In vivo effects of low molecular weight heparins on experimental thrombosis and bleeding

Recent studies with heparin fractions indicate that it is possible to dissociate the antithrombotic and hemorrhagic effects of heparin and so improve its therapeutic potential. Heparin inhibits blood coagulation by 3 independent mechanisms by augmenting the effect of antithrombin III (the major effect), by augmenting the inhibitory effect of thrombin or heparin cofactor II, and by disrupting the activation of blood coagulation on the platelet surface; it has an additional effect on hemostasis through its interaction with blood platelets. Some insight into the mechanism of heparin-induced bleeding has been provided by studies with low molecular weight heparins. These heparins have reduced antithrombin activity but retain anti-Xa activity and have antithrombotic properties in animals with a reduced risk of bleeding. There is evidence that the reduction in the bleeding risk is unrelated to the anticoagulant effect of these low molecular weight heparins, but that it may be related to the observation that they inhibit platelet function less than standard heparin. The very low molecular weight heparins (molecular weight 3,000 daltons), have virtually no anti-IIa activity and are relatively weaker antithrombotic agents than low molecular weight heparins of 5,000 daltons. A minimal amount of anti-IIa activity is required for full expression of the antithrombotic activities of these low molecular weight heparins.     

The new heparins]

Low molecular weight heparins are increasingly prescribed in France. Prepared from standard heparin by depolymerisation, they show a markedly decreased anti IIa activity and a anti Xa/anti IIa ratio ranging from 2 to 4. Their mode of action in the coagulation system is still not well known and it is difficult to explain the mechanism of their antithrombotic effect, demonstrated in vivo. They seem to inhibit the first traces of thrombin and then counteract the priming and amplification of coagulation. Their fibrinolytic activity is also a disputed question, but seems to be lower than that of standard heparin. The pharmacological studies show a venous as well as arterial antithrombotic activity of a low molecular weight heparin on several animal models, a lower but not negligible bleeding risk as compared to unfractionated heparin. Furthermore heparin fragments have a weak interaction with platelets, which allow to foresee a greater efficacy of LMWH than standard heparin in arterial thrombosis. Some very rare cases of thrombocytopenia in patients treated with LMW heparins have been recently reported. The compared pharmacokinetics of heparins gave proof of a renal elimination of low molecular weight heparin and a bio availability of about 90% after subcutaneous injection. Many clinical studies allowed to define indications of heparin fragments in prophylactic treatment after surgery as well as in medical patients and in curative treatment in case of deep vein thrombosis. However, others studies must be carried out to define the real efficacy of such a treatment during pulmonary embolism, disseminated intravascular coagulation and myocardial infraction, or during thrombotic complications after vascular surgery.     

The platelet proaggregating and potentiating effects of unfractionated heparin,low molecular weight heparin and heparinoid in intensive care patients and healthy controls

Abstract: Heparin binds to platelets and can cause platelet proaggregating and potentiating effects, possibly causing thrombocytopenia, particularly in patients in intensive care with hyperaggregable platelets. In this study we compared the platelet proaggregating and potentiating effects of unfractionated heparin (UH), 2 low molecular weight (LMW) heparins, enoxaparin and dalteparin, and a heparinoid, danaparoid sodium (orgaran), to platelets of an ICU patient population and a normal control group. In both populations UH caused platelet aggregation in a dose-dependent manner. This occurred in the therapeutic range of the drug, with as little as 0.5 U/ml UH. The LMW heparins caused less and the heparinoid least platelet aggregation. Generally, the aggregation observed in ICU patients was greater than in the normal population. The potentiating effects of the 4 drugs in association with physiological agonists was examined. Similar patterns of potentiation were observed in both populations, with UH causing significant enhancement of platelet aggregation, the LMW heparins intermediate and heparinoid least enhancement. There was substantial variability in the individuals' platelets' reactions to the drugs, in particular to UH. Our findings suggest that UH has the greatest effect, the low molecular weight heparins an intermediate effect and the heparinoid the least propensity to cause platelet activation.     

Differential response of human and bovine platelets to bovine von Willebrand factor and vascular subendothelium

Human platelets undergo agglutination when stirred with bovine plasma (BP), but bovine platelets do not. The present study has shown that exposure of washed bovine platelets to subthreshold concentrations of adenosine diphosphate or thrombin before stirring restores their sensitivity to BP, and the cells undergo rapid agglutination. This agglutination was prevented by a monoclonal antibody, to glycoprotein GPIb. Flow cytometry studies revealed that exposure of bovine platelets to thrombin caused an increase in their ability to bind antibodies known to react with human GPIb or GPIIb-IIIa receptors. Interaction of bovine and human platelets with vascular subendothelium revealed additional differences in reactivity. Bovine platelets in citrate anticoagulant reacted poorly with subendothelium under flow conditions compared with human platelets. In contrast, bovine platelets in blood with low molecular weight heparin as anticoagulant adhered more readily than human cells. These findings suggest that different mechanisms are involved in hemostasis in human and bovine species.     

Are all low molecular weight heparins equivalent in the management of venous thromboembolism?

Low molecular weight heparins are replacing unfractionated heparin in a number of clinical indications because of their improved subcutaneous bioavailability and more predictable antithrombotic response. Clinical trials have demonstrated that low molecular weight heparins are at least as safe and effective as unfractionated heparin for the initial treatment of venous thromboembolism, and unfractionated heparin and warfarin for primary and secondary thromboprophylaxis. The mechanism behind the antithrombotic action of low molecular weight heparins is not fully understood but is likely to involve inhibition of coagulation factors Xa and IIa (thrombin), release of tissue-factor-pathway inhibitor, and inhibition of thrombin activatable fibrinolytic inhibitor. Different low molecular weight heparins have been shown to have various effects on coagulation parameters. Seven low molecular weight heparins are currently marketed worldwide, each demonstrated distinct chemical entities with unique pharmacokinetic and pharmacodynamic profiles. Each low molecular weight heparin is approved for specific indications based on the available efficacy and safety data for that product. The relative efficacy and safety of the low molecular weight heparins are unclear because there have been very few direct comparisons in randomized clinical trials. While recommending low molecular weight heparins for the prevention and treatment of venous thromboembolism, clinical guidelines have not specified individual agents. National and international organizations recognize that low molecular weight heparins are distinct entities and that they should not be used interchangeably in clinical practice. Each low molecular weight heparin should be used at the recommended dose when efficacy and safety data exist for the condition being treated. When these data are not available, the dosing and administration of low molecular weight heparins must be adapted from existing data and recommendations.     

Differential response of human and bovine platelets to bovine von Willebrand factor and vascular subendothelium

Human platelets undergo agglutination when stirred with bovine plasma (BP), but bovine platelets do not. The present study has shown that exposure of washed bovine platelets to subthreshold concentrations of adenosine diphosphate or thrombin before stirring restores their sensitivity to BP, and the cells undergo rapid agglutination. This agglutination was prevented by a monoclonal antibody, to glycoprotein GPIb. Flow cytometry studies revealed that exposure of bovine platelets to thrombin caused an increase in their ability to bind antibodies known to react with human GPIb or GPIIb–IIIa receptors. Interaction of bovine and human platelets with vascular subendothelium revealed additional differences in reactivity. Bovine platelets in citrate anticoagulant reacted poorly with subendothelium under flow conditions compared with human platelets. In contrast, bovine platelets in blood with low molecular weight heparin as anticoagulant adhered more readily than human cells. These findings suggest that different mechanisms are involved in hemostasis in human and bovine species.     

Inhibition of heparanase-mediated degradation of extracellular matrix heparan sulfate by non-anticoagulant heparin species

Incubation of human platelets, human neutrophils, or highly metastatic mouse lymphoma cells with sulfate-labeled extracellular matrix (ECM) results in heparanase-mediated release of labeled heparan sulfate cleavage fragments (0.5 less than Kav less than 0.85 on Sepharose 6B). This degradation was inhibited by native heparin both when brought about by intact cells or their released heparanase activity. Degradation of heparan sulfate in ECM may facilitate invasion of normal and malignant cells through basement membranes. The present study tested the heparanase inhibitory effect of nonanticoagulant species of heparin that might be of potential use in preventing heparanase mediated extravasation of bloodborne cells. For this purpose, we prepared various species of low-sulfated or low-mol-wt heparins, all of which exhibited less than 7% of the anticoagulant activity of native heparin. N-sulfate groups of heparin are necessary for its heparanase inhibitory activity but can be substituted by an acetyl group provided that the O-sulfate groups are retained. O-sulfate groups could be removed provided that the N positions were resulfated. Total desulfation of heparin abolished its heparanase inhibitory activity. Heparan sulfate was a 25-fold less potent heparanase inhibitor than native heparin. Efficiency of low-mol-wt heparins to inhibit degradation of heparan sulfate in ECM decreased with their main molecular size, and a synthetic pentasaccharide, representing the binding site to antithrombin III, was devoid of inhibitory activity. Similar results were obtained with heparanase activities released from platelets, neutrophils, and lymphoma cells. We propose that heparanase inhibiting nonanticoagulant heparins may interfere with dissemination of bloodborne tumor cells and development of experimental autoimmune diseases.     

Inhibition of thrombin-catalyzed reactions in blood coagulation and platelet activation by heparin fractions in the absence of antithrombin III

The antithrombin-III-independent effect of heparin was studied in the following thrombin-catalyzed reactions: activation of purified plasma factor V and partially purified plasma factor VIII:C, generation of factor Va from the platelets and, in the presence of collagen, of the platelet procoagulant activity. Five heparin fractions and a heparinoid were compared to crude heparin. Crude heparin was a more potent inhibitor of these reactions than the fractions or the heparinoid. The inhibitory action of heparin (fractions) appeared to be the result of the formation of a complex between heparin and thrombin that alters the specificity of thrombin towards high molecular weight substrates. The inhibition of these thrombin-dependent feedback reactions in blood coagulation might be of importance in the mechanisms for the dissociation between the antithrombotic and hemorrhagic properties of low molecular weight heparins.     

Mechanism and control of platelet-platelet interactions : II. Action of thrombin on membrane-associated and purified platelet and rabbit skeletal muscle myosins

Thrombin action on intact human platelets was studied using SDS-acrylamide gel electrophoresis. Analysis of purified platelet membranes from thrombin-treated intact platelets showed the rapid disappearance of a peptide with a molecular weight of 200,000 and the simultaneous appearance of three smaller peptides with molecular weights of 171,000, 85,000, and 78,000. Isolated human platelet myosin is degraded by thrombin to peptides identical with those produced in platelet membranes. ATPase activity was retained in the thrombin-cleaved platelet myosin fragments and these fragments also contained peptides similar to rabbit heavy meromyosin. Thrombin produced similar changes in rabbit skeletal muscle myosin. We conclude that the thrombin-sensitive protein of platelet membranes is identical with isolated platelet myosin and that thrombin action produces heavy meromyosin-like and possibly light meromyosin-like fragments.     

低分子肝素在人工肝血浆置换治疗重型肝炎中的应用观察

观察低分子肝素在人工肝(ALSS)血浆置换(PE)治疗重型肝炎中的临床效果。将30例重型肝炎患者随机分为两组,应用低分子肝素抗凝组(LMWH组)15例,应用普通肝素抗凝组(SH组)15例,两组行ALSS-PE治疗前后均记录出血、凝血情况及有关凝血功能指标变化。30例患者行60例次PE治疗后,LMWH组在出血倾向、血浆分离器及管路凝血发生率方面均优于SH组(P<0．05),且其检测有关凝血功能指标的改善方面均明显优于SH组(P<0．05)。低分子肝素在重型肝炎PE治疗中的有效性和安全性均优于普通肝素。     

Human endothelial cells are stimulated and vascular smooth muscle cells are inhibited in their proliferation and migration by heparins.

The actions of two natural heparins and a semi synthetic low molecular weight heparin with low anticoagulant activity have been studied on the migration and proliferation of human vascular endothelial and vascular smooth muscle cells in vitro. In a migration assay non irradiated confluent cultures of endothelial cells and smooth muscle cells were "wounded" with a sharp razor blade in such a way, that the migration of individual cells from the wound edge into a region void of cells could be measured. The maximum distance migrated (perpendicularly to the wound edge) three days after wounding, was taken as an index for migratory activity. All tested heparins reduced migration of vascular smooth muscle cells and enhanced migration of vascular endothelial cells in a concentration dependent manner. Cell proliferation was studied in clone culture. All heparins tested were found to inhibit smooth muscle cell growth. The number of clones, as well as the size of single clones, were smaller with increasing concentrations of natural and low molecular weight heparin. The endothelial cells, however, exhibited contrary responses; with increasing concentrations of heparin, the cloning efficiency and the cell number of individual endothelial cell clones increased. This opposite effects of natural heparins and low molecular weight heparin could be of importance in preventing secondary stenosing intimal proliferations after angioplasty, bypass operation and embolectomy or even atherogenesis. Since the low molecular weight heparin has only a low anticoagulatory activity, it may be more appropriate than natural heparins for long term therapy to prevent artery stenoses caused by intimal SMC proliferation.     

Randomized, controlled trial of low molecular weight heparinvs. no deep vein thrombosis prophylaxis for major colon and rectal surgery in Asian patients

PURPOSE: Routine deep venous thrombosis prophylaxis is controversial in Asian patients, because deep venous thrombosis incidence was considered negligible. Because of recent reports of significantly higher incidences, a randomized, controlled trial was conducted to assess the effectiveness and complications of enoxaparin prophylaxis (low molecular weight heparins) in major colorectal surgery. METHODS: Three hundred twenty consecutive patients were randomly assigned to control or low molecular weight heparins groups. Patients in the low molecular weight heparins group were given perioperative enoxaparin starting 12 hours before surgery. The surgeon (blinded) assessed for difficulties related to possible enoxaparin administration. Independent blinded observers performed daily clinical assessments and Doppler studies (at the 3rd and 5th postoperative day). Deep venous thrombosis was confirmed by duplex ultrasound, and pulmonary embolism was confirmed by lung scans or postmortem examinations. RESULTS: Deep venous thrombosis developed in 5 of 169 (3 percent) controls and 0 of 134 low molecular weight heparins patients (P=0.045). Three of the deep venous thrombosis patients had pulmonary embolism, which was fatal in one patient. The surgeons were unable to perceive any increased surgical difficulties in the low molecular weight heparins group. The bleeding-related complications were significantly higher in the low molecular weight heparins patients (controls, n=3 (1.8 percent); low molecular weight heparins, n=9 (6.7 percent)). However, apart from one subdural hematoma and two abdominal hemorrhages needing re-exploration, which also occurred in one of the controls, these complications were minor bruises at the wounds, drains, or injection sites. CONCLUSION: Deep venous thrombosis prophylaxis is needed in Asian patients undergoing major colorectal surgery.     

低分子肝素钙联用曲美他嗪治疗不稳定型心绞痛的疗效观察

目的观察低分子肝素钙联用曲美他嗪治疗不稳定型心绞痛的疗效。方法将106例冠心病不稳定型心绞痛患者随机分为两组,其中治疗组56例,对照组50例;两组均常规给予硝酸酯类、阿司匹林肠溶片、ACEI、β受体阻滞剂、降脂等治疗;治疗组加用低分子肝素钙4000IU,1次/12h,皮下注射,连用7d;曲美他嗪20mg/次,3次/d,连用30d。结果治疗组总有效率达92.85%,对照组总有效率68.00%,治疗组明显优于对照组。结论 低分子肝素钙联用曲美他嗪治疗不稳定型心绞痛安全有效,值得临床推广应用。     

Comparative inhibition of LPS-activated human monocyte-induced thrombin generation by unfractionated heparin and low molecular weight heparins

Monocyte tissue factor may play a role in the physiological or pathological triggering of blood coagulation. It is well known that unfractionated heparin and low molecular weight heparins inhibit extrinsic thrombin generation. However, this notion has never been confirmed in a physiological model using tissue factor obtained from stimulated human monocytes. This is the purpose of this study. It was important to obtain a pure preparation of monocytes with no platelet contamination. This was possible by leukapheresis and elutriation. Under sterile and endotoxin-free conditions, the process does not activate tissue factor expression by monocytes. We adapted the technique of thrombin generation on an automatic analyzer and used human monocyte tissue factor to trigger thrombin generation. Our results show that unfractionated and low molecular weight heparins potently inhibit monocytic tissue factor induced thrombin generation. The comparison of low molecular weight heparins suggests that molecules with higher anti-IIa/anti-Xa ratios exert a stronger inhibitory effect. These data may be relevant to explain the therapeutic effects of unfractionated and low molecular weight heparins in cardiovascular disorders such as unstable angina.     

The effect of molecular weight on heparin binding to platelets

Low molecular weight heparin is reported to be less reactive with platelets than larger heparins. We probed the molecular basis for this pattern of reactivity by characterizing the saturable platelet binding of [3H]heparin in plasma using heparins of different molecular weights (Mr approximately 3000, approximately 5000, approximately 10,000, approximately 15,000). Binding affinity increased with increasing molecular weight, as expressed by decreasing apparent dissociation constants (Kdapp approximately 1.3 microM for Mr approximately 3000, to Kdapp approximately 0.31 microM for Mr approximately 15,000). After adjusting for the effect of antithrombin III in the plasma, true dissociation constants (Kd) could be calculated and these showed the same trend with molecular weight (Kd approximately 1.1 microM for Mr approximately 3000 to Kd approximately 0.096 microM for Mr approximately 15,000). Platelet binding capacity for the different heparin fractions also increased with molecular weight, although this correlation appeared to lessen with the largest species. Heparin antithrombin III affinity was shown not to affect heparin binding to platelets. We propose a model in which heparin binding to platelets is mediated by charge interaction. Larger molecules with more charge bind with greater affinity and to sites with a broader range of electronegativity than do smaller, less     

In Vivo Platelet Activation Induced by Bothrops jararaca Venom in Rabbits

Disturbances of platelet morphology, coagulation and fibrinolysis were studied 3, 6, and 24 h following administration of Bothrops jararaca snake venom to rabbits (80 μg/kg, i.v.). The activation of coagulation and fibrinolytic systems was demonstrated by a significant decrease in fibrinogen concentration, and an increase in fibrin(ogen) degradation product concentration, respectively. However, the prothrombin activity remained within normal limits throughout. Significant thrombocytopenia was observed 3 h following venom administration. A decrease in platelet dense body numbers was observed until 24 h. 'Exhausted' platelets and evidence of granular secretion were frequently observed in envenomed rabbits. The open canalicular system was only dilated in extensively degranulated platelets. The mean platelet area and boundary values were not significantly different from control group. Therefore, B. jararaca venom can stimulate platelets in vivo, inducing release of platelet granular content. The etiology of thrombocytopenia in B. jararaca envenoming seems to be a multifactorial process, causing platelet sequestration.     

Impairment of primary haemostasis by low molecular weight heparins in rats

Different low molecular weight (LMW) heparins were tested on primary haemostasis in rats. Four preparations were studied; one was devoid of any effect on the bleeding time, while the other three prolonged the bleeding time to varying extents. As a consequence we studied the effect of these heparins on platelet aggregation. The fractions which prolonged the bleeding time, also inhibited the ex vivo and in vitro platelet aggregation, whereas the one devoid of any effect on the bleeding time did not affect platelet aggregation. Similar results were obtained using both platelet-rich plasma (PRP) and gel-filtered platelets. The in vitro response of platelets to aggregating agents may offer a parameter to detect the presence of 'bleeding factor(s)' in some LMW heparin preparations.     

Plasmin effect on platelet glycoprotein Ib-von Willebrand factor interactions

We have studied the effect of streptokinase on platelets in platelet- rich plasma (PRP) and of plasmin on washed platelets. By three and one- half minutes after the addition of 50,000 IU/mL streptokinase to PRP, the maximum rate of ristocetin-induced platelet agglutination declined 40%, and by 60 minutes, it declined 70%. During the same time interval, the thrombin time increased from 20 seconds to over 120 seconds. At a concentration as low as 50 IU/mL, streptokinase reduced the maximum rate of ristocetin-induced platelet agglutination by 50% and prolonged the thrombin time to 1.5 times control value. Streptokinase added to PRP also caused inhibition of platelet aggregation following stimulation by 2.9 mumol/L adenosine diphosphate, 0.25 U/mL thrombin, and 0.025 mg/mL collagen. Plasmin, 0.05 to 1.0 CU/mL, reduced ristocetin-mediated agglutination of washed platelets in the presence of von Willebrand factor (vWF) from 66% of control to 2% of control, following a one-hour incubation. Autoradiograms produced following sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) of plasmin- treated 125I-surface-labeled platelets demonstrated progressive loss of a protein with a molecular weight (mol wt) of 180,000; simultaneously, a protein with mol wt 135,000 appeared on autoradiograms produced following SDS-PAGE of the surrounding platelet medium. These proteins are similar in molecular weight to glycoprotein (gp) Ib, a platelet surface receptor for vWF, and glycocalicin, a proteolytic fragment of gpIb. By use of an enzyme-linked immunosorbent assay (ELISA) based immunoinhibition assay for glycocalicin, we were able to demonstrate that plasmin treatment of washed platelets released a glycocalicin- related antigen into the surrounding medium and that appearance of this material corresponding to loss of vWF-dependent, ristocetin-induced agglutination.     

Modulatory effects of Escherichia coli capsular-derived sulfaminoheparosans and heparins on tissue factor-mediated activation of platelets: flow cytometric analysis.

Sulfaminoheparosans (alternatively known as bioheparins) represent sulfated derivatives obtained from the K5 capsular polysaccharide of Escherichia coli. Previous studies have shown that these agents are structurally comparable to heparins and capable of exerting anticoagulant and antiprotease effects like heparins. Furthermore they are also able to release tissue factor pathway inhibitor (TFPI). Tissue factor (TF) plays a vital role in the pathogenesis of thrombotic and cardiovascular disorders. Anticoagulants such as heparins and bioheparins inhibit this thrombogenic mediator and thereby downregulate the activation of prothrombin and factor X. This study was carried out to determine the effects of several bioheparin fractions and heparins on TF-mediated platelet activation and their direct effect on platelets using human whole blood flow cytometry. Four different sulfaminoheparosan fractions with mean molecular weights of 20, 9, 7, and 6 kDa were tested for their inhibitory effects on platelet activation at two different concentrations (100 and 10 microg/mL). Unfractionated heparin and a low-molecular-weight heparin, tinzaparin, were also tested under the same experimental conditions for comparative modulatory responses. Fresh whole blood from healthy female and male volunteers (n = 5) was mixed with each of these agents and incubated with TF (diluted thromboplastin C) to activate platelets. Platelets were labeled with the antibodies CD61 FITC (GP IIIa) and CD62 PE (P-selectin). The data were analyzed in terms of percent platelet aggregation and platelet P-selectin expression. At 100 microg/mL, all of these agents strongly and significantly inhibited (approximately 40%) the platelet activation induced by TF in comparison to saline control. The inhibitory effects of each of these agents were slightly weaker (approximately 24% inhibition) at 10 microg/mL. The inhibitory effects of these agents on P-selectin expression correspond to their effects on platelet aggregation. At 100 microg/mL all the agents produced greater than 80% inhibition of P-selectin expression whereas at 10 microg/mL, the inhibition is greater than 70% except for bio-20 kDa, which produced less than 50% of inhibition. No molecular weight dependence was observed with bioheparin fractions in terms of inhibitory effects on platelet aggregation or P-selectin expression. None of the bioheparins and heparins exhibited any direct effects on platelets. These observations suggest that both the bioheparins and heparins are capable of inhibiting TF-mediated activation of platelets. Thus the therapeutic effects of bioheparins in the TF-mediated pathogenesis of platelet activation may be similar to those of heparins.     

A Thrombin-Sensitive Protein of Human Platelet Membranes

The action of thrombin on intact human platelets has been studied with the aid of polyacrylamide gel electrophoresis in sodium dodecylsulfate. A single major membrane protein band with a molecular weight of 190,000 disappears after thrombin treatment, while a new membrane protein with a molecular weight of 107,000 appears. This may represent hydrolysis of the thrombin-sensitive protein. When platelets are disrupted or when the thrombin-sensitive protein is solubilized from membranes prior to thrombin treatment, no hydrolysis occurs. The effect of thrombin on the platelet membrane protein is complete within 2 min which suggests that hydrolysis of this membrane protein may trigger the physiological effects of thrombin on platelets.