Kenichi Ogiwara

New assays for monitoring haemophilia treatment

Summary.  Precise measurements of factor VIII (FVIII) or factor IX (FIX) activity are believed to be essential for clinical management in haemophilia, although discrepancies between factor levels and clinical severity have been recognized. Clot wave form analysis has demonstrated that different wave form patterns may be evident in severe haemophilia A patients with levels of FVIII activity <1 IU dL−1, and this might explain, in part, the phenotypic heterogeneity seen in these patients. In addition, the relatively new technique of computer‐assisted thrombelastography (TEG), in which coagulation is initiated by tissue factor, has revealed a considerable degree of variability in different patients in the presence FVIII levels, which are sufficient to normalize TEG parameters. In contrast, a global thrombin generation test (TGT) has been proposed as a sensitive and reliable method for assessing overall clotting function in haemophilia patients. Several studies have demonstrated a significant correlation between TGT and FVIII/FIX levels, and these measurements also appear to correlate with the clinical phenotype. The TGT may be very useful, therefore, for evaluating overall haemostasis in different clinical situations, although substantial inter‐assay and inter‐individual variations have been reported. Both the TEG and TGT have been found to be particularly helpful for monitoring haemostatic therapy with bypassing agents or conventional FVIII or FIX concentrates in patients with inhibitors. These global tests enable the selection of appropriate therapeutic agents in individual circumstances and offer the opportunity to tailor the most effective haemostatic treatment even during severe bleeding or major surgery.

Correlations between global clotting function tests, duration of operation, and postoperative chest tube drainage in pediatric cardiac surgery.

Background:  Systemic coagulation disorders after cardiac surgery represent serious postoperative complications. There have been few reports, however, identifying preoperative coagulation tests that predict postoperative bleeding. The aim of the present study was to investigate the relationship between postoperative hemorrhage and coagulation parameters determined by global coagulation assays, to define potential predictive markers.

The Factor VIIIa C2 Domain (Residues 2228–2240) Interacts with the Factor IXa Gla Domain in the Factor Xase Complex

Factor VIIIa functions as a cofactor for factor IXa in the phospholipid surface-dependent activation of factor X. Both the C2 domain of factor VIIIa and the Gla domain of factor IXa are involved in phospholipid binding and are required for the activation of factor X. In this study, we have examined the close relationship between these domains in the factor Xase complex. Enzyme-linked immunosorbent assay-based and surface plasmon resonance-based assays in the absence of phospholipid showed that Glu-Gly-Arg active site-modified factor IXa bound to immobilized recombinant C2 domain (rC2) dose-dependently (Kd = 108 nm). This binding ability was optimal under physiological conditions. A monoclonal antibody against the Gla domain of factor IXa inhibited binding by ∼95%, and Gla domainless factor IXa failed to bind to rC2. The addition of monoclonal antibody or rC2 with factor VIIIa inhibited factor IXa-catalyzed factor X activation in the absence of phospholipid. Inhibition was not evident, however, in similar experiments in the absence of factor VIIIa, indicating that the C2 domain interacted with the Gla domain of factor IXa. A fragment designated C2-(2182–2259), derived from V8 protease-cleaved rC2, bound to Glu-Gly-Arg active site-modified factor IXa. Competitive assays, using overlapping synthetic peptides encompassing residues 2182–2259, demonstrated that peptide 2228–2240 significantly inhibited both this binding and factor Xa generation, independently of phospholipid. Our results indicated that residues 2228–2240 in the factor VIIIa C2 domain constitutes an interactive site for the Gla domain of factor IXa. The findings provide the first evidence for an essential role for this interaction in factor Xase assembly.

A putative inhibitory mechanism in the tenase complex responsible for loss of coagulation function in acquired haemophilia A patients with anti-C2 autoantibodies

Acquired haemophilia A (AHA) is caused by the development of factor (F)VIII autoantibodies, demonstrating type 1 or type 2 inhibitory behaviour, and results in more serious haemorrhagic symptoms than in congenital severe HA. The reason(s) for this remains unknown, however. The global coagulation assays, thrombin generation tests and clot waveform analysis, demonstrated that coagulation parameters in patients with AHA-type 2 inhibitor were more significantly depressed than those in patients with moderate HA with similar FVIII activities. Thrombin and intrinsic FXa generation tests were significantly depressed in AHA-type 1 and AHA-type 2 compared to severe HA, and more defective in AHA-type 1 than in AHA-type 2. To investigate these inhibitory mechanism(s), anti-FVIII autoantibodies were purified from AHA plasmas. AHA-type 1 autoantibodies, containing an anti-C2 ESH4-epitope, blocked FVIII(a)-phospholipid binding, whilst AHA-type 2, containing an anti-C2 ESH8-epitope, inhibited thrombin-catalysed FVIII activation. The coagulation function in a reconstituted AHA-model containing exogenous ESH4 or ESH8 was more abnormal than in severe HA. The addition of anti-FIX antibody to FVIII-deficient plasma resulted in lower coagulation function than its absence. These results support the concept that global coagulation might be more suppressed in AHA than in severe HA due to the inhibition of FIXa-dependent FX activation by steric hindrance in the presence of FVIII-anti-C2 autoantibodies. Additionally, AHA-type 1 inhibitors prevented FVIIIa-phospholipid binding, essential for the tenase complex, whilst AHA-type 2 antibodies decreased FXa generation by inhibiting thrombin-catalysed FVIII activation. These two distinct mechanisms might, in part, contribute to and exacerbate the serious haemorrhagic symptoms in AHA.

A New Drug Delivery System for Intravenous Coronary Thrombolysis With Thrombus Targeting and Stealth Activity Recoverable by Ultrasound

OBJECTIVES The purpose of this study was to develop a new intelligent drug delivery system for intracoronary thrombolysis with a strong thrombolytic effect without increasing bleeding risk. BACKGROUND Rapid recanalization of an occluded coronary artery is essential for better outcomes in acute myocardial infarction. Catheter-based recanalization is widely accepted, but it takes time to transport patients. Although the current fibrinolytic therapy can be started quickly, it cannot achieve a high reperfusion rate. Recently, we generated nanoparticles comprising tissue-type plasminogen activator (tPA), basic gelatin, and zinc ions, which suppress tPA activity by 50% with 100% recovery by ultrasound (US) in vitro. METHODS The thrombus-targeting property of nanoparticles was examined by an in vitro binding assay with von Wilbrand factor and with a mouse arterial thrombosis model in vivo. The thrombolytic efficacy of nanoparticles was evaluated with a swine acute myocardial infarction model. RESULTS Nanoparticles bound to von Wilbrand factor in vitro and preferentially accumulated at the site of thrombus in a mouse model. In a swine acute myocardial infarction model, plasma tPA activity after intravenous injection of nanoparticles was approximately 25% of tPA alone and was recovered completely by transthoracic US (1.0 MHz, 1.0 W/cm(2)). During US application, plasma tPA activity near the affected coronary artery was recovered and was higher than that near the femoral artery. Although treatment with tPA alone (55,000 IU/kg) recanalized the occluded coronary artery in only 1 of 10 swine, nanoparticles containing the same dose of tPA with US achieved recanalization in 9 of 10 swine within 30 min. CONCLUSIONS We developed an intelligent drug delivery system with promising potential for better intravenous coronary thrombolysis.

Novel FV mutation (W1920R, FVNara) associated with serious deep vein thrombosis and more potent APC resistance relative to FVLeiden.

Factor V (FV) appears to be pivotal in both procoagulant and anticoagulant mechanisms. A novel homozygote (FVNara), a novel mechanism of thrombosis associated with Trp1920→Arg (W1920R), was found in a Japanese boy and was associated with serious deep vein thrombosis despite a low level of plasma FV activity (10 IU/dL). Activated partial thromboplastin time-based clotting assays and thrombin generation assays showed that FVNara was resistant to activated protein C (APC). Reduced susceptibility of FVaNara to APC-catalyzed inactivation and impaired APC cofactor activity of FVNara on APC-catalyzed FVIIIa inactivation contributed to the APC resistance (APCR). Mixtures of FV-deficient plasma and recombinant FV-W1920R confirmed that the mutation governed the APCR of FVNara. APC-catalyzed inactivation of FVa-W1920R was significantly weakened, by ~11- and ~4.5-fold, compared with that of FV-wild-type (WT) and FVLeiden (R506Q), respectively, through markedly delayed cleavage at Arg506 and little cleavage at Arg306, consistent with the significantly impaired APC-catalyzed inactivation. The rate of APC-catalyzed FVIIIa inactivation with FV-W1920R was similar to that without FV, suggesting a loss of APC cofactor activity. FV-W1920R bound to phospholipids, similar to FV-WT. In conclusion, relative to FVLeiden, the more potent APCR of FVNara resulted from significant loss of FVa susceptibility to APC and APC cofactor activity, mediated by possible failure of interaction with APC and/or protein S.

A modified thrombin generation test for investigating very low levels of factor VIII activity in hemophilia A.

Discrepancies between low levels of FVIII:C and clinical symptoms in severe hemophilia A are well-known. We have recently demonstrated that levels of FVIII:C < 0.2 IU/dl were consistent with clinical phenotype by clot waveform analysis, suggesting that precise measurement of very low levels of FVIII:C was clinically important. Thrombin generation tests (TGTs) triggered by tissue factor (TF) have been recently utilized to monitor coagulation function in hemophilia A. We examined whether TGT was useful for evaluating hemophilia A patients with very low levels of FVIII:C. TGTs in 40 hemophilia A plasmas with FVIII:C < 0.2–17 IU/dl (measured by clot waveform analysis using MDA-II™) were performed using TF and/or ellagic acid (ELG). The lagtime in ELG-TGT at very low levels of FVIII:C was shortened dose-dependently, whilst this parameter in TF-TGT was not significantly affected. Other parameters (endogenous thrombin potential, peak thrombin, time to peak) correlated with FVIII:C levels to some extent in both assays (r = 0.4–0.7). Using a TF/ELG mixture in TGT, however, the correlation coefficients increased to ~0.85. TGT parameters correlated well with levels of FVIII:C > 0.2 IU/dl, although the lagtime was not especially informative. We conclude that modified TGT, using a TF/ELG mixture as the trigger, is useful for monitoring coagulation function at very low levels of FVIII:C in hemophilia A.

Plasmin-induced procoagulant effects in the blood coagulation: a crucial role of coagulation factors V and VIII.

Plasminogen activators provide effective treatment for patients with acute myocardial infarction. However, paradoxical elevation of thrombin activity associated with failure of clot lysis and recurrent thrombosis has been reported. Generation of thrombin in these circumstances appears to be owing to plasmin (Plm)-induced activation of factor (F) XII. Plm catalyzes proteolysis of several coagulant factors, but the roles of these factors on Plm-mediated procoagulant activity remain to be determined. Recently developed global coagulation assays were used in this investigation. Rotational thromboelastometry using whole blood, clot waveform analysis and thrombin generation tests using plasma, showed that Plm (≥125 nmol/l) shortened the clotting times in similar dose-dependent manners. In particular, the thrombin generation test, which was unaffected by products of fibrinolysis, revealed the enhanced coagulation with a ∼two-fold increase of peak level of thrombin generation. Studies using α2-antiplasmin-deficient plasma revealed that much lower dose of Plm (≥16 nmol/l) actually contributed to enhancing thrombin generation. The shortening of clotting time could be observed even in the presence of corn trypsin inhibitor, supporting that Plm exerted the procoagulant activity independently of FXII. In addition, using specific coagulation-deficient plasmas, the clot waveform analysis showed that Plm did not shorten the clotting time in only FV-deficient or FVIII-deficient plasma in prothrombin time-based or activated partial thromboplastin time-based assay, respectively. Our results indicated that Plm did possess procoagulant activity in the blood coagulation, and this effect was likely attributed by multicoagulation factors, dependent on FV and/or FVIII.

Regulation of coagulation factors during liver regeneration in mice: Mechanism of factor VIII elevation in plasma

INTRODUCTIONS The profiles of coagulation factor production during liver regeneration process remains to be fully elucidated. The present study was aimed to perform a comprehensive analysis whether hepatic gene expression was differentially regulated relative to the secretion of biologically active coagulation factors using a mouse model of liver regeneration. MATERIALS AND METHODS Liver regeneration was induced by performing a 2/3 partial hepatectomy (PHx). Plasma samples were assessed for coagulation factor activities (fibrinogen, prothrombin, V, VII, VIII, IX, X, XI, XII, and XIII) and the liver mRNA levels of coagulation, anti-coagulation, and fibrinolytic factors were quantified by real-time RT-PCR during the phase of liver regeneration. RESULTS At the peak of liver regeneration, the expression levels for all of the genes analyzed were found to be reduced in a time-dependent manner. Consistent with the gene expression levels, plasma activities of all coagulation factors, except for FVIII, were temporally declined during the same time frame. FVIII paradoxically demonstrated a significant increase (P<0.05) in plasma activities concomitant with the decrease of liver mRNA expression levels. We found that the increase in plasma FVIII activities might be associated with (1) a delay in the inactivation of plasma FVIII caused by increased VWF in plasma and decreased FVIII clearance in the liver, (2) the rapid release of FVIII from the storage sites, and (3) the alteration of intracellular trafficking pathway of FVIII. CONCLUSIONS The present study demonstrated that the process of liver regeneration involves a general reduction for many of the coagulation cascade proteins, but there are paradoxical increases in plasma levels of FVIII and VWF.

Protein S down-regulates factor Xase activity independent of activated protein C: specific binding of factor VIII(a) to protein S inhibits interactions with factor IXa.

Protein S functions as an activated protein C (APC)‐independent anticoagulant in the inhibition of intrinsic factor X activation, although the precise mechanisms remain to be fully investigated. In the present study, protein S diminished factor VIIIa/factor IXa‐dependent factor X activation, independent of APC, in a functional Xa generation assay. The presence of protein S resulted in an c. 17‐fold increase in Km for factor IXa with factor VIIIa in the factor Xase complex, but an c. twofold decrease in Km for factor X. Surface plasmon resonance‐based assays showed that factor VIII, particularly the A2 and A3 domains, bound to immobilized protein S (Kd; c. 10 nmol/l). Competition binding assays using Glu‐Gly‐Arg‐active‐site modified factor IXa showed that factor IXa inhibited the reaction between protein S and both the A2 and A3 domains. Furthermore, Sodium dodecyl sulphate polyacrylamide gel electrophoresis revealed that the cleavage rate of factor VIIIa at Arg336 by factor IXa was c. 1·8‐fold lower in the presence of protein S than in its absence. These data indicate that protein S not only down‐regulates factor VIIIa activity as a cofactor of APC, but also directly impairs the assembly of the factor Xase complex, independent of APC, in a competitive interaction between factor IXa and factor VIIIa.