In Vitro Characterization Of Low Molecular Weight Fractions Of Heparin

1981 ◽  
Author(s):  
Jawed Fareed ◽  
Harry L Messmore ◽  
Daniel A Walz ◽  
Jean Choay ◽  
J C Lormeau
Keyword(s):  
Molecular Weight ◽  
The Other ◽  
Low Molecular Weight ◽  
In Vitro Test ◽  
Thrombin Time ◽  
Specific Test ◽  
Platelet Factor ◽  
At Iii ◽  
Heparin Fractions

Numerous extraction, chromatographic (ion exchange, gel, and affinity), chemical and enzymatic degradation methods have been employed to obtain heparin fractions. The present assays to evaluate potency (e.g. pharmacopeial and coagulant) do not truly reflect the antithrombotic properties of these fractions. In addition, the synthetic peptide substrate based assays to measure the anti Xa activity do not correlate with the coagulant anti Xa assays. We have developed an in vitro test battery to evaluate low molecular weight heparin fractions. Porcine mucosal heparin fractions are assayed for anti Xa activity in coagulant and amidolytic assays and the results are expressed as a ratio. The effect of these fractions on coagulant assays such as prothrombin time (PT), partial thromboplastin time (PTT), thrombin time (TT), Stypven time (ST) on freshly prepared normal human plasma (NHP) is determined The retention characteristics of these fractions on platelet factor 4 and AT-III bound sepharose columns were also determined. We have compared the extracted and chemically depolymerized heparin fractions and found that the anti Xa activity doesn’t always correlate with the other parameters studied. The extracted fractions were slightly stronger in the USP assays and showed a biphasic retention on the PF-4 column whereas the chemically depolymerized product showed only one peak. On the other hand, on the AT-III column both fractions showed similar elution patterns. Our studies suggest that heparin and its fractions exhibit differential behavior on various assays and a specific test may not be used as an index of the potency of their antithrombotic effects. Furthermore, the potency of these fractions should be stated on a weight basis when evaluated in the in vivo animal models rather than in terms of a specific test (e.g. anti Xa activity and US Pharmacopeial assays).

In Vivo Studies on the Inhibition of Coagulation by Fractionated Heparin and by a Heparin Analogue I. Effects of Heparin Fractions

1981 ◽  
Vol 46 (03) ◽  
pp. 612-616 ◽  
Author(s):  
U Schmitz-Huebner ◽  
L Balleisen ◽  
F Asbeck ◽  
J van de Loo
Keyword(s):  
Molecular Weight ◽  
Day Treatment ◽  
Gel Filtration ◽  
Weight Fraction ◽  
In Vivo Studies ◽  
Low Molecular Weight ◽  
High Molecular Weight Fraction ◽  
Platelet Factor ◽  
Heparin Fractions

SummaryHigh and low molecular weight heparin fractions obtained by gel filtration chromatography of sodium mucosal heparin were injected subcutaneously into six healthy volunteers and compared with the unfractionated substance in a cross-over trial. Equal doses of 5,000 U were administered twice daily over a period of three days and heparin activity was repeatedly controlled before and 2, 4, 8 hrs after injection by means of the APTT, the anti-Xa clotting test and a chromogenic substrate assay. In addition, the in vivo effect of subcutaneously administered fractionated heparin on platelet function was examined on three of the volunteers. The results show that s.c. injections of the low molecular weight fraction induced markedly higher anti-Xa activity than injections of the other preparations. At the same time, APTT results did not significantly differ. Unfractionated heparin and the high molecular weight fraction enhanced ADP-induced platelet aggregation and collagen-mediated MDA production, while the low molecular weight fraction hardly affected these assays, but potently inhibited thrombin-induced MDA production. All heparin preparations stimulated the release of platelet Factor 4 in plasma. During the three-day treatment periods, no side-effects and no significant changes in the response to heparin injections were detected.

scholarly journals Anticoagulant Properties of a Green Algal Rhamnan-type Sulfated Polysaccharide and Its Low-molecular-weight Fragments Prepared by Mild Acid Degradation

Marine Drugs ◽  
2018 ◽  
Vol 16 (11) ◽  
pp. 445 ◽  
Author(s):  
Xue Liu ◽  
Peng Du ◽  
Xiao Liu ◽  
Sujian Cao ◽  
Ling Qin ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Anticoagulant Activity ◽  
Sulfated Polysaccharide ◽  
Low Molecular Weight ◽  
Thrombin Time ◽  
Acid Degradation ◽  
Molecular Weights ◽  
Green Algal

The active sulfated polysaccharide from seaweed possesses important pharmaceutical and biomedical potential. In the study, Monostroma sulfated polysaccharide (MSP) was obtained from Monostroma angicava, and the low-molecular-weight fragments of MSP (MSP-Fs: MSP-F1–MSP-F6) were prepared by controlled acid degradation. The molecular weights of MSP and MSP-F1–MSP-F6 were 335 kDa, 240 kDa, 90 kDa, 40 kDa, 24 kDa, 12 kDa, and 6.8 kDa, respectively. The polysaccharides were sulfated rhamnans that consisted of →3)-α-l-Rhap-(1→ and →2)-α-l-Rhap-(1→ units with partial sulfation at C-2 of →3)-α-l-Rhap-(1→ and C-3 of →2)-α-l-Rhap-(1→. Anticoagulant properties in vitro of MSP and MSP-F1–MSP-F6 were evaluated by studying the activated partial thromboplastin time, thrombin time, and prothrombin time. Anticoagulant activities in vivo of MSP and MSP-F4 were further evaluated; their fibrin(ogen)olytic activities in vivo and thrombolytic properties in vitro were also assessed by D-dimer, fibrin degradation products, plasminogen activator inhibitior-1, and clot lytic rate assays. The results showed that MSP and MSP-F1–MSP-F4 with molecular weights of 24–240 kDa had strong anticoagulant activities. A decrease in the molecular weight of MSP-Fs was accompanied by a decrease in the anticoagulant activity, and higher anticoagulant activity requires a molecular weight of over 12 kDa. MSP and MSP-F4 possessed strong anticoagulant activities in vivo, as well as high fibrin(ogen)olytic and thrombolytic activities. MSP and MSP-F4 have potential as drug or helpful food supplements for human health.

NEUTRALIZING EFFECT OF PLATELET FACTOR 4 OR HISTIGINE-RICH GLYCOPROTEIN AGAINST LOW MOLECULAR WEIGHT HEPARIN AND UNFRACTIONATED HEPARIN

1987 ◽  
Author(s):  
K Takahashi ◽  
M Niwa ◽  
N Sakuragawa
Keyword(s):  
Molecular Weight ◽  
Low Molecular Weight Heparin ◽  
Antithrombin Iii ◽  
Platelet Factor 4 ◽  
Low Molecular Weight ◽  
Bleeding Tendency ◽  
Human Origin ◽  
Platelet Factor ◽  
At Iii ◽  
Antifactor Xa

Purpose: Low molecular weight(LMW) heparin shows stronger antifactor Xa(F-Xa) and weaker anti-thrombin(TH) activities compared with unfractionated(UF) heparin, and shows less bleeding tendency in the cases of clinical use. Platelet factor 4(Pf-4) and histidine-rich glycoprotein(HRG) neutralize heparin. We investigated on the heparin neutralizing effects of them to both kinds of heparinMaterials and methods: LMW heparin(Kabi and Pharmuka) and UF heparin(Novo) were used. Antithrombin III(AT-III), HRG(human origin ) and pf-4( bovine origin ) were purified by our methodsTH(Green-Cross) and F-Xa(Sigma) were used. Reaction mixtures for anti-TH or anti-F-Xa were as follows: 1 vol of AT-III( 0.1 U/ml)+ 1 vol of heparin( 10 ug/ml)+l vol of pf-4 or HRG(varied)→incubated for 5 min→+l vol of TH(5 U/ml) or F-Xa( 7 nKat/ml)→incubated for 5 min→ + S-2238 or S-2222→ recorded at 405 nm.Results: (1) Pf-4 showed the equivalent anti-TH effect on both kinds of heparin, and 3 ug of pf-4 neutralized 1 ug of heparinOn F-Xa neutralizing effect, 13 ug of pf-4 neutralized 1 ug of UF heparin, but could not neutralize LMW heparin. (2) HRG showed the same results on anti-TH effect of both kinds of heparin, but could not neutralize the anti-F-Xa effect of LMW heparin on the same amount of HRG which neutralized that of UF heparin. Conclusion: Anti-F-Xa effect of. LMW heparin could not be easily neutralized by pf-4 or HRG compared with that of UF heparin.

EFFECT OF VARIOUS HEPARIN(OID)S ON HEPARIN COFACTOR II MEDIATED ANTI-THROMBIN ACTIVITY AND INHIBITION OF THROMBIN GENERATION IN VITRO

1987 ◽  
Author(s):  
T G van Dinther ◽  
F Hol ◽  
D G Meuleman
Keyword(s):  
Molecular Weight ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Weight Fraction ◽  
Blood Platelet ◽  
Low Molecular Weight ◽  
Heparin Cofactor Ii ◽  
Thrombin Activity ◽  
At Iii

The effects of various heparin(oid)s, standard heparin VII (SH), dermatan sulphate (DS), a low molecular weight fraction of heparin (UMW-H), FragminR (FRA), Org 10172 = low molecular weight heparinoid, the fraction of Org 10172 with high affinity for AT-III (HA-10172) and the low affinity fraction (LA-10172) respectively were examined on in vitro thrombin generation and inactivation.Thrombin inactivation in the presence of either heparin cofactor II (HC-II) or anti-thrombin III (AT-III) was assessed with two newly developed assays using the purified cofactors, thrombin and chromogenic substrate S2238 on microtiterplates. Thrombin generation in the presence of HC-II and AT-III was studied using purified factor Xa, prothrombin and blood platelet lysate and the residual thrombin activity was assessed amidolytically.The inhibition of the compounds on thrombin activity are summarized in the tableThe following conclusions can be drawn:- SH, LMW-H, HA-10172 and FRA potentiate the AT-III mediated inactivation of Ha more strongly than the HC-II mediated inactivation.- DS and LA-10172 show the reverse pattern of inactivation, while Org 10172 potentiates both inactivaton pathways to a similar extent.Thrombin generation in the presence of HC-II is inhibited by mw-heparin(oid)s at approx. 2-5 times lower concentrations than the HC-II mediated thrombin inactivation, while the inhibiting effect of SH in both assays is comparable.AT-III mediated thrombin generation inhibition and AT-III mediated thrombin inactivation is comparable as well for SH, LMW-H and FRA. In contrast, Org 10172 and its subfractions are approx. 10 times more potent on AT-III mediated thrombin generation inhibition than on AT-III mediated thrombin inactivation.Org 10172 shows low anti-thrombin activity and this activity is mainly mediated via FC-II.

Comparative Studies on An Anti-Xa Enriched Ultra Low Molecular Weight Heparin with Bemiparin

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4394-4394
Author(s):  
Debra Hoppensteadt ◽  
Angel Gray ◽  
Josephine Cunanan ◽  
Walter Jeske ◽  
Jeanine M. Walenga ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Platelet Aggregation ◽  
Whole Blood ◽  
Anticoagulant Activity ◽  
The Other ◽  
Positive Interactions ◽  
Low Molecular Weight ◽  
Significant Activity ◽  
Thrombin Time ◽  
Low Molecular Weight Heparins

Abstract Abstract 4394 Most low molecular weight heparins (LMWHs) have a mean molecular weight in the range of 4–6 kDa and anti-Xa/IIa ratios of 3–6. Further depolymerization of porcine mucosal heparin results in the generation of Ultra low molecular weight heparins (ULMWHs) with a molecular weight range of 2–4 kDa with proportionately decreased anti-Xa and anti-IIa activities. Bemiparin (Rovi, Madrid, Spain) represents one such ULMWH. AVE 5026 (Sanofi-Aventis, Paris, France) is a unique ULMWH (2.5 kDa) which exhibits higher affinity to antithrombin (AT) and therefore, enhanced anti-Xa activity. Because of the compositional differences between these two agents, it was hypothesized that each of these agents will have distinct anticoagulant, antiprotease and thrombin generation effects. Each of these agents was supplemented to native whole blood. Anticoagulant activity was measured using ACT, TEG, PT, APTT, thrombin time and Heptest assays. Similar studies were carried out in plasma. Amidolytic assays were used to determine the anti-Xa and anti-IIa activities. Both agents were also tested for the interactions with heparin cofactor II (HC II) and AT and were compared in the HIT antibody screening assay using platelet aggregation. In whole blood clotting assays bemiparin showed a strong anticoagulant activity in comparison to AVE 5026. Both agents also exhibited assay dependent differences in the APTT, heptest and thrombin time assays. AVE 5026 exhibited a higher anticoagulant activity in the heptest whereas bemiparin showed a stronger anticoagulant effect in the other clot based assays. In the amidolytic anti-Xa assay, AVE 5026 showed an activity of 156U/mg compared to 86 U/mg for bemiparin. In the anti-IIa assay bemiparin showed a higher activity (10 U/mg) in comparison to AVE 5026 (3.2 U/mg). The calculated Xa/IIa ratio of AVE 5026 was > 48, whereas it was 8.6 for bemiparin. While bemiparin exhibited interactions with HC II, AVE 5026 did not show significant activity in the tested concentrations (anti-IIa – IC50: 1.10±.45 μ M and >3.44±.00 μ M, respectively). On the other hand, AVE 5026 exhibited stronger interactions with AT in comparison to bemiparin (anti-FXA – IC50: .223±.03 μ M and .894±.06 μ M, respectively). Interestingly, heparinase digestion of the two products resulted in a complete loss of anti-IIa activity, but residual anti-Xa activity was found. AVE 5026 exhibited stronger anti-Xa interactions even after heparinase digestion. In the heparin induced platelet aggregation assay at 2.5 μ g/ml, bemiparin showed a relatively higher prevalence of positive interactions with HIT antibodies, whereas AVE 5026 showed a much lower prevalence (slope; AVE 5026 compared bemiparin, p=0.012). Bemiparin exhibited greater platelet factor 4 neutralization in comparison to AVE 5026. These studies clearly demonstrate that while bemiparin behaves like a typical ULMWH, AVE 5026 behaves differently in the different assays. Moreover, the oligosaccharide composition of the two products, in terms of distribution profile structure, is also different. Therefore, AVE 5026 does not represent a typical depolymerized ULMWH and is expected to exhibit a distinct pharmacologic and clinical profile. Disclosures: Hoppensteadt: Sanofi-Aventis: Research Funding.

Platelet Aggregation Induced In Vivo By Heparin And Heparin Fractions

1981 ◽  
Author(s):  
M Silane ◽  
J N Lindon ◽  
B J Ransil ◽  
R D Rosenberg ◽  
E W Salzman
Keyword(s):  
Molecular Weight ◽  
Platelet Aggregation ◽  
Reciprocal Relationship ◽  
Low Molecular Weight ◽  
Arterial Blood ◽  
Platelet Aggregate ◽  
Heparin Fractions ◽  
Platelet Aggregates

As we have reported, heparin-induced platelet aggregation in vitro varies among heparin subfractions, being generally less with lower molecular weights and having a reciprocal relationship with antithrombin affinity.We now have studied heparin-induced platelet aggregates in vivo by the technique of Wu and Hoak using arterial blood from unanesthetized rabbits. Porcine mucosal heparin was fractionated by gel filtration into high molecular weight (ave. 15,000 Daltons) or low molecular weight (ave. 6,000 Daltons) preparations. IV administration of commercial porcine mucosal heparin (spec. act. 150 u/mg) or high (spec. act. 183 u/mg) or low (spec. act. 208 u/mg) molecular weight fractions was followed by an increase in the platelet aggregate ratio compared with preinjection control values. The rise in platelet aggregate ratio with heparin was significantly different from the effect of a saline placebo (n=8) but was not significantly different among rabbits receiving the commercial heparin (n=9) or the high (n=8) or low (n=8) molecular weight preparations. Peak rise in circulating aggregate ratio occurred 2 minutes after the injection, and values returned to control levels within 15 to 30 minutes. There was no change in platelet count in blood collected in EDTA, suggesting that the aggregates were not removed from the circulation in vivo.Heparin fractions of low molecular weight were further separated according to antithrombin affinity by an antithrombin binding technique. In 8 rabbits low molecular weight/high antithrombin affinity heparin (spec. act. 480 u/mg) did not cause formation of platelet aggregates. The results were significantly different from those with commercial heparin (p=0.05) or with the other heparin fractions (p=0.06).Clinical use of low molecular weight heparin of high antithrombin affinity may lead to fewer heparin-induced platelet effects and to an improvement in anticoagulant therapy.

A Comparative Study Of Heparin And Heparin Fractions In Preventing Experimental Venous Thrombosis

1981 ◽  
Author(s):  
D P Thomas ◽  
T W Barrowcliffe ◽  
U Lindahl ◽  
L Thunberg ◽  
R E Merton ◽  
...  
Keyword(s):  
Venous Thrombosis ◽  
Specific Activity ◽  
Binding Capacity ◽  
Weight Basis ◽  
Blood Levels ◽  
Thrombin Time ◽  
High Affinity ◽  
At Iii ◽  
Heparin Fractions

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.

DIFFERENT LOW MOLECULAR WEIGHT HEPARINS (LMWHs) ARE NOT IDENTICAL IN THE IN VITRO SCREENING REGARDLESS OF POTENCY ADJUSTMENT

1987 ◽  
Author(s):  
D Hoppensteadt ◽  
J Fareed ◽  
J M Walenga ◽  
R M Emnuele ◽  
A Racanelli
Keyword(s):  
Molecular Weight ◽  
Test System ◽  
Low Molecular Weight ◽  
In Vitro Test ◽  
Low Molecular Weight Heparins ◽  
Relative Safety ◽  
Considerable Controversy ◽  
Vitro Test ◽  
Compositional Variations

There is considerable controversy in the standardization and potency evaluation of low molecular weight heparins (LMWHs).Since these agents are produced by fractionation or depolymerization procedures, the resulting material shows marked compositional variations regardless of the similarity in molecular weight. In order to address the question “are different LMWHs the same?”, we utilized a uniform multiparametric in vitro test battery. Seven commercial LMWHs Choay Fraxiparine (CY 216), Choay CY 222, Novo LHN, Kabi Fragmin, Opocrin 2123 (OP), Hepar RD 11885 (RD), Pharmuka Enoxaparine (PK) and an unfractionated porcine mucosal heparin (PMH) were studied at equigravimetric amounts in established assays. PMH and LMWHs were ranked acdording to the dose-dependent effects and the results are summarized in the following:As evident from the above data, each of the agents studied showed a distinct profile in the in vitro test system studied. This data suggests that LMWHs are individual drugs with marked differences in in vitro actions, which may not be the true determinants of the relative safety/efficacy in clinical settings.

scholarly journals The metabolic heterogeneity of rabbit ear cartilage chondroitin sulphate

1970 ◽  
Vol 116 (3) ◽  
pp. 329-336 ◽  
Author(s):  
J. Peter Bentley ◽  
Bohumila Rokosová
Keyword(s):  
Molecular Weight ◽  
Chondroitin Sulphate ◽  
The Other ◽  
Low Molecular Weight ◽  
Rabbit Ear ◽  
Ear Cartilage ◽  
Metabolic Heterogeneity ◽  
Papain Digestion

The only glycosaminoglycans that can be isolated from the ear cartilage of 2-month-old rabbits are chondroitin 4-sulphate and chondroitin 6-sulphate. These chondroitin sulphates exhibit molecular-weight polydispersity when isolated from tissue by papain digestion. The chondroitin sulphate is metabolically heterogeneous in that radioactive precursors [14C]glucose or [35S]sulphate are preferentially incorporated into the higher-molecular-weight polymers both in vivo and in vitro. No transfer of radioactivity from the high-molecular-weight chondroitin sulphate to the low-molecular-weight chondroitin sulphate was seen during 15 days in vivo. It is suggested that there are at least two pools of proteoglycan in the tissue. One of these pools is metabolically active whereas the other is not.

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