Low Molecular Weight Heparins: Anti-Xa/Aptt And Platelet Aggregation

1981 ◽  
Author(s):  
F Fussi ◽  
M R Smith ◽  
A Girolami ◽  
L Visentini ◽  
F Fabris
Keyword(s):  
Molecular Weight ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Significant Inhibition ◽  
Significant Rise ◽  
Low Molecular Weight ◽  
Inhibiting Activity ◽  
Low Molecular Weight Heparins ◽  
Platelet Release ◽  
Mean Molecular Weight

Porcine mucosal Heparin (mean molecular weight= 15.000 Dalton) has been chemically depolymerized in presence of peroxides and N-sulphate groups have been re-introduced by reaction with sulpho- trioxides of amines. The depolymerization has been stopped at different times and the products are essayed for Anti-Xa, APTT and their ratio. A rise in the ratio Anti-Xa/APTT has been observed.In order to check for an activity on the platelet aggregation, two sets of experiments have been carried out: in a first model, the 5-hydroxytrip- tamine (5-HT) release has been measured after addition of different quantities of products to washed platelets. One commercil Heparin gave a significant rise in 5-HT release whereas a normal porcine mucosal Heparin did not show any modification in basal values, nor significant modifications have been observed for low molecular weight heparins (10.000 to 7.000 D). Heparansulphate (HS) and Dermatansulphate (DS) show a slight anti-aggregating activity in this test.In a second model, the percentage inhibition in collagen induced platelet release has been measured in a platelet rich plasma (PRP). All the tested heparins show a very significant inhibition, whereas HS and DS have no inhibiting activity in this test.

scholarly journals Heparin-induced thrombocytopenia: studies with a new low molecular weight heparinoid, Org 10172

Blood ◽  
1989 ◽  
Vol 73 (6) ◽  
pp. 1592-1596
Author(s):  
BH Chong ◽  
F Ismail ◽  
J Cade ◽  
AS Gallus ◽  
S Gordon ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Reaction Rate ◽  
Platelet Rich Plasma ◽  
Inhibitory Effect ◽  
Cross Reaction ◽  
Low Molecular Weight ◽  
Heparin Induced Thrombocytopenia ◽  
Normal Platelet

Studies were performed to determine the cross-reaction rate of the heparin-dependent antibody with Org 10172, a new low molecular weight heparinoid, and to investigate the effect of Org 10172 on platelet activation induced by the antibody. The plasmas of 17 patients with thrombocytopenia induced by standard heparin were shown, by platelet aggregation studies, to contain the heparin-dependent antibody. Of these 17 patient plasmas, only three cross-reacted with the heparinoid, producing a cross-reaction rate of 18%. When Org 10172 was added to a reaction mixture containing normal platelet-rich plasma, patient plasma, and standard heparin with non-cross-reacting plasmas, it inhibited platelet aggregation and thromboxane B2 production induced by the antibody, provided that the ratio of Org 10172 concentration (anti- Xa U/mL) to standard heparin concentration (IU/mL) exceeded 2.5 to 5.0. This inhibitory effect was observed only with platelet activation mediated by the antibody, but not by collagen (2 micrograms/mL) or ADP (5.0 mumol/L). Additionally, three of 17 patients with serious thrombosis, whose plasma showed no cross-reaction with the heparinoid, received Org 10172 treatment with a good response in each case. These findings suggest that Org 10172 may be a useful drug for the treatment of heparin-induced thrombocytopenia.

In Vivo Effects of a Low Molecular Weight Heparin Fragment on Platelet Aggregation and Platelet Dependent Hemostasis in Dogs

1988 ◽  
Vol 60 (02) ◽  
pp. 232-235 ◽  
Author(s):  
B Ljungberg ◽  
H Johnsson
Keyword(s):  
Molecular Weight ◽  
Platelet Aggregation ◽  
Low Molecular Weight Heparin ◽  
Platelet Rich Plasma ◽  
Skin Flap ◽  
Low Molecular Weight ◽  
Dog Plasma ◽  
In Vivo Effects ◽  
Induced Aggregation

SummaryPlasma defibrinogenated dogs were used to study the influence of conventional heparin and a low molecular weight heparin fragment (Fragmin®, mean MW 5,000 d) on platelet dependent hemostasis. The heparins were given intravenously in gravimetri- cafly equal doses. The bleeding from standardized skin flap wounds and platelet aggregation (ADP and thrombin) was studied. In comparison, higher doses of the fragment than of heparin were required to increase the bleeding. ADP-induced aggregation in defibrinogenated platelet rich plasma (after addition of normal dog plasma) was potentiated by both heparins. After injection of heparin or the fragment, ADP induced platelet aggregation without prior addition of normal plasma to the testtube.In conclusion the heparin fragment affected bleeding to a less extent than conventional heparin. One explanation might be a weaker inhibition of thrombin-induced platelet aggregation.

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.

Lack of In Vitro Cross-Reactivity Predicts Safety of Low-Molecular Weight Heparins in Heparin-Induced Thrombocytopenia

1997 ◽  
Vol 3 (1) ◽  
pp. 58-62 ◽  
Author(s):  
Sherif S. Farag ◽  
Heten Savoia ◽  
Cindy J. O'Malley ◽  
Katherine M. McGrath
Keyword(s):  
Molecular Weight ◽  
Platelet Count ◽  
Platelet Aggregation ◽  
Unfractionated Heparin ◽  
Cross Reactivity ◽  
Low Molecular Weight ◽  
Low Molecular Weight Heparins ◽  
Aggregation Assay ◽  
Heparin Induced Thrombocytopenia

Alternative anticoagulation in patients with heparin-induced thrombocytopenia (HIT) is often problematic. The relatively high cross-reactivity rate reported for the low-molecular-weight heparins (LMWH) has discouraged their use in this setting. This study has investigated the safety of using the LMWH Fragmin, based on a negative heparin-dependent platelet aggregation test using the latter, in patients with proven HIT. Fifty-three evaluable patients with clinical and laboratory evidence of HIT were evaluated for cross-reactivity with Fragmin using a Fragmin-dependent platelet aggregation test. In 20 of 38 patients who showed no in vitro cross-reactivity. Fragmin was substituted for unfractionated heparin. The outcome of these 20 patients was evaluated and compared to that of the remaining 33 patients, in whom anticoagulates were ceased or warfarin or Orgaran was used. Eighteen of 20 patients treated with Fragmin increased their platelet count by ≥50 x 109/l from a mean nadir of 57.9 ± 4.7 x 109/l within 2.8 ± 0.29 days following substitution of Fragmin for unfractionated heparin. Twenty-eight of the 33 remaining patients who did not receive Fragmin increased their platelet count by ≥50 x 109/l from a mean nadir of 53.0 ± 4.8 x 109/l within 3.0 ± 0.29 days. In seven patients (two treated with Fragmin), response could not be evaluated due to death within 36 h of cessation of heparin or discharge from hospital. The results indicate that in vitro cross-reactivity testing employing a heparin-dependent platelet aggregation assay can be safely used to select patients with HIT for further anticoagulation with LMWH. Key Words: Fragmin—Crossreactivity—Heparin-induced thrombocytopenia.

scholarly journals Heparin-induced thrombocytopenia: studies with a new low molecular weight heparinoid, Org 10172

Blood ◽  
1989 ◽  
Vol 73 (6) ◽  
pp. 1592-1596 ◽  
Author(s):  
BH Chong ◽  
F Ismail ◽  
J Cade ◽  
AS Gallus ◽  
S Gordon ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Reaction Rate ◽  
Platelet Rich Plasma ◽  
Inhibitory Effect ◽  
Cross Reaction ◽  
Low Molecular Weight ◽  
Heparin Induced Thrombocytopenia ◽  
Normal Platelet

Abstract Studies were performed to determine the cross-reaction rate of the heparin-dependent antibody with Org 10172, a new low molecular weight heparinoid, and to investigate the effect of Org 10172 on platelet activation induced by the antibody. The plasmas of 17 patients with thrombocytopenia induced by standard heparin were shown, by platelet aggregation studies, to contain the heparin-dependent antibody. Of these 17 patient plasmas, only three cross-reacted with the heparinoid, producing a cross-reaction rate of 18%. When Org 10172 was added to a reaction mixture containing normal platelet-rich plasma, patient plasma, and standard heparin with non-cross-reacting plasmas, it inhibited platelet aggregation and thromboxane B2 production induced by the antibody, provided that the ratio of Org 10172 concentration (anti- Xa U/mL) to standard heparin concentration (IU/mL) exceeded 2.5 to 5.0. This inhibitory effect was observed only with platelet activation mediated by the antibody, but not by collagen (2 micrograms/mL) or ADP (5.0 mumol/L). Additionally, three of 17 patients with serious thrombosis, whose plasma showed no cross-reaction with the heparinoid, received Org 10172 treatment with a good response in each case. These findings suggest that Org 10172 may be a useful drug for the treatment of heparin-induced thrombocytopenia.

Biological Activities of Two Ultra-Low Molecular Weight Heparins, Semuloparin and Bemiparin, Reveal That a Common Potency Standard Is Inadequate

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4324-4324
Author(s):  
Walter Jeske ◽  
Debra Hoppensteadt ◽  
Angel Gray ◽  
Jeanine M. Walenga ◽  
Josephine Cunanan ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Platelet Aggregation ◽  
Confidence Interval ◽  
Thrombin Generation ◽  
Anticoagulant Activity ◽  
Serotonin Release ◽  
Low Molecular Weight ◽  
Low Molecular Weight Heparins ◽  
Safety And Efficacy ◽  
Response Data

Abstract Abstract 4324 Introduction: Ultra-low molecular weight heparins (U-LMWHs) are being developed to improve the safety and efficacy of antithrombotic therapy. Two of these agents, bemiparin (Rovi, Madrid, Spain) and semuloparin (Sanofi-Aventis, Paris, France), are produced by distinct methods. Bemiparin is produced by esterification of porcine mucosal heparin followed by alkaline hydrolysis and fractionation. Semuloparin is prepared by a highly selective depolymerization reaction using a phosphazene base which preserves the AT binding sequences from destruction. While both bemiparin and semuloparin are effective at reducing the incidence of post-surgical VTE, some biological differences have been observed in humans. The objective of this investigation was to determine whether a common standard could be used to define their potency. Methods: Activities were compared using typical clinical coagulation assays and pharmacological assays required for potency assessment. Anticoagulant activity was assessed using aPTT and ACT assays. Anti-Xa and anti-IIa activities were determined using amidolytic assays. Constrained and unconstrained curves were determined for the anti-Xa and anti-IIa data to assess parallelism of the concentration-response curves. Thrombin generation was measured using a fluorometric substrate kinetic method (Technothrombin TGA assay; Technoclone GmbH, Vienna, Austria). Platelet function was assessed using platelet aggregometry and the serotonin release assay (SRA). Results: A significantly larger prolongation of the aPTT was observed with bemiparin at concentrations >1 μ g/ml. Differences in anticoagulant activity measured by the Heptest assay were not observed at concentrations below 2.5 μ g/ml. When supplemented to whole blood samples at higher concentrations, bemiparin and semuloparin are able to prolong the kaolin ACT. Anti-FXa activity for bemiparin and semuloparin was the same over the concentration range tested. Bemiparin produced more anti-FIIa activity at each concentration tested. Depletion of AT led to a complete loss of anti-FXa and anti-FIIa activities for both agents while depletion of heparin cofactor-II had minimal impact on anti-IIa activity. Parallelism assessment is used to identify similarity or difference of products in biological assays. The constrained and unconstrained models using anti-FXa concentration-response data were equivalent (p=0.422), indicating that bemiparin and semuloparin were equivalent (IC50 ratio = 0.97, confidence interval= 0.95–0.98, CV = 0.7%). In contrast, the constrained and unconstrained models using anti-FIIa concentration-response data were not equivalent (p<0.001), indicating that the in vitro anti-FIIa activities of the two agents is not equivalent (IC50 ratio = 1.25, confidence interval = 1.17–1.34, CV = 3.4%). At clinically relevant concentrations, bemiparin more effectively inhibited thrombin generation than semuloparin. Platelet aggregation in response to collagen, ADP and arachidonic acid was not affected by either agent at concentrations up to 10 μ g/ml. At a concentration of 5 μ g/ml, bemiparin was able to completely inhibit thrombin-induced aggregation (0.5 U/ml), whereas no effect was observed with semuloparin. Cross-reactivity of bemiparin and semuloparin with anti-PF4/heparin HIT antibodies was tested using platelet aggregation and the SRA. The level of maximal aggregation was comparable in the presence of bemiparin and semuloparin, however, the rate of aggregation was significantly slower in the presence of semuloparin compared to bemiparin. The typical bell-shaped concentration-response curve was observed with both drugs in the SRA. Although the peak release was comparable for the two drugs (65.7 ± 16.0% for semuloparin vs. 75.5 ± 6.6% for bemiparin), the peak serotonin release was observed at lower concentrations for bemiparin (1 μ g/ml) than for semuloparin (10 μ g/ml) suggesting a higher sensitivity of bemiparin to (stronger interaction with) the anti-PF4/heparin antibodies. Conclusions: These data demonstrate that the molecular profiling of heparin-derived agents is insufficient to determine drug class. Additional structural characterization and multiple biologic activities relevant to clinical safety and efficacy need to be considered as well. As such, the use of a common reference standard for potency determination of ULMWH is not valid. Disclosures: Rigal: Sanofi-Aventis: Employment. Bayol:Sanofi-Aventis: Employment. Viskov:Sanofi-Aventis: Employment.

THE ACTION OF HEPARIN IN PLASMA

1987 ◽  
Author(s):  
H Coenraad Hemker
Keyword(s):  
Molecular Weight ◽  
Platelet Rich Plasma ◽  
Cooperative Effect ◽  
Low Molecular Weight ◽  
Antithrombotic Drugs ◽  
Clotting Factors ◽  
Low Molecular Weight Heparins ◽  
Platelet Factor ◽  
P Type ◽  
Thrombin Formation

Much is known about the influence of heparin on the interaction between coagulation proteases and antithrombin III. In the complex milieu of clotting plasma and a fortiori in platelet rich plasma many proteins compete for the heparin, the proteases are partly protected from AT III (-heparin) action and several antiproteases compete for one protease. Also the concentrations of the reactant vary in the course of the clotting process. This makes it difficult to obtain a clear insight in the action of heparin in plasma from studies on purified system only. We therefore developed a method that allows us a) to measure the breakdown constant of endogenously generated thrombin in plasma and to determine what part of it is due to bindingα2 macroglobulin, b) derive the time course of prothrombinase activity from the thrombin generation curve. This method allows us to study separately the action of heparin on prothrombinase and on thrombin.Using this method we could show that: a) Classical heparin acts primarily on thrombin. Its action on other activated clotting factors is of secondary importance,b) The product activation of thrombin isespecially important in the intrinsic pathway, the thrombin dependent activation of factor VIII is the process that determines the onset of explosive thrombin formation. This explains why the APTT is much more sensitive to heparin than the PT is. c) The low molecular weight heparins that we. studied can be divided into two classes: the S type heparins, that, like unfractionated heparin, act practically only on thrombin and the P type heparins that do not act on thrombin but do inhibit prothrombinase. The synthetic pentasaccharide of Choay et al. is a typical example ofthis last group, d) We demonstrated a cooperative effect between thromboplastin and platelets that is mediated via thrombin.It is expressed as a shortening of the agphase of thrombin formation in plateletrich plasma, e) Heparin, but also other antithrombotic drugs inhibit the cooperative effect mentioned above. This suggests that this effect is a good candidate for the screening of antithrombotic drugs, f) The amount of thrombin formed in platelet rich plasma is hardly influenced by concentration of heparin that completely inhibits the formation of thrombin in platelet poor plasma. This is caused by the release of platelet factor 4. g) S type low molecular weight heparins can be conveniently standardized with a standard of classical heparin. P type low molecular weight heparins require a P type standard preparation.

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