Yoichi Sakata

Significance of Cross-Linking of α2-Plasmin Inhibitor to Fibrin in Inhibition of Fibrinolysis and in Hemostasis

When blood is clotted, alpha(2)-plasmin inhibitor (alpha(2)PI) is cross-linked to fibrin by activated fibrin-stabilizing factor (activated coagulation Factor XIII, plasma transglutaminase). The amount of cross-linked alpha(2)-PI is proportional to the amount of alpha(2)PI present at the time of clotting. Plasma from a patient with congenital deficiency of alpha(2)PI was supplemented with various amounts of purified alpha(2)PI. Clots were prepared from these plasmas and were suspended in plasma containing a normal concentration of alpha(2)PI, and spontaneous clot lysis was observed. When the clot was formed in the presence of calcium ions and thereby allowing cross-linking to occur, the rate and extent of fibrinolysis were found to be inversely proportional to the concentrations of alpha(2)PI present in the clot at the time of clotting. When the clot was formed in the absence of calcium ions so that no cross-linking occurred, the clot underwent fibrinolysis at similar rates, regardless of the concentrations of alpha(2)PI in the clot. When the clot formed in the presence of calcium ions was squeezed and washed to remove unbound proteins before being suspended in plasma, the extent of fibrinolysis was also inversely proportional to the amount of alpha(2)PI cross-linked to fibrin. Similar results were obtained when the clot was suspended in buffered saline instead of plasma. These observations suggest that spontaneous fibrinolysis is mainly carried out by plasminogen/plasminogen activator bound to fibrin, and this fibrinolysis caused by fibrin-associated activation of plasminogen was mainly inhibited by alpha(2)PI cross-linked to fibrin. To further support this concept, alpha(2)PI treated with activated fibrin-stabilizing factor and that had lost most of its cross-linking capacity was used in similar experiments. This modified alpha(2)PI had the same inhibitory activity on plasmin as the native inhibitor, but gave significantly less inhibition of fibrinolysis in every experiment, particularly when the clot was compacted by platelet-mediated clot retraction or by squeezing. Thus, it was concluded that alpha(2)PI cross-linked to fibrin plays a significant role in inhibition of physiologically occurring fibrinolysis. It is further suggested that the absence of cross-linked alpha(2)PI contributes to accelerated fibrinolysis and hemorrhagic tendency in patients with congenital deficiency of fibrin-stabilizing factor.

Abnormal Plasminogen: A HEREDITARY MOLECULAR ABNORMALITY FOUND IN A PATIENT WITH RECURRENT THROMBOSIS

A patient who suffered a recurring thrombosis over the last 15 yr has been investigated. The only abnormality found in this patient was a significantly depressed level of plasminogen activity in plasma. In spite of the depressed plasminogen activity, the patient was found to have a normal level of plasminogen antigen concentration. It was calculated that the activity per milligram of plasminogen of the patient was approximately one-half the values of normal subjects. The same discrepancy between biological activity and antigen concentration was found in the other members of the kindred. A niece was found to have practically no plasminogen activity but possessed a normal concentration of plasminogen antigen. Both her parents were found to have approximately half the normal plasminogen activity and normal antigen levels. These studies suggested that the molecular abnormality was inherited as an autosomal characteristic, and the family members who had half the normal levels of activity with normal plasminogen antigen were heterozygotes whereas the one with practically no plasminogen activity was homozygote. Subsequent studies showed that the pattern of gel electrofocusing of purified plasminogen of the heterozygotes consisted of 10 normal bands and 10 additional abnormal bands, each of which had a slightly higher isoelectric point than each corresponding normal component. This indicates that plasminogen of the heterozygote is a mixture of normal and abnormal molecules in an approximately equal amount, which was substantiated by active site titration of purified plasminogen preparations obtained from the propositus and a normal individual. The gel electrofocusing pattern of the homozygote consisted of abnormal bands only. The defect is a hereditary abnormality of plasminogen.

Congenital Deficiency of α2-Plasmin Inhibitor Associated with Severe Hemorrhagic Tendency *

alpha(2)-Plasmin inhibitor (alpha(2)PI) is a recently characterized, fast-reacting plasmin inhibitor in human plasma that appears to play an important role in regulation of in vivo fibrinolysis. We report here a case of complete deficiency of alpha(2)PI in man. The patient, a 25-yr-old Japanese man, had a life-long severe bleeding tendency (hemarthrosis and excessive bleeding after trauma). The following tests were within normal limits: platelet count, bleeding time, thrombin time, prothrombin time, partial thromboplastin time, titers of known clotting factors, platelet glass bead retention, Factor VIII-related antigen, platelet aggregation by ADP, collagen and ristocetin, and clot retraction. Routine liver function tests were also normal. The only abnormal finding was that whole blood clot lysis was extemely rapid and was complete in 4-8 h. The concentration of plasma protease inhibitors, including alpha(2)-macro-globulin, antithrombin III, alpha(1)-antitrypsin, and C1INH, were all normal. The concentration of alpha(2)-PI in the patients plasma, assayed by immunological methods, was <0.1 mg/100 ml (normal concentration, 6.1+/-0.88 mg/100 ml [mean+/-SE]) and functional assays showed a complete deficiency of alpha(2)PI. Addition of purified alpha(2)PI to the patients whole blood completely corrected the accelerated fibrinolysis. The patients parents, four siblings, and four other members of this family were asymptomatic, but the titers of alpha(2)PI in their plasmas were congruent with50% of normal pooled plasma. There were three consanguineous marriages in this family, and the alpha(2)PI deficiency appears to have been inherited as an autosomal recessive trait. We speculate that alpha(2)PI deficiency in this patient has led to uninhibited in vivo fibrinolysis that probably causes the severe hemorrhagic tendency. Thus, this study indicates the important role of alpha(2)PI in hemostasis.

Essential roles of sphingosine 1-phosphate/S1P1 receptor axis in the migration of neural stem cells toward a site of spinal cord injury

Neural stem/progenitor cells (NSPCs) migrate toward a damaged area of the central nervous system (CNS) for the purpose of limiting and/or repairing the damage. Although this migratory property of NSPCs could theoretically be exploited for cell‐based therapeutics of CNS diseases, little is known of the mechanisms responsible for migratory responses of NSPCs. Here, we found that sphingosine 1‐phosphate (Sph‐1‐P), a physiological lysophospholipid mediator, had a potent chemoattractant activity for NSPCs, in which, of Sph‐1‐P receptors, S1P1 was abundantly expressed. Sph‐1‐P‐induced NSPC migration was inhibited by the pretreatment with pertussis toxin, Y‐27632 (a Rho kinase inhibitor), and VPC23019 (a competitive inhibitor of S1P1 and S1P3). Sph‐1‐P does not act as intracellular mediator or in an autocrine manner, because [3H]sphingosine, incorporated into NSPCs, was mainly converted to ceramide and sphingomyeline intracellularly, and the stimulation‐dependent formation and extracellular release of Sph‐1‐P were not observed. Further, Sph‐1‐P concentration in the spinal cord was significantly increased at 7 days after a contusion injury, due to accumulation of microglia and reactive astrocytes in the injured area. This locally increased Sph‐1‐P concentration contributed to the migration of in vivo transplanted NSPCs through its receptor S1P1, given that lentiviral transduction of NSPCs with a short hairpin RNA interference for S1P1 abolished in vivo NSPC migration toward the injured area. This is the first report to identify a physiological role for a lipid mediator in NSPC migration toward a pathological area of the CNS and further indicates that the Sph‐1‐P/S1P1 pathway may have therapeutic potential for CNS injuries.

Aspirin resistance detected with aggregometry cannot be explained by cyclooxygenase activity : involvement of other signaling pathway(s) in cardiovascular events of aspirin-treated patients

Summary.  Objectives: Although the concept of aspirin resistance is extensively reported in medical literature, its precise mechanisms and clinical outcomes are largely unknown. In this study, we examined individual thromboxane biosynthesis and platelet aggregation in aspirin‐treated patients, and whether the results of a platelet aggregation test influenced clinical outcomes. Results: Subjects taking 81 mg of aspirin (n = 50) and controls (n = 38) were evaluated for platelet aggregation and platelet cyclooxygenase‐1 (COX‐1) activity by measuring collagen‐induced thromboxane B2 production. For aggregometry, both light transmission (LT) and laser‐light scattering methods were employed to quantitatively evaluate aggregate sizes and numbers. Aspirin treatment resulted in the inhibition of collagen‐induced platelet aggregation, particularly the transition from small to large platelet aggregates. Although platelet COX‐1 activity seemed to be uniformly inhibited in all patients, platelet aggregation studies showed great inter‐individual differences; variation in platelet COX‐1 activity only accounted for 6–20% of the individual aggregations. Factor analysis revealed the existence of a common factor (other than platelet COX‐1) that explained 48.4% of the variations in platelet aggregation induced by collagen, adenosine diphosphate (ADP), and collagen‐related peptide. We then prospectively enrolled 136 aspirin‐treated patients in our study, and we found that being in the upper quartile level of LT, or with large aggregate formation induced by collagen, was an independent risk factor for developing cardiovascular events within 12 months [hazard ratio (HR) = 7.98, P = 0.008 for LT; HR = 7.76, P = 0.007 for large aggregates]. On the other hand, the existence of diabetes mellitus was an independent risk factor for overall outcomes (HR 1.30–11.9, P = 0.015–0.033). Conclusions: Aspirin resistance expressed as unsuppressed platelet COX‐1 activity is a rare condition in an out‐patient population. Other factor(s) affecting collagen‐induced platelet aggregation may influence early outcomes in aspirin‐treated patients.

Plasminogen Activator Inhibitor 1 Promotes a Poor Prognosis in Sepsis-Induced Disseminated Intravascular Coagulation

Sepsis-induced disseminated intravascular coagulation (DIC) is a serious condition because it is closely linked to the development of multiple organ dysfunctions.We compared molecular fibrinolysis markers for 117 patients with sepsis-induced DIC and 1627 patients with nonseptic DIC. Levels of fibrinogen and fibrin degradation products and D-dimer were significantly lower in sepsis-induced DIC cases than in nonseptic DIC cases. In septic DIC cases, plasma plasminogen activator inhibitor 1 (PAI-1) levels were significantly higher than in nonseptic DIC cases. D-dimer levels were negatively correlated with plasma PAI-1 levels in septic DIC cases. Multiple Organ Dysfunction Scores were significantly higher in septic DIC patients with PAI-1 levels >90 ng/mL than in the group with PAI-1 levels <30 ng/mL. The Kaplan-Meier survival functions until 28 days after DIC diagnosis were significantly lower in the group with PAI-1 levels >90 ng/mL than in the other groups. In a multivariate analysis, plasma PAI-1 levels at DIC diagnosis were an independent risk factor for mortality in sepsis-induced DIC (hazard ratio, 1.012; P = .008). These data suggest that plasma PAI-1 plays an important role in sustaining DIC in septic DIC cases and contributes to multiple organ failure and decreased survival in such patients.

Il2rg Gene-Targeted Severe Combined Immunodeficiency Pigs

A porcine model of severe combined immunodeficiency (SCID) promises to facilitate human cancer studies, the humanization of tissue for xenotransplantation, and the evaluation of stem cells for clinical therapy, but SCID pigs have not been described. We report here the generation and preliminary evaluation of a porcine SCID model. Fibroblasts containing a targeted disruption of the X-linked interleukin-2 receptor gamma chain gene, Il2rg, were used as donors to generate cloned pigs by serial nuclear transfer. Germline transmission of the Il2rg deletion produced healthy Il2rg(+/-) females, while Il2rg(-/Y) males were athymic and exhibited markedly impaired immunoglobulin and T and NK cell production, robustly recapitulating human SCID. Following allogeneic bone marrow transplantation, donor cells stably integrated in Il2rg(-/Y) heterozygotes and reconstituted the Il2rg(-/Y) lymphoid lineage. The SCID pigs described here represent a step toward the comprehensive evaluation of preclinical cellular regenerative strategies.

Antagonism of Sphingosine 1-Phosphate Receptor-2 Enhances Migration of Neural Progenitor Cells Toward an Area of Brain Infarction

Background and Purpose— We have previously shown that the sphingosine 1-phosphate (S1P)/S1P receptor-1 (S1P1R) axis contributes to the migration of transplanted neural progenitor cells (NPCs) toward areas of spinal cord injury. In the current study, we examined a strategy to increase endogenous NPC migration toward the injured central nervous system to modify S1PR. Methods— S1P concentration in the ischemic brain was measured in a mouse thrombosis model of the middle cerebral artery. NPC migration in vitro was assessed by a Boyden chamber assay. Endogenous NPC migration toward the insult was evaluated after ventricular administration of the S1P2R antagonist JTE-013. Results— The concentration of S1P in the brain was increased after ischemia and was maximal 14 days after the insult. The increase in S1P in the infarcted brain was primarily caused by accumulation of microglia at the insult. Mouse NPCs mainly expressed S1P1R and S1P2R as S1PRs, and S1P significantly induced the migration of NPCs in vitro through activation of S1P1R. However, an S1P1R agonist failed to have any synergistic effect on S1P-mediated NPC migration, whereas pharmacologic or genetic inhibition of S1P2R by JTE-013 or short hairpin RNA expression enhanced S1P-mediated NPC migration but did not affect proliferation and differentiation. Interestingly, administration of JTE-013 into a brain ventricle significantly enhanced endogenous NPC migration toward the area of ischemia. Conclusions— Our findings suggest that S1P is a chemoattractant for NPCs released from an infarcted area and regulation of S1P2R function further enhances the migration of NPCs toward a brain infarction.

Prevalence of genetic mutations in protein S, protein C and antithrombin genes in Japanese patients with deep vein thrombosis

INTRODUCTION Genetic deficiencies of PROS1, PROC, and SERPINC1 (antithrombin) are risk factors for deep vein thrombosis (DVT). Diagnosis of the inherited deficiencies of these three genes is sometimes difficult because of the phenotypic variability. This study was undertaken to reveal the frequency of nonsynonymous mutations of these three genes in Japanese DVT patients. PATIENTS/METHODS One hundred seventy-three DVT patients were registered by the Sub-group of Blood Coagulation Abnormality, from the Study Group of Research on Measures for Intractable Diseases. We sequenced the entire coding regions of the three genes in all DNA samples and identified the nonsynonymous mutations. RESULTS AND CONCLUSIONS For PROS1 we identified 15 nonsynonymous mutations in 28 DVT patients; for PROC, 10 nonsynonymous mutations in 17 patients; and for SERPINC1, 13 nonsynonymous mutations in 14 patients. Five patients had two mutations in PROS1 and PROC, and all of them had PROS1 K196E mutation. We previously identified one patient with a large PROS1 gene deletion. Thus, 55 out of 173 patients (32%) carried at least one genetic defect in the three genes. The PROS1 K196E mutation found in 15 Japanese DVT patients was the most prevalent. Mutations of PROC K193del and V339M were the second, each found in four patients. Our data suggested that the PROC K193del mutation caused the loss of the anticoagulant activity but not the amidolytic activity. Our effort is the first DNA resequencing study to identify the genetic variations in DVT patients without any consideration of their plasma activities and antigens. To minimize selection bias in a future evaluation of the contribution of genetic deficiency to DVT, we must recruit patients consecutively.

Efficient expression of a transgene in platelets using simian immunodeficiency virus-based vector harboring glycoprotein Ibα promoter: in vivo model for platelet-targeting gene therapy

Platelets release several mediators that modify vascular integrity and hemostasis. In the present study, we developed a technique for efficient transgene expression in platelets in vivo and examined whether this targeted‐gene‐product delivery system using a platelet release reaction could be exploited for clinical applications. Analysis of luciferase reporter gene constructs driven by platelet‐specific promoters (the GPIIb, GPIbα, and GPVI) revealed that the GPIbα promoter was the most potent in the megakaryoblastic cell line UT‐7/TPO and human CD34+‐derived megakaryocytes. Transduction of UT‐7/TPO;CD34+‐derived megakaryocytes; and c‐Kit+, ScaI+, and Lineage− (KSL) murine hematopoietic stem cells with a simian immunodeficiency virus (SIV)‐based lentiviral vector carrying eGFP resulted in efficient, dose‐dependent expression of eGFP, and the GPIbα promoter seemed to bestow megakaryocytic‐specific expression. Transplantation of KSL cells transduced with SIV vector containing eGFP into mice showed that there was preferable expression of eGFP in platelets driven by the GPIbα promoter [7–11% for the cytomeglovirus (CMV) promoter, 16–27% for the GPIbα promoter]. Furthermore, transplantation of ex vivo‐transduced KSL cells by SIV vector carrying human factorVIII (hFVIII) driven by the GPIbα promoter induced the production of detectable transcripts of the hFVIII gene and the hFVIII antigen in bone marrow and spleen for at least 90 days and partially corrected the hemophilia A phenotype. Platelet‐targeting gene therapy using SIV vectors appears to be promising for gene therapy approaches toward not only inherited platelet diseases but also other hemorrhagic disorders such as hemophilia A.—Ohmori, T., Mimuro, J., Takano, K., Madoiwa, S., Kashiwakura, Y., Ishiwata, A., Niimura, M., Mitomo, K., Tabata, T., Hasegawa, M., Ozawa, K., Sakata, Y. Efficient expression of a transgene in platelets using simian immunodeficiency virus‐based vector harboring glycoprotein Ibα promoter: in vivo model for platelet‐targeting gene therapy. FASEB J. 20, E769–E779 (2006)