Recombinant Hirudin Research Articles

Purification and Identification of Recombinant Hirudin and Its Degradation Products Expressed in Pichia pastoris

The purification and identification of recombinant hirudin (r‐hirudin) (rHV2‐Lys47) and its several C‐terminal proteolytic degradation derivatives, produced by Pichia pastoris, were described. The high‐purity rHV2‐Lys47 of above 99% and its three degradation products were obtained by a straightforward two‐step chromatography procedure, a combination of cation exchange and reverse phase chromatography, with a recovery yield of 74% for hirudin. The purified rHV2 had the predicted N‐terminal amino acid sequence and the derivatives were the degradation products of hirudin, short of one to three amino acid residues at C‐terminal.

Direct thrombin inhibitors for anticoagulation.

To review the progress in developing direct thrombin inhibitors (DTIs) for anticoagulation within the context of existing anticoagulation therapies. Searches of MEDLINE (1993-June 2003) were conducted. We examined English-language articles, human studies, and relevant animal studies, and obtained additional citations from the references of these articles. Because of its pivotal role in hemostasis, thrombin is a key therapeutic target in the treatment and prevention of thromboembolic disorders. Conventional anticoagulant therapies, such as warfarin, unfractionated heparin, and low-molecular-weight heparin, exert their pharmacologic action by indirect thrombin inhibition. Although these agents are effective, each has limitations, prompting a search for more effective, specific, better-tolerated, and convenient anticoagulants. The efficacy and safety of factor Xa inhibitors are being investigated. Furthermore, the development of DTIs such as recombinant hirudin (lepirudin), bivalirudin, and argatroban continues. Challenges in the development of DTIs include establishing a binding affinity for thrombin that is not associated with excessive bleeding, attaining high thrombin specificity, achieving inhibition of both unbound and clot-bound thrombin, and producing an effective, fixed-dose oral anticoagulant to improve the practicality of anticoagulation therapy. Ximelagatran, an oral DTI designed to meet these standards, is currently in Phase III clinical trials. Significant progress has been made in developing DTIs. The recent emergence of orally administered DTIs may simplify the prevention and treatment of thrombosis.

Thrombopénies et thromboses induites par l’héparine: Physiopathologie, diagnostic et traitement

Introduction. – Heparin-induced thrombocytopenia is due to the development of IgG antibodies specific to platelet factor 4. More frequently observed after administration of unfractionated heparin, this complication is characterized by a delayed decrease in platelet count (after the 5th day of treatment) associated with venous and/or arterial thromboses. Current knowledge and key points. – It is often difficult to discard another potential cause for thrombocytopenia and to demonstrate by Elisa or platelet activation tests the presence of heparin-dependent antibodies is therefore mandatory in every patient. Withdrawal of heparin treatment is always necessary as well as the administration of an alternative antithrombotic agent. Danaparoid sodium (mixture of glycosaminoglycanes mainly with anti-Xa activity) or lepirudin (a recombinant hirudin with only antithrombin activity) are both recommended but these two drugs are associated with a significant risk of bleeding in case of renal failure. Oral anticoagulants such as coumadin can only be given when platelet count is normalized and if the clinical evolution is favorable. Potent antiplatelet agents (ilomedine or tirofiban) can be used in specific situations necessitating heparin such as extracorporeal circulation. Future prospects and projects. – Early administration of coumadin for the treatment of venous thromboembolic disease efficiently prevent the occurrence of heparin-induced thrombocytopenia, which could disappear in the future with a wider use of the new antithrombotic agents, fondaparinux and ximelagatran.

Prevention of Venous Thromboembolism Following Orthopaedic Surgery

Patients undergoing total hip or total knee replacement are at high risk of venous thromboembolism (VTE), and are therefore considered to be populations well suited for the evaluation and dose optimisation of new anticoagulants. Deep vein thrombosis may lead to life-threatening pulmonary embolism, disabling morbidity in the form of the post-thrombotic syndrome, and risk of recurrent thrombotic events. There is increasing evidence that anticoagulant treatment for the prevention of VTE should be extended from 1 to at least 4 weeks after surgery. Anticoagulation with vitamin K antagonists (such as warfarin), low molecular weight heparin or unfractionated heparin effectively lowers the risk of VTE, but these anticoagulants have limitations such as the need for coagulation monitoring and subsequent dose adjustment (vitamin K antagonists), difficulty of continuing prophylaxis out of hospital because of the requirement for parenteral administration, and risk of heparin-induced thrombocytopenia. The development of new anticoagulants has been pursued with the aim of finding more effective, safer and/or more convenient therapies. Thrombin is a central regulator in the coagulation and inflammation process and several direct thrombin inhibitors (DTIs) with distinct pharmacological profiles, as well as pharmacological differences from the conventional anticoagulants, are currently in clinical use for certain indications or are under development. Clinical experience with parenterally administered DTIs has accumulated since the mid 1990s, although only desirudin (a recombinant hirudin) is currently approved for use in patients undergoing orthopaedic surgery. Two oral DTIs, ximelagatran and dabigatran etexilate, are in clinical development. Dabigatran etexilate has recently been evaluated in phase II clinical trials in patients undergoing total hip replacement. Several large phase III trials have now demonstrated the efficacy and safety of ximelagatran in the prevention of VTE following total hip or knee replacement. Ximelagatran can be used with an oral fixed dose without the need for coagulation monitoring or dose adjustment. Hence, it offers significant potential to facilitate the management of anticoagulation in or out of hospital.

Lepirudin bei heparininduzierter Thrombozytopenie

ZusammenfassungDie heparininduzierte Thrombozytopenie (HIT) Typ II ist eine antikörpervermittelte unerwünschte Arzneimittelwirkung auf Heparin, die paradoxerweise mit einem Abfall der Thrombozytenkonzentration und einem erhöhten Risiko für thromboembolische Komplikationen einhergeht.Die Antikörper reagieren gegen ein Neoepitop des Plättchenfaktors 4 nach Bindung mit Heparin. Die Inzidenz der HIT II ist geringer unter niedermolekularem Heparin im Vergleich zu unfraktioniertem Heparin und bei nicht operierten Patienten im Vergleich zu Patienten nach großen operativen Eingriffen. Bei der HIT II ist die unverzügliche Einleitung einer alternativen Antikoagulation wegen der hohen, durch Antikörper vermittelten Thrombogenität erforderlich. Das rekombinante Hirudin Lepirudin (Refludan®) ist Antikoagulanz der Wahl. Eine langfristigere Antikoagulation kann in Abhängigkeit von begleitenden Faktoren mit subkutanem Lepirudin und mit oralen Antikoagulanzien erfolgen.

The effect of anticoagulation with subcutaneously delivered polyethylene glycol conjugated hirudin and recombinant tissue plasminogen activator on recurrent stenosis in the rabbit double-balloon injury model

Myointimal hyperplasia is the condition usually responsible for recurrent stenosis (restenosis) after endarterectomy, bypass grafting and angioplasty. Its cause is still not known. The present study examined whether inhibition of thrombin by tissue plasminogen activator (r-TPA) or polyethylene glycol recombinant hirudin (PEG-hirudin) could reduce restenosis in an animal model. Restenosis was induced in 20 cholesterol-fed rabbits. The right carotid artery underwent a double-balloon injury while left carotid artery acted as a control. Recombinant tissue plasminogen activator (1 mg kg −1 s.c.) and PEG-hirudin (0.7 mg kg −1 s.c.) were given subcutaneously with normal saline acting as a control. Blood levels of PEG-hirudin were measured by both ELISA and an Ecarin (activity) assay. Vessel dimensions were measured in histological sections, obtained from perfusion-fixed tissue, using computerised planimetry. The model reproduced many of the histological changes found in human restenosis, such as intramural thrombus, rupture of the elastic lamina, macrophage infiltration and smooth muscle migration. Reinjury caused an almost three-fold reduction in the area of the lumen (median 0.25 mm 2) compared with uninjured vessels (median 0.72 mm 2). The mean plasma levels of PEG-hirudin and r-tPA achieved were 291 ng/ml (S.E.M. 28 ng/ml) and 34 IU/ml (S.E.M. 12 IU/ml), respectively. PEG-hirudin significantly inhibited the effect of balloon injury on luminal area compared with saline-treated controls (0.21 versus 0.44 mm 2, respectively, P<0.05). Recombinant tPA also had a similar inhibitory affect, but this did not reach statistical significance (0.16 versus 0.44 mm 2, respectively, P>0.05). The magnitude of luminal narrowing was significantly reduced by subcutaneous injection of PEG-hirudin. Further studies are required to determine whether this effect can be enhanced by other antithrombins or improved methods of delivery.

Heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT), next to bleeding complications, is the most important side-effect of heparin therapy in cardiac patients and the most frequently found thrombocytopenia induced by medication. Two types of HIT are distinguished on the basis of both severity of disease, and pathophysiology: type I HIT is an early, transient, clinically harmless form of thrombocytopenia, due to direct heparin-induced platelet aggregation. Thromboembolic complications are usually not seen. No treatment is required. A normalization of platelet count even if heparin is continued is a usual observation. Type II HIT is more severe than type I HIT and is frequently complicated by extension of preexisting venous thromboembolism or new arterial thrombosis. The thrombocytopenia is caused by a pathogenic heparin-dependent IgG antibody (HIT-IgG) that recognizes as its target antigen a complex consisting of heparin and platelet factor IV. Type II HIT should be suspected when the platelet count falls to less than 100,000 per cubic millimeter or less than 50% of the base line value 5 to 15 days after heparin therapy is begun, or sooner in a patient who received heparin in the recent past. The clinical diagnosis of type II HIT can be confirmed by several sensitive assays. In cases of type II HIT, heparin must be stopped immediately. However, if the patient requires continued anticoagulant therapy for an acute event such as deep venous thrombosis, substitution of an alternative rapid-acting anticoagulant drug is often needed. In the authors experience Danaparoid sodium, a low-sulfated heparinoid with a low cross-reactivity (10%) to heparin, can be regarded as an effective anticoagulant in patients with type II HIT. Preliminary experiences with intravenous recombinant hirudin are also encouraging and suggest that this direct thrombin inhibitor will emerge as a valuable alternative treatment for patients who suffer from HIT.

The role of recombinant hirudins in the management of thrombotic disorders.

Native hirudin is the most potent natural direct thrombin inhibitor currently known; it is capable of inhibiting not only fluid phase, but also clot-bound thrombin. Recombinant technology now allows production of recombinant hirudins (r-hirudins), which are available in sufficient purity and quantity with essentially unaltered thrombin-inhibitory potency. As thrombin is known to play a key role in a number of thrombotic disorders, numerous studies focused on the impact of r-hirudins on the clinical course in these diseases. R-hirudins provided significantly more stable anticoagulation than standard heparin, but demonstrated a relatively narrow therapeutic range with relevant bleeding risk even at clinically effective doses. In doses that are not associated with an increased bleeding risk, r-hirudins often failed to demonstrate significant superiority to heparin. To date, r-hirudins have a definite role in the treatment of heparin-induced thrombocytopenia, where they markedly reduce the high risk of thrombosis. For prophylaxis of deep vein thrombosis, r-hirudins have been shown to be superior to both unfractionated and low molecular weight heparin, but are not extensively used in this indication. In acute coronary syndromes, a definite role of r-hirudins has not yet been firmly established. When applied in an appropriate dose as adjunct to thrombolysis in patients with acute myocardial infarction, randomized, controlled trials did not show a consistent benefit of r-hirudins, especially in the long-term. In patients undergoing coronary balloon angioplasty for acute coronary syndromes, promising effects in the early postprocedural phase did not translate to an improved outcome after 6 months. In patients with unstable angina pectoris, efficacy and safety of r-hirudins as primary antithrombotic therapy are still under debate. In the future, r-hirudins are to be compared with alternative or additional potent antithrombotic agents or treatment strategies. This comparison will ultimately lead to their final placement in the management of thrombotic disorders.

Transport of proteins and peptides across human cultured alveolar A549 cell monolayer

An in vitro cultured monolayer system of alveolar epithelial cells was used as a model to investigate the transport pathway of the peptides and proteins, salmon calcitonin (sCT), insulin (INS), recombinant hirudin (rHAV2), and recombinant human growth hormone (rhGH), in pulmonary epithelium. Human lung adenocarcinoma A549 cells formed continuous monolayers when grown on the polycarbonate filters of Transwell plates. The transport of the peptides and proteins having MW of 3400–22,000 Da was studied under different conditions. The results showed that the apparent permeability coefficients ( P app) of these macromolecules across A549 cell monolayers ranged from 2×10 −6 to 5×10 −6 cm s −1 and exhibited a good inverse correlation with molecular weight. No concentration, direction, or temperature dependence was observed in the permeation of sCT, INS, and rHAV2. While the P app of rhGH in the BA direction (2.25×10 −6 cm s −1) was less than that in the AB direction at both concentrations (3.20×10 −6 and 3.29×10 −6 cm s −1). The P app values of rhGH were concentration and temperature independent in the AB direction. These findings suggest that the hydrophilic peptides and proteins used in this study, sCT, INS, rHAV2, and rhGH, appear to cross the A549 cell monolayers via a paracellular pathway by a passive diffusion mechanism.

Directed evolution towards protease-resistant hirudin variants

Hirudin, a thrombin-specific inhibitor, is efficiently digested and inactivated by proteases with pepsin- and chymotrypsin-like specificity. Using a combination of phage display selection and high-throughput screening methods, several variants of recombinant hirudin were generated. Only very few variants comprising amino acid substitutions in the amino-terminal domain (residues 1–5) and in the carboxyl-terminal tail (residues 49, 50, and/or 56, 57, 62–64) were identified that showed thrombin inhibition activities similar to those of the wild-type polypeptide. Analysis of protease susceptibility, however, revealed that mutations, which conferred protease resistance, simultaneously diminish thrombin inhibition activity. This is particularly apparent for substitutions in the region of residues 56–64, which forms a large number of electrostatic and hydrophobic interactions with thrombin in the crystal structure of the complex. Unlike wild-type hirudin, the variant comprising Pro 50– ⋯ –His 56–Asp 57– ⋯ –Pro 62–Pro 63–His 64 is completely resistant to pepsin and chymotrypsin cleavage; however, this is at the expense of thrombin inhibition activity where there is a 100-fold increase in the IC50 value. The frequent replacement of wild-type amino acids by proline at major protease cleavage sites indicates that at least pepsin- and chymotrypsin-like enzymes may exhibit a (conformational) specificity concerning the P1 and P2 positions. On the basis of these results, proline substitutions appear to be a general strategy to design polypeptides that are not susceptible to digestion by a broader range of different proteases.

Novel Anticoagulants for the Prevention and Treatment of Venous Thromboembolism

Unfractionated heparin, low molecular weight heparin and vitamin K antagonists are anticoagulants currently used for the prevention and treatment of deep vein thrombosis and pulmonary embolism. Considerable limitations of these agents, such as a narrow therapeutic window, a variable dose response or lack of oral bioavailability, created the need for new anticoagulants. Numerous new compounds with different mechanisms of action have been developed and some have been already approved for clinical use. Quite recently, fondaparinux, an indirect anti-factor Xa inhibitor, has been licensed in Europe and in the US for prevention of VTE in patients undergoing hip or knee replacement surgery. In addition, lepirudin, a recombinant hirudin derivative, and the heparinoid danaparoid, have been approved in Austria for treatment of heparin-induced thrombocytopenia. Recombinant nematode anticoagulant protein c2, the orally available thrombin inhibitor ximelagatran and ART-123, a recombinant soluble thrombomodulin, are in advanced stages of clinical development. This article reviews mechanisms and sites of action, and the current state of preclinical and clinical research of these and various other agents with respect to the prevention and treatment of venous thromboembolism).

Selectin-mucin interactions as a probable molecular explanation for the association of Trousseau syndrome with mucinous adenocarcinomas.

Trousseau described spontaneous, recurrent superficial migratory thrombophlebitis associated with occult cancers, and this was later correlated with disseminated microangiopathy (platelet-rich clots in small blood vessels). Trousseau syndrome often occurs with mucinous adenocarcinomas, which secrete abnormally glycosylated mucins and mucin fragments into the bloodstream. Since carcinoma mucins can have binding sites for selectins, we hypothesized that selectin-mucin interactions might trigger this syndrome. When highly purified, tissue-factor free carcinoma mucin preparations were intravenously injected into mice, platelet-rich microthrombi were rapidly generated. This pathology was markedly diminished in P- or L-selectin-deficient mice. Heparin (an antithrombin-potentiating agent that can also block P- and L-selectin recognition of ligands) ameliorated this platelet aggregation, but had no additional effect in P- or L-selectin-deficient mice. Inhibition of endogenous thrombin by recombinant hirudin also did not block platelet aggregation. Mucins generated platelet aggregation in vitro in hirudinized whole blood, but not in platelet-rich leukocyte-free plasma nor in whole blood from L-selectin-deficient mice. Thus, Trousseau syndrome is likely triggered by interactions of circulating carcinoma mucins with leukocyte L-selectin and platelet P-selectin without requiring accompanying thrombin generation. These data may also explain why heparin ameliorates Trousseau syndrome, while vitamin K antagonists that merely depress thrombin production do not.

Production of recombinant hirudin in galactokinase-deficientSaccharomyces cerevisiae by fed-batch fermentation with continuous glucose feeding

The artificial gene coding for anticoagulant hirudin was placed under the control of theGAL10 promoter and expressed in the galactokinase-deficient strain (Δgal1) ofSaccharomyces cereivisiae, which uses galactose only as a gratuitous inducer in order to avoid its consumption. For efficient production of recombinant hirudin, a carbon source other than galactose should be provided in the medium to support growth of the Δgal1 strain. Here we demonstrate the successful use of glucose in the fed-batch fermentation of the Δgal1 strain to achieve efficient production of recombinant hirudin, with a yield of up to 400 mg hirudin/L.

Antihirudin antibodies following low-dose subcutaneous treatment with desirudin for thrombosis prophylaxis after hip-replacement surgery: incidence and clinical relevance

Recombinant hirudin has been found to be immunogenic in patients treated with lepirudin following heparin-induced thrombocytopenia (HIT). We assessed the incidence of immunoglobulin G (IgG) antihirudin antibodies by enzyme-linked immunosorbent assay in 112 patients enrolled in a dose-finding study with desirudin. Patients received desirudin subcutaneously following orthopedic hip surgery at 10 mg twice a day (n = 17), 15 mg twice a day (n = 75), and 20 mg twice a day (n = 20). Of 112 patients, 11 (9.8%) developed antihirudin antibodies independently of the dose. The rate of immunization did not differ from that observed in HIT patients treated with lepirudin (P =.113). Plasma concentrations of desirudin did not differ between antihirudin antibody-positive and -negative patients. Antihirudin antibodies had no impact on incidences of deep vein thrombosis and/or pulmonary embolism, allergic reactions, and hemorrhage. However, the total number of immunized patients observed was low and so infrequent (but severe) effects of antihirudin antibodies cannot be excluded.

Finding of an isoleucine derivative of a recombinant protein for pharmaceutical use

Protein modification generally occurs by addition to the amino acid side-chains of protein at the post-translational stage, for example, by enzymatic or chemical reactions after polypeptide synthesis. Recently, the recombinant hirudin analog CX-397, a potent thrombin inhibitor, was found to contain methylated Ile residues when it was overproduced by Escherichia coli in the absence of amino acids in the culture medium. The Ile derivatives, deduced to be β-methylnorleucine [βMeNle; (2 S, 3 S)-2-amino-3-methylhexanoic acid] by systematic chromatographic analysis, do not appear to be normal post-translational modifications of the protein because Ile has no functional group in its side-chain. We, therefore, propose that βMeNle is biosynthesized by E. coli, activated by E. coli isoleucyl-tRNA synthetase (IleRS), then incorporated into the overproduced recombinant hirudin analog. The biosynthesis of βMeNle in E. coli is thought to occur as follows: α-ketovalerate is synthesized from α-ketobutyrate by three Leu biosynthetic enzymes, α-isopropylmalate synthase (IPMS) (EC 4.1.3.12), α-isopropylmalate isomerase (ISOM) (EC 4.2.1.33) and β-isopropylmalate dehydrogenase (IPMD) (EC 1.1.1.85), which have broad substrate specificities. α-Ketovalerate is then converted to α-keto-β-methylcaproate by three Ile and Val biosynthetic enzymes, acetohydroxy acid synthase (AS) (EC 4.1.3.18), acetohydroxy acid isomeroreductase (IR) (EC 1.1.1.86) and dihydroxy acid dehydratase (DH) (EC 4.2.1.9). Finally, this is converted to βMeNle by branched-chain amino acid transaminase (EC 2.6.1.42), one of the Ile and Val biosynthetic enzymes.

Effects of ximelagatran, an oral direct thrombin inhibitor, r-hirudin and enoxaparin on thrombin generation and platelet activation in healthy male subjects

ObjectivesThe effects of ximelagatran, an oral direct thrombin inhibitor (DTI), recombinant hirudin (r-hirudin) and enoxaparin on thrombin generation and platelet activation were studied in humans. BackgroundRecombinant hirudin (parenteral DTI) and enoxaparin (low molecular weight heparin) have been demonstrated to be clinically effective in acute coronary syndromes. Ximelagatran is currently under investigation for the prevention and treatment of thromboembolism. The shed blood model allows for the study of thrombin generation and platelet activation in humans in vivo. MethodsThis was an open-label, parallel-group study involving 120 healthy male volunteers randomized to receive one of three oral doses of ximelagatran (15, 30 or 60 mg), r-hirudin (intravenous) or enoxaparin (subcutaneous) at doses demonstrated to be clinically effective in acute coronary syndromes, or to serve as a control. Thrombin generation (prothrombin fragment 1+2 [F1+2] and thrombin-antithrombin complex [TAT]) and platelet activation (β-thromboglobulin [β-TG]) biomarkers were studied using a shed blood model involving blood collection from skin incisions made using standardized bleeding time devices. ResultsOral ximelagatran, intravenous r-hirudin and subcutaneous enoxaparin rapidly and significantly (p < 0.05) decreased F1+2, TAT and β-TG levels in shed blood, indicating inhibition of thrombin generation and platelet activation. Statistically significant concentration (melagatran, the active form of ximelagatran)-response relationships for F1+2 (p = 0.005), TAT (p = 0.005) and β-TG (p < 0.001) levels, with IC50s of 0.376 (F1+2), 0.163 (TAT) and 0.115 (β-TG) μmol/l, were detected. Melagatran showed dose-proportional pharmacokinetics with low variability. All drugs were well tolerated. ConclusionsOral administration of the DTI ximelagatran resulted in a rapid inhibition of both thrombin generation and platelet activation in a concentration-dependent manner using a human shed blood model. The inhibition of thrombin generation by 60 mg ximelagatran was comparable to that observed with doses of r-hirudin and enoxaparin demonstrated to be effective for the treatment of acute coronary syndromes.

Sensitively Detecting Recombinant Hirudin Variant-2 with Capacitive Immunoassay Based on Self-Assembled Monolayers

Recombinant hirudins have a definite role in patients with heparin-induced thrombocytopenia, and prevent coronary thrombosis and recurrences. Here, capacitive immunosensors based on self-assembled monolayers (SAMs) were developed to immobilize polyclonal recombinant hirudin antibodies on gold electrodes for detecting recombinant hirudin variant-2 (r-HV2), a low-weight protein, directly and sensitively. A fast electrochemical measurement, potentiostatic step method, was applied to measure the capacitance changes caused by the interaction between the antigens and antibodies. The insulating properties of the immunosensors were investigated by cyclic voltammetry in a buffer solution containing (Fe(CN)6)3−/4−. The detection capability of the immunosensors modified by short-chain alkanethiol, thioctic acid (TA), or mercaptoacetic acid (MAA), were compared. Based on TA SAMs, the immunosensor could detect r-HV2 in the range from 100 pg/mL to 5 ng/mL, with a detection limit of 50 pg/mL. For the MAA modified immunosensor, a linear detection range from 5 pg/mL to 1 ng/mL was obtained, with a detection limit of 2.5 pg/mL. This indicated capacitive immunosensors with thinner SAMs could detect the antigens with higher sensitivity. The success of detecting r-HV2 showed that the capacitive immunoassay might be employed in the practical application with characteristics of simplicity and sensitivity.

Microvascular repair following crush-avulsion type injury with vein grafts: effect of direct inhibitors of thrombin on patency rate.

The aim of this study was to develop a standardized effective thrombogenic arterial anastomosis model, as usually encountered in clinical practice, and to offer a detailed evaluation of the antithrombotic effect of thrombin's direct inhibitors, antithrombin III and hirudin, as locally applied. Wistar rats were divided into four groups of 12 animals each. The carotid artery sustained a standardized crush-avulsion-type injury (groups B-D). A segment of the afflicted area was removed and replaced by a microvenous graft. Group A had no crush-avulsion injury inflicted; a microvenous graft replaced a simple resection from the center of the carotid artery. During microvascular anastomoses, normal saline (groups A and B), recombinant hirudin (group C), or antithrombin III (group D) were locally applied. Bleeding times were recorded, and patency tests were performed 20 min, 48 h, and 1 week after blood flow reestablishment. All grafts were harvested and examined histologically. Patency tests, 1 week postrevascularization, demonstrated that this experimental crush-avulsion injury model ensured low patency in group B (25%), whereas group A, which had no injury inflicted, achieved a 100% patency rate. The local application of hirudin and antithrombin III significantly increased bleeding times as well as the patency rate (92% and 75%, respectively) compared to group B. These findings indicate the efficiency of the experimental model and the potential use of thrombin's direct inhibitors in microvascular surgery.

Antithrombin activity of lepirudin adsorbed to silicone (polydimethylsiloxane) tubing

Long-term intravenous catheters, or venous access deviallows them to adsorb to both polar and hydrophobic ces (VADs), are widely used in the care of chronically ill patients to administer medications and blood products and to obtain blood samples for laboratory testing [1]. Although the intravascular component of VADs is made of elastomers (e.g., silicone rubber, polyurethane) selected for their physical and chemical properties to be nonreactive with blood, a major complication of VADs continues to be thrombosis [2,3]. The pathogenesis of VAD-related thrombosis is undoubtedly multifactorial, but there is evidence that the catheter material itself is thrombogenic. Within minutes, for example, the catheters adsorb coagulation proteins from whole blood [4], and adsorption of fibrinogen leads to platelet adhesion [5]. Furthermore, VADs removed from patients are rich with thrombin, which presumably supports ongoing coagulation and platelet aggregation at the catheter surface [6]. In an attempt to reduce the thrombogenicity of VADs, catheter polymers have been chemically modified to bind heparin [7,8]. Heparin was the only practical anticoagulant available for treating catheters until the direct thrombin inhibitors, recombinant hirudins and argatroban, were introduced into clinical use [9,10]. The potential for the newer anticoagulants for promoting the thromboresistance of VADs has received limited attention [7,11]. Because the direct thrombin inhibitors are proteins or amino acid derivatives rather than glycosaminoglycans, their physical interaction with the surface of catheters is different from that of heparin. The amphipathic property of proteins in particular

Effect of non-heparin thrombin antagonists on thrombin generation, platelet function, and clot structure in whole blood.

Platelet contractile force (PCF), which is absent in blood obtained during cardiopulmonary bypass, significantly recovers after protamine sulfate administration. In vitro studies reveal this effect to be primarily caused by heparin. Because many of heparin's effects are mediated by suppression of thrombin generation and activity, this study assessed the influence of thrombin inhibition on PCF. The effects of natural and synthetic antithrombins were measured. Clots were formed by the addition of batroxobin (0.21 microg/mL) to whole blood (platelet count 200,000/microL). Force development was measured from the moment of batroxobin addition. After 1200 s of clotting, purified antithrombin III (22 microM) reduced PCF by 74%. Thrombomodulin (0.014 microM) reduced PCF by 60%. At 0.040 microM, PCF was reduced by 82% (6.5-1.2 Kdynes). Hirudin decreased PCF in a dose-dependent fashion, with complete suppression at concentrations > or = 0.30 microM. At concentrations between 0.04 and 0.29 microM, Lepirudin (Refludan, a recombinant therapeutic hirudin) produced dose-dependent delay and suppression of PCF. Above 0.29 microM Lepirudin, PCF was totally suppressed. At 1.60 microM, bivalirudin (a synthetic, 20 amino acid peptide) delayed and reduced PCF by 50%. At 6.40 micro;M, PCF was completely suppressed. Although 20 microM of P-PACK II (d-Phenylalanyl-L-Phenylalanylarginine- chloro-methyl ketone 2 HCl) had little effect, 40 microM delayed onset of force development from 300 to 600 s and reduced PCF at 1200 s from 5.2 to 3.3 Kdynes. At 120 microM, force development was totally suppressed. Four micromol Thromstop (BNas-Gly-(pAM)Phe-Pip) delayed force development by greater than 800 s and PCF at 1200 s was reduced by 70%. At 0.20 microM, Argatroban (a synthetic polypeptide direct thrombin antagonist) delayed onset of PCF from 300 to 540 s and decreased PCF by 40%. At a concentration of 0.40 microM and above, Argatroban totally suppressed PCF. These results indicate that some of the antiplatelet effects of heparin are the result of thrombin inhibition and that low-level thrombin generation is essential for clot retraction. The sensitivity of PCF to the presence of thrombin may permit monitoring of antithrombin agents via this assay.