bluuebunny/crossref_metadata_2025 huggingface dataset:
Interrelationships Between the Complement System and the Coagulation Kinin-Forming and Fibrinolytic Systems
Authors: Cooper Neil R.
Published: May 2019

During contact activation, the coagulation, kinin-forming and fibrinolytic systems are initiated following the attachment of Factor XII to a surface by a complex series of protein-protein interactions that lead to the activation of serine proteases by limited proteolysis. The activation processes are modulated by protein cofactors and by inhibitors. These molecular processes are similar to those involved in triggering of the complement (C) system, another major plasma effector system. Multiple interrelationships of these four plasma effector systems that have been suggested will be summarized and their functional significance considered.

We have begun a systematic investigation of such potential relationships employing highly purified proteins of the four systems. Initial efforts have been directed at determining whether plasma kallikrein, plasmin, thrombin or Factor Xa activate the first reacting component of the classical C pathway, Cl (C) is a calcium-dependent complex of three proteins: CIq , involved in binding ta antibody or other activators, generally on a surface; and Clr and Cls, zymogens of serine proteases. Both kallikrein and plasmin directly activate Clr and also Cls while thrombin and Factor Xa do not. Kallikrein and plasmin cleave the single polypeptide chain of Cls at a site near to or identical to that attacked by

and also cleave Cls at another, distant site. These evolving studies confirm and extend earlier work and indicate that the four plasma effector systems are closely interrelated.
Mechanism of Activation of Human Plasma Prekallikrein
Authors: Bouma Bonno N. , Vlooswijk Riek A.A.
Published: May 2019

Human prekallikrein (PK) is in plasma complexed to High Moleculair Weight Kininogen (HMWK). The formation of a complex of PK with HMWK was demonstrated by cross-immunoelectrophoresis. Purified PK alone did not migrate upon electrophoresis, however when mixed with HMWK or Fletcher plasma, PK migrated with the same mobility as PK present in normal plasma. Also addition of PK to HMWK resulted in a reduction of the electrophoretic mobility of HMWK on cross-immunoelectrophoresis using anti-HMWK. Fitzgerald plasma which is deficient in HMWK has a PK clotting activity of 26% but no detectable PK-antigen level. Reconstitution with HMWK yielded PK-antigen levels corresponding to the PK-clotting activity. Addition of HMWK to normal plasma did not increase the PK-antigen levels.

PK was activated by purified human (β-XIIa (28, 000 mol.wt) and by bovine α-XIIa (80, 000 mol.wt). Activation by αXIIa but not by by β-XII was enhanced by the presence of HMWK and dextran sulphate. After complete activation by α-XIIa in the presence of HMWK kallikrein remained complexed to HMWK. The generation of kallikrein activity paralelled the extent of proteolytic cleavage. SDS-polyacrylamide gel electrophoresis in the presence of reducing agents showed that both α-XIIa and β-XIIa generated fragments with apparent mol.wt’s of 52,000, 42,000 and 37,000. These fragments are initially held together by disulfide bonds, a second relatively slow cleavage generates fragments with similar mol. wt’s but observed on non reduced SDS-gels. This cleavage is not accompanied by a decrease in kallikrein activity.
Replacement Therapy in Hemophilia Patients with Inhibitors
Authors: Shapiro S.S.
Published: May 2019

Approximately 15% of patients with hemophilia A develop inhibitors to Factor VIII. These inhibitors have a high degree of specificity for Factor VIII procoagulant activity. Inhibitor patients seem to fall into 2 groups: roughly 3/4 are “strong” responders, whose Factor VIII antibody titer rises substantially after exposure to Factor VIII, while the remainder are “weak” responders, whose antibody level remains below 3-5 Bethesda units/ml despite exposure to Factor VIII. The latter group can be treated successfully with Factor VIII, although the dose required may be greater than in non-inhibitor patients. The “strong” responders can be treated with Factor VIII if their antibody titer is very low, although anamnesis will ensue within 3-4 days, reaching a maximum in 10-20 days. When antibody titers are high, treatment can be extremely difficult. Use of Factor VIII may still be possible, when combined with massive plasmapheresis; otherwise treatment with vitamin K-dependent factor concentrates may be attempted. A variety of such concentrates is available, both “non-activated” and “activated”. The former type of product may have become less useful for the treatment of inhibitor patients in recent years. The NTH Cooperative Study of Factor VIII Inhibitors in Hemophilia A has recently conducted a double-blind study of Konyne and Proplex in the treatment of Inhibitor patients. These results will be presented.
Platelet Adherence to Human Artery Subendothelium in Pulsatile and Non-Pulsatile Flow
Authors: Sakariassen K. S. , Bolhuis P. A. , Sixma J.J.
Published: May 2019

Platelet adhesion to human artery subendothelium was investigated in a perfusion chapter model according to Baumgartner. A non-occlusive roller pump was used to recirculate the perfusate and obtain pulsatile flow. Non-pulsatile flow was produced by means of gravity The flow measurements were carried out by an electromagnetic flow assay. In the non-pulsatile flow system the shear rate at the wall was calculated at 840 sec-1 and in the pulsatile flow system at 804±205 sec-1. Platelets were labeled with 51Cr and treated with aspirin to prevent platelet-platelet interaction. Platelet adhesion increased with perfusion time but twice as many platelets adhered in non-pulsatile flow between 2 and 5 min. This difference diminished after prolonged perfusion. The presence of red cells(Ht 40%)caused a 6-fold increase of adherence in the non-pulsatile system and 9-fold in the pulsatile system. At counts below 200.000/ul at 5 min perfusion time more platelets adhered in the non-pulsatile system. This difference disappeared at higher platelet counts. Increasing shear rates caused increased adherence, but more adherence was in general found in the non-pulsatile system. These results Indicate that increased adherence is observed in a non-pulsatrle system, if saturation effects are avoided.
Effect of pH on Platelet Aggregation and Leakage of β-Thromboglobulin from Platelets
Authors: Lloyd J.V. , Tunbridge L.J. , Rudzki Z. , Watts S.E.
Published: May 2019

Experiments were carried out to determine whether there is a relationship between leakage of β-thromboglobulin (BTG)from platelets and changes in response to ADP occurring during storage or changes in pH. Blood or platelet rich plasma (PRP) collected in 0.1M citrate was incubated at 37°C and plasma BTG estimated by radioimmunoassay; further leakage of BTG was effectively prevented by adding PGE1 and cooling to 0-4°C.

Plasma BTG concentration was 21ng/ml at venesection and 95ng/ml 60 minutes later when pH of PRP or whole blood was maintained at 7.35-7.50 by a 5% CO2 atmosphere. Further leakage was slight, plasma BTG being 123ng/ml at 120 minutes, but when pH was allowed to drift to 8.6 at 120 minutes after venesection plasma BTG was 325ng/ml (3.5% of total platelet BTG) and this was accompanied by liberation of 8.6% of H3'-serotonin from the platelets. Similar results were obtained when pH of whole blood was allowed to drift to 7.7. At pH 7.7-8.0 platelet aggregation was consistently enhanced.

The experiments demonstrated leakage of BTG from platelets within 30 minutes of venesection and before deterioration of response to ADP. Further leakage of BTG and serotonin was induced by a shift in plasma pH and may be due to a pH related stimulus causing both changes in sensitivity to ADP and release of granule contents. Initial leakage after venesection may reflect small unavoidable changes in pH.
Enhanced Affinity of Fibrinogen and Factor XIII Induced by Limited Proteolysis
Authors: Chung S.I. , Folk J.E. , Finlayson J.S.
Published: May 2019

Previous studies have shown that tissue transglutaminase has strong affinity for fibrinogen. In this study, the binding of protransglutaminase, i.e., plasma Factor XIII(a2b2) and platelet Factor XIII (a2), to fibrinogen was examined by ultracentrifugation, exclusion and affinity chromatography, Immunoelectrophoresis, and salt precipitation. Affinity was detected only by salting out and affinity chromatography. Limited proteolysis to form Factor XIIIa (a′2b2 and a′2) enhanced the affinity for fibrinogen. Limited proteolysis of fibrinogen by thrombin or ancrod enhanced affinity for both a2b2 and a2. Fibrin also bound a′2b2 and a′2. The b2 subunit exhibited no affinity for fibrinogen or fibrin. Thus, in the presence of fibrin(ogen), a′2b2 gave rise to a fibrin(ogen)-a′2 complex, and the b2 subunit was liberated.

The enhanced affinity for fibrinogen induced by limited proteolysis of Factor XIII suggests that fibrinogen could serve to adsorb active transglutaminase(s) released or generated in the circulation.
Fibrinolytic States in a Patient with Congenital Deficiency of Alpha 2-Plasmin Inhibitor
Authors: Aoki N. , Sakata Y. , Matsuda M. , Yoshida N.
Published: May 2019

A patient with congenital deficiency of α2-plasmin inhibitor (α2PI) was studied for his fibrinolytic system. The patient’s whole blood clot formed in vitro was lysed rapidly in several hours on incubation. This accelerated in vitro fibrinolysis was blocked by an addition of purified α2PI. In spite of the accelerated in vitro fibrinolysis, the increases of fibrinogen/fibrin degradation products and Fragment A were not detected when the blood was mixed with tranexamic acid-aprotinine mixture immediately after drawing blood, suggesting no increased in vivo fibrinogenolysis. When the patient’s citrated plasma was incubated in vitro, no or little progressive degradation of fibrinogen was observed in spite of the accelerated conversion of native Glu-plasminogen to modified Lys-plasminogen in the patient’s plasma. When the patient’s plasma was clotted by recalcification, fibrin formed was rapidly lysed. These results may suggest that α2PI is effective in inhibiting fibrinolysis but other protease inhibitors in plasma such as α2-macroglobulin are ineffective in inhibiting fibrinolysis although they are effective in inhibiting plasmin in plasma milieu thereby prevent fibrinogenolysis. Deficiency of α2PI in this patient may have led to uninhibited in vivo fibrinolysis resulting in friable hemostatic plugs causing severe hemorrhagic tendency.
Antiplatelet Drugs, Controlled by Platelet Survival
Authors: Bremer G. , Richter O. , Jacobi E.
Published: May 2019

The mean Platelet survival is an in-vivo parameter in the analysis of platelet function. Thromboembolic diseases are associated with shortened platelet survival, because platelets are consumed in the thrombus generanation. Platelet survival therefore is an parameter for controlling the effectiveness of antiplatelet drugs. In order to investigate the effect of several antiplatelet drugs, platelet survival is measured in Patients suffered from long-standing diabetes mellitus with secondary complications with and without the following antiplatelet drugs: 3 times of 500 mg per day SH 1117 (Sulfinylaceticacidemethylester), 3 times of R 149 per day (75 mg Dipyridamole + 330 mg Acetyl-Salicylic-Acid) and a third group treated with 3 times per day 150 mg Anturano + 150 mg Dipyridamole. The shortened platelet survival in Diabetes mellitus can be normalized by R 149, whilest SH 1117 increases the platelet survival only moderately. Under the treatment with Anturano combined with Dipyridamole platelet survival is prolonged, but not normalized.
Modifications of the Freeze-Cleaved Circulating Platelets Membrane in Hypercholesterolemic Rabbits, Prevented by AD6.
Authors: Weber G. , Bianciardi G. , Pierli C. , Toti P.
Published: May 2019

Circulating platelets present on their membrane some protuberances, placed among the plasma-membrane particles, when examined with F.E. technique. Statistic tests have shown a significant increase in the average number of such protuberances in the platelets of hypercholesterolemic (15 and 45 days) rabbits.

After i.v. administration of AD6, (8-mono-chloro carbochromen, a substance with a supposed anti-aggregating action) the number of the protuberances of circulating platelets in the hypercholesterolemic rabbits approaches to the average values of the normocholesterolemic ones.
Effects of Dietary Fatty Acids on Platelet Aggregation and Platelet Thromboxane B2 Production in the Rabbit.
Authors: Agradi E. , Tremoli E. , Petroni A. , Colombo C. , Galli C.
Published: May 2019

Semisynthetic isocaloric diets containing 25% of either butter or corn oil were fed to male rabbits for periods of 21 days and 2½ months. Platelet aggregation induced by threshold concentrations of collagen and sodium arachidonate and the conversion of labelled arachidonic acid were then studied. Response of platelets from butter-fed animals was considerably higher to arachidonate, whereas the thresholds for collagen-induced aggregation were similar in both groups of animals. Conversion of labelled arachidonic acid to thromboxane B2, studied at different substrate concentrations in washed platelets, was significantly higher, at low substrate concentration, at 21 days in the butter fed group, and the difference was still present after 2½ months of treatment. Conversion of arachidonic acid to the hydroxy fatty acids by lipoxygenase activity was not affected by the dietary treatments. In conclusion, the metabolic conversion of exogenous arachidonic acid to thromboxane B2 appears to be selectively affected by the type of dietary fatty acids and this effect is associated with changes in the response of platelets to aggregating concentrations of arachidonic acid.
Acquired Thrombasthenia in Non Hodgkin’S Lymphoma
Authors: Colvin B.T. , Lloyd M.J. , Mills M.J. , Bainbridge D.R. , Bateman S. , Turnbull A.L.
Published: May 2019

A patient with non-Hodgkin’s lymphoma developed a typical thrombasthenic defect confirmed by platelet aggregation. The patient’s plasma was capable of inducing an identical defect in normal platelets. Patient’s platelets were found to be coated with IgGK and IgAK when studied by immunofluorescent techniques. Immunoglobulin of similar specificity was demonstrated in tumour at autopsy. IgAK specific antibody to normal platelets was found in the plasma.

The finding of this autoantibody mirrors the observation of Levy-Toledano et al (1978) Blood, 51, 1065 in which a similar isoantibody was demonstrated in a multitransfused thrombasthenic patient.
Venom Coagglutinin and Lyophilized Platelets for Assay of Plasma Von Willebrand Factor: Comparison With Ristocetin
Authors: Brinkhous K.M. , Read M.S. , Dombrose F.A.
Published: May 2019

A new procedure for assay of plasma von Willebrand factor (vWF), employing venom coagglutinin, a recently discovered “activator” of vWF, and lyophilized human or animal platelets has been developed (Brinkhous, K.M., Potter, J.Y., Read, M.S., Fed. Proc. 38, 1979). Comparison was made of vWF levels as assayed by ristocetin (0.6 mg/ml) and venom coagglutinin (1.25 u/ml) with fixed and lyophilized human and canine platelets. A unit of venom coagglutinin is that amount which causes 4+ aggregation of a standard canine platelet-plasma mixture in 15 sec. The vWF values of 20 normal subjects fell in the range of 80-125% and for subjects from vWD kindreds the range was <5-60%. There were no significant differences in vWF levels with either the ristocetin or venom coagglutinin assay. The latter assay has two advantages: no false positive results due to protein precipitation and animal platelets can be used.
Binding of Ristocetin to Platelets
Authors: Jenkins C.S.P. , Clemetson K.J. , Lüscher E.F.
Published: May 2019

Ristocetin induces platelet agglutination in the presence of von Willebrand factor (VIIIR:WF), part of the factor VIII complex. The role of ristocetin in platelet agglutination is not yet. Clear. To follow the interaction of ristocetin with platelets and/or VIIIR:WF, ristocetin was labelled with[3H] by reductive methylation. [3H]-Ristocetin agglutinates platelets in the presence of VIIIR:WF in a manner indistinguishable from unlabelled ristocetin. The binding of[3H]-ristocetin to normal and chymotrypsin-treated platelets (which ore not agglutinated by ristocetin/VIIIR:WF) was studied in the presence and absence of VIIIR:WF (normal pool plasma and plasmas from patients with von Willebrand’s disease) and at non-agglutinating and agglutinating concentrations of ristocetin. Virtually no difference in binding was seen whether VIIIR:WF was present or not. Chymotrypsin-treated platelets do not bind less ristocetin than control platelets. A pronounced direct relationship was found between [3H]-ristocetin bound by normal platelets and total ristocetin concentration. This implies that at the higher concentrations of ristocetin either more binding sites are exposed or that aggregation of ristocetin occurs.
Platelets Cross-Linked with Dimethyl Adipimidate Hydrochloride (DMA) Used in Ristocetin Assay For F VIII: Rcof.
Authors: Qazzaz M. , Podolsak B. , Wenzel E.
Published: May 2019

When PRP or washed platelets are incubated with dimethyl adipimidate (DMA)(final concentration 2 mg/ml, 30 - 60 min.), the aggregation of the platelets by ADP, collagen or thrombin is clearly reduced. Aggregation by ristocetin plus F VIII: Rcof is however not affected.

DMA is a bifunctional imidoester which acts as a cross-linking reagent. SDS Polyacrylamide gel electrophoresis of platelet membranes shows a different pattern for membrane glycoproteins from the platelet treated with DMA when compared to untreated platelets. This different pattern is due to cross-linking of the glycoproteins.

The DMA - treated platelets can be stored for at least three weeks at 4° without losing their ability to aggregate in the ristocetin F VIII: Rcof test system. Both freshly -treated and stored DMA - treated platelets could be used to assay F VIII: Rcof reproducible in normal pooled plasma and in defective plasma (von Willebrand’s disease patients; N = 8). The preparation of DMA-fixed platelets takes less than two hours.
Purification and Properties of a Porcine Tissue Plasminogen Activator and its Comparison with Activators from Human Uterine Tissue, Blood and Urine
Authors: Kok P.
Published: May 2019

A plasminogen activator was prepared from porcine ovaries by a simple procedure described earlier (Kok and Astrup, Biochemistry 8:79, 1969) and further purified by ion exchange chromatography on SE- or SP-Sepharose and affinity chromatography on Sepharose-lysine.

The preparation was homogeneous by gel electrophoresis at pH 3.5 and SDS-polyacrylamide electrophoresis(PAGE). The specific activity was about 4.5 x 106 A and A tissue activator units per mg protein (one tissue activator unit is comparable to 0.1 CTA urokinase unit in a fibrinolytic assay under specified conditions). The molecular size estimated by SDSPAGE corresponded to a Mw of 67000 dalton. After reduction with dithiothreitol the activator produced a single band corresponding to a Mw of 33500 dalton, indicating cleavage into two chains of equal size. Isoelectric focusing revealed several bands, which were active and with an isoelectric pH range of 7-8.

An activator from human uterine tissue and an activator in the euglobulin fraction from human plasma were similar to the porcine tissue activator in molecular size, estimated by gel filtration, and immunochemically. The porcine tissue activator differed from the activator from human urine in reactivity, molecular size and immunochemically.

(This work was supported by USPHS Grant HL-05020, NIH, National Heart and Lung Institute, to dr. Tage Astrup and by the Swedish Medical Research Council (project no. 13X 03906 07A).)
Purification of Porcine and Human Tissue Activator
Authors: Wallen P , Rånby M , Bergsdorf N , Kok P
Published: May 2019

Tissue activator from pig heart: A highly purified activator preparation from pig heart has been prepared, essentially using two affinity adsorbtion steps. 1) Affinity adsorbtion to fibrin and elution with potassium thiocyanate. 2) Affinity chromatography on Sepharose-arginine. The final product, which is obtained by gel filtration on Sephacryl S-200 contains according to SDS-polyacrylamide gel electrophoresis one main band with Mw 64000. Reduced samples still appear as one component but with Mw 31000. The specific activity is about 500000 IU/mg (WHO Reference Preparation for Urokinase) and the yield 15-25 %.

Tissue activator from human uterus: A highly purified preparation of human tissue activator has been prepared from uterus by an immunosorbent technique using antibodies produced in goats against the porcine tissue activator and coupled to Sepharose. A crude preparation from 1 kg uterus tissue and containing about 100000 IU tissue activator was adsorbed on 30 g of the immunosorbent. The activity was eluted with a KSCN-gradient. Further purification was obtained by affinity chromatography on Sepharose-arginine. The yield in the active fraction was 30-35 % and the specific activity 200000 to 300000 IU/mg. SDS-polyacrylamide gel electrophoresis showed one main band and 1-2 additional trace components.
Correlation of in Vivo Clot Lysis with other Thrombolytic Variables Following Administration of abbokinase ®(Tissue Culture Urokinase), to Anesthetized Dogs
Authors: Martin D. , Cain J. , Chmiel J. , El Masry S.E.
Published: May 2019

An anesthetized dog model, using an extracorporeal loop containing an autologous radioactive clot, was utilized to test the effects of various doses of ABBOKINASE® on the rate of clot lysis and on plasma levels of urokinase, plasmin, antiplasmin, plasminogen and fibrinogen. The effects of ABBOKINASE® on hematocrit, euglobulin lysis time and125 I-clot lysis, in vitro, were also determined. Correlations were sought between plasma urokinase, plasmin, antiplasmin and the rate of clot lysis. Kinetic evaluations of half-lives of urokinase and plasmin and of the rate of regeneration of antiplasmin were made. Some of the conclusions reached were: 1) plasma fibrinogen does not decrease until antiplasmin is depleted and free plasmin appears in blood. 2) plasma urokinase levels are related to the dose infused and decrease with a half-life of about 8 minutes following infusion. 3) the rate of clot lysis in the loop is proportional to the dose of ABBOKINASE® over a defined range of doses and can be fitted to a mathematical model. 4) at lower doses, clot lysis occurs in the absence of measurable free plasmin.
Control of Heparin Conformation and Activity by Calcium Binding
Authors: Boyd J. , Long W.F. , Williamson F.B.
Published: May 2019

The conformations of heparins in aqueous solutions were studied in the presence, of various metal ions, using nuclear magnetic resonance and chiroptical techniques. These studies indicate that a specific, intramolecular, cooperative binding of calcium ions induces a major change in the conformation of the polysaccharide. This change involves the locking of specific tetrasaccharide units into a rigid conformation, in which N-sulphato groups of glucosamine and O-sulphato groups of iduronate appear to be maintained in close proximity. Such an arrangement would increase the ability of these groups to interact electrostatically with cationic residues of other macromolecules such as antithrombin III. Our findings suggest that the relative effectiveness of different acidic polymers in antithrombin III-mediated anti-coagulation may be related to the rigidity of the polymer and to the suitable disposition of anionic substituents along the polymer chain.
Administration of Fractionated Heparins to Man
Authors: Lane D.A. , MacGregor I.R. , Cella G. , Kakkar V.V.
Published: May 2019

A commercial mucosal heparin preparation (Leo Pharmaceuticals) has been fractionated by gel filtration into five different MW fractions of approximate mean MWs 9,500-25000. These five hep rins were randomly administered to 5 normal volunteers by intravenous and subcutaneous injection. The anticoagulant effects of the fractions were then determined by KCCT, anti-Xa clotting and anti-Xa chromogenic substrate assays. Following intravenous injection the high MW fraction produced identical elimination curves in all three assays, but with decreasing MW there was a divergence between results obtained by KCCT and anti-Xa assays. The lowest MW fraction produced between 2-3 times greater heparin levels when measured by anti-Xa assay. This divergence in assay results was not caused by the heparin induced release of an inhibitor of the Xa reaction. This was shown by performing neutral isation experiments with purified PF4 that demonstrated that both low and high MW fractions released this component, which accounted for a third of the anti-Xa heparin level. The divergent results were caused rather by the previously reported difference in specific activities when fractionated heparins were assayed in vitro by different assay methods A MW dependence was observed for the absorption of the fractions into the circulation following subcutaneous injection, with low MW heparin producing higher heparin levels determined by all the assays.
Thrombopoietin-Induced Stimulation of Megakaryocyte- Enriched Bone Marrow Cultures
Authors: Kellar K. L. , Evatt B. L. , McGrath C. R. , Ramsey R. B.
Published: May 2019

Liquid cultures of bone marrow cells enriched for megakaryocytes were assayed for incorporation of 3H-thymidine (3H-TdR) into acid-precipitable cell digests to determine the effect of thrombopoietin on DNA synthesis. As previously described, thrombopoietin was prepared by ammonium sulfate fractionation of pooled plasma obtained from thrombocytopenic rabbits. A control fraction was prepared from normal rabbit plasma. The thrombopoietic activity of these fractions was determined in vivo with normal rabbits as assay animals and the rate of incorporation of 75Se-selenomethionine into newly formed platelets as an index of thrombopoietic activity of the infused material. Guinea pig megakaryocytes were purified using bovine serum albumin gradients. Bone marrow cultures containing 1.5-3.0x104 cells and 31%-71% megakaryocytes were incubated 18 h in modified Dulbecco’s MEM containing 10% of the concentrated plasma fractions from either thrombocytopenic or normal rabbits. In other control cultures, 0.9% NaCl was substituted for the plasma fractions. 3H-TdR incorporation was measured after cells were incubated for 3 h with 1 μCi/ml. The protein fraction containing thrombopoietin-stimulating activity caused a 25%-31% increase in 3H-TdR incorporation over that in cultures which were incubated with the similar fraction from normal plasma and a 29% increase over the activity in control cultures to which 0.9% NaCl had been added. These data suggest that thrombopoietin stimulates DNA synthesis in megakaryocytes and that this tecnique may be useful in assaying thrombopoietin in vitro.
Familial Deficiency of a Plasma Factor Stimulating Vascular Prostacyclin (PGI2) Activity
Authors: Remuzzi G. , Marchesi D. , Misiani R. , Mecca G. , de Gaetano G. , Donati M.B.
Published: May 2019

We previously reported successful treatment with plasma in patients with thrombotic micro angiopathy and defective vascular PGI2 activity and suggested that a defect in a plasma factor stimulating PGI2 synthesis might be implicated in the pathogenesis. We report here that in one of these patients the plasma defect was still detectable 1 year after clinical remission (without recurrencies). Two of this patient’s 4 children had a simile,though less severe, plasma defect. The proposita is a 54-year-old woman admitted with a clinical and laboratory picture of haemolytic uraemic syndrome. Unlike normal plasma, the patient’s plasma had a low capacity to stimulate PGI2 production by rat aortic rings (previously washed until their endogenous PGI2 activity was exhausted). After plasma treatment the patient’s plasma behaved normally in this respect, but again appeared deficient at outpatient follow-up. PGI2 stimulating activity was normal in 2 daughters but consistently low (20-60% of control) in both patient’s sons. None of them had any history or clinical signs of microangiopathic disorders. Detection of this plasma defect in apparently healthy subjects and in patients who have recovered from thrombotic microangiopathic episodes could have clinical implications. Our observations support previous suggestions that some people have a congenital (or hereditary) predisposition to thrombotic microangiopathy.

(Supported by Ass. Berg. Mal. Ren. and Italian CNR, Rare disease Project).
Tissue Thromboplastin: Relationship between Procoagulant Activity Lipid Composition and Morphology on Storage
Authors: Stevenson K. , Slater P. , Poller L.
Published: May 2019

Liquid suspensions of tissue extract thromboplastin gradually deteriorate on storage. Successive batches of standardised extracts of human brain prepared in this laboratory over a ten-year period have been studied by high pressure liquid chromatography. With storage, changes in lipid composition took place. Significant reductions in relative concentrations of phosphatidyl choline, phosphatidyl ethanolamine and phosphatidyl serine were found, while an increase in free fatty acids ana malonaldehyde was observed coincidental with decreasing procoagulant activity. Transmission electron microscopy showed changes in thromboplastin membrane structure which correlated with the observed biochemical characteristics and reducing procoagulant activity of extracts.
A Novel Chromogenic Assay Equivalent to the One Stage Prothrombin Time (PT)
Authors: Baughman D. J. , Lytwyn A.
Published: May 2019

A chromogenic assay equivalent to the PT has been developed which, it is expected, will allow the standardization of therapeutic limits. Assays were performed by combining 0.25 ml S-2238 (2mM), 0.40 ml tissue thromboplastin, and 0.050 ml plasma in 1.40 ml TRIS buffer (pH=8.50, I=0.15). The rate of thrombin generation was obtained by automatically computing the slope of In (A405nm/min. ) vs time. The results indicated that thromboplastin does not interfere with S-2238 hydrolysis. Thrombin production (A/min.) occurs just before clotting, rises rapidly to a maximum and falls. During its rise, the thrombin concentration increases as a positive exponential, exp(bt). The exponential rate, called b, is constant from the initial appearance to the maximum thrombin concentration, b is proportional to plasma concentration and to log Factor (X, VII, V) concentration for dilutions with deficient plasma. Log 1/b correlates with log PT using dicoumarolized plasmas. Thrombin appearance is quite delayed (12 min.) in diluted, deficient plasma. Thus, the assay appears sensitive to the extrinsic system and can be used to monitor dicoumarolized plasmas. Unlike the standard PT, this assay directly measures the rate of thrombin production and could be standardized by comparing the exponential rate to specific Factor concentrations.
Fibrinolytic Activity of the Arterial Wall in Occluded Arteries With Thrombi
Authors: Donner L. , Klener P. , Vodáková L.
Published: May 2019

The plasminogen activator in 117 specimen of 20 coronary and 20 pulmonary arteries completely occluded by thrombi, or emboli was studied within the adventitia and intima using Todd s histochenncal method. 39 cadavers were used, 1-18 hours post mortem from subjects aged 45 to 88 years.

In occluded arteries both coronary and pulmonary the plasminogen activator activity was decreased in comparison of patent normal and atherosclerotic coronary and pulmonary arteries. In arterial thrombi a low grade focal activity of plasminogen activator was detected. It is suggested that the decrease of plasminogen activator in occluded human arteries might be due to the impaired oxygen supply of the vessel wall and to the consumption of plasminogen activator for lysis of the thrombus. These mechanisms must probable influence the plasminogen activator for a certain and prolonged time, since there were no changes of fibrinolysis within the vessel wall of arteria carotis of rats where a peracute thrombosis by means of an electric current was provoked.
Effects of Tranexamic Acid, CIS-AMCHA, and 6-Aminohexanoic Acid on the Activation Rate of Plasminogen by Urokinase in the Presence of Clot
Authors: Takada A. , Takada Y.
Published: May 2019

When human plasma was mixed with 50 units of urokinase (UK) and clotted with thrombin in the presence of S-2251, the hydrolysis of S-2251 in the clot was higher than that in the plasma. The presence of 10 μM of tranexamic acid resulted in decrease in the activation rate of plasminogen in the clot but not in the plasma. Further increase in its concentration resulted in marked increase in plasminogen activation in both clot and plasma. The addition of 6-aminohexanoic acid or cis-AMCHA showed changes in the rate of plasminogen activation similar to that shown in the presence of tranexamic acid. Fifty μM of tranexamic acid totally inhibited urokinase induced clot lysis, while 1 mM of cis-AMCHA and 6-aminohexanoic acid totally inhibited clot lysis. The presence of 1 mM of thesewaminoacids remarkably increased the activation of plasminogen by UK, but no clot lysis was observed. Hydrolysis of 5-2251 was higher in the presence of tranexamic acid than in its absence or clot formation without tranexamic acid. Tranexamic acid seems to be more effective in enhancement of activation of plasminogen by UK than clot formation. Plasmin formed by UK was coexistent with α2PI. The presence of tranexamic acid or clot formation resulted in smaller extent of interaction of plasmin with α2PI, thus saving the exhaustion of α2PI and preventing instant inactivation of plasmin by α2PI.
Plasmin Activity, Plasminogen Levels and Concentrations and Functional Activities of Antiplasmins During Septicemia
Authors: Gallimore M. J. , Aasen A.O. , Lyngaas K. , Larsbraaten M. , Amundsen E. , Smith Eriksen N.
Published: May 2019

Recent studies have indicated that the fibrinolytic system becomes activated during endotoxemia in animals and septicemia in man. In the present study we have used chromogenic peptide substrate assays to measure plasmin activity and “immediate” and “total” antiplasmin activities in plasma samples from normal subjects and patients with septicemia. Plasma concentrations of plasminogen (Pg), α2 antiplasmin (α2AP), α2-macroblobulin (α2M) and α1-antitrypsinla (α1AT) were also determined. In samples from patients with fatal sepsis low levels of Pg, α2 AP and α2 M were found together with reduced “immediate” and “total” an antiplasmin activities. Plasmin activity and α1 AT levels were higher than normal. Three patients who recovered following septicemia had higher Pg and α2 AP concentrations than those who died and functional antiplasmin activities were within the normal ranges. Out results confirm that the fibrinolytic system becomes activated during septicemia and in sepsis of fatal outcome a marked consumption of Pg and α2 AP occurs. In our limited number of recovery patients the fibrinolytic system appeared to have been less activated than in those who died.
A Study of Intravascular Platelet Aggregation in the Rat
Authors: Duncan G. G. , Smith G. M.
Published: May 2019

Intravascular platelet aggregation can be studied by measuring the fall in the circulating platelet count induced by aggregating agents in anaesthetized animals. The Technicon Auto-counter was modified and connected via a double cannula to an anaesthetized rat to give a continuous count of the number of circulating platelets (1). Adenosine diphosphate (ADP), Collagen, Arachidonic acid (AA) and 5-Hydroxytryptamine (5-HT) were given at 15 minute intervals over a period of 2-3 hours. Aspirin (10 mg/Kg IV ) and Indomethacin (1-8 mg/Kg IV) partially inhibited collagen-induced aggregation and Indomethacin (2 mg/Kg IV) completely inhibited AA-induced aggregation. Adenosine (0.25 mg/min) inhibited the ADP-induced aggregation but did not inhibit aggregation produced by collagen or the residual response to collagen that remains after the addition of indomethacin.

Reproducible responses to ADP and collagen were obtained but responses to AA and 5-HT were not reliable. Collagen-induced aggregation is thought to be mediated by the liberation of ADP, 5-HT and the formation of prostaglandin (PG ) endoperoxides and thromboxane A2. This study has shown that collagen-induced aggregation is reduced by inhibition of PG synthesis but the involvement of ADP or 5-HT could not be shown.
Tachyphylaxis in Human Blood Platelets Induced by Various Agonists and the Effect on Response To other Agonists
Authors: Ruggles P.A. , Scrutton M.C.
Published: May 2019

Tachyphylaxis of human platelets to ADP, adrenaline, thrombin, 5-HT and vasopressin (VP) was induced by preincubation of stirred citrated PRP with an agonist concentration which induced primary reversible aggregation. The effect was demonstrable within 2 mins, after addition of some of the agonists and persisted for at least 30 mins. The extent of tachyphylaxis showed a positive correlation with agonist concentration used to induce the initial reversible response. Partial aganists at the ADP (2’, 3’-dialcohol ADP) or α-adreno-(clonidine) receptors did not induce significant tachyphylaxis to subsequent addition of the full agonist. In most instances platelets iaade tachyphylactic to a given agonist showed an unchanged or enhanced response to all other agonists including arachidonate. However tachyphylaxis to ADP, 5HT or thrombin was associated with an attenuated response to collagen. Furthermore tachyphylaxis to thrombin also caused attenuation of the response to VP and arachidonate. Induction of tachyphylaxis to VP, or addition of oxytocin (an inhibitor of aggregation induced by VP) had no effect on the response to thrombin. Thus the region of the thrombin receptor responsible for induction of tachyphylaxis to this agonist is not identical with that at which VP interacts. If stimulus-response coupling involves a common pathway for most agonists these data suggest that development of tachyphylaxis depends on events which preceed the effect of the agonists en this common pathway.
Hepar Inconcentration, Antithrombin III and Antiplasmin in Elective Surgery with Low Dose Heparin Prophylaxis
Authors: Kruse-Blinkenberg H.O. , Gormsen Johs
Published: May 2019

Eighty patients underwent abdominal surgery in low dose heparin (Novo) 5.000 units subcutanously 2 hours preoperatively and every 8 hours until full mobilization. Clinical end points were 125J fibrinogen uptake test and venography. Heparin concentrations, antithrombin III(AT III) against IIa and Xa and antiplasmin, were measured on synthetic substrates (Kabi Diagnostica, Sweden). Deep vein thrombosis (DVT) developed in 16 patients. Heparin concentrations were identical in patients ± DVT preoperatively but the concentrations were significantly lower postoperatively on day 1-5 in the patients with DVT, AT III (against IIa)was lower pre- and postoperatively in patients with DVT, but the differences were only significant lower when measuring AT III against Xa, pre- and postoperatively. The antiplasmin level was higher in patients with malignency and in patients with DVT, and the differences were significant postoperatively (p < 0.01).
Hemorrhagic Complications of Heparin Therapy in Elderly People
Authors: Kher A. , Hervy M.P. , Henry J.F. , Forette F. , Berthaux P.
Published: May 2019

The most important side effect of heparin therapy is bleeding. The reported incidence has varied widely and the relationship of bleeding to dose, route of administration, age, sex, underlying haemostatic defect and hypertension has been suggested. A review of one years experience with subcutaneous heparin in 113 elderly patients is presented in this paper. They are divided into three groups : Group I without laboratory control, Group II - dosage of heparin being controlled with recalcification plasma test, and Group III-heparin requirement controlled by thrombo-elastography. Group I - 24 women (average age 82.1 years) receiving 5000 I.U. every 8 or 12 hours, mean duration of treatment 24 days ± 13.3 : one fatal retroperitoneal haemorrhage. Group II - 35 females, 10 males (average age 81.3 years), mean daily dose of heparin 13.250 I.U.(TID), duration of treatment 30.3 ± 24 days, 10 patients sustained bleeding complications. Group III -38 females, 6 males (average age 81.6 years), average daily heparin requirement 12.250 I.U. (TID), mean duration of treatment 42.2±55.5, 4 patients developed minor bleeding.

The relationship between age, sex, control of heparin dosage and existence of hypertension will be discussed in detail.
Heparin Anticoagulation:A Systems Approach
Authors: Shepherd R. , Rodvien R. , Mielke C. H.
Published: May 2019

Failure to give heparin in a repeatable manner has prevented definition of conditions which optimize heparin’s use. We have set up an algorithm (AL) for continuous IV heparin to be given in a standard manner to pts. with suspected venous thrombolic disease(TED). AL’s intent was to allow intermittent adjustment of heparin to keep the activated clotting time (ACT) between 170-200 secs. (1½-2 x normal). Other pts. not on AL who received continuous IV heparin and were monitored by the ACT served as a non-random concomitant control population. Five community hospitals provided 120 pts. managed by nurses using AL. AL kept the ACT in the goal range (76 % vs 54 %), and bleeding was diminished but not eliminated (<2 % per day exposure). Bleeding, in spite of good control of the ACT, occurred only between days 2 and 9. This decrease in bleeding occurred despite increased amounts of heparin (1767 u/hr. mean vs 1200 u/hr., literature’s mean). No clinical recurrences were recognized but efficacy could not be tested because TED was not always documented by phlebography. Physicians’ acceptance of the nurses role has been excellent. With AL, hypotheses concerning infusion conditions to optimize heparin’s safe and efficacious use can now be tested.
Plasma Heparin Levels and Patient Acceptability with Subcutaneous Heparin Sodium and Heparin Calcium
Authors: Bonnar J. , Ma P.T.S.
Published: May 2019

A double-blind crossover trial in 40 normal subjects was carried out using 5,000 and 10,000 USP units of heparin sodium and heparin calcium given subcutaneously. Plasma heparin was measured by the antifactor Xa method and also using the substrate S-2222. Heparin sodium was found to have a longer duration of action and a greater total absorption than heparin calcium. A second double-blind crossover study in 30 subjects was carried out to determine patient acceptability of self administered subcutaneous heparin (5000 USP units). A preference for heparin sodium was expressed by 77.4% of the subjects and 9.7% preferred heparin calcium, the remainder found no difference. Heparin calcium was found to be significantly more painful during injection than heparin sodium (p< 0. 001). Plasma levels of heparin after subcutaneous administration of 5,000, 7,500 and 10,000 units of heparin sodium and heparin calcium were studied in late pregnancy between 30 and 40 weeks gestation. A wide individual variation was found but none had plasma levels above 0.40 units per ml after 10,000 units 12 hourly. During late pregnancy a regimen of 5000 units of heparin sodium given 8 hourly is safe; a higher dosage of heparin calcium is required to achieve similar levels of plasma heparin.
I - High Molecular Weight Urokinase (UK) and Different Molecular Weight Heparin (HP) Fractions in Fibrinolysis
Authors: Villaverde C. , Badimón L. , Valdecasas F.
Published: May 2019

Following our previous works on the interaction of UK and Hp (Thromb. Haem, 38, 105, 1977), we have carried out research to clarify the possible influence of the specific molecular weights characteristics in that interaction, We have studied the relationship of HMW-UK (54,000) to HMW and LMW Hp in rat blood. Inhibitors were evaluated by chromogenic substrate 5-2251 and by our method on an activated fibrin plate, and fibrinolysis by euglobulins. The HMW-UK was tested in a dosage range of 100 to 300 PI.U/ml and Hp ones were: from 0.001 to 1,000 I.U/ml. Hp did not alter the basal inhibitor levels measured on fibrin substrate but we obtained on synthetic substrate an inhibition of 12% on the basal value for all Hp dosages. HMW-Hp produced an increase in the fall of the inhibitory activity induced by HMW-UK for low dosages of Hp, whilst high dosages produced an increase of inhibition, suppressing the UK indeed reduction of inhibitor levels. For LMW-Hp the low dosages did not alter the UK effect while high ones imppreased it. Euglobulins activity HMW-UK induced were not modified by low dosages but high ones induced a highly significant inhibition (for both Hp fractions).
An Inhibitor to Factor VIII Antigen Presenting as Acquired Von Willebrand Disease
Authors: McGrath K.M. , Johnson C.A. , Stuart J.J.
Published: May 2019

A patient with clinical and laboratory evidence of acquired von willebrand disease is described. He presented with the recent onset of spontaneous hemorrhageand demonstrated a prolonged bleeding time, reduced factor VIII. C, and undetectable factor VIII:AG and factor VIIIR:WF. Following transfusion of cryoprecipitate, there was a smaller than expected immediate increase in factor VIII:C, factor VIIIR. WF and factor VIIIR. AG, with a rapid return to baseline levels, and no secondary rise in factor VIII:C. An inhibitor could be demonstrated in the patient’s plasma which markedly decreased the level of faotor viriRiAG in normal plasma while only weakly decreasing the activity of factor VIIIR. WF. Activity against factor VII1:C was demonstrable in vitro using a concentrated inhibitor preparation. The inhibitor was contained in the IgG fraction of plasma and lacked precipitating properties. This inhibitor, which demonstrates a specificity not previously seen in spontaneous anti-factor VIII antibodies, provides further evidence for the separate identity of the antigen and von Willebrand factor sites of the factor VIII molecule. It also indicates that inhibition of ristocetin-induced aggregation of platelets is not the most sensitive method for the detection of an inhibitor in all cases of acquired von Willebrand disease.
Homologous Antibodies to Factor IX Shorten the Bovine Thromboplastin Coagulation Time of Human Plasma
Authors: Örstavik K.H.
Published: May 2019

We have previously demonstrated that neutralization of factor IX in normal plasma by heterologous antisera shortens the one stage prothrombin time determined with bovine thromboplastin. In this study we demonstrate a similar effect of ahomologousantibody. Addition of 1/5 volume of plasma irom a patient with hemophilia B- and an acquired inhibitor to factor IX (titre 5 U/ml) gave a shortening of the prothrombin time of plasma from 7 normal persons [mean 34 sec) compared to the prothrombin time determined atter addition of 1/5 volume of control plasma from a patient with hemophilia B- and no acquired inhibitor [mean 39 sec). Addition of inhibitor plasma had no eftect on the prothrombin time of plasma from 6 patients with hemophilia B-. Complexes between factor IX and the human inhibitor could be demonstrated both before and after the coagulation with bovine thromboplastin. These complexes Were demonstrated as a factor IX antigen with a reduced electrophoretic mobility in crossed Immunoelectrophoresis against a rabbit antiserum to factor IX, The results indicate that normal tactor IX loses the ability to act as an inhibitor in the coagulation with bovine thromboplastin after having formed a soluble complex with a homologous antibody.