Affinity For Thrombin Research Articles

Potent and selective bicyclic lactam inhibitors of thrombin. Part 4: transition state inhibitors

Bicyclic piperazinone based thrombin inhibitors of general structure 2 were prepared and evaluated in vitro and in vivo. These inhibitors, having in common an electrophilic basic trans-cyclohexylamine P1 residue, displayed high thrombin affinity, high selectivity against trypsin and good in vivo efficacy in the rat arterial thrombosis model.

Both the High Affinity Thrombin Receptor (GPIb-IX-V) and GPIIb/IIIa Are Implicated in Expression of Thrombin-induced Platelet Procoagulant Activity

Platelets activated by alpha-thrombin express surface procoagulant activity (PCA) that accelerates the conversion of prothrombin to alpha-thrombin. Following activation with 10 nM alpha-thrombin, the PCA of normal platelets was approximately five-fold higher than that of Bernard-Soulier platelets (lacking GPIb). Normal platelet PCA was inhibited approximately 50% by activation in the presence of the anti-GPIb MoAbs LJIb10 or TM60. Moreover, normal platelet PCA was completely abrogated in the presence of a combination of both LJIb10 and c7E3, a MoAb directed against alphaIIbbeta3 (GPIIb/IIIa). In contrast. PCA expressed by Bernard Soulier or Glanzmann platelets was not inhibited by either LJIb10 or c7E3 MoAb. The platelet activating peptide SFLLRN at 10 microM, a concentration which fully activates platelet aggregation and Ca2+ mobilization, generated PCA activity one fifth of that generated by alpha-thrombin at 10 nM but anti-PAR1 antibodies did not affect thrombin-induced PCA expression. These results demonstrate that GPIb mediates, at least in part, the thrombin-induced activation of platelets that leads to PCA, and that alphaIIbbeta3 is also involved in PCA generation, but these results do not support a major role for PAR1 in this activation.

The Amino Acid Sequence in Fibrin Responsible for High Affinity Thrombin Binding

Human fibrin has a low affinity thrombin binding site in its E domain and a high affinity binding site in the carboxy-terminal region of its variant gamma' chain (gamma'408-427). Comparison of the gamma' amino acid sequence (VRPEHPAETEYDSLYPEDDL) with other protein sequences known to bind to thrombin exosites such as those in GPIbalpha, the platelet thrombin receptor, thrombomodulin, and hirudin suggests no homology or consensus sequences, but Glu and Asp enrichment are common to all. Tyrosine sulfation in these sequences enhances thrombin exosite binding, but this has not been uniformly investigated. The fibrinogen gamma' chain mass determined by electrospray ionization mass spectrometry, was 50,549 Da, a value 151 Da greater than predicted from its amino acid/carbohydrate sequence. Since each sulfate group increases mass by 80 Da, this indicates that both tyrosines at 418 and 422 are sulfated. A series of overlapping gamma' peptides was prepared for evaluation of their inhibition of 125I-labeled PPACK-thrombin binding to fibrin. gamma'414-427 was as effective an inhibitor as gamma'408-427 and its binding affinity was dependent on all carboxy-terminal residues. Mono Tyr-sulfated peptides were prepared by substituting non-sulfatable Phe for Tyr at gamma'418 or 422. Sulfation at either Tyr residue increased binding competition compared with non-sulfated peptides, but was less effective than doubly sulfated peptides, which had 4 to 8-fold greater affinity. The reverse gamma' peptide or the forward sequence with repositioned Tyr residues did not compete well for thrombin binding, indicating that the positions of charged residues are important for thrombin binding affinity.

Pass the protease: new interactions between GPCRs

When is a G-protein-coupled receptor (GPCR) not a GPCR? When, according to the report by Nakanishi-Matsui et al.1xPAR3 is a cofactor for PAR4 activation by thrombin. Nakanishi-Matsui, M. et al. Nature. 2000; 404: 609–613Crossref | PubMed | Scopus (364)See all References1, it instead acts as a non-signalling cofactor, aiding the activation of another GPCR. Such novel cooperativity appears to occur during thrombin activation of platelets in the mouse.Thrombin, a serine protease and the main enzyme of the blood coagulation cascade, acts directly on platelets via protease-activated receptors (PARs). By slicing off the N-terminal portion of the PAR protein, thrombin releases ‘tethered’ ligand sequences that auto-activate the receptor and its associated G protein. Mouse platelets express subtypes PAR3 and PAR4, PAR3 having greater affinity for thrombin. But surprisingly, when expressed alone, PAR3 is unresponsive to thrombin, whereas PAR4 responds only to unnaturally high thrombin concentrations. Might PAR3 and PAR4 therefore act together to mediate thrombin’s effects?Nakanishi-Matsui et al. showed that expressing the genes encoding PAR3 and PAR4 together drastically lowered the threshold for thrombin activation of phosphoinositide by increasing the rate of PAR4 cleavage. To test the theory that PAR3 might be contributing its bound thrombin to cleave a neighbouring PAR4, chimaeric PAR molecules were constructed with the thrombin-binding region of PAR3 inserted into the N-terminal domain of PAR4. On expression, these too displayed low thresholds for thrombin activation. A final convincing piece of evidence, this time from actual mouse platelets, was that desensitization of PAR4 by a PAR4-specific agonist abolished thrombin signalling.The authors propose a mechanism, novel for GPCRs, whereby PAR3, PAR4 and thrombin form a ternary complex. Thrombin first binds to and cleaves PAR3, which then unselfishly donates its captured thrombin to PAR4 without itself becoming activated. The role of PAR3 as a cofactor for PAR4 activation might serve to enhance the specificity of thrombin’s actions. Although this mechanism appears to offer an additional stage for pharmacological regulation of coagulation, cooperativity appears to be absent from human platelets, which express PAR1 and PAR4, but not PAR3. One straightforward verification of the cofactor model will be a complete absence of thrombin signalling in platelets derived from the awaited mPAR4 knockout mice.

Characterization of Proexosite I on Prothrombin

Activation of prothrombin by factor Xa is accompanied by expression of regulatory exosites I and II on the blood coagulation proteinase, thrombin. Quantitative affinity chromatography and equilibrium binding studies with a fluorescein-labeled derivative of the exosite I-specific peptide ligand, hirudin(54-65) ([5F]Hir(54-65) (SO(3)(-)), were employed to identify and characterize this site on human and bovine prothrombin and its expression on thrombin. [5F]Hir(54-65)(SO(3)(-)) showed distinctive fluorescence excitation spectral differences in complexes with prothrombin and thrombin and bound to human prothrombin and thrombin with dissociation constants of 3.2 +/- 0.3 micrometer and 25 +/- 2 nm, respectively, demonstrating a 130-fold increase in affinity for the active proteinase. The bovine proteins similarly showed a 150-fold higher affinity of [5F]Hir(54-65)(SO(3)(-)) for thrombin compared with prothrombin, despite a 2-5-fold lower affinity of the peptides for the bovine proteins. Unlabeled, Tyr(63)-sulfated and nonsulfated hirudin peptides bound competitively with [5F]Hir(54-65)(SO(3)(-)) to human and bovine prothrombin and thrombin, exhibiting similar, 40-70-fold higher affinities for the proteinases, although nonsulfated Hir(54-65) bound with 7-17-fold lower affinity than the sulfated analog. These studies characterize proexosite I for the first time as a specific binding site for hirudin peptides on both human and bovine prothrombin that is present in a conformationally distinct, low affinity state and is activated with a approximately 100-fold increase in affinity when thrombin is formed.

Recombinant fragment of von Willebrand factor AR545C inhibits platelet binding to thrombin and platelet adhesion to thrombin-treated endothelial cells.

Activated, but not resting, platelets are capable of adhering to intact endothelial cells (ECs). We evaluated the effect of a recombinant von Willebrand factor (VWF) fragment AR545C, which inhibits glycoprotein Ib (GPIb)/VWF binding, on platelet adhesion to human ECs under static or flow conditions. Incubation of resting platelets with intact endothelium under flow conditions (350/s) resulted in minimal platelet adhesion. The adhesion was enhanced two- to threefold after either platelet activation by thrombin receptor agonist peptide (TRAP) or EC pretreatment with thrombin. The enhancing effect of thrombin was abolished by addition of either hirudin (10 u/ml) or PGE1 (1 microg/ml). Preincubation of resting platelets with increasing concentrations of AR545C under static or flow conditions resulted in a dose-dependent inhibition of thrombin-induced enhanced adhesion to ECs. AR545C (0.3 microM) completely abolished the effect of thrombin, reducing platelet adhesion to the control level observed with non-treated ECs. In contrast, the same concentration of AR545C had no effect on the adhesion of TRAP-activated platelets to ECs. AR545C also inhibited thrombin-induced platelet aggregation and binding in a dose-dependent manner. In addition, 0.3 microM of AR545C reduced thrombin-induced serotonin release by 57%, whereas monoclonal antibody AN51, which inhibits ristocetin-induced platelet aggregation, had no effect on either thrombin-induced platelet aggregation or binding or on serotonin release. Similarly, AR545C had no effect on TRAP-induced serotonin release. These findings suggest that (i) AR545C inhibits platelet activation mediated by thrombin and this inhibition occurs through blocking the high-affinity thrombin binding sites on the GPIb/IX complex and (ii) AR545C has no effect on the moderate affinity thrombin receptor (seven transmembrane domain thrombin receptor; STDR).

Bivalirudin: a new generation antithrombotic drug

Bivalirudin (Angiomax, The Medicines Company) is a synthetic 20 amino acid peptide rationally designed on the basis of structural studies of hirudin, a naturally occurring anticoagulant. Bivalirudin represents a new class of anticoagulant drugs that directly inhibits thrombin, a key component in blood clot formation and extension. With its high binding affinity and specificity for thrombin, bivalirudin acts directly on thrombin, rather than via other clotting factors. The compound has a variety of potential uses as an alternative to heparin in the management of cardiovascular disease and related medical procedures i.e., unstable angina (UA), myocardial infarction (MI) and percutaneous transluminal coronary angioplasty (PTCA).

Biphasic kinetics of activation and signaling for PAR1 and PAR4 thrombin receptors in platelets.

Thrombin activates platelets in an ordered sequence of events that includes shape change, increase in cytoplasmic Ca(2+), activation of the alphaIIbbeta3 integrin, granule secretion, aggregation, and formation of a stable hemostatic plug. Activation of this process has also been implicated in the pathogenesis of atherosclerosis, stroke, and thrombosis. There are two identified thrombin-activated receptors on the surface of human platelets. PAR1 is a high-affinity thrombin receptor, and PAR4 is a low apparent affinity thrombin receptor of uncertain function. The goal of these studies is to determine the kinetics of thrombin activation of PAR1 and PAR4 and to relate the individual inputs from each receptor to platelet Ca(2+) signaling, secondary autocrine stimulation, and aggregation. Using a combination of PAR-specific peptide ligands and anti-PAR1 reagents, we separated the biphasic thrombin Ca(2+) response of platelets into two discrete components-a rapid spike response caused by PAR1, followed by a slower prolonged response from PAR4. Despite having a 20-70-fold slower rate of activation, PAR4 produces the majority of the integrated Ca(2+) signal that is sustained by the continuous presence of catalytically active thrombin. Surprisingly, PAR4 activation is much more effective than PAR1 activation in mounting secondary autocrine Ca(2+) signals from secreted ADP. The strong ADP response due to activated PAR4, however, requires prior activation of PAR1 as would normally occur during treatment of platelets with thrombin. Thus, the late signal generated by activated PAR4 is not redundant with the early signal from PAR1 and instead serves to greatly extend the high intracellular Ca(2+) levels that support the late phase of the platelet aggregation process.

Extending the lifetime of anticoagulant oligodeoxynucleotide aptamers in blood

We have investigated (123)I and (125)I DNA aptamer analogs of anticoagulant DNA aptamers to thrombin exosite 1 and exosite 2 for thrombus imaging potential. Two severe problems are rapid clearance from circulating blood and blood nuclease. With aptamers (unlike antisense) the nucleotide analogs used in polymerase chain reaction-selection cycles also must be used in the radiotracer. We investigated 3'-biotin-streptavidin (SA) bioconjugates of the aptamers to alleviate these problems. Blood nuclease assays and biodistribution analysis were used in the mouse and rabbit. We found that 3'-biotin protected the aptamers significantly from blood nuclease in vitro, but it did not slow in vivo clearance. In contrast, the 3'-biotin-SA bioconjugates were resistant to blood nuclease in vitro and were also longer-lived (10-20 times) in vivo. Bioconjugate aptamers retained affinity for thrombin. Two solutions emerge: 1) In noncirculating blood (within a thrombus) 3'-biotin extends aptamer lifetime, whereas 2) in circulating blood (the transport medium), where more aggressive clearance is encountered, 3'-SA extends aptamer lifetime.

Effect of loop sequence and size on DNA aptamer stability.

The thrombin aptamer is a 15-mer oligodeoxyribonucleotide that folds into a unimolecular quadruplex consisting of a stack of two guanine quartets connected by two external loops and one central loop and possesses a high affinity for thrombin. We have undertaken a systematic examination, in KCl, of the thermodynamic stability of thrombin aptamer analogues containing sequence modifications in one or more of the loops, as well as in the number of quartets. UV melting studies have been carried out to obtain the relevant thermodynamic parameters for these aptamers. van't Hoff analysis of these data, with a two-state model for unimolecular denaturation, gave excellent fits to the experimental observations. Thermodynamic analysis indicates that the central loop sequence in the parent aptamer is optimal for stability. Modifications in this or other loops can effect either DeltaH degrees, DeltaS degrees, or both. Addition of a single G at the 5'-end decreases stability while addition of a G at the 3'-end increases stability. Differential scanning calorimetry experiments on the thrombin aptamer reveal that a heat capacity change, not detected by UV measurements, accompanies the unfolding of the aptamer.

Peptide modified gold‐coated polyurethanes as thrombin scavenging surfaces

Thin layers of gold were deposited on polyurethane film and chemisorbed with three peptides having an N-terminal cysteine: Cys-Pro-Arg, Cys-(L)Phe-Pro-Arg, and Cys-(D)Phe-Pro-Arg. The ability of these surfaces to act as thrombin scavengers was evaluated. The peptides are related to the known thrombin inhibitor Phe-Pro-Arg chloromethyl ketone and were shown to have significant thrombin inhibitory activity in solution. Attachment of the peptides to gold was confirmed by water contact angle and X-ray photoelectron spectroscopy measurements. Thrombin adsorption from a buffer and plasma was investigated, and chromogenic substrate assays were carried out for thrombin activity on the surfaces and in the supernatant following adsorption. The data suggest that the peptide-modified surfaces are able to adsorb thrombin with high affinity from a buffer and that thrombin is taken up selectively from plasma. The Cys-(D)Phe-Pro-Arg modified surfaces showed particularly high affinity for thrombin. It was also found that the activity of thrombin adsorbed on the peptide surfaces was inhibited, and inhibition was greatest on the Cys-(D)Phe-Pro-Arg surface. We concluded that the peptide surfaces may have potential as antithrombogenic materials via their ability to scavenge and inhibit thrombin generated as a result of blood–material contact. © 2000 John Wiley & Sons, Inc. J Biomed Mater Res, 49, 66–78, 2000.

Peptide modified gold-coated polyurethanes as thrombin scavenging surfaces.

Thin layers of gold were deposited on polyurethane film and chemisorbed with three peptides having an N-terminal cysteine: Cys-Pro-Arg, Cys-(L)Phe-Pro-Arg, and Cys-(D)Phe-Pro-Arg. The ability of these surfaces to act as thrombin scavengers was evaluated. The peptides are related to the known thrombin inhibitor Phe-Pro-Arg chloromethyl ketone and were shown to have significant thrombin inhibitory activity in solution. Attachment of the peptides to gold was confirmed by water contact angle and X-ray photoelectron spectroscopy measurements. Thrombin adsorption from a buffer and plasma was investigated, and chromogenic substrate assays were carried out for thrombin activity on the surfaces and in the supernatant following adsorption. The data suggest that the peptide-modified surfaces are able to adsorb thrombin with high affinity from a buffer and that thrombin is taken up selectively from plasma. The Cys-(D)Phe-Pro-Arg modified surfaces showed particularly high affinity for thrombin. It was also found that the activity of thrombin adsorbed on the peptide surfaces was inhibited, and inhibition was greatest on the Cys-(D)Phe-Pro-Arg surface. We concluded that the peptide surfaces may have potential as antithrombogenic materials via their ability to scavenge and inhibit thrombin generated as a result of blood-material contact.

Purification of a biologically active recombinant glyceraldehyde 3-phosphate dehydrogenase from Candida albicans.

We report here the purification of a functionally active recombinant glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from Candida albicans. The GAPDH protein encoded by the TDH1 gene was obtained as a glutathione S-transferase fusion protein by expression in the vector pGEX-4T-3, and purified by affinity chromatography and thrombin digestion. The purified protein displays GAPDH enzymatic activity (42 micromol NADH min(-1) mg(-1)) as well as the laminin and fibronectin binding activities previously described. In addition, the recombinant GAPDH is covalently modified by NAD linkage; this modification is stimulated by nitric oxide and probably involves a sulfhydryl group (cysteine) residue since it is inhibited by Hg(2+) and cysteine.

Potent bivalent thrombin inhibitors: replacement of the scissile peptide bond at P(1)-P(1)' with arginyl ketomethylene isosteres.

We have designed highly potent synthetic bivalent thrombin inhibitors, which consist of an active site blocking segment, a fibrinogen recognition exosite blocking segment, and a linker connecting these segments. The bivalent inhibitors bind to the active site and the fibrinogen recognition exosite simultaneously. As a result, the inhibitors showed much higher affinity for thrombin than the individual blocking segments. Various arginyl ketomethylene isosteres ArgPsi[CO-CH(2)-X]P(1)' were incorporated into the bivalent inhibitors as P(1)-P(1)' segment to eliminate the scissile bond. The P(1)' residue is a natural or unnatural amino acid; specifically, the incorporation of mercaptoacetic acid exhibited superiority in synthesis and affinity for thrombin. Inhibitor 16, (D-cyclohexylalanine)-Pro-ArgPsi[CO-CH(2)-S]Gly-(Gly)(4)-Asp-Tyr-G lu- Pro-Ile-Pro-Glu-Glu-Tyr-cyclohexylalanine-(D-Glu)-OH, showed the lowest K(i) value of 3.5 +/- 0.5 x 10(-13) M, which is comparable to that (K(i) = 2.3 x 10(-13) M) of recombinant hirudin. Consequently we successfully reduced the size of the inhibitor from approximately 7 kDa of recombinant hirudin to approximately 2 kDa without losing the affinity.

Identification of Bothrojaracin-like proteins in snake venoms from Bothrops species and Lachesis muta

Bothrojaracin, a 27 kDa protein isolated from Bothrops jararaca venom, forms a non-covalent complex with thrombin, thus blocking its activity. We have previously identified a bothrojaracin-like protein in B. alternatus venom [Castro, H.C., Dutra, D.L.S., Oliveira-Carvalho, A.L., Zingali, R.B., 1998. Bothroalternin, an inhibitor of thrombin from the venom of Bothrops alternatus. Toxicon 36, 1903-1912]. In this report, we have examined snake venoms from six different Bothrops species (B. atrox, B. cotiara, B. jararacussu, B. moojeni and B. neuwiedi), from Lachesis muta and from Crotalus durissus terrificus for the presence of bothrojaracin-like proteins, which we define here as 27 kDa proteins that are immunologically related to bothrojaracin and that inhibit thrombin-induced platelet aggregation. The immunological analysis of these venoms by different techniques indicated the existence of at least one protein recognized by anti-bothrojaracin serum in all venoms tested. Bothrojaracin-like proteins were purified from all crude venoms, except for C. d. terrificus, by a single-step procedure using a thrombin affinity column (PPACK-thrombin-Sepharose). Retained material that inhibits thrombin-induced platelet aggregation was found in a different proportion in each species. Under non-reducing conditions, SDS-PAGE of this material revealed several bands between 20-60 kDa; only those bands corresponding to 27 kDa were recognized by anti-bothrojaracin serum. ELISA confirmed the greater bothrojaracin immunoreactivity of proteins present in B. atrox and B. cotiara as compared to other Bothrops species. Smaller amounts of proteins related to bothrojaracin were found in L. muta venom and were absent from the venom of C. d. terrificus. Our results thus suggest that bothrojaracin-like proteins are widely distributed among Bothrops genera.

The interaction of thrombomodulin with Ca2+.

Thrombomodulin (TM) is a cofactor for protein C activation by thrombin and each residue of a consensus Ca2+ site in the sixth epidermal growth factor domain (EGF6) is essential for this cofactor activity [Nagashima, M., Lundh, E., Leonard, J.C., Morser, J. & Parkinson, J.F. (1993) J. Biol. Chem. 268, 2888-2892]. Three soluble analogs of the extracellular domain of TM, solulin (Glu4-Pro490), TME1-6 (Cys227-Cys462) and TMEi4-6 (Val345-Cys462) were prepared for equilibrium dialysis experiments by exhaustive dialysis against Ca2+-depleted buffer. However, all three analogs still contained one tightly bound Ca2+ (Kd approximately 2 microm), which could only be removed by EDTA. Epitope mapping with Ca2+-dependent monoclonal antibodies to EGF6 provided further localization of this tight Ca2+ site. Equilibrium dialysis of the soluble TM analogs in [45Ca2+] between 10 and 200 microm revealed a second Ca2+ site (Kd = 30 +/- 10 microm) in both solulin and TME1-6, but not in TMEi4-6. Ca2+ binding to this second site was unaffected by bound thrombin and we attribute it to the consensus Ca2+ site in EGF3. A 75-fold decrease in the binding affinity of thrombin to TM was observed with immobilized solulin treated with EDTA to remove the high affinity Ca2+ by measuring kassoc and kdiss rates in a BIAcoretrade mark instrument. Ca2+-dependent conformational transitions detected by CD spectroscopy in the far UV indicate a more ordered structure upon Ca2+ binding. Bound Ca2+ stabilized soluble TM against protease digestion at a trypsin-like protease-sensitive site between Arg456 and His457 in EGF6 compared with protease treatment in EDTA. Finally, TM containing EGF domains 4-6, but lacking the interdomain loop between EGF3 and 4 (TME4-6), has an identical Ca2+ dependence for the activation of protein C as found for TMEi4-6, indicating this interdomain loop is not involved in Ca2+ binding.

Design and synthesis of potent and selective 5,6-fused heterocyclic thrombin inhibitors

Thrombin, a serine protease, plays a central role in the initiation of thrombotic events. We report the design, synthesis, and antithrombotic efficacy of XU817 (7), a nonpeptide 5-(amidino) indole thrombin inhibitor. Utilizing the co-crystal structure of XU817 bound in the active site of thrombin we were able to synthesize analogs with enhanced thrombin affinity.

Argatroban as a potential anticoagulant in cardiopulmonary bypass-studies in a dog model

Argatroban is a selective thrombin inhibitor synthesized in Japan. Argatroban, which has a high affinity for thrombin, and markedly inhibits thrombin-induced reactions, has been used in patients with artherosclerosis obliterans. The efficiency of argatroban, instead of heparin, as an anticoagulant in dog models of cardiopulmonary bypass was explored. In the first study, argatroban was administered as a bolus plus infusion for 1 h during cardiopulmonary bypass at doses of 1.0 mg + 10 microg/kg per min, 2.0 mg + 10 microg/kg per min and 3.0 mg + 10 microg/kg per min (n = 2 per group). Activated clotting time and arterial gas analyses were performed beforehand and 10 min thereafter. In the second study, there were four groups. In the first group (n = 5), no coated extracorporeal circuit was used and heparin (2 mg/kg) was used as an anticoagulant. In the second group (n = 5), a coated extracorporeal circuit was used and heparin was used (2 mg/kg) as an anticoagulant. In the third group (n = 3), no coated extracorporeal circuit was used and argatroban (2.0 mg + 10 microg/kg per min) was used as an anticoagulant. In the fourth group (n = 5), a coated extracorporeal circuit was used and argatroban was used (2.0 mg + 10 microg/kg per min) as an anticoagulant. All animals were perfused for 120 min at 40 ml/kg per minute. Platelet count, activated clotting time, thrombin-antithrombin III complex, antithrombin III, fibrinogen, fibrinogen degradation products and C3a were measured to evaluate platelet, coagulofibrinolytic and the complement system. Activated clotting time values and the effect of argatroban during cardiopulmonary bypass indicated a dose-dependent response. The next highest dosing group (2.0 mg + 10 microg/kg per minute) had activated clotting time values of 250-300 seconds during cardiopulmonary bypass, and fell after reaching near-normal levels within 60 minutes. No clots were noted in the extracorporeal circuit. The argatroban group showed lower levels in their coagulofibrinolytic system compared with the heparin group. The platelet count remained at a high level in the argatroban group. It is concluded that the combination of heparinized cardiopulmonary bypass circuits, and the use of argatroban as an anticoagulant, is safe and reduces the activation of coagulation and fibrinolytic systems and preserves platelet count.

Argatroban as a Potential Anticoagulant in Cardiopulmonary Bypass-Studies in a Dog Model

Argatroban is a selective thrombin inhibitor synthesized in Japan. Argatroban, which has a high affinity for thrombin, and markedly inhibits thrombin-induced reactions, has been used in patients with artherosclerosis obliterans. The efficiency of argatroban, instead of heparin, as an anticoagulant in dog models of cardiopulmonary bypass was explored. In the first study, argatroban was administered as a bolus plus infusion for 1 h during cardiopulmonary bypass at doses of 1.0 mg + 10 μg/kg per min, 2.0 mg + 10 μg/kg per min and 3.0 mg + 10 μg/kg per min ( n = 2 per group). Activated clotting time and arterial gas analyses were performed beforehand and 10 min thereafter. In the second study, there were four groups. In the first group ( n = 5) no coated extracorporeal circuit was used and heparin (2 mg/kg) was used as an anticoagulant. In the second group ( n = 5). a coated extracorporeal circuit was used and heparin was used (2 mg/kg) as an anticoagulant. In the third group ( n = 3), no coated extracorporeal circuit was used and argatroban (2.0 mg + 10μg/kg per min) was used as an anticoagulant. In the fourth group ( n = 5), a coated extracorporeal circuit was used and argatroban was used (2.0 mg + 10 μg/kg per min) as an anticoagulant. All animals were perfused for 120 min at 40 ml/kg per minute. Platelet count, activated clotting time, thrombin—antithrombin III complex, antithrombin III. fibrinogen, fibrinogen degradation products and C3a were measured to evaluate platelet, coagulofibrinolytic and the complement system. Activated clotting time values and the effect of argatroban during cardiopulmonary bypass indicated a dose-dependent response. The next highest dosing group (2.0 mg + 10 μg/kg per minute) had activated clotting time values of 250–300 seconds during cardiopulmonary bypass and fell after reaching near-normal levels within 60 minutes. No clots were noted in the extracorporeal circuit. The argatroban group showed lower levels in their coagulofibrinolytic system compared with the heparin group. The platelet count remained at a high level in the argatroban group. It is concluded that the combination of heparinized cardiopulmonary bypass circuits, and the use of argatroban as an anticoagulant, is safe and reduces the activation of coagulation and fibrinolytic systems and preserves platelet count.

X-Ray Crystallographic Analyses of Human α-Thrombin Complexed to Peptidyl Aminophosphonates: Evidence of a Binding Mechanism

We sought to study O,O-Diphenyl dipeptidylaminophosphonates and their interaction with the coagulation protease, human α-thrombin. The ‘fibrinogen-like’ recognition sequence D-Phe-Pro-Arg, is a template of high affinity for thrombin. This was modified by replacing the ‘PI’ Arg by a neutral side chain, and the unnatural amino acid diphenylalanine was used in place of the ‘P3’ Phe. The tripeptides of general formula D-Dpa-Pro-NHCHRP(OPh)2 are highly selective for thrombin amongst other serine proteases; and, more importantly for the design of an efficacious anti-thrombotic agent, show particularly low activity toward other coagulation serine proteases of the cascade. However the kinetics of thrombin inhibition were seen to be dependent on the compounds' structure. To explain this we have studied compounds (1), where R is pentyl and compound (2) where R is (3-methoxy)propyl, for which the kinetics of inhibition were analysed as competitive (2 μM) and slow, tight-binding (final Ki 20nM) respectively. Analysis of the X-Ray crystal structure of these compounds complexed to human α-thrombin, unusually shows no covalent bond formed between the phosphorus nucleus and the serine 195 in the catalytic site of the enzyme. These observations are at variance to the proposed mechanism of action of other phosphorus based serine protease inhibitors.