Anticoagulant and antithrombotic effects of heparin and low molecular weight heparin fragments in rabbits

Heparin and heparin fragments of different molecular weight and with different anti-factor X_a/APTT activity ratios were studied with respect to their ability to inhibit thrombus formation in an animal model. It is concluded that: a) Neither anti-factor X_a nor the APTT activity alone is a good reflector of the antithrombotic activity. b) Anti-factor X_a active fragments must have a minimum molecular weight in order to elicit good antithrombotic activity. c) High affinity for antithrombin III is important for good antithrombotic activity. d) A heparin fragment of molecular weight 4 000 has the same antithrombotic activity as heparin but less effect on the clotting time.     

Pravastatin potentiates the anticoagulant effects of low molecular weight heparin

Introduction: Statins have been shown in randomized trials to reduce coronary events independent of baseline LDL-C level. In the case of pravastatin sodium (PS), there is conflicting evidence as to what is the actual mechanism of its non-lipid lowering beneficial effects. Because pravastatin has been found to prolong the clotting time in vitro, we conducted a study to determine if pravastatin plus low molecular weight heparin (LMWH) would result in a synergistic effect on the in vitro clotting time, thus supporting the hypothesis that pravastatin exerts antithrombotic effects through reduction of fibrin formation. Materials and methods: Aliquots of PS were combined with dalteparin, a LMWH, in 500 μl of human whole blood. The clotting time in seconds was analyzed on a Sonoclot® Coagulation Analyzer, a miniviscometer that is sensitive to early fibrin generation. Results: PS and LMWH, each resulted in a significant prolongation of the clotting time compared with control. The combination of PS and LMWH resulted in a significantly prolonged clotting time compared with either given alone. All values were significantly different from each other (p<0.05). Our results showed that the combination of PS and a LMWH prolongs the clotting time to a significantly greater degree when compared to either administered alone. Conclusions: The synergistic effect of PS and LMWH on prolongation of the clotting time suggests that PS exerts its effect by inhibition of the coagulation cascade and fibrin formation.     

普通肝素间断静脉滴注治疗脑梗死的临床研究

目的观察普通肝素间断静脉滴注治疗脑梗死的疗效,并与常规治疗、巴曲酶、低分子肝素进行对比。方法将入院患者按入院先后顺序半随机分为常规治疗组,普通肝素组,巴曲酶组,低分子肝素组,每组200例;记录在该患入院时,入院后第一周,及出院时的神经功能缺损评分分值;记录每个患者的牛津郡社区脑卒中规划分型;仅对于普通肝素及巴曲酶两组进行凝血象监测。结果（1）第一周及出院时评分差值比较：普通肝素组与其他三组比较均有统计学意义（P〈0．05）,出院时评分差值巴曲酶组与常规治疗组比较差异有统计学意义（P〈0．05）;（2）疗效比较（按疗效等级）：普通肝素组与常规治疗组及低分子肝素组相比疗效差异有统计学意义,肝素组与巴曲酶组疗效差异无统计学意义;（3）各治疗组出血事件发生率：常规治疗组4％,普通肝素组11．5％,巴曲酶组13．5％,低分子肝素组9％,几乎均为尿隐血及牙龈出血事件,其中常规治疗与其他三组比较差异均有统计学意义（P〈0．05）;（4）肝素组与巴曲酶组组间AVIT值比较及FbgC值比较差异均有统计学意义（P〈0．001）;（5）各梗死类型间评分差值比较无统计学意义;（6）各梗死类型问疗效比较：腔隙性梗死（LACI）与全前循环梗死（TACI）、部分前循环梗死（PACI）、后循环梗死（POCI）比较差异均有统计学意义;TACI与POCI比较差异有统计学意义。TACI与PACI比较差异无统计学意义;PACI与POCI比较差异无统计学意义;（7）各梗死类型间出血倾向比较：TACI与PACI、POCI、LACI比较差异有统计学意义,PACI与LACI比较有统计学意义;（8）男女间评分差值、疗效、出血倾向比较差异均无统计学意义。结论静脉内使用普通肝素间断静脉滴注较低分子肝素及常规活血类药物有更好的疗效,有助神经功能改善,方法简便,且并不比低分子肝素及巴曲酶具更多的出血风险,相对安全。     

Cerebral hemorrhage due to heparin limits its neuroprotective effects: studies in a rabbit model of photothrombotic middle cerebral artery occlusion

We investigated the efficacy of heparin on cerebral ischemic damage in a rabbit model of middle cerebral artery (MCA) photothrombosis and in the same model, cerebral hemorrhage induced by heparin as its side effect was also investigated. Using a photothrombosis model in rabbits, 38 animals were divided into four groups, heparin low-dose I and II, heparin high-dose and vehicle. In heparin low-dose I (n=10) or II (n=7), heparin was administered for 23.5 or 22 h, respectively, starting 30 or 120 min after the start of photo-irradiation to induce thrombosis. In high-dose (n=7), heparin was administered 30 min after the start of photo-irradiation for 23.5 h. In the vehicle treated group (control), 14 animals were infused continuously with saline for 23.5 h. Heparin at low and high doses prolonged Activated partial thromboplastin time (aPTT) by about 3 and 10 times compared with control group. The results show that cerebral hemorrhage was present in all animals, gross hemorrhage was observed in one animal each of the heparin low-dose I and high-dose groups, and in three animals of the heparin low-dose II group, while no gross hemorrhage was observed in control group. In heparin low-dose I, the size of cerebral infarction was significantly (P<0.01) reduced and neurological deficits were significantly (P<0.01) improved. In contrast, in heparin high-dose, the infarct size significantly increased, especially in the cortex (P<0.0001), and neurological deficits were significantly (P<0.01) worsened. In heparin low-dose II, the size of cerebral hemorrhage significantly (P<0.001) increased compared with the control group. In conclusion, using a photothrombotic model in the rabbit MCA, we have investigated the antithrombotic benefits and hemorrhagic risks associated with heparin. Of unique feature of our model is the fact that in a single animal model, we could evaluate doses of heparin which reduce cerebral infarction and doses which can promote cerebral hemorrhage. This model can be extended to determine both benefits and risks of antithrombotic agents.     

The effects of heparin and low molecular weight heparins on bone

Recent clinical trials have shown that the risk of developing osteoporosis is substantially lower when low molecular weight heparins (LMWHs) are used in place of unfractionated heparin. While the reason(s) for this difference has not been fully elucidated, studies with animals have suggested that heparin causes bone loss by both decreasing bone formation and increasing bone resorption. In contrast, LMWHs appear to cause less bone loss because they only decrease bone formation. Whether all LMWHs decrease bone formation and therefore cause bone loss is unknown. For example, preliminary in vitro studies with the synthetic pentasaccaride, Fondaparinux, have suggested that it may not decrease bone formation and thus, may have no deleterious effects on bone. Further studies are required in order to determine if all LMWHs cause bone loss equally.     

Regional distribution of high-affinity [H]somatostatin binding sites in the human brain

The distribution of high-affinity binding sites for [³H]somatostatin has been studied in membrane preparations from a number of regions of normal human brain. The highest densities of binding sites (> 48 fmol/mg protein) were found in the cerebral and cerebellar cortices and the hippocampus, with intermediate binding densities (30–46 fmol/mg protein) being present in the basal ganglia, amygdala, septum and claustrum. The lowest densities of binding sites (<14 fmol/mg protein) were observed in the hypothalamus, thalamus and substantia nigra. The binding of [³H]somatostatin in both the frontal cortex and cerebellar cortex demonstrated pharmacological specificity, since somatostatin-28, but not somatostatin-28_1–12 or Des AA^{1,2,4,5,12,13}, -Trp⁸-somatostatin, competed for the binding sites. Scatchard analysis of the binding in both frontal cortex and cerebellar cortex revealed the presence of two classes of high-affinity binding sites.     

Influence of heparin;of different heparin fractions and of a low molecular weight heparin-like substance on the mechanism of fibrinolysis

The influence of different heparin fractions and of a synthetic polysulfated polysaccharide (SP54) on the fibrinolytic mechanism was examined. In vitro,a significant shortening of the euglobulin lysis time (ELT) was found after addition of standard mucosa heparin, of high MW heparin and of SP 54 respectively. Low MW heparin fractions had no influence on the ELT. Simultaneously with the shortening of the ELT an activation of factor XII and of kallikrein was observed. A similar effect was found in groups of volunteers after i.v. or s.c. injections and even after oral administration of SP 54. The effect of venous occlusion on the ELT and on the activation of factor XII was considerably increased when heparin or SP 54 was injected 2 h prior to the test. When a comparable concentration of the test substances was added to plasma samples before and after venous occlusion,the effect on the ELT was much less pronounced than after injection whilst the effect on factor XII was comparable in both tests. From these results the conclusion was drawn that activation of fibrinolysis by polysulfated polysaccharides is achieved by an endogenous pathway as well as by an increased availability of the vascular activator. The magnitude of the activation of fibrinolysis partly depends on the MW of the substance but apparently also on the degree of sulfatation since a low MW substance with a high number of sulfate bonds such as SP 54 was considerably more active than the low MW fraction of standard heparin.     

Comparison of the in vivo hemorrhagic and antithrombotic effects of a low antithrombin-iii affinity heparin fraction

Antithrombin III (ATIII) affinity chromatography of commercial grade heparin yields fractions of high and low affinity for ATIII. In vitro, the high affinity fraction accounts for most of the anticoagulant effect while there is evidence that the low affinity material interferes with platelet function. We have studied the relative antithrombotic and hemorrhagic effects of low affinity heparin. The low affinity heparin fraction, specific activity 43 USP units/mg, was compared with standard heparin (150 USP units/mg) in rabbit experimental models. A residual 12.5% by weight of this low affinity heparin showed high affinity for ATIII. Inhibition of thrombosis in a stasis-hypercoagulability model was directly related to the weight (mg) of high affinity material in each of the heparins. In the bleeding model, when similar weights (mg) of high affinity material were infused, significantly more bleeding was demonstrated with the low affinity fraction which contained a 5-fold excess by weight of low affinity material. We have demonstrated that a low affinity heparin depleted of in vitro anticoagulant and in vivo antithrombotic activity significantly contributes to hemorrhage.     

Clinical evaluation of low-molecular-weight heparin (FR-860) on disseminated intravascular coagulation (DIC) - a multicenter co-operative double-blind trial in comparison with heparin -

This study was evaluated the effectiveness, safety and utility of FR-860 and to compare those with heparin in patients with Disseminated intravascular coagulation (DIC). A diagnosis of DIC was made based on the criteria proposed by the Research Committee on DIC in the Ministry of Health and Welfare of Japan. FR-860 (FR group,75 anti-factor Xa international units/kg/day) and Heparin (HP group, 240 units/kg/day) were administered for 5 days by continuous intravenous infusion. The total number of enrolled patients was 126 cases, and after excluding 1 case a total of 125 cases. Moderate or higher improvement of bleeding symptoms was 33.3% in the FR group and 18.5% in the HP group. On the organic symptoms, FR group showed a significantly higher improvement rate than the HP group, 20.5% and 8.2% respectively. On the overall efficacy of cases with pretreatment plasma AT III levels of less than 21 mg/dl or less than 70%, FR group showed a significantly higher improvement rate than the HP group. The safety rate of FR-860 (93.4%) was a significantly higher than that of the HP group (79.7%). Our report demonstrates that FR-860, as a therapeutic agent for the treatment of patients with DIC, is significantly higher safety and clinical utility as compared with heparin.     

Enhancement by heparin of thrombin-induced antithrombin III proteolysis: Its relation to the molecular weight and anticoagulant activity of heparin

Previous findings indicated that binding of heparin to antithrombin III (AT III) facilitates thrombin-induced proteolysis of the inhibitor. We now studied this property of heparin in regard to its molecular weight and anticoagulant activity. Commercial heparin was resolved on Sephadex G-200 into six fractions of decreasing molecular weight. From each fraction high affinity (HA) heparin was isolated by chromatography on AT III-Sepharose and examined in reaction of α-thrombin with a molar excess of ¹²⁵I AT III. Proteolysis of the inhibitor was assessed by SDS polyacreylamide gel electrophoresis. In the presence of the HA heparin from 18% to 38% of AT III participating in reaction appeared in the form of inactive 50,000-dalton fragment, as opposed to 7% of AT III fragmented in the absence of heparin. Although the ability to potentiate proteolysis was at its peak in the medium-molecular-size heparin fraction, the amount of degraded inhibitor relative to anticoagulant activity increased with decreasing molecular weight of the polysaccharide. These findings are consistant with the possibility that the ability of bound heparin to facilitate the cleavage of AT III by thrombin is generally less contingent upon secondary characteristics of the polysaccharide than the anticoagulant activity.     

In vitro and in vivo studies of the anti-Xa activity of heparin

Several heparin preparations have been assayed using the Denson and Bonnar anti-Xa assay. The heat defibrination step involved in this assay has been shown to introduce discrepancies into the results, due mainly to co-precipitation of the heparin with fibrinogen. The amount of heparin lost is dependant on the molecular weight. The anti-Xa activity of ex vivo samples from subjects given heparin was much more resistant to loss during defibrination than in vitro samples, resulting in artificially high potency estimates. A modified assay has been proposed, omitting the defibrination step. The results provide further evidence that the anti-Xa activity observed after subcutaneous injection of heparin differs from that measured when the drug is added to plasma in vitro.     

History of the dopamine hypothesis of antipsychotic action

The dopamine hypothesis of how antipsychotic drugs exert their beneficial effect in psychotic illness has an interesting history that dates back to 1950. This hypothesis is not to be confused with the dopamine hypothesis of schizophrenia; the aim of the latter is to explain the etiology of schizophrenia. The present review does not deal with schizophrenia but, rather, with the historical development of our current understanding of the dopamine-associated actions of the drugs that reduce the symptoms of psychosis. This historical review begins with the serendipitous discovery of chlorpromazine, a drug synthesized around a chemical core that initially served to produce man-made dyes. This molecular core subsequently contributed to the chemistry of antihistamines. It was with the aim of producing a superior antihistamine that chlorpromazine was synthesized; instead, it revolutionized the treatment of psychosis. The first hypothesis of how this drug worked was that it induced hypothermia, a cooling of the body that led to a tranquilization of the mind. The new, at the time, discoveries of the presence of chemical transmitters in the brain soon steered investigations away from a temperature-related hypothesis toward questioning how this drug, and other drugs with similar properties and effects, modulated endogenous neurotransmission. As a result, over the years, researchers from around the world have begun to progressively learn what antipsychotic drugs do in the brain.     

The effect of heparin on endothelial stability

Heparin in ultra low doses had a stabilizing effect on endothelial lining in rats. This effect was demonstrated as prevention of increases in counts of circulating endothelial cells after a standard intravenous citrate injection. The same effect of heparin was observed even with other provoking agents /adrenaline, hypotonic saline/. The duration of heparin effect was very prolonged, a 50 % effect having been observed 5 hours after the administration. Subcutaneous heparin was effective only in relatively high doses and had a lower maximum effect compared with intravenous heparin.     

A whole blood flow cytometric determination of platelet activation by unfractionated and low molecular weight heparin in vitro

The influence of unfractionated (Heparin–Natrium) and low-molecular heparin (Fragmin®) on platelet activation in whole blood was investigated by FACS analysis in vitro using antibodies against glycoprotein (gp) IIb/IIIa (CD 41), GMP 140 (CD 62P), gp 53 (CD 63) and fibrinogen. Samples were also labeled with anti-gp Ib (CD 42b). Neither unfractionated heparin (UFH) nor low molecular weight heparin (LMWH) led to significant (i.e., p<0.05) changes in fluorescence intensities of platelets labeled with anti-gp IIb/IIIa or anti-gp 53. Significant platelet activation due to unfractionated heparin could be observed by labeling with anti-GMP 140 (UFH: p=0.009; LMWH: p=0.16). The proportion of platelets with surface-bound fibrinogen was significantly increased (UFH: p=0.00006; LMWH: p=0.008). After incubation with heparins, activation ability of platelets by adenosine diphosphate (ADP) was significantly increased. The potentiating action of unfractionated heparin was larger. Therefore, flow cytometric results of platelet activation in patients receiving heparin should be interpreted carefully.