Demetra Callas

Antithrombin Agents: The New Class of Anticoagulant and Antithrombotic Drugs

The plasma levels of soluble thrombomodulin (TM) were measured in 44 patients with chronic myeloprolif erative disorder, 15 with polycythemia vera (PV), 29 with es sential thrombocythemia (ET), and a group of 62 matched healthy controls. The younger patients had significantly lower TM levels (mean: 15.6 ± 4.8 ng/mL) than the older patients (mean: 28.6 ± 8.2 ng/mL, p < .001). Moreover, a significant negative correlation between platelet counts and plasma TM levels in healthy persons was noted (r = 0.317, p < .05). The only significant difference we found in plasma TM levels be tween patients and controls or among patients was between the young patients with ET (mean: 29.0 ± 19.2 ng/mL) and young healthy controls (mean: 15.6 ± 4.8 ng/mL). It is possible that younger ET patients with more active platelets are more sus ceptible to earlier vascular damage. The lack of any significant difference compared with the older patient population supports this hypothesis. Key Words: Thrombomodulin—Essential thrombocythemia—Polycythemia vera.

Fibrinolytic compromise by simultaneous administration of site-directed inhibitors of thrombin

Newly developed synthetic and recombinant thrombin inhibitors possess strong anticoagulant effects. Despite these effects, interactions of these agents with enzymes in the fibrinolytic network result in the modulation of such proteases as t-PA, u-PA and streptokinase. The inhibitory spectrum of several thrombin inhibitors [D-Phe-Pro-Arg-H(GYKI 14166), D-MePhe-Pro-Arg-H(GYKI 14766), Boc-D-Phe-Pro-Arg-H (GYKI 14451), Ac-D-Phe-Pro-boroArg-OH (DuP 714), recombinant hirudin (r-Hir) and unfractionated porcine mucosal heparin complexed with antithrombin III (Heparin/AT-III)] was studied towards various serine proteases such as tissue plasminogen activator (t-PA), plasmin, plasminogen/streptokinase complex, urokinase and kallikrein. Aprotinin was also studied in the same systems as the thrombin inhibitors. All four tripeptide derivatives were found to inhibit t-PA, plasmin and plasminogen/streptokinase complex at micromolar concentrations (IC50: 0.57 mM-3.3 microM). Boc-D-Phe-Pro-Arg-H and Ac-D-Phe-Pro-boroArg-OH also inhibited urokinase, while Ac-D-Phe-Pro-boroArg-OH inhibited kallikrein as well (IC50: 0.15 mM-16 microM). In contrast, r-Hir and Heparin/AT-III did not inhibit any of these enzymes at millimolar concentrations (IC50 > or = 1 mM). Aprotinin inhibited plasmin, plasminogen/streptokinase complex and kallikrein at micromolar concentrations (IC50: 3.1-0.85 microM). In a rabbit thrombolysis model, where pre-formed clots are lysed by streptokinase, simultaneous administration of D-MePhe-Pro-Arg-H or Ac-D-Phe-Pro-boroArg-OH, at concentrations approximately 1 mumol/kg, i.v. resulted in complete inhibition of the fibrinolytic process. Aprotinin at 0.1 mumol/kg, i.v. produced similar inhibition. These results demonstrate that thrombin inhibitors may exert significant antiprotease actions against various fibrinolytic enzymes.

Section Review—Cardiovascular & Renal: Recent Developments in Antithrombotic Agents

During the past decade, many significant developments in the clinical management of thrombotic and vascular disorders have occurred. In particular, several newer approaches for the prophylactic and therapeutic management of such disorders as venous thrombosis, acute myocardial infarction and stroke have been introduced. This has only been possible due to the understanding of the molecular mechanisms involved in the thrombogenic process which plays a key role in the pathophysiology of thrombotic and vascular disorders. With the increased knowledge of the pathophysiology of thrombosis have come advances in drug treatment possibilities. Advances in biotechnology and separation techniques have contributed to the development of many newer antithrombotic, anticoagulant and thrombolytic drugs. Many new drugs and devices based on newer concepts are currently being tested in various clinical trials. Hirudin, hirulog, Gpllb/llla targeting antibodies and tissue factor pathway inhibitor (TFPI), are some examples. Fro...

ANTITHROMBIN AGENTS AS ANTICOAGULANTS AND ANTITHROMBOTICS: Implications in Drug Development

Synthetic and recombinant thrombin inhibitors have undergone several clinical evaluations for thrombotic and cardiovascular indications. While the initial trials were focused in coronary indications, more recently, these agents are also developed for the prophylaxis and therapeutic management of thromboembolic disorders. Hirudin, PEG-hirudin and argatroban are in advanced clinical development. Recombinant hirudin has been approved in Europe as a substitute anticoagulant for the management of HIT patients. Several additional clinical trials are currently carried out to demonstrate the usefulness of these agents in thrombotic and cardiovascular indications. Despite these developments such issues as dosage optimization, laboratory monitoring, neutralization and drug interactions require additional studies for the optimal development of these drugs.

Direct inhibition of protein Ca by site directed thrombin inhibitors : implications in anticoagulant and thrombolytic therapy

Abstract Several synthetic and recombinant antithrombin agents are currently developed for anticoagulation during thrombolytic therapy. Many of these agents are designed to inhibit thrombin at the active site. This active site is similar in enzymes belonging to the family of serine proteases. Since many of the thrombolytic enzymes are serine proteases (the class of enzymes in which thrombin and most of the coagulation enzymes are included), the newly developed antithrombin agents may also inhibit their pharmacologic actions and compromise thrombolytic efficacy. For this reason, it is important to compare the relative antithrombin and antifibrinolytic actions of these agents. A systematic investigation of the antiprotease profile and inhibitory spectrum against fibrinolytic enzymes is also important. Although several thrombin inhibitors have been compared in their ability to inhibit various enzymes involved in the activation of fibrinolysis (1), no reports on the interactions of these inhibitors with activated protein C are available. Since protein Ca has a dual role, both as an anticoagulant in the coagulation cascade (by inactivating factors Va and VIIIa) and as a profibrinolytic enzyme (by inactivating PAI-1 and PAI-III and thus stimulating the activity of t-PA) (2), it is important that its activity is not compromised by thrombin inhibitors, for optimal coagulation and fibrinolytic state. The current studies are therefore designed to investigate the direct inhibitory effects of synthetic and recombinant antithrombin agents on the amidolytic action of protein Ca. Human activated protein C has been made available by plasma fractionation and thus the inhibitory effects of thrombin inhibitors in a defined biochemical system was possible to investigate.

Modulation of platelet function and vascular smooth muscle contractile actions by a novel, selective, highly potent 5-HT2 antagonist (SR46349)

SR46349 is a novel, selective 5-HT2 receptor antagonist with the chemical structure {trans, 4-[(3Z)3-(2-dimethylaminoethyl) oxyimino-3(2-flurophenyl) propen-1-yl]phenol hemifumarate}. This agent has been found to exhibit antithrombotic actions in animal models of thrombosis. In order to investigate the effects of this agent on agonist induced vascular smooth muscle contraction, we utilized rabbit aortic ring and rat aortic strip preparations. Serotonin and platelet rich plasma (PRP) activated with arachidonic acid (AA) were used to determine the modulatory effect of SR46349. The IC50 for SR46349 was found to be: 1) rabbit aortic ring: 0.4 +/- 0.1 ng/ml for 5-HT and 0.25 +/- 0.05 ng/ml for PRP/AA. 2) rat aortic strip: 0.5 +/- 0.1 ng/ml for 5-HT and 0.3 +/- 0.1 ng/ml for PRP/AA. These results indicate that SR46349 is a highly potent inhibitor of aortic smooth muscle contraction. To further study the structure-activity relationship, we utilized the cis derivative of this agent, SR46615. This agent was found to be a relatively weaker inhibitor of the agonist induced aortic smooth muscle contraction. The studies reported here provide also comparative data on ketanserin, ritanserin and two new serotonin antagonists on the smooth muscle modulatory actions.

Synthetic and recombinant antithrombin drugs.

As the final enzyme in the activation of the coagulation system, the serine protease, thrombin, is believed to be an important target for the development of new anticoagulant/antithrombotic drugs. Direct thrombin inhibitors are either derived from natural sources, such as hirudin or are chemically synthesised, such as argatroban. The coupling of hirudin or parts of it with other entities leads to novel agents with different pharmacokinetic and pharmacodynamic characteristics, such as polyethylene glycol (PEG)-hirudin or the hirulogs. Due to the reversible or irreversible inactivation of the enzyme, thrombin inhibitors exert strong anticoagulant effects that can be measured in global clotting assays. Furthermore, these compounds inhibit thrombin-induced platelet reactions and influence other cellular, receptor-mediated actions of thrombin, e.g., on vascular cells. Directly acting thrombin inhibitors prevent blood clotting and are also capable of inhibiting clot-associated thrombin; however, they do not effectively block the further generation of the enzyme. Comprehensive experimental studies suggest that thrombin inhibitors may be effective drugs in a wide range of intravascular thrombus formation, also including the inhibition of vascular restenosis. Recent clinical trials revealed the effectiveness of direct thrombin inhibitors in various thrombotic and cardiovascular indications, but also a tendency to an increased risk of bleeding complications. At present, thrombin inhibitors are the most promising class of drugs for the initial therapy of patients with heparin-induced thrombocytopaenia (HIT) or the heparin-induced thrombocytopaenia and thrombosis syndrome (HITTS). They are also useful for the management of venous thrombosis and for acute ischaemic syndromes as well as for invasive procedures. However, with regard to the long-term outcome, a superiority of thrombin inhibitors over heparin has not yet been demonstrated. Several important issues, such as monitoring, pharmacological antagonism and drug interactions will also play an important role in the development of these new drugs. Further clinical trials are required to confirm the effectiveness of direct thrombin inhibitors in the prophylaxis and treatment of various thromboembolic and cardiovascular disorders.

Fibrinolytic Compromise by Synthetic and Recombinant Thrombin Inhibitors: Implications in the Treatment of Thrombotic Disorders

Site-directed synthetic and recombinant antithrombin agents are widely used to prevent reocclusion during thrombolytic therapy in acute myocardial infarction and other vascular occlusive disorders. However, minimal studies have been conducted to examine the interactions between these anticoagulant and thrombolytic agents. Because of the structural homology of serine proteases, some of these newly developed antithrombin agents are also capable of inhibiting fibrinolytic enzymes, which may lead to fibrinolytic compromise during thrombolytic therapy. In addition, inhibition of thrombomodutin-bound thrombin may also result in fibrinolytic deficit. Several thrombin inhibitors were studied in in vitro systems to assess whether their inactivating properties extend to fibrinolytic and profibinolytic enzymes, such as kallikrein, plasmin, urokinase, streptokinase, and tissue plasminogen activator. The thrombin inhibitors studied included hirudin, hirulog-I, argatroban, D-MePhe-Pro-Arg-H, and Ac-D-Phe-Pro-boroArg-OH. Their activities were compared with those of aprotinin, which is currently used clinically as an antifibrinolytic agent. Although argatroban, hirulog-I, and hirudin exhibited minimal inhibition of the nonthrombin enzymes studied, the tripeptide derivatives showed variable inhibitory activities, with the boronic acid derivative being the most potent and universal inhibitor. The in vivo antifibrinolytic activities of these thrombin inhibitors were also studied in a rabbit model of jugular vein clot lysis. In agreement with the in vitro studies, argatroban, hirulog-I, and hirudin exhibited minimal antifibrinolytic activities, while Ac-D-Phe-Pro-boroArg-OH and D-MePhe-Pro-Arg-H showed marked inhibition of the thrombolytic process. The results of these studies indicate that newly developed thrombin inhibitors with a broader serine protease spectrum may exhibit fibrinolytic compromise resulting in diminishment of the expected thrombolytic outcome. Key Words: Thrombolysis—Fibrinolysis—Thrombin inhibitors—Argatroban—Hirudin—Hirulog-I—Aprotinin.

Molecular weight and biochemical profile of a chemically modified heparin derivative, Suleparoide®

Recently, a new chemically modified derivative of heparin (Suleparoide, Syntex Laboratories Buenos Aires, Argentina) was introduced for the prophylaxis of thrombosis and treatment of vascular disorders. This agent is claimed to contain a depolymerized, chemically modified, heparin derivative with similar biologic actions as heparan sulfate. To study the pharmacologic profile of this agent, we have defined its molecular weight distribution profile, utilizing a computerized gel permeation chromatographic system equipped with ultraviolet and refractive index detectors. Suleparoide exhibited a normal molecular distribution profile with no contaminants. It exhibited a weight average of 9.3 K DA and an apparent peak MW of 8.0 K DA. Approximately 50% of the molecular components were < 5.0 K DA and 40% > 5.0 K DA. The results from these studies on the mechanisms show that Suleparoide has anticoagulant activity primarily mediated through Heparin Cofactor-II (HC-II) and because of its novel mechanism of action, further investigations on the biochemical profile of Suleparoide are carried out. Global clotting tests such as Activated Partial Thromboplastin Time (APTT), Heptest and Thrombin Time (TT) revealed a concentration dependent effect in all assays. Plasma samples supplemented with Suleparoide exhibited no significant anti-Xa and anti-IIa activities. However, in the HC-II mediated inhibitory assay for IIa, Suleparoide exhibited significant activity. In contrast, the Antithrombin-III (AT-III) mediated inhibition of IIa was much weaker.