Takehiko Koide

A novel automated microchip flow-chamber system to quantitatively evaluate thrombus formation and antithrombotic agents under blood flow conditions

Summary.u2002 Background and aims:u2002In the present study, we describe a newly developed microchip‐based analytical system to evaluate white thrombus formation (WTF). Efficacies of various antithrombotic agents were compared under different flow conditions. Methods:u2002Whole blood containing corn trypsin inhibitor was perfused over a microchip coated with collagen and tissue thromboplastin at the lower and higher shear rates of 240 and 600u2003s−1, and WTF process inside the microchip was quantified by monitoring a flow pressure. Parameters of T10 (time to 10u2003kPa), T10–80 (time from 10 to 80u2003kPa) and OT (occlusion time; time to 80u2003kPa) were used to evaluate the onset and the growth rate of WTF, and the capillary occlusion, respectively. Results:u2002After perfusion was started, white thrombus composed of activated platelets and fibrin was formed on the coated surface. Thrombus gradually increased in size and eventually occluded the capillary. Among anticoagulants, heparin (0.5–1.0u2003Uu2003mL−1) potently prolonged T10 at both shear rates, whereas low molecular weight heparin (1.0–2.0u2003IUu2003mL−1) inhibited the growth of WTF at the lower shear rate. Among antiplatelet agents, abciximab (1–2u2003μgu2003mL−1) significantly reduced the size and number of thrombi, which was additively enhanced in the presence of heparin (0.5u2003Uu2003mL−1). OS‐1 (specific GPIbα‐antagonist) prevented the complete capillary occlusion. Conclusion:u2002The novel monitoring system of WTF may be useful in preclinical and clinical evaluations of different types of antithrombotic strategies, and their effects in combination.

A microchip flow-chamber system for quantitative assessment of the platelet thrombus formation process.

As the pathogenesis of arterial thrombosis often includes platelet thrombus formation (PTF), antiplatelet agents are commonly used for the prevention of thromboembolic events. Here, using a novel microchip flow-chamber system we developed to quantitatively analyze the PTF process, we evaluated the pharmacological efficacies of antiplatelet agents under different arterial shear rates. Hirudin-anticoagulated whole blood was perfused over a collagen-coated microchip at shear rates of 1000, 1500, and 2000s(-1), and PTF in the absence and presence of various antiplatelet agents was observed microscopically and quantified by measuring flow-pressure changes. The onset of PTF was measured as T(10) (time to reach 10 kPa), and AUC(10) (area under the flow pressure curve for the first 10 min) was calculated to quantify the overall stability of the formed thrombus. Aspirin and AR-C66096 (P2Y(12)-antagonist) at high concentrations (50 μM and 1000 nM, respectively) prolonged T(10) only modestly (AR-C66096>aspirin), but effectively decreased AUC(10), resulting in unstable PTF at all examined shear rates. With dual inhibition using both aspirin (25 μM) and ARC-66096 (250 nM), AUC(10) was drastically reduced. Nearly complete suppression of AUC(10) was also observed with abciximab (2 μg ml(-1)) and beraprost (PGI(2)-analog; 4 nM). Although OS-1 (GPIbα-antagonist; 100 nM) prevented complete capillary occlusion, significant amounts of microscopic thrombi were observed on the collagen surface. In contrast to abciximab and beraprost, OS-1 differentially affected PTF under higher shear conditions. Our novel analytical system is capable of distinguishing the pharmacological effects of various antiplatelet agents under physiological shear rates, suggesting that this system may aid in the determination of the appropriate type and dose of antiplatelet agent in the clinical setting.

High affinity interaction between histidine-rich glycoprotein and the cell surface type ATP synthase on T-cells

Histidine-rich glycoprotein (HRG) is a plasma protein implicated in the innate immune system. In recent studies, we showed that either HRG, or the Arg23-Lys66 glycopeptide derived from HRG, in concert with concanavalin A (Con A), promotes a morphological change and adhesion of the human leukemic T-cell line MOLT-4 to culture dishes, and that cell surface glycosaminoglycan or Fcgamma receptors do not participate in this cellular event. In the present study, we identified the alpha-subunit of ATP synthase as one of the HRG-binding proteins on the surface of T-cells by HRG-derived glycopeptide affinity chromatography and by a peptide mass finger printing method. HRG specifically interacted with mitochondrial ATP synthase with a dissociation constant of 66 nM. The presence of alpha- and beta-subunits of ATP synthase on the plasma membrane of MOLT-4 cell was demonstrated by immunofluorescent staining and FACS analysis. The HRG/Con A-induced morphological changes of MOLT-4 cells were specifically inhibited by a monoclonal antibody against the beta-subunit of ATP synthase. These results strongly suggest that the cell surface ATP synthase functions as a binding protein for HRG on MOLT-4 cells, which is required for the morphological changes observed in MOLT-4 cells following treatment with HRG/Con A.

N-Linked oligosaccharide processing, but not association with calnexin/calreticulin is highly correlated with endoplasmic reticulum-associated degradation of antithrombin Glu313-deleted mutant

Previously we showed that two antithrombin mutants were degraded through an endoplasmic reticulum (ER)-associated degradation (ERAD) pathway [F. Tokunaga et al., FEBS Lett. 412 (1997) 65]. Here, we examined the combined effects of inhibitors of glycosidases, protein synthesis, proteasome, and tyrosine phosphatase on ERAD of a Glu313-deleted (DeltaGlu) mutant of antithrombin. We found that kifunensine, an ER mannosidase I inhibitor, suppressed ERAD, indicating that specific mannose trimming plays a critical role. Cycloheximide and puromycin, inhibitors of protein synthesis, also suppressed ERAD, the effects being cancelled by pretreatment with castanospermine. In contrast, kifunensine suppressed ERAD even in castanospermine-treated cells, suggesting that suppression of ERAD does not always require the binding of lectin-like ER chaperones-like calnexin and/or calreticulin. These results indicate that, besides proteasome inhibitors, inhibitors of ER mannosidase I and protein synthesis suppress ERAD of the antithrombin deltaGlu mutant at different stages, and processing of N-linked oligosaccharides highly correlated with the efficiency of ERAD.

Histidine-rich glycoprotein and concanavalin A synergistically stimulate the phosphatidylinositol 3-kinase-independent signaling pathway in leukocytes leading to increased cell adhesion and changes in cell morphology.

Histidine-rich glycoprotein (HRG) promoted the adhesion and morphological changes of human T-cell line MOLT-4 in a Con A-dependent manner. This morphological change-promoting activity was specific for HRG and the Arg23-Lys66 glycopeptide from human HRG. The carbohydrate chain at Asn45 was essential for this activity. The morphological changes of MOLT-4 cells caused by HRG and Con A (HRG/Con A) were not inhibited by phosphatidylinositol 3-kinase inhibitor, wortmannin or LY294002, while the changes by Con A alone were completely inhibited by these reagents, suggesting that HRG/Con A cooperate to activate leukocytes via a signaling pathway distinct from that by Con A alone. The morphological changes by Con A were associated with pseudopodia like structure. On the other hand, the morphological changes caused by HRG/Con A were associated not only with pseudopodia like structure but also with an increase of the F-actin-rich surface protrusions. Wortmannin inhibited only the formation of pseudopodia like structure.

Inhibition of the 20S Proteosome by a Protein Proteinase Inhibitor: Evidence That a Natural Serine Proteinase Inhibitor Can Inhibit a Threonine Proteinase

The 20S proteasome (20S) is an intracellular threonine proteinase (Mr 750,000) that plays important roles in many cellular regulations. Several synthetic peptide inhibitors and bacteria-derived inhibitors such as lactacystin and epoxomicin have been identified as potent proteasome inhibitors. However, essentially no protein proteinase inhibitor has been characterized. By examining several small size protein proteinase inhibitors, we found that a well-known serine proteinase inhibitor from bovine pancreas, basic pancreatic trypsin inhibitor (BPTI), inhibits the 20S in vitro and ex vivo. Inhibition of the 20S by BPTI was time- and concentration-dependent, and stoichiometric. To inhibit the 20S activity, BPTI needs to enter into the interior of the 20S molecule. The molar ratio of BPTI to the 20S in the complex was estimated as approximately six BPTI to one 20S, thereby two sets of three peptidase activities (trypsin-like, chymotrypsin-like and caspase-like) of the 20S were all inhibited. These results indicate that an entrance hole to the 20S formed by seven alpha-subunits is sufficiently large for BPTI to enter. This report is essentially the initial description of the inhibition of a threonine proteinase by a protein serine proteinase inhibitor, suggesting a common mechanism of inhibition between serine and threonine proteinases by a natural protein proteinase inhibitor.

Chemically modified thrombin and anhydrothrombin that differentiate macromolecular substrates of thrombin.

Summary.u2002 Background: Thrombin is a primary inducer of thrombus formation by activations of coagulation cascade and platelet aggregation. Hitherto, several types of thrombin inhibitors have been developed for therapeutic purpose. Objectives: We prepared modified thrombin (M‐thrombin) and modified anhydrothrombin (M‐anhydrothrombin) by chemical modification of carboxyl groups of thrombin and anhydrothrombin, respectively, to present a new strategy for a potent antiplatelet–anticoagulant agent and new tools for investigation of thrombin functions. Results: M‐anhydrothrombin retained high affinity for factor VIII (FVIII), but demonstrated lower affinity than anhydrothrombin for fibrinogen and factor V (FV). Both M‐anhydrothrombin and anhydrothrombin prolonged activated partial thromboplastin time (APTT) without affecting prothrombin time, and M‐anhydrothrombin prolonged APTT much more than anhydrothrombin. M‐anhydrothrombin also retained affinity for the recombinant extracellular domain peptide of protease‐activated receptor 1 (PAR1). M‐thrombin exhibited marginal clotting activity (4% of thrombin), but induced platelet aggregation in platelet‐rich plasma without forming a fibrin clot, which was completely suppressed by anti‐PAR1 antibody (ATAP2) and by M‐anhydrothrombin, but not by anhydrothrombin. These results indicate that M‐thrombin induced platelet aggregation through the activation of PAR1, and M‐anhydrothrombin inhibited this process completely. In contrast, neither M‐anhydrothrombin nor anhydrothrombin apparently inhibited thrombin‐induced platelet aggregation. Only in the presence of the Gly‐Pro‐Arg‐Pro (GPRP) peptide that inhibits polymerization of fibrin, M‐anhydrothrombin completely inhibited thrombin‐induced platelet aggregation. Conclusion: M‐thrombin is PAR1‐specific and M‐anhydrothrombin is FVIII‐ and PAR1‐specific derivatives, and thereby, are new tools as specific agonist and antagonist, respectively, of PAR1. Furthermore, M‐anhydrothrombin may be an attractive model for development of a potent anticoagulant–antiplatelet agent.

M-AAA-Thrombin: Potent anticoagulant and antiplatelet thrombin derivative with differential affinity for factor VIII and PAR1

BACKGROUNDnThrombosis is a major cause of morbidity and mortality, and thrombin is a major inducer of thrombus formation. Thus, several antithrombotic agents targeting thrombin have been developed. We previously reported on a thrombin derivative prepared by dual chemical modifications designated as M-anhydrothrombin, which possessed both anticoagulant and antiplatelet properties. In order to obtain a more potent antithrombotic thrombin derivative, we prepared a recombinant thrombin mutant and its chemically-modified derivative, and examined their antithrombotic efficacies.nnnMETHODS AND RESULTSnWe prepared a thrombin mutant, 65A43A205A-Th (designated as AAA-Th) in which Lys65(70)(1), and His43(57) and Ser205(195) were replaced by Ala, and its chemically-modified derivative at the carboxyl groups (designated as M-AAA-Th). M-AAA-Th possessed no enzymatic activity, but retained high affinity and specificity for factor VIII, and prolonged the APTT with a slight effect on PT and no effect on TT. Platelet aggregation induced by PAR1 activation was also suppressed by M-AAA-Th. In contrast, conventional thrombin inhibitors, argatroban and hirulog, substantially prolonged the TT rather than the APTT and the PT. In thromboelastgraph assays, M-AAA-Th suppressed whole blood clotting in a dose-dependent manner, and its effect was synergistically enhanced in the presence of soluble thrombomodulin (s-TM). M-AAA-Th also demonstrated a potent antithrombotic property in the FeCl(3)-induced carotid arterial thrombosis model in guinea pigs with minimum effects on the APTT and PT and no prolongation of the TT.nnnCONCLUSIONnM-AAA-Th may be a potent anticoagulant and antiplatelet thrombin derivative with differential affinity for factor VIII and PAR1.

Antithrombin and Heparin Cofactor II: Structure and Functions

Antithrombin (AT) and heparin cofactor II (HOI) are plasma serpins that function as principal regulators of blood coagulation. These serpins inhibit their target proteinases by forming an inactive enzyme-inhibitor complex through an interaction between their reactive center and the active site of the proteinase. Among the coagulation proteinases, AT mainly inhibits factor Xa and thrombin, and HCII exclusively inhibits thrombin. However, both AT and HCII by themselves are poor inhibitors of coagulation proteinases as these reactions proceed at a slow rate and are time-dependent. These serpins also require glycosaminoglycans for their physiological functions. By binding to glycosaminoglycans, a conformational change occurs in the reactive center loop (RCL), which activates these serpins to become a more efficient inhibitor of coagulation proteinases. The physiological cofactor for AT is heparan sulfate proteoglycans on endothelial cells, and that for HCII is dermatan sulfate proteoglycans on vascular smooth muscle cells. The physiological importance of AT as an anticoagulant in the circulation is well supported by a number of thrombotic disorders in patients with AT deficiency. However, the physiological function of HCII as an anticoagulant is unclear, as HCII deficiency is not a significant risk factor for venous or arterial thrombosis. Recent crystallographic analyses of monomeric native AT variant, AT-thrombin, or factor Xa-pentasaccharide complexes, as well as HCII-thrombin complex, have revealed more detailed mechanisms of proteinase inhibition by these serpins and mechanisms of glycosaminoglycan-dependent enhancement of the reaction rates, together with some revisions of our previous knowledge.