to 2.26±0.42) and was significantly less on days 2 thru 5 than in animals with grafts (p <. 05–.005). Urinary 5HIAA excretion, measured for 2 months before and after graft placement, increased from 2644.9±80.6 to 3091.2±96.4 μg/gm creat./day (p <.001). In these animals, we conclude that platelets interact with the prosthetic surface, release 5HT and recirculate. Reutilization of released 5HT occurs but may be limited by depressed uptake. The net effect is a reduction in platelet 5HT.
Induction of DIC by Russell’s Viper Venom in the Rat. Preventive Activity of Ticlopidine on Consumption Coagulopathy
Authors: Lacaze B. , Ferrand C. , Pepin O.
Published: May 2019

The intraperitoneal administration of the LD50 dose of Russell’s viper venom to Sprague Dawley rats induces a DIC with consumption coagulopathy. The PT, TT and APTT are increased and the recalcification time and platelet count is considerably decreased. Also the thromboelastogram is quite perturbed. In order to follow the modifications of the coagulation parameters induced by the venom in the presence and absence of Ticlopidine. Four groups of each eight animals are investigated : Control, Ticlopidine 150 mg/kg/d. P.O. 4 days, Venom LD5O of Russell’s venom viper (Sigma), i.e. 400 μg/kg by I.P. route, 15 hrs before sacrifice, Ticlopidine + Venom T. 150 mg/kq/d. P.O. 4 days and the LD50 venom 15 hrs before sacrifice. The venom showed versus control a significant increase of PT, and APTT a significant decrease of Recalcification time and platelet count. The prevention of the DIC by Ticlopidine is characterized by the normalization of the coagulation factors. In comparison with the venom the Ticlopidine group has an increase of rocalci-fication time and PT, TT, APTT and platelet count were identical with the control group. The fibrinogen level increase significantly. The histopathological examination of kidneys and lunqs show the characteristic lesions of DIC in the venom animals, and the protective activity of Ticlopidine versus the intravascular aggregats in the Ticlopidine venom group. No protection by Ticlopidine however, was shown against the neurotoxicity of the venom. The inhibition of platelet aggregats and the antithrombotic activity of a drug can be evaluated by this experimental model of an acute DIC.
Isolation from Blood Platelets and Polymorphs of A Ca2+-Dependent Regulator Protein [CDR] which Activates Cyclic Nucleotide Phosphodiesterase
Authors: Young N. J. , Crawford N.
Published: May 2019

In recent years a low molecular weight (15-20,000) heat stable, calcium binding protein [CDR, or modulator protein] has been identlfied and isolated from a wide variety of tissues (heart, brain, testis, gizzard, red cells and platelets). This protein has been shown to have a multi-functional role in controlling the activities of cyclic nucleotide phosphodiesterase, adenylate cyclase, muscle protein kinase, Ca2+Mg2+ ATPase and also the phosphorylation of membrane proteins and assembly-disassembly of microtubules. We have isolated a similar protein from human platelets and guinea pig PMN leucocytes by solubilisation of the cells in urea and precipitation with methanol followed by DE 52 cellulose and G100 Sepharose chromatography. Both the isolated proteins purified to homogeneity have molecular weights on dodecyl sulphate/polyacrylamide gel electrophoresis of 16-17000 and they comigrate with the CDR protein similarly isolated from bovine brain. Tested against activator-deficient brain cyclic nucleotide phosphodiesterase the platelet protein showed a Ca2+-dependent 4 fold enhancement of activity comparable to that shown by polymorph CDR [4 fold] but less than that achieved with bovine brain CDR [5-9 fold]. A number of other physico-chemical properties of the three regulatory proteins have been compared.
Purification of a Highly Potent Heparin-Like Anticoagulant from Healthy Human Plasma
Authors: Yue Robert H. , Starr Toby , Gertler Menard M.
Published: May 2019

A highly potent-heparin-like anticoagulant (accelerator) has been purified from citrated healthy human plasma. After heat defibrination and BaSO4. treatment on the plasma, the accelerator was adsorbed onto a DEAE-cellulose column and elution was achieved using a high ionic strength linear buffered salt gradient. The eluted accelerator was further purified by polyacrylamide gel electrophoresis. The purified accelerator can accelerate the inhibition of thrombin by antithrombin III and has a specific activity of 226 heparin units/mg of carbohydrate. The accelerator contains a small amount of protein-like material. The amount of accelerator present in healthy human blood is extremely small and can only be first detected in the concentrate after the DEAE-cellu-lose chromatography. A mere 0.5 mg of the purified accelerator is obtained from 100 ml of human plasma. Concomitant with this investigation, a second heparin-like substance also has been purified but has very low anticoagulant activity in terms of heparin units. The naturally occurring accelerator may function as heparin in the circulating blood and its level in blood may have a clinical significance in thrombotic vascular disease. Further work on its physical and chemical properties is now in progress.
Heparin Induced Platelet Aggregation Presentation and Surgical Consequences
Authors: Bernhard V.M. , Hussey C.V. , Towne J.B.
Published: May 2019

Heparin induced thrombocytopenia and its vascular complications were identified in 25 patients. Aggregation studies performed using ADP and/or heparin in these patients were compared with responses in 18 normal. individuals. The specific aggregating factor was demonstrated in plasma. Other findings of note were normal fibrinogen, minimal elevation of fibrin split products, a normal or slightly prolonged prothrombin time and severe thrombocytopenia. Arterial and venous thrombosis required surgery in seven patients, four of whom required major amputation. Although an antibody has not been identified, the time of development of this phenomenon suggests an immune response. Only those patients who had previous exposure to heparin developed this phenomenon in less than six days. Thrombotic complications occurred with either bovine lung or porcine gut preparations from different manufacturers and appeared following both subcutaneous and intravenous administration. Electron microscopy of clots removed from four patients revealed characteristic platelet fibrin thrombi. Early recognition, prompt discontinuance of heparin and the use of platelet antiaggregants will prevent disastrous consequences. All patients undergoing heparin therapy should be carefully observed for thrombocytopenia with specific platelet aggregation studies when appropriate.
The Role of Prostaglandins and Thromboxanes in Platelets
Authors: Samuelsson B. , Granström E.
Published: May 2019

Recent work on the function of prostaglandins and thromboxanes in platelets will be discussed as well as methods for assay of cyclo-oxygenase catalyzed products.
Automated Microdensitometry and Protein Assays as Measures for Platelet Adhesion and Aggregation on Collagen Coated Slides Under Controlled flow Conditions
Authors: Muggli R. , Baumgartner H.R. , Tschopp B.
Published: May 2019

Microscope slides were homogeneously coated over a length of 2 cm with a mixture of soluble and fibrillar collagen and exposed at 37°c and under laminar flow to citrated whole rabbit blood at a flow-rate of 100 ml/min. Surface coverage with platelets (adhesion) and platelet accumulations higher than about 5 μm in height (aggregation) were determined by automated microdensitometry of fuchsine stained ‘en face’ preparations. The platelet mass per unit surface was measured witha modified Lowry technique whose sensitivity was equivalent to 5×105 platelets. Platelet number, amount of protein and surface coverage with platelet accumulations correlated. After a per fusion time of 10 min thrombi up to 30 μm in height and oriented in the direction of flow had developed on the collagen coated area. Surface coverage with platelets was 75% and the amount of deposited protein 1.4 μg/mm2 (2x106 platelets/mm2). On the uncoated surface single platelets predominated; the surface coverage was 20% and the density of platelets 8x104/mm2. Acetylsalicylic acid at 100 μM decreased platelet aggregation by about 80% without affecting adhesion.

The new parallel plate per fusion system offers rapid quantitation of platelet-surface and platelet-platelet interaction after exposure to flowing blood and may also be diagnostically useful.
Clot Promoting Effect of Platelet-Vessel wall Interaction
Authors: Hornstra G.
Published: May 2019

When a piece of vascular tissue (rat, rabbit) is incubated in plasma, clotting occurs. The presence of platelets accelle rates this clotting process. Inhibition of vascular prostacyclin (PGI2) production, either by drugs or by inducing arachidonic acid deficiency, causes the platelets to become activated by the vessel wall, resulting in platelet shape change and -aggregation and in a further enhancement of the coagulation response. Hirudin very actively inhibits both aggregation and clotting whereas ADP scavenging has only a weak effect. This indicates that the aggregation response, occurring upon plateletvessel wall interaction is for the greater part mediated by thrombin. Saturated fat feeding increases the clot promoting effect of the platelet-vessel wall interaction. This appeared not to be due to differences in the production of vascular PGI2 or platelet endoperoxides and thromboxanes (measured as malondialdehyde). These findings indicate that:

1. Thrombin, locally generated upon the interaction between damaged vessel wall and blood constituents, contribute to arte rial thrombus formation;

2. Prostacyclin regulates local thrombin formation viaits inhibiting effect on platelet activation;

3. The existence of another, platelet bound affector is postulated.
m-[o-(2-Chloro-5-Fluorosulfonylphenylureido)Phenoxybutoxy]Benzamidine: Affinity Labeling Reagent Which Can Identify Secondary Binding Sites of Coagulation, Complement and Fibrinolytic Serine Proteases
Authors: Bing D.H. , Robison D. , Andrews J.M. , Laura R.
Published: May 2019

We have determined that m-[o-(2-chloro-5-fluorosulfonylphenylureido)phenoxybutoxy]benza-midine [mCP(PBA)-F) is an affinity labeling reagent which labels both polypeptide chains of thrombin, factor Xa, complement component Cls and plasmin. As this means it is reacting outside of the catalytic center, we have called this reagent an exo-site affinity labeling reagent. Progressive irreversible inhibition of these enzymes by this reagent is rapid (k1st 2.5-4.6 × 10-3 sec-3), the kinetics of inactivation are consistent with Inhibition proceding via formation of a specific enzyme-i-nhibitor complex analogous to a Michaelis-Menton complex (KL = 115-26 μM), and diisopropyIfluorophosphate or p-amidino-phenylmethanesulfonylfluoride prevent labeling by [3H]mCP(PBA)-F. A molecular model of mCP(PBA)-F shows that the reactive SO2F group can be 17 A from the cat ionic amidine. The data are consistent with the hypothesis that both peptide chains are required for the specific proteolytic activity exhibited by these proteases and that the peptide chain which does not contain the active site serine is close to the catalytic center. (Supported by NIH and AHA grants)
Purification of α2-Antiplasmin by a Single-Step Affinity Chromatographic Procedure
Authors: Wiman B.
Published: May 2019

A new and efficient single-step purification method for human α2-antiplasmin has been elaborated. The method is based on the interaction between α2-antiplasmin and a fragment (LBSI) constituting the three NH2-terminal triple-loop structures in plasminogen produced by elastase digestion. This fragment has been purified and coupled to Sepharase and used for affinity chromatographic purification of α2-antiplasmin using plasminogen depleted plasma as starting material. After adsorption and washing at high ionic strength the α2-antiplasmin is specifically eluted with 6-aminohexanoic acid. The inhibitor preparation obtained in this way is over 90% pure as judged from SDS Polyacrylamide gel electrophoresis and activity measurements. About 40-45 mg pure α2-antiplasmin per liter plasma is obtained representing a yield of about 60%. LBS-I Sepharose has much higher capacity for α2-antiplasmin and is also much more specific than plasminogen-Sepharose. Repetitive treatment of plasma with LBS I-Sepharose failed to adsorb the last 20% of α2-antiplasmin as judged by Laurell electrophoresis. This supports the recent finding of Clemmensen (1979) on partially purified α2-antiplasmin that a form of the inhibitor with less affinity for the lysine-binding sites in plasminogen may exist, even in unfractionated plasma. The major part of this type of α2-antiplasmin is also a functional antiplasmin since it can form a complex with plasmin.
Decreased Reactivity of Plasmin-Streptokinase Towards α-2-Antiplasmin
Authors: Cederholm-Williams S.A. , Lijnen H.R. , De Cock F. , Collen D.
Published: May 2019

Plasmin (P) is very rapidly (rate constant 2 × 107 M-1s-1) inactivated by α-2-antiplasmin (A), and blocking of the lysine binding sites (LBS) of plasmin reduces this rate 50 fold (Eur. J. Blochen. 84, 573-578, 1978). Using purified reactants and the plasmin substrate D Val-Leu-Lys-pNA (S 2251) the influence of streptokinase (SK) on the reaction between P and A was examined. Addition of SK to P reduces the reaction rate with A in a sigmoidal fashion. Assuming the formation of a reversible bimolecular P:SK complex which is un-reactive towards A then the dissociation constant Kd of this complex is less than 10-9 M. The LBS of P does not play a role in this interaction since similar Kd values are found with modified P, lacking LBS and in the presence of 0.3 mM 6-aminohexanoic acid which blocks the LBS. The P:SK complex is only very slowly inhibited by A with a rate constant < 5 x 102 M-1s-1.

It is concluded that the α-2-antiplasmin is less reactive towards the plasmin-strepto-kinase complex due to a modification of the active centre of plasmin rather than an effect upon the lysine binding sites.
Affinity Chromatography of Factor VIII Subunits
Authors: Lane J.L. , Ekert H.
Published: May 2019

Gel filtration of factor VIII resulted in elution of a fraction which possessed procdegulant activity (VIII:C), ristocetin cofactor (VIII:RCF) and antigenic activity (VIII:Rag). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of this preparation showed that no protein entered the gel. Eight bands ranging from 30,000 to 230,000 molecular weight (mw) were seen when 400μg reduced protein was loaded onto the SDS-PA geL Attempts to assay the functional properties were made in order to investigate whether the multiband structure was a property of factor VIII or due to contaminants. These proved unsuccessful, as reduction and SDS-PAGE of factor VIII destroyed its functional properties. Affinity chromatography with human and rabbit antibodies to factor VIII provided an alternative method of relating the subunit structure to functional properties. In-solubilized human antibody inactivated VIII:C and did not bind VIII. Rag. The protein bound to human antibody was dissociated with NH4 SCN and reduced. SDS-PAGE showed heavy protein staining, with 95% of the protein present in 35,000 and 62,000 mw bands. Similar treatment of a control human IgG column showed minimal protein in the same mw region. Using insolubilized rabbit antibody to human factor VIII, all 8 bands were observed after NH4 SCN dissociation, reduction and SDS-PAGE. These results suggest that it is unlikely that the low mw bands observed on heavily loaded gels are due to protein contamination.
The Pathogenesis of Thrombotic Thrombocytopenic Purpura
Authors: Kwaan H.C.
Published: May 2019

The vascular lesions with microthrombi were studied in 12 patients with thrombotic thrombocytopenic purpura (TTP), diagnosed by the characteristic clinical and laboratory findings and confirmed histologically in each case. While defibrination was not observed, and with only minimal changes in the circulating levels of fibrinogen, fibrin degradation products and plasminogen activator, the microthrombotic lesion was invariably present. Immunofluorescent and histochemicaL studies indicated that both platelet and fibrin were present In the mitrothrombi with the platelet components dominant In many cases. Using the fibrin slide method, plasminogen activator was demonstrated in the uninvolved blood vessels but totally absent in the vessels occluded by microthrombi. In contrast, fibrinolysis is always present in the vessels afflicted with other types of thrombosis, such as the microthrombi in disseminated intravascular coagulation. Since circulating fibrinolytic activity was normal in TTP, the absence of vascular fibrinolysis is a local defect due to either inhibition by the platelet deposits or by local vascular damage. The inability of thrombolysis may explain the absence of systemic defibrination and the severity of the disease.
Evidence for a Coagulopathy Related to Endotoxaemia in Patients with Obstructive Jaundice
Authors: Hunt D.R. , Allison M.E.M. , Forrester A. , Prentice C.R.M. , Blumgart L.H.
Published: May 2019

Animal studies suggest that endotoxaemia may contribute to tbe disturbances of coagulation in obstructive jaundice. Two groups of patients, 14 controls (C) and 28 jaundiced (J) having pancreatic or biliary surgery were studied prospectively. Endotoxaemia, soluble fibrin (s. f.) by tanned red cell agglutination, FDP and DVT were measured for comparison with outcome of surgery. In group J more complications occurred with 7 deaths and 10 DVT; by comparison, only one death occurred in group C and no DVT, fever or haemorrhage.S. f. was found in none of group C but in 9 of 24 in group J pre-operatively. Presence of s. f. did not influence outcome of surgery. Endotoxaemia and raised FDP were also seen more frequently pre-operatively in group J. Of the group J patients 11 had endotoxaemia or FDP before operation and 7 died. An association between endotoxaemia and FDP was shown in both groups and in group J endotoxaemia appears related to s. f. although s, f. and FDP are independent. A coagulopathy associated with endotoxaemia is present in some jaundiced patients and they fair badly after surgery.
Pulmonary Insufficiency in the Rat After Intravascular Coagulation and Inhibition of Fibrinolysis - Studies on Pulmonary Microcirculation and Pathophysiology
Authors: Gerdin B. , Saldeen T. , Sandler H. , Sedin G.
Published: May 2019

In posttraumatic pulmonary insufficiency microembolism of fibrin to the lungs and inhibited fibrinolysis have been given key-roles and the term “the delayed microembolism syndrome” has been designated to those cases. The pathogenetic mechanisms involved are only to a limited extent known. Those were investigated in a rat model. Fibrinolysis was inhibited with AMCA and intravascular coagulation induced by a 5-min injection of thrombin, 500 NIH/kg b.w.. Within 5 min a profound consumption of fibrinogen occurred and 125-la-belled fibrin was embolized to the lungs. At that time the alveolar circulation was decreased, vasoconstriction and platelet-red cell aggragates appeared as seen by in vivo microscopy and arterial PO2 was diminished irrespective of fibrinolysis. After restitutio for tha progressive pulmonary insufficiency with PO2 decrease and PCO2 retention occurred. Trasylol did not influence on pulmonary damage. Converting enzyme activity was not diminished. A retention of fibrin degradation products (fdp) occurred in the lungs and infusion of low molecular weight fdp aggravated pulmonary oedema, l.ung mast cells were partly degranulated and mepyramine maléate slightly counteracted pulmonary oedema. It is concluded that release of vasoactive low molecular weight fdp probably plays a causal role in oedema formation; probably in part by releasing histamine from lung mast cells.
An Experimental study of the Pathogenesis of Venous Thrombosis Versus DIC: The Role of Intrinsic and Extrinsic Coagulation Pathways
Authors: Gurewich V. , Nunn T. , Lipinski B.
Published: May 2019

Clinically, UIC is rarely accompanied by venous thromboembolism. These two entities were studied in rabbits in order to explain this paradox. Ellagic acid (0.1M, 10ml/kg), aged serum (1.3-10ml/kg), Factor IXa concentrate (0.5-3. Oral/kg), Xa concentrate(1.0-5. Oml/kg) tissue (rabbit lung) extracts or simplastin. (l-4ml/kg) and purified thrombin(10-200u/kg) were infused. Venous segments were ligated is seconds after infusion, examined after 10 minutes stasis and thrombosis was graded (0-4+). Fibrinogen concentration, PTT, TT, PT and fibrin monomer (FM) were measured. Ellagic acid, serum and IXa each caused 4+ thrombi at low doses whereas even at the highest doses, they caused no change in fibrinogen concentration and only moderate FM (2+) elaboration. The PTT shortened after all these infusions. By contrast, Xa, thrombin and the tissue extracts caused fibrinogen consumption (approx. 25-50%). substantial FN (4+) elaboration but no venous thrombosis. The PTT, but not the TT or PT, was prolonged £1-1/2 baseline) for 2-3h. after the infusions. Mixing post-infusion and baseline plasmas (1:1), prolonged the PTT of the latter. These findings suggest that activation of the intrinsic pathway readily induces thrombosis in areas of stasis but not DIC, The latter is more readily induced via the extrinsic pathway and is associated with the elaboration of a potent, labile inhibitor of intrinsic coagulation which may be responsible for the absence of stasis thrombi.
An assay for Activators of Platelet Adenylate Cyclase Present in Rabbit Blood: Evidence that Prostacyclin (PCI2) is not A Circulating Hormone
Authors: Haslam R.J. , McClenaghan M.D.
Published: May 2019

Measurement of cyclic [3H]AMP in washed rabbit platelets labelled by preincubation with [3H] adenine permitted the detection and assay of activators of platelet adenylate cyclase (e.g. PCI2). Cyclic [3H)AHP was extracted from the platelets with TCA and isolated by chromatography on neutral alumina and Dowcx 50. In 30 s incubations at 37°C, increases in platelet cyclic [3H]AMP were detected with ≥ 0.4 nM PGI2 in the absence of a cyclic AMP phosphodiesterase inhibitor and with > 0.04 nM PGI2 in the presence of 1 mM 3-isobutyl-1-methylxanthine (MIX). Log/log plots of the increases in cyclic [3H]AMP in the presence of MIX were linear with 0.04-4.0 nM PGI2. Unlike bioassays for PGI2 based on inhibition of platelet aggregation, this assay could be applied to whole blood. Mixtures of 0,4 ml citrated rabbit blood, 0.04 ml labelled platelet suspension (108 platelets; 1 μCi 3H) and 0.06 ml of other additions including MIX were incubated for 30 s at 37°C. The same values for platelet cyclic [3H]AMP were obtained with arterial blood assayed 30 s or 30 min after its removal from rabbits, indicating the absence of detectable PGI2 which had a half-life of about 7 min in blood at 37°C. The effects of different concentrations of added PGI2 on the cyclic [3H]AMP in citrated rabbit arterial blood containing labelled platelets showed that fresh blood contained less than 0.1 pmol PCl2/ml. As > 0.4 nM PGI2 was required to inhibit aggregation of rabbit platelets in plasma-containing systems, the results suggest that the circulating concentration of PGI2 in rabbits is too low to affect platelet function.
Evidence for two Separate Receptors Mediating the Stimulation of Platelet Adenylate Cyclase by Prostaglandin I2 (PCI2), PGD2 and PCE1
Authors: Mills D.C.B. , Macfarlane D.E.
Published: May 2019

Prostacyclin (PGI2), and prostaglandins D2 and E1, all inhibit aggregation of human platelets by stimulating adenylate cyclase. In platelets prelabelled with 14C adenine, PGI2 has a higher intrinsic activity than either PGD2 or PGE1α, as well as a higher apparent affinity. PGE1, but not PGD2, inhibits the action of high concentrations of PGI2, both when added simultaneously with PGI2 and when added 3 min later. In the latter case the slow spontaneous fall in intracellular cyclic AMP Is accelerated by PGEj but not by PCD2. PGF2, at concentrations up to 100 μM neither stimulated the cyclase by itself, nor did it Inhibit the effects of PGI2, PCE, or PCD2. PCF2α which did cause a small increase in cyclic AMP, inhibited PGD2 strongly, PGE1 not at all, and PCI2 slightly at high concentrations. N0164, an inhibitor of aggregation induced by bls-enoic prostaglandins, inhibited cyclase stimulation by PGD2 but not by PGE1, PGE2, PGD1, or PGI2, though it reduced PGI2-induced inhibition of platelet aggregation. Preaddition of PGE1, but not of PG2, or PGD2, at submaximal concentrations, inhibited subsequent response to high dose PGI2. The results suggest chat PGE1 and PCD2 probably act on the same enzyme, but through a different receptor. PGE1 acts as a partial agonist for the receptor for PCI2, but In addition causes a tachyphylaxis not seen with PCI2, or with, PGD2.
Prostaglandins (PG) Inhibit Human Platelet Aggregation by PG Receptor Antagonism as well as Adenylate Cyclase Stimulation
Authors: MacIntyre D.E. , Salzman E.W.
Published: May 2019

11-Deoxy and/or 15 or 16 alkyl substitution confer platelet aggregating activity to bis-enoic PG's: e.g., 11-deoxy-PGE2 (threshold (T)-5μM); ll-deoxy-15(S)-15-methyl-PGE2 (T=0.1μM);ll-deoxy-15(S)-16-methyl-PGE2(T=0.1μM). Responses induced by such PG’s mimic those induced by PGH2 and PGH2 analogues (e.g., U44069). N0164 competitively inhibits aggregation induced by PG’s and suppresses “secondary” responses induced by low concentrations of ADP or arachidonic acid, suggesting involvement of a specific PG stimulatory receptor in platelet aggregation. We compared non-aggregatory PG’s as inhibitors of pri mary aggregation induced by ADP or U44069. PG’s containing 11-deoxy and/or 16-alkyl groups selectively inhibited aggregation induced by U44069. Mean I50 values (n-4) against ADP and U44069 respectively were PGA2 (>100μM; 0.3μM); PGB2 (>100μM; 5μM); PGD2 (6nM; 2nM); PGE1(10nM; 5nM); 11-deoxy PGE1 (60μM; 2μM); ll-deoxy-15-(R)-16-methyl (100μM; 2μM); 13,14-dihydro-16-methyl-PGE2 methyl ester(750nM; 15nM). A low ADP: 406 I50 ratio (e.g., PGD2) indicates that inhibition is mainly due to adenylate cyclase stim ulation, and a high ratio (e.g., PGA2) that inhibition is mainly due to PG receptor antagonism. We have demonstrated a PG stimulatory receptor on the platelet, and its involvement in “secondary” aggregation. PG’s inhibit aggregation by combining with this receptor and/or by stimulating adenylate cyclase.
Isolation and Partial Characterization of a Novel Circulating Antithrombin
Authors: Messmore Harry , Pervez Zaheer , Fareed Jawed
Published: May 2019

We have previously reported on a novel circulating anticoagulant (Clin. Res. 22:396 A, 1974. Thromb. and Haem. 38:77, 1977 (abstr) in a 42 year old female who has had repeated episodes of life threatening hemorrhage since the age of 3. Our preliminary studies showed it to be a protein with some of the properties of a heparin activated antithrombin III. This report is on additional studies to further characterize the Inhibitor«

The patient’s plasma was fractionated on Sephadex G-200, and the fraction showing immediate acting antithrombin activity was further purified on heparin-sepharose and conconavalin A-sepharose affinity columns. The patient’s inhibitor did not bind to heparin sepharose, and was thus separable from her antithrombin III. It did bind to conconavalin A, and was eluted from the conconavalin A with α - D (+) methylglucoside. It has very broad serine protease inhibitor activity, blocking the hydrolysis of synthetic chromogenic substrates by thrombin, factor Xa, plasmin and trypsin. It did not inhibit Reptilase.

Immunochemical assays show it to be α1 antitrypsin. Isoelectric focusing shows it to he a variant of normal, with its isoelectric point being different from a normal control, and pure α1 antitrypsin (commercial, human).

The total α1 antitrypsin level in this patient is about 50% of normal, and It has potent immediate acting antithrombin activity. It appears to be similar to a phenotype previously reported as Antithrombin Pittsburgh (Blood 51: 129, 1978).
Evidence of Abnormal Factor: VIII Related Protein in Diabetes Mellitus
Authors: Cacciola E. , Giustolisi R. , Lombardo T. , Musso R. , Russo M. , Sortino G.
Published: May 2019

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

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

In diabetic patients, particularly in the ones at higher thrombogenic risk, the evaluation of VIII:C/VIIIR; AG and VIII:vWF/VIIIR;AG ratios showed a significant (P<0.001) increased VIIIR:AG with a higher co-ristocelin activity than the procoagulant one. Furthermore, biophysical and bio chemical modifications of diabetic Factor VIIIR;AG, mainly a lower EM on 2 DCIE, were found. These results suggest that the Factor VIIIR:AG abnormality, detectable in diabetic plasma, can be related with an abnormal glycoprotein biosynthesis.
Congenital Antithrombin III Deficiency in 3 Families (7 Affected Members).
Authors: Conard J. , Samama M. , Horellou H.H. , Cazenave B. , Griquer P. , Barsotti J. , Herreman G. , Godeau P.
Published: May 2019

A congenital Antithrombin III (AT III) deficiency affecting 7 members of 3 families is reported.

The first thrombo-embolic accidents were observed between the age of 22 and 35: they were spontaneous or occured after delivery or oral contraception. In one patient, a deep vein thrombosis was observed during heparin treatment. In 2 cases, recurrent pulmonary embolic episodes required vena cava ligation. No thromboembolic accident was observed during oral anticoagulation.

AT III was measured by an amidolytic method and by the Mancini method on plasma and serum ; the antithrombin activity was determined on serum by the von Kaulla method. In 7 patients, a decreased AT III was found by all the methods performed. The AT III level was around 50% in patients treated or not by oral anticoagulants.

One patient was studied during heparin treatment and then under oral anticoagulants: AT III levels were lower under heparin.
Mechanism of Heparin Rebound
Authors: Fabian I. , Aronson M.
Published: May 2019

Heparin rebound is occasionally encountered following protamin sulfate administration for the neutralization of excess of heparin. In these situations the anticoagulatory properties of heparin are initially abolished, but within several hours the blood display again an increased clotting time. The purpose of this work was to try to reproduce the phenomenon under in vitro conditions, and to provide a working hypothesis for its explanation. Under the condition used the following parameters were obtained (according to the APTT method): clotting time of untreated plasma 50-55 seconds; with the addition of 4 units heparin/ml plasma>3 minutes; and with the addition of 50-100 μg of protamin to the heparinized plasma the clotting time reverts to 50-55 seconds. It was, however, found that incubation of heparin-protamin complex with the plasma at 37° for several hours, reduced the effectivity of the Protamin, in other words, a longer coagulation time was observed. Subsequently, we found by electrophoretical methods that (heparin bound) protamin is proteolitically degraded upon incubation in plasma, the anticoagulatory properties of the heparin remaining intact. In summary, our results are compatible with the hypothesis that heparin rebound can be explained by the degradation of Protamin by plasma enzymes and the release of this newly available heparin in to the circulation. The importance of this phenomenon in conjunction with other observations previously described by us are discussed.
Heparin-Coated Surfaces that bind Antithrombin have Reduced Platelet Reactivity
Authors: Lindon J. , Brier-Russcll D. , Salane M. , Smith M. , Labarre B. , Dincer A. , Rosenberg R. , Merrill E. , Salzman E.
Published: May 2019

Surfaces with heparin bound by covalent linkage react with platelets but are passivated by prior exposure to platelet free plasma (PFP), provided they are capable of adsorbing antithrombin III (AT). For example, heparin-agarose beads induce platelet adhesion and aggregation, which are reduced by preincubation of the beads with PFP, but PFP depleted of AT is not effective. The degree of passivation of the beads parallels their removal of AT from PFP. The beads are also passivated by a solution of purified AT.

Furthermore heparin-polymethyl acrylate copolymers made by radical polymerization initiated by Ce4+ bind AT, as demonstrated by measurement of AT remaining in solution after adsorption or eluted from the surface after plasma incubation. On such a surface, AT is activated just as by heparin in solution, measured by the ability of heparinized beads to inactivate thrombin (S-2160 chromogenic tripeptide substrate assay) in the presence of AT and by lack of inactivation of thrombin with AT-poor plasma and return of inactivating effect when AT is added back.

Aggregation of platelets by specific heparin fractions in solution appears to be an analogous phenomenon, which is inhibited by antithrombin.
Non-Thrombogenic Characteristics of Hybrid Vascular Prostheses
Authors: Sawyer P.N. , Stanczewski B.
Published: May 2019

In our continuing efforts to develop a compound, coated vascular prosthesis, combining the best characteristics of biologic and nonbiologic materials, we have produced a hybri graft with pseudo biological coating. Five types of velour and knit dacron prostheses, have been cross linked subsequently with negatively charged proteins. Albumin and gelatin were used to coat the prosthesis or the combination of both. Following electrochemical characterization coated prostheses were implanted into the carotid and femoral arteries of dogs for 1 sec. 2 hr. and 1 mo. Preparations that showed promising short term results were implanted in the abdominal aorta of dogs for 18 months. Controls included uncoated prostheses and prostheses that had been coated but not negatively charged. Upon removal, the grafts were examined for thrombi and other luminal narrowings (photography, light and scanning electron microscopy). Fifty two uncoated and coated grafts have been examined. These exhibit a range of thrombus formation, from none to complete occlusion, in the various knit/coating combinations. We believe these hybrid prostheses will prove to be very useful in vascular reconstructive surgery due to (i) the nonthrombogenic characteristics of the negatively charged protein structure, (ii) more uniform healing and (iii) ease of implantation since preclotting is not necessary.
The Formation of Fibrin Around Large Platelet Aggregates (PA’s) in Heparinized Platelet Plasma (PRP)
Authors: Wurzinger L.J. , Blasberg P. , Vehr H.
Published: May 2019

In concavities on the surface of large PA’s (107-108 μm3) which were produced “spontaneously” or upon addition of ADP 10-6 M in a homogenous shear field in a Couette-type aggregometer (40 s-1), we observed the formation of a fibrin network within 5 mins after sampling. This fibrin network, which could be identified with fluorescent antibodies, formed despite anticoagulation with 7.5 II heparin/ml. The formation of this fibrin network was less pronounced and delayed when the heparin concentration was raised to 20 U/mL 50 U/ml completely suppressed the formation of the network. Concomitantly we found masses of degranulated and ballooned platelets around PA’s with fibrin formation. As we suspected secreted’PF 4 (Antiheparinfactor) being responsible for this phenomenon, we analysed the β-thromboglobulin (β-TC)in heparinized PRP, which is secreted along with PF 4.

We found the formation of fibrin not so much correlated to the extent of platelet aggregation in the whole sample, but to the release of β-TG. The samples wherein fibrin was formed, were characterized by β-TG levels in plasma that amounted to up 5% of total platelet protein. In our view this explains the fact, that despite heparinization during extracoporeal circulation the local formation of fibrin-consolidated white thrombi and emboli is observed.
The Action of Calciparine in the Prevention of Premature Delivery
Authors: Eglin G. , GAUTIER G. , Maillet R. , Colette C.
Published: May 2019

In the study of the causes of premature delivery, interest in blood vessel-related mechanisms is related to platelet secretion of prostaglandin, whose activity on uterine muscle fiber is well established.

The action of indometacine on this secretion of prostaglandin is used clinically but its lack of harmful effects on the fetus has not been shown, demanding great caution.

Our clinical study of subcutaneous calcium heparin, with well known action on platelets, was undertaken in this framework. All threats of premature delivery from the 26th to 34th weeks, except in cases of cerclage, were considered in a double-blind study with 2 randomized groups. The first group was treated with Calciparine (7 500 IU b.i.d.) and beta-mimetics, the second group with a placebo (solvant) and beta-mimetics.

Conclusions are based on prematurity in the 2 groups.