Keiko Wanaka

Development of plasma kallikrein selective inhibitors.

During the course of the development of active center-directed plasmin inhibitors, it was found that N-(trans-4-aminomethylcyclohexanecarbonyl)-lysine-4-methoxycarb onylanilide inhibited plasma kallikrein more potently than other enzymes such as plasmin, urokinase, and thrombin, although the inhibitory activity was not as potent and enzyme selectivity not as high. Based on studies of structure-activity relationship, we designed and synthesized the plasma kallikrein selective inhibitor, N-(trans-4-aminomethylcyclohexanecarbonyl)-phenylalanine-4-carboxy methyl- anilide (Tra-Phe-APAA). Tra-Phe-APAA inhibited plasma kallikrein with a Ki value of 0.81 microM, while it inhibited glandular kallikrein, plasmin, urokinase, tissue plasminogen activator, factor Xa, factor XIIa, and thrombin with Ki values of > 500, 390, 200, > 500, > 500 > 500, and > 500 microM, respectively. We designated Tra-Phe-APAA as PKSI-527. Using PKSI-527 as an affinity ligand, we synthesized a new affinity gel (PKSI-Toyopearl) and employed it for the rapid purification of plasma kallikrein from human plasma. Human plasma activated with kaolin after acid treatment was applied to a PKSI-527-Toyopearl column. Adsorbed protein was eluted with 50 mM glycinehydrochloric acid buffer (pH 3.0). Plasma kallikrein was purified 181-fold with a yield of 85% from the kaolin-activated plasma.

Development of plasmin and plasma kallikrein selective inhibitors and their effect on M1 (melanoma) and ht29 cell lines

trans-4-Aminomethylcyclohexanecarbonyl-Tyr(O-Pic)-octylamide (YO-2) inhibited plasmin (PL) selectively, while trans-4-aminomethylcyclohexanecarbonyl-Phe-4-carboxymethylanili de (YO-1) inhibited plasma kallikrein (PK). YO-2 induced apoptosis of M1 (melanoma) cell line and HT29 colon carcinoma cells during 24 h through activation of caspase-3, while YO-1 did not affect either cell line even during 48 h.

Management of Uremic Patients With Heparin-Induced Thrombocytopenia Requiring Hemodialysis

Medical records of 122 patients with suspected heparin-induced thrombocytopenia on dialysis were reviewed. Method of dialysis in heparin-induced thrombocytopenia patients with bleeding from various causes (including surgical interventions) and how to cope with blood access occlusion induced by heparin-induced thrombocytopenia were studied. Of 122 patients, 17 who met the criteria of >30% thrombocytopenia, clots in the extracorporeal circulation, positive for heparin/PF4 complex antibodies, and improvement from heparin-induced thrombocytopenia with the use of an alternative anticoagulant or another strategy for heparin-induced thrombocytopenia were chosen. Argatroban was uneventfully introduced in 12 patients having neither bleeding nor blood access failure. In all, 2 of 5 patients were treated with peritoneal dialysis. The others requiring a regional anticoagulant were given nafamostat mesilate. Argatroban as an alternative provides effectively anticoagulation in patients with heparin-induced thrombocytopenia on dialysis. In patients with heparin-induced thrombocytopenia with bleeding and its associated risk, nafamostat mesilate was an alternative. Peritoneal dialysis also was applied in cases of blood access failure due to heparin-induced thrombocytopenia.

Effect of a synthetic thrombin-inhibitor MD805 on the reaction between thrombin and plasma antithrombin III

MD805, a synthetic thrombin-inhibitor, effectively retarded the time-dependent inactivation of thrombin which was generated endogeneously or added exogeneously in human plasma. The kinetical study of the time-dependent inactivation indicated that the type of inhibition was competitive and the obtained Ki of MD805 for thrombin was 3 x 10(-8)M. MD805 also inhibited the formation of thrombin-ATIII complex. These results indicated that the active site of thrombin was involved in the reaction between thrombin and ATIII, and that MD805 competed with ATIII for thrombin in exactly the same manner as it competed with fibrinogen or synthetic peptide substrates. As a result, MD805 would serve as a protective agent for ATIII from being consumed, in addition to its potent thrombin-inhibitory activity without the aid of ATIII. By contrast, heparin accelerated the time-dependent inactivation rate of thrombin and the formation of thrombin-ATIII complex, which indicates that heparin accelerates the consumption of ATIII.

Murine Monoclonal Antibody to Platelet Factor 4/Heparin Complexes as a Potential Reference Standard for Platelet Activation Assays in Heparin-Induced Thrombocytopenia

Quality control of the platelet activation assays to diagnose heparin-induced thrombocytopenia (HIT), 14C-serotonin release assay (SRA) and platelet aggregation test (PAT) has yet to be established due to lack of reference standards and the difficulty of obtaining significant amounts of HIT antibodies from patients with HIT. We prepared a murine monoclonal antibody to human platelet factor 4 (hPF4)/heparin complexes (HIT-MoAb) and investigated the platelet activating action of HIT-MoAb by using SRA and PAT. The HIT-MoAb activated human platelets at low heparin concentration and the platelet activations were inhibited at high heparin concentration in both SRA and PAT. The HIT-MoAb produced a concentration-dependent effect. Moreover, the platelet activation at low heparin concentration was inhibited by anti-FcγRIIa antibody. These results indicated that HIT-MoAb has characteristics similar to human HIT antibodies regarding heparin-dependent platelet activation. Therefore, it is suggested that HIT-MoAb has the potential to be a positive control or reference standard in platelet activation assays.

Synthesis and evaluation of tripeptidic plasmin inhibitors with nitrile as warhead.

Plasmin is best known as the key molecule in the fibrinolytic system, which is critical for clot lysis and can initiate matrix metalloproteinase (MMP) activation cascade. Along with MMP, plasmin is suggested to be involved in physiological processes that are linked to the risk of carcinoma formation. Plasmin inhibitors could be perceived as a promising new principle in the treatment of diseases triggered by plasmin. On the basis of the peptidic sequence derived from the synthetic plasmin substrate, a series of peptidic plasmin inhibitors possessing nitrile as warhead were prepared and evaluated for their inhibitory activities against plasmin and other serine proteases, plasma kallikrein and urokinase. The most potent peptidic inhibitors with the nitrile warhead exhibit the potency toward plasmin (IC50 = 7.7–11 μM) and are characterized by their selectivity profile against plasma kallikrein and urokinase. The results and molecular modeling of the peptidic inhibitor complexed with plasmin reveal that the P2 residue makes favorable contacts with the open binding pocket comprising the S2 and S3 subsites of plasmin. Copyright

Identification of novel plasmin inhibitors possessing nitrile moiety as warhead

Lysine-nitrile derivatives having a trisubstituted benzene, which belongs to a new chemical class, were prepared and tested for inhibitory activities against plasmin and the highly homologous plasma kallikrein and urokinase. The use of the novel chemotype in the development of plasmin inhibitors has been demonstrated by derivatives of compound 9.

Use of an active center-directed plasmin inhibitor elucidates the multiplicity of plasmin actions

In our studies, designed to synthesize an active center-directed plasmin (PL) inhibitor, N-(4-aminomethylbenzoyl)-4-(3-picolyloxy)-L-phenylalanine n-hexylamide dihydrochloride (PASI-535) was found. We characterized PASI-535 and analyzed the actions of PL, comparing with those of PASI-535 and tranexamic acid (t-AMCHA). (1) PASI-535 strongly inhibited not only fibrinolysis (IC50: 2.9 x 10(-6) M) but also amidolysis (Ki value: 2.9 x 10(-6) M) and fibrinogenolysis (IC50: 4.5 x 10(-6) M) induced by PL. While t-AMCHA which strongly inhibited fibrinolysis (IC50: 6.0 x 10(-5) M), rarely inhibited amidolysis (Ki value: 4.0 x 10(-2) M) and fibrinogenolysis (IC50: 1.0 x 10(-2) M). (2) PL is able to liberate kinins by degrading kininogen. This kinin-generation by PL was inhibited by 2 x 10(-5) M PASI-535. However, it was little inhibited even by 1 x 10(-3) M t-AMCHA. (3) The inhibitory effect of PASI-535 and t-AMCHA on tumor growth was studied. In sarcoma-180 bearing mice, ascites retention and the increase of tumor cells were markedly suppressed by subcutaneous injection of PASI-535, either 30 mg/kg/day or 50 mg/kg/day, for 5 days, and the inhibitory effect was dose-dependent. Although t-AMCHA also reduced both ascites retention and the increase of tumor cells, it needed approximately 40 times (2 g/kg/day) the amount of PASI-535 to exert these effects. PASI-535 may be a useful tool in analyzing the multiplicity of PL actions. Moreover, PASI-535 can be used as an antifibrinolytic drug which has a mechanism of function different from that of t-AMCHA.

Pyrrolopyrimidine-inhibitors with hydantoin moiety as spacer can explore P4/S4 interaction on plasmin.

In the development of plasmin inhibitors, a novel chemotype, pyrrolopyrimidine scaffold possessing two motifs, a hydantoin-containing P4 moiety and a warhead-containing P1 moiety, is uncovered. A unique feature of the new line of the plasmin inhibitors is that the interaction between the plasmin inhibitors and key subsites in plasmin can be controlled by a spacer like hydantoin. The application of the novel chemotype is demonstrated by 1n and provides further evidence on the importance of hydantoin as the spacer.

Evaluation of Circuit and AV Fistula Clotting and Detection of Anti-PF4/Heparin Complex Antibodies in Hemodialysis Patients Suspected of Having Heparin-Induced Thrombocytopenia:

A retrospective study was performed to elucidate the characteristics of heparin-induced thrombocytopenia (HIT) in newly treated hemodialysis (HD) patients who essentially required anticoagulation with unfractionated heparin (UFH). Seventy-eight patients suspected of having HIT within 3 months of starting HD with UFH were selected for this study. Their platelet counts were routinely followed, and anti-PF4/heparin complex antibodies (HIT antibodies) were measured with enzyme-linked immunosorbent assay (ELISA) until the titer became negative. The characteristics of thrombocytopenia were a platelet count of ≤150 × 109/L and/or decrease of ≥30% and as caused by the intermittent use (3 times/a week) of UFH during HD. Fifty-five patients showed unexpected clotting in the extracorporeal circuit and/or arteriovenous fistula (AVF) thrombosis, while 23 patients had neither of these complications. The patients were classified into HD-related and non-HD-related thrombus groups. The impact of various combinations of the 3 clinical factors (thrombocytopenia, timing, and HD-related thrombus) and the results of ELISA as a laboratory factor were examined. A combination of 2 platelet factors (thrombocytopenia and timing) and ELISA positivity did not reveal the presence of HIT, while a combination of the 3 clinical factors and a positive ELISA improved the accuracy of HIT diagnosis. The findings on the 4-factor combination were supported by high rates of seroconversion in a serotonin release assay. Combining appropriate clinical factors and a positive ELISA may lead to the proper management of HD patients suspected of having HIT. In conclusion, while HD patients showed a drop of ≤150 × 109/L or ≥30% on days 7 to 30, unexpected clotting in the circuit and/or AVF thrombosis was considered as a sign of HIT development.