Effects of unfractionated heparin, dermatan sulfate and low molecular weight heparin on vessel wall permeability in rabbits.
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Animals↗
Biomedical subjects
F A Ofosu
Publications and source records attributed to F A Ofosu.
Effects of dermatan sulfate and heparin on inhibition of thrombus growth in vivo.
The effects of dermatan sulfate and heparin on inhibition of fibrin accretion onto existing thrombi as related to their ex vivo anticoagulant activity and abilities to inhibit increased prothrombin clearance induced by thrombi were investigated. Our results indicate that for equivalent anti-thrombin activities, dermatan sulfate is a more effective inhibitor of fibrin accretion onto existing thrombi than is heparin. These observations raise the possibility that in some clinical conditions dermatan sulfate, rather than heparin, may be a better antithrombotic agent of choice. This beneficial effect of dermatan sulfate appears to be unrelated to anti-factor Xa activity either endogenous to dermatan sulfate itself (which is unlikely since it does not catalyze factor Xa inhibition) or to anti-factor Xa activity associated with other glycosaminoglycans released into the circulation following dermatan sulfate administration since this activity is less than that associated with heparin treatment. It is more likely that dermatan sulfate mediates this beneficial effect by more effectively inhibiting thrombin within a thrombus than can heparin. This possibility is supported by the ability of dermatan sulfate to normalize prothrombin consumption in animals with existing thrombi.
Animals↗
Anticoagulant effects of heparin in neonatal plasma.
Available data on the anticoagulant effects of heparin in neonatal plasma are scarce and conflicting: relative to adult plasma, neonatal plasma has been reported to show both resistance as well as sensitivity to heparin. We explored this apparent paradox by comparing how well heparin accelerated inhibition of exogenous thrombin and prevented thrombin generation in defibrinated neonatal and adult plasmas. Using amidolytic assays, we determined the effects of heparin on 1) the neutralization of exogenous human alpha-thrombin and on 2) the formation of endogenous thrombin activity after contact activation and recalcification. Neonatal plasma proved resistant to heparin (0.05 U/mL) during inhibition of added thrombin (15 NIH U/mL). Inhibition of thrombin in heparinized neonatal plasma became as efficient as in adult plasma only after raising the AT III activity to normal adult values. However, de novo generation of thrombin activity was very susceptible to inhibition by heparin, even in neonatal plasmas with physiologically low AT III levels. Peak thrombin activity generated in neonatal plasma in the absence of heparin was 50% or less of peak adult activity, and this already reduced ability of neonatal plasma to generate thrombin activity upon prothrombin activation was further decreased by heparin (0.05-0.2 U/mL). We conclude that due to the neonatal AT III deficiency, added thrombin is inactivated less effectively by heparin in neonatal than in normal adult plasma. Yet, the generation of thrombin activity is impaired in neonatal plasma and easily suppressed by heparin. We speculate that newborn infants may be resistant to heparin therapy during overt thrombotic disease, when neutralization of abnormal thrombin activity is the therapeutic goal.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
An approach to assigning in vitro potency to unfractionated and low molecular weight heparins based on the inhibition of prothrombin activation and catalysis of thrombin inhibition.
Unfractionated and low molecular weight (LMW) heparins with good antithrombotic activity invariably catalyze thrombin inhibition and inhibit the appearance of thrombin activity in contact-activated plasma. Conversely, the antithrombotic efficacy of LMW heparins decreases as their ability to catalyze thrombin inhibition and to inhibit the appearance of thrombin activity in plasma decrease. The activated partial thromboplastin time (APTT) has proven a reliable test for assaying unfractionated heparin. We therefore compared 2 unfractionated and 3 LMW heparins on the basis of the minimum concentrations required to double the APTT of normal plasma and by then determined how this anticoagulant effect was achieved. The amount of unfractionated and LMW heparin which doubled the APTT was found to be equivalent to approximately 0.25 antithrombin units. This concentration of each glycosaminoglycan completely inhibited prothrombin activation for 45 s after CaCl2 was added to contact-activated plasma; accelerated thrombin inhibition by purified antithrombin III by approximately 50-fold; and accelerated thrombin inhibition equally by antithrombin III in undiluted plasma. This concentration of the three LMW heparins increased, by approximately 70-fold, the rate of factor Xa inhibition by purified antithrombin III compared to the 50-fold increase seen with the two unfractionated heparins. These results thus suggest that tests based on the inhibition of prothrombin activation and/or on the catalysis of thrombin inhibition provide a useful basis for assigning in vitro potency to both unfractionated and LMW heparins.
Catalysis↗
Further studies on the mechanisms for the antithrombotic effects of sulfated polysaccharides in rabbits.
A recent study (Fernandez et al., Thromb. Haemostas. 1987; 57: 286-93) demonstrated that when rabbits were injected with the minimum weight of a variety of glycosaminoglycans required to inhibit tissue factor-induced thrombus formation by approximately 80%, exogenous thrombin was inactivated approximately twice as fast in the post-treatment plasmas as the pre-treatment plasmas. In this study, we investigated the relationship between inhibition of thrombus formation and the extent of thrombin inhibition ex vivo. We also investigated the relationship between inhibition of thrombus formation and inhibition of prothrombin activation ex vivo. Four sulfated polysaccharides (SPS) which influence coagulation in a variety of ways were used in this study. Unfractionated heparin and the fraction of heparin with high affinity to antithrombin III potentiate the antiproteinase activity of antithrombin III. Pentosan polysulfate potentiates the activity of heparin cofactor II. At less than 10 micrograms/ml of plasma, all three SPS also inhibit intrinsic prothrombin activation. The fourth agent, dermatan sulfate, potentiates the activity of heparin cofactor II but fails to inhibit intrinsic prothrombin activation even at concentrations which exceed 60 micrograms/ml of plasma. Inhibition of thrombus formation by each sulfated polysaccharides was linearly related to the extent of thrombin inhibition achieved ex vivo. These observations confirm the utility of catalysis of thrombin inhibition as an index for assessing antithrombotic potential of glycosaminoglycans and other sulfated polysaccharides in rabbits. With the exception of pentosan polysulfate, there was no clear relationship between inhibition of thrombus formation and inhibition of prothrombin activation ex vivo.
Animals↗
Mechanisms of action of heparin: applications to the development of derivatives of heparin and heparinoids with antithrombotic properties.
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Heparin↗
Mechanisms for the anticoagulant effect of heparin and related polysaccharides.
The anticoagulant actions of heparin and related polysaccharides can best be addressed by answering the following 2 questions: to what extent do unfractionated heparin, low molecular weight heparins, dermatan sulfate and heparan sulfate inhibit the activation of prothrombin in plasma depleted of antithrombin III and heparin cofactor II? What roles do antithrombin III and heparin cofactor II play in the expression of the overall anticoagulant effects of heparin, low molecular weight heparins, dermatan sulfate and heparan sulfate? Since only heparin can, but only weakly at best, inhibit the activation of prothrombin in plasma depleted of both antithrombin III and heparin cofactor II, it is obvious that the anticoagulant effects of heparin, low molecular weight heparins, dermatan sulfate and heparan sulfate are mediated primarily by their catalytic effects on the antiprotease actions of antithrombin III (heparin, low molecular weight heparins and heparan sulfate) and heparin cofactor II (dermatan sulfate). The catalytic efficiencies of various glycosaminoglycans (GAGs) on the inhibition of thrombin by undiluted plasma follow in the order of the ability of each GAG to inhibit the intrinsic pathway activation of prothrombin. The reasons for this observation probably follow from the effects of GAGs on the 2 positive amplification reactions of thrombin during blood coagulation. Factor VIIIa and factor Va, which directly or indirectly contribute to the rapid formation of prothrombinase, arise from the limited proteolysis of factor VIII and factor V by thrombin and/or factor Xa. On depletion of thrombin by enhanced formation of thrombin-antithrombin III or thrombin-heparin cofactor II, factor Xa provides the only mechanism by which factor VIII and factor V activation in plasma will proceed.(ABSTRACT TRUNCATED AT 250 WORDS)
Anticoagulants↗
Antithrombin-III-Hamilton: a gene with a point mutation (guanine to adenine) in codon 382 causing impaired serine protease reactivity.
Antithrombin-III-Hamilton is a structural mutant of antithrombin III with defective serine protease reactivity, demonstrable in three members of a French Canadian family. The propositus, a 54-year-old man with a history of recurrent thromboembolic events, and his two asymptomatic grown children are heterozygous for the mutant antithrombin III gene. In all three individuals, the immunoreactive antithrombin III level is normal, while the antithrombin and antifactor Xa activity is approximately 50% of the control value. Two dimensional immunoelectrophoresis of antithrombin-III-Hamilton in the presence of heparin is normal. Purified antithrombin-III-Hamilton did not form thrombin-antithrombin III complex when incubated with thrombin for up to 30 minutes. The normal and mutant antithrombin III alleles of the propositus could be distinguished by linkage to Pstl restriction fragment length polymorphisms (RFLP). Genomic DNA from the propositus was cloned into EMBL 3 phage vectors and two clones containing nearly complete copies of the antithrombin-III-Hamilton allele were identified. Exon 6 of both clones was subcloned into M13 phage vector and sequenced, revealing a G----A point mutation in the first base of codon 382. Codon 382 codes for alanine in the normal allele and for threonine in the antithrombin-III-Hamilton allele. Alanine-382, 12 residues from the reactive center, is a highly conserved amino acid in the family of serine protease inhibitors known as the serpins. We postulate that, as a result of the substitution of threonine for alanine in antithrombin-III-Hamilton, either the tertiary structure or the hydrophobicity of the thrombin-binding region is altered, causing aberrant conformation of the Arg-393-Ser-394 bond at the reactive center impairing the interaction between antithrombin-III-Hamilton and the activated serine proteases.
Alleles↗
Increased sulphation improves the anticoagulant activities of heparan sulphate and dermatan sulphate.
Heparan sulphate and dermatan sulphate have both antithrombotic and anticoagulant properties. These are, however, significantly weaker than those of a comparable amount of standard pig mucosal heparin. Antithrombotic and anticoagulant effects of glycosaminoglycans depend on their ability to catalyse the inhibition of thrombin and/or to inhibit the activation of prothrombin. Since heparan sulphate and dermatan sulphate are less sulphated than unfractionated heparin, we investigated whether the decreased sulphation contributes to the lower antithrombotic and anticoagulant activities compared with standard heparin. To do this, we compared the anticoagulant activities of heparan sulphate and dermatan sulphate with those of their derivatives resulphated in vitro. The ratio of sulphate to carboxylate in these resulphated heparan sulphate and dermatan sulphate derivatives was approximately twice that of the parent compounds and similar to that of standard heparin. Anticoagulant effects were assessed by determining (a) the catalytic effects of each glycosaminoglycan on the inhibition of thrombin added to plasma, and (b) the ability of each glycosaminoglycan to inhibit the activation of 125I-prothrombin in plasma. The least sulphated glycosaminoglycans were least able to catalyse the inhibition of thrombin added to plasma and to inhibit the activation of prothrombin. Furthermore, increasing the degree of sulphation improved the catalytic effects of glycosaminoglycans on the inhibition of thrombin by heparin cofactor II in plasma. The degree of sulphation therefore appears to be an important functional property that contributes significantly to the anticoagulant effects of the two glycosaminoglycans.
Anticoagulants↗
Catalysis of thrombin inhibition provides an index for estimating the antithrombotic potential of glycosaminoglycans in rabbits.
Previous studies have demonstrated that standard anticoagulant tests are poor indices of the antithrombotic potential of glycosaminoglycans which are weak catalysts of the thrombin-antithrombin III reaction. In this study we investigated whether the catalysis of thrombin inhibition by plasma could serve as a reliable index for assessing the antithrombotic effectiveness of glycosaminoglycans. Equal volumes of 125I-thrombin and control or test plasma were incubated for up to 10 min at 37 degrees C. Inactivation of thrombin was then determined after 7.9% SDS-polyacrylamide gel electrophoresis and subsequent autoradiography. Increasing concentrations of heparin (greater than 0.066 micrograms/mL or 0.01 USP units/mL) and dermatan sulfate (greater than 0.1 micrograms/mL) could be readily demonstrated in undiluted plasma by enhanced formation of complexes of thrombin with antithrombin III and heparin cofactor II respectively. However, the detection of any catalytic effect of the two glycosaminoglycans decreased significantly with increasing plasma dilutions. When ex vivo plasmas obtained from rabbits that had been injected with the minimum dose of any one of seven glycosaminoglycans required to achieve their optimal antithrombotic effect were assessed for their ability to catalyse thrombin inhibition, there was approximately a 2-fold increase in the amount of thrombin inactivated 30 s after the thrombin had been added to the plasma. The enhanced inhibition of thrombin was achieved by catalysis of antithrombin III and/or heparin cofactor II activities. These results suggest that measurement of the catalysis of thrombin inactivation in undiluted plasma is a sensitive and reliable index for estimating the antithrombotic potential of glycosaminoglycans in rabbits.
Animals↗
The inhibition of thrombin-dependent positive-feedback reactions is critical to the expression of the anticoagulant effect of heparin.
Heparin catalyses the inhibition of two key enzymes of blood coagulation, namely Factor Xa and thrombin, by enhancing the antiproteinase activities of plasma antithrombin III and heparin cofactor II. In addition, heparin can directly inhibit the activation of Factor X and prothrombin. The contributions of each of these effects to the anticoagulant activity of heparin have not been delineated. We therefore performed experiments to assess how each of these effects of heparin contributes to its anticoagulant activity by comparing the effects of heparin, pentosan polysulphate and D-Phe-Pro-Arg-CH2Cl on the intrinsic pathway of coagulation. Unlike heparin, pentosan polysulphate catalyses only the inhibition of thrombin by plasma. D-Phe-Pro-Arg-CH2Cl is rapid enough an inhibitor of thrombin so that when added to plasma no complexes of thrombin with its inhibitors are formed, whether or not the plasma also contains heparin. Heparin (0.66 microgram/ml) and pentosan polysulphate (6.6 micrograms/ml) completely inhibited the intrinsic-pathway activation of 125I-prothrombin to 125I-prothrombin fragment 1 + 2 and 125I-thrombin. On the addition of thrombin, a good Factor V activator, to the plasma before each sulphated polysaccharide, the inhibition of prothrombin activation was demonstrable only in the presence of higher concentrations of the sulphated polysaccharide. D-Phe-Pro-Arg-CH2Cl also completely inhibited the intrinsic-pathway activation of prothrombin in normal plasma. The inhibitory effect of D-Phe-Pro-Arg-CH2Cl was reversed if thrombin was added to the plasma before D-Phe-Pro-Arg-CH2Cl. The inhibition of the activation of prothrombin by the three agents was also abolished with longer times with re-added Ca2+. Reversal of the inhibitory effects of heparin and pentosan polysulphate was associated with the accelerated formation of 125I-thrombin-antithrombin III and 125I-thrombin-heparin cofactor complexes respectively. These results suggest that the anticoagulant effects of heparin and pentosan polysulphate are mediated primarily by their ability to inhibit the thrombin-dependent activation of Factor V, thereby inhibiting the formation of prothrombinase complex, the physiological activator of prothrombin.
Calcium↗
Factor VIII concentrate for hemophilia: comparison of two heat-treated products.
In a controlled trial of heat-treated factor VIII concentrate from Cutter Laboratories and from Connaught Laboratories Limited, 25 patients with hemophilia received Cutter factor VIII and 24 patients with hemophilia received Connaught factor VIII. The mean increase in factor VIII level was similar for the two products (1.88% and 1.98% per unit/kg of body weight respectively [p greater than 0.1], or 89% and 95% respectively of the expected increase [p greater than 0.1]). In-vivo survival (the duration of factor VIII in the circulation) was compared after seven infusions of each product. The mean half-disappearance times were 10.7 and 9.1 hours respectively (p = 0.1), and the mean biologic half-lives were 11.6 and 9.9 hours respectively (p = 0.04); the clinical significance of the latter difference is dubious. Both products could be reconstituted satisfactorily from the dried state. Two subjects experienced relatively minor side effects following infusion of one lot of Cutter factor VIII. Individual responses to both products showed considerable variation. Therefore, when it is critical to attain predetermined levels of factor VIII, such as before and after surgery, factor VIII assays should be performed to check the patient's response.
Acute Disease↗
Use of plasma segments for estimating factor VIII activity in pools of fresh frozen plasma.
This study describes a procedure for estimating the factor VIII activity of pools of fresh frozen plasma destined for fractionation into factor VIII concentrates. This information is useful for estimating overall yields. We compared the factor VIII activity of plasma packs and the attached segments shortly after the production of fresh frozen plasmas, during storage at -30 degrees C for up to 4 weeks, after shipping at -20 degrees C to the fractionation site, after storage at -30 degrees C at the fractionation site, and after cryoprecipitation. The factor VIII activity of the segments and the plasma packs were indistinguishable at all stages except after cryoprecipitation. Our results suggest that, prior to the cryoprecipitation stage, a pool of a representative number of plasma segments can be used to determine the factor VIII activity of the plasma pool to be fractionated.
Blood Preservation↗
The blood transfusion effect: characterization of T cell alloantigen-specific receptors by sera from transfused mice.
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Animals↗
Hemorrhagic doses of heparin and other glycosaminoglycans induce a platelet defect.
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Animals↗
Lack of relationship between enhanced bleeding induced by heparin and other sulfated polysaccharides and enhanced catalysis of thrombin inhibition.
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Animals↗
Induction of antiidiotypic antibodies by blood transfusions. Characterization of T cell alloantigen-specific receptors by sera from transfused mice.
Balb/c (H-2d) mice were transfused weekly with 0.1 ml of whole blood from C3H (H-2k) mice. One week after 3 blood transfusions (BT), the mice were bled and the sera collected and pooled. The 3BT serum was absorbed twice with C3H lymphocytes and IgG isolated by ion-exchange chromatography. Balb/c anti-C3H, Balb/c anti-Balb/c, and Balb/c anti-SJL (H-2s) lymphocytes were generated in the mixed lymphocyte cultures and metabolically labeled with 35S-methionine. Cell lysates were prepared from labeled lymphocytes and precleared by absorption with normal mouse serum. Immunoprecipitation was carried out by 3BT-IgG and NMS-IgG. 3BT-IgG specifically precipitated 7 molecules (30K, 60K, 72K, 86K, 92K, 97K, 145K) from Balb/c anti-C3H lymphocytes. In contrast, 3BT-IgG did not precipitate these molecules from Balb/c anti-Balb/c or from Balb/c anti-SJL lymphocytes. The data suggest that BT induces antibodies directed against the blood donor alloantigen-specific receptors on recipient's T lymphocytes.
Animals↗