Anticoagulant Molecules Research Articles

ENHANCED TISSUE FACTOR PATHWAY INHIBITOR RESPONSE AS A DEFENSE MECHANISM AGAINST ONGOING LOCAL MICROVASCULAR EVENTS OF LEGG-CALVE-PERTHES DISEASE

The precise pathogenetic basis of Legg-Calve-Perthes disease (LCPD) is currently unknown. Hemostatic abnormalities, i.e., hypercoagulability and/or hypofibrinolysis, were proposed in the genesis of the LCPD. Deficiency of tissue factor pathway inhibitor (TFPI), a critical natural anticoagulant molecule, may lead to a prothrombotic state in a wide variety of conditions. The aim of this study is to assess the circulating TFPI pool in the LCPD. Group I consisted of 44 patients with LCPD and group II comprised 38 healthy children. Median (IQR) TPFI concentration was significantly higher in the group I (p <. 0001). Enhanced TFPI response could be regarded as a compensatory defense mechanism against ongoing local microvascular events of occlusion and revascularization of LCPD. TFPI molecule may be an important link between the crossroads of the LCPD genesis and pathogenetic microvascular changes in the disease course. Further investigations are needed to shed light on the endothelial anticoagulant kinetics, the unique microvascular compromise, and the self-limiting nature of the disease.

The effect of anticoagulant drugs on cancer

The effect of anticoagulant drugs on cancer

Blood coagulation in cystic fibrosis: modulating inflammation?

Cystic fibrosis (CF) is a devastating chronic inflammatory disease caused by a mutation in the gene encoding cystic fibrosis transmembrane regulator (CFTR), which is a chloride channel in epithelia cell membranes [1]. The phenotype is dominated by pathological changes occurring in organs that express CFTR, particularly the airways, where infection contributes to severe chronic pulmonary disease. The still unavoidable result is a markedly reduced life expectancy (median age of survival 32 years in 2000). Chronic infection is a hallmark of this disease, so that the question whether infection elicits inflammation or vice versa has never been answered unequivocally. Colonization with specific pathogens, particularly Pseudomonas aeruginosa ,o ccurs in about 100% of cases. Contributing factors are reduced interleukin-10 production in the lungs and reduced sialysation of glycoconjugates on epithelial cell membranes. CFTR specifically acts as receptor for P. aeruginosa, facilitating its clearance by internalization under normal conditions. The F508-deleted CFTR cannot bind this bacterium and impaired clearance is the result. Finally, epithelial cells produce defensins which are only partially active in a high salt milieu which is typical of CF, diminishing the antimicrobial barrier [1]. All these factors probably contribute to the chronic infection in CF. The resulting inflammatory state triggers a host response reaction, which also involves the blood coagulation system. In past decades much attention has been given to the possible role that blood coagulation might play in the inflammatory response, as an integral part of the innate immune system [2]. In experimental models of sepsis it has been demonstrated that proinflammatory mediators including tumor necrosis factor (TNF)-a, interleukin (IL)-1 and interleukin (IL)-6 induce tissue factor expression and subsequent thrombin generation. TNF-a impairs fibrinolytic activity by inducing plasminogen activator inhibitor-1 (PAI-1), while the same mediators downregulate the level of expression of natural

Thrombomodulin and Tissue Factor Pathway Inhibitor in Endocardium of Rapidly Paced Rat Atria

Atrial fibrillation (AF) is well known as one of the cardiogenic causes for thromboembolism. Although decreased flow and hypercoagulable state of the blood in the fibrillating atrium have been emphasized as the underlying mechanisms, endocardial dysfunction in maintaining the local coagulation balance could also contribute to the thrombogenesis in AF. The paroxysmal AF model was created by rapid atrial pacing in anesthetized rats. To test the hypothesis that AF induces local coagulation imbalance by disturbing the atrial endocardial function, the gene expression of intrinsic anticoagulant factors, thrombomodulin (TM) and tissue factor pathway inhibitor (TFPI), were determined by means of ribonuclease protection assay, Western blotting, and immunohistochemistry. Rapid atrial pacing for 8 hours significantly decreased TM and TFPI mRNA levels in the left atrium but not in the ventricle, leading to the downregulation of their immunoreactive proteins. Immunohistochemical analysis revealed that TM and TFPI were expressed predominantly in the endocardial cells of the normal atrium, presumably preventing local blood coagulation, and that rapid atrial pacing induced the loss of TM and TFPI expression in the endocardium, leading to deficiency in anticoagulant barriers between the atria and the blood. Rapid atrial pacing acutely downregulated the gene expression of TM and TFPI in the atrial endocardium, thereby inducing local coagulation imbalance on the internal surface of the atrial cavity. These results would support the validity of supplement of anticoagulant molecules deficient in AF.

Les nouveaux anticoagulants dans la maladie thromboembolique veineuse

Objective. – Treatment of deep venous thrombosis is founded on the association of low molecular heparins and oral anticoagulants for 3–6 months. In spite of their uncontested efficacy, these therapeutics bring an hemorrhagic risk and the need of regular laboratory controls for the whole duration of the treatment. The clinical need to extend the indications of anticoagulants have made necessary the development of new antithrombotic treatments. New features. – Two new molecules, fondaparinux and ximelagatran, have been recently developed and are, at present, in very advanced phase of clinical research. Already published phase III studies on venous thromboembolism (VTE) prophylaxis show the efficacy of these new molecules towards this indication. The results of phase III study in patients with acute VTE have not been published yet. Projects and perspectives. – The new anticoagulant molecules fondaparinux and ximelagatran could open the way to new therapeutic possibilities that could simplify the managing of patients under anticoagulant treatment.

Experimental and clinical results with the thrombin inhibitor Argatroban

Argatroban is the smallest anticoagulant molecule of direct thrombin inhibitors. The main attributes of this synthetic drug are its rapid onset of anti-thrombin action, the rapid reversibility of its anticoagulant effect, potent inhibition of clot-bound thrombin, the absence of antibody formation and no need for dosage adjustment in patients with renal impairment. It is eliminated by hepatic metabolism. These properties make argatroban a predictable anticoagulant with intravenous use in a routine clinical setting. Argatroban is approved in the US and Canada for both prophylaxis and treatment of thrombosis in patients with heparin-induced throm-bocytopenia (HIT) and in the US as an antithrombotic agent during percutaneous coronary interventions in patients with HIT or a history of HIT. Preliminary reports document the feasibility of using argatroban for anticoagulation during hemodialysis and as adjunct to thrombolysis for treatment of myocardial infarction. Current recommendations for argatroban monitoring are to use the aPTT (activated partial thromboplastin time) for low doses and the ACT (activated clotting time) for high doses. The specific inhibition of thrombin can be measured with the ECT (ecarin clotting time).

Targeted gene disruption of natural anticoagulant proteins in mice.

The blood coagulation system is a complicated cascade of reactions and feedback regulations that executes a rapid response to vascular injury, yet avoids occlusion of the vessel. There are several key components of this system in the regulation of blood clot propagation, such as antithrombin (AT), tissue factor pathway inhibitor (TFPI), thrombomodulin (TM) and protein C (PC), of which defect causes thromboembolic diseases. In recent years, targeted gene disruption technique by homologous recombination has been introduced to investigate the physiological roles of those natural anticoagulant molecules, not only in thrombogenesis but also in embyrogenesis. We have studied the natural anticoagulantion system in a decade, and recently established AT knockout mice as well as ryudocan (syndecan-4) knockout mice. Ryudocan is a cell surface heparan sulfate proteoglycan, which bears heparin-like glycosaminoglycan (heparan sulfate) cahins, originally cloned from rat microvascular endothelial cells. We have demonstrated that ryudocan deficiency impairs the control of coagulation in fetal vessels of the placenta in mice. We have also reported that complete antithrombin deficiency in mice results in embryonic lethality, with severe fibrin deposition in the myocardium and the liver, accompanied with extensive subcutaneous hemorrhage. In this presentation, recent advances in understanding roles of natural anticoagulant molecules through the researches of targeted gene-knockout mice, including our experiences in antithrombin deficient mice and ryudocan deficient mice, will be discussed.

Platelet adhesion to commercial and modified polymer materials in animals under psychological stress and in a no-stress condition

It is well known that stressful stimuli change blood functions and that protein and platelet parameters are altered in humans and animals subjected to stress. We have examined the influence of psychological stress on the morphological responses of platelets on commercially available materials [polyester (VP), fluoropassivated polyester (VPF), non-woven benzylic ester of hyaluronic acid (Hyaff11)] and on materials synthesised (PUPA) and/or surface modified by sulphation (Hyaff11S) or by immobilisation of the anticoagulant molecules heparin and sulphated hyaluronic acid (PUPA-Heparin, PUPA-HyalS, HyalS-PET). Moreover, the anticoagulant activity (i.e. thrombin inactivation) of the materials was analysed. In the no-stress condition, the surfaces with a low degree of platelet adhesion were Hyaff11S, HyalS-PET, PUPA-Heparin and PUPA-HyalS. Hyaff11, PET and PUPA had the highest number of adherent platelets within the series. VP and VPF exhibited an intermediate behaviour. The exposure of animals to stress induced a dramatic change in platelet number and morphology on PET, HyalS-PET, PUPA, PUPA-HyalS and Hyaff11: there was a higher degree of platelet adhesion, increased platelet spreading and the appearance of pseudopodia. In VP, VPF, Hyaff11S and PUPA-Heparin, there were no changes in platelet adhesion in stress conditions with respect to the no-stress condition; the latter two materials, the only ones able to prolong thrombin time, had a very low number of adherent platelets.

Recombinant adeno-associated virus vector-transduced vascular endothelial cells express the thrombomodulin transgene under the regulation of enhanced plasminogen activator inhibitor-1 promoter.

We were able to facilitate plasminogen activator inhibitor 1 (PAI-1) promoter activity approximately by 14-fold using an enhancer element. This enhanced PAI-1 promoter has a strong basal activity, comparable to CAG promoter activity, and has a response similar to the PAI-1 promoter with respect to TGFbeta 1 and TNFalpha stimulation. The characteristics of the enhanced PAI-1 promoter are thought to be suited to timely and tissue-specific expression of anticoagulant molecules in the vascular cells. Thus, we developed recombinant adeno-associated virus (rAAV) vectors using the enhanced PAI-1 promoter and were successful in transducing vascular endothelial cells to express the thrombomodulin transgene under the regulation of the enhanced PAI-1 promoter in vitro. Thromobomodulin transgene expression driven by the enhanced PAI-1 promoter in rAAV vector-transduced cultured endothelial cells was between 600- and 1000-fold higher than constitutive thrombomodulin gene expression in cultured human umbilical vein endothelial cells and was up-regulated by TGFbeta1 and TNFalpha stimulation which may down-regulate endogenous thrombomodulin gene expression in endothelial cells. The brain vascular endothelial cells of Mongolian gerbils could also be transduced by the same rAAV vector in vivo. Transduction of endothelial cells by rAAV vectors to express enhanced PAI-1 promoter-driven transgenes may be a useful gene therapy approach for vascular diseases.

Activation de la coagulation par la greffe intraportale d’îlots de Langerhans chez le porc

Study aim: Intraportal islet allograft appears to be one of the promising treatments for type I diabetes. However, many limiting factors persist. An activation of the coagulation cascade upon contact with islets, has been reported recently in vitro and could play a crucial role in a non specific inflammatory reaction and favour the specific immune reaction. The aim of this experimental study was to confirm in vivo this activation of the coagulation cascade. Material and methods: An allogenic islets preparation or a material control (inert microbeads) was injected intraportally, in Large White pigs ( n=26), associated with or without an anticoagulant treatment (heparin). Systemic markers of haemostasis were measured in pigs for 72 hours following injection of the studied material. Results: The thrombin–antithrombin complex increased and platelet count decreased in groups receiving preparation of islets, both indicators of an activation of the coagulation cascade. This activation was proportional to the injected volume and was partially attenuated by heparin. No activation was observed in pigs receiving the material control. Conclusion: The activation of the coagulation cascade and the non specific inflammatory reaction could be one of the obstacles to the success of the islet allografts. The use of anticoagulant and anti-inflammatory molecules could potentially allow an improvement of the present results of islet allograft.

Inhibition of invasion and experimental metastasis of murine melanoma cells by human soluble thrombomodulin

Thrombomodulin (TM) is an anticoagulant molecule expressed on the endothelial cell surface and soluble TM antigen, which is present in human plasma and urine, represents the products of limited proteolytic cleavage of cell-surface TM. Recently, it was demonstrated that TM is also expressed on the surface of several tumor cells and the expression level of TM negatively correlated with malignancy in cancer. We investigated the effect of soluble TM isolated from human urine (uTM) on the invasion and metastasis of murine melanoma cells (B16F10 cells) through a reconstituted basement membrane (Matrigel) and in a murine model of experimental lung metastasis. Matrigel reconstituted with uTM inhibited the invasion of B16F10 cells in a dose-dependent manner in a range from 10 to 1000 ng/ml uTM as compared with the control Matrigel without uTM. The inhibitory action of uTM was not altered in the presence of an excess amount of hirudin, an inhibitor of thrombin proteolytic activity, but abolished in the presence of anti-human TM IgG. Matrigel reconstituted with thrombin (1 NIH unit/ml) enhanced the invasion level of cells by 1.5-fold relative to the control Matrigel without thrombin. The thrombin-enhanced invasion of B16F10 cells was repressed by addition of hirudin (10 units/ml) or uTM (100 ng/ml) into the Matrigel. Matrigel reconstituted with hirudin (10 units/ml) and uTM (100 ng/ml) additionally accelerated the inhibitory activity of hirudin or uTM on the thrombin-enhanced invasion of B16F10 cells. Moreover, metastatic colonies formed in the lungs of mice injected intravenously with B16F10 cells were significantly reduced by injection of uTM once a day up to 2 days after co-injection of uTM with the cells. These results suggested that Matrigel reconstituted with uTM inhibited the invasion of B16F10 cells in vitro through a thrombin-independent mechanism and the injection of uTM suppressed experimental lung metastasis of the cells in mice.

In vitro study of blood-contacting properties and effect on bacterial adhesion of a polymeric surface with immobilized heparin and sulphated hyaluronic acid

The blood-contacting properties and the effect on bacterial adhesion of a material based on polyurethane and poly(amido-amine) (PUPA), both in its native form and with the anticoagulant molecules heparin or sulphated hyaluronic acid (HyalS3.5) electrostatically bonded to its surface, were evaluated and compared in vitro. The presence of the biological molecules on the surface was revealed by a dye test and ATR/FTIR analysis. Bound heparin was found to maintain its physiological action, in terms of thrombin inactivation, as well as did free heparin. Moreover, it reduced the degree of platelet adhesion. On the contrary, bound HyalS3.5 lost its anticoagulant activity, though it reduced platelet adhesion. The number of platelets on both modified surfaces was low. Their shape distribution, as determined by SEM, did not differ significantly on the two modified surfaces or with respect to the bare PUPA surface. HyalS3.5 and heparin also inhibited adhesion of Staphylococcus epidermidis to the material. A possible relationship between the platelet and bacterial adhesion is ascribed to the mediating role of plasma proteins.

Regulated inhibition of coagulation by porcine endothelial cells expressing P-selectin-tagged hirudin and tissue factor pathway inhibitor fusion proteins.

Thrombotic vascular occlusion resulting in infarction occurs during hyperacute rejection of allografts transplanted into sensitized patients and remains a major problem in experimental xenotransplantation. A similar process is also found in disorders of diverse etiology including atherosclerosis, vasculitis, and disseminated intravascular coagulation. We have previously constructed two membrane-tethered anticoagulant fusion proteins based on human tissue factor pathway inhibitor and the leech anticoagulant hirudin and demonstrated their functional efficacy in vitro. These constructs have now been modified by the addition of a P-selectin sequence to the cytoplasmic tail to localize them in Weibel-Palade bodies. They have been transfected into Weibel-Palade body-positive endothelial cells isolated from the inferior vena cava of normal pigs. In resting endothelial cells, fusion protein expression colocalized with P-selectin and was confined to Weibel-Palade bodies. These cells had a procoagulant phenotype in recalcified human plasma. However, after activation with phorbol ester the anticoagulant proteins were rapidly relocated to the cell surface where they specifically inhibited the clotting of human plasma. Novel anticoagulant molecules may prove useful therapeutic agents for gene therapy in thrombotic disease and postangioplasty or for transgenic expression in animals whose organs may be used for clinical xenotransplantation. Expression in vascular endothelial cells may be regulated by inclusion of P-selectin cytoplasmic sequence, to restrict cell surface expression to activated endothelium.

Inhibition of tissue factor-dependent and -independent coagulation by cell surface expression of novel anticoagulant fusion proteins.

Thrombotic vascular occlusion occurs in disorders of diverse etiology, including atherosclerosis, vasculitis, and disseminated intravascular coagulation. The same process results in hyperacute rejection of renal allografts transplanted into sensitized patients and remains a major problem in experimental xenotransplantation. We have previously described the design and expression of several genetic constructs encoding novel fusion proteins with anticoagulant properties. They are based on two naturally occurring soluble anticoagulant proteins, human tissue factor pathway inhibitor (hTFPI) and the leech protein hirudin, which act early and late in the clotting cascade, respectively. We report the expression of human hTFPI-CD4 on the surface of immortalized porcine endothelial cells (IPEC), and show that it functions across the species divide as evidenced by the binding of membrane-expressed porcine tissue factor (pTF)-human factor VIIa complexes. Using a human plasma recalcification clotting assay, we distinguished between pTF-dependent and pTF-independent fibrin generation, and we have demonstrated that expression of hTFPI-CD4 on IPEC effectively prevented pTF-dependent clotting. Moreover, we show that when hTFPI-CD4 was co-expressed with the hirudin construct, the procoagulant properties of in vitro cultured, activated IPEC were almost completely abolished. These results suggest that these novel anticoagulant molecules may prove useful therapeutic agents for gene therapy or for transgenic expression in animals whose organs may be used for cliniCal xenotransplantation.

Heparin Increases Protein S Levels in Cultured Endothelial Cells by Causing a Block in Degradation

Heparin is a natural anticoagulant molecule that can alter the activity and/or levels of other molecules involved in blood coagulation. Protein S, an anticoagulant protein, is synthesized and released into the plasma by endothelial cells. Immunological assays revealed a significant increase in protein S found in the media and in the endothelial cells after heparin treatment. Time assays revealed a rapid heparin effect on protein S levels in the media. Upon treatment with chondroitin sulfate, a small increase in the amount of protein S in the conditioned medium was also detected but the change in the cell-associated protein S levels after chondroitin sulfate treatment was a decrease rather than the increase implying that heparin and chondroitin sulfate are operating through different mechanisms. Radioimmunoprecipitations and cycloheximide treatments indicated no significant difference in protein S synthesis in heparin treated cells. In experiments comparing heparin and ammonium chloride effects, heparin seems to mimic the ammonium chloride effect on the levels of protein S in the media. Together, the data indicate that heparin increases the levels of protein S found in the media of cultured endothelial cells by producing a specific block in protein S degradation.

Loss of ATP diphosphohydrolase activity with endothelial cell activation.

Quiescent endothelial cells (EC) regulate blood flow and prevent intravascular thrombosis. This latter effect is mediated in a number of ways, including expression by EC of thrombomodulin and heparan sulfate, both of which are lost from the EC surface as part of the activation response to proinflammatory cytokines. Loss of these anticoagulant molecules potentiates the procoagulant properties of the injured vasculature. An additional thromboregulatory factor, ATP diphosphohydrolase (ATPDase; designated as EC 3.6.1.5) is also expressed by quiescent EC, and has the capacity to degrade the extracellular inflammatory mediators ATP and ADP to AMP, thereby inhibiting platelet activation and modulating vascular thrombosis. We describe here that the antithrombotic effects of the ATPDase, like heparan sulfate and thrombomodulin, are lost after EC activation, both in vitro and in vivo. Because platelet activation and aggregation are important components of the hemostatic changes that accompany inflammatory diseases, we suggest that the loss of vascular ATPDase may be crucial for the progression of vascular injury.

13. Development of a clot-targeted anticoagulant

The effectiveness of anticoagulant therapy is limited by the inability of many anticoagulant molecules to inhibit completely clot bound thrombin. Despite the development of direct, potent thrombin inhibitors such as hirudin, there remains significant opportunity to improve the treatment efficacy. Research in this area has provided evidence to indicate that the failure of existing anticoagulant therapies is due to the procoagulant nature of the clot itself. Clot-targeted anticoagulants offer the potential to: (1) simplify the administration methods; (2) reduce the quantity required for treatment, thus reducing the potential cost of treatment; (3) increase the local therapeutic concentration and (4) to prevent systemic side-effects. A model system has been developed for preparation of targeted anticoagulants using avidin and biotin for coupling. The Fab fragment of the monoclonal antibody DD3B6/22 which binds to the D-dimer component of cross-linked fibrin was prepared and conjugated to avidin. This conjugate was then reacted with the biotinylated specific active site thrombin inhibitor, biotin-PPACK (D-Phe-Pro-Arg-chloromethyl ketone). This targeted anticoagulant (TAC) molecule retained both the ability to bind D-dimer and inhibit thrombin. The reagent binds clots prepared in vitro, which correlates well with the good binding properties observed in an in vivo imaging trial carried out with DD3B6/22. The inhibition of clot procoagulant activity by the TAC reagent versus conventional inhibitors, biotin-PPACK, PPACK and hirudin was assessed in an in vitro assay developed in this study. Clots were pretreated with inhibitor and then thoroughly washed before the procoagulant activity was assayed. The TAC reagent was able to inhibit 90% of the procoagulant activity whereas the conventional inhibitors were only able to inhibit a maximum of 60%. This novel approach to targeting biological active molecules to the clot surface could be extended to include lytic agents or other anticoagulant molecules.

Binding of activated protein C to a specific receptor on human mononuclear phagocytes inhibits intracellular calcium signaling and monocyte-dependent proliferative responses.

Upon activation, mononuclear phagocytes (Mphi) play key roles in the development of septic shock and multiple host immune responses, but details of the regulation of Mphi activation are little understood. We recently showed that the physiologic anticoagulant molecule, activated protein C (APC), blocks responses of human blood Mphi, alveolar Mphi, or THP-1 cells induced by LPS, IFN-gamma, or PMA, including TNF-alpha production and down-regulation of several LPS binding-related proteins. We now report a possible mechanism of action through inhibition of the rapid intracellular calcium signaling that occurs at the onset of Mphi activation, and characterization of a specific Mphi receptor for APC. Flow cytometry studies using Fluo-3 showed that Mph activation by Fc-receptor cross-linking or rIFN-gamma caused a rapid increase in free intracellular calcium, a primary event in multiple signal transduction pathways, which was blocked by pretreatment with APC. Consistent with this, addition of APC inhibited PHA-induced T cell proliferation in a dose- and time-dependent manner. Peak suppression (> 70%) required addition of APC within the first hour of 72 hr cocultures of Mphi and lymphocytes, and proliferative responses were not restored by addition of IL-2 or TNF-alpha. Biochemical studies showed that 125I-labeled APC bound specifically to M phi in a time-dependent and saturable manner. Scatchard analysis indicated there were 180,690 binding sites for APC per cell, which were of high affinity (Kd value of 12.9 mM). Binding of 125I-APC was doubled by activation of Mphi with LPS, and bound APC was not displaced by the zymogen, protein C (PC), or by enzymatically inactive (diisopropyl fluorophosphate- or PPACK-treated) APC, indicating an absolute requirement for the active site of APC in its binding to Mphi. APC binding was blocked by a polyclonal Ab to human PC/APC, but not by protein S, factor Va or Xa, or a polyclonal antithrombomodulin antibody. When 125I-APC was crosslinked to its receptor, immunoprecipitated and analyzed by SDS-PAGE under reducing conditions, a covalent complex (110-115 kD) of 125I-APC (62 kD) and its receptor was seen. In addition, a Mphi membrane protein of 50-55 kD, as determined by SDS-PAGE, was affinity-purified using an APC-Affigel column, and confirmed by ligand binding. Taken together, our findings document the presence of a M phi surface receptor for APC, which appears distinct from a recently described endothelial receptor for PC and APC, and which may be involved in the inhibitory effects of APC on activation of human Mphi, including Mphi-dependent T cell proliferation.

The interaction of leukocytes with platelets in blood coagulation.

The concept that leukocytes play an active role in hemostasis and thrombosis has only recently been accepted. Leukocytes may influence coagulation directly, by the production of procoagulant and anticoagulant molecules, or indirectly, by actions on vascular cells including platelets, endothelial cells, and other leukocytes. This review examines the role of leukocytes in coagulation with an emphasis on regulation of leukocyte function by interactions, with platelets. Activated platelets may serve both to localize leukocytes in areas of thrombosis and to modulate their function. Over the past year, several in vitro studies further defined molecular mechanisms by which leukocytes may regulate coagulation. Further, in vivo studies have provided support for the relevance of these mechanisms in pathophysiologic coagulation.

The influence of low molecular weight heparin on neointimal proliferation in cultured human saphenous vein

To investigate the effect of low molecular weight heparin (LMWH) on neointimal proliferation in cultured human saphenous vein, a model of human vein graft intimal hyperplasia. Dose ranging LMWH concentration study. Culture Laboratory, Department of Surgery. Fifteen segments of human long saphenous vein were incubated at 37 degrees C for 14 days in culture medium with 30% foetal calf serum. LMWH was added to one of the paired segments at 1, 10 and 100 micrograms/ml (five veins each dose). 5-bromo-2-deoxyuridine (Brd-U) was used to label proliferating cells. Neointimal thickness (micron and proliferation index (% labelled neointimal cells). Neointimal thickness and proliferation index were both significantly reduced by LMWH at 100 micrograms/ml [control vs. LMWH, reduction in thickness 21 microns vs. 7 microns (median difference 12 microns, 95% conf. int. 6-18), reduction in proliferation index 33% to 6% (median difference 19%, 95% C.I. 4-32)]. High dose LMWH reduces neointimal proliferation in cultured human saphenous vein. The practical clinical application of these results may require the use of non anticoagulant heparin-like molecules and/or local drug delivery systems.