bluuebunny/crossref_metadata_2025 huggingface dataset:
The Role Of Calcium In Platelet Microtubule Assembly- Disassembly
Authors: Kikuchi M , Ikeda Y , Handa M , Matsuda S , Muraki H , Toyama K , Yamamoto M , Watanabe K , Ando Y
Published: July 2018

Microtubules exist in a dynamic equilibrium between polymerized and depolymerized forms in human platelets, playing a major role to maintain the discoid shape of platelets. It has been previously shown that the interaction of aggregating agents with platelets leads to a rapid but transient disassembly of microtubules. ( Steiner and Ikeda, J.Clin. Invest. 63:443,1979 ) In this paper, the role of calcium in the equilibrium between assembled and disassembled microtubules was investigated. The respective pools of soluble and polymerized tubulin were “frozen” by addition of a glycerol-dimethyl sulfoxide-containing medium to platelet rich plasma, preincubated with 2 µM A23187 for various time intervals. The two pools of tubulin were estimated by measuring the colchicine binding activities of total and polymerized tubulin according to the method of Wilson.

Resting platelets were found to contain 56.2 ± 2.7 µg tubulin per 109 platelets, of which 56.7 % was in polymerized form. Addition of A23187 to platelet rich plasma produced a transient decrease in the pool of polymerized tubulin within 30 sec., followed by a return to base-line values within 2 min.. TMB-8, a known intracellular calcium antagonist, abolished this transient decrease in polymerized tubulin induced by A23187 in a concentration dependent manner, while indomethacin or acetylsalycylic acid did not.

These findings may indicate the important role of intracellular calcium in microtubule assembly-disassembly.
Role Of Cyclic Amp In The Inhibition Of Human Platelet Functions By Quercetin, A Flav0N0Id That Potentiates PGI2 Effect
Authors: Cazenave J P , Beretz A , Stierlé A , Anton R
Published: July 2018

Injury to the endothelium (END) and subsequent platelet (PLAT)interactions with the subEND are important steps in thrombosis and atherosclerosis. Thus,drugs that protect the END from injury and also inhibit PLAT function are of interest. It has been shown that some flavonoids(FLA), a group of compounds found in plants, prevent END desquamation in vivo, inhibit cyclic nucleotide phosphodiesterases(PDE)and inhibit PLAT function. We have studied the structure-activity relationships of 13 purified FLA on aggregation and secretion of 14c-5HT of prelabeled washed human PLAT induced by ADP, collagen(COLL) and thrombin(THR). All the FLA were inhibitors of the 3 agents tested. Quercetin(Q), was the second best after fisetin. It inhibited secretion and aggregation with I50 of 330µM against 0.1 U/ML.THR, 102µM against 5µM ADP and 40 µM against COLL. This inhibitory effect is in the range of that of other PDE inhibitors like dipyridamole or 3-isobutyl-l- methylxanthine. The aggregation induced by ADP, COLL and THR is at least mediated by 3 mechanisms that can be inhibited by increasing cAMP levels. We next investigated if Q, which is a PDE inhibitor of bovine aortic microsomes,raises PLAT cAMP levels. cAMP was measured by a protein-binding method. ADP- induced aggregation(5µM) was inhibited by PGI2 (0.1 and 0.5 nM) . Inhibition was further potentiated(l.7 and 3.3 times) by lOµM Q, which alone has no effect on aggregation. The basal level of cAMP(2.2 pmol/108PLAT) was not modified by Q (50 to 500µM). Using these concentrations of Q,the rise in cAMP caused by PGI2(0.1 and 0.5nM) was potentiated in a dose dependent manner. Q potentiated the effect of PGI2 on the maximum level of cAMP and retarded its breakdown. Thus Q and possibly other FLA could inhibit the interaction of PLAT with the components of the vessel wall by preventing END damage and by inhibiting PLAT function through a rise in cAMP secondary to PDE inhibition and potentiation of the effect of vascular PGI2 on PLAT adenylate cyclase.
Adhesion Of Human Platelets In Steady Shear Flow: Effects Of Drugs
Authors: Richardson P D , Kane R , Aggarwal K
Published: July 2018

Adhesion of platelets to a transparent wall can be observed and recorded continuously by using a shear flow chamber built into a microscope slide. A field of area 50,000-100,000 εm2 can be viewed by phase contrast. Adhesion (and subsequent detachment and departure) events can be monitored as functions of site position in the field and of elapsed time. Fresh anticoagulated human platelet-rich plasma (PRP) was pumped steadily through a flow chamber so that a wall shear rate of 25 sec-1 was obtained at the field of view. Videotapes were analysed for stationary platelets, with the locations of sites and their state of occupancy being digitized for computer data logging and analysis. As the PRP was pumped steadily through the chamber, it was found that there was a 2-3 min delay before adhesion events occurred. Then the cumulative number of adhesion sites grew linearly with time, the least-squares fit giving the cumulative site growth rate (CSGR) with a high correlation coefficient r> 0.98. Some platelets detached, but later the same sites were re-occupied; this process often repeated, so that a few sites were occupied by as many as eight different adherent platelets in fifteen minutes. An index was also found for the re-use of sites (RUS), based on the frequency of re-use. Pre-incubation of platelet-rich plasma 40-105 minutes with aspirin, 5-10 mM, reduced CSGR and RUS. Injection of adenosine, resulting in 50 µM in the flow, and of dipyridamole, 250 µM, both reduced CSGR. Chlorpro- mazine, 10 µM, did not cause significant differences from controls. Preincubation with an analog of cIMP reduced CSGR and RUS markedly, especially in the presence of ADP.
Platelet Interaction With Basement Membrane Proteins
Authors: Balleisen L , Rauterberg J , Risteli J , Rohde H , Timpl R
Published: July 2018

Basement membranes consist mainly of two components: non-fibrillar type IV collagen and the non-collagenous glycoprotein laminin (m.w.900.000) which is capable to interact with cell surfaces. The collagenous protein was studied in form of two major fragments comprising together the total mass of the molecule. 7-S collagen which resembles the crosslinking domain of type IV collagen was isolated after collagenase digestion and consisted of four triple helices aligned in a parallel fasion (m.w.360.000). The major triple helical domain of type IV collagen (m.w.450.000) could be obtained by a acid extraction but had lost most of the 7-S domain.

Interaction with platelets was examined in aggregation, cell spreading and fibrin clot retraction assays including the determination of malondialdehyde formation. 7-S collagen was the most active component in all three assays. Aggregation was induced by as little as 25 µg/ml and was confirmed by electronmicroscopy. When compared to interstitial collagens, however, 10-20 fold higher amounts of 7-S collagen are required to produce the same effects. Acid extracted type IV collagen possessed virtually no activity. Laminin did not aggregate platelets but promoted strongly their attachment and spreading on a plastic substrate. Thus both basement membrane proteins apparently interact with platelets in different ways via distinct domains of the molecules.
The Effects Of Ticlopidine Hydrochloride On Bleeding Time And Platelet Function In Man
Authors: Ellis D J , Roe R L , Bruno J J , Cranston B J , McSpadden M M
Published: July 2018

Ticlopidine hydrochloride (T) was given to healthy male and female subjects in doses ranging from 125-500 mg/day for up to 8 days to study the rate of onset and offset of action and to define the optimal dosage for inhibition of platelet function tests.

Template bleeding time (BT) using the Simplate® device and ex vivo platelet aggregation were done every day from the start of dosing through the post-dosing period until platelet function had returned to baseline.

Achievement of maximal bleeding time prolongation and maximal inhibition of platelet aggregation induced by ADP, collagen, and epinephrine required 3-5 days of dosing. Rate of onset was higher at higher drug doses. Post-dose return to baseline took 4-8 days.

BT prolongation was dose-dependent with 2.0 × prolongation at 125 mg/d, 2.5 × at 250 mg/d, 3.4 × at 375 mg/d and 4.7 × at 500 mg/d.

At steady state, ADP aggregation (1st and 2nd phase) was inhibited by 50-60%, epinephrine aggregation was inhibited by 40-80%, and collagen aggregation was inhibited by 5580%. Inhibition was dose-dependent. Arachidonic acid (1 × 10-3M) induced aggregation was not inhibited. ATP release during collagen aggregation was inhibited by 20-50% and malondialdehyde production induced by thrombin was inhibited 20-55%, both in a dose-related fashion.

Ticlopidine hydrochloride is a potent inhibitor of platelet function with a broad spectrum of activity and a delayed onset and offset of action. The drug is neither a prostaglandin synthetase nor a phosphodiesterase inhibitor and appears to act by a novel mechanism.
The Binding Of Thrombin By Clots Formed From Fragment X
Authors: Liu C Y , Nossel H L , Kaplan K L
Published: July 2018

Previous studies showed that: 1. thrombin was specifically
and reversibly bound by fibrin, 2. Scatchard analysis of the data suggested high and low affinity binding sites, and 3. the bound thrombin was quantitatively released following proteolysis of the fibrin by plasmin. In the present study thrombin binding to clots formed from fragment X was studied. The binding of thrombin to fibrin decreased progressively in relation to the original fibrinogen concentration as the fibrin was formed from fibrinogen progressively degraded with plasmin and thus progressively less clottable. Fragment X was then isolated by Sephadex G-200 filtration of partially proteolysed fibrinogen. The fragment X preparation exhibited 76% clottability with thrombin, was heterogeneous with an average molecular weight of 292,000 ±36,000, and contained 2 moles fibrinopeptide A and 0.25 moles fibrinopeptide B per mole. Fibrin formed from clottable fragment X bound thrombin with a molar binding ratio of 0.32 compared to 0.35 for fibrin formed from intact fibrinogen and a binding constant of 7.5 × 105 M-1 compared to 6.6 × 105 M-1 for the high affinity site on fibrin from intact fibrinogen. The data indicate that the NH2-terminal end of the Bβ chain and the COOH-terminal portion of the Aα chain arenot required for high affinity thrombin binding. Because the demonstrated binding is to clottable plasmin degradation products and the molar binding ratio is less than one, it is suggested that the higher affinity thrombin binding site is not present in the fibrinogen molecule but is formed by two or more fibrin molecules present in a polymer.
Substrate Specificity Of Bovine Thrombin And Factor Xa Toward Peptide Para-Nitroanilide Substrates
Authors: Lottenberq Richard , Jackson Craig M
Published: July 2018

Kinetic parameters were obtained for commercially available chromogenic substrates with bovine alpha thrombin and Factor Xa. Enzyme concentrations were determined by active site titration using p-nitrophenyl- p-guanidinobenzoate. Initial rate studies were performed at 25 deg. C at pH 7.8. Non-linear least squares procedures were utilized to determine Michaelis constants (Km’s) and catalytic constants (kcat’s) for an assumed simple Michaelis-Menten model for the hydrolytic reaction. For several substrates with very high Km’s, the second order rate constant, kcat/Km, was the only parameter that could be accurately obtained, although estimates for the two kinetic parameters could be calculated from linearized forms of the Michaelis-Menten equation. Twenty peptide-anilide substrates were evaluated with thrombin. For those substrates for which completely valid parameters could be determined, Km estimates ranged from 0.43 to 360 uM; kcat values varied from 0.060 to 130/second. The kcat/Km values ranged from 500 to 93,000,000 liter/mole/second. Twenty-one peptide-anilide substrates were evaluated with Factor Xa. The Km range was 35 to 1300 uM; kcat range 0.071 to 260/second. The kcat/Km values ranged from 15 to 1,900,000 liter/mole/second. Effects of pH, ionic strength, temperature and concentration of polyethylene glycol in the reaction mixture were also determined.

Inferences regarding the substrate specificity of thrombin and Factor Xa that can be drawn from these data will be discussed.
Kinetic Description Of Conversion Of Bovine Prethrombin 2 To Thrombin
Authors: Carlisle Thomas L , Jackson Craig M
Published: July 2018

Bovine Factor Xa slowly converts Prethrombin 1 to thrombin plus Fragment 2. Inclusion of Ca2+ increased the rates of Prethrombin 1 consumption, Prethrombin 2 production and thrombin formation detected by SDS polyacrylamide gel electrophoresis or by thrombin assay. Ca2+ also increased the rate of thrombin formation from equimolar mixtures of Prethrombin 2 and Fragment 2 (Prethrombin 2/Fragment 2) by approximately 1.8 fold. Calcium ion thus increases the rate of both proteolyses required to generate thrombin from Prethrombin 1. Studies using Factor Xa (des light chain residues 1-44) indicated that this effect of Ca2+ required the region of Factor Xa containing gamma-carboxyglutamic acid.

Factor Va markedly lowered the apparent Km of Factor Xa for Prethrombin 2/Fragment 2, with decreases greater than 20 fold observed under some conditions. The apparent maximum velocity also increased by up to 50 fold. The extent of increase was greater at higher concentrations of Factor Va, and was about 6 fold greater in the presence of Ca2+ than in its absence. Factor Va binding to Factor Xa (forming XaVa with enhanced substrate binding and/or catalytic efficiency), and Factor Va binding to Prethrombin 2/Fragment 2 (forming a substrate more readily bound and/or cleaved) must be considered among the possible explanations for these effects. Previous qualitative observations suggest that these effects of Factor Va on activation of Prethrombin 2/ Fragment 2 are important in understanding the activation of prothrombin.
Oral Administration Of Eicosapentanoic Acid (EPA) Stimulates Production Of Prostacyclin By Rat Aorta
Authors: Hamazaki T , Hirai A , Terano T , Tamura Y , Kumagai A , Sajiki J
Published: July 2018

Prostacyclin (PGI2) which is mainly produced in blood vessels has the most potent platelet antiaggregatory activity of any substance yet found. It is therefore thought that PGI2 is a preventive agent for cardiovascular diseases (CVD). We found that EPA orally administered to rats stimulated rat thoracic aorta to produce a PGl2like substance at 3 times normal levels. While there are several reports suggesting that EPA may contribute to the prevention of CVD by inhibiting platelet functions and improving plasma lipid concentrations, this is the first report that shows the relationships between orally administered EPA and PGl2~like substance production from aorta. METHODS: Two groups of Wistar rats were fed either a normal diet (group A) or a normal diet plus 74% pure EPA, 60 mg/kg/day (group B), for 8 weeks. Thoracic aortas were excised, cut into rings, and incubated in 50 mM Tris-HCl buffer, pH 7.5, for 10 min. The activity of the PGl2-like substance was measured by inhibition of human platelet aggregation. Platelet aggregability of rats in both groups was also checked with ADP as an aggregant. RESULTS: The rate of production of the PGl2~like substance by the aortas of group B was 4.0±1.4 ng/mg wet aorta/10 min (calibrated with authentic Na-PGI2), while that of group A was 1.4±0.5 (P<0.001). Platelet aggregability was depressed in group B rats. There was no significant difference in concentrations of plasma lipids such as total and HDL-cholesterol, triglycerides, and phospholipid. DISCUSSION: It has been suggested that EPA could prevent CVD through its antiaggregatory property. Now we suggest a new possibility that EPA could prevent CVD by stimulating blood vessels to produce more PGI2-like substance as well as by inhibiting platelet aggregation directly.
Increased Vascular PGI2-Formation And Increased Plasma 6-OXO-PGI1α After Calcitonin In Human And Experimental Animals
Authors: Sinzinger H , Clopath P , Staehelin A , Silberbauer K , Kovarik J
Published: July 2018

5 normolipemic minipigs treated daily for 28 days with 40µg/kg h-calcitonin synthetized in thoracic (4,0±0,8 pgPGI2/mg/min) and abdominal (3,2±0,7 pg) aorta significantly more prostacyclin than in 5 control animal^ receiving the vehicle only (3,4 ± 0,6 pg and 2,3±0,7 pg) respectively. In 8 healthy males aged 26-32 years, a single intramuscular injection of 0,5 mg calcitonin led to an increase and measurable levels of 6-oxo-PGF1α (about 100pg/ ml) between 15 and 30 minutes after application (lower detection limit: 70 pg/ml). Calcitonin levels after 5 minutes increased to more than 1000pg/ ml and started to decline after 60-120 minutes. In two healthy volunteers (physicians) 30 minutes after i.m. application of 0,5 mg calcitonin, PGI2- formation in the forearm vein was examined by the bioassay technique of Moncada. The values after calcitonin application (x = 9,86 pg) were found to be enhanced in comparison to age and sex matched controls (x = 7,11 pg). The same 8 healthy males received also a depot-calcitonin and salmon-calcitonin (100 MRC-units), and the time course of 6- oxo-PGF.x in unextracted plasma and caltitonin was was estimated. In 50 patients with peripheral vascular disease, after a single calcitonin administration,the 6-oxo-PGF1αlevels remained under the level of detection. 11: is discussed, whether the temporarily enhanced vascular PGI2-synthesis as well as the increase of 6-oxo-PGF1α-levels could be an explanation for the beneficial role of calcitonin seen in the treatment of this disease.
The Structural Error And Its Relation To The Malfunction In Some Abnormal Fibrinogens
Authors: Henschen A , Southan C , Kehl M , Lottspeich F
Published: July 2018

The primary structure elucidation of normal human fibrinogen has recently been completed. Thus, it is now possible to analyse the structure- function relationship in detail. In abnormal fibrinogens the function is altered. It is obvious that information about the corresponding structural errors would be of great importance. Functional abnormality of fibrinogen has been described for more than 70 cases. However, only in a single case, Fibrinogen Detroit, the structural error was identified by Blombäck’s group. Recently, our group has analysed several abnormal fibrinogen by separating the three peptide chains and performing direct sequence analysis of the chains. The first fibrinogen (obtained from R. Marx and W. Schramm) had an Arg→Asn substitution in position 19 of the Aa-chain, i.e. in the same position as in Fibrinogen Detroit, and was characterised by delayed A- peptide release and monomer polymerisation. The second fibrinogen (obtained from C. and J. Soria) had an Arg→Cys exchange in position 16 of the Aα- chain, and was characterised by absence of releasable A-peptide. The third fibrinogen (obtained from V. Hofmann) had the same substitution as the previous, but in the heterozygous state, and was characterised by delayed A-peptide release. It is obvious that when the Arg residue in position 16, i.e. at the thrombin cleavage site, is replaced by a neutral residue thrombin will not cleave. When the Arg residue in position 19 is exchanged the cleavage is slowed down. Several other fibrinogens with delayed A-peptide release showed, however, no primary structure error in this section of the molecule.
Carbohydrate Composition And Catabolism Of Five Abnormal Fibrinogens
Authors: Lane D A , Allen A K , Markwick J , Mackie I , Thompson E , Owen J
Published: July 2018

An investigation has been made of the carbohydrate composition and catabolism of five abnormal fibrinogens. These were fibrinogens, London, Manchester, Oslo II, a fetal fibrinogen isolated from pooled cord blood and a newly discovered case of hypodysfibrinogenemia which has been tentatively designated fibrinogen London II. The carbohydrate composition of fibrinogen (mol sugar/mol of fibrinogen) was studied by amino acid analysis techniques for amino sugars and methanolysis followed by GLC for neutral sugars and sialic acid. The adult variants did not differ significantly from the normal adult fibrinogen which typically contained 22 mannose, 18 galactose, 20 N-acetylglucosamine, 4 N-acetyl- galactosamine and 12 sialic acid residues. However, fetal fibrinogen was markedly different, containing 15 mannose, 21 galactose, 14 N-acetylglucosamine, 20 N-acetylgalactosamine and 20 sialic acid residues. The catabolism of the I125- labelled fibrinogens was studied in New Zealand White rabbits. Normal fibrinogen was eliminated with a mean t1/2 of 50.2 h (n = 5, range 42-60). The mean t1/2 of fetal fibrinogen was similar 47.7 h (n = 5, range 41-70) and fibrinogens London, London II, Manchester and Oslo II had t1/2’s of 39, 42, 38 and 44 h respectively. It is concluded that (a) the increased sialic acid on fetal fibrinogen is probably due to there being more O-glycosylated serine or threonine residues and this does not alter catabolism, (b) the reduced plasma fbgen of London II is caused by impaired synthesis rather than hypercatabolism, and (c) the catabolism of fibrinogen is only minimally dependent upon the full integrity of the regions of the molecules involved in polymerisation and fibrinopeptide releasing functions.
Isolation And Characterization Of Fragments Of Human Fibrinogen Generated By Chemical Cleavage At Cysteine Residues
Authors: Benabid Y , Doolittle R F
Published: July 2018

Human fibrinogen and its constituent S-sulfo chains were treated with 2-nitro-5-thiocyanobenzoic acid (NTCB) under conditions appropriate for cleaving on the amino-terminal sides of all cysteine residues. The fibrinogen or chains were first reduced with dithiothreitol (DTT) and the excess reducing agent removed by dialysis under nitrogen. The course of fragmentation was followed by SDS polyacrylamide gel electrophoresis and the purification of major fragments accomplished by gel filtration under a variety of solvent conditions. Our primary aim was to isolate and study the 112/113-residue polypeptides that exist between disulfide rings in the native molecule and that are thought to comprise the “coiled-coils” that connect the central and terminal domains. We also attempted to purify the other major fragments produced by NTCB-treatment, however, including 277-residue and 139-residue fragments from the α-chain, and a 108-residue β-chain polypeptide and a 144-residue fragment from the γ-chain. Of all of these, the 277-residue peptide from the α-chain is the easiest to isolate and was readily obtained in pure form. The 112/113-residue interdomainal connectors presented a number of technical problems with regard to their isolation, including a decided propensity to self-associate. Accordingly, we resorted to circular dichroism as a tool for following the residual α-helix that is an index of the presence of these segments, a technique we had planned to use to study the re-association of the separate segments. Preliminary evidence suggests that we were able to reform α-helical structures, presumably coiled- coils, to various degrees with different combinations of segments from the different chains.
Sequential Formation Of Des-A Fibrin And Des-Ab Fibrin And Binding Of Fibrinogen To Des-Ab Fibrin
Authors: Müller-Berghaus G , Bernhard J C , Hofmann H , Mahn I , Ostheimer F R , Selmayr E , Thiel W
Published: July 2018

Thrombin cleaves the fibrinopeptides A and B from fibrinogen in a consecutive manner, producing first des-A fibrin and consequently, des-AB fibrin. Physico-chemical and in-vivo properties of both types of fibrin were comparatively studied by gel filtration, affinity chromatography and in-vivo behavior. At 37°C and in the presence of fibrinogen and Ca++, des-A fibrin formed soluble fibrin-fibrin dimers. Des-AB fibrin, however, formed oligomers at 37°C. Fibrin-fibrinogen interactions were studied by affinity chromatography. At 37°C, des-A fibrin did not interact with immobilized fibrinogen, whereas des-AB fibrin stuck to it. Small amounts of des-A fibrin iv injected were cleared from the circulation much faster than des-AB fibrin. Large amounts of des-A fibrin injected into animals aggregated to microclots. These results suggest that des-A fibrin in a low concentration (0.01 mg/ml) does not form circulating oligomers and can very fast be cleared from the circulation possibly because of its weak interaction with fibrinogen. Aggregated des-A fibrin, however, is cleaved to form des-AB fibrin. Des-AB fibrin binds fibrinogen making it accessible to further thrombin cleavage. Thus, the threshold of clot formation would be the concentration of des-A fibrin in plasma. The preferential sequence of reactions is: fibrinogen → des-A fibrin → des-A fibrin aggregates → des-AB fibrin aggregates. Des-AB fibrin + fibrinogen → des-AB fibrin-fibrinogen → des-AB fibrin-des-A fibrin etc.
The Role Of Heparin In Antithrombin III Proteolysis
Authors: Marciniak E
Published: July 2018

Recent reports indicate that thrombin is capable of degrading part of the uncomplexed antithrombin III (AT III). Presence of heparin in the reaction of AT III and thrombin, in addition to enhancement of the binding rate, causes an unexplained, excessive decrease in the total AT III binding capacity. This suggests that heparin plays a major role in thrombin-catalyzed AT III proteolysis. Addition of heparin to reaction mixtures of human AT III and α- thrombin, conducted with a large excess of inhibitor, increased the ratio of AT III utilization by more than 50% and induced a release of inactive 50,000-dalton AT III fragment. This fragment, detectable by SDS-polyacrylamide gel electrophoresis with SDS concentrations not exceeding 0.1%, showed a decreased affinity for heparin. It was released from unbound AT III by small quantities of thrombin simultaneously with the formation of AT III-thrombin complex, if at least 1 μg/ml of polydispersed heparin participated in the reaction. To detect a similar degree of proteolysis in heparin-free reaction about 10 times more of α-thrombin was required. The extent of AT III proteolysis promoted by individual heparin fractions obtained in gel filtration correlated inversely with the anticoagulant activity of these fractions. Excessive decrease of residual inhibitory activity and changes in two-dimensional immunoelectrophoresis suggested that part of AT III in plasma is also subjected to proteolysis following addition of thrombin and heparin. These data indicate that the effect of heparin on AT III is more complex than generally recognized. On the one hand, heparin contributes to a rapid neutralization of the enzyme; on the other, heparin facilitates proteolytic degradation of unbound inhibitor even by small quantities of thrombin, causing reduction of the overall binding potential of AT III.
A Novel Anti-Thrombotic Heparinoid (ORG 10172) Devoid Of Bleeding Inducing Capacity: A Survey Of Its Pharmacological Properties In Experimental Models In Rats
Authors: Meuleman D G , Dedem G Van , Hobbelen P M J , Moelker H C T
Published: July 2018

The pharmacological profile of a heparinoid (Org 10172) was assessed In experimental thrombosis and bleeding models In rats. Org 10172 Inhibited thrombus formation In arteriovenous shunts dose dependently (ID50=5 mg/kg i.v.); heparin USP had an ID50 of 0,4 mg/kg I.v. in the same model. Taking the effective dose ratio of the compounds into account both Org 10172 and heparin USP similarly reduced the 125I-fibrin content In thrombi; Org 10172 however affected less the 51Cr-labeled platelet content in the thrombi than heparin USP.

Heparin USP progressively prolonged bleeding at doses of 1 mg/kg i.v. and higher, whereas Org 10172 showed no increase in bleeding over a dose range of 50-200 mg/kg i.v. The benefit (anti-thrombotic)/risk (bleeding) ratio is at least 20-fold higher than for heparin USP.

In vitro Org 10172 did not Inhibit the collagen-Induced release of serotonin from platelets, in contrast to heparin USP. Org 10172 was at least 500 times less active In enhancing the thrombin-AT III Interaction than heparin USP, it showed a very small increase In APTT and had about 6% of the factor Xa- inhibiting activity of heparin USP. The improved profile of the drug may be attributed to these quite different effects on blood platelet function and the coagulation pathway.
A Comparative Study Of Heparin And Heparin Fractions In Preventing Experimental Venous Thrombosis
Authors: Thomas D P , Barrowcliffe T W , Lindahl U , Thunberg L , Merton R E , Hiller K F , Eggleton C A
Published: July 2018

We have compared the relative efficacy in preventing venous thrombosis of an ordinary mucosal heparin, a low molecular weight (LMW) heparin fraction and a decasaccharide fragment with high affinity for AT III. We examined the extent to which all three preparations impaired the formation of serum-induced stasis thrombi in New Zealand White rabbits. The LMW fraction, despite having an in vitro potency by APTT half that of ordinary heparin (but comparable anti-Xa activity) was as effective as heparin on a weight basis in preventing thrombosis.

Two minutes after intravenous injection of 30 μg/kg of the LMW fraction the mean blood level by anti-Xa clotting assay was 0.12 i.u./ml (range 0.08-0.21), which was sufficient to prevent thrombosis. In contrast, the decasaccharide fragment, which had a specific activity in vitro by anti-Xa assays of 1000-1300 i.u./mg, but essentially no activity by APTT or thrombin time assays, prevented stasis thrombi only when given at a dose of 100 μg/kg, giving blood levels in excess of 0.3 i.u./ml by anti-Xa assays.

It is concluded that in this experimental model a decasaccharide fragment, despite having a very high affinity for AT III, was less effective on a weight for weight basis than either ordinary heparin or a LMW fraction in preventing venous thrombosis. This suggests that while a sufficiently high anti-Xa activity can alone prevent venous thrombosis, the effectiveness of heparin as an antithrombotic drug does not depend solely on its AT III-binding capacity.
Antiheparin Antibody: Preparation And Use As A Functional Probe
Authors: Gitel S N , Medina V M , Wessler S
Published: July 2018

Antiheparin antibodies were raised in 5 of 6 rabbits immunized sequentially with covalent complexes of ovalbumin-heparin and bovine IgG-heparin. Confirmation of the presence of heparin-specific antibodies in the antisera was based on: (1) antisera precipitation of BSA-heparin but not BSA alone, (2) antisera inhibition of heparin anticoagulant activity without precipitating heparin, (3) antisera precipitation of heparin-heparin complexes, and (4) antibody removal from the antisera by heparin- Sepharose. The antisera had hemagglutination titers between 1:10,000 and 1:50,000 when tested against tanned red cells labeled with BSA-heparin complex; whereas no hemagglutination occurred with tanned erythrocytes tagged with BSA. Heparin-heparin oligomers, prepared by carbo- diimide coupling in aqueous solution and retaining anticoagulant activity, precipitated in the presence of the antiheparin antibodies. Data indicating that heparin has separate binding sites for antithrombin 111 and thrombin were obtained by quantitating the heparin oligomer- antibody precipitate in the presence and absence of these two purified, heparin-binding, coagulation proteins.
Carrier Detection In Haemophilia A By Measurement Of Factor VIII Clotting Antigen (VIIICAg) And Factor VIII Related Antigen (VIIIRAg
Authors: Peake I R , Newcombe R G , Davies B L , Furlong R A , Ludlam C A , Bloom A L
Published: July 2018

In order to assess the value of measurement of VIIICAg in the detection of carriers of haemophilia A, plasma samples were obtained on three separate occasions from each of 23 obligate carriers of mild and severe haemophilia, and 26 normal females. At each visit each sample was divided into three and each aliquot was then assayed for VIIICAg (immunoradiometric assay), clotting factor VIII (VIIIC) (two stage assay) and VIIIRAg (Laurell immu noelectrophoresis). After calculating median values at each visit, and for the three visits, a comparison of the ratios VIIIC/VIIIRAg and VIIICAg/VIIIRAg was made. Likelihood ratios (of being a carrier) were calculated using an unequal variance predictive method for both ratios. These showed that laboratory data calculated on the median of the three-visit medians had greater discriminatory power than a single-visit median value. Using the median of three visits both VII IC/VIIIRAg and VIIICAg/VIIIRAg gave the same proportional misclassification of carriers as normals (4 of 23- 17%). However the ratios VII ICAg/VIIIRAg were more discriminatory due to the greater reproducibility between visits of VIIICAg results than those of VIIIC. There was no statistically significait difference between VII ICAg/VIIIRAg (or VII IC/VIIIRAg) ratios obtained from carriers of mild or severe haemophilia. The ratio VII ICAg/VIIIRAg was therefore shown to be the method of choice for carrier detection except theoretically in the rare CRM+ families.
VIII: C And VIII: CAg Response In Haemophilia A After Infusion Of F VIII Concentrates, Fresh Citrated Plasma Or Heparin Plasma
Authors: Nilsson I M , Holmberg L , Borge L , Kristoffersson A C
Published: July 2018

VIII:C (one-stage) and VIII:CAg (solid phase IRMA using both two homologous nonhaemophilic antibodies and a haemophilic antibody against VIII: C, Holmberg et al 1979, Scand. J. Haemat. 23, 17) were studied in 8 patients with severe, and 2 with mild, haemophilia A after administration of 3 different f VIII concentrates (AHF-Kabi, Hemofil, high purity Factorate) or fresh citrated plasma or heparin plasma. The recovery of VIII: C was about the same for all the preparations (70–100 %), but that of VIII: CAg considerably less (30–50 %). When the concentrates were added in vitro to the haemophilia plasma (the patients all multitransfused) the recovery of VIII:C was about 100 % but that of VIII: CAg only 30–50 %. In normal plasma the recovery of VIII: CAg was 100 %. When the concentrates were added to haemophilia plasma depleted from IgG (protein A Sepharose) the recovery of VIII: CAg was about 100 %. This indicates that most haemophiliacs have antibodies reacting with the f VIII coagulant protein with little or no interference with the coagulant activity. The half-life of VIII:C was about 12 h, but that of VIII:CAg only 3 h or less. We also infused fresh citrated plasma into 2 haemophiliacs, but the results were the same. The response to transfusion observed in haemophilia indicates a change in the native properties of f VIII already on collection of blood. This might be due to the decalcification with citrate. Thus, in preliminary experiments using fresh heparin plasma the curves for the disappearance rate of VIII: C and VIII: CAg were parallel.
Factor VIII Complex In Non-Hemophilic Patients With Antibody To Factor VIII: C
Authors: Croissant M P , Allain J P
Published: July 2018

An antibody to Factor VIII in non-hemophiliacs is a rare occurrence. The presence of low but detectable Factor VIII: C (VIII: C) and high Factor VIII antigen (VIIIR: Ag), together with high antibody levels, suggests a complex influence of anti- VIII: C on Factor VIII metabolism.

We have studied Factor VIII complex in 16 non-hemophilic patients with anti-VIII: C. VIII: C, VIIIR: Ag and VIII: Rcof were measured by standard techniques and VIII: C antigen (VIII: CAg) by a liquid phase immunoradiometric assay using radio labelled anti-VIII:C Fab’ fragments purified from a hemophiliac.

The plasma from all patients had low VIII: C (0.01 - 0.24 u/ml) and high VIIIR:Ag (1.4 - 8.8 u/ml) content. VIII:Rcof was approximately 50% of the VIIIR:Ag level. An increase in electrophoretically fast-moving VIIIR: Ag was observed in some cases

VIII: CAg was tested in 13 patients. Two patterns of the dose response were observed. In most samples obtained from patients with low affinity anti-VIII:C whose VIII: CAg ranged from 0.19 - 2.45 u/ml, the dose response paralleled that of the control. In patients with high affinity antibodies the dose response curve was less steep than control, suggesting a competition between the patient’s antibody and the hemophilic anti-VIII: C Fab’ fragments for the VIII: C antigenic sites

The Factor VTII complex was studied sequentially in 3 patients during immunosuppression. The results showed that : 1) when VIII: C normalizes, VIIIR: Ag levels and electrophoretic mobility progressively return to normal ; 2) levels of VIIIR: Ag are inversely correlated to the VIII: C irrespective of VIII: CAg levels ; 3) high VIII: CAg levels correspond to VIII:C-anti-VIII: C circulating immune complexes.

In non-hemophilic patients low VIII: C, high VIII:CAg and high VIIIR: Ag indicate the presence of an anti-VIII: C inhibitor. Measurements of various components of Factor VIII in these patients are not only a means for monitoring the effect of therapy but also provide a rare opportunity to study the metabolism of Factor VIII in man.
Intraoperative Anticoagulation In Cardiovascular Surgery
Authors: Effeney D , Chin D , Goldstone J , Krupski W , Ellis R
Published: July 2018

The optimal heparin dose during the occlusive phase of cardiovascular operations to prevent intraoperative thrombosis or excessive bleeding has not been determined Therefore, we studied the kinetics of heparin effect in 28 patients undergoing peripheral vascular and cardiac operations. The activated clotting time(ACT)was measured in seconds by an electronic clot timer. The maximum ACT after initial heparin administration and the time for decay of half the heparin effect (t 1/2) were determined.

The data show that the anticoagulant effect of a given dose of heparin varies among patients. No correlation was found between the t 1/2 and the dosage of heparin given. Despite higher doses of heparin given to cardiac patients, the t 1/2 in cardiac and vascular patients were not significantly different.

Four patients were given the same dose of heparin pre and intra-operatively in an effort to predict the intraoperative effect. The times to maximum effect were the same but t 1/2 intraoperatively was longer (p<.01).

These results indicate: (1) maximum heparin effect occurs later than previously believed and is different for cardiac and peripheral vascular patients; (2) the recommendation to give more heparin based on the 5-minute ACT is not valid; (3) individual response to a standard dose of heparin is unpredictable, both in duration and maximum effect; and (4) intraoperative monitoring of heparin effect is practical and is the only way that any consistent specific end point of heparin can be achieved.
Sequential Pneumatic Compression-An Effective Method Of Preventing Deep Venous Thrombosis
Authors: Scurr J H , Bucknall T E , Ellis H , Wastell C
Published: July 2018

195 patients undergoing major surgical operations over 40 years of age were randomly allocated into 3 groups. In Group 1 the patients were given subcutaneous Heparin 5000 units b.d. for 7 days. Group 2, graduated compression stockings were applied on admission to hospital, being removed for the duration of surgery and replaced by a pneumatic sequential compression device. Group 3, graduated compression stockings were applied on admission to hospital, were removed during surgery and replaced with a seouential compression device which remained until the patient was fully ambulant and then replaced with a graduated compression stocking. Patients were studied daily using the I125 fibrinogen uptake test, doppler, strain guage plethysmography and venography to confirm positive or doubtful results. The incidence of DVT in the Heparin and extended compression group is 9%. The incidence in the compression group is 14.2%. Blood loss and haematoma were significantly greater in the Heparin group. Although there is no significant difference between the incidence of DVT in either group, there were significantly less problems in the compression groups. Sequential compression presents an effective method of preventing DVT.
The Contribution Of Increased Fibrinolysis Induced By Intermittent Pneumatic Compression Of The Legs In The Prevention Of Deep Vein Thrombosis
Authors: de Boer A C , Turpie A G G , Butt R , Genton E
Published: July 2018

Intermittent pneumatic compression (IPC) consistently prevents venous stasis by increasing venous return of the legs but fails to prevent deep vein thrombosis (DVT) in some patients, especially patients with malignancy and subarachnoid haemorrhage (SAH). This suggests a mechanism in addition to preventing stasis is required for the prophylactic effect of IPC. IPC increases fibrinolysis and this may contribute to DVT prevention. In 74 patients with SAH treated with anti fibrinolytic drugs, 21% of 42 randomized to IPC prophylaxis developed DVT and 19% of 32 controls. In 143 neurosurgical patients aged >55 years and not treated with antifibrinolytic drugs, 30% of controls (n=76) developed DVT compared to 16% treated with IPC (n=67; p>0.05), in contrast to 187 patients <55 years in whom 20% of controls (n=88) developed DVT, compared with none in the IPC group (n=99; p< 0.001). The effect of IPC on fibrinolytic activity in 80 of these patients was evaluated using a modification of the dilute blood clot lysis time (mean ± SEM, hrs). In 64 medical control patients there was a correlation between age and lysis time (r=0.33; p<0.05). In operative patients aged <55 years, lysis time was significantly shortened in patients given IPC compared with age-matched controls (6.3 + 0.6 v 8.8 + 0.8; p<0.05). In patients aged >55, there was no difference in lysis time in IPC patients compared with age-matched controls (10.2 ± 1.4 v 9.7 ± 0.8; p> 0.1). In all patients treated with antifibrinolytic drugs, lysis times were markedly prolonged. The data show that ineffectiveness of IPC in certain patient groups was related to failure of enhancement of fibrinolytic activity and this is consistent with the hypothesis that activation of fibrinolysis contributes to the prevention of DVT with IPC.
Interaction Of Fibrinogen With Platelets In Plasma
Authors: Marguerie G , Larrieu M J , Plow E F
Published: July 2018

Fibrinogen binds to specific receptors on washed human platelets and these sites are induced by ADP. This interaction is assumed to be the basis for the participation of the molecule in ADP-stimulated platelet aggregation, but fibrinogen binding to platelets in plasma has not been directly demonstrated. In this study, we have characterized the binding of 125 I-fibrinogen to platelets in the platelet rich plasma (PRP) of afibrinogene- mic patients. In either citrated or heparinized PRP, association of fibrinogen with platelets was demonstrable and was dependent on ADP dose. This binding was time dependent and reached equilibrium in 10 to 15 min. At 22°C, the rate constants of association were kon = 0.9×10-6 M-1 min-1 in plasma compared to kon = 0.6×l0-6M-1 min-1 in Tyrode’s buffer indicating that initial rate of reaction was similar in both milieu. The kinetics of binding in plasma were the same at 37°C and 22°C but fewer molecules were bound at the higher temperature. In the PRP, saturable binding of fibrinogen to the platelet was achieved at concentrations greater than 0.5 μM. A linear Scatchard plot was derived which indicated a single class of binding site with an affinity constant of Ka = 1.8×106 M-1 and 32,000 fibrinogen molecules were maximally bound per platelet. These values are essentially identical to those obtained with washed platelets. In buffer, platelet-bound fibrinogen becomes progressively nondissociab1e , and this stabilization occurred to a partial extent in plasma. In sum, these results establish that the previously defined steps in ADP-induced binding of fibrinogen to platelet occur in plasma.
ADP And Epinephrine-Induced Release Of Platelet Fibrinogen
Authors: Kaplan K L
Published: July 2018

Added fibrinogen is said to be essential for induction of platelet aggregation and release by ADP and epinephrine. Furthermore, both ADP and epinephrine induce binding of fibrinogen to the platelet surface. In the present study gel-filtered human platelets were examined to determine whether they would aggregate and release platelet fibrinogen in response to ADP or epinephrine without exogenous fibrinogen. Platelets gel-filtered into Tyrode’s buffer containing ImM Mg++, no added Ca++, and 0.35% bovine serum albumin had a fibrinogen concentration in the supernatant of less than 1 nM. After aggregation by ADP or epinephrine the fibrinogen concentration in the supernatant ranged from 11 to 41 nM. ADP and epinephrine each induced biphasic aggregation and release of platelet factor 4 and β-thromboglobulin as well as of fibrinogen. Protein release by epinephrine was coincidental with dense granule adenine nucleotide release. Because Ca++ ions affect fibrinogen binding to platelets, the effect of Ca++ on aggregation and protein release was examined and it was found that both were inhibited by added Ca++ at 1-2 mM. The ability of gel-filtered platelets to undergo ADP and epinephrine induced aggregation and release in the absence of exogenous fibrinogen suggests that released platelet fibrinogen can support these processes. The concentrations of released fibrinogen in these experiments were lower than those reported to be necessary for exogenous fibrinogen for support of aggregation, suggesting that released fibrinogen may interact more efficiently with the platelet membrane. The amount of released fibrinogen in these experiments is similar to the Kd for high-affinity fibrinogen binding reported by Niewiarowski et al. (30nM). Finally, although inhibition of ADP and epinephrine induced aggregation and release by physiologic Ca++ concentrations implies that these processes do not occur in vivo, it is likely that platelet fibrinogen released by collagen or thrombin does function physiologically.
Characterization Of The ADP-Induced Fibrinogen Btnd1Ng Sites On Human Platelets Using Photoaffinity Techniques: Some Preliminary Findings
Authors: Peerschke Ellinor I , Zucker Mariorie B , Rotman Avner
Published: July 2018

The interaction of fibrinogen with its, platelet membrane receptor was investigated using 125-labeled fibrinogen which was photoaffinity labeled with a light-sensitive azide. This photoreactive material (125I-NPA-fibr) was indistinguishable from unlabeled fibrinogen as well as from iodinated fibrinogen on SDS-PAGE. It bound specifically to platelets stimulated with ADP and was crosslinked to the platelet membrane after exposure to light ( λ >300 nm) for 4 min. No crosslinking was observed in the presence of EDTA or with platelets that failed to aggregate with ADP either due to the congenital deficiency thrombasthenia or following incubation with EDTA for 8 min at 37° , pH 7.8 and recalcification. SDS-PAGE of platelets bearing crosslinked 125I-NPA-fibr revealed a radiolabeled band of about 450,000 daltons in addition to the 340,000 dalton radioactive band of fibrinogen, suggesting that fibrinogen had been covalently bound to a platelet membrane component with an intact apparent molecular weight of approximately 110,000 daltons. Following reduction, an extra radioactive band was noted at 80,000 daltons. As the A∝-chains of fibrinogen were too weakly labeled to be detected by autoradiography, this indicated that either the Bβ or γchain of fibrinogen was attached to a 25,000-35,000 molecular weight platelet membrane fragment. We conclude that the additional radioactive bands observed after electrophoresis of platelets bearing specifically bound-photoaffinity labeled 125I-fibrinogen most likely represent the binding of the B β or γ chains of fibrinogen to the platelet fibrinogen receptor which may be GPIIb.