bluuebunny/crossref_metadata_2025_split huggingface dataset:
DEMONSTRATION OF Cl-INHIBITOR COMPLEXES IN PLASMA BY HIGHLY SENSITIVE RADIOIMMUNOASSAYS USING A MONOCLONAL ANTIBODY AGAINST A NEODETERMINANT ON COMPLEXED Cl-INHIBITOR
Authors: Nuijens J H , Huijbregets C C M , Thijs L G , Hack C E 
Published: August 2018

Levels of factor XIIa- and kallikrein-Cl inhibitor (Cl-Inh) complexes in plasma reflect activation of the contact system in vivo. Here, we report the development of radioimmunoassays (RIAs) for these complexes using a monoclonal antibody (mAb K0K12) that reacts with a neodeterminant exposed on Cl-Inh after interaction with proteases. mAb K0K12 was obtained by a fusion experiment with spleen cells of a mouse hyperimmunized with Cl-Inh complexes.Experiments with purified Cl-Inh incubated with either Cls or elastase revealed that the determinant for mAb KOK12 is exposed on complexed as well as proteolytically inactivated (modified) Cl-Inh.Radioimmunoassays (RIAs) for the detection of factor Xlla-Cl-Inh and kallikrein-Cl-Inh complexes were performed as follows: mAb K0K12 was coupled to Sepharose and incubated with the sample to be tested. Binding of Cl-Inh complexes was detected by a subsequent incubation with 125I-antibodies against factor XII or (pre)kallikrein.With these RIAs, activation of 0.1% of factor XII or prekal-likrein in plasma is easily detected.Optimal conditions for blood sampling and processing were established, i.e. conditions that prevented any in vitro activation of factor XII and prekallikrein. Levels of factor XIIa-Cl-Inh and kallikrein-Cl-Inh complexes in plasma samples from normal donors were less than 0.1 U/ml (100 U/ml is the maximal amount of Cl-Inh complexes generated in pooled plasma by DXS). Considerably higher, and fluctuating levels were observed in patients with diseases such as septicaemia. These highly sensitive RIAs will facilitate studies concerning the role of the contact system in human pathophysiology.
INTACT MICROTUBULES ARE NECESSARY FOR COMPLETE PROCESSING, STORAGE AND REGULATED SECRETION OF VON WILLEBRAND FACTOR BY ENDOTHELIAL CELLS
Authors: Sinha S , Wagner D D 
Published: August 2018

The importance of intact microtubules in the processing, storage and regulated secretion of von Willebrand factor (vWf) from Weibel-Palade bodies in endothelial cells was investigated. Human umbilical vein endothelial cells treated for one hour with colchicine (10-6M) or nocodozole (10-6M) lost their organized microtubular network. Stimulation of these cells with secretagogues (A23187, thrombin) produced only 30% release of vWf in comparison to control cells containing intact microtubules. The nocodazole treatment was reversible. One hour incubation in the absence of the drug was sufficient for microtubules to reform and to restore the full capacity of the cells to release vWf.Long-term incubation (24 hours) of endothelial cells with microtubule destabilizing agents had a profound effect on vWf distribution. In control cells vWf was localized to organelles in the perinuclear region (i.e. endoplasmic reticulum and Golgi apparatus) and to Weibel-Palade bodies. In drug-treated cells vWf staining was dispersed throughout the cytoplasm and Weibel-Palade bodies were absent. The vWf synthesized in the absence of microtubules contained significantly less large multimers than that produced by control cells. This was not due to possible side effects of the drugs on the cells because the presence of lumicolchicine, an inactive analogue of colchicine, had no effect on vWf processing. Since Weibel-Palade bodies specifically contain the large multimers (Sporn et al, Cell 46:185-190, 1986), we hypothesize that the structural defect in vWf secreted by cells in the absence of microtubules is due to the lack of Weibel-Palade bodies in these cultures.In summary, the intact microtubular cytoskeleton in the endothelial cells in culture, appeared to be crucial for normal release of Weibel-Palade bodies after stimulation with secretagogues, for reformation of new Weibel-Palade bodies and for the efficient intracellular multimerization of vWf dimeric molecules.
THE ROLE OF GPIb IN PLATELET ACTIVATION
Authors: Wyler B , Clemetson K J 
Published: August 2018

Platelet membrane glycoprotein lb (GPIb) is known to contain a receptor for von Willebrand factor (vWf) and for thrombin. The role of GPIb in platelet activation was investigated by comparing the effect of removal of the binding sites by specific proteolysis with that of blocking them using specific antibodies. Specific removal of the 45 kDa outer part of GPIb by treatment of platelets with human leukocyte elastase caused loss of platelet agglutination response to von Willebrand factor (vWf) but also a weaker activation by thrombin similar to that found with Bernard-Soulier syndrome platelets which lack GPIb. Removal of the major part of the external region of the GPIba chain, including the 45 kDa domain, by calcium activated protease or by chymotrypsin, produced similar effects. The 45 kDa region of GPIba was purified by tryptic cleavage of isolated glycocalicin followed by gel filtration. Antibodies to this fragment were produced in rabbits and showed high specificity and affinity for the 45 kDa polypeptide. Fab fragments of IgG prepared from this anti serum affect platelet response to vWf and thrombin in a dose-dependent way. In both protease-treated and antibody-treated platelets the reduced response to thrombin, but not to vWf could be overcome by increasing the dose of thrombin. In contrast to removal of the 45 kDa domain, antibody treatment affected not only the platelet response to vWf and thrombin but also to collagen, ADP, PAF and arachidonic acid. Only the response to the calcium ionophore A23,187 was unaltered.Though not the essential receptor GPIb is clearly involved in platelet response to thrombin. A possible mechanism could involve a conformational change in the cytoplasmic/inter-membranous part of GPIb induced by binding of thrombin to the 45 kDa domain. Binding of antibodies might affect platelet activation by other agonists via a general down regulation effect. These results support the two receptor models for thrombin activation of platelets as proposed for other cell -types. In platelets the first receptor appears to be GPIb but the second receptor has not yet been identified.
EFFECT OF CACL2 ON ANTI-Xa ACTIVITIES OF HEPARIN AND LMW HEPARINS
Authors: Barrowcliffe T W , Le Shirley Yvonne 
Published: August 2018

Assays of anti-Xa activity of heparin are generally carried out using plasma or AT III, in the absence of Ca++ ions. We studied the effect of physiological (3 mM) Ca++ on the relative anti-Xa activities of heparin and LMW heparin, using human AT III, bovine Xa and S-2222.Potencies of a LMW heparin standard vs. the International Standard (IS) for unfractionated heparin (UEH) were decreased by 45% in the presence of CaCl2. This was shown to be due to a differential effect of CaCl2 on the two substances, with a 93% potentiation in activity of the UEH standard, and only 7% increase for the LMW standard. Addition of CaCl2 to 8 other LMtf heparins gave increases in anti-Xa activity ranging from 0 to 60%. Studies on heparin fractions ranging from 4,000 to 30,000 M.Wt., prepared by gel filtration, showed that a minimum M.Wt. of > 6,000 is required for CaCl2 potentiation of anti-Xa activity.In kinetic studies, the influence of UEH on second order rate constants was increased by 55% in the presence of CaCl2. However, CaCl2 had no effect on the rate enhancement by three high-affinity oligosaccharides, of 5, 10 and 20 saccharide units.These studies show that the effect of CaCl2 on the anti-Xa activity of heparin is highly dependent on M.Wt. Anti-Xa activities of LMW heparins determined in the absence of CaCl2 could be misleading.
ROLE OF DISULFIDE BONDS NEAR THE CALCIUM BINDING SITES IN FIBRINOGEN
Authors: Procyk R , Blomback B 
Published: August 2018

Incubation of fibrinogen with 0.5 mM dithiothreitol in the presence of .20 mM calcium chloride cleaved disulfide bonds located at: the N-terminal end of the Aα-chain (either Aα28-Aα28 or Aα45-γ23), the C-terminal end of the Aα-chain (Aα442-Aα472) and the N-terminal end of the γ-chain (either of the symmetrical γ8, γ9 disulfides or the Aα45-γ23 disulfide bond). In the absence of calcium ions two additional disulfides, γ326-γ339, and one in the N-terminal end of the γ-chain were reduced.Plasmin digestion of the reduced fibrinogens in buffers containing calcium chloride produced fragments D and E, except that smaller fragments of D were generated from the fibrinogen in which the γ326-γ339 disulfide bonds were reduced and alkylated. In these samples calcium did not protect the C-terminal end of the γ-chain from extensive digestion.Addition of thrombin to partially reduced and alkylated fibrinogen prepared in the presence of calcium gave a clotting time similar to control unreduced fibrinogen. However, when the γ326-γ339 disulfide bonds and another γ-chain disulfide bond most likely in the N-terminal region were cleaved in reduced fibrinogen prepared in the absence of calcium, the thrombin clotting time was extremely prolonged. Apparently the disulfide bonded structure supported by γ326-γ339 was important both for binding of calcium and also for normal clotting.
A NEW AT III VARIANT WITH DEFECTIVE PROTEASE BINDING SITE
Authors: Aiach M , Roncato M , Sorin G , Dezellus P , Fiessinger J N 
Published: August 2018

A decreased plasma anti thrombin activity in presence heparin cofactor (hep-cof) or in absence of heparin (AT) was discovered in a 47 year-old patient presenting with recurrent venous thromboembolism. The immunoreactive material (AT III-IR) was normal. The same biological abnormalities were found in two relatives of the patient, leading to the diagnosis of hereditary qualitative AT III deficiency.The propositus'AT III was coeluted with normal AT III from an heparin sepharose column. An additional step of ion-exchange chromatography on a Mono Q column using a FPLC system (Pharmacia, Bois d'Arcy, France) allowed the purification of a protein which was homogenous in SDS - 10 % polyacrylamide electrophoresis gel (PAGE). AT III purified from propositus'plasma, normal plasma and the plasma of a patient known to have an AT III variant with defective protease binding (1) were compared. The specific activities measured as hep-cof AT or factor Xa inhibition in absence of heparin (anti Xa) were respectively 6.4 and 4.8 U/mg for the propositus 'AT III and 13.6 and 8.5 U/mg for the normal AT III (one unit is the activity of 1 ml of a plasma pool prepared from 30 normal subjects). The formation of protease inhibitor complexes was studied by incubating purified AT III with purified thrombin (in molar ratio 1:4, 1:2, 1:1) during 5 minutes at 37 °C and submitting the mixture to PAGE. The densitometric scan showed that in equimolar ratio the percentage of an AT III-thrombin complex (with 92 kA Mr) reaches 70 for normal AT III and respectively 30 and 23 for the propositus'AT III and the already described variant AT III Charlevillé (1). A 70 % proportion of free AT III (58 kA) remained for the propositus. As previously observed an unidentified 63 kA compound appeared for AT III Charleville. This results strongly suggest that in the patient described here, half th the AT III molecules are normal, the others having a defective protease binding site. We propose to call this new variant AT III Avranches, the town where the propositus was born.(1) Thrombosis Research 1985, 39, 559-570.
PREHOSPITAL THROMBOLYTIC TREATMENT: A FEASIBILITY STUDY
Authors: Bossaert L , Demey H , Colemont L , HRM H 
Published: August 2018

Aim of the study: Thrombolysis is the treatment of choice in acute myocardial infarction (AMI). The delay between onset of symptoms and administration of thrombolytic drugs is critical for successful reperfusion and myocardial salvage. We studied the feasibility and safety of early prehospital thrombolytic treatment of AMI “at home”Methods: Eminase(R)(APSAC;BRL 26921 ) was used as thrombolytic agent: its long half-life allows administration as a single IV bolus. The study was performed in collaboration with a well organised group of GP's, extensively retrained in ECG and CPR for the purpose of this study. Whenever a GP made the tentative diagnosis of AMI of less than 2 hours, inclusion and exclusion criteria were reviewed using a check-list, and the mobile intervention team of the hospital (MITUZA), consisting of CCU physicians and emergency nurses, was activated. After rechecking all criteria, including a 12 lead ECG, brief medical history and physical examination, a single IV bolus of 30 U Eminase(R) was given, followed by transferral to the CCU. Follow-up included estimation of infarct-size and LV function using biochemical, ECG, radionuclide and angiographic methods.Results: Up till now, 15 male patients (age=57±9) have been treated using this protocol. Initial prehospital treatment consisted of Eminase(R) IV in 10 (reperfusion 9/10). Subsequent in-hospital treatment was streptokinase in 3 (reperfusion 2/3). In the 11 reperfused patients, PTCA was performed in 7 and CABG in 4. After onset of symptoms, the GP arrived after 55±24 min (15-90 min), Eminase(R) was administered after 95±28 min (75-130 min). The total calculated time gain (interval between treatment at home and admission in the CCU) was 42±15 min (20-75 min). There were no adverse events.Conclusion: This pilot study indicates that prehospital thrombolytic treatment of AMI is feasible and safe, resulting in a considerable time gain. Early reperfusion was obtained in 90%. Collaboration with a well organised and trained group of GP's, clearly defined inclusion and exclusion criteria and administration of the thrombolytic drug exclusively by experienced critical care physicians are mandatory.
DECREASED FIBRINOLYTIC CAPACITY IN SURVIVORS OF ACUTE MYOCARDIAL INFARCTION
Authors: Schoenfeld P , Therville E , Bethume P , Leautaud P , Wachel D , Rodzynek J J 
Published: August 2018

Disturbances of several markers of fibrinolytic capacity have been reported in patients with an history of thrombotic pathologies. In this work we assessed Euglobulin Lysis Time (ELT) before and after venous, stasis, t-PA release (t-PAr) expressed as the ratio of t-PA antigen after versus before venous stasis, and presence of t-PA inhibitor (t-PAi) in 2 populations: group 1, normal controls (n=50) and group 2, survivors of acute myocardial infarction (AMI)(n = 17).The results were as follows :1) ELT shortening after venous stasis = 46/50 in group 1 compared to 3/17 in group 2 (p < 0.01, X2 test);2) t-PAr insufficient in 1/50 patients of group 1 compared to 15/17 patients of group 2 (p < 0.01, X2 test);3) presence of t-PAi 3/50 in group 1 compared to 9/17 in group 2. The 3 normal controls with t-PAi were also characterized by absence of ELT shortening after venous stasis. In group 2, the relationship between absence of ELT shortening and other parameters of fibrinolysis appeared as follows :In patients of group 2 without ELT shortening after venous stasis, insufficiency of t-PAr was consistently more pronounced in those without detectable t-PAi.We conclude that :1) our data confirm the marked abnormalities of fibrinolysis in post-AMI patients;2) these abnormalities appear to be related to insufficency of t-PAr, either alone or in combination with the presence of t-PAi.
THROMBELASTOGRAPHIC EVALUATION OF THE EFFECT OF ADDED PLASMINOGEN TO PLASMA ON FIBRINOLYSIS USING VARIOUS FIBRINOLYTIC ACTIVATORS
Authors: Summaria L , Yang G G , Holloway D S , Caprini J A 
Published: August 2018

When lysis of peripheral thrombi requires infusion of plasminogen activators for longer than 24 hours duration, slow resolution of an obstructive thrombus can result in tissue injury and loss of limb. This study evaluated the effects of increasing the plasminogen concentration on the kinetics of clot lysis induced by plasminogen activators. By adding varying concentrations of any plasminogen activator to plasma, clot lysis can be continuously monitored using thrombelasto-graphy (TEG). Parameters measured during the lysis portion of the pattern are the final amplitude reached (Af), various time parameters (T, T’ and T50% ) and the absolute change in amplitude ( Δ A). By adding different amounts of Lys-Plasminogen (LYS-PLG) to plasma, we were able to measure the effect of added LYS-PLG on the lysis pattern with different activators (streptokinase, the plasminogen-streptokinase complex, or the B-chain-streptokinase complex). Statistical analysis of the plots of T50%(time for 50% lysis, where the TEG amplitude is reduced to naff that of the maximum amplitude) versus LYS-PLG concentration showed that a second order polynomial regression gave R values of better than 0.969. The addition of a small amount of LYS-PLG (20% of the normal plasma concentration) resulted in a two-fold decrease in T50% with all activators. Maximum clot-lysis enhancement was reached when 60% excess LYS-PLG was added. This amount caused a 2.9 to 3.9 fold decrease in T50%. Values above 60% did not significantly reduce clot- lysis time any further when very low levels of plasminogen activator were added to plasma. Plots of clot-lysis (T50%) versus activator or LYS-PLG concentration showed that tne amount of activator could be decreased by at least two-fold if between 40 to 60% extra LYS-PLG is addedThus, the addition of LYS-PLG to a plasma clot produced a two- to three-fold increase in the rate of clot lysis (2 to 3 fold decrease in T50%) or permitted a two-fold lesser amount of activator to produce the same lysis pattern.
INCREASED THROMBOTIC TENDENCY IN A FAMILY WITH HEREDITARY ANGIONEUROTIC EDEMA ( HANE )
Authors: Kirschstein W , Hoffmann U , Simianer S , Dempfle E , Kortsik C , Heene D 
Published: August 2018

Biochemical hallmark of HANE is a reduction of C1-inhibitor. We observed a family with type II disease ( non-functional protein ), in which 3 of 6 affected members had arterial thromboembolic events at young ages. For evaluation of alterations in the hemostatic system analysis included: fibrinogen, FVII, FVIII, FIX, FXI, FXII, prekallikrein PK, antithrombin III ATIII, protein C PC, α2 antiplasmin α2AP, cl α2 macroglobulin α2 MG, plasminogen activator inhibitor PAI, plasminogen PG, euglobulin clot lysis time ECLT and tissue plasminogen activator tPA at baseline and after venous occlusion. The results are shown in part in the table:There is evidence of nearly no response to venous occlusion in 2 and a diminished response in 1 out of 4 patients.We conclude, that the increased thrombotic tendency in this family is related to the increased potential of prephase coagulation factors and impaired fibrinolytic response to venous occlusion concomitantly with the reduction of the main inhibitor of the contact activation system.
LOCAL ACTIVATION OF COAGULATION AND FIBRINOLYSIS IN LUNG DISEASE IS REFLECTED IN BR0NCH0ALVE0LAR LAVAGE FLUID
Authors: Nakstad Britt , Lyberg Torstein 
Published: August 2018

Fibrin deposition in the alveolar space and the lung interstitium is a prominent feature of many types of pulmonary inflammatory diseases.Ce11s of the monocyte/ macrophage lineage have a dualistic role in the balance between coagulation and fibrinolysis having the ability to synthesize both procoagulant factors and plasminogen activators. In the present studies bronchoa1veo1 ar lavage were performed on 128 patients with various lung diseases and 29 healthy controls.Both lung alveolar macrophages (LAM) and the supernatant bronchoalveolar lavage fluid (BALF) expressed two types of procoagulant activities a)thromboplastin and b) a direct factor X activator.The procoagulant in BALF was associated with membrane vesicles which sedimented at 100000g,lh.By electron microscopy the BALF ultrasediment was seen to consist for a large part of membrane material and this was confirmed by monitoring the content of different marker enzymes for specific subcellular structures. LAM from patients had significantly higher specific thromboplastin activity than LAM from controls (4,36 -0.98(SEM)vs.0.81± 0.14 U/mg cell protein).BALF collected from patients had significantly higher levels than BALF from controls of a ) thrombop1 astin(0.66-0.18vs.0.07 ±0.01 U/ml) b ) factor VII activity(1.33±0.31vs.0.48±0.06 U/ml) c)fibrin degradation products(presentin 28,7vs.0% of the cases) and d) fibronectin(491±103 vs.35±5 ng/ml ) In addition,the level of plasminogen activator was higher in controls than in patients(294±68 vs.!02±14 m U/m1).These studies show that activation products of the coagulation and fibrinolytic systems can be detected in BALF and that lung disease often is associated with abberations in the balance between these systems.Fibrin serves as a substrate for fibronectin and the increases in lavage fibronectin may reflect the development of lung fibrosis.
HOW DOES CALCIUM ALGINATE ACHIEVE HAEMOSTASIS IN SURGERY?
Authors: Jarvis P M , Galvin D A J , Blair S D , McCollum C N 
Published: August 2018

Calcium alginate (Kaltostat, Cair Ltd) is a new absorbable material for topical haemostasis in surgery. The possible mode of action, release of calcium ions in exchange for sodium was investigated in human blood.Calcium release was measured in 15 mg samples of calcium alginate placed in 20 ml of 0.9% saline, for intervals of 1, 3 or 10 minutes. To assess the effect on platelets, 3 mg of calcium alginate or surgical gauze were added to 5 ml of Heparinised (100 units) fresh blood for 2 minutes and platelet counts then made using plain blood as a control. Finally using a thrombelastograph, the activation of whole blood coagulation was assessed after a 2 minute contact with 3 mg of calcium alginate, surgical gauze or no additive as control.When calcium alginate was placed in saline, 26% of calcium ions were released in 1 minute giving a calcium ion concentration of 4.62 t 0.02 mmol/L, with only slight further release after 10 minutes to 4.82 ± 0.004 mmol/L. There was a corresponding decrease in sodium ion concentration. Adding calcium alginate to whole blood reduced the platelet count from a control value of 248 i 16 × 109/L to 222 f 15 × 109/L (p< 0.05) compared to 241 ± 15 × 109/L for surgical gauze. Similarly calcium alginate shortened whole blood coagulation time from 17-7 i 1.0 minutes control, to 12.9 ± 1-32 mins (p< 0.001) compared to 15.0 ± 1.5 mins (p< 0.02) for surgical gauze.Calcium alginate rapidly releases calcium ions in exchange for sodium on contact with blood stimulating both platelet activation and whole blood coagulation, significantly more than simple contact activation by surgical gauze.