bluuebunny/crossref_metadata_2025 huggingface dataset:
The Host’s Response To Endotoxin: Inflammation, Thrombosis And Hemorrhage
Authors: Movat H Z , Wasi S , Jeynes B J
Published: July 2018

The purpose of this study was to investigate the kinetics of lesions induced by endotoxin. New Zealand White rabbits were given 2 or 3 of the following radiolabelled cells or proteins to quantitate changes in the microcirculation: 131I-serum albumin (vascular permeability) 125I-fibrinogen (thrombosis), 51Cr-leukocytes (leukocyte infiltration or leukocyte “thrombi”), 59Fe-erythrocytes (hemorrhage), 111In-platelets (thrombosis). When the animals were injected intradermally with E. coli or S. minnesota endotoxin, they developed a transient inflammatory reaction in which increase in vascular permeability and leukocyte and platelet accumulation peaked in 2-3 hour old and hemorrhage in 4-6 hour old lesions. By 16-18 hours the inflammatory process had subsided. When at this time endotoxin was administered intravenously thrombo-hemorrhagic lesions were superimposed on the subsiding inflammatory lesions. The thrombo-hemorrhagic lesions were characterized by rapid accumulation of 111In-platelet or 125I-fibrinogen (the 131I-albumin given simultaneously did not increase) and 59Fe-erythrocytes, all of which subsided in 6-hour old lesions. Electron microscopy demonstrated phagocytosis of endotoxin by macrophages and PMN-leukocytes. The latter showed regressive changes and necrosis followed by their phagocytosis by macrophages. Endotoxin injected intradermally induces an inflammatory reaction. Unlike other inflammatory stimuli, endotoxin “prepares” the injected site for the elicitation of a thrombo-hemorrhagic lesion. Indirect evidence supports the view that monocyte-macrophages may be responsible for the formation of tissue thromboplastin and the initiation of thrombosis in the microcirculation and polymorphonuclears for the hemorrhage.
Factor VIII Related Antigen (FVIIIRAG) In Porcine Endothelial Cells
Authors: Giddings J C , Jarvis A L , Bloom A L
Published: July 2018

Porcine aortic and umbilical vein endothelial cells were maintained in culture and examined for their ability to synthesise or release factor VIII related antigen (Willebrand factor, FVIIIRAG). Two different antisera raised against purified porcine FVIIIRAG were used in conventional immunodiffusion, immunoelectrophoresis and immunofluorescence techniques. Both antisera were adsorbed with porcine von Willebrand's disease (vWd) plasma and gave identical monospecific immunoprecipitin reactions when tested against normal porcine plasma or concentrated porcine FVIIIRAG and failed to show any precipitin reaction against vWd pig plasma. Primary and extended cultures of umbilical vein endothelial cells showed positive intracellular immunofluorescence with either antiserum and supernatant culture medium from these cells contained FVIIIRAG when tested in immunoprecipitin tests. However, cultures derived from aorta demonstrated a different pattern of results. Antiserum one showed homogenous intracellular immunofluorescence but antiserum two gave an immunofluorescence pattern which was extracellular and fibrillar, similar to that seen with human endothelial cells stained with anti-fibronectin. Culture supernatant and lysedaortic endothelial cells did not contain FVIIIRAG in immunoprecipitin tests with either antiserum. The results indicate either that the antisera were not specific and that the aortic cells were not endothelial or that porcine endothelial cells derived from aorta may be different from those obtained from umbilical vein in their ability to synthesise FVIIIRAG. Immunohistological demonstration of “FVIIIRAG” cannot therefore be used as the only criterion for identifying porcine aortic endothelial cells.
Quantitative Studies On The Fibrinolytic Activity Of Normal Intima And Atherosclerotic Plaque
Authors: York J L , Benjamin W S
Published: July 2018

This study was undertaken to determine if the fibrinolytic capacity of the intimal-medial layer of the human aorta decreases with increasing severity of atherosclerosis. Aortas were obtained from 32 normal healthy subjects who died from a sudden traumatic incident. Paired controls consisted of samples of grossly healthy tissue which were removed from sites adjacent to the plaque. Intima and media containing atherosclerotic lesions and lesion free control sections were stripped from the thoracic aorta and were homogenized. Plasminogen, α2-macroglobulin and α1-antitrypsin levels in the homogenate supernatants were determined by radial immunodiffusion. Plasmin activity was determined on plasminogen free fibrin plates. No significant differences were found in the content of any of the moieties measured between the paired control intima-media and the lesions. The plasminogen content of the intima-media did not change as a function of the lesion grade; however, the streptokinase activated plasmin, as measured by fibrinolysis on fibrin plates, was decreased six times in tissue containing lesions of grades 4-6 when compared to lesions of grades 1 and 2. The mean age of both groups was not significantly different. In these same samples the plasmin inhibitors α2 macroglobulin and α1-antitrypsin were increased 3 times and 2 times, respectively in the intima-media of the aortas containing grade 4-6 lesions as compared to the aortas containing grade 1 plus 2 lesions. These data suggest that there is a decreased fibrinolytic capacity of the normal intimamedia of aortas containing lesions of grade 4-6. This decrease in plasmin activity is not due to a decrease in amounts of plasminogen but rather to an increase in α2- macroglobulin, α1-antitrypsin, and possibly other plasmin inhibitors.
Identification Of A Cell-Surface Cofactor For Antithrombin III On Cultured Murine Endothelium
Authors: Busch P C , Owen W G
Published: July 2018

Cultures of mouse brain capillary endothelium grown on microcarrier beads were used as a model for the study of interactions of plasma proteins with the endothelium in the microcirculation, where the ratio of surface area to volume is large. Use of the beads increased the relative cell surface area about 100 fold over that in monolayer cultures. Cells obtained from capillary explants were grown to confluence on 140 μ (mean diameter) beads of cross-linked dextran. The cell-covered beads were packed into 0.3 ml columns in silicone-coated glass tubes, and were used within 2 hours. Cells remained viable during all experimental periods. Radioiodinated thrombin was bound to the cells, and unlabeled diisopropyl phosphoryl-thrombin eluted most of the bound radioactivity, which was identified by electrophoresis as α-thrombin. Likewise, bound thrombin was eluted with anti thrombin III, and most of the radioactivity was identified as thrombin-anti thrombin III complex. In contrast, when thrombin and anti thrombin III were incubated for comparable intervals in the absence of cells, little complex formation occurred. Similar enhancement of complex formation could not be readily detected over monolayer cultures because of the low ratio of surface area to volume coupled with a significant reaction rate without cells. It is concluded that a substance on the cell surface catalyzes the reaction of thrombin with anti thrombin III and that this catalyst becomes significant when cell surface concentration approaches that in the microcirculation.
Effect Of Tissue Culture Irradiation On Orientation Of Cytoskeletal Fibers In Migrating Sheets Of Endothelial Cells
Authors: Gotlieb A I
Published: July 2018

Irradiation of endothelial cells (EC) in tissue culture has been used to markedly reduce cell proliferation in order to study the effects of substances on the regulation of EC migration. Since irradiated EC (IRR-EC) migrate the same distance as do non-irradiated EC (NIRR-EC) over periods of up to six days, it is assumed that EC migration is not effected by irradiation. The purpose of this study was to examine the effect of irradiation on the cytoskeleton of migrating EC. The in-vitro experimental wound technique was used as a model system. A linear wound was made in confluent cultures of porcine thoracic aortic EC. EC were observed to migrate into the wound as a cohesive sheet of cells with only a few free unattached cells being present along the front edge of the wound. Cultures were irradiated one hour before wounding with 1500 rads. NIRR-EC migrated the same distance as did the IRR-EC although the latter were much flatter and each IRR-EC covered a larger surface than did the NIRR-EC. The orientation of cytoskeletal fiber bundles localized by immunofluorescence microscopy using antisera produced against electrophoretically purified porcine uterine myosin and chicken gizzard tropomyosin were different when comparing migrating IRR and NIRR-EC in the first row of the endothelial sheet. At 44 hours after wounding myosin localization showed that the main myosin stained fibers in 89% of the IRR-EC were roughly parallel to the wound edge while 11% were roughtly perpendicular to the wound edge. In NIRR-EC the figures were 54% and 46% respectively. Tropomyosin localization showed a similar diffence, 73% and 27% in IRR-EC and 45% and 55% in NIRR-EC. The data shows that there are differences in the extent of spreading of and in the orientation of cytoskeletal fiber bundles in migrating IRR-EC. These differences may reflect different cytoskeletal processes involved during migration of IRR and NIRR-EC.
Prostacyclin Synthesis By Cultured Vascular Smooth Muscle Cells : Influence Of Growth And Ageing
Authors: Larrue J , Rigaud M , Leroux C , Crockett R , Bricaud H
Published: July 2018

The ability of arterial smooth muscle cells (SMC) to produce prostacyclin (PGI2) is well documented both in vivo and under culture conditions. Advanced atherosclerosis has been associated with a severe decrease in PGI2 synthesis by arteries and we recently demonstrated that PGI2 synthesis by cultured SMC from atherosclerotic aorta was significantly depressed. In the present experiments, changes in arachidonic acid (AA) cascade in cultured SMC were studied as a function of cell proliferation and ageing.

SMC derived from explants of the medial layer of 2 years old rabbit aorta were grown under standard conditions (HAM F10 medium + 10 % Foetal Calf Serum). Prostaglandin synthetic activity was investigated by cell free homogenate radiochemical assays and confirmed by gaz chromatography - mass spectrometry determinations after 10 mn incubation with 2 nM AA.

An age related decrease in PGI2 biosynthetic activity (expressed as pM/10PGI6 cells) was found in confluent cells from passages 5 to 22 (31.4 ± 3 vs 15.4 ± 5.6 respectively - p≤0,01 -). On the contrary, PGI2 generation was enhanced during the proliferative phase (day 2-5) (55.5 ± 11.8) when compared to confluent cells at the same (5th) passage (p≤0,01).

These results are consistent with the enhanced PGI2 formation in early arterial proliferative lesion and the inhibition of SMC proliferation by cyclooxygenase inhibitors recently reported and indicate that PGI2 synthetic activity in arterial SMC may be a significant age-dependant endogenous factor involved in the pathogenesis of atherosclerosis.
Comparative Aspects Of The Occluding Junctions In The Pulmonary And Bronchial Microcirculation
Authors: Bartels H
Published: July 2018

Compared with the pulmonary microcirculation the bronchial microcirculation reacts with a large increase in permeability after administration of vasoactive substances (Pietra et al., Cire. Res. 29, 323, 1971). Thus the occluding junctions between the endothelial cells of the rat pulmonary and bronchial microcirculation were investigated by means of freeze-fracture replicas in order to establish differences in their substructure which could be responsible for the different permeability properties. The prevailing feature of the occluding junctions between the endothelial cells of the pulmonary microvascular bed was a continuous system of anastomosing membrane foldings,carrying rows of intramembranous particles, which were usually located on face E of the split plasma membrane. This type of occluding junction is characteristic of capillaries (Simionescu et al., J. Cell Biol. 67, 863, 1975) . In the bronchial microcirculation many of the endothelial occluding junctions showed discontinuous assemblies of membrane foldings, which were lacking particles on face E. This type of “open” junction is typically localized at the venular end of the microvascular bed and susceptible to vasoactive substances (Simionescu et al., J. Cell Biol. 78, 27, 1978). It is suggested that the different permeability properties of the pulmonary and bronchial microcirculation is due to the large amount of postcapillary venules with their morphologically and functionally characteristic occluding junctions.
Antithrombin III Progressive Function: A Biochemical Analysis
Authors: Gomperts E D , Izadi P
Published: July 2018

Antithrombin III (AT III) was measured in the various fractions obtained during the partial purification of AT III-Heparin Cofactor (AT III-HCF) via a heparin-bound affinity chromatography system. AT III antigen and HCF activity was present to some extent in all fractions, but significant progressive function was observed only in that obtained via PEG-4000 precipitation. This precipitate was applied to a Sephacryl-200 column. Fractions were collected and those demonstrating maximum AT III antigen and progressive thrombin inhibition were pooled and reapplied to the washed Sephacryl S-200 column. Fractions were collected and assayed via specific antisera for AT III, α1AT, α2M and α1 acid glycoprotein (α1AG) . AT III antigen and progressive function were confined primarily to one peak containing virtually no α2M, a low level of α1AT, and moderate quantities of α1AG. PAGE of the component showed AT III antigen separating into two bands. Assessment of the two other prominent bands showed no reactivity with antisera to α2M, α1AT or α1AG. PAGE-SDS showed a different pattern with the AT III doublet moving as a single band. These electrophoretic patterns were identical with that of the AT III-HCF purified by the heparin-affinity system. AT III antigen-two dimensional immunoelectrophoresis in the presence of heparin of both the Sephacryl and heparin- affinity purified components was very different with the Sephacryl purified AT III protein showing both a fast peak and a very prominent slow moving hump. The AT III heparin purified component showed primarily a fast component. These two fractions differed in one other respect in that AT III inhibition of thrombin was activated, heparin-like, by EDTA. This effect was totally absent in the heparin affinity purified AT III-HCF. On the basis of these observations it is postulated that AT III purified by the affinity system is biochemically altered resulting in an inability to respond functionally to non-heparin activating stimuli.
Inactivation Of Thrombin By Antithrombin III In The Presence Of Surface Bound Heparin
Authors: Goosen M F A , Sefton M V , Hatton M W C
Published: July 2018

The inactivation of thrombin by antithrombin III was studied in the presence of immobilized heparin. It was found that heparin accelerated the inactivation of thrombin by first forming a complex with thrombin which was then inactivated by antithrombin III contrary to the hypothesis of Rosenberg and Damus (J. Biol. Chem., 248, 6490, 1973) and the results of Salzman et al. Heparin was immobilized onto polyvinyl alcohol (PVA) by acetal coupling with glutaralde- hyde to give a very stable bond at pH 7.4. Using small columns of heparin-PVA beads eluted by 0.14M NaCl buffered at pH 7.4 both thrombin and antithrombin III bound to the immobilized heparin. If thrombin was loaded before an excess of antithrombin III, significant inactivation of thrombin was observed; however, loading antithrombin III before thrombin did not measurably inactivate thrombin. The results suggest that the covalently-bound heparin effectively participates in the inactivation of thrombin through the formation of surface bound heparin-thrombin, which then reacts with antithrombin III to yield a surface bound thrombin-antithrombin III complex. Heparin-PVA is potentially useful as a nonthrombogenic coating in the preparation of small diameter vascular prostheses and blood sampling catheters.
Relative Contributions Of Thrombin And Antithrombin III Affinities Of Heparin Fractions To The Rate Of Inactivation Of Thrombin By Antithrombin III
Authors: Cerskus A L , Birchall K J , Ofosu F , Blajchman M A , Hirsh J
Published: July 2018

Heparin enhances the rate of inactivation of thrombin (IIa) and other coagulant proteases by antithrombin III (AT III). The anticoagulant activity of heparin is associated with heparin moieties which have high affinity to AT III. However, the contribution of the IIa affinity to the anticoagulant activity has not been as clear. Standard porcine mucosal heparin was fractionated on affinity columns consisting of purified human AT III and IIa immobilized on agarose to determine the effect of heparins of various affinities on the second order rate constant (K") for the inactivation of IIa by AT III. Results are expressed as the rate enhancement factor (REF) which is defined as the ratio of the K" in the presence of 50 ng/ml heparin to the K" in the absence of heparin. The REF for unfractionated heparin was 9.5. Chromatography on either AT III-agarose or IIa-agarose resulted in elution of three heparin fractions corresponding to a void volume fraction, a low affinity fraction and a high affinity fraction. The REF’s of the fractions eluted from AT III-agarose were 1.3, 1.4 and 18.8, respectively while the REF’s for the corresponding IIa fractions were 5.6, 14.9 and 18.1, respectively. Rechromatography of the high affinity fraction from AT III-agarose on the IIa-agarose column resulted in the elution of three fractions with REF’s of 17.9, 21.1 and 27.0, respectively. Conclusions: 1. Heparin affinity to AT III is critical for anticoagulant activity as all of the activity is found in the high affinity fraction. 2. Heparin affinity to IIa also contributes to anticoagulant activity but is not critical as significant activity is observed in low affinity fractions. 3. High affinity to both IIa and AT III results in greater activity than that seen with fractions of high affinity to either protein alone.
Effect Of pH On The Stability And Conformation Of α-Thrombin
Authors: Villanueva German B
Published: July 2018

It is known that storage at pH 6 stabilizes thrombin against inactivation. In order to determine whether structural changes accompany this stabilization, the conformation of human α-thrombin at pH 6.0 and 7.5 was investigated by chemical modification, solvent perturbation, UV difference spectroscopy and circular dichroism. It was shown that the CD spectra of α-thrombin at 230-200 nm peptide region were indistinguishable at two pH values indicating no difference in the secondary structure. However, differences were observed in the 320-250 nm aromatic region suggesting some changes in the microenvironment of the aromatic chromophores. Solvent perturbation in 20% ethylene glycol indicated 3.7 ± 0.5 Trp and 7.8 ± 0.5 Tyr were exposed to the solvent at pH 6.0 while 4.3 ± 0.4 Trp and 8.4 ± 0.5 Tyr were exposed at pH 7.5. Chemical modification of tryptophan residue by dimethyl(2-hydroxy- 5-nitrobenzyl)sulfonium bromide in a 100-fold molar excess of the reagent showed 3 reactive residues at pH 6.0 and 6 at pH 7.5. These results suggest that when thrombin is exposed to low pH, structural changes occur that decrease the relative degree of exposure of tryptophan and tyrosine residues. Furthermore, UV difference spectroscopy showed the development of a positive differential spectrum when thrombin at pH 6.0 was exposed to pH 7.5. From this study, it is concluded that the stability of thrombin at pH 6.0 is due to a more compact structure of the enzyme which is probably a result of reduced charge interaction at low pH.
Clotting And Chromogenic Substrate Assays Measure Separate Thrombin Activities
Authors: Milad M P , Hassouna H I
Published: July 2018

Thrombin, the final serine protease responsible for the conversion of fibrinogen to fibrin, is known to have the ability to cleave other peptides in addition to those involved in blood coagulation. Many synthetic substrates were devised to be acted on by thrombin reflecting esterolytic, amidolytic and other cleaving properties. Antithrombin III, the major protease inhibitor of plasma, has been shown to bind the proteolytic properties of thrombin irreversibly. The purpose of this study was to determine whether all enzymatic properties are destroyed simultaneously or whether the inhibitor binds the active site leaving esterolytic functions intact.

Purified α-thrombin was examined with fibrinogen, and H-D-Phe-Pip-arg-p-nitroanilide assays to prepare standard working curves relating units thrombin to the rate of fibrin endpoint or to the rate of production of chromophore. Then progressive antithrombin inactivation of seryl residue was examined by increasing concentrations of ATIII incubated with thrombin for five minutes and aliquots simultaneously examined for the ability to clot a standard fibrinogen solution and to cleave the synthetic substrate.

The clotting activity decreased steadily with increasing antithrombin, whereas the esterase activity remained constant. The study provides further evidence to the observations of Chang et al (Biochemistry 18: 113, (1979)) that thrombin contains two functional sites, one highly specific for the fibrinopeptide region of fibrinogen and the second site relatively nonspecific, responsible for thrombin’s esterolytic activity. An important implication of this study is that clotting assays and chemical assays that use synthetic peptides reflect separate functional properties of the enzyme thrombin.
Enzymatic Properties Of Staphylocoagulase-Human Prothrombin Complex (Staphylothrombin)
Authors: Kawabata S , Morita T , Igarashi H , Iwanaga S
Published: July 2018

Staphylocoagulase(SC) is an extracellular protein produced by Staphylococcus aureus. This protein is known to coagulate several mammalian plasmas, by forming an active molecular complex with prothrombin. However, there is not enough knowledge of the molecular mechanism of prothrombin activation by SC. The present work was undertaken to elucidate the enzymatic properties of SC-human prothrombin (HP) complex. A highly purified SC was prepared from a culture filtrate of Staphylococcus aureus, strain st-213, by a bovine prothrom- bin-Sepharose affinity column developed in our laboratory. This SC could activate HP but not bovine prothrombin. A SC-HP complex formed with the molar ratio of 1 to 1 had a strong amidase activity towards α-thrombin fluorogenic substrate, Boc-Val-Pro-Arg-4-methylcoumary1-7-amide. The activity of the complex was stable at 0°C for at least 8 hrs, while the activity was rapidly decreased with incubation at 37°C. SDS-polyacrylamide gel electrophoretic analysis on the complex formed at 37°C indicated not only a proteolysis of SC portion to small derivatives, but also the fragmentation of HP portion to prethrombin 1 and profragment 1. This proteolytic fragmentation seemed to be due to a self-catalytic reaction of the SC-HP complex, since no endogeneous proteolytic activities were detected in each preparation of SC and HP. Bovine prothrombin, prethrombin 1 and prethrombin 2 inhibited the complex formation between SC and HP, but bovine profragment 1 and 2 had no effect on the complex formation. These results indicate that a high affinity site of HP to SC must be located in the prethrombin 2 region. Like α-thrombin, the amidase activity of SC-HP complex was competitively inhibited with benzamidine (Ki=1.2 x 10-1 M). However, both clotting and amidase activities of the complex could not be inhibited by 4 molar excess of hirudin or antithrombin III in the presence of heparin.
Significant Differences In Bleeding Time(BT) ᶓ Blood Loss (BL) Between Males ᶓ Females Following Aspirin(ASA) Ingestion
Authors: Young V P , Giles A R , Pater J , Corbett W E N
Published: July 2018

Recent clinical studies suggest that ASA may reduce the incidence of thromboembolic events in males but not females. Similarly, studies in experimental animals have demonstrated sex differences in response to thrombotic stimuli ᶓ modifications of this by ASA. We have determined the effect of ASA on both BT ᶓ BL during the performance of a strictly standardised test in 40 normal male ᶓ female subjects.

Duplicate 5mm incisions (1mm depth) were made on the volar surface of the forearm below a cuff maintained at constant pressure (40mm Hg) for exactly 45" prior to injury induction. Blood from the incisions was drawn into graduated micropipettes. Thus both BT (measured from time of injury induction to the time no more blood could be drawn into the pipette) ᶓ BL were measured. At the conclusion of the test, ASA (650mg) was ingested ᶓ the BT(BL) determined exactly 2 hrs. later. There were no differences in the means of BT or BL between the male and female groups preASA. Following ASA there was a significant increase in both BT ᶓ BL for each individual (p<0.01) although in females the post-ASA values frequently fell within the pre-ASA range for the group. In males the effect was more pronounced ᶓ the increase in both BT ᶓ BL was significantly more in males than females (p<0.01). There was a highly significant correlation (p<0.001) between BT ᶓ BL measured both pre- ᶓ post-ASA for both sexes.

The basis for the differences noted remains obscure but it would appear unlikely that they relate to differences in the degree of inhibition induced in prostaglandin synthesis either in the platelet or vessel wall. It is possible that in males, platelet-vessel wall interactions are more dependant on ASA-inhibitable prostaglandin pathways than females or, alternatively, that ASA is modulating this interaction in a way that has yet to be identified.
Recovery Of Platelet Function After Aspirin Ingestion: In Vivo And In Vitro Studies
Authors: Bradlow B A , Chetty N , Birnbaum M
Published: July 2018

Blood was taken from normal volunteers before and two hours after a 600mg oral dose of aspirin (ASA). Platelet rich plasma (PRP) was prepared from both samples. Mixtures of normal and ASA treated PRP were tested for aggregation responses and for production of thromboxane B2 (TXB2) by RIA (New England Nuclear). Normal aggregation responses were found when the mixture contained 26% of normal platelets (NP) with ADP, 40% NP with adrenalin, 54% NP with collagen and 52% NP with arachidonic acid (AA)(Mean Values). TXB2 production in the mixtures increased when the proportion of normal platelets was raised above 10 to 30%. These findings indicate partial restoration of platelet function by an admixture of 10-20% NP to ASA treated platelets although 50% NP was necessary to restore all functions tested. In vivo studies on seven volunteers given 300mg ASA showed partial recovery of malondialdehyde production after 72 hours, partial return of aggregation responses to collagen, ADP, adrenalin and AA at 96 hours and partial or full return of aggregation responses in all subjects by 120 hours. Since new platelets enter the circulation at the rate of 10-15% per day the results of our in vitro studies suggest that some recovery of platelet function might occur as soon as 24 hours after a dose of ASA and full recovery within 3-5 days. The longer recovery period indicated by the in vivo studies may be attributable to the effect of ASA on megakaryocyte cyclooxygenase.

Conclusion: ASA administered once every 24 hours may be sufficient to suppress platelet function adequately and continuously but partial recovery of function might occur 48-72 hours after ASA ingestion.
Comparison Of The Effects Of Aspirin In Humans Ex Vivo In Platelet-Rich Plasma And Whole Blood
Authors: Nunn Barbara
Published: July 2018

The effect of aspirin on human platelet function is usually assessed using platelet-rich plasma (PRP). Some preliminary results in vitro suggested that the effect of aspirin appears to be greater in PRP than whole blood. To explore this possibility further, a comparison of the effect of aspirin in humans ex vivo has been made taking measurements simultaneously in whole blood and PRP at 2 platelet concentrations. Blood samples (36ml) were drawn from 7 male volunteers after a light breakfast. Each took 300mg soluble aspirin and blood samples were drawn again 2 hours later. Blood was mixed with 0.1 volumes 129nM trisodium citrate. Some (30ml) was then centrifuged to prepare PRP and platelet -poor plasma (PPP) by standard techniques. Platelet concentration of some PRP was adjusted with PPP to equal that of the corresponding blood sample; the rest was adjusted to 350,000 per μl. Aggregation in response to collagen (Horm, Munich) was measured photometrically at 37°. Aggregation in 0.5ml aliquots of whole blood was measured after 4 min stirring with 154mM NaCl (control) or collagen at 37° as the fall in single platelet count determined using an Ultraflo- 100 whole blood platelet counter (Clay Adams). The concentrations of collagen producing a 50% maximal response (EC50) in PRP and blood were determined. Dose-ratios for each volunteer were calculated by dividing the EC50 obtained after aspirin by the corresponding value obtained before aspirin.

The effect of aspirin was significantly (p<0.001) less in blood than PRP. Whether or not the results in whole blood more closely reflect the effect of aspirin in vivo remains to be determined.
Experimental Study Of The Antithrombotic Effect Of Aggregation Inhibitors, Heparin And Combinations With Heparin
Authors: Schlag G , Grünwald M , Redl H
Published: July 2018

Experimental thrombosis was produced by the freezing method to investigate in a randomized prospective trial the antithrombotic effect of the platelet aggregation inhibitors EhO 26644 (EMD; 0,1 mg/kg b.w.), acetylsalicylic acid (ASA; 30 mg/kg b.w.), heparin (50 I.U./kg b.w.), the combination of heparin (50 I:U:/kg b.w.) and EMD (0,1 mg/kg b.w.) and the combination of heparin and dihydroergotamine (HDHE; 50 I.U. heparin/kg b.w. 0,005 mg DHE/kg b.w.). Experimental thrombosis was caused under general anesthesia by freezing the jugular veins for 2 minutes. Incidence and weight of thrombi were studied under control (saline 1ml/kg b.w.) and the antithrombotic regimes. Each group consisted of 20 rats with an average weight of 355 g.

Results: All antithrombotic drug series differed significantly from the control series. The minimal rate of thrombus formation was found in the HDHE group (2/20 vs 18/20 in control), while heparin (4/20) alone showed better results than the series with the aggregation inhibitors (EMD 8/20, ASA 6/20) or the combination with EMD (5/20). The lowest weight of thrombi was found in the series heparin EMD, differing not significantly from HDHE. Thus regarding frequency of thrombosis and weight of thrombi the antithrombotic combination HDHE showed the best results from the 5 regimes.
Mechanisms Of Coagulopathy After Envenomation By The Eastern Diamondback Rattlesnake
Authors: Kitchens C S , Van Mierop L H S
Published: July 2018

All 34 patients seen at this hospital during the 19781980 period who were envenomated by poisonous snakes were studied in a prospective manner with respect to their hemostatic system. Blood was drawn on the patient’s arrival to the emergency room and every 6 h thereafter. Blood was analysed for platelet count; routine coagulation tests; levels of factors II, VIII, IX, XII; clottable fibrinogen; fibrinogen antigen; fibrin degradation products (FDP); plasminogen (PI); antithrombin III (AT III); α2 plasmin inhibitor (API); and plasminogen activator (PA). Twelve of the 34 patients underwent a coagulopathy as described below. These patients included all 10 patients envenomated by the Eastern diamondback rattlesnake (Crotalus adamanteus) and 2 of 17 patients bitten by the pygmy rattlesnake (Sistrurus miliarius). Values of all the above coagulation tests in 15 pygmy rattlesnake and 7 moccasin (Agkistrodon piscivorus) victims were indistinguishable from normal. Patients undergoing coagulopathy rapidly developed noncoagulable blood as defined by a thrombin time (TT) >120 s; blood remained incoagulable for an average of 18 h. The nadir clottable fibrinogen (0 mg/dl), fibrinogen antigen (99 mg/dl), Pl (20% of normal), API (17% of normal), and maximal levels of FDP (1:4096) and PA (20 times normal) were all significantly (p < 0.001) altered when compared with normal values. The platelet count and AT III levels were only midly decreased. Factors II, VIII, IX, and XII were normal. Because venom from the Eastern diamondback rattlesnake does not directly activate Pl, we conclude that the coagulopathy following envenomation by that reptile appears to be due to partial proteolysis of the fibrinogen with secondary activation of Pl by PA released from the endothelium. The resulting defibrination is distinguishable from disseminated intravascular coagulation.
Antigenic Properties Of Human Fibrin Plasmic Fragment D-Dimer And Its γ-γ Chain Remnant
Authors: Cierniewski C S , Nowak P , Krajewski T
Published: July 2018

Immunochemical analyses of human D-dimer and its γ-γ chain remnant were performed to identify their antigenic markers which would be of value in distinguishing between disseminated intravascular coagulation and primary fibrinogenolysis. Cross-linked fibrin was obtained by direct clotting of the fresh citrated plasma after adding excess CaCl2. The plasmic digests of the cross-linked fibrin were fractionated by ion-exchange chromatography and gel filtration. Thus, high molecular weight D-dimer was purified. The γ-γ peptide remnant was purified by ion-exchange chromatography on CM-52 cellulose from the reduced D- dimer. Purity of both polypeptide fragments was determined by standard techniques. They were used to immunize rabbits. Antisera to D-dimer and γ-γ chain remnant were characterized in binding assays and in equilibrium competitive inhibition assays in order to analyze the expression of their antigenic determinants in intact fibrinogen and its plasmic degradation products. Antisera to D-dimer preferentially bound D-dimer and γ-γ chain remnant and to a lesser extent fragment D and fibrinogen. Similarly, antisera to γ-γ chain remnant bound mostly γ-γ , D-dimer as well as γ poiypeptide chain. There was no binding of the intact human fibrinogen. The results obtained in the competitive inhibition assays employing antisera to D-dimer and γ-γ chain remnant, before and after adsorption with fragment D or γ polypeptide chain respectively, as well as fibrin fragments are discussed in this report.
Anticoagulant And Calcium-Binding Properties Of High Molecular Weight Derivatives Of Human Fibrinogen, Produced By Plasmin (Fragments X)
Authors: Nieuwenhuizen W , Gravesen M
Published: July 2018

Early plasmin degradation products (fragments X) of human fibrinogen were prepared and purified on Sepharose 6B-CL. X-fragments were characterized with respect to amino-terminal amino acid sequences, polypeptide-chain composition, anticlotting properties and calcium-binding. Amino-terminal amino acids were alanine and tyrosine. The molecular weights of the chains were about 26,000, 58,000 and 48,000 for Aα-, Bβ- and γ-chains, respectively. Fragments X were about 6 times as potent in anticlotting behaviour as D-fragments prepared in the presence of calcium ions. Calcium-binding properties were similar to those of fibrinogen: No differences were observed between fragments X prepared in the presence of calcium ions and those prepared in the presence of EGTA. Results indicate that the carboxy-terminal parts of the Aα-chains of fibrinogen are not involved in calcium-binding and that differences in chain-remnants as observed in late plasmic degradation products (which depend on the presence of calcium ions or EGTA in the incubation medium) are introduced beyond the stage of fragment X-formation.
Terminal Plasmin Degradation Products Of Duck Fibrinogen
Authors: Krajewski T , Nowak P , Cierniewski C S
Published: July 2018

Previously we have found that duck or goose fibrinogen are built up of Aα, Bβ and γ chains like mammalian one. However, they are more sensitive to proteolytic action than pig fibrinogen and rapidly form the D-E complex. The aim of this report is to characterize terminal plasmic products of duck fibrinogen. The protein was digested in the presence of calcium ions with human plasminogen activated by streptokinase. Degradation products were firstly analysed by polyacrylamide gel electrophoresis, secondly, isolated by DEAE-cellulose chromatography and gel filtration on Sephadex G-100, thirdly, characterized in order to establish their peptide composition and amino acid content. Three fragments, namely fg-DH, fg-DM and fg-DL as well as single fragment E were identified among final degradation products. Under conditions used, fg-DH was the major form of fragment D with molecular weight of about 100,000. For fg-DM and fg-DL, fragments Mr 89,000 and Mr 80,000 have been found, respectively. All fragments differed in the length of gamma chain remnants which varied from Mr 42,000 to Mr 24,000. Only a single population of fg-E with Mr 43,000 was identified in plasmic degradation products. The constituent peptide subunits of that fragment were also characterized. Fg-D appeared to be a strong inhibitor of fibrin monomer polymerization both in homologous /duck fibrin monomers/ and heterologous /pig fibrin monomers/ systems.
Rapid Radioimmunoassay For Fibrinopeptide A In Human Plasma
Authors: Woodhams B J , Kernoff P B A
Published: July 2018

The originally-described method for assay of fibrino- peptide A (FPA) in human plasma includes alcohol precipitation and dialysis steps which are complex, time consuming, and limit the applicability of the assay. The purpose of this work was to devise an assay for FPA which could be more easily applied to clinical studies, and to use this assay to study some of the factors which might cause artefactual results on blood samples obtained from patients. A rapid method for FPA assay has been developed which is simple, robust and sensitive and allows results to be obtained within 2.5 hrs. of blood collection. The assay depends on the use of bentonite to absorb fibrinogen from plasma, and gives a normal range for plasma FPA (0.3 - 1.5 pmol/ml) which is similar to that measured using the originally-described method. There is an equally good correlation between results obtained by the two methods on samples obtained from patients with various levels of circulating FPA and/or fibrinogen/fibrin degradation products (FDPs). Following venepuncture, FPA levels in sequential samples of blood drawn through 19-gauge ‘Butterfly’ needles became progressively lower in successive samples, indicating that a larger than usual discard of blood is necessary if basal levels are to be accurately assessed. Different antisera showed differences in affinity for FPA and cross reaction with des amino tyrosyl FPA, but such differences are unlikely to cause problems when assaying clinical samples. Generation of FPA from whole blood in vitro is completely suppressed by heparin/Trasylol anticoagulant in conventionally-used concentration, and we therefore find no evidence in favour of the suggestion that this anticoagulant mixture is unsuitable for sample collection for FPA assay.
PGI2 Production In Human Endothelial Cells Cultured In Diabetic And Nondiabetic Serum
Authors: Paton R C , Guillot R , Passa Ph
Published: July 2018

Reduced levels of prostaglandin I2 (PGI2) may contribute to the platelet hyper-reactivity and vascular complications found in diabetes mellitus. This study compared PGI2 production (PGI2-like activity and 6-keto-PGF1α levels) by vascular endothelial cells cultured in the presence of serum from 15 diabetics with proliferative retinopathy (5 treated by surgical hypophysectomy) and 15 sex-matched nondiabetic controls. Endothelial cells from human umbilical veins were cultured in M199 with either 20 % diabetic or control serum. At confluence, cultures were washed and stimulated with 0.1 NIH u/ml bovine thrombin. After 2 min incubation, the supernatant was tested for i)PGI2-like activity on ADP- induced platelet aggregation, results expressed as % inhibition and ii) 6-keto-PGF1α by radioimmunoassay, results expressed as nmol/ml. There was a significant correlation between PGI2-like activity and 6-keto-PGF-1α levels (r 0.78, p<0.001). The liberation of PGI2 from endothelial cells from different umbilical cords varied, but both PGI2-like activity (mean± SEM 21.9± 4.8 vs 28.3± 5.1 p<0.05) and 6-keto-PGF-1α (3.15± 0.68 vs 3.95 ±0.91 nmol/ml, p <0.05)were significantly lower in superantant from cells cultured in the presence of diabetic compared to control serum. PGI2 production was not significantly different in cells cultured with serum from hypophysectomised and nonhypophysectomised diabetics.

These results suggest that serum from diabetics with proliferative retinopathy contains factors which impair the release or production of PGI2 by endothelial cells and that this effect is not mediated by the pituitary.
Effects Of Soluble Immune Complexes From Diabetics On Platelet Aggregation And Release
Authors: Colwell J , Van Zile J , Kilpatrick M , Virella G
Published: July 2018

Soluble immune complexes (IC) can be detected in the serum of diabetic patients in frequencies ranging from 25 to 46%, depending on the screening technique used. Insulin-treated diabetics show a high frequency of insulin- anti-insulin IC (61% vs 4% in non-insulin treated patients) while other non-specific screening techniques show similar positivity frequencies in these two populations of diabetics. Soluble IC were isolated from the sera of 6 patients (5 under insulin treatment) using several combinations of polyethylene glycol precipitation, gel filtration and affinity chromatography. Sera from two normal donors was processed similarly and trace amounts of protein were recovered and used as controls. Platelet aggregation and release of ATP were studied with a Chronolog Lumi- aggregometer using platelet-rich plasma and low concentrations of ADP. All preparations of purified IC were found to induce platelet aggregation and ATP release of ADP sensitized platelets, while similarly obtained preparations from normal sera were inactive. Previous investigations had shown that sera from some diabetic patients contain factor(s) with platelet-activating properties. The present experiments suggest that soluble IC may be at least one of the factors.
The influence of microangiopathy in Diabetics with antiplatelet drugs
Authors: Bremer G , Richter O , Jacobi E , Koschinsky T
Published: July 2018

It is well kown that microangiopathy is associated with shortened platelet survival. Therefore a pilot study was performed to test several antiplatelet drugs with respect to their ability to increase platelet survival. The study was confined to 18 patients with long standing Diabetes mellitus and microangiopathv of I.- II. degree, exhibiting platelet survival times less than 7 days. The patients were treated with antiplatelet drugs. Dipyridamole with several combination of Acetylsali- cylicacid (ASA) and Sulfinpyrazon.

1. 225 mg Dipyridamole and 990 mg ASA per day,
2. 225 mg Dipyridamole and 300 mg ASA per day,
3. 150 mg Dipyridamole and 150 mg Sulfinpyrazon
per day.

Platelet survival was determined by the method described by Harker. The resultant survival curves were analysed by a computer program written by Murphy and Francis which is based on the multine hit model for platelet survival.

The results of the study are: After 10 days treatment the platelet survival was markedly increased under each of the combination.

If one assumes that there exists a causal relationship between microangiopathy and platelet survival,a long-term treatment by antiplatelet drugs may be a prophylaxis against microangiopathy in diabetics.
Exposure To Dextran During Isolation Enhances The Chem0- Tactic Response Of Human Peripheral Polymorphonuclear Leukocytes To Synthetic N-F-Met-Leu-Phe But Not To Zymosan Activated Serum (ZAS)
Authors: Stewart G J , Dall’Olio A , Stewart C R
Published: July 2018

The effect of the method of isolation of human peripheral polymorphonuclear leukocytes (PMN) on their chemotactic response to ZAS and N-f-met-leu-phe was studied. Chemotoxis was measured in Boyden chambers by counting the cells at consecutive 10 μM depths of the filters and calculating the chemotactic index (CI). PMN isolated by two-step ficoll-hypaque gradients and resuspended in medium 199 with 0.035% human albuman had a control CI of 9.75. With ZAS (1:2) as the chemoattractant in the lower chamber (1 hr at 37°C, CO2 incubator) the CI was 31.44 (222% increase). Exposure of gradient isoalted PMS to a
1-1000 dilution of DMSO increased the baseline CI to 24.72 when the medium remained the same. N-f-met-leu-Phe as the chemoattractant in the lower chamber illicited a very modest (19% increase) over a range of 10-10 to 10-5 M. In contrast, PMN isolated by a combination if ficoll- hypaque cushion and dextran sedimentation responded to N-f-met-leu-phe with an increase from baseline CI of 18.75 to stimulated CI of 33.18 at 10-8 M (77% increase). After exposure to dextran PMN isolated by the two-step gradient also responded to N-f-met-leu-phe. Thus, a low concentration of dextran or other glucose polymer may be required for the response of PMS to synthetic chemotactic peptides. This is consistent with the wide spread use of PMN isolated by dextran sedimentation or PMN from pero-tineal exudate elicited by the injection of glycogen
solution. Glucose polymers might replace bacterial surface carbohydrates and function to absorb the peptides thereby making an active polyvalent ligand.
Adherent Mononuclear Cells And Activated Protein C (APC)
Authors: Taylor F B , Esmon C T , Carroll R C , Lockhart M L
Published: July 2018

Addition of bovine APC to dilute whole blood or euglobulin assays of fibrinolytic activity produces little effect on rates of clot lysis. However, addition of APC to undiluted non anti coagulated or to 3.2% citrated whole blood followed by 125I-fibrinogen and thrombin produces clots which release fibrin degradation products and lyse within 24 hours. Ordinarily clotted non-anticoagulated whole blood does not lyse.

Addition of increasing amounts of APC from 12-100ug/ml increases the rate of whole blood clot lysis at least 20 fold over a 3.2% citrate control. This response is dose dependent with no evidence of saturation. Addition of increasing amounts of APC (25-200ug/ml) to platelet poor plasma (PPP) increases the rate of clot lysis only twofold. This increase is also dose dependent but is saturated at 80-100ug/ml. Addition of APC (60ug/ml) to 3.2% citrated PPP plus adherent mononuclear cells (1,000-2,000/MM3) again increases the rate of clot lysis at least 20 fold as with the whole blood system. However, this increase in rate is delayed. Substitution of platelet rich plasma (PRP) for PPP eliminates the lag phase. Substitution of non-adherent cells (1,000 MM3), RBC (3 million/MM3) or polymorphonuclear cells produces no acceleration of PPP clot lysis above the PPP-APC control. When deoxyglucose (1.4 × 10-1M) and anti-mycin (2.9 × 10-5M) are added to the PPP plus adherentxells before APC, the cell dependent acceleration of clot lysis is abolished. Removal of plasminogen activator (PA) and plasminogen (P) from PPP by lysine agarose adsorbtion also inhibits the effect of APC and adherent cells whereas reconstitution restores activity to 60%.

We conclude that adherent mononuclear cells are required for expression of optimal APC pro-fibrinolytic activity and that this may require both P and PA. This suggests another role for mononuclear cells (monocytes) found in inflamatory and thrombotic lesions.