Glycoprotein IIb/IIIa receptors play an important role in platelet aggregation by binding fibrinogen. Drugs that antagonize this binding have been developed for the treatment of thrombotic diseases including acute coronary syndrome. These drugs are of four main types: [1] monoclonal antibody, [2] synthetized peptide, [3] non-peptide, and [4] oral antagonist. Since these drugs inhibit the final step in platelet aggregation, i.e., binding of fibrinogen to the platelet membrane, they were expected to 1) have high specificity for inhibiting platelet aggregation, 2) inhibit any kind of aggregation regardless of stimulation, 3) have high potency in aggregation suppression. As a result of large clinical trials, however, monoclonal antibody alone was revealed to be effective, but non-peptide and oral antagonists were ineffective. The possible mechanisms that lead to such inefficiency are: 1) difference in binding duration to platelets, 2) concomitant suppression of other adhesion molecules beside IIb IIIa, 3) difference in binding site in the receptor and presence of a partial agonist effect, 4) aggregation promotion or induction of apoptosis via caspase-3 activation, 5) modified aggregation by gene polymorphism. New findings on platelet physiology through studying ways to overcome the above problems will lead to the development of new drugs and treatment.

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The action of lipoprotein lipase on chylomicrons (CM) and very low density lipoproteins (VLDL) produces remnant lipoproteins (RLP) that are rich in triglycerides, cholesterol and apolipoprotein E (apo E). Apo E serves as a ligand for the LDL receptor and mediates uptake of RLP by macrophages, vascular wall and other cells that express the LDL receptor. Uptake of RLP can profoundly alter the physiology of cells and promote atherosclerosis and thrombosis. Like RLP, blood platelets also have roles in atherosclerosis and thrombosis; hence it is likely that RLP can influence platelet activity as well. Platelet aggregation was assessed by measuring the loss of single platelets. Apo E3/3-rich RLP derived from normal human plasma VLDL and CM were prepared by an immunoseparation method. At 2.5 to 10 μl, RLP induced platelet aggregation that increased with the dose of RLP, but decreased it at 25 to 200 μl. Unlike apo E3/3-rich RLP, apo E4/3 (heterozygous phenotype) rich RLP caused platelet aggregation in a dose-dependent manner, without producing a bell-shape dose-response relationship. Scanning electron microscopy revealed that activated platelets had adhered to and formed aggregates on the red cell membrane. The platelet response was unaffected by aspirin, but was inhibited by apyrase (an ADP scavenger), 2-chloroadenosine (a platelet ADP-receptor antagonist) and cilostazol, a phosphodiesterase type III inhibitor. It is thought that RLP cause leakage of ADP from red cells, which then mediates platelet aggregation.

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The quest to develop new antithrombotic agents has been stimulated by clinical needs and by advances in biotechnology that have made it possible to produce drugs that target specific steps in thrombogenesis. Established anticoagulants such as unfractionated heparin and the coumarins are effective, but have two major limitations: narrow therapeutic windows and highly unpredictable dose-response relationships. Consequently, these drugs often cause complications such as serious bleeding that require close monitoring of their use by laboratory tests to balance safety and effect. These limitations provided the impetus for the development of new anticoagulants that inactivate thrombin, factor Xa, factor IXa or the factor VIIa/tissue factor complex. Similarly, agents that enhance the protein C anticoagulant pathway have also been developed. Of these, direct thrombin inhibitors, soluble thrombomodulin, protein C, and activated protein C have been evaluated clinically for parenteral administration. However, there is enormous interest in the development of safer and more effective oral anticoagulants. In the future, such orally active direct inhibitors of thrombin and factor Xa, if they can be given safely without the need for laboratory monitoring, may replace the coumarins for the long-term treatment of thromboembolic disorders. To achieve these goals, these compounds need high, consistent oral bioavailability.

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Thrombomodulin (TM), a membrane-bound receptor for thrombin on the endothelial cell surface, contributes to the regulation of the coagulation system. TM is known to exist in human plasma and urine as soluble forms. We purified soluble TM from human urine (MR-33) and investigated the anticoagulant effects of MR-33 in vitro and in vivo. In human plasma, MR-33 inhibited not only the procoagulant activity of thrombin, but also the thrombin generation via accelerating the thrombin-catalyzed protein C activation. In rat disseminated intravascular coagulation (DIC) models, intravenous infusion of MR-33 improved the hematological abnormalities without excessive prolongation of APTT and bleeding time. Benefit (dose required for 50% inhibition of fibrinogen decrease: ED50) to risk (minimum dose required for significant prolongation of bleeding time) ratio was 1:27 for MR-33. Furthermore, the anticoagulant activities of MR-33 was independent of AT III activity, and MR-33 was effective on heparin-resistant DIC models with low AT III level in rats. Intravenous injection of MR-33 prevented the endotoxin-induced increases in TAT, TNF-α and IL-6 level and pulmonary vascular permeability in mice. These results indicate that MR-33 may be a clinically useful antithrombotic agent with reduced risk for hemorrhage, and this drug also has antiinflammatory effects. Clinical trials of MR-33 for the treatment of DIC are now in progress in Japan.

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Protein C (PC) is an important anticoagulant protein in blood and converted to its active form, activated protein C (APC), by thrombin bound with thrombomodulin. APC exhibits an anticoagulant effect by the inactivation of FV a and FVIII a. In addition, APC exerts a profibrinolytic effect by inactivation of PAI-1 and inhibition of TAFI activation. APC is strongly anti-thrombotic because of its anticoagulant and profibrinolytic effect. APC has γ-carboxyglutamic acid residues that bind to acidic phospholipids expressed on activated plateles or injured endothelial cells. Thus APC works only at the site where clots are formed and has a weak effect in primary hemostasis; this means that the use of APC is expected not to have any hemorrhagic risk. In both DIC animal models and clinical studies, we confirmed safer amelioration by APC than heparin. Recently, a specific receptor for PC/APC was found on endothelial cell membrane and antiinflammatory effects of APC were also reported. Thus APC is thought to play an important regulatory role in blood coagulation, fibrinolysis and inflammation, especially in thrombotic diseases.

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Thrombin-activatable fibrinolysis inhibitor (TAFI) is a 60-kDa plasma protein that has been shown to be identical to plasma carboxypeptidase B (CPB) and carboxypeptidase U (CPU). TAFI is activated by thrombomodulin (TM)-bound thrombin and specifically removes the C-terminal Lys and Arg by its CPB activity. One of its target substrates is the C-terminal Lys residue in the α-chain of plasmin-digested fibrin, which is critical for plasminogen binding to fibrin. Thus, its removal seems to be the main mechanism through which TAFI inhibits fibrinolysis. In this article, relevance of C-terminal Lys of plasmin-digested fibrin in fibrinolysis is described, and then possible roles of TAFI and TM-bound thrombin in a cross-talk between coagulation and fibrinolysis are discussed.

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Hibernation in mammalians such as hamsters is a physiological state characterized by an extreme reduction of various functions such as body temperature and metabolism. Under such severe conditions, the central nervous system (CNS) activity is maintained at a functionally responsive level. Although hibernation is an interesting behavioral state, the physiological mechanisms of the introduction to and/or the arousal from hibernation have not been clearly defined. Intracerebroventricularly (i.c.v.) injected adenosine produces hypothermia in various animals. The effect of adenosine is generated by A1-receptors and is caused by the suppression of the thermogenesis center of the posterior hypothalamus. At on ambient temperature of 5, i.c.v. injected N⁶-cyclohexyladenosine (CHA) adenosine A1-receptor agonist induces profound hypothermia in hamsters. Although the time course of the descent of body temperature coincided with that of entry into natural hibernation, the effect was not antagonized by 8-cyclopentyltheophyllin (CPT), an adenosine A1-receptor antagonist. However, i.c.v. injection of CPT elevated the body temperature and interrupted hibernation, albeit the deep-phase (post-entry 30 h) was unaffected. This result suggests that a different system may suppress the thermogenesis center in the deep hibernation phase. Interestingly, i.c.v. injected thyrotropin releasing hormone (TRH) elevated the body temperature in both hibernation phases in hamsters. These findings suggest that the central adenosine and TRH play important roles in thermoregulation and that the new thermogenesis system, activating in low-body temperature, is induced in naturally hibernating animals.

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The occurrence of delayed emesis induced 24 h after the administration of a non-platina chemotherapeutic agent, doxorubicin hydrochloride (doxorubicin), as well as behaviors such as feeding, drinking and defecation were examined in dogs. A single intravenous administration of 2 mg/kg doxorubicin induced emesis within 24 h of administration in some dogs, while delayed emesis was observed 24 h after administration in all dogs. This delayed emesis emerged strongly at day 3 or 4 and decreased at day 5. Hypophagia, the decreased frequency of drinking and the increased frequency of defecation were induced shortly after delayed emesis. Twenty-four hours after the administration of doxorubicin, a daily dose of 0.3 and 1 mg/kg/ day, p.o. azasetron, a 5-HT₃ antagonist, was administered for 4 days. Doxorubicin-induced delayed emesis was observed to decrease by about 30 and 50%, respectively. This result suggests that 5-HT₃ receptors play a role in the mechanism of delayed emesis. Azasetron was found to improve the increased frequency of defecation, but exerted no obvious effect on hypophagia or on the decreased frequency of drinking. Taken together, we suggest that doxorubicin-induced emesis in dogs is a useful method to study further the mechanisms of delayed emesis and to investigate novel therapeutic agents against delayed emesis.

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